Publications

A highly unstable allele has been isolated at the white locus of Drosophila mauritiana, a sibling species of D. melanogaster. This allele, white-peach (wpch), mutates spontaneously in males and females to give both wild-type and bleached-white derivatives. The mutation frequency is about 10(-3) mutations/generation. There is no evidence for clustering among mutant progeny, and phenotypically wpch flies with mosaic patches of wild-type tissue in the eyes are frequently recovered. Another X-linked locus, plum, is destabilized when wpch is on the same X chromosome.

A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurrence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 +/- 0.8; the comparable figure for IS5 was 3.7 +/- 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements. The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.

Many enzymes in intermediary metabolism manifest saturation kinetics in which flux is a concave function of enzyme activity and often of the Michaelis-Menten form. The result is that, when natural selection favors increased enzyme activity so as to maximize flux, a point of diminishing returns will be attained in which any increase in flux results in a disproportionately small increase in fitness. Enzyme activity ultimately will reach a level at which the favorable effect of an increase in activity is of the order 1/(4Ne) or smaller, where Ne is the effective population number. At this point, many mutations that result in small changes in activity will result in negligible changes in fitness and will be selectively nearly neutral. We propose that this process is a mechanism whereby conditions for the occurrence of nearly neutral mutations and gene substitutions can be brought about by the long-continued action of natural selection. Evidence for the hypothesis derives from metabolic theory, direct studies of flux, studies of null and other types of alleles in Drosophila melanogaster and chemostat studies in Escherichia coli. Limitations and complications of the theory include changes in environment or genetic background, enzymes with sharply defined optima of activity, overdominance, pleiotropy, multifunctional enzymes and branched metabolic pathways. We conclude that the theory is a useful synthesis that unites many seemingly unrelated observations. The principal theoretical conclusion is that the conditions for the occurrence of neutral evolution can be brought about as an indirect result of the action of natural selection.

Fifteen independently isolated complementary DNA clones that contain T-cell receptor (TCR) V beta genes were sequenced and found to represent 11 different V beta genes. When compared with known sequences, 14 different V beta genes could be defined from a total of 25 complementary DNA's; 11 clones therefore involved repeated usage of previously identified V beta's. Based on these data, we calculate a maximum likelihood estimate of the number of expressed germline V beta genes to be 18 with an upper 95 percent confidence bound of 30 genes. Southern blot analysis has shown that most of these genes belong to single element subfamilies which show very limited interstrain polymorphism. The TCR beta-chain diversity appears to be generated from a limited V beta gene pool primarily by extensive variability at the variable-diversity-joining (V-D-J) junctional site, with no evidence for the involvement of somatic hypermutation.

Chemostat studies of bacteria that harbour the prokaryotic transposable elements Tn5 and Tn10 and the temperate phages lambda, Mu, P1 and P2 have shown that these accessory DNA elements confer a selective advantage on their hosts. We propose that similar selective effects provided the initial impetus for the evolution of nascent accessory DNA elements in primitive bacterial populations. In subsequent evolution the elements acquired or perfected the 'selfish' characteristics of over-replication and horizontal transmission. Such selfish traits led to the dissemination of accessory DNAs among commensal strains, species and genera, genetically interconnecting them to create a 'commonwealth' of species that potentially share a common gene pool. The involvement of accessory DNAs in genetic exchange provides selection at the population level for refinement and diversification of the elements and for regulation of their replication, transposition and transfer among cells. The diversity of intracellular environments encountered by the elements imposes constraints on their evolution while at the same time altering the selection pressures operating on conventional chromosomal genes. This process of coevolution of accessory DNAs with the genomes of their diverse hosts has led to a unique population structure and mechanism of genetic exchange among bacteria, which constitutes the most effective adaptive strategy yet devised by selection.

DNA from Escherichia coli strains in a reference collection of 72 recent natural isolates (ECOR strains) and 25 natural isolates from the "pre-antibiotic" period 1930-1940 (Murray strains) were studied to determine the genomic abundance of insertion element IS5 and the size of genomic restriction fragments carrying sequences homologous to IS5. Among the ECOR strains, nearly two-thirds lack DNA sequences that hybridize with IS5, and one-half of the remainder have only one copy. Among strains in which IS5 is present, extensive variation in the size of IS5-bearing restriction fragments occurs, in many cases allowing distinction among strains that are judged to be nearly identical in genotype because of the identical electrophoretic mobility of the enzyme coded by each of 11 chromosomal loci. Among the Murray strains in which IS5 is present, the average number of elements per strain is larger, but not markedly so, than among recent isolates. Comparison between duplicate strains in the Murray collection suggests that the rate of accumulation of IS5 elements in prolonged storage in stab tubes corresponds to an apparent probability of transposition of approximately 0.008 +/- 0.002 per IS5 element per year. Because of the extensive genetic variation among strains, insertion elements such as IS5 would seem to be convenient genetic markers with which to detect recent common ancestry among strains.

A DNA fragment that includes the wild-type rosy (ry+) gene of Drosophila melanogaster has been introduced by microinjection into the germ line of the reproductively isolated species Drosophila simulans and incorporated into the D. simulans genome. Transformation was mediated by the transposable element P, which occurs in the genome of most natural populations of D. melanogaster but not in D. simulans. Rubin and Spradling [Rubin, G.M. & Spradling, A.C. (1982) Science 218, 348-353] have previously shown that the ry+ DNA fragment, which is flanked by recognition sequences of P element, can transform the germ line of D. melanogaster. Successful transformation in D. simulans indicates that the P element continues to function as a transposable element in the D. simulans genome. Moreover, the ry+ gene of D. melanogaster functions in the genome of D. simulans to produce normal eye color, despite the estimated 1 to 5 million yr of reproductive isolation since the evolutionary divergence of these species. Interspecific DNA transformation provides a useful method for the study of genetic differences affecting gene expression among related but reproductively isolated species.

E. coli is a successful and diverse group of organisms, well defined by DNA hybridization within the Enterobacteriacae and including the closely related organisms Shigella and the Alkalescens-Dispar biogroup. The primary habitat of E. coli is the lower intestinal tract of warm-blooded animals, which is colonized shortly after birth. At any one time, most normal individuals carry several strains of E. coli in their intestinal tract, including a small number of resident clones exhibiting a rate of replacement measured in weeks or months and a much larger number of transient clones that are replaced in a matter of days or weeks. The secondary habitats of E. coli are soil, sediment, and water, where its half life is thought to be only a few days. Pathogenic forms of E. coli are associated with diarrheal diseases, urinary tract infections, neonatal meningitis, nosocomial infections, and in infections of domesticated animals. E. coli populations contain much genetic diversity, more than is found in most eukaryotes. Genetic diversity has been studied from the standpoint of (a) serology with respect to surface antigens, (b) biogrouping with respect to variable characters such as nutritional versatility, antibiotic resistance, and bacteriophage susceptibility, (c) electrophoresis of enzymes of intermediary metabolism or outer membrane proteins, (d) DNA hybridization, (e) restriction-fragment length polymorphisms, (f) DNA sequences, (g) insertion sequences, and (h) plasmids. However identified, strains of E. coli appear to have a wide, but not totally indiscriminate, host range. Aside from genes directly associated with virulence, genetic divergence between pathogenic and nonpathogenic strains, although statistically significant, is not pronounced. Electrophoretic studies indicate that, while some serotypes may represent a single genetic clone almost exclusively, other serotypes may represent two or more genetically unrelated clones. Unrelated clones may therefore converge to the same or very similar serotypes. Electrophoresis has also been used to define three groups of clones among natural isolates, perhaps corresponding to subspecies of E. coli. These groups are worldwide in distribution and have a wide host range. E. coli populations exhibit great linkage disequilibrium, which occurs as highly nonrandom combinations of alleles at different loci. Reproduction is evidently largely asexual, with insufficient genetic recombination to dissipate linkage disequilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

Six naturally occurring alleles representing four electromorphs of the enzyme glucose-6-phosphate dehydrogenase were transferred by P1-mediated transduction from natural isolates of Escherichia coli into the genetic background of E. coli K12 and were studied in pairwise competition in chemostats limited for glucose in order to estimate differences in growth rate associated with the alleles. Although the level of resolution of such experiments is a growth rate differential of approximately 0.002 h-1, no significant differences among the strains were found. Studies of apparent Km and Vmax in crude enzyme extracts of the strains also failed to reveal any significant differences among the electromorphs. These results support the view that the alleles are selectively neutral or nearly neutral under these conditions.

A novel genetic change leading to increased activity of 6-phosphogluconate dehydrogenase (6PGD) in E. coli has been observed. The mutation is a deletion of approximately 0.4 kilobase pairs occurring between the structural gene of 6PGD (gnd) and one copy of an insertion element (IS5) found normally in E. coli K12 a few hundred base pairs upstream (counterclockwise) from gnd at 44 minutes on the conventional genetic map. The deletion is associated with a threefold higher activity of 6PGD and a 57% increase in the maximum growth rate when cells are grown in gluconate.

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.

Twelve diverse strains of Drosophila melanogaster have been examined with respect to their individual fitness components and with respect to their relative performance under competitive and noncompetitive conditions. Individual fitness components included estimates of time until successful copulation (t), fecundity (f) and egg-to-adult viability (v), and a composite index of overall fitness of the form fv/t was used for comparisons among strains. Noncompetitive performance was assessed in terms of the biomass (standing crop) and productivity of equilibrium experimental populations. Competitive performance was assessed in terms of relative competitive ability vis-a-vis a standard compound-autosome-bearing strain in single-generation tests. A significant correlation was found between the composite index of individual fitness components and the competitive compound-autosome test. Although the biomass and productivity of equilibrium populations were correlated with each other, neither of these noncompetitive measures was correlated with individual fitness components or with the composite index. We suggest that the performance of strains in such noncompetitive tests may be related to what Wright has called the "mean selective value" of the populations. Judging from their association with the composite index of individual fitness components, competitive tests such as the compound-autosome test seem to be related more nearly to the average Darwinian fitness of the populations.

Five alleles representing three electromorphs of phosphoglucose isomerase (PGI) have been transferred from natural isolates of E. coli into the genetic background of E. coli K12 and examined for their effect on growth rate in chemostats limited for glucose or fructose. With glucose limitation, all alleles are selectively neutral or nearly neutral within the limit of resolution of the technique, whether the genetic background is nonmutant or whether it contains a deletion of the locus of glucose-6-phosphate dehydrogenase, the enzyme that provides an alternative metabolic pathway for the substrate of PGI. With fructose limitation, one of the naturally occurring alleles has a small but reproducible detrimental effect on growth rate. A kinetic difference in this detrimental allozyme, apparently relating to an inhibition constant, has been observed in some, but not all, lots of substrate, and a similar difference has also been noted in one of the rare electromorphs that could not be transferred into E. coli K12. These results support a model of genetic variation in which the alleles are neutral or nearly neutral in the prevailing environment but have a potential for selection that can be expressed under the appropriate conditions of environment or genetic background. This hypothesis is discussed in the context of allozyme polymorphisms observed in other organisms.

Insertion sequence IS50R, which encodes the transposase and an inhibitor of transposition of the kanamycin-resistance transposon Tn5, increases the growth rate of E. coli K12 cells relative to that of their otherwise isogenic counterparts during competition in continuous culture. Most clones isolated from chemostats in which selection had occurred retain their original number of copies of IS50R at their original genomic locations, implying that the increased growth rate is not mediated by transposition. The selective advantage due to a single IS50R element averages about 5% per hour. When the number of copies of IS50R is small, the growth-rate advantage is approximately proportional to the number of copies of IS50R. These results imply that IS50R has effects on cells that are independent of both position and transposition and may be important in the initial selection leading to the appearance of such elements in bacterial populations.

A model of selection involving two selectively equivalent classes of alleles at a locus is considered. One class consists of normal alleles A1, A2, A3,. . .; the other class consists of detrimental alleles a1, a2, a3, . . . . Mutation within and between allelic classes can occur without restriction, but selection operates in such a way as to maintain an approximately constant overall frequency of A-type and a-type alleles is derived, and it is shown that the distribution of allele frequencies in a sample of detrimental alleles depends on the forward (A to a) mutation rate but not on the selection coefficient, degree of dominance, or mutation rate among a-type alleles. Recurrent mutation therefore generates allelic multiplicity among detrimental alleles, and this is discussed in the context of clinical heterogeneity in simple Mendelian disorders.

Males of Drosophila melanogaster that are heterozygous for the segregation distorter (SD) chromosome produce a gross excess of SD-bearing offspring because most of the non-SD-bearing sperm are dysfunctional. These dysfunctional sperm exhibit abnormalities in chromatin condensation and compaction during spermiogenesis. Use of the fluorescent dye sulfoflavine, which is specific for basic proteins, has now revealed that the dysfunctional sperm are also defective in the normal transition from somatic to spermatid-specific histones.

Strains of D. melanogaster, representing a range of genetic diversity, were systematically subjected to each of several techniques that have been devised to assess total or net fitness. All of these techniques operationally define fitness in terms of reproductive success under competitive conditions. For the set of strains tested, an excellent correlation was obtained between the competitive indices produced by two single-generation intraspecific competitions, despite fundamental differences in the strains used as competitive standards. The results of two interspecific tests were also correlated, but the intraspecific vs. interspecific test result comparisons ranged from highly significant to nonsignificant correlations. For the fitness estimates, the overall variances produced by the intraspecific tests were greater than those obtained in the interspecific competitions. No significant correlations were found between the results of a long-term (multi-generation) competitive assessment of fitness and any of the single-generation tests. Therefore, although all of these tests refer to a net parameter called "fitness", they are clearly not all measuring the same thing. Differences in the competitive interactions operative in each technique are suggested to account for these results.