Polymorphism and stability in the histone gene cluster of Drosophila melanogaster

Histone genes in Drosophila melanogaster are organized into repeats of 4.8 and 5.0 kb (Lifton et al., 1978). We find these repeat sizes in every one of the more than 20 Drosophila strains we have examined. Strains differ in the relative amounts of the two repeat types, with ratios varying from 11 to 14, the 5.0 kb repeat always present in equal or greater amounts than the 4.8 kb repeat. Restriction enzyme digestion and blotting analysis reveals that the strains also differ in a number of far less abundant fragments containing histone DNA sequences. In the Amherst and Samarkand strains, there are, in addition, many copies of 4.0 and 5.5 kb repeat-like fragments respectively. A series of stocks were made isogenic for single second chromosomes from the Amherst strain. The hybridization patterns of the histone DNA from these stocks containing different Amherst chromosomes are very similar but a number of differences in the minor fragments were seen. The stability of the histone locus restriction pattern was tested by following the DNA derived from a single second chromosome of the b Adhn² pr cn strain over a two year period. The restriction pattern of major and minor bands remained identical. Finally, histone loci distinguishable by their restriction pattern on blots were recombined with visible markers. These chromosomes will be useful in tracing the fate of specific histone loci during genetic manipulations.     

The evolution of the novel Sdic gene cluster in Drosophila melanogaster

The origin of new genes and of new functions for existing genes are fundamental processes in molecular evolution. Sdic is a newly evolved gene that arose recently in the D. melanogaster lineage. The gene encodes a novel sperm motility protein. It is a chimeric gene formed by duplication of two other genes followed by multiple deletions and other sequence rearrangements. The Sdic gene exists in several copies in the X chromosome, and is presumed to have undergone several duplications to form a tandemly arrayed gene cluster. Given the very recent origin of the gene and the gene cluster, the analysis of the composition of this gene cluster represents an excellent opportunity to study the origin and evolution of new gene functions and the fate of gene duplications. We have analyzed the nucleotide sequence of this region and reconstructed the evolutionary history of this gene cluster. We found that the cluster is composed by four tandem copies of Sdic; these duplicates are very similar but can be distinguished by the unique pattern of insertions, deletions, and point mutations in each copy. The oldest gene copy in the array has a 3' exon that has undergone accelerated diversification, and also shows divergent regulatory sequences. Moreover, there is evidence that this might be the only gene copy in the tandem array that is transcribed at a significant level, expressing a novel sperm-specific protein. There is also a retrotransposon located at the 3' end of each Sdic gene copy. We argue that this gene cluster was formed in the last two million years by at least three tandem duplications and one retrotransposition event.     

Entropy and GC Content in the beta-esterase gene cluster of the Drosophila melanogaster subgroup

We perform spectral entropy and GC content analyses in the beta-esterase gene cluster, including the Est-6 gene and the psiEst-6 putative pseudogene, in seven species of the Drosophila melanogaster species subgroup. psiEst-6 combines features of functional and nonfunctional genes. The spectral entropies show distinctly lower structural ordering for psiEst-6 than for Est-6 in all species studied. Our observations agree with previous results for D. melanogaster and provide additional support to our hypothesis that after the duplication event Est-6 retained the esterase-coding function and its role during copulation, while psiEst-6 lost that function but now operates in conjunction with Est-6 as an intergene. Entropy accumulation is not a completely random process for either gene. Structural entropy is nucleotide dependent. The relative normalized deviations for structural entropy are higher for G than for C nucleotides. The entropy values are similar for Est-6 and psiEst-6 in the case of A and T but are lower for Est-6 in the case of G and C. The GC content in synonymous positions is uniformly higher in Est-6 than in psiEst-6, which agrees with the reduced GC content generally observed in pseudogenes and nonfunctional sequences. The observed differences in entropy and GC content reflect an evolutionary shift associated with the process of pseudogenization and subsequent functional divergence of psiEst-6 and Est-6 after the duplication event.     

Sequence and heterogeneity in the 5S RNA gene cluster of Drosophila melanogaster.

The sequence of the entire 5S RNA gene of Drosophila melanogaster was determined by sequencing collectively 23 copies contained in a cloned fragment of Drosophila DNA and by sequencing individually four subcloned gene copies. A repetitive heptamer (GCTG CCT) present in variable numbers immediately following the coding sequence, is responsible for the length heterogeneity in the spacer region. Some of the gene copies contain a nucleotide change in the coding region which results in a new site for the restriction enzyme Mn1 I. The variant 5S RNA produced by these gene copies has not been detected in vivo. Two other single nucleotide variations were identified in the spacer region.     

Detection of a novel DNA polymorphism in the beta-globin gene cluster

Analysis of DNA from the beta-globin gene cluster in an Albanian family identified a novel RsaI site approximately 550 base pairs 5' to the beta-globin gene. In this family, two chromosomes carrying otherwise identical beta-globin haplotypes were found to differ at the RsaI site. Population screening demonstrated the presence and absence of the site in DNA from individuals of northern European, Mediterranean, Middle Eastern, Southeast Asian, African, and Asian Indian descent, indicating that this site is a DNA polymorphism common in many ethnic groups. The polymorphism is also present in DNA from individuals carrying different beta-globin alleles. Additional nucleotide sequence changes identified in an RsaI (+) genomic clone in the region immediately 3' to the RsaI site suggest a mechanism for the randomization of the site with respect to haplotype.     

A gene responsible for a cuticular hydrocarbon polymorphism in Drosophila melanogaster.

Drosophila melanogaster is polymorphic for the major cuticular hydrocarbon of females. In most populations this hydrocarbon is 7,11-heptacosadiene, but females from Africa and the Caribbean usually possess low levels of 7,11-heptacosadiene and high quantities of its position isomer 5,9-heptacosadiene. Genetic analysis shows that the difference between these two morphs is due to variation at a single segregating factor located on the right arm of chromosome 3 near map position 51.5 and cytological position 87C-D. This is precisely the position of a desaturase gene previously sequenced using primers derived from yeast and mouse, and localized by in situ hybridization to the polytene chromosomes of D. melanogaster. Alleles of this desaturase gene may therefore be responsible for producing the two hydrocarbon morphs. Mating tests following the transfer of these isomers between females of the two morphs show that, in contrast to previous studies, the hydrocarbon profiles have no detectable effect on mating behaviour or sexual isolation.     

Enzyme polymorphism in Drosophila melanogaster populations in Iraq

Electrophoretic studies of the degree and pattern of polymorphism at two third-chromosome loci, esterase-6 (Est-6) and phosphoglucomutase (PGM), were carried out in three Drosophila melanogaster populations collected from different localities in Iraq: Mosul, Tuwaitha, and Basrah. The results show that only the Tuwaitha population was polymorphic for both loci; the other two populations were polymorphic for Est-6 and monomorphic for PGM. The allele frequency changes at both loci were followed for 20 generations in an experimental cage derived from the Tuwaitha population; it was found that there is a deviation from Hardy-Weinberg equilibrium at both loci toward the homozygote.     

Small circular DNA in Drosophila melanogaster

Covalently closed small circular DNA isolated from Drosophila melanogaster is described. The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2. It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2 cells ranges from 0.09-7.3 μm, with a mean contour length of 1.1 μm. This DNA has a buoyant density of 1.703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells. The restriction enzyme EcoRI cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes.Both logarithmic and stationary phase cells contain approximately 3–40 average sized small circular DNA molecules per cell, representing a maximum of 0.03% of the total cellular DNA. Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0.3 μm. The drug-amplified DNA has a buoyant density in the range of 1.698-1.703 g/cc. No amplification was seen in cells treated with either inhibitor for 3.5 hr. Ethidium bromide, cytosine arabinoside, β-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA.     

Manifestations of the eyeless-dominant gene in Drosophila melanogaster

A phenogenetic study was carried out of the eyD-mutation in Drosophila melanogaster which reduces the eye and quantitatively affects different regions of the head capsule originating from the eye imaginal disc. It was shown that the change in eye size is connected causally with changes in certain regions of head capsule: increase in chaetae number or duplication in different regions of the head capsule may be due to the degeneration of presumptive ommatidia. It is supposed that the variable response of chaetae number to the decrease in eye size depends on the origin of chaetae and their topography at different stages of the disc development.     

DNA sequence variation at a duplicated gene: excess of replacement polymorphism and extensive haplotype structure in the Drosophila melanogaster bicoid region

The bicoid (bcd) gene of Drosophila has played an important role in understanding the system of developmental regulatory genes that controls segmentation in the fruit fly. Several studies in Drosophila and closely related insects suggest that bcd may be the result of a gene duplication in the Dipteran lineage. In addition, the presence of a large, conserved secondary structure in the 3' untranslated region (UTR) makes the bcd gene a good candidate for studying compensatory evolution and the relationship between RNA secondary structure and patterns of standing variation in natural populations. Despite these interesting aspects, a population-level analysis has until now not been performed on bcd. In this study, DNA sequence variation was examined for a 4-kb region of the bcd gene, including a portion of the 5' UTR, the entire coding region, and the 3' UTR, for 25 Drosophila melanogaster isofemale lines from Zimbabwe and one allele from D. simulans. Statistical tests revealed a significant excess of replacement polymorphisms in the D. melanogaster lineage that are clustered in two putative linker regions of the Bicoid protein. This result is consistent with a relaxation of selective constraints in these regions. In addition, we found a distinct haplotype structure and a significantly smaller number of haplotypes than predicted by the standard neutral model. It is unlikely that the haplotype structure is maintained by epistatic selection acting on the secondary structure in the 3' UTR or by the association of the bcd gene with polymorphic inversions. Instead, our two main observations, namely the occurrence of a haplotype structure and the excess of replacement polymorphisms, may indicate that the selective history of this gene is rather complex, involving both the relaxation of purifying selection in some parts of the protein and the action of positive selection in other parts of the gene region.