Institute of Molecular Genetics, Moscow, Russia
Abstract
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 ßCK2tes encoding the putative testis-specific ß-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 510 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.
Introduction
The processes of the origins of multigene families in eukaryotic genome, as well as mechanisms supporting identity of divergence of their members, has been a topic of numerous discussions (Liao 1999
). Special attention might be given to the homologous (paralogous) repeats which have been shown to be functionally related. An unusual and puzzling example of such repeats is represented by paralogous Stellate and Su(Ste) tandem repeats localized on the X and Y chromosomes, respectively, in the Drosophila melanogaster genome (Hardy et al. 1984
; Livak 1990
). Both of these repeats contain open reading frames (ORFs) with extensive homology to the ß-subunit of protein kinase CK2 (Livak 1990
; Kalmykova, Dobritsa, and Gvozdev 1997
). The Su(Ste) repeats are considered suppressors of testis-specific Stellate transcription, since their deletion causes Stellate hyperexpression, coupled with male sterility (Hardy et al. 1984
). The Stellate repeats are located in the euchromatin of the X chromosome, in the 12E region of polytene chromosome map, as well as in constitutive heterochromatin of the X chromosome (Shevelyov 1992
; Palumbo et al. 1994
; Tulin et al. 1997
). The polymorphism of Stellate repeats and their homogenization in the heterochromatic X-linked cluster has been studied (Tulin et al. 1997
). The Stellate and Su(Ste) repeats were related to the special "parasitic system" (Bozzetti et al. 1995
) or to the pair of "self-promoting elements" driving a balance of male and female progenies in a population (Hurst 1996
; Hurst and Smith 1998
). It was shown recently that the unique euchromatic testis-expressed gene ßCK2tes, encoding the testis-specific ß-subunit of protein kinase CK2, might be considered a precursor of the X-linked Stellate and the Y-linked Su(Ste) repeats (Kalmykova et al. 1997
). Here, further study of Su(Ste) repeats and detection of a Su(Ste) orphon allow us to suggest that the Su(Ste) Y-linked repeats and their coding capacities, at least at some periods during their evolution, evolved on a Y chromosome under the pressure of translational selection. We accentuate that the Y-linked Su(Ste) repeats carry at least stretches of ORFs evolved under selection on a level of translation. The origin of the Y chromosome is usually thought to occur as a result of degeneration of the X chromosome (Charlsworth 1996
). In contrast, our observations might support a view that at least some segments of the Y chromosomes of Drosophila evolved acquiring repeats capable of affecting fertility of individuals (Hackstein et al. 1996
). Here, we also report some peculiarities of comparative intralocus concerted evolution of paralogous Stellate and Su(Ste) clusters.
Materials and Methods
The bacteriophage P1 library was screened by hybridization to Stellate and a Y-specific Su(Ste) probe to obtain P1E8 and P13H1 phages (Kalmykova, Dobritsa, and Gvozdev 1998
). The orphon was subcloned from P1E8 into the PstI site of pTZ19R. To obtain Su(Ste) oligomers, the fragments of phage P13H1 were subcloned into pTZ19R after partial XbaI restriction. The 300-bp BglII/HindIII fragments of clone 72, comprising three tandem Su(Ste) copies, were subcloned into pTZ19R to obtain the 72-1, 72-2, and 72-3 subclones. The orphon and Su(Ste)-carrying plasmid subclones were sequenced on both strands from universal plasmid or Su(Ste)-specific primers by Sanger's procedures (Sambrook, Fritsch, and Maniatis 1989
) using Sequenase, version 2.0, T7 DNA polymerase (U.S. Biochemical).
Results
Su(Ste) Orphon
The 6.5-kb PstI fragment with extensive homology to the Su(Ste) unit was isolated by subcloning of the P1E8 Stellate-positive clone (fig. 1
). Direct sequencing of the regions including both XbaI sites revealed the presence of anonymous sequence at the 5' XbaI site and the AT-rich repeat (610) at the 3' XbaI site, similar to that which was shown to specifically interact with nuclear lamins (Baricheva et al. 1996
). This result allows us to consider the Stellate-like sequence an orphon, localized outside of the cluster and flanked by unassigned repeats. The orphon, included in the PstI fragment, was easily detected by Southern analysis as the 6.5-kb PstI fragment in several randomly chosen stocks (not shown) because the regular Stellate and Su(Ste) repeats contain no PstI sites and are revealed in the DNA bands of high molecular weights (exceeding 15 kb). Comparative Southern analysis of DNA from males and females revealed that this Stellate-like fragment is located on the Y chromosome (not shown). The detected orphon contains 52 diagnostic nucleotide substitutions (fig. 2 ) coinciding with the Su(Ste) sequence, as well as the downstream Y-specific sequence peculiar to the Su(Ste) repeats, starting from position 1029. At the same time, only 12 diagnostic nucleotide positions peculiar to Stellate sequence were detected in the orphon sequence (fig. 2
). The orphon contains the remnant (~80 bp, upstream of position 212, including 37 bp of inverted repeat) of transposon 1360 (hoppel), inserted in each tandemly repeated Su(Ste) unit (Balakireva et al. 1992
). Thus, as a whole, the orphon is much more related to the Su(Ste) repeats than to the Stellate ones. The orphon also carries two typical diagnostic features of Stellate repeats: the position of the 3' splice site of the first intron is similar to the Su(Ste) copies, and the 10-bp deletion of the main exon (positions 826835) peculiar to the Su(Ste) repeat is absent, as in Stellate copies. The nucleotide Stellate-like positions in the orphon sequence are randomly distributed along the orphon sequence, and no clusters of these positions were observed. Thus, the origin of similarities between the orphon and the Stellate repeat seems improbable as a result of ectopic conversion events. Our observations suggest that the orphon, still carrying a set of diagnostic features of the X-linked Stellate repeats, represents the damaged dead derivative of an ancestor of amplified heterochromatic Y-linked Su(Ste) repeats (see below).
|
|
|
As a whole, pairwise comparison of the Su(Ste) and Stellate sequences reveals traces of translational selection which might be responsible for the maintenance of a definite level of translational efficiency of these repeats. The Su(Ste) ORF containing the conservative zinc finger is maintained, whereas it is damaged in the orphon. The putative conservative zinc finger CPX3CX22CPXC is also damaged as a result of a 5-bp deletion (positions 710714) and elimination of the second Cys residue. Thus, the orphon might be considered a profoundly damaged pseudogene, since numerous nucleotide deletions, as well as G and C insertions, impair the ORF. The initiator ATG codon is transformed to the ATT Ile codon. The 5' splice site in the second intron is damaged as a result of a T deletion in the canonical GT dinucleotide (fig. 2 ). The orphon is much more damaged than the Su(Ste) repeats that have been sequenced (Balakireva et al. 1992
; Kalmykova, Dobritas, and Gvozdev 1998
). Detection of the orphon accentuates the existence of selective pressure and homogenization maintaining stretches of ORFs in the clustered Su(Ste) repeats.
Concerted Evolution of the Y-Linked Su(Ste) Repeats
The Su(Ste) size variants, as well as Su(Ste) units with similar restriction sites (McKee and Satter 1996
; Kalmykova et al. 1997
), appear to be clustered in particular subintervals of the locus. The randomly chosen Su(Ste) repeats differ at 6.4% of nucleotide size, as compared with 2.5% of divergence for homologous Stellate repeats (McKee and Satter [1996]
; calculated from the previously reported data of Balakireva et al. [1992]
and Shevelyov [1992]
). To directly evaluate the efficiency of concerted evolution of Su(Ste) repeats, the fragments of adjacent clustered Su(Ste) repeats were sequenced. The presence of a driver effect of concerted evolution was shown, since all three repeats (72-1, 72-2, and 72-3) contain six diagnostic nucleotide substitutions, as well as three shared single-nucleotide deletions, along the sequenced regions (fig. 2
). The earlier randomly chosen and sequenced Su(Ste) copies or cDNA clones do not contain these substitutions or deletions (Balakireva et al. 1992
; Kalmykova, Dobritsa, and Gvozdev 1998
). Results of the pairwise sequence comparison of three adjacent Su(Ste) repeats show three to four substitutions per 330 nt, that is, more than 1% of divergence. At the same time, intralocus divergence of the adjacent heterochromatic Stellate repeat does not exceed 0.1%0.2% (Tulin et al. 1997
). Thus, the efficiency of homogenization of Su(Ste) repeats is 510 times as low as than that of Stellate repeats.
Discussion
The Stellate repeats and their suppressors, Su(Ste) repeats, were considered an evolved parasitic system capable of maintaining itself (Bozzetti et al. 1995
). This system was thought by others to contain self-promoting genetic elements (Hurst 1996
), in which the original Stellate gene affected DNA packing of the X chromosome and its successful transmission to progeny. It was proposed that Stellate repeats act as a driver causing preferential transmission of the X chromosome, thus biasing the sex ratio toward females. The evolved Su(Ste) repeats on the Y chromosome were proposed to act in a dose-dependent fashion (Hurst 1996
), causing a balance between transmission of the X chromosome and Y chromosome efficiency. The secondary origin of Su(Ste) as a suppressor of driver effects exerted by Stellate was suggested (Hurst 1996
). However, Stellate repeats do not exert any driver effect (Palumbo et al. 1994
; L. Robbins, personal communication). Alternatively, the Y-linked Su(Ste) repeats can represent an immediate derivative of euchromatic ßCK2tes-like gene, and possibly one that is more ancient than the X-like Stellate repeats. The Stellate-like sequence is present only on the Y chromosome of Drosophila simulans (Livak 1984
). We propose the presence of the Y-linked ancestors of the Su(Ste)like repeats before the split of the melanogaster and simulans lineages. Actually, the rough estimation (Kalmykova et al. 1997
) of the period of divergence of Stellate and Su(Ste) repeats (6 Myr) exceeds the time (2 Myr) elapsed since the split of the melanogaster and simulans lineages (Russo, Takezaki, and Nei 1995
). In this report, evidences was presented that Su(Ste) repeats underwent translation selection for the more preferred codons, whereas codon bias for the Stellate units declined. This latter relaxation of selection was demonstrated for D. melanogaster since its split from D. simulans (Akashi 1997
).
We detected the Y-linked Su(Ste) orphon detached from the main cluster of tandemly repeated units. The orphon sequence combines diagnostic traits of both types of diverged Stellate and Su(Ste) paralogous repeats. Thus, detection of this "molecular fossil" on the Y chromosome allows us to suggest that the Su(Ste) repeated cluster really evolved on the Y chromosome. The orphon carries the same evidence of translational selection as the Su(Ste) repeats, although this orphon repeat was drastically damaged, escaping the pressure of concerted evolution which has continued to be operative in the Su(Ste) cluster, at least in the region encoding the zinc finger motif of protein kinase CK2. The putative ancestor on the Y chromosome of both the X-linked Stellate and the Y-linked Su(Ste) repeats might be represented by the close ancient relative of the orphon sequence, comprising the Stellate-like 3' splice site of the first intron, the inserted transposon hoppel (1360), and the ORF still subjected to the pressure of translational selection. We propose that this Y-linked repeated ancestor engendered the contemporary Stellate repeats, as well as Su(Ste) ones. These latter repeats contained partially damaged ORFs, possibly acquiring a new function as suppressors of Stellate repeats when a period of translational selection was transformed to episodic changes and damage to the Su(Ste) ORFs.
We want to accentuate the probability of translational selection on a Y chromosome, which might be opposed to the commonly accepted and established view of genetic erosion and degeneration of the Y chromosomes (Charlesworth 1996
). The Su(Ste) repeats conserved stretches of Stellate ORFs and the possibility of their translation was discussed (Kalmykova, Dobritsa, and Gvozdev 1997
). In contrast, the orphon shows numerous damages to this ORF, including the mutation of the Cys residue in the region of the conservative putative zinc finger responsible for dimerization of protein kinase CK2 subunits (Chantalat et al. 1999
). Comparison of the ORFs of the Su(Ste) and Stellate repeats indicates the bias of Su(Ste) ORF preferred codon usage. The Su(Ste) ORF contains a cluster of G- and C-ending codons (interval encompassing positions 751837) (fig. 2
) that were shown to be preferred in the D. melanogaster genome (Powell and Moriyama 1997
). We also note that the evolution of Su(Ste) repeats on the Y chromosome has been driven by translational selection maintaining an unaltered conservative zinc finger motif. Selection for preferred codons suggests that effective recombination events occur. Actually, selection at single nucleotide positions is more effective in regions of high recombination (Powell and Moriyama 1997
). The nature of this process in the Y chromosome remains obscure. Transposon insertion may possibly affect the peculiarities of evolution of Su(Ste) repeats. In the course of their evolution on a Y chromosome, Su(Ste) repeats acquired the transposon hoppel (1360), since the remnant of the transposon was detected in the orphon, a damaged copy of an intermediate in the course of Su(Ste) evolution. Interestingly, experiments indicate that transposon activity might be an important factor driving the concerted evolution of repetitive DNA (Thompson-Stewart, Karpen, and Spradling 1994
). It has been suggested that excision of the transposon leaves a double-strand break, causing subsequent events of recombination (Spradling 1994
). The Stellate sequences, which are extensively homogenized and also were maintained by natural selection (Tulin et al. 1997
), require the usage of more rare codons as compared with the Su(Ste) sequence. This observation indicates that clustered Su(Ste) units were maintained under selection pressure and concerted evolution takes a part in this process.
The role of gene conversion or unequal crossing over between repeats in the Su(Ste) (Balakireva et al. 1992
) and Stellate clusters (Tulin et al. 1997
) was deduced earlier and might be considered as a force driving concerted evolution of these repeats. Here we have shown by direct sequencing of adjacent Su(Ste) repeats, as well as by their comparison to the orphon, that concerted evolution drives clustered Su(Ste) sequences. Nevertheless, the level of divergence of the Y-linked Su(Ste) repeats is 510 times as high as than that of the Stellate repeats. Interestingly, X-linked rDNA spacers are significantly more similar to each other than are Y-linked rDNA spacers (Williams et al. 1987
). These differences can reflect distinct efficiencies of recombination events responsible for concerted evolution of repeats on the X and Y chromosomes. The single spontaneous events causing divergence of the Su(Ste) repeats may entail the further decrease of recombination efficiency. It was thought that polymorphism between tandemly repeated genes suppresses recombination, thereby preventing sequence homogenization of the gene family (Parniske et al. 1997
). Actually, it was shown that conversion in D. melanogaster was highly sensitive to single-base mismatches within the homologous region (Nassif and Engels 1993
). Thus, Stellate repeats on the X chromosome are suggested to be more susceptible to recombination events than more diverged Su(Ste) repeats on the Y chromosome. However, the differences in recombination frequencies among the X- and Y-linked repeats may be explained by unknown profound disparities of the intimate mechanisms of recombination, rather than by mere differences in repeat divergence.
Acknowledgements
We thank Y. Shevelyov for critical comments and V. E. Alatortsev for improvement of the text. We also thank two anonymous reviewers, through whose comments the manuscript was considerably improved. This work was supported by grants from the Russian Foundation of Basic Research (98-04-49107 and 99-04-48561) and the Russian program Frontiers in Genetics (99-1-069).
Footnotes
1 Keywords: heterochromatin
tandem repeats
Drosophila melanogaster,
concerted evolution
Y chromosome
translational selection
2 Address for correspondence and reprints: Vladimir Gvozdev, Institute of Molecular Genetics of RAS, Kurchatov sq., Moscow 123182, Russia. E-mail: gvozdev{at}img.ras.ru
literature cited
Akashi, H. 1997. Codon bias evolution in Drosophila. Population genetics of mutation-selection drift. Gene 205:269278.
Balakireva, M. D., Y. Y. Shevelyov, D. I. Nurminsky, K. J. Livak, and V. A. Gvozdev. 1992. Structural organization and diversification of Y-linked sequences comprising Su(Ste) genes in Drosophila melanogaster. Nucleic Acids Res. 20:37313736.
Baricheva, E. A., M. Berrios, S. S. Bogachev, I. V. Borisevich, E. R. Lapik, I. V. Sharakhov, N. Stuurman, and P. A. Fisher. 1996. DNA from Drosophila melanogaster beta-heterochromatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ. Gene 171:171176.
Bozzetti, M., S. Massari, P. Finelli et al. (11 co-authors). 1995. The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the ß subunit of casein kinase 2. Proc. Natl. Acad. Sci. USA 92:60676071.
Chantalat, L., D. Leroy, O. Filhol, A. Nueda, M. Benitez, E. M. Chambaz, C. Cochet, and O. Dideberg. 1999. Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization. EMBO J. 18:29302940.
Charlesworth, B. 1996. The evolution of chromosomal sex determination and dosage compensation. Curr. Biol. 6:149162.[ISI][Medline]
Hackstein, J. H. P., R. Hochstenbach, E. Hauschteck-Ungen, and L. W. Beukeboom. 1996. Is the Y chromosome of Drosophila an evolved supernumerary chromosome? BioEssays 18:317323.
Hardy, R. W., D. L. Lindsley, K. J. Livak, B. Lewis, A. L. Silverstein, G. L. Joslyn, J. Edwards, and S. Bonaccorsi. 1984. Cytogenetic analysis of a segment of the Y chromosome of the Drosophila melanogaster. Genetics 107:591610.
Hurst, L. D. 1996. Further evidence consistent with Stellates involvement in meiotic drive. Genetics 142:541643.
Hurst, L. D., and N. G. C. Smith. 1998. The evolution of concerted evolution. Proc. R. Soc. Lond. B Biol. Sci. 265:121127.[ISI]
Kalmykova, A. I., A. A. Dobritsa, and V. A. Gvozdev. 1997. The Su(Ste) repeat in the Y chromosome and ß CK2 tes gene encode predicted isoforms of regulatory ß-subunit of protein kinase CK2 in Drosophila melanogaster. FEBS Lett. 416:164166.
. 1998. Su(Ste) diverged tandem repeats in a Y chromosome of Drosophila melanogaster are transcribed and variously processed. Genetics 148:243249.
Kalmykova, A. I., Y. Y. Shevelyov, A. A. Dobritsa, and V. A. Gvozdev. 1997. Acquisition and amplification of a testis-expressed autosomal gene, SSL, by the Drosophila Y chromosome. Proc. Natl. Acad. Sci. USA 94:69276302.
Liao, D. 1999. Molecular evolution '99. Concerted evolution: molecular mechanism and biological implications. Am. J. Hum. Genet. 64:2430.[ISI][Medline]
Livak, K. J. 1984. Organization and mapping of a sequence of Drosophila melanogaster X and Y chromosoms that is transcribed during spermatogenesis. Genetics 107:611634.
. 1990. Detailed structure of the Drosophila melanogaster Stellate genes and their transcripts. Genetics 124:303316.
McKee, B., and M. T. Satter. 1996. Structure of the Y chromosomal Su(Ste) locus in Drosophila melanogaster and evidence for localized recombination among repeats. Genetics 142:149161.
Nassif, N., and W. Engels. 1993. DNA homology requirements for mitotic gap repair in Drosophila. Proc. Natl. Acad. Sci. USA 90:12621266.
Palumbo, G., S. Bonaccorsi, L. G. Robbins, and S. Pimpinelli. 1994. Genetic analysis of Stellate elements of Drosophila melanogaster. Genetics 138:11811197.
Parniske, M., K. E. Hammond-Kosack, C. Goldstein, C. M. Thomas, D. N. Jones, K. Harrisson, B. B. H. Wulff, and D. G. Jones. 1997. Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato. Cell 91:821832.
Powell, J. R., and E. N. Moriyama. 1997. Evolution of codon usage bias in Drosophila. Proc. Natl. Acad. Sci. USA 94:77847790.
Russo, C. A. M., N. Takezaki, and M. Nei. 1995. Molecular phylogeny and divergence times of drosophilid species. Mol. Biol. Evol. 12:391404.[Abstract]
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Sharp, P. M., and G. Matassi. 1994. Codon usage and genome evolution. Curr. Opin. Gen. Dev. 4:851860.[Medline]
Shevelyov, Y. 1992. Copies of a Stellate gene variant are located in the X heterochromatin of Drosophila melanogaster and are probably expressed. Genetics 132:10331037.
Spradling, A. C. 1994. Transposable elements and the evolution of heterochromatin. Pp. 7683 in Molecular evolution of physiological process. Rockefeller University Press.
Thompson-Stewart, D., G. H. Karpen, and A. C. Spradling. 1994. A transposable element can drive the concerted evolution of tandemly repetitions DNA. Proc. Natl. Acad. Sci. USA 91:90429046.
Tulin, A. V., G. L. Kogan, D. Filipp, M. D. Balakireva, and V. A. Gvozdev. 1997. Heterochromatic Stellate gene cluster in Drosophila melanogaster: structure and molecular evolution. Genetics 146:253262.
Williams, S. M., G. R. Furnier, E. Fuog, and C. Strobeck. 1987. Evolution of ribosomal DNA spacers of Drosophila melanogaster: different patterns of variation on X and Y chromosomes. Genetics 116:225232.