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DNA sequences and comparison of argininosuccinate synthetase genes from two methanogenic archaebacteria

Morris, Christina Jane, Ph.D.

The Ohio State University, 1987

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 PLEASE NOTE:

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University Microfilms International DNA SEQUENCES AND COMPARISON OF AR6IHIN0SUCCINATE SYNTHETASE

GENES FROM TWO METHANOGEN1C ARCHAEBACTERIA

DISSERTATION

Presented in Partial Fulfillment of the Requirement for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

By

Christina Jane Morris, B Sc , M.S.

The Ohio State University

1987

Reading Committee: Approved By

John N. Reeve

Charles J Daniels

Darrell R. Galloway

Berl R. Oakley Adviser

Department of Microbiology PLEASE NOTE:

Copyrighted materials in this document have not been filmed at the request of the author. They are available for consultation, however, in the author's university library.

These consist of pages:

Cartoon in pre-pages

University Microfilms International 300 N ZE EB RD . ANN AH BOB. Ml 48106 (313) 7fj 1 4 700 T o Mum and Dad

ii ACKNOWLEDGEMENTS

I uiould like to thank my adviser, Dr John Reeve, for his advice, encouragement and support throughout this project

Thanks also go to the past and present members of Dr Reeve's and Dr Daniels’ labs for their assistance, ideas, understanding in times of need and, most of all, friendship Special thanks go to Dr Chuck Daniels for the use of his computer and office.

To quote Maureen Stapleton: "I mould like to thank everyone I’ve ever met in my mhole life", but perhaps I’ll keep it short and finally thanx my family for their love, support and phone calls VITA

October 4, 1959 ...... Born - Bristol. England.

1981 ...... B.Sc. (Honours), Queen Mary Col lege, Uni vers it y of London/ London. England.

1981-1982 ...... Teaching Associate. Department of Microbiology, The Ohio State University. Columbus. Ohio, USA

1982-1987 Research Associate, Department of Microbiology, The Ohio State University

1983 ...... M S The Ohio State Un i versity

1987 ...... UCLA International School on Mo1ecu 1ar Evo1ut ion Fellowship. Los Angeles, Ca1i forn i a

PUBLICATIONS

Morris, C.J. and JN. Reeve <1984). Functional expression of an archaebacterial gene from the methanogen Methanosarcina barken in Escherichia coli and Bacillus subtilis In, Microbial Growth on Cl Compounds.

Morris, C J and J N Reeve <1985) Expression of a methanogen gene in Escherichia coli and in Bacillus subtilis In, Proceedings of the First International Symposium on Biotechnological Advances in Processing Municipal Wastes for Fuels and Chemicals

Reeve, J.N., P.T Hamilton, G.S. Beckler, C.J. Morris and C H Clarke <1986). Structure of methanogen genes. Syst Appl. Microbial. 7-. 5-12

iv Reeve, J N., G.S. Beckler, J.W Brown, D.S. Cram, E.S. Haas, P.T. Hamilton, C.J. Morris, B.A, Sherf and CF. Weil <1986). Divergence of methanogens, conservation of the his 1 gene sequence in all three biological kingdoms and the status of Methanohacterium thermoautotrophicLim Proceedings of the Fifth Inter national Symposium on Growth on Cl Compounds. Haren, Netherlands, P255-260.

FIELD OF STUDY

Molecular Biology TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT...... ni

UITA ...... iv

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF ABBREUIAT IONS x

INTRODUCTION I

LITERATURE REUIEW 4 Genome structure ...... 4 DNA-dependent RNA polymerases ...... 6 Ar chaebac terial ribosomes ...... 7 7S RNA 11 Protein-encoding genes from ar'chaebacteria 11 Computer analysis of amino acid and DNA sequences ...... 14 Argininosuccinate synthetase- genes and pseudogenes ...... 17

METHODS ...... 19 Grouith of cells ...... 19 Isolation of methanogen DNA ...... 19 Large scale isolation of plasmids from E, coli ...... 21 Large scale isolation of plasmids from £ subtilis 23 Small scale isolation of plasmids from E coli and £ subtilis ...... 24 Preparation and transformation of competent cells of £ coli...... 25 Preparation and transformation of competent cells of £ subtilis 26 Restriction and ligation of plasmid DNA...... 27 Agarose and polyacrylamide gel electrophoresis ...... 29 Minicell preparation and radioactive labeling of plasmid encoded polypeptides ...... 29 Nick translation ...... 31 vi Radioactive labeling of oligomer probes 32 DNADNA hybridization with dehydrated agarose gels ...... 33 DNADNA hybridization with Zeta-Probe membrane. 34 Maxam and Gilbert sequencing 35 6 reaction ...... 37 G + A reaction 38 C * T reaction ...... 38 C reaction ...... 38 Strand cleavage reaction ...... 39 M13 sequencing. . 40

RESULTS...... 43 Construction of plasmids pET821, pET822, pET8£3, PET824 and pET825 43 Expression of methanogen derived DNA in JL suhtilis 46 Polypeptides synthesized in DS410 minicells encoded by pET821> pET823, pET824 and pET825 47 Nucleotide sequence of pET821 53 Nucleotide sequence of pET371 ...... 68 Codon usage ...... 67 Computer analysis of carbamyl phosphate synthetase genes ...... 70 Computer analysis of argininosuccinate synthetase genes ...... 76 Construction of an evolutionary tree for argminosuccinate synthetase genes ...... 86 Occurrence of the 14 bp and 29 bp direct repeats in £3 barken MS and £3 barkeri 227 89

DISCUSSION...... 94 Expression of methanogen derived DNA in tuuo eubacterial systems ...... 94 Sequences needed for expression of genes in methanogens ...... 98 U barkeri direct repeats ...... 99 Codon usage and gene organization in £3 barkeri and £3 uannielii 101 Evolutionary relationship of ar gininosuccinate synthetase genes 103 Introns within the human argminosuccinate synthetase gene 105 Carbamyl phosphate synthetase in£3 barkeri 106

CONCLUSIONS 108

BIBLIOGRAPHY 110

vii LIST OF TABLES

Table P age

1 List of microorganisms 20

2 Codon usage 68

3 X Similarity between carbamyl phosphate synthetases of different species 77

4 X Similarity between argminosuccinate synthetases of different species. 85

viii LIST OF FIGURES

Figure Page

1 Plasmids derived from pET821 ...... 44

2. Autoradiogram of the electrophoretic separation of radioactively labeled polypeptides in plasmid containing £.. coli minicells ...... 49

3 Autoradiogram of the electrophoretic separation of radioactively labeled polypeptides in plasmid containing £.. coli mimcells ...... 51

4 Detailed restriction map of 2.1 Kbp of pET821 DNA ...... 54

5 Nucleotide sequence of 2 1 Kbp of pET821 DNA 57

6, Detailed restriction map of pET371 DNA ...... 61

7. Nucleotide sequence of pET371 DNA ...... 63

8 Diagon analysis of carbamyl phosphate synthetase ...... 71

9. Carbamyl phosphate synthetase amino acid comparison ...... 74

18. Diagon analysis of argininosuccinate synthetase ...... 78

11. Argininosuccinate synthetase amino acid comparison ...... 80

12 Amino acid residues conserved between M barken. £1 vanmelii and human argininosuccinate synthetase sequences 82

13. Evolutionary tree for argminosuccinate synthetase ...... 87

14. Autoradiogram of DNA^DNA hybridizations between fcl harkeri DNA and radioactively labeled PET371 probes . . 91

IX LIST OF ABBREVIATIONS

Abbreviation. Full Name

A adenosine

ADP adenosine diphosphate aEF archaebac teria) elongation factor ala alanine

AMP adenosine monophosphate

AMP ampicillin asn asparagine asp aspartic acid

ATP adenosine triphosphate

Aggg absorbance at 260 nm

A*5Q0 absorbance at 580 nm bp base pair

C cytosine

C- carboxyl

CAM chloramphenicol

CAT chloramphenicol acetyl transferase cDNA complementary DNA

CoM coenzyme M

(2-mercaptoethanesulfonic acid) ddNTP dideoxy NTP

DNA deoxyribonucleic acid dNTP deoxy NTP

DTT dithiothreitol

EDT A ethylenediaminete trace tic acid

EF elongation factor x EMBL European Molecular Biology

Laboratory

EtBr ethidium bromide

G guanosine g grams g gravitational force h hour

HumASPs human argininosuccinate synthetase

pseudogene lie isoleucine

1PT6 isopropyl thiogalac toside

KAN kanamycin

Kbp kilobase pairs

1 liter

LB Luria broth leu leucine

M molar mg milligram's) min minute(s) ml millili ter

MM M9 minimal medium mM millimolar mRNA messenger RNA

N any base A, C, G or T

N- amino nm nanometers

0^860 optical density at 860 nm

ORF open reading frame

PAGE polyacrylamide gel electrophoresis

XI PAUP Phylogenetic Analysis Using

Parsimony

PFP paraf luor opheny Lalanine phe phenylalanine

PIR Protein Information Resource

PPi inorganic pyrophosphate

PFO 2,b, diphenyl-l,3-oxazole pro proline

R purine A or G

RNA ribonucleic acid rRNA ribosomal RNA

S Svedberg sedimentation unit

Sfljj association coefficient

SDS sodium dodecyl sulfate ser serine

SSC standard sodium citrate

<4 41g Na citrate^l, 877g NaCl^l)

T thymidine

TA Trts acetate (40mN Tris.HCl pH 8 0,

0.2mM CH3COONa, 2mM EDTA)

TBE Tris borate (89mM Tns.borate,

89mM boric arid, 2mM EDTA, pH 8 0)

TCA trichloroacetic acid

TE 18mM Tris.HCl pH 8 5, ImM EDTA

TEMED N,N,N' ,N' ~tetramethylethylenediamine

TES 30mM Tris.HCl pH 80, 50mM NaCl,

5mM EDTA

TET tetracycline

Tris tris(hydroxymethyl>aminomethane tRNA transfer RNA trp tryptophan xii tyr tyrosine

U uracil

UU ultraviolet

U volts uj/ u weight by volume

X-gal 5-bromo-4-chloro-3 indoly 1 galacto-

pyranoside in dimethy If or mamide

Y pyrimidine C or T

U9 microgram

Ml microliter (s > ju M micromolar

Mill Introduction

The existence of three primary kingdoms, eukaryotes, eubacteria and archaebacteria, uias proposed by Noese and Fox in 1977 (143) Before 1977, the classification of living organisms had separated them into the eukaryotes, those organisms which had a nuclear membrane and cellular organelles, and the prokaryotes, those that did not Further classification within these kingdoms using morphological and physiological traits, which could not be distinguished as having arisen through divergent or convergent evolution, was in retrospect misleading.

Zuckerkandl and Pauling (153) suggested the study of

"semantides* (informational macromolecules) would provide a better indication of evolutionary history and the advent of macromolecular sequencing has allowed this hypothesis to be tested. UJoese and Fox chose the 1£S ribosomal RNA CrRNA) component of the 30S subunit of the ribosome and its eukaryotic equivalent, the 18S rRNA from the 40S ribosomal subunit as the base for their studies. The 16S^18S rRNA molecule is ideal in that it is present in all organisms, maintains the same function in each, is sufficiently large and has both highly conserved and not so highly conserved sequences which allows analysis of both distant and close phylogenetic relationships. Data obtained from the cataloguing of oligonucleotides produced by ribonuclease (RNase) T1 digestion of 1£S/'18S rRNA molecules from very diverse organisms, suggested that the prokaryotes comprise two phylogenetically 2 distinct groups, now designated the eubacteria and the archaebacteria.

Thermoplasma sp , extreme thermophiles (or sulfur dependent archaebacteria), extreme halophiles and methanogens are grouped together to form the archaebacteria. Their classif ication as a kingdom, separate from the eubacteria and eukaryotes, is supported by their cell uiall composition <67, 73,

137>, SNA-dependent RNA polymerase subunit compositions <123,

152>, ether-linked lipids <57, 81, 129), transfer RNA base modifications (10, 48), 5S rRNA structure (41) and the presence of unique coenzymes <1, 3, 21, 37, 46, 87) Further inquiry into the molecular biology of the archaebacteria has shown that although they are prokaryotic and similar in size to the eubacteria, members of the archaebacteria also have features in common with the eukaryotes Intervening sequences (introns) have been found in rRNA and tRNA genes <29, 66, 69, 78),

SNA-dependent RNA polymerase subunits are immunologically related to eukaryotic RNA polymerases <150, 151) and diphtheria toxin is capable of ADP-ribosylating archaebacterial and eukaryotic elongation factors but not eubacterial elongation factor EF-G (68) It has been proposed, therefore, that

"simple" prokaryotes have not evolved into the more complex eukaryotes but that each of the three kingdoms has arisen independently from a common ancestor, the progenote (142, 143,

144)

Methanogens, obligately anaerobic organisms which generate methane by growth on Cl compounds, are the most diverse group within the archaebacteria. Studies of genome organization and gene structure in methanogenic archaebacteria were initiated in 1982 (16, 49, 109). It uias demonstrated that cloned fragments of methanogen genomic DNA could complement 3 Escherichia coli auxotrophic mutations in the his A <26, 145), PurE

<49, 50, 51>, proC (49, 50, 52) and *caG <49, 50, 145) genes 0 continuation of the study of the Methanosarcina barkeri derived

DNA capable of complementing £. coli argG and Bacillus subtilis ar

£L barkeri DNA, clone and determine the DNA sequence of the homologous gene from Methanococcus vannielii. a methanogen but very distant relative of £L barkeri. and make an evolutionary comparison to the human argininosuccinate synthetase cDNA (13)

These experiments mere undertaken to provide information and insight into the evolutionary relationships betuieen tuio methanogens and between members of the three biological kingdoms. LITERATURE REU1EM

The proposed evolution of the archaebacteria into a

phylogenetic ally separate kingdom is supported by studies of

their molecular biology Information that is currently available

is presented here to indicate the similarities betuieen the

archaebacteria and the differences between the archaebacteria

and the eubacteria and eukaryotes Knowledge pertaining to

methanogens will be emphasized.

Genome structure.

The prokaryotic nature of archaebacteria emphasizes the

similarities with the eubacteria. The genome of

Me th ano bacterium thermoautotrophicum at 1.1 x 109 daltons is

402 the size of the E. coli genome, whereas the Halobacterium

halobium genome is equivalent in size to that of L coli at 2.5 x

109 daltons <1111. A small percentage (€%> of the M.

thermnjiitnirnohirnm genome was determined, by renaturation

kinetics, to contain repeated DNA sequences <100) 1L halobium

has been shown to have a large number of repeated DNA

sequences arranged in clusters and dispersed around its

chromosome <111, 112). These repeated DNA sequences comprise

greater than fifty families, are highly mobile and repeat

sequence-associated genomic rearrangements occur at

frequencies >4 x 10-3 events per family per cell generation

<112). Nine different insertion elements have now been identified 4 in fcL hjlnhmm and one in the methanogen Methanohreuiharter

smithii (33, 52V The majority of these insertion elements ere

structurally similar to insertion elements in eubacteria and

eukaryotes; at least one open reading frame uiithin the element,

terminal inverted repeats and duplication of the target DNA

sequences.

The m ol/t G + C of the archaebacteria covers a very broad

range Within the three orders of the methanogens.

Me thanobac. ter tales . Methanococcales, and Methanomicrobiales.

molG + C values range from 25 8X (Methanosphaera stadtmamae)

to 61.2/f

acidophilum has a molZ G+C of 46 and the other members of the

sulfur-dependent, extremely thermophilic archaebacteria have

mo 1/i G + C values that range betuieen 31 and 62. The major ON A

fraction of the extreme halophiles has a narrower range of

mo IX G + C between 60 and 68, however molX G + C of the minor DNA

fractions of repeated DNA sequences are lower, between 49 7

and 59

Extrachromosomal DNA has been found in a number of

archaebacteria, most often in halophiles (101). The first report

of a cryptic plasmid in a methanogen was from JtL

thermoautotrophicum (88) and this has been more recently

followed by others in Methanococcus isolate PL-12/M <127) and

Methanococcus iannaschii (147). Baresi and Bertani <4) reported

the presence of a phage isolated from Methanohreuibacter sp ,

and a variety of other phages have been isolated from

Halobacterium and Thermoproteus s p p <60, 116, 130) Covalently closed circular prophages have been reported in Sulfolnbus spp

<84) and fcL iannaschii (146) To date, only DNA-containing phages have been identified. 6 DNA-dependent RNA Pfllumerases

Eubacteria have a single DNA-dependent RNA polymerase with a minimal subunit composition of p.D'jtzg,? (150) whereas there are three eukaryotic RNA polymerases I, II and 111 which have much more complex subunit configurations (151, 152).

Archaebacterial RNA polymerases are more similar to the eukaryotic RNA polymerases containing at least eight subunit polypeptides (151, 152). Antisera, raised against the large subunits of Saccharomuces carevisiae DNA-dependent RNA polymerases, cross react more strongly with archaebacterial

RNA polymerases than with eubacteria RNA polymerases (45, 152)

Based on immunological cross-reactivities of subunits, functional relationships have been proposed for the largest subunit B of the thermoacidophile enzyme with eubacterial 0 subunit, the second Largest eukaryotic subunit and subunits B' and B' ' of the methanogens and halophiles. The second largest subunit of the thermoacidophile enzyme, subunit A, is related to the p* subunit of E_ coli polymerase and the largest subunit of the eukaryotic polymerase. The effects of various antibiotics have also indicated similarities between the RNA polymerases of the three kingdoms. Both eukaryotic RNA polymerase 1 and 1. acidophilum RNA polymerase are stimulated by silybin whereas eubacterial RNA polymerases and eukaryotic RNA polymerases II and 111 are not (152). Archaebacterial RNA polymerases are similar to the eukaryotic RNA polymerases in that they are insensitive to rifampin and streptolydigin, inhibitors of eubacterial RNA polymerase (152) however like eubacterial RNA polymerases archaebacterial RNA polymerases are resistant to cc-amanitin, a strong inhibitor of eukaryotic RNA polymerases II and III (152) 7 Archaebacterial ribosomes

The archaebacterial and eubacterial ribosomes resemble

each other in size (70S), are comprised of two subunits <30S

and 50S>, and contain only three ribosomal RNAs, 16S, 23S and 5S

(134). Eukaryotic ribosomes are larger in size (80S), comprised

of two larger subunits (40S and 60S), and have, in most cases

(135), a 5.8S rRNA molecule in addition to 18S, 28S and 5S rRNAs.

Cataloguing of 16S and 18S rRNA oligonucleotides, resulting from

RNase T1 digestion, was the original basis for dividing the prokaryotes into the eubacteria and archaebacteria (2, 42, 143)

As complete sequences of 16S/'18S rRNA become available (65, 77,

83, 132, 148) phylogenetic relationships continue to became clearer. The complete sequences for nine archaebacterial 16S rRNAs have supported the concept of extensive diversity within

the archaebacterial kingdom and a clear distinction between the

archaebacteria, the eubacteria and the eukaryotes (148). The

sequence data divided the archaebacteria into two phyla, one composed of the methanogens, halophiles and Thermootasma spp

and the second composed of the aerobic and anaerobic extreme

thermophiles <2, 148). In addition, the sequences indicated a

specific relationship between the halobacteria and the

Methanomicrobiales. which was unclear when only partial

sequencing data was available (2).

The pyrimidine rich 3' end of the 16S rRNA molecule in eubacteria is involved in binding of mRNA to eubacterial ribosomes to initiate translation (119) A pyrimidine rich region is also present at the 3' end of the archaebacterial 16S rRNA molecule (2) and is assumed to function in a similar manner in methanogens (52). The data are ambiguous for a similar role in

H. halobium (11, 36) The bacterio-opsin mRNA of H. halobium contains only three nucleotides at the 5' terminus preceding 8 the initiation codon, uihich is much shorter than the Leader sequences found in most eubacterial mRNAs. 8 pyrimidine rich sequence complementary to the 3' end of the halobacterial 16S rRNA does occur howeuer, downstream of the initiation codon, within the protein encoding sequence

Archaebacterial SS rRNA secondary structures haue features in common with the eukaryotic or eubacterial SS rRNAs and also haue unique features <41> Halnbacterium tnorrhuae SS rRNA has an extra 188-nucleotide sequence present in its 231 nucleotide sequence that is otherwise analogous to

Halobactenum rutirubrum. howeuer the additional bases do not seem to disrupt the secondary structure of the molecule. An intron has been identified in the 23S rRNA gene of

Desulfurococrus mobilis <69). The intron contains the consensus sequence necessary for splicing of rRNA introns (class 1) in eukaryotes (19) Other proper ties howeuer, including putatiue base pairing between the ends of the RNA and the absence of a uridine at the 3' end of the 5' exon, are charac teristic of eukaryotic class 111 introns

The gene order of structural rRNAs in archaebacteria is similar to that in eubacteria. S' 16S-23S-SS, except in L acidonhilum where the genes are not Linked <56, 59) In the methanogens and halobacteria, the rRNA genes form an operon with transfer RNA (tRNA) genes between the 16S and 23S rRNA genes and in some cases also distal to the SS rRNA gene <139,

148). fcJL uannielii contains four rRNA operons and an additional unlinked SS rRNA gene; unlinked SS rRNA genes are a feature of eukaryotes <139, 141). The unlinked SS rRNA gene is Located in a cluster of seuen tRNA genes which may all be transcribed as an operon. In contrast to the methanogens, most other

archaebacteria studied so far, have the unusual feature of

only one set of rRNA genes.

Many tRNAs genes have been characterized, from a variety

of archaebacteria <29, 47, 48, 66, 141), the most extensive

studies describe the tRNA genes from Halobactenmm uoldanii <29,

47> and £1- uannielii <141). The archaebacteria! tRNA genes are

mostly similar to eukaryotic tRNA genes in that they do not

encode the 3' CCA terminal sequences found on mature tRNAs

<138) Only one tRNA gene

gene cluster which contains the 5S rRNA gene, encodes the 3'

CCA (138). To date, seven tRNA genes from archaebacteria have

been shouin to contain introns, tuuo from Sulfolohus

solfataricus. four from Thermoproteus tenax and one from H

volcanii <29, 66, 140). The £, solf ataricus tRNAser and tRNA**u

introns are 25 and 15 nucleotides long respectively, and the

lengths of the JL tenax intron sequences range from 16 to 20

nucleotides whereas the U. volcanii tRNA*,rp intron is 185

nucleotides in length. Most of the introns are located 3' to

the anticodon, as are introns in eukaryotic tRNAs. These tRNA

molecules are capable of forming a normal cloverleaf pattern

if the introns are not included in the tRNA secondary

structure. Three of the L tenax introns are unusual in that

they are located either within or 5' to the anticodon sequence

topics under investigation

Similarities between archaebacteria and eukaryotes have been found in studies of elongation factors involved in 10 translation Diphtheria toxin catalyzes the covalent binding of

A DP (adenosine diphosphate) to the eukaryotic polypeptide elongation factor EF-2. The toxin does not houiever ribosylate

the eubacterial equivalent of EF-2, namely EF-G Like the eukaryotic translocation factor, the archaebacterial aEF-2 contains a post-translational ly modified histidinyl r esidue, diphthamide, uihich is ADP-ribosylated by diphtheria toxin (68,

100). Although the eukaryotic and archaebacterial elongation

factors are similar they cannot substitute for each other or

for eubacterial EF-G in in vitro polypeptide synthesis using ribosomes prepared from organisms from each kingdom <71) The

aminoacyl-tRNA binding factor from archaebacteria aEF-lcc is inter changeable in vitro with the eukaryotic EF-lac and probably

the eubacterial EF-Tu (71). However eukaryotic and eubacterial

aminoacyl-tRNA binding factors are rarely interchangeable

Archaebacterial and eubacterial growth is sensitive to neomycin, virginiamycin and chloramphenicol however studies in vitro

showed streptomycin and chloramphenicol, inhibitors of eubacterial 70S ribosomes, do not bind to ribosomes from M. barkeri (115). This explains the resistance of all archaebacteria

to streptomycin. Inhibition of methanogen growth by chloramphenicol is related to the presence of a nitro group on

the chloramphenicol molecule. This compound probably acts as

an oxidizing agent (6). Anisomysin, an inhibitor of eukaryotic

translation <113) and virginiamycin, an inhibitor of eubacterial ribosomes, both inhibit ribosomes from methanogens

Archaebacteria] and eubacterial ribosomes generally contain a similar number of proteins, although in

Methanosarcina, Methanococcus and Sulfolobus. there are more proteins associated with the 30S subunit <85). The number of

acidic ribosomal proteins tends to be higher in archaebacterial ribosomes and this seems to correlate with the cytoplasmic

concentrations of ions in halobacteria and some methanogens

(6!) The ribosomal ft proteins of £L thermaautotrophicum and U.

show more sequence similarity to the eukaryotic A

protein than to the equivalent eubacterial component LZ/LIS

(63). The archaebacterial 5S rRNft — binding proteins also have

more sequence similarity to the eukaryotic than the eubacterial

equivalent proteins. In contrast, archaebac terial proteins uihich

are similar to the eubacterial proteins S9 and S15 do not shouu

any similarity to eukaryotic proteins (63>

7S RNA

Archaebacteria contain large amounts of an additional

stable RNA molecule, not associated with the ribosome whose

function is currently unknown (79, 89). There are similarities

between the sequences of the H. halohium 7S RNA molecule and

the mammalian 7S-L RNA, which is a component of the mammalian

signal recognition particle (89). The E, coli 6S non ribosomal

RNA does not appear similar to the archaebacterial and

eukaryotic 7S RNfts.

Protein-encoding genes from archaetaacteria.

The first protein—encoding archaebacterial gene to be

studied in detail was bap from H. halohium which encodes

bacterio-opsin (11, 36) The amino acid sequence of

bacterio-opsin and the UNA sequences of the hup and bro

(bacterio—opsin-related protein) genes have been determined (11,

36) Their comparison indicated that the halobacteria use the

standard genetic code and that no intervening sequences are present in the bap gene. Subsequent studies of protein-encoding archaebacterial genes have confirmed these 12 observations. The lack of observed introns may be misleading as a generality due to cloning of genes in £_ call by demanding their functional expression in L coli. Genes cloned from M smithii <49, 50, 51, 52), CL barken <15, 49, 50), CL thermalithotr oohicns <136), CL uannielii <7, 25, 26),

MethanocnccLis voltae <26, 72, 145), CL thermoautotrophicum <14,

51), and Methanobreuibacter arboriohilus <16) have been seen to direct polypeptide synthesis in E. coliA number of these genes complement L coli auxotrophic mu tat ions, his A <26, 145), his I <7>, proC <49, 50, 52), PiirF <49, 50, 51), and arg 6 <49, 50, 145), and uiith the exception of aroS cloned from CL voltae <145), the cloned DMA sequences encoding these genes also contain promoters recognized by L coli RNA polymerase. Promoter sequences recognized by £. coli RNA polymerase do not appear to be the same as the sequences recognized by archaebacterial

RNA polymerase. Conserved sequences upstream of CL uannielii and CL voltae tRNA genes

RNA polymerase binding sequences upstream of the CL uannielii hisA gene that are similar to the proposed promoter consensus sequence for CL uannielii tRNA genes which also binds RNA polymerase <18).

Methyl coenzyme M

Studies have shouun that in all cases the genes are arranged in

a cluster in the order mcrB

25, ?2). DNA sequencing has located two additional open reading

frames mcrD and mcrC. between genes mcrB and julcxiG <14.

15, 25), however the function of their products is unknown

The mrr genes are thought to be transcribed as an operon into a single transcript since a probable promoter sequence exists upstream of mcrB and the translation initiation codons of the other four ORFs are located very close to the termination codon of the preceding QRF. Sequences complementary to the

3' end of 16S rRNA are found preceding all five ORFs in the mcr operons <14, 15, 25). Codon usage of the £L vannielii mcr ft ■ mcrB and mcrS genes indicates a strong preference for codons with a

C in the wobble position in the codon pairs ftflC/U

(asp), CACs'U , AUC/U <25>

The mrrC and mcrD genes within the cluster and other M. vannielii genes do not show this preference and usually have a

U or A in the wobble position The codon pairs above are likely to be translated by the same tRNA with a 6 in the first anticodon position (138) It has been suggested therefore, that the presence of a C in the wobble position might ensure maximum efficiency of translation of transcripts of highly expressed genes such as those encoding methyl reductase

Codon usage within the £L thermoautotrophicum and £1. barkeri mcr gene clusters is in accordance with this hypothesis <14, 15), although the preference for a U or A in the wobble position of codons in the genes of these organisms is not as strong as in

M_ vannielii Comparison of protein and TNA sequences of the mcr genes from the different methanogen species should provide information on conserved structures and possible 14 functional domains Since methyl CoM reductase is common to

all methanogens, comparison of sequencing data will also provide

further insight into their divergence during evolution.

Genes cloned from different methanogenic species that

complement the same L coli mutation have been compared <26,

51). The purl genes from a thermophilic methanogen, M.

thermoautotrophicum. and a mesophilic methanogen, fl. smithii are

532 identical and encode polypeptides that are 742 similar if

identical and conservative amino acid substitutions are considered <51) Genes cloned from LL vannielii and tL voltae

that complement an £. coli hisA mutation are 662 identical and encode polypeptides that are 762 similar if identical amino acids

and conservative substitutions are considered <26). In both cases the genes were cloned from methanogenic species within

the same order, tL thermautntrnohinim and tL Smithii are classified as Methanohacteriales. and tL vannielii and tL voltae are classified as Methanococcales <2) The comparative sequencing data was therefore in agreement with the proposed classification. A gene cloned from tL vannielii capable of complementing an £. coli hisl mutation has been compared to the homologous genes from JL coli. Salmonella tuphimurium.

Neurosoora crassa and S. cerevisiae (7). It was concluded that all the genes were derived from a common ancestral gene and were indicative of the overall relatedness of the eubacteria, archaebacteria and eukaryotes

Computer analysis of amino acid and DMA sequences.

The use of computers in the analysis of amino acid and

DNA sequences has increased in recent years <34). Uery simple computer programs are available that search DNA sequences for open reading frames

131) and can predict DNA and RNA secondary structures. A number of databanks are now commercially auailable that list published amino acid and DNA sequences eg. P1R (Protein

Information Resource) and EMBL (European Molecular Biology

Laboratory) databanks respectively. These databanks can be accessed through computer programs, such as the IBM Genepro program, that search through the sequences of the databank using any of a number of characteristics as a basis for the search A search of the databank titles can be made to identify sequences that encode proteins of similar function using key words such as hydrogenase or dehydrogenase.

Searches can also be made of all available sequences from a particular organism eg. human, Uicia faba or L coli. If the identity of a sequence is known eg. in this study argininosuccinate synthetase, its name can be used to search the databank for analogous sequences isolated from different organisms. Should the identity of a sequence be unknown a computer search can compare its amino acid or DNA sequence to those of the databank and determine which sequences in the databank, if any, are similar If there is a sequence within the databank that appears similar to the unknown sequence their relationship, or lack thereof, can also be determined using available computer programs An alignment program eg DNAstar

ALIGN, will compare and align two or more sequences and maximize their similarity by introducing gaps within the sequences. It is best to align amino acid sequences since DNA sequences are predicted to have at least 25% similarity by random chance as there are only four possible bases at each compared location (93). Diagon analyses of DNA and amino acid 16 sequences can also be used to determine the extent of relationships between two sequences ( 121) If a sequence is compared to itself in a Diagon analysis, the plot will indicate if internal homologies are pre' ent in the amino acid sequence or if a SNA sequence contains inverted or direct repeats.

A number of computer programs are now used to predict the secondary strueture of an amino acid sequence eg the

Beckman Microgenie program which uses the Chou and Fasman algorithm <22, 23, 24). There is controversy however as to the accuracy of these predictions and X-ray crystallography remains the preferred method (43, 64) Determination of the hydropathicity of an amino acid sequence is possible through programs using algorithms based on calculations made by Kyte and Doolittle <74) and Hopp and kloods (58). This can be useful in identifying likely antigenic determinants of proteins.

Hydrophilic regions of an amino acid sequence are more likely to be located on the exterior of a folded protein and therefore presented as an antigen Antibodies raised against a synthetic peptide, synthesized with the same hydrophilic sequence of amino acids, would therefore be likely to recognize and bind to the complete protein.

Computer programs are available which predict evolutionary relationships between DNA or amino acid sequences and hence between the organisms from which they originate A program developed by Lake (75) determines an unrooted tree for four DNA or RNA sequences by the method of evolutionary parsimony. The PAUP program, developed by Swofford, can use

DNA, RNA or amino acid sequence alignments to determine the most parsimonious tree (rooted or unrooted) for four or more taxa (44, 126) In the latter program, different weightings can be chosen by the researcher to compensate for conservative 17 amino acid substitutions, transitions verses transuersions in SNA sequences, single event deletions etc , allouiing more flexibility in the analysis of the data

Overall therefore, the extent to which a sequence can be analyzed has recently increased rapidly as more advanced computer programs have become available

Argininosuccinate sunthetase genes and pseudogenes.

Previously me showed that a gene cloned from H barken ivas capable of conferring arginine independence to £. coli X760.

The data indicated that the DNA encoded argininosuccinate synthetase, an enzyme which catalyzes the penultimate step of arginine biosynthesis in £. coli:

L-citrulline -*■ L-aspartate ♦ ATP

------> AMP + PPi + ar gininosuccinate

6enes encoding ar gininosuccinate synthetase (argG) have also been cloned from d. voltae <145), L coli (28>, Streptomuces cattleua <124), tL crassa <31), cerevisiae (31) and human (13), however only the human cDNA clone has been sequenced completely <13). The human genome contains one expressed arginmosuccinate synthetase gene and approximately 14 pseudogenes that are dispersed on at least 11 chromosomes (5)

The pseudogenes are not expressed in human cells. There are at least 13 exons contained in the expressed gene which spans a total of 63 Kbp, 7 of the intron/exon boundaries have been determined <39, 48) The pseudogenes have presumably resulted from reverse transcription of the ar gininosuccinate synthe tase mRNA and insertion of the resultant cDNA back into the human chromosomes. Some of the ar gininosuccinate synthetase pseudogenes appear to have resulted from duplication of other argininosuccinate synthetase pseudogenes (94). A number of the 18 pseudogenes have been cloned, sequenced and compared to the human argininosuccinate synthetase cDNA sequence which provides an indication of their evolutionary relationships <62,

94>

In this study, an additional argfi gene was cloned, from M. vannielii The sequences of the tL vannielii and tL frarkfeTJ argS genes were determined and compared. This represents a comparison of homologous sequences from species of different orders of methanogens, the Methanococcales and

Methanomicrobiales respectively <2> Comparisons to the human cDNA sequence and two pseudogenes were made to construct an evolutionary tree for argininosuccinate synthetase METHODS

Srouith of cells

E, coli and D subtilis strains (Table I) were grown in LB broth as rich medium which contains 10g tryptone, 5g NaCl and

5g yeast extract per liter H^O, adjusted to pH 7 0 with 10M

NaOH, 15g of agar was added per liter to solidify the medium for plates. Stock solutions were used to produce final antibiotic concentrations of 100 jug ampicillin/ml (AMP), 5 jug kanamycin/ml (KAN) and 30 jug chloramphenicol/ml (CAM)

Determination of complementing ability used plates containing M9 minimal medium (32)- NagHPO^ 6 g/L KHgPO^, 3 g^l,

NaCl, 05 g^l; NH4CI, 1 g-^1; CaClg, 01 mM; Mg SO 4, 10 mM; glucose,

0.4/i ui/vj amino acids, 40 jug-'ml; vitamins, 1 jig-'ml, agar, 15 g^l.

Clones containing pHE3-based recombinant plasmids transformed into £. coli RR28 cells were selected on M9 minimal medium containing 200 jug para-fluorophenylalanine/ml (PFP) and 30 uig

CAM/ml (55).

Isolation of methanogen DNA (49).

Frozen, pelleted cells of Methanosarcina barker! strains

MS and 227 were crushed for 10 min, with a pestle, in a mortar pre-cooled with liquid nitrogen. Repeated additions of liquid nitrogen to the cells aided their disruption. Cell debris was transferred to a screw-capped test tube containing 10 ml TE buffer <10 mM Tris.HCl pH 85, 1 mM EDTA), TE-equilibrated phenol

19 SB Table 1

Microorganisms used in this studu.

Mama Sauxt* Genotype

Mathanosarcina barken MS M.P Bryant mild type

Methanasarcina barken 227 M.P Bryant uiild type

Methanococcus uannielii ft Stadtman mild type

Q X 7 60 R Curtiss III iina-1, leu-1, an , tonftR. iAtY2, exoC119, tsx. purE 1. yaiK2, trp3. his ft 4 . ar_g636, CEil. xnl-1. mtl - 1. ili/06, thi-1, met -12

Fifhprichu coli DS418 J H Reeve jDlDfl, rnmB.

Escherichia cnli RR28 H Hennecke F~, thi-1, RCUP2, leuB6. lacVl. g*J.IC2, ara-14. mil-1. a.yi-5, A**, suoE44. hsrlR ” pbeSl?. recfl!3

Latiieru-lna coli GM272 CBSCN 6478 dcm-6. dam-3. metBl, hidS21, lacVl or lac?4. thi-1. oalKg. mtl-g. tonP2 or tonft3l. sup E44 tsx -1 or tajt-78, X®

Escherichia coli GM2L63 CGSCi 658) ara-14. leu06. tonOl3, lacVl. tSjt-78< sup E44 , aalKP. dcm-6. his64. ru tl3 E , dam-13: Tr>9. X®, isil-5. mtl-1. thi-1. h sdRg

Escherichia coli JM105 J Messing thi. CJtsL, endft , shcBlS. hsdR4. Adac-croflB). CF', traD36. prodB. laclq7^.M153

Esthenciua cob JP221 J Carbon hsdR. hidM, tne^tES, lfuB. recft

Bacillus subtilis MB97 S Baumberg ar0 0 2 . leufll

Bacillus subtilis IE6 0 Dean tCJ*C2, the-5

Bacillus subtilis CU4B3 JN Reeve thvifl. thy B. metB. dm IU 01 21

<10 ml> added and mixed gently uiith the lysate on a rotary shaken for 30 mm. The mixture uuas transferred to a 30 ml

Corex tube and the phases separated by centrifugation at 1000g for 5 min in the SS34 rotor. The upper aqueous phase was transferred to another screw-capped test tube, the phenol extraction repeated twice, the final aqueous layer transferred to a beaker, ls 9 volume of 3M C^COONa and 25 volumes of cold 95K ethanol added to precipitate nucleic acids which were harvested by centrifugation at 12,000g, 4°C for 30 min in the

SB34 rotor The pellet was redissolved in a minimal volume <<15 ml) of TE and treated with bovine pancreatic RNase <50 m g/'ml) for 30 min at 37°C.

7 5 at a concentration of 1 mg/'ml and heated at 80°C for 15 min to inactivate DNases) The phenol extraction and ethanol precipitation steps were repeated. The DNA was finally dissolved in a minimal volume <

Large scale isolation of Plasmids from E_ coli

E. coli cells, containing the desired plasmid, were grown at

37°C in 500 ml LB containing AMP <100 jig/ml) to an A580=0 6

CAM was added to a final concentration of 200 U 9 / mt and incubation continued overnight. Cells were harvested by centrifugation in a GSA rotor at 5,800g, 4°C for 10 min, washed by resuspension in 10 ml 25Z ui/v sucrose, 50 mM Tris HC1

8 0), transferred to a SS34 polypropylene tube and centrifuged at 4300g, 4°C for 10 min Cells were then resuspended in 3 ml 22 25% ui/w sucrose, 50 mM Tris.HCl, the mixture placed on ice for subsequent additions, 0 6 ml of lysozyme <10 mg/ml) added and the mixture incubated at 4°C for 5 min EDTft (16 ml, 250 mM) uias added, incubation continued for 5 min at 0°C, 4 8 ml of lytic mix <255i w/v Triton X-100, 50 mM EDTA, 50 mM Tris.HCl pH

8.0) added slowly and the mixture swirled gently. Complete lysis was obtained by a 20 min incubation at 0°C. The crude lysate uias spun at 27,000g for 30 min at 4°C to pellet cell debris and chromosomal DNA The cleared lysate supernatant containing the plasmid DNA, was carefully decanted into a beaker and the volume measured. An addition of lg CsCl uias made for every 1 ml of cleared lysate and the solution mixed to dissolve the CsCl. The volume was remeasured and 0.8 ml of ethidium bromide solution <10 mg/'ml) added for every 10 ml of

CsCl solution, bringing the final density to approximately 1.55 g/'ml. The solution was transferred to polyallomer UTi 65 centrifuge tubes, expelling all air. These tubes were heat sealed, capped and spun in a UTi 65 rotor, in a Beckman model

L8-70 ultracentrifuge for 16-20 h at 20°C at 180,000g Plasmid and chromosomal DNA bands were visualized by illumination with ultraviolet light <300 nm) The top of the tube was pierced with a syringe needle and the lower plasmid band recovered by inserting a 21 gauge needle, attached to a 1 ml syringe. Just below the band and withdrawing the syringe plunger An equal volume of isopropanol, saturated with an aqueous solution of

5M NaCl, 10 mM Tris.HCl (pH 8.5) and 1 mM EDTA, was added to remove the ethidium bromide from the plasmid DNA. The mixture separated into two phases and the upper phase was discarded.

Repeated extractions were made until all color had been 23 removed from the lower, DNA-containing phase Two volumes of sterile water and six volumes of cold 95/i ethanol were added and the mixture placed at -20°C overnight to precipitate DNA

A white DNA pellet, obtained by centrifugation at 4°C, 12,000g for 30 min in the SS34 rotor, was washed once with cold 70/i ethanol, dried and dissolved in 100-200 jul TE

Large scale isolation of Plasmids from B. sub tilts.

B subtilis cells were grown in 4x 300 ml LB or M9 minimal medium plus KAN (5 ug/'ml) at 37°C, until an A580 of 0 5 was reached Cells were harvested by centrifugation in a GSA rotor at 8,000g, 4°C for 20 min, washed by resuspension in 100 ml cold TES <30 mM Tris HC1 pH 8 0, 50 mM NaCl, 5 mM EDTA) and centrifuged in the 6 SA at 8,000g, 4°C for 20 min The washed cells were resuspended in 20 ml TES containing 20/i w/v sucrose and 1 mg lysazyme^ml and incubated with shaking at 37°C for lh to produce protoplasts. TES containing 8/i ws'u SDS <20 ml) was added slowly to induce lysis and 20 ml aliquots then transferred to 12 x 50 ml polypropylene tubes NaCl <5M) in

TES <5 ml) was added to each aliquot, the mixture incubated for 5 min at 6 8°C, 4°C for at least 8h, and then loaded into a pre-cooled SS34 rotor at 4°C and centrifuged at 39,000g for 30 m»n The resulting supernatants were decanted into four GSA centrifuge bottles. Two volumes of cold 95/i ethanol were added and the bottles placed at -20°C for lh DNA was precipitated by centrifugation in the GSA rotor at 16,000g, 0°C for 30 min, the ethanol decanted and the pellets dissolved in 24 25 ml TES Aliquots <12.5 ml) uuere transferred to 125 ml

Erlenmeyer flasks and mixed with an equal volume of phenol

equilibr a ted with TE The mixtures were t ransf erred to 30 ml

Corex tubes and incubated on ice far 15 min with gentle shaking

T wo phases were separ a ted by centnf ugation in the SS34 rotor

at 0°C, 12,000g for 10 min, the upper aqueous phase removed

and transferred to 125 ml Erlenmeyer flasks and an equal

volume of chlorof ormisoamylalcohol (241) mix added The

mixtures were transferred to 30 ml Corex tubes, incubated on

ice for 15 min with gentle shaking and the two phases

separated by centrifugation as described above The upper

aqueous phase was removed to 30 ml polypropylene tubes, two

volumes of cold 95X ethanol were added and DNA precipitated by

incubation at -20°C for at least lh. The DHA was sedimented by

centrifugation in the SS34 rotor, at 4°C and 27,000g for 20 min,

dried and then dissolved in TE for CsCl-EtBr centrifugation in

the UTi 65 as described above (see Large scale isolation of plasmids from £. coli.)

Small scale isolation of plasmids from E £.ali-4tfld fl—subtilis

C82).

Cells were grown overnight at 37°C in 5 ml of LB plus the appropriate antibiotic. Aliquots (15 ml) were transferred to

Eppendorf tubes and centrifuged for 1 mm in an Eppendorf model 5412 centrifuge (12,000g), the supernatants discarded, an additional 1.5 ml aliquot added to each tube and the centrifugation repeated A pellet obtained from a total of 3 ml of suspension, was resuspended in 100 jul of 50 mM glucose*

10 mM EDTA* 25 mM Tris.HCl (pH 8 0) containing 2 mg lysozyme^ml. 25 This mixture uias incubated on ice for 30 min,, 200 u 1 of 0 2N

NaOH containing i/i uj/u SDS unarmed to 56°C added, the tube inverted to mix the solutions and placed on ice for a further 5 min of incubation. To this mixture 150 jul of 3M CH3COONa

4.8) uias added, the solution mixed and the incubation continued for lh on ice. The crude lysate uias centrifuged for 5 min in the Eppendorf centrifuge ( 12,0009 ) to pellet cell debris The supernatant uias transferred to a clean tube for DNA precipitation by addition of tuio volumes of cold 9 57i ethanol and 15 min incubation at -70°C Tubes mere centrifuged for 5 min in the Eppendorf centrifuge to pellet DNA, uihich uias mashed once uiith cold 70/i ethanol and dried. Each pellet mas dissolved in 50 jul TE containing 1 pg RNase A/ml A 10 jul aliguot of DNA solution mas normally sufficient for visualization of plasmid DNAs folloming electrophoresis through horizontal

0.7/i ujs'v agarose gels (see belom)

Preparation and transformation of competent cells of E coli (9.

7fiX

Cells mere grouin in 580 ml of LB at 37°C until an A580 of

0.2 mas reached, then chilled by incubation for 20 min on ice.

All subsequent steps mere belom 4°C Cells mere harvested by centrifugation in a 6 SA rotor at 7,500g, for 15 min, resuspended in 200 ml 100 mM CaClg and incubated for 20 min Cells mere sedimented by centrifugation at 7,500g for 15 min. Resultant cell pellets mere resuspended in 5 ml 100 mM CaCl^ and incubated for 20-24h Cold glycerol mas added (final concentration I05i m/'v) and aliquots (500 jul) transferred to 1.5 ml Nunc (Uangard International, Neptune, Nem Jersey) storage 26

tubes and stoned frozen at - 70°C.

For transformation, aliquots mere thauied on ice and 100

M 1 of competent cells added to transforming DNA <1 jug in 20

.uI) in 1.5 ml Eppendorf tubes The tubes uuere placed on ice

for 10 min, incubated at 37°C for 2 mm and the cells then

inoculated into 2 ml LB. Cultures were grown for l-2h at 37°C

with gentle shaking to allow expression of plasmid-encoded

genes Aliquots <100 Ml) were plated from the undiluted

cultures and from serial dilutions onto LB plus antibiotic or

MM-argimne

selection of transformants Plates were incubated at 37°C

ouernight

Preparation and transformation of competent cells of B.

suh tills

Cells were plated for confluent growth on TBAB plates (S

g NaClzl, 8 g agar/1, 17 5 g tryptose blood agar basest) and

incubated overnight at 37°C. The resultant lawn of cells was

suspended in 20 ml HS medium Cper 100 ml: 66.5 ml H 20, 10 0 ml

10X S-Base <20 g

g Na citrate.2H2(Vl), 0 1 ml 100 mM MgSG4, 2.5 ml 20/i ui/y

glucose, 0.5 ml 1 mg tryptophan/ml, 1.0 ml SZ casamino acids, 5 0

ml 2Ji yeast extract, 10 ml QZ w^v arginine and 4 Y. w/ v

histidine!?, to give a final concentration of l-3xl0 7 cells/ml

The suspension was incubated in a 250 ml Erhlenmeyer flask with vigorous agitation at 37°C until the culture approached the stationary growth phase 61gceral was added to 55i v

tubes and stored frozen at -70°C.

An aliquot (1 ml) of frozen cell suspension was allowed to

thaw and added to 20 ml LS medium, 2 ml 10X S-Base, 0.02 ml

100 mM MgSO^, 0.5 ml 20/i w/'v glucose, 0.02 ml 5 mg

tryptophan/ml, 0 1 ml 22 w^v casamino acids, 1.0 ml 22 w/u

yeast extract, 0.1 ml 50 mM spermidine, 0.5 ml 100 mM MgClg,

15 66 ml HjjO The cell suspension was incubated at 37°C and

200 ul aliquots removed at 15 min intervals after an initial lh

period of growth These aliquots were added to ice-cold solutions of transforming DNA dissolved in 100 Mi SSC <4 41 g Na citrate/1, 8.77 g NaCl''l>. After mixing, the cells plus DNA were incubated at 37°C for 30 min, LB (2 ml) added, growth allowed

at 37°C for 2h, 100 m 1 aliquots spread on selective plates and the plates incubated overnight at 37°C Each batch of cells was so assayed to determine the time after inoculation into LS medium

transformation.

The minicell producing strain, Jl. subtilis CU403, required

the addition of thymine <20 jug^ml) and methionine <20 jug^'ml) to all growth media.

R e s tr ic tio n and liaa.ti.QnL o f plasm id JHdiL Digestion of DNA by restriction enzymes used the buffers recommended by the commercial suppliers In each case 1 unit of enzyme per jug of DNA was used and reactions incubated at the appropriate temperature for t-2h. Treatment at 6 8°C for

10 min terminated most enzyme digestions and sep ar a ted cleaved 28 sites, eliminating apparent partial digestion products due to non-disjunction of fragments Preparations of digested DNA were visualized following agarose or polyacrylamide gel electrophoresis (see below)

Ligation of sticky-ended DNA in ligase buffer <10 mM

Tris.HCl pH 7.5, 10 mM MgCle, 50 mM NaCl, 1 mM ATP, 10 mM p-mercaptoethanol) used T4 ligase at a concentration of 0.2 units per jug of DNA Incubations lasted 2-12h at 17°C Blunt end ligations used the same buffer plus 2 units T4 ligase per jug of DNA for 2-12h at 14°C The products of ligations were visualized following agarose gel electrophoresis (see below)

Agarose and polyacrylamide gel electrophoresis.

Electrophoresis of DNA in agarose used horizontal slab gels of 0.7X w^v agarose in TA buffer (40 mM Tris MCI pH 8 0,

0.2 mM CH3C00Na, 2 mM EDTA) An electrophoresis chamber was sealed with tape, the well forming comb placed in position and

100 ml of boiled agarose solution dispensed into the chamber

Hhen the gel had solidified the tape was removed, the gel immersed in TA buffer to cover the gel surface and the comb carefully removed. One tenth volume of tracking dye <105i w-'v sucrose, 0.25X bromophenol blue) was added to each DNA sample before loading into the gel. Electrophoresis at room temperature was carried out at 100U for 3h or 20U overnight

Following electrophoresis the gel was removed and DNA in the gel stained by immersion of the gel in ethidium bromide solution

(1 ,ug''ml in TA) for 15 min. The DNA was visualized by UU transillumination (300 nm) and photographed using a Polaroid

MP-4 Land camera, Kodak Wratten gelatin filter 23A and Polaroid

667 film. Exposures were at f 16 for 15 sec 29 Separation of DNA fragments less than 1000bp in length

required electrophoretic separation through polyacrylamide gels

Gels of 20?i, 85i or 5 V. w/v polyacrylamide uiere run in TBE

buffer <89mM Tris borate, 89 mM boric acid, 2mM EDTA pH 8 0)

Glass plates <24 x 16 cm) were arranged with spacers to form

a pocket, clamped and the edges sealed with 2 .V, agar

Acrylamide solution <50 ml) was de-gassed prior to the addition

of 30 u 1 N,N,N ,N ,-tetramethylethylenediamine (TEMED) to initiate polymerization The solution was poured between the glass plates, a comb inserted, polymerization allowed

TA) for 45 min and then the gels destained in water for 10 min.

DNA bands were visualized as described aboue. Photography used Polaroid 665 film at f;5.6 for 15 sec

Minicell preparation and radioactive labeling of Plasmid-encoded polypeptides C106X

The minicell producing strains, £ coli DS410 and £. subtilis

CU403, were transformed with the appropriate plasmids DS410 was grown overnight at 37°C in 150 ml LB plus AMP <100 jug^ml) or CAM <30 jug^ml) CU403 was grown overnight at 30°C in 150 ml M9 minimal medium supplemented with 0.4/i w^u glucose, 20 30 jug thymine/ml, 20 jug methionine/ml and 5 m 9 KflN/ml Cells and minicells mere harvested by centrifugation at 0,000g at 4°C for

10 min in a 6SA rotor Pellets mere thoroughly resuspended in

3-5 ml M9 medium and the suspension layered on tap of 30 ml

10—33% m/v sucrose gradients in M9 salts, made in SW2? cellulose nitrate ultracentrifuge tubes using a gradient maker.

The gradients uiere centrifuged at 5,000g at 4°C for 20 min in a HB-4 rotor fitted with plastic adaptors to prevent the tubes from collapsing. Minicell bands were removed by insertion of a

21 gauge needle, fitted to a 10 ml syringe, just above the bottom of the mimcell band and withdrawal of the minicells into the syringe. (The lower and upper portions of the minicell band are not taken as they contain parental cells and lysed cells, respectively, mixed in with the minicells) Minicells were removed from suspension in the sucrose solution by centrifugation at 20,000g at 4°C for 10 min in a SS34 rotor

The pellet was resuspended in 1 ml of M9 medium, layered on a second 30 ml sucrose gradient, centrifuged and the minicell band removed from the gradient as before. The minicells were pelleted and checked for parental cell contamination by phase contrast microscopy If contamination levels were acceptable, 1 parental cell per 10^~^ minicells, the absorption of the minicell suspension was measured at AS80 and the suspension used to label plasmid-encoded polypeptides radioactively during their synthesis in the minicells

if) For labeling, minicells were resuspended (2x10 /ml) in M9 minimal medium supplemented with 0.4% w/v glucose, 20 ug

D-cycloserine/ml and 1/10 volume of methionine assay medium.

For each 100 jil of suspension, 05 jul L-(^^S)-methionine (970 31 Ci/mmol> uias added and the mixture incubated at 3?°C for lh.

The labeled minicells mere pelleted by centrifugation in an

Eppendorf centrifuge <12,000g) for 15 min, resuspended in 50 ml of SDS-PA6E sample buffer <0 125 ml 0 1M Tris HC1 pH 6 8. 0 3 ml

10JS uj/w SDS, 0 2 ml 502 v/w glycerol. 0.05 ml 0-mercaptoethanol per ml) per 100 jul of the original minicell suspension and incubated at 100°C for 3 mm.

Similar trichloroacetic acid

3-oxazole for fluorography, dried and then used to expose X-ray film at -70°C. Development of the film, after the appropriate exposure period, required immersion of the film for

2 mm in developer, 30 sec in water and 5 min in fixer The film was washed in water for 30 min and allowed to dry

Nick Translation (.32).

Approximately 1 jig of the plasmid DNA to be labeled, dissolved in 15 jul TE, was mixed with 2.5 jul of 10X NT reaction buffer <0.5 M Tris HC1 p H 7.5, 0 1 M MgS04, 10 mM DTT and 500 jug BSA/ml) and 2.5 jul of a solution containing 0.2 mM dCTP, 0 2 mM dTTP and 0 2 mM d 6TP in a 1.5 ml Eppendorf tube. A solution of 1 mg DNase I/ml in 50 mM Tris HC1 pH7 5, 10 mM

MgS04, 1 mM DTT and 502 u/v glycerol was diluted 1/40,000 in NT buffer and 0 5 jul of the diluted solution was added to the

■ » P nick translation reaction Additions of 5 jul of ac-< P>-dATP

<50 juCi) and 1 jul of DNA polymerase I <4 units) were made and the reaction incubated at 14°C for 3h The reaction was 32 terminated by adding 23 j_il of stop solution (0.02 M EDTA, 2 mg

salmon sperm DNA/'ml and 2% w^v SD5) and diluting to 0 5 ml with TE The reaction mixture uias loaded onto a Sephadex G-50 column (0 7 cm x 20 cm), pre-equilibrated with TE, and

subjected to column chromatography using TE as the running buffer Fractions <0.25 ml) were collected and 2 m 1 aliquots

of each fraction spotted onto 1 cm^ pieces of 3MM Whatman paper, the papers placed in scintillation vials and the r adioac tiuity in each vial counted in a Beckman model LS7500 scintillation counter Column fractions found to contain labeled

DNA were pooled and used as the probe material in hybridization experiments.

Radioactiue labeling of oligomer orohes

Approximately 30 picomoles of oligomer DNA were added to

3 ill 10X kinase buffer (0 5 M Tris.HCl pH 9 5, 100 mM MgClg, 50

mM dithiothreitol, 50Z w^v glycerol), 1 j j . 1 u *^P-ATP <100 juCi), 1 jul T4 polynucleotide kinase <30 units) and water to bring the total volume to 30 jul The solution was incubated at 37°C for lh, 10 mM Tris.HCl pH 8.0 added to giue a final volume of 100 jil ready to load on a Sephadex-G50 spin column pre-equilibrated with 10 mM Tris HC1 pH 8 0 The spin column was prepared in a 1 ml plastic tuberculin syringe stoppered with glass wool The syringe was suspended inside a 15 ml tissue culture tube and filled with equilibrated Sephadex-650 The column was packed by repeated spins at 500g for 20 sec in a table top centrifuge until a total bed volume of 1 ml was achieved and then washed six times with 10 mM Tris.HCl pH 8.0 for 20 sec at 500g Some more 10mM Tris.HCl buffer was added, the column spun at 500g for 3 min, the 100 jil radioactiue sample loaded and the column 33 respun at 500g for 3 min The eluate uias collected into a

lidless Eppendorf tube placed in the bottom of the tissue

culture tube. One uiash uuith 100 jul 10 mM Tris.HCl pH 8.0 at

500g for 3 min eluteci the radioactively labeled oligonucleotide

probe in the Eppendorf tube but left unincorporated

nucleotides in the column A 2 jul aliquot uias used, as described above, to determine the amount of radioactivity incorporated into the probe

DMA-DNA hybridization with dehudrated agarose gels <32)

DNA fragments produced by restriction endonuclease digestions uiere separated through 10/i ui/v agarose gels in TA buffer. The DNA fragments uiere stained by immersion of the gel in a solution of 1 jug ethidium bromide/'ml in TA and photographed using UU transillumination. The gel was soaked for lh in 50 mM NaOH at room temperature to denature the

DNA, rinsed with distilled water for 30 min, transferred to a sheet of 3MM Whatman paper which was placed on a Hoeffer model SE-1140 gel drier The gel was dehydrated by running the drier without heat for 30 min, with heat for 30 min, and without heat for a further 15 min. The dried gel and Whatman paper were separated from each other by flotation on distilled water The gel was then transferred to a heat-sealable plastic bag to which pre-hybridization solution CSX SSC, 58/i w/'u formamide, IX Denhardt’s reagent, 50 mM Na^HPC^ buffer pH 6 5) and 1 mg denatured salmon sperm DNA/'ml was added The bag was sealed and incubated at 42°C for 16h The pre-hybridization solution was replaced with hybridization solution <5X SSC, 50X ui^v formamide, IX Denhardt’s reagent, 20 mM Na^HPO^ buffer pH 6.5, 0.5 mg denatured salmon sperm 34 DNA/'ml and 18X wv'v SDS), heat-denatured, radioactiue probe uias

added, the bag resealed and incubated at 42°C for a further

4-16h. The gel uias mashed twice uiith 2X SSC plus IX

Oenhardt's reagent, twice with 2X SSC plus 0,1% uu^u SDS and twice with 8 IX SSC plus IX w^v SDS All washes were at 42°C for 15 min. Excess liquid was removed, the gel wrapped in

Saran Wrap and then used to expose X-ray film inside an intensifying screen cassette at -70°C

DMA-ORA hybridization with Zata-Probe membrane

DMA fragments produced in restriction endonuclease

digestions were separated through either 1.0X uj/w agarose gels in TA buffer or 0.5 X ui/w polyacrylamide gels in TBE buffer

DNA fragments were stained by immersion of the gel in a solution of 1 jug ethidium bromide^ml in TA and photographed using UU transillumination The gel was soaked for 38 min in

0 2N NaOH, 0 5M HaCl for 30 min at room temperature to denature the DNA After neutralization by three 10 mm washes in TA buffer the gel was placed on a pre-soaked wick of 3MM

Whatman paper drawing TA buffer from a reservoir Zeta-probe membrane, cut to the size of the gel and pre-soaked in TA buffer for 5 min, was placed on top of the gel and covered with two layers of pre-soaked 3MM Whatman paper Dry paper

towels were placed on top to draw the TA buffer by capillary

action and transfer the DNA to the zeta-probe membrane

Transfer was allowed to proceed overnight, the zeta-probe membrane air dried and then baked for 2h at 80°C The baked membrane was transferred to a heat-sealable bag, pre-hybridization solution <5X SSC, 50X w^v formamide, 5X 35 Denhardt's reagent, 0.1% iu/w SDS, 258 iig denatured calf thymus

DNA/'ml, 25 mM NagHP04 buffer pH 6 5) added and the bag sealed

Approximately 0 1 ml of pre-hybridization mixture uias used per square cm of membrane After incubation <42°C for 12-14h) the pre-hybridization mixture uuas replaced by an equal volume of hybridization solution <5X SSC, 50% w-" v formamide, 5X Denhardt's reagent, 0 1% SDS, 250 Mg denatured calf thymus DNA-'ml, 25 mM

NagHPO^ pH 6 5 containing denatured probe at final 2-3x10** counts/ml), the bag resealed and incubated at 42°C for a further 20-24h The membrane was washed with agitation four times, for 10 min in 2X SSC, 0 1% w^u SDS at room temperature, excess liquid removed, the membrane wrapped in Saran Wrap and used to expose X-ray film. A more stringent wash of 0.1X SSC,

0 1/1 uj/u SDS at 3?° — 42°C for 30 min was used if the signal to noise ratio was unacceptably low

Maxam and Gilbert DMA sequencing <06)

DNA <30 M9 > was digested with the appropriate restriction enzymes under the conditions specified by the enzyme supplier

One tenth volume of 1 M Tris.HCl pH 7.9 and 8 units of calf intestinal alkaline phosphatase were added and the mixture incubated at 56°C for 30 min The reaction was halted by addition of 200 m 1 8 45M CH^COOHH^ deprateinized twice by extraction with 200 m 1 TE-equilibrated phenol and once by extraction with 200 m 1 chloroformisoamylalcohol <24 = 15 The dephosphorylated DNA was precipitated by adding 750 ul of cold

95/i ethanol, vortexing and placing the mixture at -70°C (dry-ice ethanol bath) for 5 min DNA was pelleted at 12,000g for 5 min 36 in the Eppendorf centrifuge, the supernatant removed with a

Pasteur pipet, 1 ml cold 70/i ethanol added, the tube gently inverted, replaced at -70°C for 5 min and recentrifuged at

12,000g for 5 min The supernatant uias discarded, the pellet dried under vacuum, redissolved in 20 jul water plus 20 jul 2X denaturation buffer <1M Tris HCl pH 9 5, 10 mM spermidine, L mM

EDTA) and the solution heated at 65°C for 15 min and quickly chilled on ice 6 mI of 10X kinase buffer (0.5M Tris HCl pH 9 5,

0 1M MgClp, 50mM DTT, 50X glycerol), 1 jul tf 32P-ATP <180 m CO, 1 jul T4 polynucleotide kinase <30 units) and 12 Ml of water uuere added and the mixture incubated at 37°C for lh The reaction was stopped by addition of 25 M1 4 5M CH 3COONH4 and 200 jul water The radioactively labeled DNA was deproteinized, precipitated and dried under vacuum as described above. The

DNA pellet was redissolved in the appropriate buffer, 30 units of restriction enzyme added, the mixture incubated at the appropriate temperature for 4h, 1/10 volume of tracking dye

<0.25X w/v bromophenol blue, 0.25X w/v xylene cyanol, 15Z ficoll) and 1/10 volume 882 w/v glycerol added and the labeled fragments separated by electrophoresis through a 55! w/v polyacrylamide gel (55! w/v acrylamide, 0.2% w/v

N,N' -methylene-bis-acrylamide, 25/i w/v glycerol in TBE>

Following electrophoresis, the gel was marked with radioactive ink, wrapped in Saran Wrap and used to expose X-ray film for 2 min Exposed regions on the X-ray film that represented the desired DNA fragments were cut from the film with a razor blade. The Saran Wrap was removed from the gel, the film repositioned using the radioactive ink as a guide, and a razor blade used to cut the gel through the guide holes in the X-ray 37 film. The desired gel slice uias removed using a spatula,

chopped into small pieces and transferred to a 1000 jul

pipetman tip sealed at its tip and plugged with glass uiool DNA

was eluted from the polymerized acrylamide by addition of 500

jul elution buffer <500 mM CH 3COONH4, 10 mM (CT^COCD^Mg, i mM

EDTA, 0.1 H w^v SDS) sealing the tip with parafilm and incubating

overnight at 4£°C The DNA solution was removed from the tip

by piercing the tip with a hot needle and centrifuging the tip in

a test tube in a table top centrifuge at 500g for 1 min The

contents of the tip were washed with 100 jul elution buffer and

respun at 500g for 30 sec The total eluate was transferred

to an Eppendorf tube containing 800 jul cold 95/i ethanol, the

tube was vortexed, chilled at - ?0°C for 5 min and centrifuged

at l£,000g for 5 min The supernatant was discarded, the pellet washed with 1 ml cold 70Ji ethanol, dried under vacuum, redissolved in 50 jul TE, extracted twice with 50 jul

TE-equilibrated phenolchloroform (30:50), once with chloroform:isoamylalcohol (24:1), reprecipitated with cold 95!i ethanol, washed three times with cold 70/i ethanol and dried under vacuum.

The labeled DNA was dissolved in £0 jul water and subjected to the four base-specific chemical cleavage reactions:

6 reaction, cleavage at guanines, G + A reaction, cleavage at purines, C + T reaction, cleavage at pyrimidines, and C reaction, cleavage at cytosines. The reactions were performed in 15 ml

Eppendorf tubes.

B reaction.

£00 jul of DMS buffer (50 mM sodium cacodylate pH 8.0, 1 mM EDTA), 4 jul of end-labeled DNA, 3 jul of carrier DNA <1 mg sonicated salmon sperm DNA/'ml) and 1 jul dimethyl sulfate were 38 combined. The reaction uias incubated on ice for 8 min, stopped

by addition of 50 ,ul DMS stop buffer <1 5M CH 3CO ON a pH 7 0, 1M

p-mercaptoethanol) followed by 750 jul cold 95Ji ethanol The

tube was wortexed, placed at -70°C for 5 min, centrifuged at

12,000g for 5 min and the supernatant removed

6+A reaction.

12 jul of water, 3 jul of carrier DNA, 6 jul of end-labeled

DNA and 2 Ail of 1 M piperidine formate <4Ji v ' v formic acid

adjusted to pH 2.0 with piperidine) were combined The reaction

mixture was incubated at 37°C for 8 min, the reaction stopped

by freezing at -70°C and the sample dried under vacuum.

C + T reaction.

12 jul of water, 3 Ail of carrier DNA, 6 Ail of end-labeled

DNA and 30 Ail hydrazine were combined. The reaction mixture was incubated on ice for 8 min and then stopped by the addition of 200 jul hydrazine stop buffer <0.3M CHjCOONa, 0 1 mM EDTA), followed by 750 jul cold 955; ethanol The reaction mixture was vortexed, placed at -70°C for 5 min, centrifuged at 12,000g for

5 min and the supernatant removed. L. r.eat:tion. 15 jul of 5M NaCl, 3 Ail of carrier DNA, 4 Ail of end-labeled DNA and 30 jul hydrazine were combined The C reaction was then treated in the same manner as the C + T reaction

DNA pellets from the 6 reaction, C + T reaction and C reaction were dissolved in 250 jul 0.3M CH^COONa and vortexed vigorously, 750 m 1 of cold 95Z ethanol added, the tubes vortexed, chilled at -70°C, centrifuged at 12,000g for 5 min and 39 the supernatants discarded The pellets mere washed mith cold

705i ethanol, the ethanol removed and the DNA pellets dried

under vacuum

Strand cleavage reaction.

The contents of all four reaction tubes mere treated

identically for the remaining steps. DNA pellets mere mashed mith 20 jul mater, redried under vacuum, redissolved in 100 pi of 1M piperidine, incubated at 90°C for 30 min and then dried under vacuum mith heat to remove the piperidine and recover

the DNA The pellets mere mashed tmice mith 10 jul mater and redried under vacuum. Tracking dye (80% m^v formamide, 10 mM

NaOH, 1 mM EDTA, 0 1% m^u bromophenol blue, 0 1% m^v xylene cyanol) mas added to the pellets to obtain similar counts/pl.

The mixtures mere incubated at 9B°C for 3 min, quick chilled on ice and 3 jul aliquots loaded on 20/i or 6% m/v polyacrylamide

sequencing gels. Polyacrylamide gels (6% uj/v- 5 7Z acrylamide,

0 3Z N, N' ,-met hylene-bis-acrylamide or 20/i ui^v- 19% acrylamide, l/£ N, NJ,-methylene—bis — acrylamide) in TBE and urea (89mM

Tris.borate pH 8 1, 2mM EDTA, 8.3M urea) mere cast betmeen a

35 5 x 44 cm glass plate and a 35.5 x 46 cm glass plate. The glass plates mere separatedby 0.8 mm spacers and clamped to form a pocket. After polymerization, the plates mere clamped into an electrophoresis chamber Electrophoresis mas carried out using TBE buffer and 80 matts until the dye front had travelled a sufficient distance After electrophoresis, one glass plate mas removed, the gel transferred to a used X-ray film, covered mith Saran ktrap and exposed to a X-ray film at

-70°C using an intensifying screen 48 M13 sequencing 1110).

Plasmid DNA and M13mpl8 RF (or M13mpl9 RF) DNA were digested with the appropriate restriction enzymes under conditions specified by the enzyme suppliers and then ligated as described above Competent JM105 cells (100 jjl) were added to the mixture, incubated on ice for 45 mm, transferred to 37°C for 15 min, added to 3 ml molten 0 5^ ui/o B agar (10 g tryptone^l, 8 g NaCl/1, 1 ml thiamine Bl/1), 10 u 1 100 mM isoprapylthiogalactaside (IPT 6 >, 50 Ml 2/i w/v

5-bromo-4-chloro- 3-indolyl- galac topyr anoside in dimethylformamide (X-gal), mixed and poured onto B agar plates

<15?; ui/'v agar) Turbid blue or white plaques were visible after incubation overnight at 37°C White plaques (i e. those that contain inserts) were stabbed with a sterile needle and the adhering phage particles inoculated into 5 ml TE, a few drops of chloroform added and the phage suspension vortexed vigorously A sterile loop was used to spread a drop from these suspensions onto a B agar plate overlaid with 3 ml 0.5X w/v B agar, 300 ol JM105 cells <109 cell/ml), 10 jul 100mM IPTB and 50 jul 2 V. w/v X-gal. Infection of the JM105 cells produced isolated plaques after incubation overnight at 37°C. A plug of agar with a white plaque was removed using a Pasteur pipet, inoculated into 2 ml YT medium (8 g tryptone/1, 5 g yeast extract/1, 2 5 g NaCl/1) containing 10 ill JM105 cells <10 9 cells/ml) and grown at 37°C for 5-8h One ml of this culture was transferred to a 15 ml Eppendorf tube, centrifuged at

12,00 0g for 2 min and the supernatant transferred to a second tube 200 Ml of 2.5M NaCl, 20% w/v polyethylene glycol

(PEG-6000) was added, the solution vortexed, incubated on ice 41 for 30 min and centrifuged at 12,000g for 5 min to pellet the phage. The supernatant uias discarded, the phage pellet dried under vacuum and then resuspended in 100 jj 1 TE, coat proteins removed by extraction uiith 50 jil TE-equilibrated phenol

followed by extraction with 50 m 1 chloroform-isoamylalcohol

<24 1). The upper aqueous layer was collected, a 0.5 volume of

7 5M CH3COONH4 pH 7 5 and 2.5 volumes cold 95/i ethanol added and the mixture incubated at -70°C for 5 min DNA was pelleted by centrifugation at I2,000g for 5 min, washed with 1 ml cold 70X ethanol, dried under vacuum and dissolved in 10 jul

TE

Template DNA (4 jul), 1 jul of 2.5 >jg M13 DNA sequencing primer/ml, 4 jil of a 32P-dATP <40 juCi) and 1 jul of 10X buffer

<0 5M NaCl, 0.1M Tris.HCl pH 7 5, 0.1M MgCl2, 0 01M DTT) were mixed in a 0.5 ml Eppendorf tube and incubated at 56°C for 10 min.

The tube was placed at room temperature and allowed to cool slowly. 2 jul DNA polymerase I

1 unit-'jul) were added, mixed and 2 jul of the solution distributed to each of four 0.5 ml Eppendorf tubes containing 3 jul of either A mix, G mix, C mix or T mix These dideoxynucleotide mixes in IX buffer contained the following concentrations of nucleotides; A mix: 60 juM ddATP, 17 juM dATP,

30 juM dGTP, 30 juM dCTP and 30 juM dTTP; G mix: 150 juM ddGTP,

17 juM dATP, 4 2 juM dGTP, 40 juM dCTP and 40 jiM dTTP; C mix:

100 juM ddCTP, 17 juM dATP, 40 juM dGTP, 4 2 jjM dCTP and 40 uM dTTP and T mix 120 mM ddTTP, 17 juM dATP, 40 juM dGTP, 40 uiM dCTP and 1.7 jiM dTTP The mixtures were incubated at room temperature for 15 min, 1 jul of chase solution <0 2 mM of each dNTP, 0 05 units DNA polymerase I^jul) added and the mixtures incubated at room temperature for a further 15 min The reactions were stopped by addition of 10 tracking dye (0 3^ w^u xylene cyanol, 0 3/! w^u bromophenol blue, S9Y. w/'u deionized formamtde, 10 mM EDTA pH 8 3) and incubation at 100°C for 3 min.

An aliquot (3 jul) of each reaction sample was loaded onto a

SY. uj/y polyacrylamide sequencing gel (as described aboue> RESULIS,

Construction of plasmids pETSgl. pETSgg. pET8£3, pET8£4. and

p £TS£5. Methanococcus uanmeln DNA uias digested uiith Hindlll and

Ps11. mixed with Hindlll -Pst I digested pUC 8 DNA, ligated and

transformed into the multi-auxotrophic strain Escherichia coli

X760 Transformants of X760 capable of growth on M9 minimal

medium plates without arginine, were screened for recombinant

plasmids. One plasmid, designated pET821, contained a 7 0 Kbp

fragment of methanogen DNA (Fig 1>. Digestion of pET82l DNA

with Hindllf and Pstl. ligation with Hindlll-Pstl digested pUC9 DNA

and transformation of X760 resulted in plasmid pET822 which

contained the 7 0 Kbp methanogen derived DNA fragment in the

opposite orientation with respect to the vector DNA X760

cells containing pET822 were also able to grow on M9 minimal

medium plates without arginine. This indicated that the cloned

methanogen DNA encoded either a suppressor tRNA molecule or a

polypeptide capable of complementing the argininosuccinate

synthetase deficiency of £. coli X760. It also suggested that

the tL vannielii DNA contained a DNA sequence recognized as a

promoter by E_ coli RNA polymerase

To determine more precisely the region of tL uanmelii DNA in pET821 necessary for aroG complementation additional plasmids were constructed by sub-cloning DNA from pET821 (Fig. 1>

Digestion with EtjiRl, ligation and transformation into X760,

followed by selection for AMP resistance, resulted in plasmid 43 44

Figure 1 Plasmids derived from pET821 pET821 has a 7 0 Kbp fragment of fcL vanmelii DNA cloned into the Hindlll/'Pst I site of pUCS pET824 tuas obtained from pET821 by EcoRl digestion and religation. Ligation of EcoRl digested pET821 with EcoRl digested pBR32S produced pET823 pET823 and pET824 do not complement araG mutations in £. coli Ligation of Bglll^Pstl digested pET821 uiith BamHl/Pstl digested pHE3 produced pET825 which retains arginine complementing activity

Restriction enzyme cleavage sites are B-BamHl. Bo -BqIII. C-Clal.

E-FroRl. H—Hindlll and P-Pstl 45

PLASM10S DERIVED F RDM PET821

PET821

PHE3

Eco R1 LIGATION Ecd Rl \ LIGATION LIGATION UUIX PETQ25 PET824

8g

F ig u re 1 46 pET824. Complementation studies determined that pET824 uias unable to confer arginine independent grouith to X?60 The DNA situated between the two EcoRl sites of pET821 must therefore be essential for acqG complementation Sub-cloning of the 13

Kbp EcoRl fragment from pET821 into the EcoRl site of pBR322, transformation into X760 and selection for AMP resistance produced plasmid pET823 Complementation studies showed pET823 was also unable to confer arginine independence to X760 indicating that the DNA situated between the two EcoRl sites of

PET821 was essential but not sufficient for argG complementation

Pstl-Bglll digested pET821 DNA and BamHl-Pstl digested pHE3 DNA were mixed, ligated and the mixture used to transform

E. coli RR28 competent cells. Transformants containing recombinant molecules were selected directly by plating cells on M9 minimal medium supplemented with 200 jug PFP/ml and 30 jig CflM/ml Plasmids present in transformants were obtained by rapid plasmid isolation and used to transform L coli X760

Transformants were selected for CAM resistance and these clones screened for their sensitivity to AMP and ability to grow in the absence of arginine. One transformant capable of growth without arginine, contained a plasmid designated pET825 (Fig. 1)

The arginine complementing entity, either polypeptide or suppressor tRNA, encoded by the methanogen derived DNA was therefore concluded to be encoded by a DNA sequence located between the terminal Ps11 site and the internal JUglll site of the H. uannielii DNA fragment in pET821.

Expression of methanogen derived DNA in B. subtilis.

A fragment of methanogen DNA cloned from Methanosacrina barken had been shown previous 1 y to complement a deficiency in 47 argininosuccinate synthetase in both L coli X760 and IL subtilis

MB97 (90, 91> To determine if the £1. vannielii derived DNA

encoded by pET821 uias also functionally expressed in IL subtilis.

BamHl digested pET821 DNA and BamHl digested pUB110 DNA were

mixed, ligated and the mixture used to transform JL coli X760

All transformants selected for their resistance to both AMP

and KAN contained plasmids capable of complementing the

arginine deficiency. Plasmid DNAs isolated from these

transformants uiere used to transform competent cells of B.

subtilis MB97 Selection for resistance to KAN provided

transformants that were screened for their ability to grow on

M9 minimal medium Plasmid DNAs were re-isolated from

transformants capable of growth without arginine and used to re-transform L coli X760. All X760 transformants resistant to

AMP and KAN were also able to grow in the absence of arginine.

This indicated complementation of L subtilis MB97 argA2 was due

to the methanogen derived DNA and not due to reversion of the arqftg mutation The same results were obtained with a chimeric plasmid constructed from EcoRl digested pURBt DNA, containing a cloned fragment of Methanococcus voltae DNA, capable of complementing L coli X760 angG36 <145), and EcoRl digested

PUB110 DNA. Overall these results show that genes from three different methanogens, barkeri. M. vannielii, and £L voltae are transcribed and the resulting mRNAs translated in both £_ coli and IL subtilis to produce a polypeptide(s) capable of complementing argininosuccinate synthetase deficiency in two eubacterial species. 48 Polypeptides synthesized in DS410 mimcells Encoded bu pET8£1.

PET823. pET824 and pET825.

It had been shown previously that pET371 <90, 91) and pURBl (145) directed the synthesis in minicells of polypeptides with estimated molecular weights (Mr) of 51,000/49,000 and

55.000 daltons respectively. Minicells from strains transformed with plasmids derived from pET371 and pURBl incapable of arginine complemention, did not synthesize these polypeptides

011 plasmids directed the synthesis of p-lactamase or chloramphenicol acetyl transferase (CAT)

Competent cells of E. coli DS410 were transformed with

PET821, pET823, pET8£4 and pET825 DNAs. Mimcells were isolated from the transformed strains and incubated with

L-

49.000 daltons whereas pET823 and pET824, plasmids incapable of arg636 complementation, did not (Fig. 2). All three plasmids directed the synthesis of 0 -lactamase. pET82S did not direct the synthesis of p-lactamase but directed the synthesis of CAT.

In addition pET825 directed the synthesis in minicells of two polypeptides with mobilities identical to those of the polypeptides synthesized in minicells containing pET821 (Fig 3)

It appears therefore that the metabolic deficiency caused by the arqfi mutation is alleviated by either the 51,000 dalton polypeptide, the 49,000 dalton polypeptide or both of these polypeptides encoded by the 13. vannielii DNA cloned in pET821

Loss of the ability to direct the synthesis of the 51,000 dalton and 49,000 dalton polypeptides always correlated with a loss of 49

Figure E Autoradiogram of the electrophoretic separation of radioactiuely labeled polgpeptides synthesized in plasmid containing coli minicells. SB

/3 lactamase

Figure 2. 51

Figure 3 Autoradiogram of the electrophoretic separation of radioactiuelg labeled polypeptides synthesized in plasmid containing L coH minicells 5£

in — OJ CvJ rr_ ro 00 GO 03 LlJ H 1- or X LlJ LlI 3 C l CL CL C l

£ lactamase

F i g u r e 3 53 complemention ability. In all plasmid constructions bath polypeptides were either encoded or not encoded, in no case uias synthesis of the 51,000 dalton polypeptide observed in the absence of the 49,000 dalton polypeptide, or vice-versa

Nucleotide sequence of pET821.

The nucleotide sequence of 2 0 Kbp of the 7 0 Kbp M_ uannielii DNA fragment in pET821 uias determined using the sequencing strategy shouin in Fig 4B The 2098 bp sequence extends from the PstI cloning site of the pUC 8 vector DNA into the methanogen derived DNA and contains two long open reading frames CFig 4A) Open reading frame 222 C0RF222) is 671 bp long, encoding the carboxyl terminal 222 amino acid residues of a polypeptide in t err up ted at the PstI site of the methanogen DNA. 0RF222 conforms with the RNV <118) and the

6 ,non- 6 ,N rules <131) and so is presumed to represent the C terminus of a bona fide M vannielii gene A computer search of the PIR amino acid sequence bank, using the IBM Genepro program, failed to find related sequences, the function of the product of 0RF222 therefore remains unknown Following the

TAA termination codon of 0RF222 are two 15 bp identical, directly repeated sequences, separated by 1 bp Although these repeats could form a hairpin structure in mRNA, the predicted instability of the hairpin stem suggests that a classical rho-independent terminator is unlikely.

The second open reading frame

Figure 4 Detailed restriction map of 2.1 Kbp of pET821

A Restriction map of 2 1 Kbp of pET821 Hatched box represents an open reading frame

397 amino acid residues of argininosuccinate sgnthetase

S. Strategg used to determine the nucleotide sequence of 2.1

Kbp of PET821 DNA

1 II' II III III £. II £. ha m o R o jnc h a A A A h a III CFO I AHA I A L A c t i Ama I H 11 A H * III S c * I L i * I A > T ID T 00

00

1 5 0 0 ■ too ■ ■ 3 0 0 ' 1 9 0 0 -o -■ 400 ■1000 '1 2 0 0 '1300 - 1 6 0 0 -ieoo -2000 -2 -500 ' 6 0 0 ' 7 0 0 'BOO -qoo "1100 1 4■ 0 0 - 1 7 0 0 -2100 t I I 1 f i 1

Figure Sfi 56 molecular weights reported for other argininosuccinate synthetase subunits (13, 28, 31, 105) 0RF397 conforms uiith RNY and 6,non-G,N rule predictions. Comparison of the 0RF397 amino acid sequence with the previously reported cDNA encoded human argininosuccinate synthetase amino acid sequence (13) revealed regions of highly conserved sequences. It was therefore concluded that 0RF39? encodes the aroG complementing polypeptide i.e encodes the argininosuccinate synthetase of M. vannielii

Three bp 5' to the AUG translation initiation codon of

0RF397 (Fig 5) is a potential ribosome binding site, the AGGTGgT sequence should be capable of hybridizing to the 3' end of the

16S rRNA of H. vannielii <3* -CCUCCACUA-5' ) (2), Expression of a gene in £. coli and A. subtilis demands hybridization of its transcript to the 3' end of their 16S rRNfls. The sequence

AGGtGGTaAT should hybridize to the sequences found at the 3 ' ends of the 16S rRNA of both E_ coli (3'-AUUCCUCCACUA-5' ) and

B. subtilis (3'-AUCUUUCCUCCACUA-5*> (2). Further upstream, between base pairs 728 and 772 (Fig. 5), are sequences similar to those indicated in DNase I and exonuclease III footprinting to be the binding sites for RNA polymerase used to transcribe the

M. vannielii his A and methyl reductase 0 subunit genes (J.W

Brown, M. Thomm, G.S. Beckter, B.A. Sherf, personal communication) (25) The sequence upstream of 0RF397

(ACTGATATATTGGTAAAA) matches the sequence found upstream of the hisA gene (ACCAATATATATGTTAAA) (26) at 13 of 18 positions

It matches the methyl reductase 0 subunit gene upstream sequence (AACTTGAATATATCT) at 9 of 18 positions (25). A sequence, ACCGAAAANTTTATATANTA found 38-50 bp upstream of M vannielii and £L voltae tRNA genes has been suggested to be a consensus promoter sequence (141) The sequence 57

Figure 5 Nucleotide sequence of 2 1 Kbp of pET821

Termination codons of ORF222 and ORF397, and the initiation codon of 0RF397 are indicated uiith single dots. Direct repeats downstream of 0RF222 are overlined with arrows. The inverted repeat sequence downstream of 0RF397, indicated by converging arrows, could be a transcription termination signal Bases which are presumed to constitute a ribosome binding site are marked with double dots ( = >. Bases marked with asterisks (O are also found upstream of the hisfl gene of £ 1. uannielii

Restriction sites used in cloning and subcloning are indicated.

Amino acids are indicated by the single letter code under the second base of each encoding codon. SB

* + t ■ 1 3 ' 3 - ■ - L -I ( * * . , n i ijm tee

*»*■,*• T m ^ M T U T .^ rr . t i « a ; t . : . . t :::»: t t . t t t l a a a t a a a a t ■' i t t t t a m . a : * c m : u <. Ai*7(,TM(TTs a ,. a a

t T T ’ 1 1 Jl 1 3 I ‘ I

lAA*c*TT0TAiT**CT;.CiLT7-CTf. AJH,AIATATTAAiA;.lT-.^ATTi*ITCiiCT*»CT»i‘TCiiACvjA*I7TJIit*T?ATTtCAiTiJ>iiC**Aj*TA

TAliHAACCCC.-AlTTrrtLT:. TrTcllTArrfCrTAIAAATCAiTTAAATCCTAAACrTAATCCTTCrTl AAffTTcirAA**CT-TTATT:fCTTAAA*rfTl TT!T?AAl(JTAAACtTA 7 so

* * * * •*...... tt Ml * ^AA»AAtir7TJA7All<.CA7rTAA<;7c;*T*fAT1CC7AiA17TAT7TTTCA7AA**Ci*TTTAlACACCACCIiCTiittttCJlwt*Ai***TT&C*tTTTr*&CATACrCTtiJIGCSCTTj BAG ■ | | i

A T At A AOC t C C t ^ C rt A AA AT T ACTTr. A A U 4T A A'", I A T A ATT AT A A AC T JCTTT CTGTTCC ACTCC ATC 1AGGGC AGCGT G AtC AtCAC T T A *A *C A A C CtQ *AQ AAA ACCC*A AG AAAC 9 * * 1 4 I 1 < t )N J L * a * V r r W V * l*i»dT4qp*dfl i k|pt«na«« TTCCTGTTTTAAACCATrArACTATTCATGCAAAyCAACAAtTTSCACTtOACTATtTTTTTACCCCAATAAACSClilTUCCCTTTATCAACGTTACCCCTTATCAACGCCTCTTCCAA 1QBO ii*lfcriytld#pfPi'*TdTirr*iKini]T*(Tf>]»t*l*

^ACCCCTCATTGCTATAAAlArTCCA&AACTTCClMACAAArrG&^CCGGATCCAATTTCACACCCCTCTACrCGAAiAGCAAAT-SACCllTTTACATTTCAtTCCCTAATAAGAAClA 1 7C0 * p i 1 * b y I • « ] t y » L | i d < l l h f C t | f e ^ n d a l ' l ' r * * * I ' I jail A AGC A CC T C A A A T T C A A AT A A TTCC !T C C G G T 4 ACCGA TT T i l ACC f T A C A AG A A C A G A AC A A A T C { A A T A T G C A lA lC AAAAA&G A A T tC C A A T IC C A G T T G A T 7 T A G J U A lA C C A T T T A M J C k*pAI»llAp'rJid]l,r <-\*flqtlkahAlOlpnaL*l(p(

G 7 ATCG ATG AAA ACTTATGCGG rA GAAGTATTCAACG CGC A A T7TTIG AA AACCCA ATG AtTGA A AC TCCA A A AG AG TCTTTTCCATC£ ACTG TTCACCC&AA A AT AC CA A A AG ATG A AG 1 * AO i I d * r 1 y ( r *1*41 > l#*pal*Lp»*ef«wt « 4 p h ) a k d *

1 AG A G f A C C fA G A A 1 1 TC A A T T T A 1GCC A G C C C T T C C J> - T 7 ‘ C A At I t ACGCG CAAA 1 A T t TC ATGC ? ATT A ATGT AATA A A A C A A G C AA a TAA a CTTGCTCCC AC AA 1-C GO C C T T C C T A H t d

GGGTAGATATTATAGAAGATAGGGT7TTAGGCCTTAAATGAAGAGAAAATTATGAATGCCGCGCTGCAATTTTATTAATAAC7GCACATIAGGCACTTG1GGAACTC:T7TTATCAAGAC l«*0

A ACAA TT ACTAT T T AIACAAATGCTCC A T tC AA A G 7 ATGC AC 1 C 7 T A 1 TT T A CA 1 A GOACTATCCC ACCAAC CTTTA AGACATG A TT TaG A T O C A TTT G TA C A C A A AACwCA AGA A AC A A I 1 0 0 iilTrki»iditri4lirt|i>'ritp|'hdldirrakt4tr

tcaatcgaatacttcgggcaaaactttagaaaggttctttaacaattcttggaagggaaagcgaatg Tgcag 7TTATCAAgaaaacatggt 7TCATTCGagaataacgacatcgatca

TG5T7lticTt7tliTACAAA&TTAt

F i g u r e 5 5 9 ACCATTTAACTGATATATT6 found 45 bp 5J of the QRF397 initiation

codon matches this putative consensus promoter sequence at 13

of 20 positions Downstream <18-19 bp) from the putative

promoter for tRNA genes is a conserved triplet T 6C which is the

site of transcription initiation, however there is no T 6C triplet

at this position upstream of ORF397 Between base pairs 766

and 771 the sequence TTGATA matches the consensus -35 region

of £, coli promoters CTTGACA) in 5 of 6 positions <54) Overall,

the intergenic region upstream of 0RF397 contains sequences

which are similar but not identical to those reported to be

involved in transcription initiation in £L vanmeln. M. voltae and

coli. and translation initiation in £L vannielii. £. coli and JL subtilis

Upstream of genes encoding enzymes involved in arginine biosynthesis in £. coli is an 18 bp control region, the *arg box*, a partially conserved, palindromic sequence < 8, 17, 27, 28, 102,

103, 104, 133) The *acq box" 'a tNATNCANt) over laps and regulates tr anscr ip tion from the promoter s of the aro regulon in £. coli Binding of a repressor to this site is thought to cause repression of transcription through steric hindrance between the repressor and RNA polymerase. There are no sequences upstream of 0PF397 that resemble the “aro box** consensus sequence of L coli. An equivalent "arq box** may be present in the met-hanogens, but with a different consensus sequence An additional means of tr anscr ip tion regulation of amino -acid biosynthetic operons in eubacteria is hy attenuation

There are no runs of arginine codons in the upstream region of

0RF397, nor are there sequences that might form a stem and loop structure in mRNA which would be needed for attenuation

DNA sequences involved in the regulation of expression of genes 6 0 encoding arginine biosynthetic enzymes in £L vanmelii do not therefore seem likely to be the same as in eubacteria

Eighteen bp 3' to the ORF397 T A A termination codon is a sequence potentially capable of forming a stem-loop structure as mRNA with 4 6-C bp in the stem and 6 unpaired A’s in the loop, folloLued by 7 U's. This resembles a rho-independent terminator of L coli

The intergenic DMA sequences flanking 0RF222 and 0RF397 are very A^T rich C78 4X A+T) uuhereas uuithin 0RF222 and 0RF397, the A + T content <67 87i A+T) is similar to the overall 67 A+T reported for the total genome of £1. vanmelii <63)

Nucleotide sequence of pET371

The nucleotide sequence of pET371, containing the M barkeri DNA fragment cloned previously <49, 50), uuas determined using the sequencing strategy shouin in Fig. 6B The 3113 bp

Hindlll fragment contains tuio long ORFs (Fig 6A> both of which conform to the RNY and 6,non-G,N rules <118, 131). Open reading frame 396 (0RF396) is 1191 bp long and encodes a polypeptide containing 396 ammo acid residues with a calculated molecular weight 44,445. This is lower than the apparent Mrs of 51,000 and 49,800 estimated from the electrophoretic mobilities of polypeptides synthesized in minicells containing pET371. The

0RF396 amino acid sequence is very similar to the amino acid sequences encoded by the £L vannielii DNA (0RF397) in pET821 and the human cDNA known to encode argininosuccinate synthetase

Taken with evidence from sub-cloning of DNA from pET371, that resulted in derivative plasmids incapable of conferring arginine independent growth on L coli X760 (90, 91), it was concluded that 0RF396 encodes an arqG complementing polypeptide i e. the argininosuccinate synthetase of £L barkeri 61

Figure 6 Detailed restriction map of pET371 DNA

A Hatched box represents an open reading frame that encodes 398 carboxyl terminal ammo acid residues of carbamyl phosphate synthetase Solid box represents an ORF that encodes the 396 amino acid residues of argininosuccinate synthetase. Positions of the 6 x 14 bp and 3 x 2 9 bp direct repeats are indicated

B. Strategy used to determine the nucleotide sequence of

P E T 3 7 1 DNA 6 8

OOU H

UOOt J

C u b i-

uO«2- HU ____ HU 033 ----- 00£2 tm 033 ——

00«_ t ' J t 0Q£2_ 00*2 , 00*2 -

oo;c_ i : t . 0012, , , t 0002

OOW I; i t;

OOQl. 1 T *

O Q il- 4 ’ ’ * * 009*- , » * oo «_ I ¥3$----- OOM- i-T F igure F 00*1 ( ’ t I O02i-

OOii

o o o i - 006_ . I ■ OOQ 1 t t ‘ ■ t 1 I < < O O i- o* «u 4 ' * „ < ►- 009- 005-

0(J* -i NN

■■■Oil -J 1 * I * o; _

301 . 63

Figure 7 Nucleotide sequence of pET3?l DNA Termination codons of 0RF398 and 0RF396, and the initiation codon of 0RF396 are indicated uiith dots. Direct repeats downstream of 0RF398

<6x14 bp) and upstream of QRF396 <3 x 2 9 bp) are over lined with arrows. Converging arrows indicate the only palindromic sequence upstream of 0RF396 Bases which are presumed to constitute a ribosome binding site are marked with double dots

<:). Bases marked with asterisks <*) are the same as those proposed as putative promoter sequences in halophiles and 1*1. smithii (53) Restriction sites used in cloning and subcloning are indicated. Amino acids are indicated by the single letter code under the second base of each encoding codon 64

[I * * " t * •; = i ' 7 : r ; a a . : t g * a ;• g g .j r * : -1 a * c t t l . r. a a * ■ , i. a a c a a t t a l: t t g : * a a a s, 7 * 7 : g c 7 r c c ; t ;tg-l77GT... , t : : m : .• 7 a 1 r . i : . . , , 0»* J4« u ' ■ . l u t c c m u : : : * : * ' : a r :; a a a t m , a c : t t g a AC c •' T A r »7-. a a A.. AGGC ag r r acGj ?r 7 . t< * U iU '.* r r c n u ic T u * n ; n :: A T T : i'; 7 t i. agc. : n :. l t t ; : , a a I T -

, T .,', a 7 '.j r Ao T T . A .. a :f a a a j a.; , T t A T : i : : ' . / - -. a a t a * ! ...a:.-: a : * : * c j * A . , t i j j t •, 11 •' a < 7 1:: i,a ., at t 1 i : 77 7 ^ 7 a a r :: . a • ; a s a t r 7 •1 A * N •’ ¥ I * ,1 I - - I r * l» g •. 3 1 J 1 A ..

•, A % r; t 7 : it ^ [ c j ^ / a a , , ; ; : * ; t a t a . ■: i : a a a t a l. c t ' T 7 g : 7 ■ T : a t . a a a ■, •: 7 a 7 7 a a ■' A T A C a g a t g g l' a *. a G a a a g r . a a g i, t ." r a t \ ; • : ] r ., * a .: ? * .r .i > ' -7 s 1 *!--■■'►* ; 1 r- 1 ■■* * - * - 1 » h ' p ; p a

rrr-:i.TTiAAGi'A^CALA*7T r : t 7 7G 7 t 7. i i m : , : " • t 7 ” g a a t c s. •' t t j f. a a a g a t t g c a g c T a a g ; j 7 a a 7 t g t a ; i g a l a c a g c t t a a a g a g r 7 r 'j g g t a . a t a." ,a f r * * r ' I ; | 1 n ri > c L f . » * I » » h , l < g ^ S . h ^ , (l.Tlr -

a a a i 1.1 aaa<.a7:;t*7: .7 a 7 7 a a s . g a a m c :• 7 : t Ti.' •” -T:::iTA»Arr ag l A c g tg ca c1 r : r rr;7 r :• t •: ;;gC c c g C AS A1 0 A*r; a g t A r T r : v ( a g g j a a t a a t 7 ii 1 = t a - , a

T ! . . G v A A g g g C A | A 7 T A 7 A AO G C AG A G C 7 7-., L I :, G s G A7 AA T : 7 r T s ; T C 7 7 A :J A-,7, A AA A' ; 7 T I T C 7 T T C C A T A A G G A A TG C A G A C A A A A £ C g A * rTT.V.T.,:; A r:; T t G . A A *... A A A

7 7C A : a:, r A , T T . . 1 H M l T . C s , . ' 1'. J ‘ ! t H A T A T ■' 7 7 L A A •. : A . . . T : 7 T •' A T j -i A 7 •. T .. .. T A A A ft A •. . T ■ A 7 . A : . A A .. 1 . A A T , T A T * * 1 1 | ■> i '

A T - . l T ’/ o . A G A Ij A : > T T A A : . T 7 A 7 ; A T r A A T » i , ' ; A A A A As A 7 :. .j ’A-., rj, s. A T s!s, 7 7 • A A ; A 7 ! A . C' l . , I. 7 , ; 7 s, 7 1 A ' 7 7 C A A 7 s ., T A s" A T A • . ■ ■ m TP.-: I r ■ j » H ^ l - - i a i * f « ^ £

I T ^ ' A I j Cj :. CJCAA7A;;:1.. ..i-A.I.LtSCT-SCCATHjAliAl'TATsrAAGAAiiutfAipAOCAACTTALiATTiiAArLCATCAACCAGTACCACAAACjiOJil^CAA&A^AAAAv'AAAr, Tf'TAA j . ■ qt*iia#AiipT*i*h4ffj;ih!ilrfyrB»iif

CC AAA A AC rAAfl’r d c ^ A A A AT rtrAArfGiTTAJiTATr. c: *TC T G g Ia A A I f z C A A C T CC,Taia AT cTaaC r & 5 T ii A aT CTCCfC.JlA A A C CG ACT A A AG *:AG A A A TT 7C A 'L::C j AA

A T TC T'.C ITA AATTc.TfrC.A AC ^A A A A A A £ A 7 C Z i: 7 T C, Z T 7 A r ' T T T T T T ? A C A T A A T T T A T T T A I'. A C T A T 7 A A t C T A A 7 T C T-C C, C. IT A T 7 A TA TCCuTTA:AT::G 7T:;AAO TTA'7'

' 7 C S T s f T “ iTIj; AC T? A 7 -f A i f c f (T AAG r iT * ACfT AACTA !ffTA f?AA T CT CAACTT^AAC t T f l A i f 17 T c a tT aatct tTac tTTa AiTCACCT. ::ACA;77A:TTG;TT7AA:

C T lT A A ST T AAACGGT7 7A C TIA 1A G C iTGGCAAATjiiAlLTTG C AC T TGCAT A T TC 10C A GG t. C T I t I t A C C T CGGTGTGCATCCCT a t CC Tt AAuiiAAAtC 7 1 C G17 A7 K C i L u l A C 7 AG 11- fl r «aMhv*lBv*at^4t»»clpt ivtkTlrC** «n« ,

7TiC-trATTTCAi:jTCGATCTA|7UCi:. *GCCTCAAijA(j|jAAATCA*uCGGSCACACrJ CAAAAGCAG*CAAtiATCAC.CAAtAACCATTAT A v GATCCITC CA4iCGAACACTTCCT7AAACAT7 1ft OC.

A”G7' TTCTCArT-ATTAAAGs"' AACCGAiGTTACCIAAGGCTAT-.TJAT.'. C. GIACTTCAATTCcr-r GCCCGTTjATCCCGAAAAAArjTGGTCuAGGCTGC.'AijGAAAjAAG.', |i,' A .7 * ‘ 1 i,1'

•: c c r 7 n c r c t c G G c T : A •: A c si A A A A G G c A A r A r • A :;c ir G G r T T 'r'iA:JG c :j7 7 tT c :3 C i:A (j# c c .:JA C A T :iG A T0T T A rrnc:cc.7A T i:cG .:G A G A T ^A A < t T g a c f r;-.. r :. a ., 7 g g l , P ] *-'6' ij. » (''dq;rfa»»T * r**

AIATCGAGTaCijCAAAAGA:.:.A:'GCAATCr [""-.G7GGAAGT(;ACAAAG77!'AA:.CG7TGCAGCGTTGiCGAAAACATCTGGAGC('GCAGTATCGAAGGCGGCAjGrT7GAA.:jA' ■ "TT' :,7

TGA&CGG'. TAGGCCTTT i7GAAGAAAATjAfcTGAfcA77GCAGGrr;AAAACGGCCTrGG7<:GAACTGATA7GATtGAAGACC&CC7GCT7GGCTTAAAAjCCCGCGAGAAGtAf'GAAr-. AC'' .'t'lf' • y, v i ‘ | Aft»njygi*-l.(jii!l

c c Cc t g c a a :• t g r 7 r t : : g g c A j •: 1.1 h : i . r; r l. a g c T I r G A G a a a •: 7 f 0 T •: C T T Li a : c c G C a g CG a a ' t l a a g t t r a a g a a g a tc g 7 o z a c g a ■: <■ a t g c t c t g a a c t 7 g r ••; p, t t a • i. • r f p .J t r- * r t - r- 3 •, T . .4 , I b < k M I V l! <] b -I p a > ,

G G C T T G 7 G (, A C G A r., r: •: G 7 7 7 7 A T . A 7 ' 7 , » A : V ' 7 7 7 A T r'.i A ■: A A G T C f • A A A A A G 7 7 A : A C G t A T A l . 7 : A A A G 7 r. J G G r 7 r. r A C A A A .7 G r 7 r ■ 7 G A G :i A 1 7 7','"' ' G * “ " . > a . r J ' ' k ' * ^ i K ! ! > » p a . t . a -

, r T I i •: » A T T ' A T T r ” • k » .. * T 1 ■ :.TT7'_ I • I • ' A T 7 * • . * A * ' 7 •! A A A G • 7 T T G 7 i A T A 7 A r * A :. •• C A . . A T A * , I I . ' s k t - * , , * p - , a ' ' ?

TAATGGATAAGrAGTAA'j7TAAT'.AT77TG 7 7 : C 7 7 7 7 A i. I . ( G A i T G ■ G A : TT' 7AC7T7T7AA7T7AA7A77ClAGTrTTTT77AAA7AATCAA7T77- T:7AA77'7’ ;':: - * . «P V • rl H T

CCrA7JA7AA7AATTtTr, A»::CAAT::TiJAA7AGT'L:rJGAAA7ATGArTACCAGrTTT7t::ATAtAArL-:7J7ArArTTTC7AGAAiTTTCt'A'.'-TTTTCCAGCGATArA7G:A7AGTA7J' ■ ‘ fc*odin AATTCTtAAAAATGCGGvAu'TGCAAG Z. G G AT G A C r G T A * AG 7 C G T T & A A T f t C A T C T A T 7 G C T A AC t z A C T G A A C T C C C A C A 7 T T r T 7 r A 7 A 7 : : 7 C T ^ C T G C C C A C : l A C : 7 ,'

F ig u re 7 65 Six bp 3' to the translation initiation codon of 0RF396 (Fig.

7) is a potential ribosome binding site. AaGgGGTagT which should be capable of hybridizing to the 3' ends of the 16S rRNAs of M barkeri (3'-UCCUCCACUA-5' >, E_ coli <3'-AUUCCUCCACUA-5'> and £. sub tills <3'-AUCUUUCCUCCACUA-5' > Further upstream, between base pairs 1554 and 1565, the sequence TTTTAATCTATA is similar to a sequence 5J TTTTAATATAAA suggested as a possible consensus archaebactenal promoter sequence from

Methanobrevibacter smithu and Halobactenum halobium data <52,

53> Comparison of the in ter genic region upstream of the M. bar ken 0RF396 with the putative promoter sequence of methanococcal tRNA genes (ACCGAAAANTTTATATANTA) located one region

0RF396 translation initiation codon yet it has been suggested that transcription of methanococcal genes starts at a TGC triplet 18-19 bp downstream from the putatiue promoter Three

29 bp directly repeated sequences, with a

AAGTTYAAGTAYTCATCAATCTYAA6TTY consensus sequence

0RF396 translation initiation codon. There are no obvious similarities however between these sequences and sequences upstream of the £L uannielii 0RF397 initiation codon nor with the putative methanococcal promoter. None of the 0RF396 upstream sequences resemble the L coli *arg box" consensus sequence

The only palindromic sequence, AAAAGATGG< 8N)CCATCTTTT, is 215 bp upstream of the 0RF396 initation codon, 3' to the three 29 bp direct repeats. There are no runs of arginine codons in the intergenic region preceding 0RF396 nor sequences potentially capable of forming the stem and loop of an attenuator The upstream region of 0RF396 contains sequences that are more 6 6 similar to those suggested as promoters in fcl* smithii and H* halobium than those suggested to be promoters in vanmelii and H volt ae. Transcription regulation of the genes encoding

M. barken arginine biosynthetic enzymes does not therefore appear to be the same as eubacterial transcription regulation

Sequences required for translation of mRNA in H, barkeri. £. r.Q li and JL subtilis are houiever present immediately preceding

0RF396.

There are no inverted repeats downstream of the 0RF396

TAA termination codon that could identify a terminator, nor is there the start of another open reading frame

The second open reading frame in pET371 <0RF398) is 1197 bp long and is located 5' to 0RF396. 0RF398 has the potential of encoding the 398 carboxyl terminal amino acid residues of a polypeptide interrupted at a Hindlll site in the methanogen DNA

0RF398 terminates at a TAA codon and is followed 4 bp downstream by six 14 bp direct repeats

5

0RF396.

A computer search of the PIR amino acid sequence bank revealed that 0RF398 is closely related to the large subunit of yeast, rat and £, coli carbamyl phosphate synthetases <80, 95,

96, 97, 98, 104) The large subunit of £_ coli carbamyl phosphate synthetase is encoded by the carB gene which is 3819 bp long encoding 1878 amino acid residues resulting in a polypeptide with a calculated molecular weight of 117,710 <95)

The sequences of the large subunits of carbamyl phosphate synthetase in rat, yeast and L call have an internal homology

<80, 95, 98) indicating that the present forms of these genes 67 resulted from a tandem duplication of an ancestral gene, an event presumed to have occured before the divergence of the eubacteria and eukaryotes The available sequence of 0RF398 is too short to determine if the gene encoding carbamyl phosphate synthetase in £L barken MS also contains a duplication. The 3.1

Kbp methanogen derived Hindi 11 DNA fragment in pET371 had been sub-cloned from pET720 <49) which contains three Hindlll fragments cloned into pHC79 from a partial Hindi 11 digest of genomic fcL barken DNA It was possible therefore, that more of the methanogen carbamyl phosphate synthetase gene was on the other Hindlll fragments of pET720 DNA sequencing from the Hindlll ends of each fragment did not however reveal more amino acid sequences related to carbamyl phosphate synthetase.

It appears therefore that, pET7E0 DNA unfortunately does not contain the Hindlll fragment encoding the N-terminal amino acid sequence of the £L barkeri carbamyl phosphate synthetase.

The intergenic DNA sequences bordering 0RF396 and 0RF398 are A^T rich <67 3 V. A + T) whereas within 0RF396 and 0RF398, the percent A + T <49.5X) is much lower The overall percent A + T reported for the total genome of barkeri is 61Ji <63) The £L barkeri derived DNA cloned and sequenced in pET371 averages

50 4X A+T

Codon usage.

Codon usages in the £L barken and £L vanmelii ORFs show a preference for codons with A or U in the wobble position (Table

2) This is presumably necessary to accomodate the overall high A + T of their genomes. Codons containing a CG dinucleotide are rarely used in the H. vannielii ORF’s and the arginine codons C 6A and CGC are never used This is consistent with codon usage in previously sequenced methanococcal genes 68 T a h 1 (* r'

Cudon usjou

» r | C

h h ij»in GAF222 r . i * i i j

1 1 I no . s > r to, ay"

Alanine ll.t M. 3 6 . a 14.1 5 6 0,0 i fc . & 16 . I 1 . 5 t 4 ‘i 10,0 fc . A 0 •* ■ A 1 8 . 4 1 30.0

A'j A 25 ‘,8. j fi i . <: 66 . 7 AGO 2 1,7 '• * , 6 57,9 27.1 ; j . 6 I 1 - r., 16.7 7GA ■5.0 ' ? '3.6 9 . 5 : g c 1 0 1 t , 7 *4 , 9 2 1,1 0 36 , 4 2 S . t - jG 7 1 , 7 10.5 2 9 . 1 28.6 5 , 1 ; gu 6 '•0.0 5 3.6 •0,5 4 . 6 9 . 5 1:1

A)pirB(Lne AAC 4 1 5 4,7 2 b **9.1 60,0 ■1 7 5.0 81.3 6 40 . D 4 23.5 AAU 3“ a 6 . ] 29 50,9 j 40.0 25,0 18.8 i 60 , 0 ! 3 76 .5

A j p a r tite G AC 12 ^3.6 19 29.2 m2 65.6 40,0 1 8 72 . 0 62 , 5 a 31.3 5 . 9 GAkJ 4 1 56 ,2 *6 70.8 22 3*.* 52 , 0 28 . 0 37 .5 l 6 66 . 7 16 9* , 1 C y a t * 1 n e UGC - ? 33.3 2 10.2 3 57.1 50 . 0 100 00 . 0 ] 50.0 1 33.3 UGU 1 A 66 , 7 9 0 1,3 u 4 2 . 9 50.0 0 0 20.0 •j 50,0 2 66,7

G lu iis in e CAA 6 11.5 26 75.0 3 17,1 0 0 0 29-4 - 70 . 0 1 00 CAG *6 80,6 a 29 02.9 1 00 1 1 1 00 70 . 6 i 30,0 0

G l u t « p i u G A A A* “5,0 6 4 75, t 66 ^ . 5 45.6 26 68 , 4 28 . 6 38 86 . 4 14 93.3 GAG 52 S«.2 18 2'.9 26 27.5 S* . 5 l 2 31 .6 7 1,4 6 lj.6 6,7

G lycine GGA 33 29.2 5 9 . t 2 2 , m 28 . 6 25 , 0 9 . 7 1 0 38.5 5 55,6 GGC 35 3 1.0 10 15.2 • i 6 1.5 39 . 3 ' 4 50 , 0 58 . 1 4 15.4 0 3 GGG 22 19,5 17.9 4 14.3 19.4 6 23.1 2 22.2 GGU 2} 20 , 4 78 3 3 ■ i 14.3 3 )0.7 12.9 fc 21 . T 2 22.2

H i s t l d i n f CAC 50 , 0 * 3 ■ , 3 i 73.3 77.8 4 80.0 55 .6 3 50.0 C AU 50 . j i 66,0 « 36.4 22 .2 1 20 , 0 4 4 . 4 3 50.0

l i o l i u c i n * AU A 1 * H .3 1 2 i^-3 26 . 7 “ . 3 j . " 1 0 29.4 46 , 2 AUC 40 4 1.2 22 26 . 2 39 54 . 9 36 .7 1 6 69.6 66 , 7 3 8,8 7 . 7 AUU 46 4 7,4 -- 0 59,5 4 3.? 36 . 7 6 26 . 1 29 . 6 ; t 6 1,0 46 . 2

L • Li c l r. e •-j CUA T 2 9 . l " , 6 0 0 2 . 5 12,0 c u e 1 41 ’ C . 6 * , 9 M . 3 6 24.0 34 . 3 2 . 6 2 8 . 0 t, 41 OUG *-0.9 £ . 9 1 fc . 7 4 16.0 *5 , 7 2 . 8 0 C'JU 1 9 1 4 , 4 60.0 ’ 1 4 4.0 0 . 6 3 0.9 5 32.0 U U A 9 6 , 9 6 . 7 1 4 . 0 52 . a 9 36 . 0 UUG 24 1 8 . 2 3 . 3 3 12.0 5 . 7 0 3 12.0

L y a i n e 0 AAA 4 5 0 1 c, 28 . 3 fc: . 0 17 0 3 .9 45 . 5 33.3 2 6 7 2 . Z 9 0.0 A AG 7 1.7 1 0 J i 6 . 1 5 0,0 • 8 5 4.=, 66 . 7 i0 2 7.8 10.0 69 Table P continued

cirB • r 4 G

birk«ri bim«ri Hu*»n nl*l"T Q*F222 Rtildut ® ^ ® t I I 1 1 and c o d o n * 0 ‘ i d , « |n • <>- iy n *Q< i|rn No. ayn * Q * *yn Xo . i)rn • yn

" f ! m J 0 . r i i . i f * J G kO ' 0 0 1 ■' ' 3 0 TOC 1 5 1 o ^ : lfir. , s n n PPienvidianine 2 9 : 5

p o ; I n f 0 A .• C 2 9 . * s 1 L : , ; r ' S . 2 ' : H * 6 ' c , < / 3 . s * n . o 2 0 . o ■':.(! i ,5 ■ '* ,6 ' r 3. ‘ i ■ s ' - ’ . 'I 1 4 . • 2 c . Cj . ■ 2n o 0, .o C- :. o

S t r i n g ACC 1 3 1 2 . A 2 , * ‘ 10.9 N A , 19 . G 30. a - 3 7.5 13-3 a . 2 A u U 1 6 ’5,2 1 •? i 12,2 6 . 5 3 ? A . ] 2 7 . 7 i a . 2 1 2 0 . u j 12.5 UCA i ? ‘ 1 1 6 . 2 2 •'"• 5 2*2 I ‘A . 3 1 5 ,2 ■* . 2 -o .o 37.5 l! C C 2 7 . 6 ■ fc 1 8 29 18.1 39 * ’ 20,6 •j 15,4 1 0 4 1 . 7 fc . 7 j 12.5 O C 0 e> 5 . 7 * -, a 1 0 T 9 , fc i 14 ♦ 3 4 9 .2 A . 2 0 a. j UCU 2* 22 . 9 J 5 **2 , 2 1 0 2 1,7 9 - 5 - 7.7 8.3 3 20,0 fc 25 * 0

T h f io n in e AC A ' 9 30 . fc l . 7 >. 4 35 39 * a 42,9 25.0 fc . fc 36 , a *6,2 ACC 29 3 3 , 0 • o 1 fc . ‘ M 0 66.7 M 28 . 6 a 3 . 0 ‘ 3 72, z 0 7,7 ACC c fc . 7 fc , 5 11.7 * 0 3 i a . 0 5 . 6 18.2 7 . 7 ACU i 9 2 l . fc 9 4 t . a 2 20 . 3 A 2 20 . 6 12 .5 16.7 5 a 5 , 5 5 38.5

Tryptopnan l 5 OCG l 5 0 1 0 0 “ 100 0 - 100 100 3 100 Tirrcsjnf i J A C 2 3 ’ 9 22 £. * "i t 3 . 9 a 3 .2 7 * ♦ 0 3S-5 50.fi ’2 6 3.2 35 . 7 4 57 , l U A 0 - i f- b . & 29,0 □ 3* * L *- 5 6 1.5 A l . 2 36.8 i 6 A . 3 \ A* . 9 1 V a 1 l r, f , i. a -i * , 7 m . 9 J _ ;• ► j 20 . 0 1 fc . 2 35.7 fc fc . 3 0 24 . - • i 13.3 :uc iS.5 ' 2 1 0 . C i 8 . 2 - 36.7 3 . fc 7 9 4 9,7 ‘ A C , ' - 5 o . ■:■ * 5 2 7 22 . L 2^.2 '8 60.0 3 . fc d , 4 u J'JU JO 2 « . « 50 . fc - o 2 1.3 2 7.5 ’ a A3 . si 3 • 3 • 6 5 7.' i 70 <7, 26, 51, 52, 53> £L barkeri codon usage in 0RF398 and 0RF396 does not conform to this general rule In 0RF396, 63 7Ji of the arginine codons are those uihich contain a CG dinucleotide and

4745i of the proline codons are encoded by CCG The glutamine codon CAA and leucine codon CUA are not present in either of the H barkeri QRF’s, and the arginine codon AGA is employed very infrequently Codon usages determined in tuuo genes from tt. halobmm show that the arginine codon AGA, glutamine codon

CAA and isoleucine codon AUA are newer used and the leucine codon CUA used very rarely <53) £L barkeri appears therefore to have some codon usage similarities to tt. halobium

Computer analysis of carbamul phosphate synthetase genes

Diagon analyses with carbamyl phosphate synthetase genes of E. coli, S, cereuisiae and El barkeri are shown in Fig 8. A

Sequence Analysis program written by J.W. Brown for the Apple computer was the source of the Diagon analysis used. Amino acid or DNA sequences are compared by their Juxtaposition in all possible overlapping configurations, acquired through movement of one sequence relative to the other at one amino acid residue or base pair intervals. At each overlapping alignment the number of adjacent identical matches are determined A "window size" is chosen to determine the number of adjacent identical matches required before their presence is plotted on the graph. For amino acid sequences the "window size" chosen was either two or three, dependent upon the signal to noise ratio The graphs represent the amino acid sequences aligned with N termini at the intersection of the X and V axes Each pair or triplet of amino acid residues is therefore compared to every possible pair or triplet in the second sequence and a match is scored by a dot on the graph Figure 8 Diagon analysis of carbamyl phosphate synthetas

Graphs show Diagon analyses for £L barkeri carB (SARCARP), yeast CPA2

tt 5 E C o fti_ ■ R P T Ey; T

M5i Y EhCriRp1. 7EV:T W5 i' VE ^C riR P ; T

I 1

' l i l J < « ! h * , l t

* 1 f i *

*♦ 5 S Pi R C Pi R P T E '■w' T

HSi vEriCriRP. TEXT #7♦ VEriCrrFF

l t » T s •* tcocutf mt 'jwn •i brnf tut lmith 11DM»l cm * m * j

iP _ - ri R. P r E

« 5 • ECOCnrPP * TEX T « 5 i E C u C n P F , T£,-.T

F i g u r e 8 73 In this way every part of one sequence is compared to every part of the second sequence.

Diagon analysis of the previously reported L coli carB and

S cerevisiae CPP2 ammo acid sequences (80) indicated three lines of homology (Fig 8) The central line represents the homology of the amino acid sequences throughout the full lengths of the £, coli and S. cerevisiae carbamyl phosphate synthetase proteins. The tuio shorter lines above and beloui the central line represent the homologies betuieen the N- and

C-terminal halves of the sequences These shorter lines indicate the tandem duplication which has occured in these carbamyl phosphate synthetase genes Diagon analysis of the M barkeri 0RF398 amino acid sequence uiith those of the L coli carB and JL cerevisiae CPA2 gene products indicated two lines of homology (Fig 8) Stronger signals towards the 3' termini of the E. coli and S . cerevisiae sequences indicate 0RF398 has greater similarity with the C-termini of these primary sequences than the N-termini 8 stronger signal also indicate that the similarity between barkeri and E . coli primary sequences is greater than between £L barkeri and £. cerevisiae.

Alignment of the £L barkeri 0RF398, £. coli carB and S. cereuisiae CPA2 amino acid sequences was obtained using the

DNAstar Align program (a homology detecting and maximizing computer program) in conjunction with the alignments previously reported for the E . coli and E . cerevisiae sequences (80) (Fig.

9) Gaps were introduced into the sequences to maximize similarities Many amino acid residues are conserved in all three species but conservation is greater between the eubacterial and archaebacterial sequences Blocks of conserved amino acids are between residues 46 and 64, 106 and 127, 173 and 190, and 238 and 255 (Fig. 9>. 74

Figure 9 Carbamyl phosphate synthetase amino acid comparison Asterisks indicate identical amino acid residues in the sequences of carbamyl phosphate synthetase From different organisms Numbers indicate the positions of the amino acid residues within the alignment Only the carboxyl terminal ends of the sequences are shouin 75

CARBAMYL PHOSPHATE SYNTHETASE AHINO ACID COMPARISON x YEAST IDRAKNRHKESSILDSIDVDQPKVSILTSVKEAKLEASKVNYFVLIRPSYVLSGAAMSWNNK aaaaa a a «« « a a * aa aaaaaa a aa a a K COLI i nRAKnRgREQHAVgRI.KLIcqPANATVTATgHAVEIIAKKIGYPLVYRPSYVLQGRAHKIVYTm a aa a a a aa aa a aaaaaaaaaaaaaaaaaa M. BARKERI mXJYPQPKGGYAT&QKKAIEVAKKIGEPVLYRPSYVLGGRAMKIYYDE a aaa aa aa a aaa aaaaaa a aa a a YEAST - IDRAENRHKESSILDSIDVDQPEWSELTSYEKAKUASKVNYPVLIRPS YVLSGAAME WNHK a a EELKAKLTLASDVSPDHPVYMSKEISQAQKIDVDA VA YHGNYLVHAISE8YEHAGYHSGDAS LVLPFQHLSDDYK IALK a a aa a aa a a a aaaa aa a aa a aaaaaaa aa aa ADLRRYPQTAVSVSNDAPVLLOHELDDAVEYDVDAILDOEMYLICOIKEBIEQAGVHSGDSACSLPAYTLSQKIQDYHR aa aa aa aa a a a aa a a aaaa a a aa aaaaaa aaaaaaaaaa a aa a a a HDLEKYtCZAVRYSHKHPILIDDFLEAASEIDYDAYCDQEDYIIGAIHEHIEEAGVBSGDGACVIPPQSLSPSYLDQVR a a aa aa aaa aaaaaaa a aa aa a aaaaaaa a aaa aa a KELKAKLT LASDYSPDHPYVKSKE1KGAQXIDYDAVAYNGNYLVHA1SEHVENAG VHSQDAS L V LPPQHLSDDYKI ALK * s 1/1 5 DIADKYAKAWKITGPTNMQIIKDGEHTLKVIECMIRASRSEPEVSKVLGYNEIEIAVKAFLGGDIVPKPVDLMLNKKYD QQYQKLAEELQVRG LHNVQF AVKNNEVYL IKYHFRAARTVPEV81CATGVPLAKVAARYMAGKBLAKQGVTKEYIPP a a aaaaaaaa aa a aaa aa aaaaaa a aaaa aa a a aa a a a DYTRXIALALRVKQLINIQMAEEQGKYY VLEANFRSSBTIPEVSKAYGIPLAKIAAKVIVGHSLKSLGYMDEPKPK DIADKVAKAHKITGPFNMQ1IKDGEBTLKVIECNIRASRSEPEYSKYLG VNFIElAVKAFLGGDIYPKPVDLHLMKKYD a a

YVATKYPQFSETRLAGADPELGYEHASTGEV AfiFCRDLIKSYWTAIQ STMNTHVPLPPSGILEGGDTEEEYLGQVA a a a a aa aa aa aaaaa aa a a aaa a a aa___a ___ a YTSVEEV VLPEMKFPUV UPLLGFEMRSTGEVHGVGRTEAEAP AKAQLCSMSTMKKBGRALLS VRE GDCERWDLA A a aaa aaa a aa aa aaaaa aaaaaaa a a aa a a aa a aa *a a HVS1ESVLLPEDKLFGADPYLGFEICSTGEVMQI DYDfGKAYYEAELAADNVLPLTGKVELfi IRM AOKTELYDYA K a a a__a aaaa aa *a aaaaa a a a a a YYATEYPOrsrTRLAOADPrLGYlMASTaKVASFaRDLIESYVTAIQ STtMTHYPLPPSG ILEGGDTSEEYLGOVA 9

SIV AT IGYSIYTTNETTETYLOEHIEEKNAKYSLIKEPKNDKRKLKELEQEYDIKAVENLASKRAESTDDYDYIMKRNA ELLKQ GEELDATHG TAIYLGEAGI NFRLYNKYHEaSPHIODRIKHGEYTYII NTTSGKRAIE DSRYI BBSA aa aaaaaa a a a aaa a a a a aa aa a a aa a KLQAA GLEUKITEG TYMYLAQBGY EMDYYKKYHDGSFNYIIimRRDEYNLII NTFTSKQSRR DGSBI RRAA aaaaaaa a a a a a a a a SI VAT IGYRTVT TW II'H TY TJBI T KgKHAgVRri KTpywnyBKI.Rgr.rQgYTI IK AYTNLASKRAESTDDYDYIMRRNA S IDEAIPLENKPgTALLEAKCLXAEIAEK IKILEEHDVIYPPKVRSWDEFIGFKAY LQYEYHYDTTLNGGEATAHALNADATKKYISYQEiaAQIK. aa a aa aaa a VDEEVPYITTMQAAI AAAAA IETHKEGEEI-T IK S I tnrYmnPOnaCM K V aa aaaa a a IDEAIPLFNEPQTALLEAKCLKAKIAEK IKILESHDYIVPFKVRSWDKEIGEKAY

Figure 9 76 The V- similarity between carbamyl phosphate synthetase of

coil, IL cerevisiae and £L barkeri uias calculated for the DNA and amino acid sequences

6E9X for identical and conserved ammo acids) than between either of the bacterlal species and jL cerevisiae The eubactenal and archaebacterial ammo acid sequences are equally similar to those of the eukaryote, 3?.QX and 40 for

DNA and 29.8 X and 30?% for identical amino acids which is increased to 45 5 X and 46 AX. when conservative substitutions are included Amino acids that are represented in a homologous sequence by residues of similar size and charge are considered to be replaced by conservative substitutions

Computer analysis of argininosuccinate sunthetase genes

Diagon analyses of the £L barkeri. M. vanmelii and human argininosuccinate synthetase genes are shown

The DNAstar ALI 6N program was used to produce the best possible alignment of the amino acid sequences of the M barkeri. M vanmelii and human argininosuccinate synthetases

(Fig 11) 6aps were introduced into the sequences to maximize similarities Three main regions of conserved residues are apparent in all three sequences (Fig 12) There are 10 amino Table 3

7. SIMILARITY BETWEEN CARBAMYL PHOSPHATE SYNTHETASE OF DIFFERENT SPECIES

'/. SIMILARITY BETWEEN- DMA AMINO ACIDS AMINO ACIDS t CONSERUED SUBSTITUTIONS

YEAST & £. COLI 37.8/i 29 S/i 45.5/i

£ COLI & ft BARKERI 50.3Z 44.5Z 62.37,

H BARKERI I YEAST 40.9/i 36 ?Ji 46.4X

N 78

Figure 10 Diagon analysis of argininosuccinate synthetase

Graphs shoui Diagon analyses for tL barkeri artaG (SARARGP), M vanmelii ar qG (UANARGP) and human (HUMftRGP) ' i i i i O C I'^ - -ixn-e TJGWMCI"'- fc | fc v L 1 fc * fc 6 fc L*: G * I [ » 5 i v r t N A R G F ' , T E X T ' j - {' '(■' knO'- O n .k ■ • ■ I WQH ■ fc A1 . * -IE fc 0 G fc r L ' O ' V W M « . ■ 4 < i )

i. - ?♦* - ’ « [ ' ftif=ftiRGF':i TEXT . l T W I l f 1 i 1 fc v iue 10 Figure h * * G G f ft t f fc G G * i : -lW. t(>4'r - !■ + - r ' 4 O > O ( l ■ I t ’ l ( T y .M llW t- « * 5 : SftfeftRGF . : SftfeftRGF 5 * lt ► . Slit n t ' i j l j i ' u rt T E> : T : E> T 79 8 0

Figure 11 Argininosuccinate synthetase amino acid comparison

Asterisks indicate identical amino acid residues in the sequences of argininosuccinate synthetase from different organisms

Numbers indicate the positions of the amino acid residues within the alignment. 8t

ARGINIHOSUCCINATE SYNTHETASE AMIHO ACID COMPARISON s HUMAN MS&KGSYYLAYSGGLDTS CILVWLKEQ GYD V IAYLANIGQKKDFEEARKXALKLGAKKVF1 M. VAMM1KLII MQEKIAV LAYSGGLDTSCCLKL LEDKYHYK WSVAVDVGQPKD DLKKPKEK AKKLGV a * * «**«•»•»« a t «a a aa aaaaaaa a a a a H. BARKERI MAKK V ALAYSGGLDTEVCIPI LKKKYGYDEWTISVDVGQPE KKIKRADAK AEKISN a a a aaaaaaaaaa aa aaa aaa a aa a aa aaa HUMAN M8SKQSWLAYSGGLDTS C1LVWLKEQ GYD VIAYLANIGQKXDFKRAKXKALKLGAKXVFI 3s KDVSRKFYEKF IWFAIQSEALYEDRYLLGTSLARPCIARKQYEIAQREGAKYVSE1GATGKGNDQ YRFKLGCVSL aaa a aa aaaa aaa aaaa aa a a a aa aaa aaaaaaa aaa LKHYTIDAKEKFAYDYIFRAIKANALYEG YPLETALARPLIAIKIA1LAKXIQADAISBQCTGKGHDQFHFESVIRTK aaaaaaaaaaa aa a aaaa aaa a a aaaaaa a a a aa a aaaaaaaaaa aaa a a KHYTIDAKKKFYKDYVTPLIKANGSYKC YVMGTSIARPL1AKKYVEAARKXGAVALABQCTGKGNDQLRFKAVFRQT aaa a a aa a aaa aaa aa a aa a aaa aa aaaaaaa aaa KDYSKKFVKKF IWFAIQSSALYEDRYLLGTSLARPCIARKQVEIAQHKGAKYVSBGATGKGMDQVRFKLGCYSL

*, n ? s AFQIKYI APWRMPEFYHRFKGRNDLMEYAKQBG I P I PVTPKHFWSMDKNU® 1SYEAGILKHPKHQAPFGL YTKTODPA aa a aaa a aaa aaaaaa a a aaaa a a aaaaaa a a aa AFEISIIAPYRDLHLTRTEElq YAKKKGIFIFYDLEKPFStDEHLWIRSIBGGlLKMPHIKTPKECFAWTYDPK aaa a aaaa aa aaaa aaa a aa a aaa a aaaaaa aa a a a a aa a D HDVIAPHRBWLTRKWEID YAEKBQIPVKYTKSKFWSYnEllIlfSRSIKGGRLEDP&FYPPKEIFEWTTSPE aaaa a aaa aaaa aa aaa aaa a a a aa a a a a AFQ IKYIAfYRHFKFYNRFKGRHDLMKYAKQBG IPIPYTPKNPWSMDENU1BISYEAGILKNPKNQAPPGLYTKTODPA s 3 s KAPNTPDILEIEFKRGVPVKVTNYKDGTTBQTSLELFHYLHKVAGKBUYGRIDIVEHRFIGMKRRGIYETPAGTILYH a aaaaa aaaa a a aa aaaa aa a a a aaa aa a a IAKDEEEYVKIKFKAGVPVAINGQKFDAIN VIKEAHKLAGRHGYGRYDIIEDRVLOLKRRENYKCPGAILLIT a a a aaaaa a a a a aa aaaaa a aaaaaaaaa aaaaa a a a KAPEQPRWDIGFEAGYPVSLDGKXM&GYA FYnHHXIAGEHOYGRTDHIKDRYIjGLKAREKYEHPAATVLRQP aaa a a a aaaa a aa aa aaaa a a a a a a aaaaaa KAPMTPDlLKIEFKXGVPYKYTNYKDGTTBQTSLELfTfYLNEVAGKBGVGRlD IYEHRTIGMKSRGI YETPAGTILYH inm AHLDI EAFTMDRKVRKI RQGLGLKFAELVYTGLRFSPECKFYRHC IAKSQKRYEGKYQ YS Y LKQQ YYI LGRESTLBLYN aa a aa a a a a a aa a a aaa a a aa a aaaa aa AIKALEQL YLSRKEL YFKEHYD6KYAD LIYKGLWHEPLRHDLDAFYDCTQKRHNGTYRAKLYKGSLR1 VGRESKCAL Y Q a a a aa aa aa a a aa aaa aa aa aa aaa a a aaaa a a a a aaa H B SPLR N SSLTR SK LK m I VDDQWSKLAYYGLVDKPLYADLHAF IDKSQKR VTGIVKYRLYKGALTILARSSPKALYS a a a aa a aa a a aaaaaa a a a aa aa a aa aa AHLDIEAFTHDRKVRKI KQQUJLKr AEL YYTGLRFSPECSFYRHCIAKSQERYKGKYQYG VLKQQYYILGRESPLSLYN 3 KELV5MNVQGDYKPTDATGFIN1NSLELKEYHRLQSKVTAK KNKVSFEHKDMDQREIV GMVKFHG LOAA1FES [JINK EDLV5FDSQTIDQKGS1 GFAKYBGFQARMYRKVHDKQ a aaa a aa a a EELVSHNVQGDYKPTDATGFININSLRLKEYHRLQSKVTAK

Figure 11 32

Figure 12 Amino acid residues conserved between fcL barkeri.

M vanmelii and human argininosuccinate synthetase sequences

Numbers indicate residue positions within the amino acid alignment. “All sequences" indicates amino acid residues conserved in all three sequences 83

b 10 15 2U 25 50 35 4U 45 bu huMAN & tl, VANN 1 LL11 M„K__V_LAYSGGLDTS_C L Y__V GQ_ED ___ B. VANN1 ELI 1 8 fcl. BARKERI M__K__V_UYSGGLDTS_C L__KY_Y__VV„_VDVGQPE ____ fcl. barkeri 4 Human H__K V_LAYSGGLDTS_CI LKE__GYD_V ______GQ_E__EE_

All Sequences : fc! K V LAYSGGLDTS C L Y V ^60_E____

10b 111 lib 121 12b 131 13b 141 14& 151

Human 4 B. vannielii ARP_1A.K_.E_A___GA___SHG_TGKGNDQ.RFE A P . l . J A P . R

M. VANN I ELI 1 a M. BARKERI ARPLIA_K__E_A___6A_A__HGCTGKGNDQ_KFE_V_R ______IAP_R

B. BARKERI i hUMAN ARP _ I A_K _ Vt _ A _ .EGA HG.TGKGNDQ.RFE ______V1A°_R

a l l S e q u e n c e s : ARP._IA_K__E_A___GA____HG_TGKGNDQ_RFt ______IAP _ R

17b lei 18b 191 19b 201 20c 211 huMAN Q M. VANN 1 ELI 1 YAK &IPIPV P.S.DEHL S_E_6ILENP____ P fcl. VANN 1 ELI 1 a B. BARKERI YAKE _GIP _ _V KP.S.DEN.W.RSIEGGILE.P ____ P H. BARKER i 4 HUMAN YAK_HGIP__YT.__PWS.DEH S .E .G .L E .P ____ p

All Sequences ; YAK_.GiP..Y.___P_S_DEH____S_E_G_LL _P____P

F i g u r e 1B 84 acid residues conserved at the N-termini of all three sequences

and 18 of 14 residues, from position 124 to 137, are identical in

the polypeptides of all three species. A third region of

conservation, between positions 172 and 212, does not contain a

long block of identical residues but has a high level of

conservation in a short stretch of residues Although there is conservation of residues in all three amino acid sequences

there is more similarity between the tuio methanogen sequences

than between either methanogen sequence and the human

sequence There are blocks of amino acid residues conserved in

the two methanogen sequences between positions 39 and 45, 66 and 76, and 267 and 292, which are not conserved in the human sequence (Fig 11). There are similarities between the individual methanogen sequences from amino acid residues 267 through 292 and the human sequence but these are not the same sequences in all three species.

The similarity between the nucleotide and amino acid sequences of argininosuccinate synthetase of human, £ 1. barkeri and H vannielii was calculated (Table 4) The two methanogen polypeptides have 49.9X identical amino acids and if conservative substitutions are included they are 66.7X similar. The methanogen genes are 52.1^ identical. Although the methanogen polypeptides are equally similar to the human sequence for identical amino acids (38.2X and 38 5Ji> and identical and conserved amino acids (53 7/i and 54 2Ji>, the barkeri nucleotide sequence is more similar to the human DNA sequence than is the DNA sequence of £1. vannielii This is due to the more frequent use of codons that have an A or U in the third base position in £L vannielii. Table 4

Z SIMILARITY BETWEEN ARGININOSUCCINATE SYNTHETASE OF DIFFERENT SPECIES

V. SIMILARITY BETWEEH- DNA AMINO ACIDS AMINO ACIDS t CONSERUED SUBSTITUTIONS

HUMAN & U. UftNHIELlI +2 BX 38.23! 53.73! tt. UANNIELII t tl BARKERI 5P \Y. 49 93! 66.7%

£1. BARKERI 4, HUMAN 47 .27. 38.5 7. 5427.

a t in 8 6 Construction of an evolutionary tree for ar^imnosuccinate synthetase genes.

An alignment of the closely related DNA sequences of M_ vannielii 0RF39?, tL barkeri 0RF396 and the human argininosuccinate synthetase cDNA was made using the DNAstar

ALIGN computer program. DNA sequences of tuio human argininosuccinate synthetase pseudogenes, HumASPsl and

HumASPs3. were included in the computer generated alignment

The alignment of five argininosuccinate synthetase sequences was used to construet the most parsimonious evolutionary tree using the IBM computer program PAUP, version 2 91 <44, 126)

In the construction of a tree, the PAUP program determines the numbers of differences between the aligned DNA sequences and quantitates these values as a measure of evolutionary distance. The computer program then generates all possible unrooted trees that could represent the evolution of the sequences The alignment of five taxa

DNA sequences) will generate 15 possible unrooted trees The total length of each possible tree is then determined and the most parsimonious tree, i.e. the shortest passible tree accounting for all the differences between the aligned sequences, is chosen as the most probable evolutionary tree.

Should two trees be of the same length both are presented as equally probable To obtain a rooted tree, one of the aligned sequences must be defined as the ancestral sequence

The evolutionary tree generated from the arginmosuccinate synthetase genes of the human cDNA ,

HumASPsl , HumASPs3

, £L barkeri and £L tf.aDniei.il

Figure 13 Evolutionary tree for argininosuccinate synthetase

DNA sequences encoding argininosuccinate synthetase from human cDNA (hum), human pseudogene 1 (psl), human pseudogene 3

, £1. uannielii

hum (1]

psl (3)

ps3 (2)

r msb (4) -6 L mcv (5)

F iq u re

m a 89 between two sequences tL barkeri and tL vannielii DNA

sequences were found to be more closely related to each other than to the human argininosuccinate synthetase sequences and represent an outgroup relative to the human sequences Divergence of the methanogemc bacteria from each other occurred before the divergence of either pseudogene f rom the expressed gene in humans. More dif f ere nee s have occured between the tL barkeri and tL vannielii sequences than between any of the human sequences. The tree indicates that the HumASPs3 pseudogene was produced by reverse transcription of human argininosuccinate synthetase mRNA before the production of the HumASPsl pseudogene. The HumASPsl sequence is most closely related to the cDNA sequence of the expressed gene. This agrees with a previously reported evolutionary tree constructed for argininosuccinate synthetase pseudogenes (94)

The evolutionary tree produced by the PAUP computer program for the argininosuccinate synthetase genes adds further support for an ancient divergence of the archaebacteria from the eukaryotes It also indicates that the methanogen and human ar gininosuccinate synthetase genes are homologous, if homology is defined as having shared a common ancestor.

Occurrence of the 14 bu and 29 bo direct repeats in M. barkeri

MS and M. barkeri 227.

The function(s) of the 6 x 14 bp direct repeats and 3 x

29 bp direct repeats in the intergenic region separating 0RF398 and 0RF396 in the tL barkeri derived DNA cloned in pET371, is unknown Hybridization of r adioac tively labeled probes to Hindi 11 digested chromosomal DNAs of tL barkeri MS and tL barkeri 2 2 7 was used in attempts to determine the number each repetitive 90 sequence per genome. Oligomer DNAs (kindly synthesized and

provided by Lisa Alex, M I T ) unth either the sequence

AAAATCCAACT66T (14-mer), present in five of the 6 x 14 bp

repeats or the sequence AA 6TTCAASTATTCATCAATCTC (29-mer), the

exact sequence of one of the 29 bp repeats and differing at

only one or three positions from the other two repeats, were

labeled at their 5' ends using « ^P-ATP. Selected fragments

from Rsal digested pET371, radioactiuely labeled at the 5' ends

*jp with a P-ATP, gave probes, one 290 bp long encoding the 6 x

14 bp direct repeats and a second 216 bp long encoding the 3 x

29 bp direct repeats The complete plasmid pET37t, nick

translated using oc ^P-dATP as the label, was also used as a

radioactive probe. Each probe was hybridized to Hindlll digested

M. barkeri MS DNA and Hindi 11 digested tL barkeri 227 DNA

immobilized either in dehydrated agarose gels or bound to a

Zeta Probe membrane

The radioactively labeled 14-mer and the 290 bp Rsal

fragment of pET371 hybridized to the same, single DNA fragment

in Hindlll digested tL barkeri MS DNA or in Hindlll digested M. barkeri 227 DNA The Hindlll fragments from the two strains of tL barkeri were not however the same size The 14-mer and 290 bp Rsal probes hybridized to a Hindlll fragment in tL barkeri MS

DNA that was the same size as the Hindlll fragment of tL barkeri MS DNA cloned in pET371. The DNA fragment in HindITI digested tL barkeri 227 genomic DNA, which hybridized to these probes, was larger The block of 14 bp direct repeats must

therefore occur once in the genomes of both tL barkeri MS and

M. barkeri 227 but due to changes in the location of sites for the restriction enzyme Hindlll in the genomes of the two strains of tL barkeri the 14 bp repeats are within a larger 91

Figure 14 Autoradiogram of DNA DNA hybridizations between M barkeri DNA and radioactioely labeled pET37l probes.

A tt barkeri MS DNA (track 2>, tL barken 227 DMA (track 3>

and pET371 DNA (track 4), digested with H indl l l . hybridize to the radioactiwely labeled 29 base oligomer probe. pBR322 DNA (track

1) does not hybridize with the 29 base oligomer probe The 14 base oligomer, 290 bp Rsal DNA fragment and 210 bp Rsal DNA

fragment probes also hybridize with Hindlll digestions of these

DNAs to gioe the same pattern (not shown)

B Hindlll digestions of pBR322 DNA (track 1), tL barkeri MS DNA

(track 2), tL barken 227 DNA (track 3> and pET371 DNA (track 4) hybridize to nick translated pET3?l DNA A. B. Hind HI Hind JR

1 2 3 4

pBR322

UJ F i g u r e 14 ru 93 Hindlll fragment in the genome of CL barkeri 227 These data do not address the question of the number of 14 bp repeats in the genomic DNAs of £1. barkeri MS and CL barkeri 227

The r adioactively labeled 29-mer DNA and the 210 bp Rsal fragment of pET371 hybridized to the same, single DNA fragment in HindUl digested CL barkeri MS DNA or in Hindlll digested M. barkeri 227 DNA

DHA fragment probes The 29 bp repeats must therefore occur only once in the genomes of CL barkeri MS and CL barkeri 227 and are closely linked to the 14 bp repeats in both genomes

Nick translated pET371 DNA, also hybridized to the Hindlll

DNA fragments of both CL barkeri MS and CL barkeri 227 which hybridized to the oligonucleotide probes. The two DNA fragments produced by Hindlll digestion of pET371 DNA

(homologous to the probe), and the single fragment produced by

Hindlll digestion of the vector pBR322 DNA, hybridized as expected to the nick t ranslated pET37l DNA The four probes carrying the repetitive sequences did not hybridize to

PBR322 DNA DISCUSSION.

The purpose of this study uuas to determine and analyze the nucleotide sequence of tuuo DNA fragments, one cloned from

M barkeri and one from tL vannielii which, when expressed in E. coli or i . snhtilis. complement a deficiency in argininosuccinate synthetase. Determination of these sequences was expected to answer the following questions. 1) Which DNA sequences encode the polypeptide Could comparison of these genes add information to the phylogeny of the archaebacteria? 4) Were there sequences in the inter genic regions f lanking the ar qG genes similar to the putative transcription and translation control sequences described from studies of other methanogen^archaebacterial genes?

Expression of methanogen derived DMA in two eubacteriai sy stem s. The ability of the methanogen derived DNAs to complement auxotrophic mutations in two eubacteriai species indicated that the methanogen DNA sequences must contain three essential elements. First, a sequence that encodes either a suppressor tRNA, or a polypeptide capable of functioning in E. coli and ]L snhtilis to suppress a deficiency of argininosuccinate 94 95 synthetase This did not necessarily indicate that the methanogen gene encoded an argininosuccinate synthetase enzyme. The methanogen polypeptide might suppress the argininosuccinate synthetase deficiency in £» coli and JL subtilis by providing an alternative means of arginine independent grouith. The second essential element must be the presence of

DNA sequences recognized in the two eubacteriai species as transcription initiation sites. The resulting mRNAs must carry the third essential regulatory element, a sequence capable of hybridizing with the 3' end of the 16S rRHA molecules in the eubacteriai ribosomes needed to direct translation initiation.

The promoters used in L coli and I. subtilis are probably fortuitous sequences since H. vannielii and tL barkeri appear to have completely different regulatory sequences for transcription initiation from eubacteria and each other.

Previously reported H vannielii genes his A (26, 145>, hisl (7), mcrBPCGA (25), 5S rRNA <139), and various tRNA genes (139, 141) have been c o m p a r e d and conserved upstream sequences proposed as promoters <53, 138, 141). Footprinting experiments have begun to elucidate the binding sites for tL vannielii

DNA-dependent RNA polymerase (18). In the case of tL barkeri. the only genes so far sequenced are the argS reported here and the mcr operon <15, 49, 50). Sequences necessary for transcription initiation in the Methanomicrohiales order of the methanogens remain uninvestigated.

The gene products of £1. barkeri 0RF396 and EL vannielii

0RF397 uiere determined to be responsible for complementation of the argininosuccinate synthetase deficiency in both L coli and i, snhtilis The amino acid sequences of these gene products are closely related, they differ in length by only one residue, contain 49. 9¥. identical amino acids, and are 66,7Ji 9 6 identical if conservative substitutions are included. Their obvious relationship to the human argininosuccinate synthetase, determined from a cDNA clone <13), leads to the conclusion that these ORFs represent the argininosuccinate synthetase genes of

M barkeri and tt vannielii. and all three genes are derived from a common ancestral gene. The methanogen polypeptides must have remained sufficiently similar to the eubacteriai polypeptides to be functionally active in L coli and i, subtilis.

Comparison to the complete £. coli ar gininosuccinate synthetase, uihich as yet has been only partially sequenced <28), mould be valuable in determining the extent of conservation between the archaebacteria and eubacteria as opposed to that between the archaebacteria and eukaryotes.

Sequences upstream of 0RF396 and 0RF397 resemble the

-35 consensus sequences for £. coli promoters but there is little resemblance in either intergenic region to the -10 consensus sequence

0RF397 sequence, TTGflTA matches the £. coli -35 consensus sequence (TTGACA) at five of six positions, uihereas GTCACA 41 bp upstream of d. barkeri 0RF396 matches the EL coli sequence at four of six positions. Sequences capable of hybridizing mRNA to the 16S rRNA of EL coli and JL subtilis ribosomes and to the appropriate methanogen rRNAs are present upsteam of the translation initiation codons of both ORFs. Their presence presumably accounts for the translation of the methanogen mRNAs in the eubacterial cells

L*Jhat remains unexplained however is the synthesis of both a 51,000 and 49,000 dalton polypeptide in minicells containing pET371 or pET821, apparently encoded by one and the same gene.

The mRNAs synthesized in eubacteriai cells may have initiation sites different from the mRNAs synthesized in methanogens In 9 ? the eubacteriai cells there may be tuio sites of initiation of transcription and translation. Both 0RF396 and 0RF397 have in-frame TAA termination codons 15 bp upstream from their initiation codons which would seem to preclude a premature start to the translation of the mRNA at an alternative AUG

(methionine) or 6UG (valine). Downstream of the 0RF396 initiation codon, within a sequence of amino acid residues conserved in both methanogens and in the human sequence, there is however a sequence TT6 ACA which is a perfect match to the —35 £, coli consensus promoter sequence The tL vannielii DRF397 sequence at the equivalent position is TT 6 ATA and it is followed 3£ bp downstream by a sequence TATAAT that matches the E» coli -10 consensus sequence at all positions. These or other such sequences internal to 0RF396 and 0RF397 could be recognized by the E. coli RNA polymerase and cause the transcription and translation of a shorter mRNA resulting in the smaller polypeptide however there are no AUG (methionine) or 6UG

(valine) initiation codons within the immediate downstream region of these possible E coli promoter sequences. Sequencing deletion derivatives of plasmid pET371 <90, 91) to determine the extent of the deletions might aid in elucidating why two polypeptides are encoded by one ORF. An alternative method, to confirm or negate the misstart proposal, would be to isolate the two polypeptides and determine their N-terminal polypeptide sequences

The appearance of two polypeptides could be due to the experimental conditions of the SDS-PA 6E gel If the disulfide bonds are not fully reduced by p — mercaptoethanal in the loading buffer, polypeptides may not completely unfold. The purpose of the SDS in the gels is to bind to polypeptides and minimize their native charge differences, all polypeptides 9 8 migrate as anions as a result of the complex unth SDS Nhen polypeptides are completely denatured, the SDS binding holds them in a rigid structure and their separation through the

SDS-polyacry 1 amide gel is dependent upon size. Incomplete denaturation of polypeptides restricts binding of SDS ions and the charge of the polypeptides becomes a factor in their migration. Polypeptides that are complexed uhth fewer SDS ions are less positively charged and will consequently migrate at a slower rate than polypeptides of equal molecular weight that are fully denatured Two bands could therefore result if they bind different amounts of SDS even though the bands represent the same polypeptide.

Sequences needed for expression of genes in methanogens.

Expression of 0RF396 and 0RF397 in cells of the methanogens U. barkeri and EL vannielii respectively, presumably does not result in two polypeptides. Correct transcription and translation start signals should result in the synthesis of only one transcript and one polypeptide. The tL vannielii 0RF397 upstream DNA sequences are similar to conserved sequences proposed as promoters far other methanococcal genes (53).

Footprinting and SI mapping experiments will be necessary to determine the sequences which bind tL vannielii DNA-dependent

RNA polymerase and the sites of transcription initiation preceding 0RF396 and 0RF397 Sequences found upstream of tL harkeri 0RF396 bear more resemblance to the promoters proposed for tL halobium and tL smithii (53) than to the putative promoters for methanococcal genes. This is in line with established evolutionary relationships within the archaebacteria. The genus hhthanosarcina is within the order

Methanomicrohiales which is more closely related to the 99 halophiles such as tL hatohium than are the otner two orders of methanogens, the Methanobac.teriales and the

Methanococcales (21. Within the methanogens however the

Methanomicrobiales and Methanohacteriales. which includes tL smithii. are thought to be the more closely related <21 Until more DNA sequences From Methannsarcina and other members of the Methanomicrobiales. such as Methanospirilium hunqatei, are available, proposals for promoters in this order would be premature

M. barkeri direct repeats.

The function of the three 29 bp direct repeats upstream of M harkeri 0RF396 is unknown. They are not artifacts of cloning as they exist in two strains of tL barkeri. MS and 227.

A general promoter function seems unlikely as they occur only once in the genomes of tL barkeri If the consensus sequence

AAGTTYAAGTAYTCATCAATCTYAA6TTY were similar to a tL barkeri promoter consensus sequence, multiple fragments would have hybridized to the probes. The 29 bp direct repeats might be involved in the regulation of expression of argininosuccinate synthetase in tL barkeri Neither tL barkeri 0RF396 nor tL vannielii 0RF397 were preceded by obvious attenuator structures and there was no resemblance in the upstream DNA sequences to the L coli "arg box". The 29 bp direct repeats might therefore function as a binding site for a regulatory molecule to regulate the expression of the argininosuccinate synthetase gene. If, however, the synthesis of all enzymes involved in the biosynthesis of arginine were coordinately controlled in H barkeri. then similar sequences would be expected upstream of other genes involved in arginine biosynthesis. Hybridization of the radioactive probes to a single lee Hindlll fragment of £1. barkeri DNA also therefore argues

against this possibility unless all the arq genes are linked in

this organism. The £9 bp direct repeats shoui sequence

similarity to seven 22 bp direct repeats located at the origin

of replication for plasmid R 6K <122> The sequence

AAC6 TACTAA6CTCTCAT6 TTT matches the 29 bp direct repeats at 17

of 22 positions if two gaps are included to maximize identity.

One could speculate therefore that the 29 bp repeats might

represent an origin of replication in tL barkeri Advances in

the molecular biology of tL barkeri will have to be made before

such a possibility could be tested by cloning the direct

repeats, with a selectable trait incapable of autonomous

replication, and transformation of the resulting plasmid into M. barkeri.

The function of the 14 bp direct repeats also remains

unknown Their location directly downstream of 0RF398

suggests a rale in termination of transcription in TL barkeri

however, there are no similar DNA sequences downstream of

0RF396. If these repeats had a general role in transcription

termination, throughout the genome, more than one Hindlll digested DNA fragment would have hybridized with the radioactive probes containing the 14 bp direct repeats. A role

in the coordination of regulation of expression of arginine

biosynthetic genes is possible, but again more than one Hindlll

digested DNA fragment would have been expected to have

hybridized to the radioactive probes if the repeat sequences

played a coordinating role in the control of expression of arq 6

genes

The lack of hybridization of multiple bands to the radioactive probes representing the 14 bp and 29 bp direct repeats may also be due to experimental conditions. Conditions 101 were chosen to give hybridization of the probes to complimentary sequences without mismatches. It is possible that the repeats resemble control elements scattered thoughout the genome but, due to mismatches in their sequences, these have not hybridized to the probes under the conditions used.

Codon usage and gene organization in M barkeri and M vannielii

Codon usage in the two tL uannielii ORFs of pET821 is very similar to that previously reported for other methanococcal genes (53); namely preferential usage of codons which have an

A or li in the third position and infrequent use of codons containing a CG dinucleotide Although the tL barkeri ORFs of pET371 haue some similarities in codon usage with the methanococcal genes there is also similarity to codon usage in

H. halohium < 11, 36. 53). Neither tL barkeri nor H. halobium ORFs contain the glutamine codon CAA or leucine codon CUA, and both use the arginine codon AGA very infrequently This is not true for tL uannielii. The codon usage of the tL barkeri mtc. operon is similar to that reported for tL uannielii and tL thermoautotrophicum am operons <14) with a preference for a

C in the wobble position. The arginine AGA codon is used more frequently in the £L barkeri mcr genes than in the araG and carB genes reported here. Use of the glutamine CAA codon is infrequent in the tL barkeri men operon and of a total 108 leucine codons only 2 are CUA codons Therefore some of the observations made for the codon usage of tL barken genes sequenced in this study are also true for the genes of the M. barkeri mcr operon. The similarity of codon usage may be indicative of the closer relationship between the extreme halaphiles and the Methanomicrobiales than between the 102 halophiles and the Mcthanobacteriales on the Mehhanococcales

More ORFs from HalQbactermm s p , Methanosarcina s r . and

Me thanospir ilium sp. need to be analyzed to determine if there are consistent observations for codon usage

The ORFs encoded by tL vannielii DNA in pET821 and M

barkeri DNA in pET371 do not appear to be organized in operons.

Distances of 390 bp and 130 bp separate ORFs in the tL tiarkeri and tL vannielii DNAs respectively. These intergenic regions contain sequences that are similar to proposed promoter sequences Previously sequenced genes from tL vannielii <7, 85,

26), tL voltae <26, 72, 145), tL thermolithotroohicus <136) and tL smithii <51, 52) appear to be organized unthm operons. This feature therefore appears to be a feature common to methanogen and eubacteriai genomes. The L col* argG gene is, in fact, transcribed separately from arq genes which encode other enzymes involved in arginine biosynthesis in L coli which are arranged in operons. Independent control of the expression of argininosuccinate synthetase may therefore be a requirement in both eubacteria and archaebacteria for, as yet, unknown reasons. In £, coli the ar gfi gene is physically separated from the carAB operon whereas these genes are adjacent in the M barkeri genome. This clustering of genes involved in the arq biosynthetic pathway may be significant in the methanogen, although the clustering is apparently not as an operon.

Intergenic regions in both tL barkeri and tL vannielii have higher A+T contents than sequences encoding polypeptides This has been found in the intergenic regions of all methanogens studied. There is not however such a dramatic increase in the

A + T content in the intergenic regions in organisms that have a high overall molX 6+C such as tL thermoautotrophicum <49 7 X

6*0 <63). The increased frequency of A+T bases in the 1 0 3 intergenic regions of methanogen genes probably mas fortuitous

for the expression of these genes in L coli Sequences

necessary for transcription initiation in E. coli are A^T rich and

therefore occur more frequently by chance in the intergenic

regions between methanogen genes.

Evolutionary relationship of argininosuccinate synthetase genes.

Comparison of homologous sequences can be used to

determine the evolutionary relationships Until more 16S rRNA

sequences from thf? archaebacteria are completely determined,

the best available measure of the evolutionary relattonships

are comparisons of oligonucleotides produced by RNase T1

digestion of the 16S rRNAs from the various organisms <2>.

Based on oligonucleotide catalogues, £1. vannielii and EL barkeri

were determined to have an association coefficient of 0.20

and were assigned to different orders, the Methanococcales and

Methanomicrobiales respectively The DMA sequences encoding

argininosuccinate synthetase from these two methanogens are

52.IX identical. Previous comparisons of homologous genes have

indicated a correlation between the X similarity of DNA

sequences and the S^g values determined from 16S rRNA

oligonucleotide comparisons <2) The hisA genes from tL vannielii

and tL voltae. two organisms within the same genus with a

value of 0 60, have DNA sequences which are 66 0X identical (26>

Two rucE genes from tL smithii and tL thermoautotrophicum.

species in different genera within the same family, with a S^jj value of 0.49, have DNA sequences that are 53.0X identical <51).

The results show there is also a correlation between the S^g values and value for X similarity of DNA sequences for the aroS genes of tL vannielii and tL barkeri. 104 Construction of an evolutionary tree using the DNA sequences of tL, uannielii. tl* barkeri and human cDNA encoding argininosuccinate synthetases provides the following observations The two methanogen argS sequences are more closely related to each other than either is to the human cDNA sequence or the human pseudogenes. Divergence of the two methanogen argfi genes however occurred prior to the divergence of the human sequences, and all the present day sequences are derived from a common ancestral gene A better indication of the evolutionary relationships between organisms is obtained when amino acid sequences are used to construct trees. Amino acid comparisons are however only more revealing if functional polypeptides are being studied. The inclusion of human argininosuccinate synthetase pseudogenes in this comparison demanded that DNA sequences be compar ?d.

Pseudogenes are not expressed and are therefore not subject to the same selective pressures An evolutionary tree, constructed previously (94), using the DNA sequences of seven pseudogenes and the functional human argininosuccinate synthetase gene, is in good agreement with the tree presented here (Fig. 131. HumASPs3 was calculated to have diverged from the expressed gene approximately 55 million years ago and

HumASPsl to have diverged approximately 48 million years ago

(94). These calculations cannot reasonably be extrapolated however to determine the probable time of divergence of the two methanogens because of the uncertainty of different mutational rates for DNA sequences with and without the selective pressure of coding for a functional gene product.

A more complete evolutionary tree for argininosuccinate synthetase would include the two methanogen AC 90 sequences described in this study, tL voltae argG (145), tL 1 0 5 thermalithotr ophicus acgG

Introns within the human argininosuccinate synthetase gene.

It has been determined that the human argininosuccinate synthetase gene contains at least 13 exons <39, 40). Ten intron-exon boundaries of seven introns have been sequenced

<39). Two of the introns, intron 1 and intron 2, are 67 bp and

5 bp respectively, upstream of the ftTG initiation codon of the argininosuccinate synthetase gene. Of the five introns determined within the gene, four lie between two codons and are classified as phase 0 introns Cintrons 3, 4, 7 and 8) <117)

Intron 9 lies between the first and second bases of a codon and so is classified as a phase 1 intron. All the intron-exon boundary sequences resemble the consensus splice sites for nuclear mRNA precursors

5,-A/'CA6GtGTft>'6AGT. . intron .

The methanogen DNA sequences that align with the intron-exon boundary sequences of the human argininosuccinate synthetase gene show similarity to the consensus splice site sequence A similarity is seen when the methanogen sequences are compared to the sequences of either the 5' or the 3' splice site sequences. This may however be a result of conservation of amino acid sequences essential to the function or folding of the polypeptide. Introns 3, 4, and 8 are located in regions of 1 0 6 the polypeptide inhere the amino acid residues of the three argininosuccinate synthetase sequences are not conserved.

Intron 7 however, lies within a highly conserved region of amino acid residues and intron 9 is located in the first codon of the amino acid sequence 6 UPU conserved in the argininosuccinate synthetase of all three species

Carbamvl phosphate synthetase in M. barkeri.

M barkeri 0RT398 was determined to be the carboxyl terminal region of a polypeptide that is homologous to the large subunits of £. coli, £L cerevisiae and rat carbamyl phosphate synthetase. In E» coli and S. ceremsiae, the genes encoding the carbamyl phosphate synthetase large subunit (carB and CPAS respectively) are located downstream of the genes encoding the small carbamyl phosphate synthetase subunit (carA and CPA1) <80. 95). These genes are cotranscribed in £. coli as an operon In the rat the two genes have become fused resulting in one gene (CPS1) encoding both subunits (98)

Internal homology within the carbamyl phosphate synthetase large subunit indicates that a tandem duplication of an ancestral gene occurred prior to the divergence of the eubacteria and eukaryotes <80, 95). The length of the carB ORF in tL barkeri was insufficient to determine if there is an internal duplication within the methanogen gene If the carbamyl phosphate synthetase gene in tL barkeri does not have interna) homology it would suggest that the gene duplication event occurred after the divergence of the archaebacteria but before the divergence of the eubacteria and eukaryotes.

Comparison of the E_ coli. cerevisiae and rat carbamyl phosphate synthetase amino acid sequences indicates that the amino-termini of the polypeptides are more conserved than the 107 carboxyl-termini <80, 95) When the tl. barkeri carB carboxyl-terminus is compared to the amino- and carboxyl-termini of £. coli carB and S» cerevisiae CPAS there is more similarity between the carboxyl-termini of the polypeptides The lack of conservation between the carboxyl-terminus of 0RF398 and the more conserved ammo-termini suggests that there may be a more conserved amino—terminal region of the tL barkeri carB gene which would be more similar to the amino-termini of the £, coli carB and S. cerevisiae CPA2 genes If this were true, then the duplication of the ancestral gene occurred before the divergence of the archaebacteria from the eubacteria and eukaryotes Use of the pET371 DNA as a radioactive probe should identify the adjacent

DNA sequences in a DNA library and the rest of the tL barkeri carB gene could be cloned and sequenced to determine the presence or absence of an internal duplication

Comparison of the carB genes of £_ coli and tL barkeri indicated the DNA sequences available were 5B.3X identical whereas the 16S rRNA oligonucleotide catalogue comparisons determined a value of 0.10 for £, coli and tL barkeri These calculations show there is a trend between the values and

V. similarity as was observed with the results for arqfi. hisA and purE genes as stated above. 1. n. vannielii 0RF397 and tL barken 0RF396 encode argininosuccinate synthetase polypeptides capable of complementing the £* coli ac.gG36 and JL subtilis angA 2 auxotrophic mutations

2. Argininosuccinate synthetase genes in tL barkeri. M uannielii and human have diverged from a common ancestral gene and show extensive conservation of amino acid sequences. The methanogen sequences are more closely related to each other

(DNA: 52 IY. identical; amino acids: 66 .7/i identical) than either is to the human tL vannielii:

54.2Ji identical amino acids).

3. The comparison of argininosuccinate synthetase and carbamyl phosphate synthetase genes supports the established phylogenetic relationship for tL barkeri and tL vannielii in separate methanogenic orders. Similarity between upstream sequences of tL barkeri and tL halobium genes and codon usage within the genes supports the established close phylogenetic relationship of the Mathanomicmbiales and the extreme halophiles.

4 The region upstream of 0RF397 in tL vannielii contains sequences similar to those suggested as methanococcal promoters, whereas sequences upstream of tL barkeri 0RF396 188 109 show more similarity to sequences suggested to be promoters in tL haUttnum and £L smithu

5 t ± barken 0RF398 encodes the carboxyl terminus of the large subunit of carbamyl phosphate synthetase, additional sequencing is needed to determine if this gene has an internal duplication.

6 The 29 bp and 14 bp direct repeats encoded by the hi harken derived DNft in pET371 are not artifacts of cloning The blocks of repeats occur once in the genomes of both H barkeri

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