DOCSLIB.ORG

Polarity of Heteroduplex Formation Promoted by Escherichia

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Polarity of Heteroduplex Formation Promoted by Escherichia Proc. Natl Acad. Sci. USA Vol. 78, No. 8, pp. 4786-4790, August 1981 Biochemistry Polarity of heteroduplex formation promoted by Escherichia coli recA protein* (genetic recombination/homologous pairing/strand transfer/joint molecules) ROGER KAHN, RICHARD P. CUNNINGHAM, CHANCHAL DASGUPrA, AND CHARLES M. RADDING The Departments of Human Genetics and Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Aaron B. Lerner, April 24, 1981 ABSTRACT When recA protein pairs circular single strands tron microscopy, we observed that the product of this reaction with linear duplex DNA, the circular strand displaces its homolog is a joint molecule in which the circular single strand has dis- from only one end of the duplex molecule and rapidly creates het- placed its homolog, starting from one end of the duplex mole- eroduplexjoints that are thousands ofbase pairs long [DasGupta, cule (ref. 8 and this paper). In 15-30 min, some heteroduplex C., Shibata, T., Cunningham, R. P. & Radding, C. M. (1980) Cell joints become thousands ofnucleotides long (8). The rapid cre- 22, 437-446]. To examine this apparently polar reaction, we pre- ation ofthese long heteroduplex joints, and the absence ofmol- pared chimeric duplex fragments ofDNA that had M13 nucleotide ecules in which circular single strands invaded the duplex DNA sequences at one end and G4 sequences at the other. Circular sin- from both ends (unpublished data) led us to infer that recA pro- gle strands homologous to M13 DNA paired with a chimeric frag- ment when M13 sequences were located at the 3' end ofthe com- tein actively drives the formation ofheteroduplex DNA in one plementary strand but did not pair when the M13 sequences were direction. The experiments described in this paper were de- located at the 5' end. Likewise circular single-stranded G4 DNA signed to examine this apparent polarity. paired with chimeric fragments only when G4 sequences were lo- METHODS cated at the 3' end ofthe complementary strand. To confirm these Enzymes. E. coli exonuclease I was purified by John Wil- observations, we prepared fd DNA labeled only at the 5' or 3' end liams (11) according to the procedure ofLehman and Nussbaum of the plus strand, and we examined the susceptibility ofthese la- (12). E. coli exonuclease VII was a gift ofJohn Chase (13). recA beled ends to digestion by exonucleases whenjoint molecules were protein (3, 14) and A exonuclease (15) were purified as de- formed. Eighty percent of the 5' label in joint molecules became sensitive to exonuclease VII. Displacement of that 5' end by recA scribed. Bacterial alkaline phosphatase was a gift of Jan Chle- protein was concerted because it did not occur in the absence of bowsky. Polynucleotide kinase was purchased from Boehringer. single-stranded DNA or in the presence of heterologous single E. coli polymerase I (large fragment) and endonuclease Sau 96 strands. By contrast, only a small fraction of the 3' label became I were purchased from New England BioLabs. Endonuclease sensitive to exonuclease VII or exonuclease I. These observations Hpa I was purchased from New England BioLabs and Bethesda show that recA protein forms heteroduplex joints in a concerted Research Laboratories (Rockville, MD). Endonuclease BamHI and polarized way. was purchased from Bethesda Research Laboratories. Preparation of DNA. Single-stranded and replicative form The product of the recA gene ofEscherichia coli is a DNA-de- DNA from phages fd, 4X174, and G4 were prepared as de- pendent ATPase that regulates a pathway of inducible repair scribed (7, 16). Superhelical DNA from M13 Goril was pre- by means of a specific protease activity (see ref. 1 for a review) pared according to the procedures used for fd DNA (7). and that also has a remarkably versatile ability to promote the Duplex DNA 32P-Labeled at the 5' End of the Plus Strand. homologous pairing of DNA molecules. With a few exceptions, We cut form I fd [3H]DNA at a single site by digesting it with recA protein can stably pair all manner of structural variants of Hpa I and labeled the 5' ends of this DNA by treating it with DNA molecules: complementary single strands with each other, bacterial alkaline phosphatase followed by polynucleotide ki- single strands with duplex DNA, and partially single-stranded nase essentially as described by Maxam and Gilbert (17). To DNA with duplex DNA (2-9). The rule that governs stable pair- verify that the 32p label was present only at the ends, we cleaved ing appears to be that one molecule must be at least partially an aliquot ofthis DNA with BamHI which cuts Hpa I-linearized single-stranded and that afree end must exist somewhere in one fd DNA into three fragments: two end fragments and an internal of the two molecules (4, 8). We have experimentally distin- fragment 3425 base pairs long. The internal fragment, separated guished two steps in the formation of stable joint molecules by by gel electrophoresis, contained less than 1% of the total 32p recA protein: (i) synapsis, which appears to require single- recovered in the three fragments. stranded DNA but does not require a free end, and (ii) strand The linear DNA with 32p label at both ends was cleaved into transfer, which requires a homologous free end and produces two fragments by Sau 96 I: a large fragment (5725 base pairs) classical heteroduplex joints in which a strand from each pa- labeled at the 5' end ofthe plus strand and a small one (683 base rental molecule is helically interwound with its complement pairs) labeled at the 5' end ofthe minus strand. The fragments (8, 10). were separated by centrifugation through a 5-20% gradient of From an experimental point of view, one of the most inter- sucrose. According to the approximate specific activity of the esting pairings promoted by recA protein is that ofcircular sin- [y-32P]ATP used to label the ends, we estimated that 30% of gle strands with linear duplex DNA. This reaction is rapid and 5' ends of plus strands were phosphorylated. efficient; its substrates and products are well defined. By elec- Duplex DNA 32P-Labeled at the 3' End ofthe Plus Strand. This DNA, which also consisted ofthe large fragment produced I with Sau 96 I and The publication costs ofthis article were defrayed in part by page charge by cutting fd form [3H]DNA both Hpa I, payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * This is paper no. 12 in a series. Paper no. 11 is ref. 11. 4786 Downloaded by guest on September 29, 2021 Biochemistry: Kahn et aL Proc. Natl. Acad. Sci. USA 78 (1981) 4787 0~~~ ~ ~ ~ ~ ~ ~ ~ ~ 0~ A~~~~~~~~~ 0~~~~~~~~~6~~~~ 'ZO I I~~~~~~~~~3 4.~~~~~ ~ ~ ~~~~~~~~~~~*~ 4!~ ~ ~ Fig3.BndC)Cicuarsigl-srade G DA:nDN i jin mleuls it te mal ramet. D)Cicuarsigl-srade f jin molcueiththlrgfrgmntArow, ppren edsof etroupex egon frme b prtil rasfr o astandfrm helinardule moleculemoifedbyKegsrtothesingle-strandede a. 19. circle.h cndtinsusdweeReaction products wereNAat0.spread fr.t/melectroni 5%microscopyomaid,by the.1Mformamidemonumactae,000%technique of Davis etytchomal. (18) a (extratedfrmhorsheart, spred ontoa hypohase o doubl-distiled wter. Te grid were otary hadowe with ungste (20) t an agle o 80.TheDNAwasexamined with a Philips EM2O1 electron mirsoe was labeled by a method to be described elsewhere. Other cular single strands as linear duplex molecules, 80% of the du- methods were as described (7, 8, 10, 14). plex DNA appeared in joint molecules in 10 min (Figs. 1 and 2). When we heated deproteinized samples for 4 min at 41'C RESULTS prior to assayingjoint molecules, we found nearly as much prod- Time Course ofFormation ofJoint Molecules from Circular uct as in the unheated samples (Fig. 2). At 500C there was a Single-Stranded DNA and Linear Duplex DNA by recA Pro- significant loss ofjoint molecules, but the fraction ofjoint mol- tein. In a reaction mixture containing about twice as -many cir- ecules that survived heating at 50'C increased from 27% at 5 min to 91% at 30 min. This increase in thermal stability of the products of the reaction as a function of the duration of the re- action is consistent with a progressive increase in the average length of heteroduplex joints. 80 As in the formation ofjoint molecules by single-stranded frag- ments and duplex DNA (21), the kinetics of formation of the joint molecules described here resemble Michaelis-Menten -@ 60 kinetics (unpublished data), which suggests that the mechanism u 1.0 offorming joint molecules from single-stranded circles and lin- 0 ear duplex DNA is related to the mechanism offorming D loops 40 (3, 21, 22). 0.5 Specific Pairing of Circular Single-Stranded DNA with the 3' End ofIts Complementary Strand. Circular single-stranded 20 DNA isolated from phage particles consists of viral or plus strands that can pair only with the minus strand of the duplex DNA. Therefore, if the growth of heteroduplex joints is uni- directional, the single-stranded DNA should pair with only one 10 20 30 end of a duplex fragment and should displace a specific end of Time, min the plus strand from the duplex DNA. To look for pairing with. FIG. 2. Time course and thermal stability ofjoint molecules made a specific end of the duplex DNA we used Hpa I to cleave by recA protein from circular single-stranded and linear duplex DNA. M13Goril DNA into two fragments that had M13 DNA at one Reaction mixtures contained 4 yM linear duplex OX174 [3H]DNA, 4.8 end and G4 DNA at the other (Fig.
Load more

Recommended publications
  • Transcripts of the Adeno-Associated Virus Genome: Mapping of the Major Rnas MICHAEL R
    JOURNAL OF VIROLOGY, Oct. 1980, p. 79-92 Vol. 36, No. 1 0022-538X/80/10-0079/14$02.00/0 Transcripts of the Adeno-Associated Virus Genome: Mapping of the Major RNAs MICHAEL R. GREEN AND ROBERT G. ROEDER Departments ofBiological Chemistry and Genetics, Division ofBiology and Biomedical Sciences, Washington University School ofMedicine, St. Louis, Missouri 63110 The four major adeno-associated virus type 2 (AAV2)-specific RNAs were mapped on the linear viral genome by a variety of biochemical techniques, including Si nuclease and exonuclease VII mapping, RNA gel-transfer hybridi- zation, and analysis of reverse transcriptase extension products. All the major AAV2 RNAs were derived from the minus DNA strand and had 3' termini at position 96. The nucleus-specific 4.3- and 3.6-kilobase (kb) RNAs had 5' termini at positions 6 and 19, respectively. The 5' terminus of the 2.6-kb RNA mapped to position 38.5. The predominant 2.3-kb AAV2 mRNA was spliced and contained a short leader sequence (approximately 50 nucleotides) which mapped to position 38.5, coincident with the 5' terminus of the 2.6-kb RNA. The 5' end of the body of the 2.3-kb RNA mapped to position 46.5. These results are discussed in terms of the involvement of single versus multiple promoters (for transcription) and RNA splicing mechanisms in the generation of the AAV2 RNAs. Mammalian DNA viruses have provided pow- In our earlier studies ofAAV2 (19), we defined erful models for the analysis and formulation of and partially characterized four predominant mechaisms of gene expression in eucaryotic AAV2 RNAs in virus-infected cells, indicating cells.
    [Show full text]
  • Ubc 2008 Spring Li Alice.Pdf
    IDENTIFICATION OF VIRULENCE DETERMINANTS OF MYCOBACTERIUM TUBERCULOSIS VIA GENETIC COMPARISONS OF A VIRULENT AND AN ATTENUATED STRAIN OF MYCOBACTERIUM TUBERCULOSIS. by ALICE HOY LAM LI B.Sc., The University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIRMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Pathology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) MARCH 2008 Alice Hoy Lam Li, 2008 i ABSTRACT Candidate virulence genes were sought through the genetic analyses of two strains of Mycobacterium tuberculosis, one virulent, H37Rv, one attenuated, H37Ra. Derived from the same parent, H37, genomic differences between strains were first examined via two-dimensional DNA technologies: two-dimensional bacterial genome display, and bacterial comparative genomic hybridisation. The two-dimensional technologies were optimised for mycobacterial use, but failed to yield reproducible genomic differences between the two strains. Expression differences between strains during their infection of murine bone-marrow-derived macrophages were then assessed using Bacterial Artificial Chromosome Fingerprint Arrays. This technique successfully identified expression differences between intracellular M. tuberculosis H37Ra and H37Rv, and six candidate genes were confirmed via quantitative real-time PCR for their differential expression at 168 hours post-infection. Genes identified to be upregulated in the attenuated H37Ra were frdB, frdC, and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE, and Rv1571. Further qPCR analysis of these genes at 4 and 96h post-infection revealed that the frd operon (encoding for the fumarate reductase enzyme complex or FRD) was expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 was higher in the virulent strain throughout the course of infection.
    [Show full text]
  • Letters to Nature
    letters to nature Received 7 July; accepted 21 September 1998. 26. Tronrud, D. E. Conjugate-direction minimization: an improved method for the re®nement of macromolecules. Acta Crystallogr. A 48, 912±916 (1992). 1. Dalbey, R. E., Lively, M. O., Bron, S. & van Dijl, J. M. The chemistry and enzymology of the type 1 27. Wolfe, P. B., Wickner, W. & Goodman, J. M. Sequence of the leader peptidase gene of Escherichia coli signal peptidases. Protein Sci. 6, 1129±1138 (1997). and the orientation of leader peptidase in the bacterial envelope. J. Biol. Chem. 258, 12073±12080 2. Kuo, D. W. et al. Escherichia coli leader peptidase: production of an active form lacking a requirement (1983). for detergent and development of peptide substrates. Arch. Biochem. Biophys. 303, 274±280 (1993). 28. Kraulis, P.G. Molscript: a program to produce both detailed and schematic plots of protein structures. 3. Tschantz, W. R. et al. Characterization of a soluble, catalytically active form of Escherichia coli leader J. Appl. Crystallogr. 24, 946±950 (1991). peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 34, 3935±3941 29. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and (1995). the thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281±296 (1991). 4. Allsop, A. E. et al.inAnti-Infectives, Recent Advances in Chemistry and Structure-Activity Relationships 30. Meritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505± (eds Bently, P. H. & O'Hanlon, P. J.) 61±72 (R. Soc. Chem., Cambridge, 1997).
    [Show full text]
  • ( 12 ) United States Patent
    US010428349B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 , 428 ,349 B2 DeRosa et al . (45 ) Date of Patent: Oct . 1 , 2019 ( 54 ) MULTIMERIC CODING NUCLEIC ACID C12N 2830 / 50 ; C12N 9 / 1018 ; A61K AND USES THEREOF 38 / 1816 ; A61K 38 /45 ; A61K 38/ 44 ; ( 71 ) Applicant: Translate Bio , Inc ., Lexington , MA A61K 38 / 177 ; A61K 48 /005 (US ) See application file for complete search history . (72 ) Inventors : Frank DeRosa , Lexington , MA (US ) ; Michael Heartlein , Lexington , MA (56 ) References Cited (US ) ; Daniel Crawford , Lexington , U . S . PATENT DOCUMENTS MA (US ) ; Shrirang Karve , Lexington , 5 , 705 , 385 A 1 / 1998 Bally et al. MA (US ) 5 ,976 , 567 A 11/ 1999 Wheeler ( 73 ) Assignee : Translate Bio , Inc ., Lexington , MA 5 , 981, 501 A 11/ 1999 Wheeler et al. 6 ,489 ,464 B1 12 /2002 Agrawal et al. (US ) 6 ,534 ,484 B13 / 2003 Wheeler et al. ( * ) Notice : Subject to any disclaimer , the term of this 6 , 815 ,432 B2 11/ 2004 Wheeler et al. patent is extended or adjusted under 35 7 , 422 , 902 B1 9 /2008 Wheeler et al . 7 , 745 ,651 B2 6 / 2010 Heyes et al . U . S . C . 154 ( b ) by 0 days. 7 , 799 , 565 B2 9 / 2010 MacLachlan et al. (21 ) Appl. No. : 16 / 280, 772 7 , 803 , 397 B2 9 / 2010 Heyes et al . 7 , 901, 708 B2 3 / 2011 MacLachlan et al. ( 22 ) Filed : Feb . 20 , 2019 8 , 101 ,741 B2 1 / 2012 MacLachlan et al . 8 , 188 , 263 B2 5 /2012 MacLachlan et al . (65 ) Prior Publication Data 8 , 236 , 943 B2 8 /2012 Lee et al .
    [Show full text]
  • Supplementary Information
    Supplementary information (a) (b) Figure S1. Resistant (a) and sensitive (b) gene scores plotted against subsystems involved in cell regulation. The small circles represent the individual hits and the large circles represent the mean of each subsystem. Each individual score signifies the mean of 12 trials – three biological and four technical. The p-value was calculated as a two-tailed t-test and significance was determined using the Benjamini-Hochberg procedure; false discovery rate was selected to be 0.1. Plots constructed using Pathway Tools, Omics Dashboard. Figure S2. Connectivity map displaying the predicted functional associations between the silver-resistant gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S3. Connectivity map displaying the predicted functional associations between the silver-sensitive gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S4. Metabolic overview of the pathways in Escherichia coli. The pathways involved in silver-resistance are coloured according to respective normalized score. Each individual score represents the mean of 12 trials – three biological and four technical. Amino acid – upward pointing triangle, carbohydrate – square, proteins – diamond, purines – vertical ellipse, cofactor – downward pointing triangle, tRNA – tee, and other – circle.
    [Show full text]
  • The Effect of Nucleic Acid Modifications on Digestion by DNA Exonucleases by Greg Lohman, Ph.D., New England Biolabs, Inc
    be INSPIRED FEATURE ARTICLE drive DISCOVERY stay GENUINE The effect of nucleic acid modifications on digestion by DNA exonucleases by Greg Lohman, Ph.D., New England Biolabs, Inc. New England Biolabs offers a wide variety of exonucleases with a range of nucleotide structure specificity. Exonucleases can be active on ssDNA and/or dsDNA, initiate from the 5´ end and/or the 3´ end of polynucleotides, and can also act on RNA. Exonucleases have many applications in molecular biology, including removal of PCR primers, cleanup of plasmid DNA and production of ssDNA from dsDNA. In this article, we explore the activity of commercially available exonucleases on oligonucleotides that have chemical modifications added during phosphoramidite synthesis, including phosphorothioate diester bonds, 2´-modified riboses, modified bases, and 5´ and 3´ end modifications. We discuss how modifications can be used to selectively protect some polynucleotides from digestion in vitro, and which modifications will be cleaved like natural DNA. This information can be helpful for designing primers that are stable to exonucleases, protecting specific strands of DNA, and preparing oligonucleotides with modifications that will be resistant to rapid cleavage by common exonuclease activities. The ability of nucleases to hydrolyze phosphodi- of exonucleases available from NEB can be found Figure 1: ester bonds in nucleic acids is among the earliest in our selection chart, Common Applications of Examples of exonuclease directionality nucleic acid enzyme activities to be characterized Exonucleases and Non-specific Endonucleases, at → (1-6). Endonucleases cleave internal phosphodiester go.neb.com/ExosEndos. 3´ 5´ exonuclease bonds, while exonucleases, the focus of this article, What about cases where you only want to degrade 5´ 3´ must begin at the 5´ or 3´ end of a nucleic acid some of the ssDNA in a reaction? Or, when you 3´ 5´ strand and cleave the bonds sequentially (Figure 1).
    [Show full text]
  • B Number Gene Name Strand Orientation Protein Length Mrna
    list list sample) short list predicted B number Operon ID Gene name assignment Protein length mRNA present mRNA intensity Gene description Protein detected - Strand orientation Membrane protein detected (total list) detected (long list) membrane sample Proteins detected - detected (short list) # of tryptic peptides # of tryptic peptides # of tryptic peptides # of tryptic peptides # of tryptic peptides Functional category detected (membrane Protein detected - total Protein detected - long b0001 thrL + 21 1344 P 1 0 0 0 0 thr operon leader peptide Metabolism of small molecules 1 b0002 thrA + 820 13624 P 39 P 18 P 18 P 18 P(m) 2 aspartokinase I, homoserine dehydrogenase I Metabolism of small molecules 1 b0003 thrB + 310 6781 P 9 P 3 3 P 3 0 homoserine kinase Metabolism of small molecules 1 b0004 thrC + 428 15039 P 18 P 10 P 11 P 10 0 threonine synthase Metabolism of small molecules 1 b0005 b0005 + 98 432 A 5 0 0 0 0 orf, hypothetical protein Open reading frames 2 b0006 yaaA - 258 1047 P 11 P 1 2 P 1 0 orf, hypothetical protein Open reading frames 3 b0007 yaaJ - 476 342 P 8 0 0 0 0 MP-GenProt-PHD inner membrane transport protein Miscellaneous 4 b0008 talB + 317 20561 P 20 P 13 P 16 P 13 0 transaldolase B Metabolism of small molecules 5 b0009 mog + 195 1296 P 7 0 0 0 0 required for the efficient incorporation of molybdate into molybdoproteins Metabolism of small molecules 6 b0010 yaaH - 188 407 A 2 0 0 0 0 PHD orf, hypothetical protein Open reading frames 7 b0011 b0011 - 237 338 P 13 0 0 0 0 putative oxidoreductase Miscellaneous 8 b0012 htgA
    [Show full text]
  • SUPPLEMENTARY INFORMATION Sequence Analysis of Hypothetical Proteins from Helicobacter Pylori 26695 to Identify Potential Virule
    SUPPLEMENTARY INFORMATION Sequence Analysis of Hypothetical Proteins from Helicobacter pylori 26695 to Identify Potential Virulence Factors Ahmad Abu Turab Naqvi1§, Farah Anjum2§, Faez Iqbal Khan3, Asimul Islam1, Faizan Ahmad1, Md. Imtaiyaz Hassan1* 1Center for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India, 2Female College of Applied Medical Science, Taif University, Al-Taif 21974, Kingdom of Saudi Arabia, 3School of Chemistry and Chemical Engineering, Henan University of Technology, Henan 450001, China http://www.genominfo.org/src/sm/gni-14-125-s001.pdf. Supplementary Table 4. List of annotated function of 340 hypothetical proteins (HPs) from Helicobacter pylori using BLASTp, STRING, SMART, InterProScan and Motif Motif found Predicted functional partner No. UniProt ID Major BLAST hit SMART (STRING) InterProScan Motif 1 O24859 Valyl-tRNA synthetase DNA primase No result No result Protein similar to CwfJ C-terminus 1 2 O24860 TrbC/VIRB2 family VirB4 homolog TrbC/VIRB2 family Conjugal transfer TrbC/VIRB2 family protein TrbC/type IV secretion VirB2 (pfam) 3 O24861 ComB3 competence VirB4 homolog Transmembrane region Membrane-bound protein Photosystem I psaA/psaB protein protein Predicted membrane protein 4 O24863 No result Lipoprotein signal peptidase No result Prokaryotic membrane Prokaryotic membrane lipoprotein lipid lipoprotein lipid attachment site profile attachment site profile 5 O24869 No result No result No result No result No result 6 O24871 No result Isocitrate dehydrogenase
    [Show full text]
  • Identification of Rnase T As a High-Copy Suppressor of the UV
    Copyright 1999 by the Genetics Society of America Identi®cation of RNase T as a High-Copy Suppressor of the UV Sensitivity Associated With Single-Strand DNA Exonuclease De®ciency in Escherichia coli Mohan Viswanathan, Anne Lanjuin and Susan T. Lovett Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454-9110 Manuscript received August 6, 1998 Accepted for publication November 24, 1998 ABSTRACT There are three known single-strand DNA-speci®c exonucleases in Escherichia coli: RecJ, exonuclease I (ExoI), and exonuclease VII (ExoVII). E. coli that are de®cient in all three exonucleases are abnormally sensitive to UV irradiation, most likely because of their inability to repair lesions that block replication. We have performed an iterative screen to uncover genes capable of ameliorating the UV repair defect of xonA (ExoI2) xseA (ExoVII2) recJ triple mutants. In this screen, exonuclease-de®cient cells were transformed with a high-copy E. coli genomic library and then irradiated; plasmids harvested from surviving cells were used to seed subsequent rounds of transformation and selection. After several rounds of selection, multiple plasmids containing the rnt gene, which encodes RNase T, were found. An rnt plasmid increased the UV resistance of a xonA xseA recJ mutant and uvrA and uvrC mutants; however, it did not alter the survival of xseA recJ or recA mutants. RNase T also has amino acid sequence similarity to other 39 DNA exonucleases, including ExoI. These results suggest that RNase T may possess a 39 DNase activity capable of substituting for ExoI in the recombinational repair of UV-induced lesions.
    [Show full text]
  • Product Sheet Info
    Master Clone List for NR-19280 Yersinia pestis Strain KIM Gateway® Clone Set, Recombinant in Escherichia coli, Plates 1-43 Catalog No. NR-19280 Table 1: Yersinia pestis Gateway® Clone, Plate 1 (UYPVA), NR-195971 Clone Well Locus ID Description ORF Accession Average Position Length Number Depth of Coverage 38250 A01 NTL02YP3655 transcriptional activator of nhaA 900 AAM87251.1 3.90531915 38268 A02 NTL02YP0999 apbA protein 912 AAM84595.1 6.01785714 38344 A03 NTL02YP3653 putative regulator 939 AAM87249.1 5.6680286 38375 A04 NTL02YP3668 homoserine kinase 951 AAM87264.1 5.57214934 38386 A05 NTL02YP1006 cytochrome o ubiquinol oxidase subunit II 957 AAM84602.1 5.45937813 38408 A06 NTL02YP2211 suppressor of htrB, heat shock protein 963 AAM85807.1 5.40877368 38447 A07 NTL02YP3649 penicillin tolerance protein 978 AAM87245.1 5.34086444 38477 A08 NTL02YP0988 thiamin-monophosphate kinase 990 AAM84584.1 6.8223301 36045 A09 NTL02YP3670 hypothetical protein 159 AAM87266.1 4.53266332 36094 A10 NTL02YP3672 hypothetical protein 174 AAM87268.1 4.72429907 36136 A11 NTL02YP1000 hypothetical protein 189 AAM84596.1 5.25327511 36241 A12 NTL02YP2577 hypothetical protein 225 AAM86173.1 3.88301887 membrane-bound ATP synthase, F0 36280 B01 NTL02YP4086 240 AAM87682.1 4.79285714 sector, subunit c 36342 B02 NTL02YP3654 30S ribosomal subunit protein S20 264 AAM87250.1 3.86513158 36362 B03 NTL02YP2570 hypothetical protein 270 AAM86166.1 5.40322581 38504 B04 NTL02YP2573 putative ABC transporter permease 996 AAM86169.1 6.65444015 38516 B05 NTL02YP0234 putative heat shock
    [Show full text]
  • Nostoc Commune UTEX 584 Gene Expressing Indole Phosphate Hydrolase Activity in Escherichia Coli
    JOURNAL OF BACTERIOLOGY, Feb. 1989, p. 708-713 Vol. 171, No. 2 0021-9193/89/020708-06$02.00/0 Copyright © 1989, American Society for Microbiology Nostoc commune UTEX 584 Gene Expressing Indole Phosphate Hydrolase Activity in Escherichia coli WEN-QIN XIE,1 BRIAN A. WHITTON,2 J. WILLIAM SIMON,2 KARIN JAGERt DEBORAH REED,' AND MALCOLM POTTSl* Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061,1 and Department ofBotany, University ofDurham, Durham City DHJ 3LE, United Kingdom2 Received 18 August 1988/Accepted 9 November 1988 A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in XgtlO. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed whef the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an M, of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP0O5, pBH6, pB8) the cyanobacterial iph gene conferred an Iph' phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo- 4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N.
    [Show full text]
  • Oral Metapangenomics Daniel Utter 13 January 2020
    Oral Metapangenomics Daniel Utter 13 January 2020 Contents Overview . .1 Organization & Workflow . .2 Step 1 - Collecting and annotating genomes . .2 Getting the genomes from NCBI . .2 Anvi’o contigs database . .3 Whole thing . .3 Functional annotation . .4 Step 2 - Mapping . .5 Data acquisition - HMP Metagenomes . .5 Recruiting HMP metagenomes to the contigs . .6 A short statement on competitive mapping and its interpretation . .7 Step 3 - Profiling the metagenome recruitment . .8 Step 4 - Pangenome and metapangenome construction . .9 Some background on the methods wrapped by the anvio programs . 10 Combining metapangenomes . 11 Anvi’o interactive display choices for metapangenomes . 13 Supplemental tables . 13 Querying the gene cluster annotations for enriched functions . 13 Granular analysis of mapping results at the per-gene, per-sample level . 17 Differential function analysis . 20 Nucleotide level coverage plots (Figure 4A, 4B) . 21 Rothia candidate driver of adaptation to BM . 21 Investigating the effect of non-specific mapping . 25 Overview This is the long-form, narrative version of the metapangenomic methods used for our study Metapange- nomics. Our goal in writing this is to make the workflow be as transparent and reducible as possible, and also to provide a step-by-step workflow for interested readers to adapt our methods to their own needs. Most of the code here is based off of Tom Delmont and Meren’s Prochlorococcus metapangenome workflow, and we are deeply indebted to their project and their dedication to sharing a reproducible workflows. Our project investiated the environmental representation of the various bacterial pangenomes to learn more about the ecology and evolution of microbial biogeography.
    [Show full text]