Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

A Novel Human Lipid Binding Protein Coding Gene: PERF15, Sequence and Cloning

Authors
1 Department of Genetics, Faculty of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran; Fertility-Infertility Health Research Center (IRHRC), Taleghani Hospital, Shaheed Beheshti University of Medical Sciences,
2 Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
3 Molecular Biology Research Center, Faculty of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran
4 Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Abstract
Introduction: PERF15 is a testicular germ-cell specific fatty-acid binding protein (FABP) isolated from mammals, originally from rats. It encodes one of the most abundant proteins of rat spermatozoa localized in the perinuclear theca. Northern blot analysis has demonstrated that rat PERF15 mRNA is exclusively transcribed during meiosis and post-meiosis. In this study, we cloned and sequenced human PERF15 gene.Materials and Methods: According to the open reading frame of automated computational analysis of Homo sapiens similar to testis fatty acid binding protein nine, two specific Primers were designed to amplify human PERF15 gene. To confirm the identity of the amplified gene, PCR products of PERF15 were cloned into appropriate plasmid vectors followed by sequencing of the inserts.Results: A unique band of ~3kb was obtained after PCR amplification. Restriction enzyme digestion using PvuII confirmed that the fragment was related to PERF15. Gene alignment, direct sequencing and application of specific primers to the gene showed 100% similarity between this gene and the computational data by gel extraction of the ~3 kb band. The human PERF15 gene contained four exons and three introns. Exons one, two, three and four, respectively, coded for 24, 57, 34 and 17 amino acids. The existing three introns were composed of 2113, 461, and 168 nucleotides.Conclusion: In spite of the homology between exonic regions and exon-intron boundaries of human PERF15 gene and that of animals, human PERF15 gene is different in size and sequence from corresponding introns in rat and murine PERF15.
Keywords

  1. Russell LD, Chiarini-Garcia H, Korsmeyer SJ, Knudson CM. Bax-dependent spermatogonia apop-tosis is required for testicular development and spermatogenesis. Biol Reprod. 2002;66(4):950-8.
  2. Pouresmaeili F, Morales CR, Oko R. Molecular cloning and structural analysis of the gene encoding PERF 15 protein present in the perinuclear theca of the rat spermatozoa. Biol Reprod. 1997;57(3):655-9.
  3. Ward WS, Kimura Y, Yanagimachi R. An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development. Biol Reprod. 1999;60(3):702-6.
  4. Oko R, Moussakova L, Clermont Y. Regional dif-ferences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry. Am J Anat. 1990;188(1):64-73.
  5. Liu RZ, Li X, Godbout R. A novel fatty acid-binding protein (FABP) gene resulting from tandem gene duplication in mammals: transcription in rat retina and testis. Genomics. 2008;92(6):436-45.
  6. Kido T, Namiki H. Expression of testicular fatty acid-binding protein PERF 15 during germ cell apoptosis. Dev Growth Differ. 2000;42(4):359-66.
  7. Grogan WM, Farnham WF, Szopiak BA. Long chain polyenoic acid levels in viably sorted, highly en-riched mouse testis cells. Lipids. 1981;16(6):401-10.
  8. Kido T, Arata S, Suzuki R, Hosono T, Nakanishi Y, Miyazaki J, et al. The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice. Dev Growth Differ. 2005;47(1):15-24.
  9. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162(1):156-9.
  10. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory; c1989. Chapter 30, Nucleic Acids Research.
  11. Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Michigan: Cold Spring Harbor Laboratory Press; c2001. p. 6.28-6.30.
  12. Steven A. Williams, Barton E. Slatko, John R. McCarrey. Laboratory investigations in molecular biology.1st ed. Canada: Jones & Bartlett Publishers; c2006. p. 213.
  13. Zalata AA, Christophe AB, Depuydt CE, Schoonjans F, Comhaire FH. The fatty acid com-position of phospholipids of spermatozoa from infertile patients. Mol Hum Reprod. 1998;4(2): 111-8.
  14. Oko R, Morales CR. A novel testicular protein, with sequence similarities to a family of lipid binding proteins, is a major component of the rat sperm perinuclear theca. Dev Biol. 1994;166(1): 235-45.
  15. Oko R, Maravei D. Distribution and possible role of perinuclear theca proteins during bovine sperm-iogenesis. Microsc Res Tech. 1995;32(6):520-32.
  16. Korley R, Pouresmaeili F, Oko R. Analysis of the protein composition of the mouse sperm peri-nuclear theca and characterization of its major protein constituent. Biol Reprod. 1997;57(6):1426-32.
  17. Schmitt MC, Jamison RS, Orgebin-Crist MC, Ong DE. A novel, testis-specific member of the cellular lipophilic transport protein superfamily, deduced from a complimentary deoxyribonucleic acid clone. Biol Reprod. 1994;51(2):239-45.