1. ABSTRACT Isolation and phylogeny of endogenous retroviral elements belonging to the HERV-K LTR in cDNA library of human fetal brain and X q 21.3 region linked to psychosis Joo-Young Choi, Seung-Heui Jeon, Joo-Mi Lee, Jun-Seop Kim, Kyoung-Mi Shin, Won-Ho Lee, and Heui-Soo Kim HTTP://mpl.biology.or.kr Division of Biological Sciences, College of Natural Sciences, Pusan National University HERV-K family of human endogenous retroviral sequences has been originally cloned from Syrian hamster intra-cisternal A type particles, has homology to mouse mammary tumour virus (MMTV), and includes sequences that are expressed in normal placenta. The HERV-K LTR elements have randomly transposed across the chromosomes in the course of human evolution. We identified five HERV-K LTR elements from the cDNA library of human fetal brain. Two of them (FB-1 and FB-5) are closely related to the human specific HERV-K LTR elements by phylogenetic analysis. We also investigated such an elements within the Xq21.3 region linked to psychosis that was replicated on the Y chromosome after the separation of the chimpanzee and human lineages. Four element of HERV-K LTR were identified in that region. Two of these elements (K-X10-5 and K-X13-1) have a high degree of sequence similarity to the human specific HERV-K LTR. To locate such elements and determine their possible relationship to genes that have contributed to late developments in human evolution provides a strategy for investigating the role of retroviruses/retrotransposons in species-related hominid characteristics. MATERIALS & METHOD Cloning of PCR Products Qiagen II gel extraction kit, T-khs307 vector, High pure Plsmid Isolation kit cDNA Library of Human Fetal Brain YAC Clone Analysis DNA Sequencing Automated DNA sequencer (Model 373A) BLAST Search Phylogenetic Analysis GCG & MEGA programs STRUCTURE of HERV-K LTR INTRODUCTION Long Terminal Repeat (LTR) 1. Play a role in regulating gene located nearby 2. U3 element contains a specific protein binding site and transcription control elements (enhancer and promoter) Research Purpose 1. To isolate HERV-K LTR elements that were expressed in cDNA library of human fetal brain and Xq21.3 region. 2. To determine nucleotide sequences of the LTR elements in cDNA library of human fetal brain and Xq21.3 region. 3. To examine phylogenetic relationships with those of human specific HERV-K LTRs. CONCLUSION 1.Two element (FB-1 and FB-5) were expressed in human fetal brain and proliferated recently in human genom by the analysis of sequence similarity and molecular phylogeny. 2.Two element (K-X10-5 and K-X13-1) in Xq21.3 region are members of the HERV-K LTRs that are known to have proliferated most recently in the human genome. Fig. 2. Nucleotide sequence alignments of HERV-K LTR family. Dashes(-) indicate no change to the consensus sequence and dots indicate gaps. Table 1. Percentage similarity of nucleotide sequences of HERV-K LTR family RESULTS & DISCUSSION M Human Fetal Brain cDNA K-X7 K-X10 K-X13 (A)(B) Fig. 1. PCR analyses of HERV-K LTR elements from cDNA library of human fetal brain (A) and YAC clone panels of the human Xq21.3 region (B). Consensus CATGTGATAG T-CTGAAATA TGGCCTCGT- GGGAAGGGAA AGACCTGACC K10LTR ---------- -.-------- ---------. ---------- ---------- AC002400 ---------- -.-------- ---------. ---------- ---------- AC002508 ---------- -.-------- ---------. ---------- ---------- AL034407 ---------- -.-------- ---------. ---------- ---------- L47334 ---------- -.-------- ---------. ---------- ---------- U47924 ---a------ -.-------- ---------. ---------- ---------- Z80898 -------g-- -.-------- ---------. ---------- ---------- FB-1 ---------- -.-------- ---------. ---------- ---------- FB-2 ---------- -.-------- ---------g ------a--- ---------- FB-4 -----a---- -.-------- ---------a --a----a-- gac-tgactg FB-5 ---------- -c-------- ---------. ---------- ---------- FB-6 ---------- -.-------- -------a-. ---------- ---------- K-X7-3 ---------- -.----g--- -------c-. ---------- --g------- K-X10-5 ---------- -.-------- ca-------. ---------- ---------- K-X13-1 ---------- -.-------- ---------. ---------- ---------- K-X13-2 ---------- -.-------- ---------. ---------- ---------- Consensus TCTCCTGCCT GTCCCTGGGC AATGGAATGT CTCGGTATAA AACCC----G K10LTR ---------- ---------- ---------- ---------- -----....- AC002400 ---------- ---------- ---------- ---------- -----....- AC002508 ---------- ---------- ---------- ---------- -----....- AL034407 ---------- ---------- ---------- ---------- -----....- L47334 ---------- ---------- ---------- ---------- -----....- U47924 --a------- ---------- ---------- --t------- -----....- Z80898 ---------c ---------- ---------- ---------- -----....- FB-1 ---------- ---------- ---------- ---------- -----....- FB-2 ---------c ---------- ---------- ---------- -----....- FB-4 ---------- ------a--- ---------- ---a------ -----....- FB-5 ---------- ---------- ---------- ---------- -----....- FB-6 ---------c ---------- ---------- ---------- -----gatt- K-X7-3 --------tc a--------- ---------- ---a--g--- -----....- K-X10-5 ---------- ---------- ---------- ------g--- -----....- K-X13-1 ---------- ---------- ---------- ---------- -----....- K-X13-2 ---------c ---t------ ---------- ---a------ ----a....- K10LTR AC002400 AC002508 AL034407 L47334 U47924 Z80898 FB-1 FB-2 FB-4 FB-5 FB-6 K-X7-3 K-X10-5 K-X13-1 K-X13-2 72 97 81 40 58 50 36 0.02 Fig 4. Phylogenetic trees obtained by neighbor-joining method for the HERV-K LTR family. (A) (B) K10LTR AL034407 K-X13-2 AC005562 AF164611 AC006029 AC002508 AC006039 AF164620 AL031654 M57950 AC002400 AC007326 AF164609 AC007325 L47334 AF017229 AC007227 K-X13-1 X72790 Hkl9-8 Hkl9-5 Hkl17-9 Hkl17-4 Hkl17-2 Hkl8-5 Hkl8-6 AF164615-1 AF164615 AB022756 AC006035 AF164614-1 AF164614 Y17832-1 Y17832 AB022753 AB022755 AC006366 AC005799 AF148679 AC016577 M12851 M12854 K-X10-5 AB022741 AB022746 AP001748 AC008062 Hkl9-3 Hkl9-4 Hkl9-7 Hkl8-4 Hkl9-6 Hkl8-2 Hkl8-7 Hkl18-7 Hkl18-8 AL109763 AF240627 AF164610 AC008553 U73641 Z80898 AC006432 AC004924 AF164612-1 AB022743 AB022749 AC000401 AC008996 AF017215 AC000387 AC007690 AL009176 AB022757 AC005820 AB022758 AF017226 AP000353 Hkl17-1 Hkl17-10 Hkl17-3 Hkl9-2 Hkl8-1 Hkl9-9 Hkl9-1 Hkl8-3 Hkl17-5 Hkl17-8 Hkl17-6 Hkl17-7 Hkl18-4 Hkl18-6 AC007684 AL133448 Hkl18-3 Hkl18-5 Hkl18-2 Hkl18-1 AC005091 AC004448 AL031283 AL121928 AL049570 AC011494 AF017227 AF164616 AF164619 AC004876 AF017221 AF017220 AC005524 AL009179 AF134984 AF012335 M12852 X87344 Y18890 AL163285 L35659 M12853 K-X7-3 AF164618 AL121985 FB-1 FB-2 FB-4 FB-5 FB-6 99 53 96 91 99 95 97 82 95 94 89 94 87 71 80 81 52 60 91 76 67 91 55 92 83 61 57 54 65 53 52 0.02 AL031774 AB023916 Z80898 U91328 82 64 92 REFERENCE 1. Akopov, S. B., Nikolaev, L. G., Khil, P. P., Lebedev, Y. B., & Sverdlov, E. D. (1998). Long terminal repeats of human endogenous retrovirus K family (HERV-K) specifically bind host cell nuclear proteins. FEBS Letters 421, 229-233. 2. Kim, H.-S. & Crow, T. J. (1999). Presence and phylogenetic analysis of HERV-K LTR on human X and Y chromosomes: Evidence for recent. Gene Genetic System 74, 267- 270. 3. Kim, H.-S., Wadekar, R. V., Takenaka, O., Winstanley, C., Mitsunaga, F., Kageyama, T., Hyun, B.-H., & Crow, T. J. (1999). SINE-R.C2 (a Homo sapiens specific retroposon) is homologous to cDNA from post-mortem brain in schizophrenia and to two loci in the Xq21.3/Yp block linked to handedness and psychosis. American Journal of Medical Genetics (Neuropsychiatric Genetics) 88, 560-566. FB-1 (AB046582) FB-5 (AB046585) FB-2 (AB046583) FB-6 (AB046586) FB-4 (AB046584) K-X7-3 (AB0022774) K-X13-1 (AB0022776) K-X10-5 (AB0022775) K-X13-2 (AB0022777)