The Genetical Society of Great Britain

Heredity 57 (1986) 125—142 The Genetical Society of Great Britain

THEGENETICAL SOCIETY

(Abstracts of Papers presented at the TWO HUNDRED AND THiRD MEETING of the Society on the 15th and 16th November 1985 at UNIVERSITY COLLEGE, LONDON)

1. Reverse-phase high- resolution andreproducibilitythan ID- performance liquid UREA! PAGE. chromatography of the 60S subunit proteins of the fungus 2.An analysis of the cyt.oplasmic Coprinus cinereus: evidence for an ribosomal proteins of altered 60S protein in a cycloheximide resistant and cycloheximide-resistant mutant sensitive monokaryotic stra;' ns and J. SardharwaIIa and J. North heterozygous dikaryons of Coprinus cinereus using 2— Cityof London Polytechnic, Calcutta House, Old Castle Street, London El 7NT. *Department of dimensional P.A.G.E. Biochemistry, King's College London (KQC), Strand, London WC2R2LS. J. D. Traynor and J. North Invitro synthesis studies of cycloheximide- Departmentof Biological Sciences, City of London protein Polytechnic, Calcutta House, Old Castle Street, resistant mutant of Coprinus cinereus have shown London El 7NT. that resistance to the drug is associated with the 60S subunit of the 80S ribosomes. Reverse-Phase 2-Dimensionalpolyacrylamide gel electrophoresis High-Performance Liquid Chromatography (RP- (P.A.G.E.) was used to study the cytoplasmic ribo- HPLC) has been explored as an approach for the somal proteins of Coprinus cinereus. The 40S ribo- separation, analysis and comparison of the pro- somal subunit had a complement of 24-30 proteins teins of the 60S subunits from the cycloheximide- which are identical in cycloheximide resistant sensitive and -resistant strains CY8 and CY8.2 strain CY8.2 and its corresponding wild-type CY8. respectively. Using a -Bondapak C18 column and In contrast, the proteins of the 60S ribosomal sub- a trifluoroacetic acid/acetonitrile solvent system, units of these strains showed a number of differen- the proteins from this subunit have been resolved ces, but comparison of these with 60S proteins into approximately 27 peaks. Comparison of the from CY9.23 and CY9, another pair of strains protein chromatograms of the two strains showed resistant and sensitive to cyclohexirnide showed two major differences: peak X, seen in the resistant that only one protein, Ln, was consistently associ- strain, is almost completely absent in the sensitive ated with resistance. strain; whereas peak V is reduced considerably in Analysis of 60S proteins from dikaryons intensity in the resistant strain. These differences heterozygous for resistance failed to confirm the correspond to the pattern shown after One- role of Ln in resistance, but did reveal the presence Dimensional Urea-Polyacrylamide Gel Elec- of 9 extra proteins which were probably derived trophoresis (ID-UREA/PAGE) of these proteins. from the second parents used to make the dikary- However, only 11 protein bands were resolved by ons, CY3 and CY13, which have a different genetic the latter technique. background from CY8 and CY9. In addition, there These results provide further evidence that were 5 proteins identified in the monokaryons resistance to cycloheximide is a property of the which were absent from the dikaryons in a pattern 60S subunit involving a protein component. They suggesting some type of regulation. also clearly establish RP-HPLC as a method of In all, 62 proteins were observed in the large choice for the rapid analysis and comparison of subunit, the range associated with any one strain eukaryotic ribosomal proteins, offering far better being 38-50. 126 THE GENETICAL SOCETY—ABSTRACTS OF PAPERS 3. Cloning of the arg-12 gene and Little is known about the congenital consequences of paternal exposure to mutagenic and clastogenic cross-pathway regulation of arg- 12 agents, despite the existence of two large amenable mRNA in Neurospora crassa groups of individuals who have been so exposed: (a) Men who have undergone chemotherapy and H. J. Flint*, J. Wilkening** and I. B. radiation treatment for cancer, and Barthelmess** (b) Atomic bomb survivors. *Department of Genetics, King's Buildings, Studies of the latter groups have failed to demo n- University of Edinburgh, Edinburgh EH9 3JN; strate an increase in the frequency of birth defects **lnstitut für Angewandte Genetik, Universität or chromosome aberrations (Awa, In: Radiation- Hannover, Herrenhäusesstr. 2 3 Hannover 21, West induced chromosome damage in Man, 1983, p. 433- Germany. 453, publ. A.R. Liss). Evidence for the former Ornithinecarbamyl transferase (OCTase), which group is far less copious. is encoded by the arg-12locus,is one of many We have applied a heterospecific in vitro fertili- amino acid synthetic enzymes whose activity is sation procedure, the sperm chromosome assay regulated through cross-pathway amino acid con- (SCA) (Tomkins eta!., Heredity 50: 211, 1983), to trol in Neurosporacrassa. Thisenzyme is encoded a number of men who are or were on individual by the arg-Blocusin Aspergillus nidulans (Berse or combined cytotoxic and radiation regimes. The et aL, Gene 25,109).Probes derived from the arg-B majority of such individuals initially become gene have now been used to identify and clone a azoospermic and those that recover spermatogenic partially homologous fragment from Neurospora function after treatment ceases, generally pass genomicDNA that carries the arg-12 gene. through an oligospermic phase. These samples arg-12 DNA probes hybridise to a single poly behave poorly in the SCA and we have utilised A transcript whose concentration was found to specific sperm selection and capacitation tech- increase greatly in vivo under conditions of amino niques to combat this problem. Further, batch vari- acidlimitationthat elicitcross-pathway ation in human serum albumin and foetal calf derepression of OCTase activity. This response serum used in the culture media also affect the was abolished in cpc-1 mutant strains that are efficiency and outcome of the SC. We have recently impaired in cross-pathway regulation, showing succeeded in conducting the entire assay in the that the product of the cpc-1 locus is involved in absence of both of these components using fully controlling OCTase mRNA levels, probably via defined media with a concomitant improvement in the rate of transcription. The level of NADP gluta- metaphase yield and quality. The advantage of this mate dehydrogenase mRNA (coded by the am interventionist approach to the attainment and gene—Kinnaird et aL, Gene 26, 253) was analysis of sperm chromosomes from genotoxic unaffected either by amino acid limitation or by exposed oligospermic samples will be discussed. the cpc-1 mutation. We have also investigated the rapidity and kinetics of the response of OCTase mRNA levels to amino acid limitation in cpc-1 strains, which have further implications for the regulatory mechanisms involved. 5.Control of DNA synthesis in bacteriophage Ti C. L. A. Paiva, D. A. Ritchie and G. Hope* 4.The analysis of sperm Department of Genetics, University of Liverpool, chromosomes in individuals P.O. Box 147, Liverpool L69 3BX. *lnstitute of Virology, University of Glasgow. Church Street, exposed to genotoxic agents Glasgow Gil 5JR. P. T. Tomkins, C. V. Carroll, J. A. Houghton The Ti genome encodes about 31 proteins, of and M. Hurley* which six are involved with DNA replication and Microbiology Department, University College, recombination. Genes 1 and 2 are implicated in Ga/way, Ireland. *Radiotherapy Department, Cork the initiation of Ti DNA synthesis; 2.5 in degrada- Regional Hospital, Cork, Ireland. tion of host DNA and shut off of host replication; THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 127 3.5 and 4 in the maintenance of viral DNA syn- 7. Biased inheritance of optional thesis and genetic recombination; and das in the partial reversal of Ti "DNA arrest" phenotype DNA insertions following caused by mutations in either gene 3.5 or 4. mitochondrial genome Mutations in genes 3.5 and 4 produce a recombination in Coprinus pleiotropic phenotype which includes: premature cinereus arrest of Ti DNA synthesis, failure to form con- catemers, reduced recombination, reduced ATP A. Economou, M. E. Zolan, P. J Pukkita and independent 5'-exonuclease activity, production of L. A. Casselton shorter than unit length progeny DNA molecules. School of Biological Sciences, Queen Mary College, We have identified a low molecular weight Mile End Road, London El 2EY and Department of peptide (called X) which seems to be a strong Biology & Curriculum in Genetics, University of candidate for the primary cause of the premature North Carolina, Chapel Hill, N.C. 27514,U.S.A. arrest of DNA synthesis observed for gene 3.5 and Twovariant mitochondrial DNAs in Coprinus 4 mutations. cinereus, H and J, are distinguished by having The possible roles of peptide X on the pre- alternative 1 23 Kb insertions at sites approxi- mature arrest of DNA synthesis and also its poss- mately 2 Kb apart in, or adjacent to, the co-i gene. ible involvement with the control of replication in Genetic recombination between J and H genomes Ti will be discussed. nearly always results in generation of a genome that contains both these insertions. Recombination was detected using the mitochondrial gene mutations acu-lO, which causes a cytochrome oxidase defect, and cap-i which confers resistance to chloramphenicol. In one series of crosses recombi- 6.Molecular studies on the nation gave rise to the wild type genotype acu- iOcap-1t Fourteen of 15 independently derived response of Brassica campestris recombinants were found to have both DNA inser- to challenge by pathogenic and tions. In a second series of crosses intragenic re- non-pathogenic mutants of combination within the cap-i gene was detected. Xanthomonas cam pestris pv. cap-i is assumed to be the structural gene for the LrRNA and is some 6 Kb distant from the nearest campestris and pv. vitians insertion site. Recombination within this gene must D. B. Collinge, J. M. Dow, D. E. Milligan and therefore be independent of any event leading to M. J. Daniels recombination of the insertions. Eight indepen- dentlyderivedcap-it recombinants were John Innes Institute, Colney Lane, examined and in all both DNA insertions were Norwich NR4 7UH. present. Hybridisation studies have shown that the Xanthomonascampestris pv. campestris is the insertions are non-homologous. The biased causative agent of black rot of Brassicas. Inocula- inheritance of these sequences suggests that they tion of mature leaves of Brassica campestris with encode a function similar to that of the w intron X.c. pv. vitians (a pathogen of Lactuca sativa) in the mitochondrial DNA of Saccaromyces causes hypersensitive reaction (HR) which is cerevisiae and that we are observing analogous localised to the region of inoculation. Physiologi- conversion events. cal and biochemical changes can be observed in inoculated areas which differ from those caused by virulent forms of X.c. pv. campestris. Various 8.Ammonium ion toxicity and non-pathogenic mutants of pv. campestris have ribosoma I mutations in Aspergillus been identified which give responses which range from something similar to HR to no detectable nidulans response. J. Hudson, R. Bratt and S. MartineHi mRNA preparations have been prepared from B. campestris leaves inoculated with X. campestris Department of Botany, Birkbeck College, University pathovars and mutants, and in vitro translocations of London, Malet Street, London WC1E7HX. have been performed using these which indicate Ammoniumtartrate or chloride are the most some differences in plant gene expression. frequently used nitrogen sources in our laboratory. 128 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS A chance observation that some suppressor- 11. Identification of a cDNA clone containing strains grew poorly on ammonium medium but almost normally on nitrate medium for the cyanogenic /3-glucosidase has led us to screen all translational mutants for gene from Trifolium repens L. ammonium sensitivity. (white clover) Strains with mutations in the putative tRNA genes, suaB and suaD, grew like wild-type on A. L. Sharif, E. Oxtoby, M. A. Dunn, D. B. ammonium medium whereas those strains with Collinge and M. A. Hugh ribosomal suppressors (suaA and suaC) behaved Department of Genetics, University of abnormally. Several suaC alleles have been Newcastle upon Tyne, Ridley Buildings, examined, and one of these, suaClO9, has been Newcastle upon Tyne NE1 7RU. tested in several genetic backgrounds All these strains are inhibited by nitrogen source. Increasing Activityof the white clover cyanogenic f3- the ammonium concentration gives a more pro- glucosidase, linarmarase, is controlled by the Li nounced effect. This is in contrast to normal growth locus. Affinity purified antibodies raised to a on proline, sodium nitrate or glutamate. Any denatured form of linamarase will immunoprecipi- ammonium salt has the same effect. Further anti- tate a 59 x iO Mr in vitro translation product from suppressor mutations which partially compensate mRNA extracted from developing leaves of Li Li for the hypersensitive reaction of suaClO9 strains and Li ii plants. No such polypeptide is produced to antibiotics and low temperature also correct to when mRNA from liii genotypes is used. The various degrees the ammonium sensitivity. 59 x iO Mr nascent polypeptide is processed to a Since the antisuppressor genes and suaC are 62 x i03 Mr protein (the subunit molecular weight thought to code for ribosomal proteins, one poss- of native, active linamarase) by unwashed dog ible explanation is that the ammonium ions inter- pancreas microsomes in a wheat germ in vitro fere directly with ribosome function or structure. translation system. A cDNA library was made in Although magnesium is the element which springs pBR322 using poly mRNA templates extrac- to mind in the context of ribosomes, monovalent ted from developing leaf tissue, genotype Li Li. ions also play a part in the association (or opposite) Putative linamarase clones were selected by of ribosomal subunits. We are examining the differential colony screening using ss cDNA probes effects of ammonium and other ions, in vitro, on synthesised using either Li Li or liii mRNA as a ribosome dissociation. template. A linamarase clone was identified We are also trying to exploit the toxic effect of amongst the putative clones by the hybrid select ammonium ions to isolate new types of ribosomal translation of a 59 x iO Mr polypeptide which is mutant. immuno-precipitated by affinity purified denatured—linamarase antibodies.

12.A cell-free system for examining ribosomal mutants in 10.Genetic controls of genome Aspergillus nidulans instability in Escherichia co/i K-i 2 A. Zamir and S. Martinetli A. P. Jessop and F. Rodger Department of Botany, Birkbeck College, University Institute of Genetics, University of Glasgow, of London, Malet Street, London WC1E 7HX. Glasgow Gil 5JS. Acell-free system has been developed for syn- Homologousrecombination not only brings about thesising polypeptides in vitro, using extracts of A. recombination in genetic crosses, but also acts on nidulans. Since it is an homologous system, it can intrachromosomal homologous sequences to gen- be used for studying translation mutants. The sys- erate chromosome rearrangements. We present tem is a modified version of that used for Podospora data on a recA-dependent instability in the 77' anserinaby M. Picard-Bennoun and colleagues. region of E. coli K-12, where a genetic component For this work we have to purify the elongation in one of our strains dramatically increases the factors partially from the S100 extract. The factors frequency of occurrence of the instability. are very labile and have to be used quickly. THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 129 Preliminary studies have been made on wild- 14. Electrophoresis of the type ribosomes and their susceptibility to ribosomal antibiotics which inhibit growth of Aspergil- ribosomal proteins of translation lus. Cycloheximide, an inhibitor of the large sub- mutants of Aspergilus nidulans unit simply and dramatically inhibits polypeptide A. Bratt and 5. Martinelli chain elongation. Several aminoglycoside antibiotics have a complex effect since they inhibit amino Department of Botany, Birkbeck College, University acid incorporation at low concentrations and of London, Malet Street, London WC1 7HX. stimulate it at higher concentrations. This effect is Usingthe "4 Corner Method" of electrophoresis being studied in conjunction with an assay for for proteins developed by Madjar et a!. (Anal. misincorporation. Biochem. 92, 174, 1979) we are analysing the pro- Several antibiotic resistant mutants have been tein components of cytoplasmic ribosomes of the examined, in vitro, without finding any with altered fungus Aspergillus nidulans. Using strain biAl ribosomal resistance. However some ribosomal (Glasgow) as wild type the 40S, 60S and 80S pro- suppressor mutants have ribosomes with altered teins have been electrophoretically separated. sensitivity to aminoglycoside antibiotics, both in Most of the proteins can be assigned to a sub-unit vitro and in vivo. but others are as yet uncertain. Strains containing translational suppressor mutations are being compared with isogenic strains and results will be presented which show that they have altered electrophoretic migration patterns in a protein. Mutations at the suaA and suaC loci (Roberts et a!., Molec. gen. Genet. 177, 57, 1979) 13.Recombination of were used for this study. mitochondrial genomes in Coprinus cinereus: its physico- chemical characterisation 15.Biochemical genetics of nitrate assimilation in yeast J. L. Baptista-Ferreira and A. Vidal Silva Centro do Micologia da Universidade do Lisboa, c. P. Jones, J. L. Wray and J. R. Kinghorn Faculdade do Ciencias, 1294 Lisboa Codex, Portugal. Department of Biochemistry and Microbiology, University of St. Andrews, Irvine Building, North Dikaryonformation, the production of sexual Street, St. Andrews, Fife, Scotland KYI69AL. mycelium from two compatible monokaryons in the basidiomycete Coprinus cinereus, is achieved Thenitrate-utilising haploid yeast, Hansenula win- by nuclear migration independent of cytoplasmic gei offers the rare opportunity to study the bio- movement; as a result, the reciprocal dikaryons chemical genetics of nitrate assimilation in yeasts. formed at the periphery of the opposed monokary- We isolated spontaneous nitrate assimilation ons only have the resident cytoplasm. However, it mutants in strains (ATCC 28162 and SH-00016) was shown (Baptista-Ferreira, Ph.D. Thesis, Uni- of opposite mating type, at an overall-frequency versity of London, 1981) that cytoplasms mixed as of 20 per cent, by selecting for resistance to chlorate a result of hyphal anastomoses and suggested that in the presence of glutamate as sole nitrogen recombination, rather than heterozygosity, of cyto- source. Four classes of chlorate-resistant mutant plasmic genes occurred in the zone of contact of were found, viz: mated monokaryons. Class I: nitrate non-utilising, nitrite utilising; Using appropriate mitochondrial gene muta- Class II: both nitrate and nitrite non-utilising; tions carried in compatible monokaryons, it was Class III: nitrate utilising, nitrite non-utilising; shown, from evidence produced by somatic segre- Class IV: nitrate and nitrite-utilising. gation analysis, that recombinant mitochondria Class III mutants were unstable and reverted to arise in the area where mated monokaryons had either Class II or Class IV phenotype. The Class anastomosed (Baptista-Ferreira, Economou and III phenotype has not previously been described Casselton, Current Genetics 7, 407, 1983). in any organism. Genetic analysis revealed a num- An investigation at the molecular level by phy- ber of complementation groups in Class I. All sico-chemical analysis of the mitDNA is currently representative alleles of each complementation underway and the results are presented. group lacked NR activity. Attempts to determine 130 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS which might be molybdenum cofactor (Mo-co) of the transformants (60-100 per cent) can incor- mutants were frustrated by the inability to assay porate other, non-selected plasmids which are xanthine dehydrogenase by any of the six pre- present in the transforming DNA mixture. This viously published procedures. Although all could high cotransformation frequency enabled the grow on hypoxanthine as sole nitrogen source introduction into A. nidulans of cosmids, plasmids some were Mo-co defective since (a) NR activity and linear DNA fragments carrying genes and and growth could be restored in some cases, in the sequences for which direct selection is impossible. presence of unphysiologically high levels of To illustrate that the cotransformation molybdate and (b) the Mo-co of other groups was phenomenon can be applied to introduce in vitro unable to reconstitute NADPH-NR activity in the modified genes at specific, pre-determined loci we Neurospora crassa nit-i reconstitution assay. Class used a model system based on the A. nidulans II contained three inter-allelic complementation trpC-E. coli lacZ fusion gene (van Gorcom et a!., groups which had low or zero NR and NiR activity Gene, in press). as well as Jow levels of functional Mo-co. We Experiments will be described which show that suggest that these three groups may represent a by cotransformation of A. nidulans the wild type complex locus involved in the regulation of NR trpC gene can be replaced by the trpC-lacZ hybrid and NiR and perhaps also the Mo-co. gene, resulting in tryptophan auxotrophic colonies with a 1acZ phenotype which are easily recog- nised by their blue colour on X-gal containing 16.Molecular analysis of the medium. These trpC mutants can be retransformed complex arom locus in Aspergillus to TrpC using the wild type trpC gene. A detailed analysis of this system will be pres- nidulans ented. I. G. Charles and A. R. Hawkins* Department of Biochemistry, University of Leicester, Leicester LE1 7RH; *Department of Genetics, Ridley Building, University of Newcastle on Tyne, Newcastle on Tyne NE1 7RU. 18.Structure and regulation of the Thearom locus of A. nidulans and other filamen- pyruvate kinase gene of tous fungi encode a multi-functional polypeptide Aspergillus nidulans. L. de Graaff that catalyses five consecutive enzymatic steps leading to chorismic acid in the polyaromatic W. J. van den Broek and J. Visser amino acid biosynthetic (arom) pathway. Department of Genetics, Agricultural University, Arom DNA sequence data will be presented Generaal Foulkesweg 53, 8703 BM Wageningen, The and the evolutionary relationships between the Netherlands. sequence of the dehydroquinase activity and that Thepyruvate kinase (pki) gene of Aspergillus of its isoenzyme, catabolic dehydroquinase nidulans was isolated from a genomic library of (encoded by the QutE gene in the quinic acid A. nidulans in bacteriophage A by heterologous utilisation gene cluster) discussed. Analysis of hybridisation with a 18 kb EcoRI fragment from arom specific mRNA will be described. the yeast gene (Kawasaki and Fraenkel, Biochem. Bioph. Res. Comm. 108, 1107, 1982). Analysis of the selected clones by Southern blotting revealed 17.Gene replacement/disruption a 29 kb EcoRI/BamHI fragment which exclusively hybridised with the yeast pki gene. This frag- in Aspergillus by cotransformation ment containing the gene was subcloned, and T. Goosen, K. Wernars, L. M. J. Wennekes, sequence analysis data will be presented. With the C. A. M. J. J. van den Hondel* and H. W. J. 29 kb EcoRI/BamHT fragment as a probe for the van den Broek wild type gene, we were able to demonstrate that a pki mutant, which lacks the pki gene product, Department of Genetics, Agricultural University, Wageningen, The Netherlands. *Medical Biological contains a 150-200bp deletion. Pyruvate kinase Laboratory TNO, Rijswijk, The Netherlands. activity is stimulated by growth on glycolytic substrates. The regulation of the gene expression is Inour studies on the transformation of Aspergillus studied both at the mRNA and the gene product nidulans we observed that a very high proportion level. THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 131

19. Analysis of regulation signals sumptive upstream activator sequence, and terminator of the A. niger glucamylase gene were in Aspergillus functionally linked to the gene encodingproren- R. F. M. van Corcom, P. Punt, W. E. nm, and inserted as a Clal fragment into plasmid Timberlake*, H. W. J. van den Broek**, H. pDJB3 (Ballance and Turner, Gene, in press). Fur- Pouwels and A. M. J. J. van den Hondel ther modifications involved the addition of various secretion signals to the prorennin gene. Medical Biological Laboratory TNO, Rijswijk, The Transformants of strain G191 were selected by Netherlands; *Department of Plant Pathology, University of California, Davis, USA; **Department pyr4 complementation and found to secrete active of Genetics, Agricultural University, Wageningen, rennin in submerged culture. The identity of rennin The Netherlands. and prorennin was confirmed by Western blots of one and two dimensional acrylamide gels. The Regulationof gene expression in Aspergillus mechanism of processing may be autocatalytic or nidulans has been investigated extensively by may involve endogenous proteases. Rennin was genetic methods. However, little is known about secreted by transformants grown on starch, xylose the control of gene expression at the molecular and glucose indicating that the A. niger glucoamy- level. A convenient and powerful system to study lase promoter is not regulated in A. nidulans. gene expression is provided by the fusion of a Southern analyses of selected transformants show promoter or regulatory region to the lacZ gene of integration of the ClaI fragment, and, for some E. coli, encoding /3-galactosidase, and determina- transformants, multiple copies were observed. tion of the /3-galactosidase activity. To test whether the lacZ-fusion system also can be used in Aspergillus we have constructed in vitro a fusion of the E. coli lacZ gene and the trpC gene of A. nidulans. The fused gene was cloned into a plasmid which contains an A. nidulans selection marker. Analysis of A. nidulans strains transformed with this plasmid shows that the E. coli 21.An Aspergillus DNA sequence lacZ gene is expressed as a functional protein. giving a high frequency of Based on the trpC-lacZfusiongene, a set of unstable transformants promoter-probe vectors was constructed to isolate and analyse transcription/regulation signals. I. L. Johnstone and A. J. Clutterbuck These vectors contain a unique Barn HI site in three Department of Genetics, Glasgow University, different reading frames in front of either the trun- Glasgow Gil 5JS. cated trpC-lazZ fusion gene or the trancated lacZ Inan attempt to construct a vector which would gene. replicate autonomously in Aspergillus nidulans, a Experiments are in progress to analyse the pro- gene bank in the pILJ16 argB vector was screened moter region of glycolytic and galactose-induce- for production of semistable transformants of an able genes in A. nidulans and Aspergillus niger. argB2 recipient. One such transformant, on coni- The results of such will be presented at the meeting. dial subculture, gave rise to approximately equal numbers of ARG and unstable ARG colonies. DNA from this strain was able to transform E. coli at high frequency to ampicillin resistance, and was shown to include both integrated and free copies of a pILJ 16-derived plasmid with a 6 kb insert. This plas mid transformed Aspergillus argB2 strains 20.Expression of bovine chymosin at high frequency (3000—5000 transformants per by Aspergillus nidulans g DNA) to give further unstable ARG colonies. Analysis of the plasmid showed it contains an D. Cullen, R. Berka, G. Grey, K. Hayenga, Aspergillus DNA insert consisting of 3 kb inverted S. Norton, M. Rey and L. Wilson repeats flanking a 376 bp unique core. Using this Genencor Inc., South San Francisco, CA 94080, plasmid as a probe in Southern blots, suggests that U.S.A. wild type Aspergillus probably contains one copy Wehave constructed several vectors for expressing of the inverted repeat, plus a few copies of the rennin in Aspergillus nidulans. The promoter, pre- single arms. 132 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 22. Genetic and molecular analysis putative splicing signals to those found in other filamentous ascomycetes. The P0K promoter has of the quinic acid utilisation gene CAAT and TATA homologies which are generally cluster in Aspergillus found in more complex eukaryotes. Additionally pyrimidine rich regions of the promoter sequence A. J. Francisco Da Silva, C. F. Roberts and share some similarities to genes of other filamen- A. R. Hawkins* tous fungi. PGK mRNA exhibits 3' heterogeneity Department of Genetics, University of Leicester, and the major polyadenylation site is positioned Leicester LEI 7RH; *Depa,ment of Genetics, Ridley 16 bp beyond the eukaryotic consensus polyadeny- Buildings, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU. lation signal AAUAAA. Potential hairpin struc- tures are also found in the 3' non-translated region Analysisof non-inducible mutants combined in of PGK mRNA which may be important in tran- heterozygous diploid strains of Aspergillus nidulans scription termination and polyadenylation. has defined two genes, qutA and qutD, which regulate the expression of a closely linked cluster of three structural genes. The product of one regu- 24.The molecular analysis of latory gene, qutD, is an activator required for the expression of all three enzyme structural genes and morphogenesis in Sordaria that of the second gene, qutA, has the characteris- brevicollis tics of a co-activator required for gene expression in the presence of quinic acid. D. J. Bond and S. J. Broxhome In order to test alternative molecular models Department of Genetics, University of Edinburgh, of the regulatory mechanism, we have cloned the Edinburgh EH93TN. three enzyme structural genes and also qutD using Themorphogenetic events which we have been probes from the homologous genes in Neurospora analysing in the fungus Sordaria brevicollis are crassa. Features of the promoter and terminator those associated with fruiting. Firstly, we have sequences flanking the enzyme coding regions will been concerned with understanding signals which be described with particular reference to the dehy- initiate the morphogenetic pathway leading to pro- droquinase gene (qutE) and in comparison with toperithecial production, and secondly we have recent data from Neurospora (Dr N. H. Giles, been analysing the morphogenetic pathway itself. personal communication). The ultimate objective is to understand how the developmental events leading to perithecial formation and ascospore production are synchronised 23.Transcription and processing and co-ordinated at the molecular level. signals revealed in the nucleotide When growing on cornmeal agar colonies of sequence of the phosphoglycerate Sordaria are barren, protoperithecia are produced only after growth is disturbed, usually following kinase (PGK) gene in Aspergillus contact with the Petri dish wall. There is a similar J. M. Clements and C. F. Roberts edge effect when the fungus is grown on chemically defined Westergaard and Mitchell's medium. On Department of Genetics, University of Leicester, Leicester LE1 7RH. Vogel's N medium, in contrast, a growing colony does produce protoperithecia. This morphogenetic The3-phosphoglycerate kinase (P0K) gene from difference is brought about by the different Aspergillus nidulans has been isolated from a nitrogen content of the two media. The addition Lambda-phage genomic library using the of ammonium ions to, or reducing the level of equivalent yeast gene as a hybridisation probe. The nitrate ions in, Westergaard and Mitchell's PGK gene has been shown to be constitutively medium separately induces a Vogel's-like response expressed at relatively high levels in comparison with respect to protoperithecial production. to other Aspergillus genes. At the molecular level protoperithecial forma- The nucleotide sequence of the entire gene and tion is characterised by low level production of its 5' and 3' flanking regions, and the transcription several phase-specific proteins (Nasrallah and Srb, start and polyadenylation site of the PGK mRNA P.N.A.S. 74: 3831-4). Further development is trig- have been determined. The gene codes for an open gered by fertilisation when some of the phase- reading frame of 421 amino acids and is interrup- specific problems are formed in increased quan- ted by two 57 bp introns both of which have similar tities. The molecular events underlying the mor- THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 133 phogenesis have been analysed by 2-D gel elec- 26. The repair of alkylation trophoresis and in vivo and in vitro labelling. Thirty-one proteins have been identified which are damage in Aspergillus nidulans present at some stage during differentiation but R. Swirski, B. M. Faulkner and P. Strike are absent from the vegetative mycelium. Department of Genetics, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. Adaptationto the killing and mutagenic effects of simple alkylating agents in Escherichia coli has 25.Dominance and epistasy in the been shown to be due to the induction, respec- context of translation mutants of tively, of a DNA glycosylase (the alkA gene prod- Aspergillus nidulans uct), and an 06-methyl guanine DNA methyl- transferase (the ada gene product). S. 0. Martinelli To determine whether such an inducible system Department of Botany, Birkbeck College, University existed in the lower eukaryote Aspergillus nidulans, of London, Malet Street, London WC1E 7HX. conidia were pretreated with low concentrations of MMS and then challenged with a higher dose. Geneticistshave used dominance tests as evidence The pre-treatment did not result in an enhanced when deciding whether mutations are in structural ability to survive the challenge dose. However, or regulatory genes and whether regulatory pro- comparisons of reversion frequency between adap- teins are positively or negatively acting entities. In ted and non-adapted conidia revealed a three-fold the context of translation, dominance tests have reduction in the pre-treated cells following a high been used to assign mutations to tRNA genes (if dose challenge. dominant) and ribosomal genes (if recessive). The Correlating these observations, and using arguments employed to assign mutants in this way suitable substrates and protein purification pro- were based on assumptions of the molecular cedures, we have demonstrated that both a 3- behaviour of the translation apparatus as well as methyl adenine glycosylase and an 06-methyl the frequency of mutations at the locus in question guanine DNA methyl-transferase activity can be and number of mutational sites. There are now an detected in such extracts. However, both of these overwhelming number of contradictions with activities appear to be constitutive, and are not recessive tRNA mutations and dominant ribo- affected by pre-treatment of mycelia with non- somal mutations. lethal levels of mutagen. It follows that, my colleagues and I have also Several mutants displaying increased sensitiv- performed this ritual, with suppressor and antisup- ity to MNNG have been isolated, and mapped on pressor mutations and have looked at all the the A. nidulans chromosomes. The most sensitive aspects of their phenotypes, not only suppression. of these, SAl, is many more times more sensitive These results will be presented and the question than wild type to NTG, but is not affected by UV posed "Are dominance tests worthwhile"? light. A less sensitive mutant, SA3, is similarly By synthesising double mutants and then unaffected by UV. These mutants, which map to examining the epistatic relationships it is often chromosome VIII and I respectively, therefore possible to work out the respective positions of appear to be defective specifically in steps con- the two relevant genes in a metabolic pathway. I cerned with the repair of alkylation damage. have made strains doubly mutant for a range of translational mutations and examined them in the hope that either predictions could be made about 27.Molecular mechanisms of order of use during translation or ribosome synthesis, or about the nature of the suppressors con- heavy metal tolerance in cerned. The results are both interesting in showing Aspergillus nidulans up mutual antagonism and disappointing in that R. N. Cooley, D. A. Thurman* and the character of the double mutant at low tem- A. B. Tomsett peratures is usually similar to the weakest of the two partners, an effect that I also found with some Department of Genetics and *Department of Botany, University of Liverpool, P.O. Box 147, developmental mutants years ago. Even so, I hope Liverpool L69 3BX. to be able to show at the meeting that the synthesis of double mutants is potentially more rewarding Theresponse of the ascomycete fungus, Aspergillus than that of dominance tests. nidulans, to a variety of metals has been tested. 134 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS

Significant variation occurs between wild-type develop. The turning on/off of the master switches strains in the extent to which their hyphal growth is executed through the exact recognition between is inhibited by metal ions. Less variation is seen specific combination of alleles. The B MT factors in their inhibition of germination. Mutants have are grouped to phenotypic classes. Six mutations been isolated which are resistant to the presence in one A factor and 27 mutations in eight B factors of metal ions as shown by their increased ability all resulted in the loss of mating specificity. Certain for hyphal growth. The evidence suggests that the MT mutations or combinations of mutations lead resistance of many of these mutants is not due to to homokaryons mimicking dikaryons carrying reduced rate of cellular uptake, either by extracel- fruiting bodies. Efforts to obtain mutations to new lular chelation or by alteration in the rate of metal specificities using effective methods and mutagens transport. Instead, the resistance may result from have significantly failed in samples exceeding iO. the induction of an intracellular mechanism, since The importance of a new level of analysis of MT a growth phase seems necessary for the resistance factors by the use of gene cloning techniques is to become apparent. emphasised. The availability of (a) all the natural Cadmium-resistant mutants have been mapped Ba and B/3 alleles, (b) almost all possible B factors, and found to be located at two loci. One locus is (c) large series of primary and secondary B muta- on chromosome VI between the nicC and lacA tions, (d) a deletion of the whole B region—all loci; the other is on chromosome IV between the this accumulated knowledge renders the B factor hisA and moB genes. The proximity of the chromo- not only an important target for the gene-cloning, some VI mutants to the methB locus is of particular but an effective candidate for such a study. Para- interest due to the relationship of sulphur- meters—genetical, cytological and chemical con- containing compounds and metal resistance. cerning the genome of Schizophyllum, relevant to Certain strains isolated as resistant to cobalt planning a strategy for cloning: 11 chromosomes ions produce snall spherical cobalt precipitates in including 7 mapped linkage groups with estimated the medium. In addition, a Cu-binding protein of genome size of 2000 CO units; some short distances similar size to yeast and animal metallothioneins involving MT genes: Ba-Bf3, class II =25kb, class has been identified. III =4.5kb; Aa-pab =8 kb. Methods for the use of the MT genes as selective markers and the phenotypes expected from this gene cloning are discussed. 28.Towards a molecular approach for dissecting the complex mating- type factors of the basidiomycetous fungi 29.An assessment of doub'e- V. Parag strand gap repair as a cause of Department of Genetics, The Hebrew University, gene conversion in fungi Jerusalem, Israel 91904. B. C. Lamb Themating type (MT) genes in fungi are master- switches of the developmental events leading to Department of Pure and Applied Biology, Imperial initiation and sequential steps of the sexual cycle. College, London SW72BB. In filamentous Ascomycetes and yeasts there is one Manymodels have been proposed to explain gene MT locus with two alternative states; in the latter, conversion in fungi. "Gene conversion" is used the frequent conversion of one state to the other here to include production of 5:3 and 3 :5 ratios is well understood in molecular terms. In the as well as 6:2 and 2:6s. In addition to Basidiomycetes the genetic control is much more hybrid-DNA (hDNA) theories, there is now the complex. In Schizophyllum commune two mating- double-strand gap repair model (Szostak et aL, type (MT) factors, A and B, each comprises two Cell 33, 25, 1983; Orr-Weaver and Szostak, Micro- discrete genes, a and /3, each with a multiple allelic biol. Rev. 49, 3, 1985). series. The combination of a and /3 alleles deter- Theproposed repair of double-strand gaps mines the mating specificity of each MT factor and does not produce 5 :3 s, 3 : 5 s aberrant 4:4 s or a mating between homokaryons carrying correction 4:4 s, but just gives 6:2 s and 2:6 s, compatible A and B MT factors lead to a normal probably in equal proportions. Within a length of dikaryotic mycelium on which fruiting bodies gap repair,there should normally be no effect of THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 135 mutational type on conversion properties and one been made to test the hypothesis that this region heterozygous mutation should not influence the serves as a preferred site for initiation of recombi- conversion properties of nearby sites. One can nation. therefore make detailed predictions about properties of gene conversion for situations with conversion: (a) almost exclusively from double-strand 31.The analysis of the gap repair; (b) mainly from gap repair, but sometimes from hDNA; (c) sometimes from gap repair chromosomes of bovine sperm but mainly from hDNA; (d) almost exclusively from hDNA. Data from fungi can then be com- P. Creighton and J. A. Houghton pared with the predictions to assess the importance Department of Microbiology, University College, of double-strand gap repair as a cause of gene Galway, Ireland. conversion. In all cases, even (d), double-strand Amethod developed in this laboratory for the gaps could initiate gene conversion processes; analysis of human sperm chromosomes (Tomkins, what is being tested is whether actual conversions Carroll and Houghton, Heredity 50,211,1983) largely arise from gap repair or from hDNA. based on the technique of in vitro fertilisation of Gene conversion data from Ascobolus immer- zona-free hamster eggs described by Rudak et a!. sus, Sordaria fimicola and Sordaria brevicollis gen- (Nature 274, 911, 1978) has now been extended erally fit predictions from situation (d) much better to the visualisation of the chromosomes of bovine than those from situation (a), although situation sperm. This communication describes recent (c) is not ruled out. Even data from yeast, which improvements in this technique involving the use were originally quoted as supporting the double- of follicular fluids and percoll gradients for the strand gap model, fit predictions from (c) and (d) capacitation of the sperm. This method yields con- more often than they fit those from (a) or (b). sistently analysable sperm metaphases with both fresh and frozen semen. Data from the study of these chromosomes will be described and the implications for cattle breeding and the A.I. industry discussed. 30.Polarity in in vitro recombination catalysed by cell extracts 32.Cone pigment polymorphism and colour vision in the primates P. Unrau, J. D. Childs, R. Aubin and M. Weinfeld H. Kalmus A. E. C. L. Radiation Biology Branch, Chalk River, Galton Laboratory, University College, London. Ontario, Canada KOJ 1JO. Arecently described photopigment polymorphism Plasmidsused for in vitrorecombinationstudies in the New World Squirrel Monkey, Saimiri (Symington et aL, Cell35,805, 1983) were modified sciureus (Mollon et a!., Proc.Roy.Soc. B 222, 373, to permit the detection of polarity in recombina- 1984) differs radically from the genetical organisa- tion. Reciprocal crosses were made between plas- tion of trichromatic colour vision in man and the mids with the mutations tetl0 and tetl4, located Old World primates. at 23 and 1246bp respectively in the tet gene. In the latter, alleles at two closely linked loci Polarised recombination was shown by the prefer- on the long arm of the X-chromosome separately ential conversion of the tetl0 mutation to wild control the photopigments of the red and green type. Mixed plasmid clones arising from recombi- sensitive cones of the retina. In Saimiri three kinds nation were frequently observed and these also of males and six kinds of females occur, of which showed the preferential conversion of the tetl0 only three—presumably heterozygotes—have tn- allele. A computer search of the pBR322 genome chromatic colour .vision, all the others being located a 13 base sequence identical to one arm dichrohiatès. Mollon et a!. explain that this rep- of the site—specific recombination sequence loxP resents a stable equilibrium, in which three alleles of P1 phage (Hoess etaL, P.N.A.S. 79,3398, 1982). are maintained by female heterozygous advantage. Within this sequence a 15 base palindrome con- In the retinae of these females, mosaics of two taining the TATAA region of the B-lactamase gene kinds of cones ("red" and "green" cones) occur was identified. A deletion of this palindrome has owing presumably to lyonisation. 136 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS

A derivation from some ancestral form of the nidulans) there is amino acid identity between A. human and Saimiri situations might be as follows: nidulans and N. crassa (McPherson, M. J. and at some stage two alleles only existed at one locus; Woottom, J. C. NAR 11, 5257, 1983),S.cerevisiae in the ancestors of Saimiri mutation then added a (Nagasu, T. and Hall, B. D. Gene in press) and third, while in the ancestry of man gene duplication E. coli(Kinnaird,J. H. and Fincham, J. R. S. Gene made subsequent differentiation possible. Such a 26, 253, 1983) of 88 per cent, 78 per cent and 79 scheme may shed a new light on the human colour per cent, respectively. Two short introns (55 and vision deficiencies. 56 bp respectively) interrupt these highly conserved A. nidulans sequences. The Aspergillus introns are found at the precise positions as the two introns (66 and 61 bp) of the 33.Incompatibility-induced cell N. crassa glutamate dehydrogenase (am) gene. death in Phanerochaete velutina (The S. cerevisiae gdhA gene is not interrupted by intervening sequences). The internal sequences are N. K. Todd, R. C. Aylmore, A. M. Ainsworth* not significantly conserved between am and gdhA and A. D. M. Rayner* introns. The two gdhA introns share an internal Department of Biological Sciences, University of consensus, ACCGTTAA, not seen in other fungal Exeter, Exeter EX4 4QG; *School of Biological or yeast introns, as well as an intact TACTAAC Sciences, University of Bath, Bath BA2 7A V. box in intron 2. Heterologous expression of gdhA Phanerochaetevelutina possesses multinucleate has been demonstrated by transformation experi- hyphal compartments and whorled clamp- ments using the N crassa deletion mutant, am 132. connexions. We have shown that it has a unifac- Information on the transcription initiation and tonal (bipolar) mating system, anastomoses abun- polyadenylation sites, codon usage, and a com- dantly and exhibits a very rapid somatic incompati- parison of A. nidulans GDH sequences with other bility reaction at sites of hyphal fusion between available GDH's will be presented. genetically different secondary mycelia. In this paper, we present the cytological details of the somatic incompatibility reaction as seen using light and electron microscopy. Our observations prompt 35.Transcription of the Aspergillus the speculation that incompatibility-induced cell nidulans mitochondrial genome death in this fungus occurs in a programmed manner. N. J. Dyson, T. A. Brown, R. B. Waring and R. W. Davies Department of Biochemistry and Applied Molecular Biology, UMIST, Manchester M60 1 aD. 34.The nucleotide sequence of Northernhybridisation, Si mapping and in vitro capping experiments indicate that the A. nidulans the Aspergillus nidulans gdhA mitochondrial genome is transcribed into RNA gene precursors that are rapidly processed into mature S. J. Gurr and J. R. Kinghorn mRNAs. We have mapped the 5'-termini of the 16S and Department of Biochemistry and Microbiology, 23S rRNA genes and located these in conserved University of St. Andrews, North Street, Irvine nucleotide sequences that are similar to possible Building, St. Andrews, Fife KY16 9AL, Scotland. transcription initiation sequences in yeast. Loosely Theentire coding sequence of the A. nidulans gdhA conserved sequences occur elsewhere in the A. gene for NADP-linked glutamate dehydrogenase nidulans genome, including at other possible tran- (GDH) has been shown to reside within a 1 8 Kb scription start points. HindIlI fragment of DNA. From the nucleotide One major surprise is the presence of at least data, the deduced amino acid sequence was found three mature multi-gene transcripts. These are rela- to be highly homologous with other microbial tively uncommon in the mitochondria of other GDH's particularly nearer the amino terminus and lower eukaryotes. Evidence will also be presented which is generally thought to be involved in for a possible role for tRNA genes in transcript catalytic activity and allosteric interactions. For processing, and for the involvement of RNA instance, in the region amino acids 43 to 161 (A. priming in genome replication. THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 137

36. tRNA genes: a transformation Laboratoire de Génétique des Ribosomes, Unite tool and a question about Associée au CNRS No. 86, Bat 400, Université conversion Paris-Sud, F.91405 Orsay Cedex, France. Inthe fungus Podospora anserina, we have V. Brygoo and R. Debuchy developed a genetic system to study the ribosomal Laboratoire do Genetique des Ribosomes, Bat. 400, control of translational accuracy (Picard-Bennoun U.P.S., 91405 Orsay Cedex, France. et a!. In: Protein synthesis (Abraham, A. K., Manysuppressors restoring prototrophy were Eikham, T. S., Pryme, I. F. eds), 1983). Ribosomal obtained from a strain leul-1 of the fungus Podos- suppressors (su), analogous to "ram" mutations pora anserina. Some, such as su4-1 and su8-1 were in E. coli, anl antisuppressors (AS), analogous to cloned in Saccharomycespombe on the basis of the bacterial "restrictive" mutations, have been suppression of opal nonsense mutations. characterised both genetically and biochemically. Sequence analysis of the suppressor genes In E. coli, mutations which modify the error rate confirmed that they encode tRNAs reading UGA in translation alter the structural genes for 5 ribo- (opal), codon (Debuchy, R. and Brygoo, Y., in somal proteins. So far 15 genes have been identified press). The sequence of the wild-type alleles in P. anserina: 7 su genes and 8 AS genes. We revealed that both encode tRNAA with a low have recently demonstrated that, at least, five of degree of homology. Furthermore, su8 contains an these genes are in fact structural genes for ribo- intron, whereas su4 does not. somal proteins of the small subunit: Si (su3), S7 As Su4 and Su8 encode isoacceptor tRNAs (sui2), S9 (AS9), S12 (AS1) and S19 (AS6) having the same anticodon their sequence differen- (Dequard-Chablat and Coppin-Raynal, Molec. ces are somewhat unusual. Intergenic conversion Gen. Genet. 195, 294, 1984; Dequard-Chablat et has been postulated as a mechanism for maintain- a!., submitted for publication; Dequard-Chablat, ing homology amongst members of a multigenic submitted for publication; Dequard-Chablat, family, and, indeed interchromosomal gene con- Molec. Gen. Genet. 200, 343, 1985). Two other version of tRNAse genes has been demonstrated ribosomal proteins are involved in translational in S. pombe. Thus, it is possible that the acquisition accuracy: S29 which is lacking in all AS3 mutants of an intron by one member of the ancestral family and S8 which is altered in the single sull mutant of tRNAser genes would have released this gene characterised till now. One protein, namely S7, from the correction mechanism of gene conversion seemsto play a key role in the ribosomal control and allows a high degree of heterology to be main- of translational accuracy. Indeed, different alter- tained in Podospora anserina; the coding sequence ations of this protein correspond to opposite differences observed between su4 and su8 may be effects. Forms more acidic than the wild-type lead correlated with the presence of an intervening to an increased accuracy, while more basic forms sequence within su8. lead to decreased accuracy (Dequard-Chablat, The cloned tRNA suppressors were used in submitted for publication). DNA transfection experiments with a strain leul-1 of P. anserina (Brygoo, Y. and Debuchy, R. Mol. Gen. Genet. 200, 128, 1985). Transformation occurs 38.J. R. S. Fincham uniquely by integration. Genetic analysis showed Department of Genetics, University of Cambridge, that integration of the transforming molecule at Downing Street, Cambridge CB2 3EH. the homologous site is not the rule: in most cases the integration point is located elsewhere. Some random integrations occur close to strategic loci as centromeres, which we hope to clone for con- 39. Incompatibility genes structing chromosome-like vectors. regulating genes for fruit-body formation in Schizophyllum 37.At least seven ribosomal commune proteins are involved in the control J. G. H. Wessels of translational accuracy in the Department of Plant Physiology, Biological Centre, fungus Podospora anserina University of Groningen, The Netherlands. M. Dequard-Chablat, E. Coppin-Raynal and Matingof two monokaryons of Schizophyllum com- M. Picard-Bennoun mune carrying different alleles at both the A and 138 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS

B incompatibility genes results in the formation 41. Vectors for isolation of genes of dikaryon with a typical mycelial morphology and the inherent capacity to form fruit-bodies by transformation of Aspergillus under proper environmental conditions. Genetic G. Turner analyses indicate that the incompatibility genes function as controlling genetic elements governing Department of Microbiology, University of the expression of genes directly involved in mor- Bristol, The Medical School, University Walk, phogenesis. Till now we have not been able to Bristol BS8 lTD. detect mRNAs from genes involved in the Genescan be isolated in Aspergillus by corn- monokaryon-dikaryon transition, possibly plementation of mutations using gene banks because their concentration is too low. However, constructed in plasmid or cosmid vectors. The during the formation of fruit-bodies by the transformation frequency is generally low, but can dikaryon, a number of genes are uniquely be enhanced by inclusion of a chromosomal repeti- expressed by producing abundant mRNAs and tive sequence ans-1 into vectors. The nature and proteins. Probing the expression of these genes behaviour of this sequence in different vectors will with cloned sequences revealed a low expression be discussed, and its use in gene isolation. of these genes in the vegetative dikaryon but not in the monokaryons. It can thus be surmised that the regulatory role of the incompatibility genes in 42.Control of gene expression in fruiting is the conditioning of fruiting genes in the chromatin in preparation for transcription, while Aspergillus nidulans full transcription of these genes is switched on by C. Scazzocchio environmental signals conducive to fruiting. lnstitut de Microbiologie, Bat. 409, Université Paris-Sud Orsay Cedex. Throughthe years the regulation of a number of dispensable metabolic pathways of A. nidulans has 40.Molecular genetics of been studied thoroughly (reviewed in Arst, H. N. pyrimidine metabolism in and Scazzocchio, C. in Bennet, J. W. and Lasure, L. L.; Gene Manipulations in Fungi, Academic Neurospora Press, 1985, in press). A number of systems have A. Radford, J. A. Glazebrook and been studied at the physiological level, structural S. F. Newbury and regulatory genes have been identified and in some cases very detailed fine structure maps are Department of Genetics, The University of Leeds, available. Leeds LS29JI We have cloned two complete systems, the Wereview the biosynthesis, uptake and salvage ethanol utilisation regulon (Lockington et aL, Gene pathways of pyrimidines in Neurospora, and the 33, 137, 1985) and a cluster comprising the three loci involved. Regulation of the pathways by both structural genes and the positive-acting regulatory nitrogen metabolite repression and pyrimidine- gene involved in proline utilisation (Green, P., specific regulatory mechanisms will be discussed. Scazzocchio, C., Arst, H. N. and Davies, R. W., The pyr-4 gene, encoding orotidine 5'-P decarboxy- unpublished). Northern blots confirm the supposi- lase, has been cloned and sequenced. The results tion that the structural genes are regulated at the of sequence comparisons with the equivalent genes level of transcription and show that the cognate of Saccharomyces cerevisiae (ura3) and Escherichia regulatory genes, alcR and prnA, are autoregu- coli (pyrF) will be presented and homologies dis- lated. Interestingly aicR is directly regulated by cussed. The pyr-4 clone has been inactivated by carbon catabolite repression which seems to work Tn 1000 insertions, and also by removal of the distal by shutting off the synthesis of this positive-acting end of the coding region and its replacement in a gene product rather than by repressing the translational fusion by lacZ. Transformation of expression of the structural genes themselves. Neurospora with these defective clones, resulting DNA sequencing of alcAandaid A, which are in complementation to pyrimidine-independence the genes coding for alcohol and aldehyde dehy- has been achieved at low frequency. These results drogenase respectively, has revealed highly con- will be discussed in the context of transformation served sequences 5' to their coding regions, which mechanisms in Neurospora. might be involved in the regulatory process. THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 139 (Gween, D. I., Piggot, M. P., Davies, R. W., Since the ts benA33 mutation blocks mitosis in Lockington, R. A., Sealy-Lewis, M. H. and conidiospores, /31- and/or f32-tubulin also must Scazzocchio, C., unpublished data). function during sporulation. Thus benomyl sensitive /33-tubulin confers benomyl sensitivity on conidiophores containing resistant /31- and/or /32- 43.The tubulin gene family of tubulin; but, when f33-tubulin is disrupted, the remaining resistant tubulins make sporulation Aspergillus nidulans resistant. These experiments demonstrate the N. R. Morris, G. May, J. Weatherbee, power of the Aspergillus system for analysing cell M. Tsang and P. Doshi specific functions of the tubulin gene family. Department of Pharmacology, UMDNJ-Rutgers Medical School, Piscataway, N.J. 08854, U.S.A. Allorganisms that have been studied have multiple 44;Position dependent and species of tubulin and multiple tubulin genes, but independent regulation of a gene very little is known about the biological implica- from the SpoCi gene cluster of tions of this multiplicity of genes and proteins. Aspergillus nidulans provides an ideal experi- Aspergillus nidulans mental system for studying this problem since it W. E. Timberlake, B. L. Miller, K. V. Miller and has relatively few tubulins and tubulin genes; there are well characterised mutations in its tubulin K. Roberti genes; it has several differentiated cell types; and Department of Plant Pathology, University of it is amenable to DNA mediated transformation. California, Davis, California 95616, U.S.A. A. nidulans has two genes for a-tubulin and Wehave previously described the structure and two genes for f3-tubulin, which together code for regulation expression of the SpoCi gene cluster a total of six polypeptides. The tubA gene codes from Aspergillus nidulans (Gwynn, D. I., Miller, for two polypeptides, 1- and a3-tubulin. TubB, B. L., Miller, K. Y. and Timberlake, W. E., J. Mo!. codes for a2-tubulin. Ben A, codes for /3i-and Biol., 180, 91, 1984). This 53 Kb segment of /32-tubulin, and tubC codes for /33-tubulin. The chromosomal DNA codes for at least 19 poly(A) question is where and how do these genes and RNAs, some of which are transcribed from over- gene products function? BenA and tubA are lapping regions. The area of developmental regula- known to function in spindle and cytoplasmic tion is approximately 37 Kb in length and is microtubules of the vegetative mycelium. The fol- delimited by 11 Kb direct repeats. With one excep- lowing evidence indicates that benA and tubC gene tion, RNAs transcribed from the central part of products are important for the development of the cluster appear late during conidiophore asexual spores. development and accumulate specifically in The two /3-tubulin genes and one of the a- spores. The exceptional transcript appears earlier tubulin genes have been cloned and sequenced. during development and accumulates specifically The /3-tubulin clones, B5 and B14, differ substan- in cells of the conidiophore. In contrast, RNAs tially with respect to nucleotide sequence and are encoded at the borders of the cluster occur in both only 80 per cent homologous with respect to pro- somatic cells and spores. The results indicated that tein sequence. Each integrates specifically in trans- if a chromatin-level control mechanism operates formation experiments. 85 integrates at the benA to regulate expression of the SpoCi gene cluster, locus. Transformation with an internal fragment as previously suggested by us (Timberlake, W. E. of B14 disrupts the tubC gene and causes the and Barnard, E. C., Cell 26,29,1981), additional disappearance of /33-tubulin from 2D gels, indicat- levels of regulation must also exist. ing that /33-tubulin is the product of the tubC gene. We have now examined the effect of reposition- Asexual sporulation is normally sensitive to ing one SpoCi gene, designated Cl-C, in the inhibition by benomyl in benA22 (benomyl resis- genome. A recipient strain of Aspergilluswascon- tant) mutants. Disruption of /33-tubulin in a structed containing a null allele of the Cl-C gene. benA22 strain by conidiation resistant mutations A plasmid containing a wild-type copy of the gene or by integration of a B14 internal fragment causes and its 5' and 3' flanking regions was then intro- sporulation to become resistant to benomyl. This duced by transformation. Several transformants demonstrates that /33-tubulin functions during were selected in which the plasmid had integrated conidiation but is not required for conidiation. at sites other than the SpoCi region. Blot analysis 140 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS

of RNA from these transformants indicated that introduction of DNA into P. chrysosporium, and the gene was expressed in somatic cells at a level the progress towards this will be discussed. An much greater than when in the normal position. objective is to obtain strains in which lignin degra- The repositioned genes nevertheless showed a con- dation proceeds during normal growth. siderable degree of developmental regulation. The This work is part of a programme supported results indicate that regulation of genes in the jointly by British Petroleum's Venture Research SpoCi gene cluster involves both position depen- Unit and the AFRC. dent and position independent mechanisms. 47.Studies on the structure and 45.H. N.Arst expression of cellulase genes from Department of Bacteriology, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane the filamentous fungus Road, London W12 OHS. Trichoderma reesei Jonathan Knowles, Päivi Lehtovaara, Tuula 46. Lignin degradation and its Teen, Merja Pentillä and Irma Salovuori control in Phanerochaete Biotechnical Laboratory, V7T, SF-02 150 Espoo, chrysosporium Finland. Cellulasesare a group of enzymes secreted by a P. Broda, 0. Birch, A. Brown, R. Haylock, number of different microorganisms all of which R. Liwicki, M. MacDonald, A. Paterson, hydrolyse /3-1,4 glycosidic bonds, though each has U. Raeder, A. Schrank and P. Sims its own substrate specificity. We have cloned genes Department of Biochemistry and Applied Biology, coding for 5 different cellulolytic enzymes and UMIST, Manchester M6O 1QD. analysed 3 of these in detail. Full length cDNA Inthe white rot basidiomycete Phanerochaete chry- clones have been isolated and characterised allow- sosporium, there is a switch determining whether ing the identification of at least two introns in each or not lignin is degraded. Depletion of carbon or gene. nitrogen sources results in increased adenyl cyclase Comparison of the DNA and putative protein activity and levels of cAMP, and the onset of sequences of the genes coding for the cellobiohy- ligninolytic activity. Of a series of phenol oxidase drolases CBH I and II and endoglucanase EG I deficient mutants, some were unable to degrade reveals a number of interesting features about the lignin; of these some, but not others, had cAMP evolution of cellulases genes. It would seem that levels that remained low on C and N depletion. exon shuffling and both divergent and covergent Others of these mutants degraded lignin more evolution have played important roles in the evol- efficiently than the parent strain; these mutants ution of these genes of this family. had abnormally high cAMP levels on C and N depletion. 48.D.A.Hopwood eDNA preparations were made from mRNA John Innes Institute, Colney Lane, Norwich NR4 7UH. isolated from ligninolytic and non-ligninolytic cultures of the parent strain. These cDNAs were 49. B. A. Bridges hybridised to genomic libraries of P. chrysosporium DNA, and a number of clones were isolated that M. R. C. Cell Mutation Unit, University of Sussex, carried sequences expressed only in the ligninolytic Falmer, Brighton BN 1 9RR. phase. These could be grouped into families by Southern blotting and by restriction site poly- 50. Restriction site polymorphism morphism segregation analysis (see Raeder and Broda, this meeting). Creation of a cDNA library segregation analysis (RSPSA) in allowed an assessment of the complexity of the Phanerochaete chrysosporium mRNA population during lignin degradation. Purification and partial sequencing of a hg- U. Raeder and P. M. A. Broda ninase protein allowed construction of oligonu- Department of Biochemistry and Applied Molecular cleotides that were used to isolate from the gene Biology, UMIST, Manchester M60 1QD. library clones containing the corresponding gene. Wehave developed a rapid method for preparing We seek to develop cloning systems allowing re- DNA from filamentous fungi and we have isolated THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 141

DNA from cultures derived from 50 independent tern of the mtDNA from these strains is generally basidiospores and the parent P. chrysosporium the same. However, three strains did show poly- strain. Labelled DNAs from genomic clones (from morphism in their restriction enzyme sites. We a A gene bank) were hybridised to Southern blots have calculated the molecular weight of rnitochon- of Sail restricted DNAs from the parent P. chryso- drial DNA from P. chrysogenum NRRL195I, iso- sporium and the basidiospores. This showed that: lated by caesium chloride density gradient centri- (a) about 80 per cent of the clones gave different fugation, to be 281 Kb. The molecular weights of hybridisation signals with parent DNA compared the mitochondrial genomes from the three other to basidiospore DNA, with 2 different types of wild type isolates are 264, 261 and 215 Kb. To patterns in the basidiospore DNAs (i.e., there date, the latter figure is the smallest mitochondrial seemed to be segregation of "restriction site poly- genome size reported for a filamentous fungus morphism"); (Sederoff, Adv. Genet., 22, 2, 1984; Minuth et a!., (b) Therefore, the basidiospores seem to be Curr. Genet., 5, 227-1982). haploid, whereas the parent P. chrysosporium strain Seventy-three per cent of the mitochondrial contains two different homologous copies of most DNA from P. chrysogenum NRRL1951 has been sequences and is probably dikaryotic; cloned into the yeast integrative vector pMa700. (c) the RSP showed a 1: 1 segregation pattern in Recombinant plasmids containing the 85, 71, 26 the basidiospores as expected from meiosis. and 24 Kb EcoRI generated fragments have been RSPSA was used to investigate genetic linkage used to transform Saccharomyces cerevisiae of genes specifically expressed during secondary MD4O/4C at high frequency. pMa700 usually metabolism of this fungus. This and the more gen- transforms at a frequency of 1-10 transformants eral uses of the method for mapping the whole per pg of plasmid DNA (Montiel eta!., Nuci. Acid fungal genome and for the detection of dispersed Res., 12, 1049, 1984) whereas the recombinant repetitive sequences will be discussed. plasmids gave a range of frequencies from 1.1 x i03 This work is part of a programme supported to 37x i03 transformants per p.g plasmid DNA jointly by British Petroleum's Venture Research and the yeast replicating vector pMa3a transfor- Unit and the AFRC. med MD4O/4C at a frequency of 27 x iO transformants per pg plasmid DNA. This strongly suggests that all four EcoRI mitochondrial DNA fragments contain a sequence which is able to confer on 51.The molecular biology of pMa700 the ability to replicate autonomously in Penicillium chrysogenum S. cerevisiae. P. Ford, G. Saunders and G. Hot School of Biotechnology, Polytechnic of Central London, 115 New Cavendish Street, 52.Analysis of mitotic London W1M8J. recombination with an excision- Ourinvestigations into the molecular biology of resynthesis model and implications Peniciilium chrysogenum have involved the construction of gene libraries and the cloning of fungal for meiotic recombination genes by complementation. In addition, analysis P. Unrau and J. R. Johnson of mitochondrial DNA from a range of wild type A. E. C. L. Health Sciences Division, Chalk River, isolates, and the isolation of autonomously rep- Ontario, Canada KOJ 1JO. licating sequences has been undertaken. Using the restriction enzymes BamHI and Hindlil, chromo- Theeffects on gene conversion of specific muta- somal DNA from P. chrysogenum NRRL1951 and tions in a known DNA sequence have been studied pBR328, two gene banks have been constructed. by Moore and Sherman (Genetics, 70, 397, 1975; Plasmid pools from recombinants have been used Genetics, 81, 1, 1977). We have analysed their data to isolate genes from P. chrysogenum able to com- in conjunction with an excision-resynthesis-driven plement the E. coli mutations proA, leuB and trpC heteroduplex model based on that of Resnick (J. (Holtet ai., Proc. 16th Workshop Conference theor. BioL, 59, 97, 1976). On our model the proba- Hoescht, Verlag Chemie, in press). bility of gene conversion is a function of the proba- Restriction enzyme analysis of the mitochon- bility, 'H', that the repair heteroduplex reaches at drial DNA from 20 wild type isolates of P. chry- least one mutant allele, the probability 'S', that the sogenum has revealed that the fragmentation pat- repair heteroduplex stops at a mismatch and the 142 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS probability 'P', that the mismatch is resolved in (both induced and meiotic) and are explicable if favour of the wild-type allele. This model can be 'S' and 'P' are near unity at the frameshift deletion. used to explain the observations of Moore and Meiosis exhibits map expansion in crosses between Sherman. For example, the observation that base substitutions but induced recombination does crosses involving alleles less than 5basepairs apart not. This is expected if the probability 'H' indepen- exhibit low induced recombination rates as com- dent of separation for induced but not meiotic pared to crosses 5ormore base pairs apart can be gene conversion (implyingpreferredre- explained if the presence of the second mismatch combination initiation sites in meiosis). That a within 4 base pairs results in a low value of 'S' single model can be used to explain the similarities (this has implications for the heteroduplex junc- and differences in meiotic and mitotic recombination). Crosses between frameshift deletions and tion rates supports an argument in favour of the base substitutions give very high recombination underlying unity of two processes.