Alexander I. Agulnik

The HLA-A*0201-Restricted H-Y Antigen Contains a Posttranslationally Modified Cysteine That Significantly Affects T Cell Recognition

A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.

Analysis of mutation rates in the SMCY/SMCX genes shows that mammalian evolution is male driven.

Mammalian evolution is believed to be male driven because the greater number of germ cell divisions per generation in males increases the opportunity for errors in DNA replication. Since the Y Chromosome (Chr) replicates exclusively in males, its genes should also evolve faster than X or autosomal genes. In addition, estimating the overall male-to-female mutation ratio (αm) is of great importance as a large αm implies that replication-independent mutagenic events play a relatively small role in evolution. A small αm suggests that the impact of these factors may, in fact, be significant. In order to address this problem, we have analyzed the rates of evolution in the homologous X-Y common SMCX/SMCY genes from three different species—mouse, human, and horse. The SMC genes were chosen because the X and Y copies are highly homologous, well conserved in evolution, and in all probability functionally interchangeable. Sequence comparisons and analysis of synonymous substitutions in approximately 1kb of the 5′ coding region of the SMC genes reveal that the Y-linked copies are evolving approximately 1.8 times faster than their X homologs. The male-to-female mutation ratio αm was estimated to be 3. These data support the hypothesis that mammalian evolution is male driven. However, the ratio value is far smaller than suggested in earlier works, implying significance of replication-independent mutagenic events in evolution.

In situ hybridization shows that Dazla expression in mouse testis is restricted to premeiotic stages IV-VI of spermatogenesis

JDepartment of Urology, University of Illinois at Chicago, Chicago, Illinois, USA 2Department of Genetics, University of Illinois at Chicago, Chicago, Illinois, USA 3Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, USA 4Department of Obstetrics and Gynecology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA 5Department of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA 6Department of Urology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA 7Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA

Mouse H-Y encoding Smcy gene and its X chromosomal homolog Smcx.

SMCY is present on the Y in mouse, human, and most mammals. Importantly, it is present on the marsupial Y showing that SMCX/ SMCY diverged at least 120 Myr years ago (Agulnik et al. 1994a). Unlike many other Y-Chromosomal genes, SMCY is present in a single copy in mouse and man. Both Y and X Chr homologs are widely transcribed in all male tissues, and the X gene is expressed in all female tissues tested including preimplantation mouse embryos (Agulnik et al. 1994a). SMCX escapes X-inactivation in human and mouse (Agulnik et al. 1994b; Carrel et al. 1996; Jegalian and Page 1998; Sheardown et al. 1996; Wu et al. 1994a, 1994b), indicating that the X and Y copies are functionally interchangeable and that transcript dose is important for the expression of this gene. Although the biological role of the genes remains unknown, it has been established that human and mouse SMCY contain epitopes of the minor histocompatibility antigen, H-Y (Ehrmann et al. 1997; Markiewicz et al. 1998; Meadows et al. 1997; Scott et al. 1995; Wang et al. 1995). In this paper we report the complete cDNA sequence of the mouse SmcyandSmcxgenes. We have established the genomic structure of the Smcygene and show that the gene is composed of 26 exons spread over 47 kb of genomic DNA. The longest open reading frame encodes 1548 amino acids forSmcyand 1551 amino acids for Smcx.The genomic structure of SMCY is entirely conserved between mouse and human. As yet, its biological role is not understood, but the homology to RBP2 (retinoblastoma-binding protein 2) and the presence of a zinc-finger domain indicate a possible involvement of the SMCX/Y (SMCX and SMCY) proteins in DNA binding and transcriptional regulation. A 1.8-kb cDNA of the mouseSmcygene and 3 kb of the mouse Smcxgenes identified previously (Agulnik et al. 1994a,b) have been used in the present study to clone and sequence full-length transcripts of the genes. RT and RACE PCR, as well as conventional screening of testis cDNA libraries, have been employed to obtain a series of the overlapping cDNA fragments representing the missing 38 and 58 ends of both genes. The Smcysequence is 5316 bp, andSmcxis 5673 bp with open reading frames (ORF) of 4647 bp and 4656 bp respectively. The transcripts are predicted to encode polypeptides of 1548aa, MW 177 kDA ( Smcy), and 1551 aa, MW 175 kDa ( Smcx). The translation start codon in Smcyis situated in the context AACATGA, which is close to the optimal ACCATG present in the Smcxsequence (Kozak 1986). In the Smcy transcript, two consensus polyadenylation signals AATAAA are at 266 bp and 18 bp upstream of poly (A) tail. InSmcxthere is only one polyadenylation signal at 55 bp upstream of the poly(A) tail. During cDNA isolation we found several splice variants of the SmcyandSmcxtranscripts. Several splice variants of the SMCY gene were also recovered from human testis and lymphocyte polyA RNA (data not shown). Different transcripts apparently utilizing different splice sites have been also reported for the human SMCX gene (Wu et al. 1994a) and for the SMCY gene (cDNA KIAA0234, GeneBank accession # D87072). Specific differences between the amino acid sequence of the SMCY and SMCX genes, coupled with their widespread expression pattern (Agulnik et al. 1994a), form the basis of the H-Y male-specific minor antigen system. Thus, Smcyhas been shown to encode several H-Y antigen epitopes, the position of which are shown in Fig. 1 (Ehrmann et al. 1997; Markiewicz et al. 1998; Meadows et al. 1997; Scott et al. 1995; Wang et al. 1995). As shown in Fig. 1, proteins derived from the translation of the open reading frames of the human and mouse SMCX/SMCY genes are highly homologous to each other. Overall amino acid sequence similarities are as follows: Smcx/SMCX 96%; Smcy/ SMCY 84%; Smcy/Smcx 84%; Smcy/SMCX 83%; and Smcx/ SMCY 86%. In all proteins the C-terminal end encoded by the last exon is the least conserved. The similarity between the amino acid sequence of SmcxandSmcyin this region is only 32% compared with an overall value of 84%. The closest identified homolog of the SMCX/SMCY gene pair is the human retinoblastoma binding protein 2 (RBP2; Fataey et al. 1993). The overall identity of the latter protein to the products of the mouse X and Y genes is 58% (67% similarity) and 57% (65% similarity) respectively. The homology is higher at the N-terminal end of the sequence. At the C-terminal end, the middle part of the twenty-third exon ofSmcyencodes 57 amino acids, which are highly conserved among mouse and human SMCX/Y proteins and RBP2 (89% identity). The RBP2 protein contains two significant domains—a zinc-finger at the N-terminal end and a homeodomain similar to the engrailed family of homeotic genes in the middle part of the sequence (Fataey et al. 1993). The zinc-finger domain is well conserved between SMCX/Y products and RBP2 protein (70% identity), but the homeodomain is less conserved (53–57% identity). The retinoblastoma product (pRB) binding domain (a stretch of amino acids shared by all retinoblastoma-binding proteins) is not conserved in any SMCX/Y protein. At the nucleotide and amino acid level, database searching revealed that both genes have several homologs: yeast hypothetical 85.0 kDa protein (Accession # P47156); mouse jumonji protein (Q62315); yeast putative 90.2 kDa zinc-finger protein (P39956); and others. SMCX/Y proteins contain a zinc-finger domain as part of a highly conserved PHD finger, a cysteine-rich region encoded by the eighth exon of the gene. Such a domain is present in a number of putative proteins derived from yeast, mammals, and plants (Aasland et al. 1995). To obtainSmcygenomic clones, we have screened a mouse Correspondence to: A.I. Agulnik

A murine TSPY

Sequences homologous to human and bovine TSPY were isolated from M. musculus testicular cDNA, and a nearly full-length gene was polymerase chain reaction (PCR) amplified from mouse genomic DNA. This gene is apparently non-functional. Contrary to the situation encountered in species along the primate and artiodactyl lineages, in which TSPY is moderately repetitive, murine Tspy appears to be single copy. Murine Tspy is located on Yp, i.e. in the same syntenic group as in man. Sequence comparisons of murine, human and bovine TSPY exons suggest that TSPY became non-functional during rodent evolution.

Smcy transgene does not rescue spermatogenesis in sex-reversed mice

Abstract. In mouse, the Sxrb deletion interval (delta Sxrb) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1–2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxrb interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxrb/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy.