Molecular evolution of two paralogous tandemly repeated heterochromatic gene clusters linked to the X and Y chromosomes of Drosophila melanogaster

Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene betaCK2tes encoding the putative testis-specific beta-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3' splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5-10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions.     

Inactivation of reporter genes by cloned heterochromatic repeats of Drosophila melanogaster is accompanied by chromatin compaction

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter gene mini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of the mini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-white have been found.     

Structural organization and diversification of Y-linked sequences comprising Su(Ste) genes in Drosophila melanogaster.

Expression of the X-linked repeated Stellate (Ste) genes, which code for a protein with 38% similarity to the beta-subunit of casein kinase II, is suppressed by the Su(Ste) locus on the Y chromosome. The structure and evolution of the Y-linked repeats in the region of the Su(Ste) locus were studied. The 2800 bp repeats consist of three main elements: the region of homology to the Ste genes, an adjacent AT-rich, Y-specific segment, and mobile element 1360 inserted in the Ste sequence. Amplification of repeats was followed by point mutations, deletions, and insertions of mobile elements. DNA sequencing shows that these repeats may be considered as Ste pseudogenes or as damaged variants of a putative gene(s) encoding a protein quite different from the Ste protein as a result of an alternative splicing pattern. A comparison of 5 variants of the Y-Su(Ste) repeats shows a number of recombination events between amplified and diverged sequences that could be due to either multiple unequal mitotic sister-chromatid exchanges or to gene conversion. It is a first demonstration on a molecular level of these processes occurring in heterochromatic non-rDNA tandemly organized sequences in an eukaryotic genome.     

Heterochromatic DNA repeats in Drosophila and unusual gene silencing in yeast cells

The 1.25-kb heterochromatic Stellate repeats of Drosophila melanogaster are capable of stably persisting in transgenic constructs and silencing the white reporter gene (mosaic position effect variegation). This system reveals an unusual form of silencing, which is insensitive to known modifiers of position effect variegation. The unusual form of silencing was studied with yeast Saccharomyces cerevisiae, a simple eukaryotic model. To be transferred into yeast cells, the D. melanogaster Stellate repeats were cloned in the pYAC4 centromeric vector (CEN4, URA3, TRP1, HIS3). The HIS3 and/or URA3 genes could be inactive in plasmids consisting of pYAC4 and the Stellate insert in yeast cells. Deletion of D. melanogaster DNA from the plasmid was found to activate the URA3 and HIS3 genes. It was assumed that the genes were repressed rather than damaged in the presence of the Stellate repeats and that a new form of gene silencing was revealed in S. cerevisiae.     

Copies of a Stellate Gene Variant Are Located in the X Heterochromatin of Drosophila Melanogaster and Are Probably Expressed

Two variants of X chromosome Stellate genes responsible for crystal formation in XO male primary spermatocytes occupy different genome positions. The majority if not all of the 1250-bp Stellate genes are located at the 12E site where the Ste locus has been mapped and almost all of the 1150-bp Stellate repeats are concentrated in the distal X heterochromatin. Sequencing of Stellate genes derived from X heterochromatin reveals the preservation of their open reading frames and precise matching with some Stellate cDNAs reported earlier. At least some heterochromatic Stellate genes are suggested to be expressed and, therefore, involved in the interaction with the Y chromosome locus Su(Ste), as are the Stellate genes from 12E.     

Mutation of Drosophila melanogaster glass-like suppress expression of mini-white transgenes and dependence on their genomic environment

Pleiotropic recessive mutation glass-like (gl-l) found in region 8C-10E of the X chromosome was shown to cause glass-like eyes having no boundaries between facets and a nonuniform pigment distribution in the presence of the endogenous white gene. The gl-l mutation completely inhibited expression of the mini-white transgene contained in several constructs, but the effect depended on the site of construct integration in the genome. The mutation had no effect on the expression of the white transgene having the enhancer and flanked by insulators. The gl-l mutation did not affect the extent of mosaic eye pigmentation when a construct with mini-white was inserted in the telomeric or pericentric region. However, in most cases it completely inhibited the mosaic mini-white expression when cloned heterochromatic repeats were adjacent to the reporter gene in a construct. The gl-l gene was assumed to play a role in the formation of the chromatin structure, because the effect of its mutation on expression of the white transgene depended on the transgene insertion site, the presence of insulators or an enhancer in the vicinity of the transgene, and on the adjacent heterochromatic repeats.     

The study of interaction between paralogous tandem repeats stellate and suppressor of stellate in the genome of Drosophila melanogaster

Testis-specific expression of tandemly repeated Stellate genes, located in eu- and heterochromatin regions of the X chromosome of Drosophila melanogaster, is suppressed by homologous Suppressor of Stellate repeats located on the Y chromosome. Using transgenic lines, we have demonstrated that three Su(Ste) copies failed to change the expression of the reporter construction carrying the bacterial beta-galactosidase gene under control of the Stellate gene regulatory sequence. Possible mechanisms of the Su(Ste) repeat suppressor activity are discussed.     

Structure of the Y Chromosomal Su(ste) Locus in Drosophila Melanogaster and Evidence for Localized Recombination among Repeats

The structure of the Suppressor of Stellate [Su(Ste)] locus on the Drosophila melanogaster Y chromosome was examined by restriction analysis of both native and cloned genomic DNA. The locus consists of short subarrays of tandem repeats separated by members of other moderately repeated families. Both size variants and restriction variants proved to be common. Most repeats fell into two size classes--2.8 and 2.5 kb--but other size variants were also observed. Restriction variants showed a strong tendency to cluster, both at the gross level where some variants were present in only one of three subintervals of the locus, and at the fine level, where repeats from the same phage clone were significantly more similar than repeats from different clones. Restriction variants were shared freely among repeats of different size classes; however, size variants appeared to be randomly distributed among phage clones. These data indicate that recombination among tandem Su(Ste) repeats occurs at much higher frequencies between close neighbors than distant ones. In addition, they suggest that gene conversion rather than sister chromatid exchange may be the primary recombinational mechanism for spreading variation among repeats at the Su(Ste) locus.     

Heterochromatic Stellate Gene Cluster in Drosophila Melanogaster: Structure and Molecular Evolution

The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 β-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (~14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.