GENETICAL SOCIETY OF GREAT BRITAIN

ABSTRACTS of Papers given at the HUNDRED AND FIFTY-FIRST MEETiNG of the Society held on 6th, 7th and 8th JULY 1966, at the UNIVERSITY OF NEWCASTLE UPON TYNE

SYMPOSIUM—GENETIC INSTABILITIES INTRODUCTION—MUTATION AND PARAMUTATION J. R. S. FINCHAM John Innes Institute, 8oy ford bury, Hertford, Herts Allknown genetic elements are subject to mutation or loss—otherwise they could never have been recognised. Unusual genetic instability, beyond what can reasonably be explained by the typically rather low frequencies of gene mutation, may be of several quite distinct kinds including the following: i. An enhanced frequency of "ordinary" mutation, as in at least one well analysed case in Escijerichia o1i; in such a case we have an enhanced mutation frequency in all genes, and a plausible explanation in molecular terms. .Moreor less extreme instability in specific genes, often under very dedicate and specific control by environmental factors and/or genetic "controlling elements ". Thisis the phenomenon covered by Brink's term "paramutation ",usedin the broad sense. One reasonable view of paramutation is that it represents, in deranged form, a normal mechanism for control of gene activity during differentiation of cells. Variegated-type position effect in Drosophila, and perhaps chromosome inactivation in mammals, may be related phenomena. We are obviously very far from being able to explain these effects in molecular terms. 3. Conditioning, in the sense of a predictable change of genotype in response to particular conditions of growth. Some kind of directed mutation may be involved here, and at least some such effects seem to be associated with changes in the nuclear genotype rather than with the cytoplasm. The underlying mechanism may have something in common with that involved in paramutation. 4. Genetic variability based on the cytoplasm underlies many of the responses to vegetative selection reported in fungi and probably also in green plants. Some results, obtained by the author and B. J. Harrison, on genetic instability of the paramutational type in Antirrhinum will be reported.

INSTABILITIESIN SALMONELLA TYPHIMURIUM OLIVER DARLINGTON Biology Laboratory, University of Sussex Inthe bacterium, Salmon€lla lyphimurium, certain slow-growing reversions of the leucine auxotroph, leu-151, exhibit a high rate of reversion back to auxotrophy. Most of these auxotrophs are themselves unstable, reverting to the slow-growing type. Both stable slow-growing and stable auxotrophic strains may be isolated from the unstable classes. The occurrence of unstable revertants of a praline auxotrophic mutant, in the unstable strain, was found to correlate with the loss of the instability from the slow- growing reversions of leu-15r. 527 528 GENETICAL SOCIETY OF GREAT BRITAIN In experiments on a proline auxotroph, pro-4o1, a peculiar type of reversion was found. The revertant colonies consisted entirely of auxotrophic cells. When the original auxotroph was used in homologous transductional crosses (selling) an unusually large number of wild-type recombinants were found. Among the reversions of a second leucine auxotroph, leu-12o, the two slow-growing classes proved to be unstable, reverting to faster growing types, termed sernjfasts. These specifically revert back to the slow-growing forms, with a very high frequency. The properties of these systems will be described and mechanisms for the in- stability will be discussed.

INSTABILITYAND SELECTIVE MUTATIONS IN THE AROMATIC GENES OF SALMONELLA TYPHIMURIUM S. RIYASATY Department of Genetics, Trinity College, Dublin Thepattern of instability which may involve one or more of the loci concerned with the biosynthesis of aromatic amino acids, and the pattern of spontaneous mutations within the tryAand tryBloci associated with a slow growing reversion of tryA-4will be described. Some models known to account for observations on other unstable systems will be discussed in relation to the results to be presented.

UNSTABLEGENES IN DROSOPHILA 1.ALDERSON Department of Genetics, Cambridge Thepaper reviews examples of unstable genes in Drosophila. Particular emphasis is directed towards the behaviour of induced mutational instabilities in Drosophila, which are used as the basis for a molecular model to explain the genetic nature of unstable genes.

THEINDUCTION OF REPLICATING INSTABILITIES IN SCHIZOSACCHAROMYCES POMBE M. ANWAR NASIM Muta genesis Research Unit, Institute of Animal Genetics, Edinburgh 9 Theinduction of instabilities of the replicating type was studied after mutagenic treatment with nitrous acid, ethylmethanesuiphonate, N-methyl-N-nitro-N-nitroso- guanidine, hydroxylamine and ultraviolet irradiation. With the exception of nitrous acid, all the mutagens tested induced such instabilities. The bearing of these results on the nature of induced replicating instabilities will be discussed. Since in these cases the induced instabilities replicate as such, "replication errors "inFreese's sense cannot explain them. It seems likely that an explanation will have to be sought for at the supramolecular level.

UNSTABLEPHENOTYPES IN ASPERGILLUS NIDULANS J. H. CROFT Department of Genetics, University of Birmingham Unstablephenotypes are very common in Aspergillus nidulans. At least three mechanisms for the control of this instability have been suggested: (i) cytoplasmic GENETICAL SOCIETY OF GREAT BRITAIN 529

" genes";(ii)metabolic steady states;(iii) chromosomal irregularities, e.g. aneuploidy. These three mechanisms will be discussed by reference to three groups of unstable morphological variants. Similarities and differences in the behaviour of these variants and methods at present available for distinguishing between the three proposed mechanisms will be emphasised.

RESPONSETO SELECTION WITHIN CLONES IN HIGHER PLANTS E. L. BREESE Welsh Plant Breeding Station, Aberystwyth Sofar there have been few reported instances of response to selection within clones of higher plants. Recent experiments with perennial ryegrass have shown that small continuous differences in growth rates between sub-clones (ramets) can be accumulated by selection to give major phenotypic differences. These differences can have a high degree of persistency and are presumed to be consequence of changes in the plasmon. Response is genotype-dependent, and somatic lability appears to be a feature of genotypes deriving from populations with histories of long-continued asexual propagation. Genetic studies with a range of persistent ryegrass population indicate that such differences may be transmitted over sexual cycles and have adaptive significance. Wider implications of these results will be discussed in relation to other reported examples of extranuclear phenomena.

ENVIRONMENTALLYINDUCED HERITABLE CHANGES IN HIGHER ORGANISMS A. DURRANT Department of Agricultural Botany, University College of Wales, Aberystwyth Thereis evidence for the occurrence of environmentally induced heritable changes that cannot be classified as mutations. They have the following characteristics: (i) They are induced by environments which are not exceptionally different from those normally experienced. (ii) All similar plants similarly treated change identically (iii) The response is quantitative, the more prolonged or the wider the range of treatment levels the greater the induced changes. Accepting only those experiments wherein the principles of Mendelian inheritance are recognised and homozygosity appreciated, the amount of information is so far limited. In other experiments the possibilities of cytoplasmic, hormonal or maternal effects have yet to be excluded. Induced changes in flax are nuclear and permanent though in some environments some reversion occurs with respect to some characters. In peas (H. R. Highkin) the permanency is dependent upon the continuation of the inducing environment but there is also evidence of pollen effects. Changes in Xicotiana (J. Hill) may be similar to those in flax. There is no evidence that the induced changes are adaptive. They may occur because the plants arc less capable of controlling their metabolism, or the changes may constitute a mechanism for promoting additional genetic variation. Lines inbred from natural populations are probably the most suitable material for the induction of heritable change.

Symposium:SummIng up—C. Auerbach 53° GENETICAL SOCIETY OF GREAT BRITAIN

OTHERPAPERS MUTATIONAL INSTABILITIES PRODUCED BY ETHYL METHANESULPHONATE IN DROSOPHILA T. ALDERSON and A. H. KHAN Department of Genetics, Cambridge Followingtreatment of Drosophila spermatozoa with ethyl methanesulphonate, fourteen lines have been isolated which continue to show apparent germinal mosaic- ism; for second chromosome recessive lethal mutations throughout succeeding genera- tions all lines originated from independent mutational events in individual sperma- tozoa from different treated males. The recurring lethal mutations originating within a line are found to be allelic both with one another and with the original mutation from which the line derived; but there is no allelisni between lethal mutations from different lines. These observations establish each continuing lethal-mosaic as a mutational instability, and suggest that a pre-inutational event can be produced by ethyl methanesuiphonate on a chromosome. Its unstable nature is subsequently characterised by its apparent behaviour in being repeatedly maintained throughout all the cell divisions which separate the fertilised ovum of one generation from that of the next generation before giving rise to a mutation. The mutational instabilities appear not to skip a generation without at least one mutation taking place, and each individual instability is found to occupy its own particular region on the chromosome. Both the lethal and the non-lethal mutational states from each of the mutational instabilities are found to revert in each case to the opposite state, and measurements of the apparent germinal lethal-mosaicism generated from lethal and non-lethal cultures suggest that the lethal mutational state is in a more stable form than its non-lethal state. A point of considerable interest which has emerged from the study of the apparent germinal lethal-mosaicisrn arising from non-lethal cultures is the marked variation in stability among the different mutational instabilities during their isolation through non-lethal cultures; yet each line shows essentially the same stability when measure- ments are made from the immediate non-lethal cultures generated from maintained lethal cultures. Storage of spermatozoa from the mutational instabilities in the female for six days seers in general to decrease the stability of both the lethal and non-lethal mutational states ; the effect is more pronounced, however, from the non-lethal mutational state.

ACHROMOSOMAL PROCESS GIVING RISE TO VEGETATIVE INSTABILITY IN ASPERGILLUS NIDULANS B. H. NGA and J. A. ROPER Department of Genetics,SheffieldUniversity Strains with duplication of a chromosome segment area readily produced in A. nidulans by crossing a normal strain to one with an unequal translocation. Bainbridge and Roper observed vegetative instability in a strain with a duplication; their results were consistent with an instability arising through loss of a terminal part of either of the segments carried in duplicate. This vegetative instability has now been observed in strains with other duplica- tions. The process has been analysed in a strain (kindly supplied by Professor R. H. Pritchard) carrying a duplication of the right arm of chromosome I and hetero- zygous for three genes. Several mechanisms have been considered as possible explan- ations of the instability. These mechanisms include (a) terminal loss, (b) mutation, (c) GENETICAL SOCIETY OF GREAT BRITAIN 53! mitotic crossing-over, (d)unequalexhange at mitosis between homologues or sister strands. It is now certain that, at least in some instances, the chromosomal loss is not terminal. If one mechanism alone is responsible for instability it can only be unequal sister-strand exchange. Such sister strand exchange gives loss hut should also give, perhaps at the same event, types with yet further duplications. Strains with extremely abnormal morph- ology have been isolated from the duplication parent. These extreme types are usually unstable. Preliminary meiotic and mitotic analyses, as well as instability patterns, are consistent with tandem duplication in these abnormal types.

INSTABILITYASSCCIATED WITH CHROMOSOME TRANSLOCATION IN ASPERGILLUS NIDULANS C. BALL Department of Genetics, University of Sheffield Amongthe revertants of meth1 (methionine requirement ; chromosome IV) were found types which showed both methionine independence and vegetative instability. One such revertant has been analysed in detail. On conidial pro- pagation, this variant repeatedly segregated a variety of phenotypes. Genetic analysis has shown that suppression of methionine requirement and morphological instability are changes jointly determined by events in a small region of the genome. Both meiotic and mitotic analyses suggest a non-reciprocal trans- location between chromosomes III and V. The data indicate that the point of translocation is the region of' the genome associated with suppression and instability.

ACHROMOSOMAL FACTOR CONTROLLING SPREAD OF INACTIVATION IN AN X-AUTOSOM E TRANSLOCATION B. M. CATTANACH M.R. C. Mutagenesis Research Unit, Institute of Animal Genetics, West Mains Road, Edinburgh 9

Genotypicselection for high and low amounts of albino-variegation induced by Cattanach's X-autosome translocation has established two lines of mice, one showing a 50 per cent, level of albino, the other a 30 per cent, level. Genetic control over the spread of the inactivating properties of the X across the associated autosomal loci was considered to be responsible. Reciprocal crosses between the two lines have shown that the controlling factor is located in the rearranged X-chrornosome. The factor may thus be the postulated X-chromosome "inactivationcentre ",butcrossing over between the factor and the autosomal insertion has not yet been detected. Both may be located in the Ta-jp region of the X-chromosome linkage map. An analysis of the selection lines in the light of the above information has revealed that the telection process had simply picked out specific rearranged X-chromosomes each of which, on the average, induces a constant level of albino-variegation. Two, or possibly three, different X-chromosornes have not been isolated and the study of the mechnism by which they came to differ can now be investigated. It is con- ceivable that, rather than the "inactivationcentre "concept,a "controlling element" system such as that found in maize may be involved.

KARYOTYPEINSTABILITY IN THE SYRIAN HAMSTER IN VIVO AND IN VITRO D. T. HUGHES and S. AUSTIN Cytogenetics Department Chester Beatty Research Institute, London, S. W.3 Chromosomeabnormalities, due to karyotype instability, have been found to form the basis of many pathological conditions. In addition, a significant increase 532 GENETICALSOCIETY OF GREAT BRITAIN in aneuploidy of sex chromosomes in human cells was found to be associated with increasing age of normal people. The karyotype of the Syrian hamster (Micro- cricetus auratus) is more suitable for studies on the X-chromosome because it can be confidently identified by its more distinctive morphology. In the present study chromosome non-disjunction and structural changes in individual chromosomes have been investigated: to determine whether the anomalies occur at random in the karyotype or affect particular chromosomes such as the X-chromosome. These features were observed in cells from two environments: (a) in vivo: bone marrow of ageing hamsters, (b) in vitro: a derivative of diploid hamster kidney cell strain (BHK 25Clone13ofMacpherson and Stoker) in suspension culture. In the karyotyped cells the X-chromosome was not preferentially involved in non-disjunction. Pseudodiploid cells (due to abnormal numbers of chromosomes per group within the karyotype and/or structurally abnormal chromosomes) were found both in vivo and in vitro. The cultured cell population was most unstable, having a pseudodiploid mode in which all the analysed karyotypes appeared to differ from one another. These findings will be discussed in relation to the mechan- ism of evolution in somatic cell populations.

DIVIDINGAND DIFFERENTIATING CELLS URSULA MIUWOCH Galton Laboratory. University College, London Oneof the most baffling problems of present-day genetics is how to account for the development of complex organisms through the division of cells into two with apparently equal chromosomal constitutions. Some recent results from measure- ments of DNA values in dividing and non-dividing cells appear to be relevant to this problem. In conjunction with Dr Kahnus, measurements were made of DNA values in the cell nuclei of honey bees, using a cytophotometric method. It was found that in young larvae, the dividing cells were diploid in females and haploid in males, but that the non-dividing cells ranged between diploid and i6-ploid in females, and between haploid and 32-ploid in males. These results show that dividing and non-dividing cells belong to different populations and that dividing cells tend to be recruited from those which have retained their basic DNA value. Results from human tissue culture appear to confirm this, although the degrees of polyploidy reached by such cells are not as high as in insects. Although the existence of somatic polyploidy is by no means a newly discovered phenomenon, the antithesis between cell division and differentiation does not seem to have been widely appreciated. It may be connected with the differential survival of diploid and heteroploid mammalian cells in tissue culture.

EVOLUTIONARYRELATIONSHIPS OF MAMMALIAN VIRUSES INFERRED FROM NEAREST NEIGHBOUR BASE SEQUENCE ANALYSIS OF THEIR DNA H. SUBAK-SHARPE Medical Research Council, Institute of Virology and J. M. MORRISON Institute of Biochemistry, University of Glasgow, Scotland DNAof mammalian cells is characteristically deficient in the CpG doublet. Provided this shortage is typical of DNA sequences that specify polypeptides, this implies that CpG-containing codons are rarely used in mammalian cells. DNA GENETICAL SOCIETY OF GREAT BRITAIN 533 of viruses adapted to use only the cell's machinery for translation should reflect its CpG shortage. However DNA of viruses that modify the cell's machinery of translation, e.g. by altering the transfer-RNA population, need not conform with the host DNA doublet pattern. These ideas were tested by nearest neighbour frequency analysis of the DNA of six mammalian viruses as well as hamster BHK2I /C13 cells. The DNAs of the two small oncogenic viruses tested (polyoma and Shope papilloma) closely reflect the host cell pattern, whereas the DNAs of the four large viruses (vaccinia, equine rhinopneumonitis, herpes simplex, pseudorabies) are totally different. As most or all polyoma virus DNA must programme polypeptides, the CpG shortage in both polyoma virus and host cell DNA must be characteristic of polypeptide- specifying DNA in mammalian cells. Doublet frequency comparisons indicate difficulty for host cells in translating the DNA of the four large viruses—unless the machinery is modified. These observations suggest different evolutionary relationships for small and large mammalian viruses.

MECHANISMOF ACTION OF PROLINE SUPPRESSORS IN ASPERGILLUS NIDULANS P. WEGLENSKI John Innes institute, Bay fordbury, Hert ford, Herts Twoclosely linked proline loci in A. nidulans are known, the mutants of which require either proline, ornithine, citrulline or arginine for growth. According to the biochemical data they are blocked between glutamate and glutamic-y-semi- aldehyde in the pathway of proline synthesis. Spontaneous revertants of the proline mutants were obtained. All revertants tested appeared to be suppressor mutations. Three suppressor loci were mapped, two of them recessive (su6pro6 and susgpro6) and one to which dominant, semidominant and recessive mutations belonged (su2pro6). All suppressors tested were unspecific, i.e. they suppressed mutations of both proline loci. The possibility that suppressor mutations affect the pathway of arginine synthesis or breakdown was tested. It was found that mutants of the su6pro6 locus are almost completely deficient in ornithine transcarbamylase activity and the mutants of the sul9pro6 locus produce ornithine -transaminase constitutively; this latter enzyme is strongly induced by arginine in the wild type. The effects of both these mutations permit the utilisation of ornithine for proline synthesis (via glutamic-y-semialdehyde). The mode of action of the dominant and recessive suppressors of the sur2pro6 locus is still not clear ; the possible mechanisms will be discussed.

INVESTIGATIONSON ENZYME DIFFERENCES BETWEEN WHITE CLOVER PLANTS GROWN IN TISSUE CULTURE MONICA HUGHES Department of Plant Science, The University, Newcastle upon Tyne Plantsof white clover were grown as undifferentiated tissue in solid and liquid culture on a cynthetic medium. These plants were selected on the strength of their cyanogenic character, as assayed with the picrate reaction. Cultured tissue, derived from plants possessing functional alleles of both genes controlling the cyanogenic character, was found to produce the enzyme -glucosidase but not the natural glucoside substrates. The production of -glucosidase by cultured tissue was studied using p-nitropheny1--D-g1ucoside as the enzyme substrate. 534 GENETICAL SOCIETY OF GREAT BRITAIN

Some physical properties of fl-glucosidase produced by single plant clones were measured. These indicated that the gene responsible for fl-glucosidase production existed as a series of alleles conditioning the formation of slightly different enzyme types. The possible nature of the locus responsible for the production of enzyme protein will be discussed.

SELF-INCOMPATIBILITYALLELES IN THE POPULATION OF OENOTHERA ORGANENSIS i. L. CROSBY Department of Botany, University of Durham Theold problem of how 45 self-incompatibility alleles could be maintained in an Oenoth8ra organensis population of about 500 individuals has been tackled by computer simulation, avoiding both the complexities of mathematical analysis and arguments about their statistical validity. Several models starting with 64 alleles at the s locus were established, to investigate the effects of perennation, mutation and population subdivision with complete or partial isolation. Mutation usually had little effect. Perennation reduced the equilibrium number of alleles, and usually increased their rate of loss. Subdivision with complete isolation allowed a high number of alleles to be retained in the whole population ; a small amount of random gene migration could destroy this effect almost completely, localised migration less so. No realistic model gave a steady state condition in any way resembling the natural state of the species. It was concluded that the species is nowhere near a steady state. Oe. organensis had once been numerous and continuously distributed with many alleles mostly occurring throughout the population. Recent disaster reduced it to a few partially isolated sub-populations with high numbers of alleles. There have been so few generations since then that there has been little allele loss.

(To be published in Evolution.)

HEADCOLOUR POLYMORPHISM IN THE GOULDIAN FINCH J. H. BENNETT Department of Genetics, University of Adelaide, South Australia TheGouldian finch which inhabits tropical and sub-tropical areas of Northern Australia has two common and well-defined head colour forms—one black and the other red. Microscopic examination of the head feathers of the red variant shows them to be morphologically distinct and spectral absorption studies suggest that a carotenoid pigment is involved. Family data are generally consistent with these two forms being controlled by a pair of alleles at a sex-linked locus, the red phase being dominant. However, the available family data from matings between red hens and cocks show a significant excess of black heads among the female progeny. Extensive population data obtained over nine years in the north-west of Australia show considerable variation between years in the frequencies of the two forms in males but much less variation in females. The proportion of black heads among males is consistently too small by comparison with that in females if a single sex- linked locus is involved and there were random mating and no selective differences. Various factors which may be important in maintaining this polymorphism will be discussed. The permissible ranges will be given for values of the differential viabilities which could result in a stable selectively balanced polymorphism having the total frequencies observed. GENETICAL SOCIETY OF GREAT BRITAIN 535 THESTRUCTURE AND FUNCTION OF B-CHROMOSOMES IN RYE R. NEiL JONES and H. REES Department of Agriculwrol Botany, University College of Wales, Aberystwyth Investigationsare described of the composition of B-chromosomes with respect to their dry mass and DNA content in comparison with the normal A-chromosome comp'ement of Rye. The influence of B-chromosomes in varying numbers upon nuclear behaviour at mitosis and meiosis, and upon various aspects of the external plant phenotype is also described, and related to the incidence and distribution of B-chromosomes in "Natural" Rye populations.