The retroposon SINE-R.C2 was first identified as a human-specific insertion in the complement C2 gene and the insertion of a closely related element has been implicated as a cause of Fukuyama type muscular dystrophy. In a previous study, we found SINE-R-type retroposons, derived from the endogenous retrovirus HERV-K family, have been found to be hominoid specific. In this report, we identified twelve SINE-R-type retroposons from the human Y chromosome by a PCR approach. Compared with other human retroposons (SINE-R.C2, 11, 14, 19, and HS307/HS408), the Y-chromosomal retroposon showed a high degree of sequence homology (86–96%). Phylogenetic analysis using the neighbor-joining method revealed that SINE-R-type retroposons on the Y chromosome were inter-related with hominoid primates, suggesting that SINE-R elements on the Y chromosome have evolved progressively in the hominoid radiation. Two elements SINE-R.Y-2 and Y-7 are more closely related to the human-specific retroposon SINE-R.C2 than other Y-chromosomal retroposons. Such elements thus are likely to be of great potential relevance to recent events in hominoid evolution and to structural change or genetic variation connected to various disease.
It has often been suggested that endogenous retroviruses and retroposons have played a role in influencing the functional organization of the human genome (Baltimore, 1985; Kazazian and Moran, 1998; Sverdlov, 1998). Approximately 1% of the human genome is comprised of such sequences, and most of them have been integrated into the primate lineage thirty-three or more million years ago (Haltmeier et al., 1995; Mager and Freeman, 1995; Steinhuber et al., 1995). The vast majority of these elements were incorporated as proviral DNA which then rapidly became replication-defective by random accumulation of mutations to be vertically transmitted as junk DNA. Therefore, particular interest attaches to families of elements that have changed recently in human and hominoid evolution. By PCR and phylogenetic analyses, Medstrand and Mager (1998) found that some solitary long terminal repeat (LTR) elements of the human endogenous retrovirus K family (HERV-K) were human specific. These elements may still be actively transposing in the human genome. For example, a HERV-K LTR element has been inserted in the antisense orientation between the gag and pol gene of the human endogenous retrovirus S71 (Leib-Mösch et al., 1993). Another solitary element has also been found at the HLA-DQB1 (Seidl et al., 1999). It has been estimated that there are 25,000 copies of such elements in the human genome (Leib-Mösch et al., 1993), and they are potentially capable of affecting the expression of closely located genes (Akopov et al., 1998). Simon et al. (1994) found that HERV-K-related LTR elements are expressed in human placenta.
Amongst the derivatives of the HERV-K10 retrovirus family is a family of Short Interspersed Nuclear Elements (SINEs) termed SINE-R for retrovirus-derived (Ono et al., 1987). SINE-R11, 14, 19 were isolated by colony blot hybridization with a HERV-K10 probe, and derive from the 3′ long terminal repeat and small upstream flanking regions. These elements entered in the genome of hominoid primates after the split of Old World monkeys in the Oligocene period (Kim et al., 1999a, b). The SINE-R.C2 element was discovered in relation to a variable number of tandem repeat (VNTR) sequence within the third intron of the gene for C2, the second component of complement, located in class III of the major histocompatibility complex on the short arm of human chromosome 6 (Carroll et al., 1984; Zhu et al., 1992). By Southern blot analysis, SINE-R.C2 is confined to Homo sapiens (Zhu et al., 1994). Within the block in Xq21.3 with homology to Yp11.2 that has been tentatively linked to shizophrenia and schizo-affective disorder (Laval et al., 1998) we previously reported two retroposons (HS307 and HS408) with 92 to 96% homology to SINE-R.C2 (Kim et al., 1999b). An element almost identical in sequence to SINE-R.C2 has recently been reported as the cause of Fukuyama-type muscular dystrophy (Kobayashi et al., 1998), and this appears to be the first association of this class of elements with disease. Influences of SINE-R elements on gene expression have been described, for example, a SINE element that acquired a role in signal transduction in the course of mammalian evolution (Shimamura et al., 1998). Here we report the presence of SINE-R-type retroposons on the human Y chromosome and their phylogenetic analysis with those of other humans and hominoids.
MATERIALS AND METHODS
Sample DNA and PCR
A human monochromosomal somatic cell hybrid DNA panel was obtained from the MRC Human Genome Mapping Project Resource Centre (HGMP-RC, Cambridge, UK, Kelsall et al., 1995). The Y chromosomal DNA sample was subjected to PCR amplification. SINE-R-type retroposons were amplified by the primer pair JS902 (5′-GAGAATGGGCCATGATGAC-3′, bases 4–22) and JS903 (5′GATCATTCTTGGATGTTTCT-3′, bases 466–485) from schizophrenic brain S11 cDNA (GenBank, accession no. AA772777) which has a high level of sequence homology with SINE-R.C2, 11, 14, 19, and HS307/HS408 elements (Ono et al., 1987; Kim et al., 1999b). The PCR conditions followed were those of Kim et al. (1996) with an annealing temperature of 56°C.
Cloning of PCR product
PCR products were separated on a 1.8% agarose gel, purified with the QIAEX II gel extraction kit (Qiagen) and cloned into the T-khs307 vector (Kim et al., 1998). Plasmid DNA was isolated by the alkali lysis method using a High Pure plasmid isolation kit (Boehringer Mannheim).
DNA sequencing and data analysis
Individual plasmid DNAs were screened for inserts by PCR. Positive samples were subjected to sequence analysis on both strands with T7 and M13 reverse primers using an automated DNA sequencer (Model 373A) and the DyeDeoxy terminator kit (Applied Biosystem). SINE-R.C2 (L09706), SINE-R11 (X07417), SINE-R14 (X07418), SINE-R19 (X07419), FMR-1 (L29074), HS307 (AB016781), HS408 (AB016782), SCHIZO (AA772777), Chi-M (AB018364), Chi-F (AB018365), Gor-M (AB018366), Gor-F (AB018367), Ora-M (AB018368), Ora-F (AB018369), Gib-M (AB018370), and Gib-F (AB018371) sequences were taken from the GenBank database. Sequence analyses were done with the aid of GAP, PILEUP, and PRETTY from the GCG program (University of Oxford). Neighbor-joining phylogenetic analysis (Saitou and Nei, 1987) was performed with the CLUSTAL W program (Thompson et al., 1994) Sequence homology was searched for in the BLAST network server (Altschul et al., 1997).
RESULTS AND DISCUSSION
The nucleotide sequences of the human endogenous retrovirus, HERV-K10, have been found to be present in hominoids and Old World monkeys by PCR analysis using genomic DNA (Zhu et al., 1994). SINE-R-type retroposons, that are clearly derived from the long terminal repeat (LTR) of the HERVK family (Ono et al., 1987; Zhu et al., 1992), have been found to be hominoid-specific by PCR analysis (Kim et al., 1999b). Comparing nucleotide sequences between SINE-R-type retroposons and HERV-K LTR elements, we showed that the 367 bp of SINE-R-type retroposons are deleted from the HERVK LTR sequences (Ono et al., 1987; Kim et al., 1999b). By BLAST search, cDNA prepared from the brain of a patient with schizophrenia (GenBank, accession no. AA772777) was found to have sequences containing a high degree of homology with the 3′ LTR of HERV-K10 (Ono et al., 1986), SINE-R11, 14, 19 (Ono et al., 1987), SINE-R.C2 (Zhu et al., 1994), and HS307/HS408 (Kim et al., 1999b). Therefore, PCR primers, JS902 and JS903, were chosen from the brain cDNA of a patient with schizophrenia. Using genomic DNA from the human Y chromosome, we identified twelve SINE-R-type retroposons by PCR amplification in the present study although we are aware that there are more SINE-R-type retroposons on the Y chromosome. Ono et al. (1987) have estimated that SINE-R elements were present at 4000 to 5000 copies per haploid human genome. So far, limited numbers of SINE-R-type retroposons (SINE-R11, 14, 19, C2, and HS307/HS408) have been reported in the human genome (Ono et al., 1987; Zhu et al., 1994; Kim et al., 1999b). As far as we know, this is the first time that the nucleotide sequences of Y-chromosomal retroposons have been demonstrated. The sequences are presented in alignment with those of the other human retroposon family in Fig. 1. The sequence data from the human Y chromosome was demonstrated as derived from SINE-R-type retroposons by comparison with other human retroposons. The hominoid specificity of the PCR products was also demonstrated by the absence of amplified rodent DNA (data not shown). As shown in Table 1, Y-chromosomal retroposons have a high degree of sequence homology with other retroposons. Sequence similarity ranged from 99.5% between SINE-R.Y-7 and SINE-R.Y-2, also between SINE-R.Y-15 and Y-17, to 85.6% between SINE-R.Y-4 and SINE-R.Y-15. The human-specific retroposon SINE-R.C2 shared 96% sequence homology with both SINE-R.Y-2 and Y-7.
Percentage similarity of nucleotide sequences in the retroposon family
To understand the evolutionary relationships among these families with hominoid retroposons, a phylogenetic tree was constructed by the neighbor-joining method. As shown in Fig. 2, the sequence of the chimpanzee male (Chi-M) is closely related to the sequences from SINE-R.Y-8 and SINE-R.Y-4, while SINE-R.Y-15 and Y-17 cluster with the sequence from the gibbon male (Gib-M) and female (Gib-F), the schizophrenic brain cDNA (SCHIZO), and the orangutan female (Ora-F). Retroposons Y-3, 11, 13, 16, 18, 19 are closely related to SINE-R14. SINE-R.Y-7 and SINE-R.Y-2 are more closely related to each other than either of them is to SINE-R11 and they cluster with SINE-R.C2. The human-specific retroposon SINE-R.C2 is closely related to the sequence of gorilla male (Gor-M) and these sequences cluster with the HS307 retroposon that we previously identified in the Xq21.3 region of homology with the Y chromosome (Kim et al., 1999b). This group with SINE-R.C2 is likely to be of great relevance to recent events in hominoid evolution. Thus the sequences of SINE-R elements on the Y chromosome are intermingled with those of similar elements in hominoid primates, suggesting that several lineages of retroposons have been evolving independently during hominoid evolution.
The non-recombining portion of the human Y chromosome includes relatively few genes and has the capacity to accumulate repetitive and other junk DNA sequences unconstrained by the functional considerations that apply elsewhere. It shows considerably greater variation between hominoid species (Glaser et al., 1997). However, particular interest attaches to regions of homology between the X and Y chromosomes. Such homologies have generally been created by X to Y transpositions and a number of such events have been tracked in the course of mammalian and primate evolution (Lambson et al., 1992; Glaser et al., 1997). Genes within these regions are thus exposed to new evolutionary pressures, and their X homologues are subject to differential patterns of inactivation (Jegalian and Page, 1998). Some evolutionary changes are known to have been influenced by retroposons, eg the LINE-LINE recombination associated with the paracentric inversion on the Y chromosome (Schwartz et al., 1997) and they may be the most recent chromosomal rearrangement in human evolution. Such X-Y homologous regions are relevant to the detection of a role of retroviruses/retroposons in recent evolution in two respects. First, examination of homologous sequences on the X and the Y may reveal new insertions and allow them to be dated. Second, investigation of the relationship between genes within these regions and such elements as may be related to them could uncover a role in gene expression since any recent change will be differentially expressed in the two sexes.
The Xq21.3/Yp11.2 region of homology is of particular interest in relation to human evolution, as it was created by a transposition after the separation of the chimpanzee and human lineages, and 3.5 Mb of the transposed region was subject to a subsequent paracentric inversion (Sargent et al., 1996; Mumm et al., 1997). We identified two retroposons (HS307 and HS408) within the Xq21.3 region with homology to the SINE-R.C2 element (Kim et al., 1999b). It will be important to examine the homologies of these elements within the Yp11.2 region to ascertain whether changes in these or other elements (eg SINE-R.Y-2 or Y-7) might be associated with genes which in turn are relevant to the sex differences that have been described in relation to Homo sapiens specific cognitive functions (Crow et al., 1998).
SINE-R11, 14 and 19 have a polypurine tract (PPT) that serves as a primer-binding-site for plus-strand DNA synthesis from retrovirally mediated reverse transcription. This indicates that SINE-R-type elements like human endogenous retroviruses have a potential functional role in the genomic DNA of human chromosomes. According to PCR and phylogenetic analyses, integration of several HERVK LTR elements have been shown to be human specific (Medstrand and Mager, 1998). We also identified a solitary HERV-K LTR element in a recently proliferated class of LTR elements, this was inserted into Xq26 after the separation of the hominid and great ape lineages (Kim et al., submitted). It seems that the LTR elements have been randomly transposed across the chromosome in the course of evolution. Apokov et al. (1998) noted that such sequences have the capacity to modify the expression of neighboring genes. These elements may also have contributed to genetic variation associated with various disease. The HERV-K sequences, to which the SINE-R elements are related, have been reported as a candidate autoimmune gene in type I diabetes (Conrad et al., 1997). Two HERV-K LTR elements have been detected in the human major histocompatibility complex locus HLA-DQ (Kambhu et al., 1990). One LTR element (DQ-LTR3) of the HERV-K family at the HLA-DQB1 locus has been associated with the rheumatoid arthritis (Seidl et al., 1999).
We thank Rekha V. Wadekar for her technical assistance. This work was supported by grants from the UK Medical Research Council and the Stanley Foundation.