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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 muta- tions 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.
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