The analysis of genetic recombination on the polaron hybrid DNA model

By H. L. K. Whitehouse and P. J. Hastings

Genet. Res. (1965), 6, 27–92

On p. 58, Table 7, in the column headed ypc, the entries relating to genotypes nos. 17 and 18, 19 and 21, 20 and 22, and 27 and 28 should be transposed within each pair, so that the column reads: 17, ⅛; 18, —; 19, ⅛*; 20, ⅛; 21, —; 22,—; 23, *; 24, ; 25, *; 26, ; 27, ⅛*; 28, —; 29, ; 30, ; 31, *; 32, . Total (wild types), *.

A mutant (Mb-s) of Escherichia coli K12 which is more sensitive than wild-type (Mb-r) to such dyes as methylene blue or acridines was studied. The Mb-s mutant can also be disinfected of F factor with lower concentrations of acridine orange than Mb-r bacteria. The Mb locus was mapped by crosses between Hfr and F−, F-duction with F13, and transduction with phage P1. It was found to be situated between the pur and T6 loci on the chromosome. Moreover, it was found to be contained in the chromosomal fragment carried by the F-prime, F13. In a heterogenote obtained by F-duction, the Mb-r gene is dominant over the Mb-s gene. The Mb gene was transduced jointly with the pur gene by phage P1.

1. The frequency of the chromosomal types of several western Mediterranean populations of D. subobscura, distributed in a zone running north–south, is analysed and compared with that from an Edinburgh (Knight, 1961) site at a similar meridian as the Mediterranean populations.

2. North–south clines are found in the frequencies of several chromosomal types. Some types are more frequent in the north, decreasing gradually southwards; others show the reverse trend of variation.

3. The comparison with a similar array of populations from Central Europe and the Central Mediterranean area, indicates that the chromosomal types more frequent in the northern populations are mostly the same, i.e. the standard orders. But chromosomal types with complex inversion orders are the most frequent in southern populations: in some chromosomes, different orders are predominant in Israel, southern Italy and southern Spain.

4. The Pyrenees, acting as an ecological barrier, strongly influence the diversity of the populations north and south of the range. This result and the latitudinal clines support the adaptive significance of the chromosomal polymorphism in D. subobscura.

5. The index of free recombination of the population from Malaga (in the south of Spain) is higher than in the populations from the northern Mediterranean area. D. subobscura apparently supports the claims of da Cunha & Dobzhansky (1954) and Carson (1955) that a higher level of chromosomal polymorphism occurs in the central areas of the distribution of a species.

The inheritance of three protein fractions of third instar larval lymph in Drosophila melanogaster as detected by starch gel electrophoresis is described. Each of the three is governed by a single autosomal gene. In two, the dominant allele determines presence, and the recessive absence of the respective fraction. The third protein fraction is governed by a single pair of co-dominant alleles.

Comparison of the starch-gel pattern to that obtained on cellulose acetate and on polyacrylamide gel fails to show definite sub-fractionation of those protein bands of which the inheritance has been studied.

Previous measurements of specific locus mutation rates in mice had all involved the seven loci, a, b, c, d, p, s and se. An experiment was performed with the same mouse stock (C3H × 101) and the same radiation dose (600 rad) to spermatogonia as had been used previously, but employing a new group of six loci, a, bp, fz, In, pa and pe. The observed mutation rate, 5·0 × 10−8 per locus per rad, was significantly lower than that for the original seven loci, but was three to four times higher than the corresponding mutation rate in Drosophila melanogaster.

Grizzled (gr) is a recessive gene with complete penetrance but reduced viability. It is linked with waltzer (v) in linkage group X, the recombination frequency being 14·6 ± 2·6%. Its visible effect is to dilute the yellow pigment of the hairs. Body weight is reduced by about 25% atbirth and remains at about 10–20% below that of non-grizzled litter mates. Viability up to the time of classification is about 50–60% of that of non-grizzled litter mates. The mortality of grizzled homo-zygotes occurs at all stages from about 10 days of gestation up to the time of classification. No specific cause of the mortality was evident.

In Neurospora crassa, a change of 2·5°C. in incubation temperature produced highly significant changes in the second division segregation frequency of asco (Linkage Group VI). A minimum value was found at 25°C. consistently increasing towards higher and lower temperatures over the range 15° to 30°C.

Where recombination frequencies in proximal and distal marked regions in Linkage Groups I and VI showed a change, this followed the same pattern as for asco with proximal intervals usually affected; but in one cross a distal region, instead of the proximal, showed a significant change. This behaviour supports the suggestion that heritable factors influencing recombination frequency in specific regions of each chromosome may show differences in temperature sensitivity.

Temperature treatments during protoperithecial development prior to meiosis as well as during meiosis had significant effects on recombination frequencies in several cases. Protoperithecia incubated at one temperature and then transferred to a different temperature after conidiation, gave second division segregation frequencies of asco intermediate in value between those obtained with continuous incubation at either of the two temperatures alone.

In the present work with Neurospora, the similarity in the relationship between temperature and recombination frequency with the reported results in Drosophila is discussed and suggests that the common temperature/recombination frequency curve is indicative of a possible direct effect of temperature on the recombination processes. Present knowledge of these processes is insufficient to permit any detailed explanation of the temperature effects found but the observed negative relationship between recombination frequency and temperature over the range 15° to 22·5°C. is discussed in kinetic terms.

The second division segregation frequency of asco was found to be increased at all temperatures tested when the asci were mounted for scoring in strong sucrose solution compared to mounting in water.

Difference in bristle-making abilities in extreme scute and in wild-type Drosophila were investigated using flies mosaic for the two kinds of tissues. It was found that both wild-type and scute tissues were autonomous with respect to bristle differentiation. From the experimental evidence it was postulated that the differences in bristle-making abilities between the two genotypes might be due to differences in tissue competence rather than differences in pre-pattern or in level of bristle-making substances.

Five allelic mutants of Neurospora which lack glutamic dehydrogenase (am mutants) were induced to revert with ultra-violet. The glutamic dehydrogenase produced by the revertants was compared to that of wild-type. Several distinct classes of revertants could be distinguished by these tests. However, genetic analysis showed that all the reversions resulted from events at or near the site of the original am mutation. The spectrum of reversion types depended on the nature of the am mutant employed. One mutant, which produces an am protein believed to be altered in its active site, yielded revertants which were all indistinguishable from wild-type. Another mutant, which produces a protein with a functional active site but altered folding properties, gave at least six classes of revertants which were different from wild-type.

A mouse antigen, MuB1, has been shown by the study of backcrosses to be inherited in a unifactorial dominant manner. Its inheritance is not linked with gamma globulin allotypes, but has been shown to be linked with the presence of the haemolytic complement system. The concentration of MuB1 in the serum of several inbred strains of mice showed sex-associated differences; however, the presence of MuBl did not depend on the sex of the parents or on the sex of the offspring. The inheritance of antibody responsiveness is discussed in terms of the inheritance of the antigen, MuB1.

A cytological study has been made, in Aspergillus nidulans, of haploid and diploid homokaryons and of heterokaryons formed between them. The effective region of growth was shown to extend about 600 μ behind the growing edge of the colony and to correspond largely with the tip cells. Acridine fluorescence gave unselective staining of all nuclei; projected nuclear areas, and their distributions in individual hyphae, were determined for the homokaryons and heterokaryons. Diploid and haploid nuclei differ in mean projected area but the overlap of their size distributions precluded unequivocal classification of individual nuclei in diploid–haploid heterokaryons. However, it was shown that the hyphae of a heterokaryon differ in nuclear ratios. This, and other evidence, strongly support the suggestion that heterokaryon maintenance and adaptation occur through interhyphal rather than intrahyphal selection.

Starting from a genetically variable stock homo- or hemi-zygous for Bar two selection lines were set up, one selected for decreased sensitivity to the effect of larval temperature on eye size (‘canalization line’), the other for increased sensitivity (‘anti-canalization line’). In each generation a sample of larvae was grown at 25°C. throughout life and another sample at 25°C. for the first 48 hours, followed by 18°C. until emergence. In the canalization lines a selection was made of individuals (five males and five females out of 100) least affected by the temperature treatment and the anti-canalization line for a similar number most affected by the treatment. These ten males and females were allowed to mate at random and from the eggs produced random samples were then treated in the next generation in a similar manner in the two temperatures. Precise counts of facet numbers on the right eyes were made at generations 4 and 9 and it was clear that selection had been effective both in decreasing and increasing temperature sensitivity.

The whole canalization line can be regarded as a population which has been subjected to disruptive selection for two different criteria: one (small eyes) being regarded as adaptive to the low-temperature regine or habitat; the other (large eyes) being regarded as adaptive to the high-temperature habitat. A similar type of analysis can be applied to the anti-canalization line in which, however, the selective values of the phenotype are regarded as reversed in value in the two habitats. The experimental procedure employed involved not only random mating between individuals selected according to these two criteria but also random allocation of the offspring of this panmictic population to the two habitats of the next generation. This corresponds to a natural population in which there is disruptive selection exerted by two different habitats but no habitat preferences exhibited by members of the population. Under these circumstances, in our experiments, disruptive selection produced only rather slight increases in phenotypic variance, which were rather larger in the anti-canalization than in the canalization lines. Possible reasons for the difference between this result and those reported by Thoday and Gibson are discussed, and it is suggested that an important reason may be the slowness of the response to selection in the first few generations in our lines.

The effects of tight linkage on the total response due to pairs of identical additive loci, segregating in a population initially in linkage equilibrium, have been studied both algebraically and by means of computer simulation. Particular attention has been given to the effects of finite population size on the probabilities of (a) the elimination from the population of the gamete carrying both ‘plus’ alleles; (b) the joint preservation of the two types of repulsion gametes; (c) the recovery of the desired combination of plus alleles through crossing-over; and (d) the fixation of the gamete in the population following its recovery.

The study is restricted to situations in which linkage is known to have an appreciable effect on total selection response, i.e. to the case of genes of large effect initially at low frequency. A comparison of regimes with the same expected response under free recombination has shown the probability of (a) to be high, and the probability of (b) to be very nearly the same for all regimes tested. Provided that the recovery of the gamete carrying both plus alleles is an unlikely event at any given point in time, the probability of the fixation of the gamete, once reconstituted, is expected to be independent of population size for genes of large effect. In this context, approximate algebraic expressions have been derived for the probability of effective recovery of the required gamete, and for the mean waiting time involved.

The spontaneous reversion to white of three purple mutants of Coprinus lagopus has been demonstrated to be due to suppression. Forty-one of the fifty suppressed purples isolated were crossed to wild-type and all crosses gave fewer than 25% purple progeny, i.e. all the suppressors appeared to be linked to the gene suppressed. Values for the percentage of purples segregated from these crosses varied continuously from 0% to nearly 20%. The functional relationships of the fifty suppressors were investigated by the technique of complementation in dicaryons. The pattern of results formally indicate two suppressor loci with extensive inter-allelic complementation at one of them. The functional allelism indicated by the complementation results is not compatible, in conventional terms, with the apparent positional spread of the suppressors. Several possible explanations are discussed but more data are required before a choice can be made between them. It seems most probable that the same genetic entity is involved in each independent event of suppression and that the characteristic segregations of purples from crosses of suppressed purples with wild-type will have to be explained in terms other than of normal recombination.

The pattern of changes of the genetic covariance between two characters on selection was examined in an effort to explain the asymmetry of correlated responses in two traits, or of the same trait in two environments, frequently observed in experimental results.

The algebraic conclusions were further examined by model selection experiments using a computer. The computer was programmed to calculate the change in gene frequency from generation to generation and to calculate from it the expected changes in genetic variances and covariance as selection proceeded. This procedure was carried out with several models of gene effects and gene frequencies.

Asymmetry of the genetic covariance, and consequently of the correlated responses, resulted when the relative change in gene frequency at the loci contributing positively and negatively to the covariance depended on the trait selected. The conditions necessary for the development of asymmetry were examined and the results suggest that any symmetry found in an experiment is perhaps more surprising than asymmetry. Probably the most frequent contribution to asymmetry in practice will be from loci contributing negatively to the covariance and having frequencies other than 0·5.

Accurate prediction of correlated response over many generations is therefore not possible without prior knowledge of the composition of the genetic covariance, as well as its magnitude. The validity of existing theory for the prediction of correlated responses is likely to be much poorer than for the prediction of direct responses. Predictions would then have to be based on the genetic parameters estimated in each generation.

A number of acridines have been tested for ability to inhibit conidia of strains of Aspergillus nidulans. The effectiveness of any one acridine in growth inhibition and killing involves interaction of genotype and conditions of treatment such as temperature, pH, treatment medium and light intensity. Mutant alleles which confer growth resistance to acriflavine are selective in their actions towards other acridines, may differ in their dominance relationships with different acridines and are even selective with regard to the conditions under which they confer acriflavine resistance. Certain pairs of acridines, used simultaneously, show additive effects, potentiation, or annulment by one of inhibition caused by the other.

Some of these findings have been applied in a study of factors affecting acridine-induced mutation in Aspergillus conidia. Under conditions which permit metabolism, acriflavine induces a high frequency of unstable morphological variants. One such variant has been shown to be a disomic. Using a system of reversion from auxotrophy to prototrophy, acriflavine-induced mutation has been obtained both with high light intensities and in the absence of light. In the latter case recombination as a feature of the mutation process is excluded.

A review is given of published accounts of mosaics in mice and other rodents, of mutations and of certain unexplained events. The cumulative weight of evidence makes a strong case for the existence of somatic crossing over in the laboratory rodents.

Polarized segregation was studied at the asco and tan spore loci of Neurospora crassa and at the gray and hyaline loci of Sordaria fimicola. Different alleles, re-isolates and temperatures of incubation of crosses were studied with the asco locus and different temperatures of incubation with the h and g loci. The mutants were crossed with the corresponding wild-types by methods giving a considerable degree of synchrony in the development of perithecia; segregation patterns were scored from perithecia at widely differing stages of ascal maturity. Significant polarized segregation was not generally observed when little or no dehiscence had occurred from a cross but usually became readily apparent as dehiscence proceeded. This indicated that the differences in frequency between the ascal classes that had previously been interpreted as showing polarized segregation at meiosis were actually due to a post-meiotic differential bursting of asci. This bursting was studied separately from the usually concurrent pigmentation of immature asci by comparing results from asci mounted in water with those from asci in the 2 M sucrose solution which was used throughout these experiments: the differential bursting produced by mounting asci in water gave similar polarized segregation results to those from natural dehiscence. A hypothesis to account for differential bursting in physiological terms was advanced and its predictions discussed. It seems likely that cases of apparent polarized segregation described in other Ascomycetes are also due to post-meiotic phenomena.

A survey of past work shows that certainty in the electrophoretic classification of mouse haemoglobins requires further understanding of the meaning of the multiple electrophoretic bands observed.

Six electrophoretic bands are observed in all. Numbered in order of mobility they give the formulae 2 for Hb-s, 1235 for Hb-d, 1246 for Hb-p and 12345 for foetal haemoglobin (Hb-f). When mercaptoethanol is added to the electrophoretic medium these formulae become: 2 for Hb-s, 23 for Hb-d, 24 for Hb-p, 234 for Hb-f. Bands 5 and 6 and a fast ultra-centrifuge fraction both increase when samples are stored.

Bands 5 of Hb-d, 6 of Hb-p, and probably 5 of Hb-f, are dimers of the whole haemoglobin molecule almost certainly joined by sulphur bridges. The monomers are bands 2 and 3 of Hb-d and 2 and 4 of Hb-p. Within each type interconversion of bands 2 and 3 and bands 2 and 4 has been observed.

The hypothesis is suggested that Hb-d and Hb-p both exist as two conformational isomers. In each case one isomer is electrophoretically indistinguishable from Hb-s: the other permits the electrophoretic recognition of their differences in bands 3 and 4, and the formation of sulphur bridges to give bands 5 and 6.

Strains with altered properties of host-induced modification (HIM) have been isolated. Someof these variants were stable on first isolation but the majority of them are unstable. They segregate during many generations two different stable HIM phenotypes and a third type which continues to segregate. Usually this instability disappears during repeated serial single colonyisolation, but in one case it still persisted after nine such single colony isolation steps. Evidence suggesting that they are unstable partial diploids is discussed.