Minisatellites are arrays of tandemly repeated DNA sequences which occur at thousands of locations in the human genome. They are frequently hypervariable with respect to allele length as a result of high rates of complex and incompletely understood recombination-based germline mutation events that alter the repeat copy number. MS1 is one of the most variable minisatellites so far isolated from the human genome. We have integrated MS1, flanked by synthetic markers, in the vicinity of a hot spot for meiotic double-strand breaks upstream of the LEU2 locus in chromosome III of Saccharomyces cerevisiae. Here we present the first tetrad analysis of mutations at a human minisatellite locus. The data showed that mutant alleles occur as single mutants in one of the spores in a tetrad, also when the mutant structure was the result of a combination of intra- and inter-allelic rearrangements. The conversional transfer of repeat units from one allele to the other was associated with flanking marker conversion which always involved the same flank of the minisatellite. The results demonstrate that conversion is the predominant mechanism by which minisatellite alleles mutate to new lengths, and also support the assumption that cis-acting elements are involved in the regulation of the mutational process in humans.

We have isolated a homologue of the period (per) gene from the Australian sheep blow fly, Lucilia cuprina, as part of a comparative approach to the analysis of dipteran circadian systems. Sequence analysis of the 4 kb per cDNA revealed the conservation of three functional domains, namely the PAS dimerization motif, and the nuclear and cytoplasmic localization domains. A fourth domain, the threonine–glycine (TG) repeat region, is also conserved in L. cuprina per but has been severely truncated. No length variation was found in the TG repeat of L. cuprina or L. sericata collected from several different latitudinal zones. Expression analysis indicated a diel oscillation in per mRNA in LD 12[ratio ]12 with a period of 24 h and a peak at Zt 12. PER-immunoreactive protein oscillations were also demonstrated, with peak immunoreactivity lagging approximately 3 h behind peak mRNA levels. These results show the existence of a Drosophila-like circadian system in a calliphorid fly. They also provide evidence for the conservation of per function across the Diptera, and confirm the relevance of the Drosophila system as a model for fly circadian rhythms.

The red imported fire ant Solenopsis invicta is the most destructive invading arthropod in the southern United States, yet little is known about its genome complexity and organization. Here we report the size, organization and GC content of S. invicta genome. DNA reassociation kinetics using S1 nuclease assay and a modified second-order kinetics model indicated that the S. invicta genome is approximately 0·62 picograms or 5·91 × 108 base pairs, composed of 36% unique, 41% moderately repetitive and 23% highly repetitive/foldback sequences. Comparison of the reassociation kinetics of short and long DNA fragments revealed that the sequence arrangement follows a pattern of short period interspersion, as in most organisms with relatively large genomes. Melting-temperature analysis showed that the GC content of the fire ant genomic DNA is 34·8%, similar to that of most eukaryotic organisms. The results reveal that the fire ant genome is much larger and more complex than those of a number of hymenopteran insects studied to date. Our study provides a foundation for further analysis and genetic manipulation of the S. invicta genome.

A lethal mutation in the Krüppel homolog (Kr h) was isolated in screens of P-element insertion mutations for modifiers of white gene expression. The mutation occurs in the 5′ untranslated region of the Kr h gene and causes a lightening of the eye colour for several alleles of white due to a decrease in white steady-state mRNA levels at pupal stages. Two related genes, scarlet and brown, were significantly affected as well in early pupae. Genetic analysis of different white alleles suggests that enhancer sequences are necessary for interaction with KR H. Thus, the Kr h gene is a member of the dosage-dependent hierarchy effective upon white.

cDNA sequence analysis of the X-linked glucose-6-phosphate dehydrogenase (G6PD) gene has shown a base difference between two subspecies of the kangaroo, Macropus robustus robustus (wallaroo) and M. r. erubescens (euro). A thymine residue in the wallaroo at position 358 in exon 5 has been replaced by a cytosine residue in the euro, which accounts for the previously reported electrophoretic difference between the two subspecies. This base difference allowed use of the Single Nucleotide Primer Extension (SNuPE) technique to study allele-specific expression of G6PD at the transcriptional level. We began by examining G6PD expression in somatic cells and observed complete paternal X inactivation in all somatic tissues of adult female heterozygotes, whereas we found partial paternal allele activity in cultured fibroblasts, thus confirming previous allozyme electrophoresis studies. In late dictyate oocytes from an adult heterozygote, the assay also detected expression of both the maternal and paternal alleles at the G6PD locus, with the maternal allele showing preferential expression. Thus reactivation of the inactive paternally derived X chromosome occurs during oogenesis in M. robustus, although the exact timing of reactivation remains to be determined.

Temporal surveys of hobo transposable elements in natural populations reveal a historical pattern suggesting a recent world-wide invasion of D. melanogaster by these transposons, perhaps following a recent horizontal transfer. To clarify the dynamics of hobo elements in natural populations, and thus to provide further data for our understanding of the hobo invasion, TPE tandem repeats, observed in the polymorphic S region of the element, were used as molecular markers. The number of TPE repeats was studied in 101 current populations from around the world, and in 63 strains collected in the past. This revealed a geographical distribution which seems to have been stable since the beginning of the 1960s. This distribution is compatible with a number of hypotheses for the dynamics of hobo elements. We propose a scenario based on an invasion in two stages: first, a complete invasion by elements with three TPE repeats, followed by the beginning of a new invasion involving hobo elements with five or seven repeats.

The rates of movement of 11 families of transposable elements of Drosophila melanogaster were studied by means of in situ hybridization of probes to polytene chromosomes of larvae from a long-term mutation accumulation experiment. Replicate mutation-accumulation lines carrying second chromosomes derived from a single common ancestral chromosome were maintained by backcrosses of single males heterozygous for a balancer chromosome and a wild-type chromosome, and were scored after 116 generations. Twenty-seven transpositions and 1 excision were detected using homozygous viable and fertile second chromosomes, for a total of 235056 potential sources of transposition events and a potential 252880 excision events. The overall transposition rate per element per generation was 1·15×10−4 and the excision rate was 3·95×10−6. The single excision (of a roo element) was due to recombination between the element's long terminal repeats. A survey of the five most active elements among nine homozygous lethal lines revealed no significant difference in the estimates of transposition and excision rates from those from viable lines. The excess of transposition over excision events is in agreement with the results of other in situ hybridization experiments, and supports the conclusion that replicative increase in transposable element copy number is opposed by selection. These conclusions are compared with those from other studies, and with the conclusions from population surveys of element frequencies.

We have isolated, characterized and mapped 33 dinucleotide, three trinucleotide and one tetranucleotide repeat loci from the four major chromosomes of Drosophila pseudoobscura. Average inferred repeat unit length of the dinucleotide repeats is 12 repeat units, similar to D. melanogaster. Assays of D. pseudoobscura and populations of its sibling species, D. persimilis, using 10 of these loci show extremely high levels of variation compared with similar studies of dinucleotide repeat variation in D. melanogaster populations. The high levels of variation are consistent with an average mutation rate of approximately 10−6 per locus per generation and an effective population size of D. pseudoobscura approximately four times larger than that of D. melanogaster. Consistent with allozymes and nucleotide sequence polymorphism, the dinucleotide repeat loci reveal minimal structure across four populations of D. pseudoobscura. Finally, our preliminary recombinational mapping of 24 of these microsatellites suggests that the total recombinational genome size may be larger than previously inferred using morphological mutant markers.

Recent experiments with Drosophila have demonstrated that the success of sperm in multiply mated females depends on the genotype of both the male and the female. To further characterize the distinction between male and female roles in sperm success, we scored variation in both sexes in sperm competitive ability among a set of chromosome replacement lines that allow identification of effects to each chromosome. We detected significant male and female effects on sperm precedence, defined as the ability of a male's ejaculate to displace resident sperm (P2) or avoid being displaced by subsequent matings (P1). Tests of effects of first, second and third chromosome substitutions revealed significant differences among third chromosomes in male sperm precedence (both P1 and P2) and a first × second chromosome interaction in female's effect on sperm precedence (only P1). We found no significant correlation between male and female effects on sperm precedence, suggesting that the variation found in both P1 and P2 has a different genetic cause in the two sexes.

Two questions were addressed: (1) What is the genetic variance–covariance structure of a suite of four female life history traits in D. melanogaster? and (2) Does the genetic architecture of these traits differ among populations? Three populations of D. melanogaster were studied. Genetic variances and covariances were estimated by sib analysis three times for each population: immediately upon establishment of populations in the laboratory, and subsequently after approximately 6 months and 2 years of laboratory culture. Entire genetic variance–covariance matrices, as well as their individual components, were compared between populations by means of likelihood ratio tests. All traits studied were significantly heritable in at least one-half of estimates. Despite large sample sizes, additive genetic covariances were for the most part not statistically significant, and only two significant negative covariance estimates were obtained throughout the experiments. Therefore, these experiments provide little support for evolutionary life history theories that are based on negative genetic correlations among life history components. Neither do they support the idea that genetic variance for fitness components is maintained by trade-offs. Evidence suggests that the G matrix of one population was initially different from those of the other two populations. Those differences disappeared after 2 years of laboratory culture. At the level of individual (co)variance components, there were relatively few differences among populations, and the overall impression was that the three populations had generally similar genetic architectures for the traits studied.

Polymerase chain reaction (PCR)-based genotyping of oocysts dissected from mosquito midguts has previously been used to investigate overall levels of inbreeding within malaria parasite populations. We present a re-analysis of the population structure of Plasmodium falciparum malaria using diploid genotypes at three antigen-encoding loci in 118 oocysts dissected from 34 mosquitoes. We use these data to ask whether mating is occurring at random within the mosquito midgut, as is generally assumed. We observe a highly significant deficit of heterozygous oocysts within mosquitoes at all three loci, suggesting that fusion of gametes occurs non-randomly in the mosquito gut. A variety of biological explanations, such as interrupted feeding of mosquitoes, positive assortative mating and outcrossing depression, could account for this observation. However, an alternative artefactual explanation – the presence of non-amplifying or null alleles – can account for the observed data equally well, without the need to invoke non-random mating. To evaluate this explanation further, we estimate the frequencies of null alleles within the oocyst population using maximum likelihood, by making the assumption that non-amplifying oocysts at any of the three loci are homozygous for null alleles. Observed levels of visible heterozygotes fit closely with those expected under random mating when non-amplifying oocysts are accounted for. Other lines of evidence also support the artefactual explanation. Overall inbreeding coefficients have been recalculated in the light of this analysis, and may be considerably lower than those estimated previously. In conclusion, we suggest that the deficit of heterozygotes observed is unlikely to indicate non-random mating within the mosquito gut and is better explained by misscoring of heterozygotes as homozygotes.

The males of six species of the Drosophila virilis group (including D. virilis) keep their wings extended while producing a train of sound pulses, where the pulses follow each other without any pause. The males of the remaining five species of the group produce only one sound pulse during each wing extension/vibration, which results in species-specific songs with long pauses (in D. littoralis about 300 ms) between successive sound pulses. Genetic analyses of the differences between the songs of D. virilis and D. littoralis showed that species-specific song traits are affected by genes on the X chromosome, and for the length of pause, also by genes on chromosomes 3 and 4. The X chromosomal genes having a major impact on pulse and pause length were tightly linked with white, apricot and notched marker genes located at the proximal third of the chromosome. A large inversion in D. littoralis, marked by notched, prevents more precise localization of these genes by classical crossing methods.

Spontaneous mutations were allowed to accumulate over 209 generations in more than 100 lines, all of them independently derived from a completely homozygous population of Drosophila melanogaster and subsequently maintained under strong inbreeding (equivalent to full-sib mating). Traits scored were: abdominal (AB) and sternopleural (ST) bristle number, wing length (WL) and egg-to-adult viability (V). On two occasions – early (generations 93–122) and late (generations 169–209) – ANOVA estimates of the mutational variance and the mutational line × generation interaction variance were obtained. Mutational heritabilities of morphological traits ranged from 2 × 10−4 to 2 × 10−3 and the mutational coefficient of variation of viability was 0·01. For AB, WL and V, temporal uniformity of the mutational variance was observed. However, a fluctuation of the mutational heritability of ST was detected and could be ascribed to random genotype × environment interaction.

Microsatellite loci have become important in population genetics because of their high level of polymorphism in natural populations, very frequent occurrence throughout the genome, and apparently high mutation rate. Observed repeat numbers (alleles size) in natural populations and expectations based on computer simulations suggest that the range of repeat numbers at a microsatellite locus is restricted. This range is a key parameter that should be properly estimated in order to proceed with calculations of divergence times in phylogenetic studies and to better investigate the within- and between-population variability. The ‘plug-in’ estimate of range based on the minimum and maximum value observed in a sample is not satisfactory because of the relatively large number of alleles in comparison with typical sample sizes. In this paper, a set of data from 30 dinucleotide microsatellite loci is analysed under the assumption of independence among loci. Bayesian inference on range for one locus is obtained by assuming that constraints on range values exist as sharp bounds. Closed-form calculations and robustness revealed by our analysis suggest that the proposed Bayesian approach might be routinely used by researchers to classify microsatellite loci according to the estimated value of their allelic range.

We present the theoretical background to a new method for measuring genetic variation for total fitness in Drosophila. The method allows heterozygous effects on total fitness of whole wild-type chromosomes to be measured under normal demography with overlapping generations. The wild-type chromosomes are competed against two balancer chromosomes (B1, B2, say), providing a standard genotype B1/B2 against which variation in the fitness effects of the wild-type chromosomes can be assessed. Fitness can be assessed in two ways: (i) at equilibrium of all three chromosomes under heterozygote advantage, and (ii) during displacement of one balancer by the other. Equilibrium with all three chromosomes present will be achieved only if the wild-type homozygote is not too fit, and if the fitnesses of the three heterozygotes are not too unequal. These conditions were not satisfied for any of a sample of 12 lethal-bearing chromosomes isolated from a random-bred laboratory population of Drosophila. At equilibrium, genotypic frequencies show low sensitivity to changes in genotypic fitness. Furthermore, where all four genotypes are viable and fertile, supplementary information from cages with only two chromosomes present and from direct measurements of pre-adult viability are required to estimate fitnesses from frequencies. The invasion method has the advantages of a greater sensitivity and of not requiring further data to estimate fitnesses if the wild-type homozygote is fertile. However, it requires that multiple samples be taken as the invasion progresses. In a discrete generation model, generation time influences fitness estimates from this method and is difficult to estimate accurately from the data. A full age-structured model can also be applied to the data from both types of experiment. For the invasion method, this gives fitness estimates close to those from the discrete generation model.

Within hybrid zones that are maintained by a balance between selection and dispersal, linkage disequilibrium is generated by the mixing of divergent populations. This linkage disequilibrium causes selection on each locus to act on all other loci, thereby steepening clines, and generating a barrier to gene flow. Diffusion models predict simple relations between the strength of linkage disequilibrium and the dispersal rate, σ, and between the barrier to gene flow, B, and the reduction in mean fitness, W¯. The aim of this paper is to test the accuracy of these predictions by comparison with an exact deterministic model of unlinked loci (r = 0·5). Disruptive selection acts on the proportion of alleles from the parental populations (p, q): W = exp[−S(4 pq)β], such that the least fit genotype has fitness e−S. Where β [Lt ] 1, fitness is reduced for a wide range of intermediate genotypes; where β [Gt ] 1, fitness is only reduced for those genotypes close to p = 0·5. Even with strong epistasis, linkage disequilibria are close to σ2p′ip′j/ rij, where p′i, p′j are the gradients in allele frequency at loci i, j. The barrier to gene flow, which is reflected in the steepening of neutral clines, is given by

formula here

where r¯, the harmonic mean recombination rate between the neural and selected loci, is here 0·5. This is a close approximation for weak selection, but underestimates B for strong selection. The barrier is stronger for small β, because hybrid fitness is then reduced over a wider range of p. The widths of the selected clines are harder to predict: though simple approximations are accurate for β = 1, they become inaccurate for extreme β because, then, fitness changes sharply with p. Estimates of gene number, made from neutral clines on the assumption that selection acts against heterozygotes, are accurate for weak selection when β = 1; however, for strong selection, gene number is overestimated. For β > 1, gene number is systematically overestimated and, conversely, when β < 1, it is underestimated.

A total of 2122 single sperm from 35 bulls belonging to six different paternal half-sib groups were analysed with respect to two markers in the bovine pseudoautosomal region (PAR) and sex- specific loci on the X and Y chromosomes, respectively. A segregation ratio significantly different from 1[ratio ]1 was observed in a test over all families, with a higher proportion of X-bearing gametes (53·5%). The analysis of recombination conducted separately for X- and Y-bearing sperm showed that X-bearing sperm cells possess highly significant individual and between-family variability in recombination rate, whereas Y-bearing sperm show linkage homogeneity. To test whether the two phenomena are related, different logistic regression models were fitted to the data. The results show that sex ratio significantly correlates with changes in recombination rate among X-bearing but not among Y-bearing sperm. Different hypotheses to explain these observations are discussed.

The present investigation deals with the genetic variability of milking speed, measured as the volume of milk collected during the first minute of milking (MD1), and its association with dairy traits. Data originated from 2589 lactations of 1421 Alpine goats, sired by 93 bucks, measured between 1985 and 1997 at the Moissac Goat Experimental Station (Lozère, France). Two genetic analyses were carried out. Firstly, a polygenic model was used to estimate genetic and phenotypic parameters among milking speed and dairy traits using a multiple-trait animal model. Secondly, segregation analysis was used to test the hypothesis of mixed model inheritance (polygenes+major gene) for MF1. Heritability and repeatability of MF1 under the polygenic model were high (0·65 and 0·82, respectively). Estimated genetic and phenotypic correlations between milking speed and dairy traits were low, positive for yields and negative for contents. Segregation analysis yielded a highly significant likelihood ratio, confirming the segregation of a major gene with two alleles with partial dominance. The difference between the mean values of the two homozygotes was around 2·3 phenotypic standard deviation units of the trait. The major gene explained more than 60% of the estimated total genetic variance. The estimate of the ‘residual’ heritability, after taking into account the effect of the major locus, was 0·30.

Correlated responses in female reproductive performance were evaluated following short-term selection within full-sib families for increased 8-week body weight in two replicates of four lines of mice: two ovine metallothionein–ovine growth hormone (oMt1a-oGH) transgene-carrier lines, one from a high-growth background (TM) and one from a control background (TC), and two non-transgenic lines, one from each of these genetic backgrounds (NM and NC, respectively). A fifth line (CC), not containing the transgene, served as a randomly selected control. The initial frequency of the oMt1a-oGH transgene construct in the TM and TC lines was 0·5. The frequency of transgenic females sampled at generations 7 and 8 of selection was 84·0% and 6·1% in the TC and TM lines, respectively. No significant female infertility differences were detected between transgene-carrier and non-transgenic lines or between transgenic and non-transgenic mice within carrier lines, whereas high-growth background lines had a higher infertility than control background lines (P < 0·05). Correlated responses in the TC transgene-carrier line were suggestive of reduced reproductive performance as indicated by increased post-implantation mortality (P < 0·05), number of dead fetuses plus implants (P < 0·05), and loss of fetuses from day 16 to parturition (P < 0·001). For the first two traits, the negative correlated responses were accounted for by the reduced performance of transgenic compared with non-transgenic females. Embryos carrying the transgene may also have a lower viability. In contrast, the NC non-transgenic line did not exhibit reduced reproductive performance for these traits. The low frequency of the transgene in the high-growth background TM line was associated with reduced fitness and a lower additive effect for 8-week body weight compared with the control background TC line.

Deterministic theory suggests that reciprocal recombination and intragenic, interallelic conversion have different effects on the linkage disequilibrium between a pair of genetic markers. Under a model of reciprocal recombination, the decay rate of linkage disequilibrium depends on the distance between the two markers, while under conversion the decay rate is independent of this distance, provided that conversion tracts are short. A population genetic three-locus model provides a function Q of two-locus linkage disequilibria. Viewed as a random variable, Q is the basis for a test of the relative impact of conversion and recombination. This test requires haplotype frequency data of a sufficiently variable three-locus system. One of the few examples currently available is data from the Human Leukocyte Antigen (HLA) class I genes of three Amerindian populations. We find that conversion may have played a dominant role in shaping haplotype patterns over short stretches of DNA, whereas reciprocal recombination may have played a greater role over longer stretches of DNA. However, in order to draw firm conclusions more independent data are necessary.