Cloning and nucleotide sequence of retroposons specific to hominoid primates derived from an endogenous retrovirus (HERV-K).

Abstract

595 ENDOGENOUS RETROVIRUSES comprise approximately 1% of the human genome.1 Although most human endogenous retroviruses (HERVs) are defective, one class, HERV-K (homologous to mouse mammary tumor virus [MMTV]), encodes viral proteins and particles3 and is highly expressed in germ cell tumors. Retroposons (a retroelementso and a retrovirus-like elements,o which multiply in the genome by retrotransposition) are distinguished from retroviruses by the absence of one or both long terminal repeats (LTRs). These repetitive elements are classified into two groupsÐshort (up to 600 bp) interspersed nuclear elements (SINEs) and long (about 6 kg) interspersed nuclear elements (LINEs)Ð that include a number of families that have diverged in the course of mammalian evolution.4 One class of SINEs, the Alu sequences, is specific to primates. Another class, termed SINE-R (for retrovirus), which is derived from the HERV-K sequence, has apparently undergone modification in the hominoid lineage.5 Such elements are a potential agency of genome change.1,6,7 The human-specific retroposon SINE-R.C2, a member of the SINE-R retroposon family (present at 4000 to 5000 copies per haploid human genome), was discovered in an investigation of the variable number of tandem repeats (VNTR) locus within intron 3 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 6.8,9 Other members of the SINE-R family in the human genome, SINER11, -14, and -19, were isolated by colony blot hybridization with an HERV-K10 probe. By BLAST search, schizophrenic brain S11 cDNA (GenBank, accession No. AA772777) was also found to have high homology with the 39 LTR of HERV-K10. Therefore, specific polymerase chain reaction (PCR) primers, JS902 and JS903, were designed from S11 cDNA. Using these primers, the sequences HS307 (GenBank, accession No. AB016781) and HS408 (GenBank, accession No. AB016782), which have a high degree of homology to the SINE-R.C2, -R11, -R14, and -R19 retroposons, were identified in an investigation of the Xq21.3 region that has been tentatively linked to schizophrenia and schizo-affective disorder by Laval et al.10 and Kim et al. Each of these retroposons derives from the 3 9 long terminal repeat and the small upstream flanking regions of the human endogenous retrovirus HERV-K10,5 the entire sequence of which has been reported as a candidate autoimmune gene in type I diabetes.11 The HERV-K10 (present at 50 copies per haploid human genome) has been found to be present in all primates except for New World monkeys, whereas SINE-R.C2 is present only in Homo sapiens. Other members of the SINER family have been found to be present in the human, chimpanzee, and gorilla genomes by Southern blot analysis.12 To obtain sequence information relating to SINE-R-type retroposons in hominoid primates, genomic DNAs from the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and agile gibbon (Hylobates agilis) were amplified by PCR using primers JS902 (5 9 -GAGA ATGGGCCATGATGAC-3 9 , bases 4±22) and JS903 (59 GATCATTCTTGGATGTTTCT-3 9 , bases 466±485) derived from schizophrenic brain S11 cDNA (GenBank, accession No. AA772777). The PCR conditions followed were those of Kim et al. with an annealing temperature of 56°C. PCR products were separated on a 1.8% agarose gel, purified with the QIAEX II gel extraction kit (Qiagen, Chatsworth, CA) and cloned into the T-khs307 vector.14 Plasmid DNA was isolated by the alkali lysis method, using the High Pure plasmid isolation kit (Boehringer Mannheim, Indianapolis, IN). Individual plasmid DNAs were screened for inserts by PCR and positive samples were subjected to sequence analyses on both strands with T7 and M13 reverse primers using an automated DNA sequencer (model 373A; Applied Biosystems, Foster City, CA) and the DyeDeoxy terminator kit. Sequence analyses were done with the aid of GAP and PILEUP from the GCG program. The neighbor-joining phylogenetic analysis was performed with the CLUSTAL W program.15 We searched for sequence homology using the BLAST network server.16 Using genomic DNA from hominoid primates, SINE-R-type retroposons were amplified and nucleotide sequences deter-