Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Characterization of the long terminal repeats of micropia elements microdissected from the Y-chromosomal lampbrush loops "threads" of Drosophila hydei

Four micropia elements from Drosophila melanogaster and D. hydei have been analysed by sequencing. Two elements, from D. hydei, micropia-DhMiF8 and -DhMiF2, were recovered by cloning microdissected Y-chromosomal lampbrush loops "threads". This method allows isolation of repetitive sequences from defined chromosomal positions, but recovery of large and overlapping inserts is difficult. In case of the Y-chromosomal micropia elements it was not possible to define the endpoints of their long terminal repeat sequences precisely. Comparison of these locus-defined micropia elements to complete micropia elements isolated from D. melanogaster allowed identification of micropia-DhMiF8 and micropia-DhMiF2 long terminal repeats (LTRs). LTR sequences from the two Drosophila species are not conserved except for a few short sequences found at comparable positions that are believed to have functional significance. In contrast, the Leu-tRNA primer binding site and plus strand primer binding site are conserved between D. melanogaster and D. hydei.     

Structural organization of the "zipper line" in Drosophila species with giant spermatozoa

The "zipper line" of Drosophila melanogaster and of Drosophila species characterized by giant spermatozoa (D. hydei, D. kanekoi and D. bifurca) was studied by electron microscopy using conventional thin-sections, lectin labeling and freeze-fracture replicas. In cross sections the membrane specializations are located either at the level of the short cistern close to the large mitochondrial derivative where a small tuft of glycocalyx is visible or, in species characterized by long spermatozoa, along a cistern beneath the plasma membrane. In correspondence of such cistern, the plasma membrane exhibits a thick and extended glycocalyx. At this level, as well as at the short tuft of D. melanogaster, alpha-mannose residues were detected. The "zipper" of D. melanogaster consists of rows of intramembrane particles longitudinally disposed along the sperm tail and associated with the external face of the plasma membrane. On the protoplasmatic face a narrow ribbon of transversal grooves is visible. Freeze-fracture replicas have revealed, in the region characterized by extended glycocalyx, the presence of a large ribbon of intramembrane particles disposed in parallel transversal rows, associated with the protoplasmatic membrane face. On the complementary external face a ribbon of parallel transversal grooves was observed. It is suggested that membrane specializations are mechanical devices to protect spermatozoa from torsion and bending in the seminal vesicles and then in the female storage organ.     

Natural selection and ribosomal DNA in Drosophila

Natural populations of Drosophila mercatorum are variable for the number of X-linked 28S ribosomal genes bearing a 5-kilobase insert. A separate polymorphic X-linked gene controls whether 28S repeats bearing the insert are preferentially underreplicated during the formation of polytene tissue. Female flies having at least a third of their 28S genes bearing the insert and lacking the ability to preferentially underreplicate inserted repeats display the abnormal abdomen syndrome. The syndrome is characterized by retention of juvenile abdominal cuticle into the adult, a slowdown in larval developmental time, and an increase in early female fecundity. The life history traits are expressed in nature and provide a basis for strong natural selection. The abnormal abdomen syndrome should be favored whenever the adult age structure is skewed towards young individuals, and field studies confirm this prediction. The closely related species, Drosophila hydei, also bears these inserts and appears to be subject to similar selection. However, D. mercatorum responds to this selection primarily through the allelic variation that controls preferential underreplication, whereas D. hydei responds primarily through adjustment of the proportion of inserted 28S genes. This is interpreted to mean that the evolution of a multigene family arises from the interaction of population-level and DNA-level processes.     

Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.     

Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila.

An evolutionary approach was applied to identify elements involved in the regulation of the segmentation gene fushi tarazu (ftz) by comparing the Drosophila melanogaster ftz gene with its Drosophila hydei homologue. The overall organization of the ftz gene is very similar in both species. Surprisingly, ftz proved to be inverted in the ANT-C of D. hydei with respect to D. melanogaster. Strong homologies extend over the entire 6 kb of the ftz upstream region with the best match in the 'upstream element'. We identified several highly conserved boxes embedded in unrelated sequences that correspond extremely well to two germ layer specific enhancers in the upstream element. Transformation experiments revealed that D. hydei ftz gene products can restore D. melanogaster ftz function and, furthermore, that trans-acting factors from D. melanogaster recognize and control D. hydei ftz regulatory elements. These findings indicate a conservation of the entire regulatory network among segmentation genes for several millions of years during the evolution of Drosophila.     

Intragenic recombination at the white locus of Drosophila hydei.

A fine-structure analysis of the white locus in Drosophila hydei was carried out by means of allele recombination. Four mutants, derived from wild type, mapped at three subloci. These are possibly homologous to the main subloci 2, 3, and 4 of D. melanogaster. Three secondary mutants, derived from the primary wiv allele, were located in the proximal part of the gene. One of them appeared as a homoallele of the original wiv, whereas the remaining two are better explained either as double mutants or as mutants which facilitate irregular exchange. Intragenic recombination at the white locus seems to be more frequent in D. hydei than in D. melanogaster. The comparatively high incidence is probably a general characteristic, common to intragenic and intergenic recombination in D. hydei.     

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.     

Adaptive evolution of Cid, a centromere-specific histone in Drosophila

Centromeric DNA is generally composed of large blocks of tandem satellite repeats that change rapidly due to loss of old arrays and expansion of new repeat classes. This extreme heterogeneity of centromeric DNA is difficult to reconcile with the conservation of the eukaryotic chromosome segregation machinery. Histone H3-like proteins, including Cid in Drosophila melanogaster, are a unique chromatin component of centromeres. In comparisons between closely related species of Drosophila, we find an excess of replacement changes that have been fixed since the separation of D. melanogaster and D. simulans, suggesting adaptive evolution. The last adaptive changes appear to have occurred recently, as evident from a reduction in polymorphism in the melanogaster lineage. Adaptive evolution has occurred both in the long N-terminal tail as well as in the histone fold of Cid. In the histone fold, the replacement changes have occurred in the region proposed to mediate binding to DNA. We propose that this rapid evolution of Cid is driven by a response to the changing satellite repeats at centromeres. Thus, centromeric H3-like proteins may act as adaptors between evolutionarily labile centromeric DNA and the conserved kinetochore machinery.     

Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled- coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.     

A novel GC-rich dispersed repeat sequence in Drosophila melanogaster.

A novel family of dispersed repeat sequences from Drosophila melanogaster is described. Sequence analysis of two members of this family show them to contain greater than 75% GC bases. These are comprised of multiple repeats of GGX triplets interspersed occasionally with CGPy and TTPy. Southern blotting shows that these repeats are not transposable elements. Twenty four homologous recombinants have been localised by in situ hybridization to seven sites in the Drosophila genome. Polyadenylated RNAs homologous to this repeat family are expressed in a complex pattern which is developmentally regulated. We suggest that this family encodes a set of glycine-rich domains in Drosophila proteins.