Chromosomal replication in Drosophila virilis

About half of the diploid genome of D. virilis is -heterochromatic (Heitz, 1934) and contains the satellite sequences found in isopycnic CsCl density gradients (Gall et al, 1971; Steinemann, 1976). The thymidine incorporation behavior of this material in the course of S phase was monitored by autoradiography. Labelled interphase nuclei show three types of labelling patterns, label exclusively confined to either eu- or -heterochromatin, and simultaneous labelling of both fractions. Using the fraction of labelled mitotic index method, the duration of the DNA-synthetic period, t_s = 11.9 ± 4.3 h and G₂ period, t_{G2 + 1/2M} = 6.9 ± 3.8 h, were determined. On the assumption that the investigated brain cells belong to an exponentially growing cell population, the cell cycle is 22.9 h long and the G₁ period lasts t_G1=4.1 h. The a-heterochromatin begins to replicate later than euchromatin and continues alone after a phase of common replication of both fractions. Noteworthy is the asynchronous termination in the proximal -heterochromatic segments of different chromosomes. Within the S phase, the first 1 h of DNA replication is exclusively confined to euchromatin, followed by 8 h of replication in both eu- and -heterochromatin and terminated by 3 h of exclusive -heterochromatin replication. Thus euchromatin has a doubling time of about 9 h and -heterochromatin of about 11 h. The -heterochromatin of D. virilis is late and slow replicating.     

Chromosomal replication in Drosophila virilis

DNA fiber autoradiography was used to determine parameters underlying the DNA replication of the eukaryotic chromosome in Drosophila diploid brain cells in organ culture. The average rate of fork movement, estimated from 4 different labelling intervals, is 0.35 μm/min at 25 ° C. Of the tandem arrays 93% show patterns which are compatible with bidirectional replication, 7% show unidirectional replication. The unidirectional mode of replication is interpreted as being a consequence of the experimental schedule (using hot-cold pulse labelling) combined with the occurrence of termination signals. — Some autoradiograms showed the expected two grain tracks of different densities; others showed only a high density track. The latter were most prominent in arrays of short replicons (<10 μm) which correlate with replicating satellite sequences. — The majority of replicons fall into size classes < 100 μm. The frequency distribution is skewed towards larger replicon sizes; it spans 2–238 μm, has a mean of ˉx = 35.6 μm and a median of = 21.0 μm. If the distribution is corrected for supposed satellite replicons, the median increases to = 31.0 μm. — In experiments using warmhot pulse labelling, arrays were scored which must have been a consequence of fixed termination signals. Furthermore, grain tracks diverging from weak labelled centers often have different lengths, indicating that these replicons contain two diverging replicating sections of unequal length. Presented to Professor Dr. Wolfgang Beermann on the occasion of his 60th birthday with my best wishes     

DNA sequence divergence in the Drosophila virilis group

DNA sequence divergence was analyzed in some sibling species of the Drosophila virilis group. Clones comprising about 0.1% of the genome DNA were selected at random from a D. virilis library for a comparative study on DNA from D. lummei, D. novamexicana, D. borealis, and D. lacicola. Blot hybridization experiments indicated that about 70% of DNA from D. lummei and D. novamexicana and less than 50% of DNA from D. borealis and D. lacicola share sequences that are homologous to DNA in D. virilis. This finding is in excellent agreement with the genealogical tree based on cytological studies (Throckmorton 1982). - Four plasmids with inserts which are present in one or a few copies per genome were hybridized in situ to polytene chromosomes. These experiments demonstrate that (1) homologous "unique" DNA sequences are localized exclusively in homologous bands and (2) homologous bands that appear to be identical in different species may contain different DNA sequences.     

Signals of demographic expansion in Drosophila virilis

Background

The antagonistic co-evolution of hosts and their parasites is considered to be a potential driving force in maintaining host genetic variation including sexual reproduction and recombination. The examination of this hypothesis calls for information about the genetic basis of host-parasite interactions – such as how many genes are involved, how big an effect these genes have and whether there is epistasis between loci. We here examine the genetic architecture of quantitative resistance in animal and plant hosts by concatenating published studies that have identified quantitative trait loci (QTL) for host resistance in animals and plants.

Results

Collectively, these studies show that host resistance is affected by few loci. We particularly show that additional epistatic interactions, especially between loci on different chromosomes, explain a majority of the effects. Furthermore, we find that when experiments are repeated using different host or parasite genotypes under otherwise identical conditions, the underlying genetic architecture of host resistance can vary dramatically – that is, involves different QTLs and epistatic interactions. QTLs and epistatic loci vary much less when host and parasite types remain the same but experiments are repeated in different environments.

Conclusion

This pattern of variability of the genetic architecture is predicted by strong interactions between genotypes and corroborates the prevalence of varying host-parasite combinations over varying environmental conditions. Moreover, epistasis is a major determinant of phenotypic variance for host resistance. Because epistasis seems to occur predominantly between, rather than within, chromosomes, segregation and chromosome number rather than recombination via cross-over should be the major elements affecting adaptive change in host resistance.     

Satellite DNA-correlated nucleosomal proteins in Drosophila virilis

Three major satellite DNAs comprise 40–45% of the genome of Drosophila virilis. Since these satellites are not substrates for most restriction enzymes, we were able to digest D. virilis nuclei with HaeIII and micrococcal nuclease and isolate chromatin fractions containing variable levels of satellite DNA. Electrophoretic analysis of these chromatin fractions revealed that the level of the acid-soluble chromosomal protein, cp17.3, was directly related to the percentage of satellite DNA in chromatin. The correlation between cp17.3 and satellite DNA abundance suggests that cp17.3 is involved in the heterochromatic condensation of satellite DNAs. cp17.3 occurs at a frequency of one molecule per 10–20 nucleosomes. It is detected in an electrophoretically distinguishable class of mononucleosomes, provisionally identified as MN1uH2A, which contains ubiquitinated histone H2A (uH2a) but lacks histone H1. It is not detected in MN1, a second class of mononucleosomes, which lacks uH2A and H1. Since cp17.3 is correlated with satellite DNAs and present in nucleosome cores, it might be a histone variant specifically associated with satellite DNAs.This work was supported by Grant GM22138 from the National Institutes of Health. G.A.V. was a predoctoral trainee supported by Grant GM07094 from the National Institutes of Health.     

A multilocus microsatellite phylogeny of the Drosophila virilis group

We used a set of 48 polymorphic microsatellites derived from Drosophila virilis to infer phylogenetic relationships in the D. virilis clade. Consistent with previous studies, D. virilis and D. lummei were the most basal species of the group. Within the D. montana phylad, the phylogenetic relationship could not be resolved. Special attention was given to the differentiation between D. americana texana, D. americana americana and D. novamexicana. Significant differences between these three groups were detected by F(ST) analyses. Similarly, a model-based clustering method for multilocus genotype data also provided strong support for the presence of three differentiated groups. This genome-wide differentiation between D. americana texana and D. americana americana contrasts with previous analyses based on DNA sequence data.     

Invasion of Drosophila virilis by the Penelope transposable element

The Penelope family of transposable elements (TEs) is broadly distributed in most species of the virilis species group of Drosophila. This element plays a pivotal role in hybrid dysgenesis in Drosophila virilis, in which at least four additional TE families are also activated. Here we present evidence that the Penelope family of elements has recently invaded D. virilis. This evidence includes: (1) a patchy geographical distribution, (2) genomic locations mainly restricted to euchromatic chromosome arms in various geographical strains, and (3) a high level of nucleotide similarity among members of the family. Two samples from a Tashkent (Middle Asia) population of D. virilis provide further support for the invasion hypothesis. The 1968 Tashkent strain is free of Penelope sequences, but all individuals collected from a 1997 population carry at least five Penelope copies. Furthermore, a second TE, Ulysses, has amplified and spread in this population. These results provide evidence for the Penelope invasion of a D. virilis natural population and the mobilization of unrelated resident transposons following the invasion.     

DNA-protein interactions in the Drosophila virilis mitochondrial chromosome.

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila virilis was investigated by Me3 psoralen photoreaction of mitochondria isolated from embryos. After photoreaction the mtDNA was purified and the pattern of DNA cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The transcribed regions of the mtDNA molecule contained some uncross-linked regions, but such regions were infrequent and randomly distributed. In contrast, the A + T-rich region around the origin of replication of the mtDNA was usually protected from psoralen cross-linking. The data were best fit by two protected sites, each approximately 400 base pairs, compared to the four 400 base pair sites observed in the equivalent region of D. melanogaster mtDNA [Potter et al. (1980) Proc. Nat. Acad. Sci. USA 77, 4118-4122]. Thus this region of the mtDNA appears to be involved in a DNA-protein structure that is highly conserved even though the DNA sequence has diverged rapidly relative to protein-coding sequences.     

The satellite bands of the DNA of Drosophila virilis

Purified DNA has been prepared from Drosophila virilis using a modification of the method derived for bacteria (Marmur, 1961). Some physical properties have been examined, a new hidden satellite discovered, and a difference found in the satellite banding pattern of different tissues. — In addition to the three satellite bands lighter than the main band previously reported (Gall et al., 1970), a new satellite heavier than the main band has been detected after thermal denaturation of the DNA (which substantially shifts the buoyant density of the main band but not that of the satellites indicating that all are fast-annealing). The satellite pattern of DNA extracted from heads alone differed from that of the entire animals: the amount of satellite I was decreased and II increased; III was unaffected; IV was increased relative to the amount in the main band. The total content of satellite material in the heads (assumed to be entirely diploid) was 42%, the highest amount reported for any organism. — Thermal transitions were determined for the DNA from adults and larvae. After preparative CsCl density gradient fractionation of adult DNA, two sets of bimodal thermal curves were obtained (in SSC) with agreement between the initial position in the preparative gradient, the thermal transitions, and the G+C content from density except for satellite III for which the T_m gave a more accurate G+C amount. DNA from satellites I and II together generated a T_m of 81.2° which was similar to a calculated T_m of 81.9° making the naive assumption that the thermal components of the two satellites would interact in a simple additive fashion. A T_m of 71.9° was ascribed to satellite III which indicates that it is not the equivalent of the poly (A-T) band found at the same density in D. melanogaster (Fansler et al., 1970). The calculated overall base composition from the density equivalents (using the value for satellite III from thermal data) gave an expected G+C content of 36.6%. The measured value was 36.0%. The possible significance of the differential satellite pattern has been discussed.     

Ovarian defects in hybrids of the species pair Drosophila virilis and Drosophila texana

In interspecific matings between Drosophila virilis and Drosophila texana female sterility is observed in F₂ hybrid females. A previous study has shown that no vitellogenin synthesis occurs in the fat body of sterile hybrid females. The results presented in this paper show that hybrid ovaries of sterile females transplanted into the abdomens of females of the parental species are not able to develop upon maturity. With few exceptions, the hybrid ovaries remained alive in the host environment, but their oocytes failed to develop to vitellogenic stages. Thus, in hybrid females between Drosophila virilis and Drosophila texana sterility is the result of defects in both the two main developmental processes of egg maturation, the synthesis of vitellogenins in the fat body and the uptake of vitellogenins by the ovary. Dev Genet 20:47–52, 1997. © 1997 Wiley-Liss, Inc.     

Vitellogenetic defects in hybrids of the species pair Drosophila virilis and Drosophila texana

In interspecific matings between the species Drosophila virilis and Drosophila texana, female sterility can be observed in F₂ backcross females and in F₂ hybrid females. The results presented in this report show that the female sterility, whenever it exists, is due to prevention of vitellogenin synthesis in the fat body, but other abnormalities such as defects with the hybrid ovaries are not excluded. The observation that sterility appears among females from backcrosses suggests that incompatibilities between interspecific genes may cause female sterility even in the presence of a complete habloid genome from one or the other species. Yet, the parallel observation that female sterility appears only in hybrid females with recombinant chromosomes indicates that sterility results when conspecific combinations of genes on the same chromosome are broken by interspecific recombination. © 1996 Wiley-Liss, Inc.     

Purification and enzyme stability of alcohol dehydrogenase from Drosophila simulans, Drosophila virilis and Drosophila melanogaster adhS.

Three alcohol dehydrogenases from Drosophila simulans, Drosophila virillis and Drosophila melanogaster adhS (which possesses an alloenzyme with slow electrophoretic mobility) were purified essentially to homogeneity. The purification procedure involves a new step of affinity chromatography, which efficiently lowers the amount of contaminants in the final preparation, producing a very stable enzyme. The purification procedure developed consists of a salmine sulphate precipitation, two CM-Sepharose CL-6B colume-chromatography steps, an affinity-chromatography step and a Sephacryl gel filtration. A minimum of 30-fold purification is obtained and the yield is not less than 34%. The isoelectric points and molar absorption coefficients were determined.     

Cytological and linkage maps of Drosophila virilis chromosomes

Cytological (photographic) maps of third-instar larvae Drosophila virilis salivary gland chromosomes were constructed; genetic maps of the chromosomes are also given together with the list of mutations known for this species.     

Drosophila virilis has long and highly polymorphic microsatellites

Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40-49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.     

RNA synthesis in embryogenesis of Drosophila virilis]

The synthesis of different RNA fractions labelled by 3H-uridine has been studied at different developmental stages in Drosophila virilis by means of thermal phenol extraction and chromatography on oligo-alphaT-cellulose. There are two periods of intensive synthesis of poly-A containing (messenger) RNA in the course of embryogenesis. The former (6--12 hrs) is the period of gastrulation and stomodeum development and the latter (15--21 hrs) is the period of intensive differentiation of larval organs. The synthesis of poly-A free RNAs predominates at the stages of blastoderm (2--5 hrs) and mesoderm segmentation and embryo shortening (12--15 hrs).     

X chromosome DNA variation in Drosophila virilis.

Comparisons of polymorphism patterns between distantly related species are essential in order to determine their generality. However, most work on the genus Drosophila has been done only with species of the subgenus Sophophora. In the present work, we have sequenced one intron and surrounding coding sequences of 6 X-linked genes (chorion protein s36, elav, fused, runt, suppressor of sable and zeste) from 21 strains of wild-type Drosophila virilis (subgenus Drosophila). From these data, we have estimated the average level of DNA polymorphism, inferred the effective population size and population structure of this species, and compared the results with those obtained for other Drosophila species. There is no reduction in variation at two loci close to the centromeric heterochromatin, in contrast to Drosophila melanogaster.