人睾丸特异的磷酸吡哆醛激酶变异剪接体PKH-T表达研究
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摘要
目的：在睾丸基因芯片与成人、胚胎睾丸及精子探针杂交发现PKH-T基因的基础上，研究其表达。方法：对目的克隆进行测序，得到其全长序列。利用生物信息学的方法分析测序结果。通过RT-PCR的方法检测出其在不同人体组织、不同生理时期男性睾丸、不同临床病理诊断的男性不育患者睾丸组织中表达的差异。结果：通过如上实验克隆一种新的人类睾丸特异全长基因PKH-T。PKH-T的cDNA全长1069bp。此cDNA序列被输入到Genbank，登录号为AY303972。蛋白质结构分析显示PKH-T包含几乎所有PKH基因的保守基序。同源比对显示由PKH-T的开放阅读框架(ORF)推导出的239个氨基酸与人类基因PKH(U89606)表达的蛋白质同源。实验结果显示PKH-T高度特异地在睾丸组织表达，并且信号强度较高。对PKH和PKH-T在不同发育阶段的男性睾丸中不同表达进行研究发现，PKH-T在成人睾丸和精子中有表达，而PKH在成人睾丸、胚胎睾丸、老年人的睾丸中有表达。用RT-PCR的方法研究发现PKH-T在唯支持细胞综合症和部分生精阻滞病人中表达缺失。结论：PKH-T是一个在成人睾丸和精子高度表达的基因。它可能在人类精子发生的过程中发挥重要作用，并可能与男性不育相关。
Aim: To identify specifically expressed genes in adult testes, fetal testes and human spermatozoa. Methods: A human testis cDNA microarray was established. Then mRNAs of human adult testis, fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as template. The microarray was hybridized with probes of adult testes, fetal testes and spermatozoa. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results: A novel human testis-specific gene, PKH-T was identified by hybridizing adult testis, fetal testis, and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1,069 bp in length. The cDNA sequence of this clone was deposited in Genbank (AY303972). PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041.) Protein analysis showed that PKH-T contained most PKH conserved motif. Homologous analysis showed that the 239 amino acid sequences deduced
from the 719 bp open reading frame (ORF) had a homology with the human gene PKH (U89606). Expression profiles showed that
PKH-T was specifically strongly expressed in testis. Comparison of the differential expressions ofPKH and PKH-T in male testis of different developmental stages indicated that PKH-T expressed in adult testis and spermatozoa, but PKH expressed in adult testis, fetal testis, and aged testis. RT-PCR result indicated that PKH-T had no expression in patient testis of SCOS and partly spermatogenic arrest. Conclusion: PKH-T is a gene highly expressed in adult testis and spermatozoa. It may play an important role in spermatogenesis in humans and may be related with male infertility.
Aim: To identify specifically expressed genes in adult testes, fetal testes and human spermatozoa. Methods: A human testis cDNA microarray was established. Then mRNAs of human adult testis, fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as template. The microarray was hybridized with probes of adult testes, fetal testes and spermatozoa. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results: A novel human testis-specific gene, PKH-T was identified by hybridizing adult testis, fetal testis, and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1,069 bp in length. The cDNA sequence of this clone was deposited in Genbank (AY303972). PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041.) Protein analysis showed that PKH-T contained most PKH conserved motif. Homologous analysis showed that the 239 amino acid sequences deduced
from the 719 bp open reading frame (ORF) had a homology with the human gene PKH (U89606). Expression profiles showed that
PKH-T was specifically strongly expressed in testis. Comparison of the differential expressions ofPKH and PKH-T in male testis of different developmental stages indicated that PKH-T expressed in adult testis and spermatozoa, but PKH expressed in adult testis, fetal testis, and aged testis. RT-PCR result indicated that PKH-T had no expression in patient testis of SCOS and partly spermatogenic arrest. Conclusion: PKH-T is a gene highly expressed in adult testis and spermatozoa. It may play an important role in spermatogenesis in humans and may be related with male infertility.
引文
1. Eddy EM. Regulation of gene expression during spermatogenesis. Semin Cell Dev Biol 1998; 9:451-7.
2. Hecht NB. Molecular mechanism of male germ cell differentiation. Bioessays 1998; 20: 555-61.
3. Sutton KA. Molecular mechanisms involved in the differentiation of spermatogenic stem cells. Rev Reprod 2000; 5: 93-8.
4. McCarrey JR. Spermatogenesis as a model system for developmental analysis of regulatory mechaniams associated with tissue-specific gene expression. Semin Cell Dev Biol 1998; 9: 459-66.
5. Okabe M, Ikawa M, Ashkenas J. Male infertility and the genetics of spermatogenesis. Am J Hum Genet 1998; 62(6): 1274-81.
6. Sha JH, Zhou ZM, Li JM, Yin L1, Yang HM, Hu GX, et al. Identification of testis development and spermatogenesis-related genes in human and mouse testes using cDNA arrays. Mol Hum Reprod. 2002; 8(6): 511-7.
7. Sha JH, Zhou ZM, Li JM, Lin M, Zhu H, Zhu H, et al. Expression of a novel bHLH-Zip gene in human testis. Asian J Androl 2003; 5(2): 83-8.
8. Yang Y, G Zhao, Winkler ME. Identification of the pdxK gene that encodes pyridoxine (vitamin B_6) kinase in Escherichia coli K-12. FEMS Microbiol Lett 1996; 141: 89-95.
9. Yong Yang, Tiffany HT, TK Man, ME Winkler. Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5'-Phosphate Biosynthesis in Escherichia coli K-12. J Bacteriol 1998; 180: 1814-21.
10. Michael CH, Anthony JT, Ewen FK. Human Pyridoxal Kinase .J Biol Chem 1997; 272(16): 10756-60..
11. TC Scott, MA Phillips. Characterization of Trypanosoma brucei pyridoxal kinase: purification, gene isolation and expression in Escherichia coli. Mol Biochem Parasitol 1997; 88(1-2): 1-11.
12. G Scholz, F Kwok. Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site. J Biol Chem 1989; 264: 4318-21.
13. P Dominici, G Scholz, F Kwok, JE Churchich. Affinity labeling of pyridoxal kinase with adenosine polyphosphopyridoxal. J Biol Chem 1988; 263:14712-6.
14. Dym M. Spermatogonial stem cells of the testis. Proc Natl Acad Sci U S A 1994;91:11287-9.
15. Wilkinson DC,Nieto MA.Detection of messenger RNA by in
situ hybridization to tissue sections and whole mounts.Methods Enzymol 1993;225:361-73.
16. Liang P,Pardee AB.Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.Science 1992;257:967-71.
17. Eickhoff H,Schuchhardt J,Ivanov I,Meier-Ewert S, O'Brien J,Malik A,et al.Tissue gene expression analysis using arrayed normalized cDNA libraries.Genome Res 2000; 10:1230-40.
18. JA Kerry, M Rohde, F Kwok. Brain pyridoxal kinase. Purification and characteriz ation. Eur J Biochem 1986; 158:581-5.
19. McCormick DB, Snell EE. Pyridoxal Phosphokinases. Ⅱ. EFFECTS OF INHIBITORS. J Biol Chem 1961; 236:2085-8.
20. F Kwok, JE Churchich. Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine. J Biol Chem 1979; 254:6489-95.
21. F Kwok, JE Churchich. The interaction of paramagnetic ions chelated to ATP with pyridoxal analogues. Fluorescence studies of pyridoxal kinase. Eur. J Biochem 1991; 199:157-62.
22. WF Wolkers, JD Gregory, JE Churchich, EH Serpersu. Arrangement of the substrates at the active site of brain pyridoxal kinase. J Biol Chem 1991; 266:20761-6.
23. Daniel Kornitzer, Aaron Ciechanover. Modes of Regulation of Ubiquitin-Mediated Protein Degradation. J Cell Physiol 2000; 182:1-11.
24. Peter Sutovsky. Ubiquitin-Dependent Proteolysis in Mammalian Spermatogenesis, Fertilization, and Sperm Quality Control: Killing Three Birds With One Stone. Microsc Res Tech 2003; 61:88-102.
2. Hecht NB. Molecular mechanism of male germ cell differentiation. Bioessays 1998; 20: 555-61.
3. Sutton KA. Molecular mechanisms involved in the differentiation of spermatogenic stem cells. Rev Reprod 2000; 5: 93-8.
4. McCarrey JR. Spermatogenesis as a model system for developmental analysis of regulatory mechaniams associated with tissue-specific gene expression. Semin Cell Dev Biol 1998; 9: 459-66.
5. Okabe M, Ikawa M, Ashkenas J. Male infertility and the genetics of spermatogenesis. Am J Hum Genet 1998; 62(6): 1274-81.
6. Sha JH, Zhou ZM, Li JM, Yin L1, Yang HM, Hu GX, et al. Identification of testis development and spermatogenesis-related genes in human and mouse testes using cDNA arrays. Mol Hum Reprod. 2002; 8(6): 511-7.
7. Sha JH, Zhou ZM, Li JM, Lin M, Zhu H, Zhu H, et al. Expression of a novel bHLH-Zip gene in human testis. Asian J Androl 2003; 5(2): 83-8.
8. Yang Y, G Zhao, Winkler ME. Identification of the pdxK gene that encodes pyridoxine (vitamin B_6) kinase in Escherichia coli K-12. FEMS Microbiol Lett 1996; 141: 89-95.
9. Yong Yang, Tiffany HT, TK Man, ME Winkler. Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5'-Phosphate Biosynthesis in Escherichia coli K-12. J Bacteriol 1998; 180: 1814-21.
10. Michael CH, Anthony JT, Ewen FK. Human Pyridoxal Kinase .J Biol Chem 1997; 272(16): 10756-60..
11. TC Scott, MA Phillips. Characterization of Trypanosoma brucei pyridoxal kinase: purification, gene isolation and expression in Escherichia coli. Mol Biochem Parasitol 1997; 88(1-2): 1-11.
12. G Scholz, F Kwok. Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site. J Biol Chem 1989; 264: 4318-21.
13. P Dominici, G Scholz, F Kwok, JE Churchich. Affinity labeling of pyridoxal kinase with adenosine polyphosphopyridoxal. J Biol Chem 1988; 263:14712-6.
14. Dym M. Spermatogonial stem cells of the testis. Proc Natl Acad Sci U S A 1994;91:11287-9.
15. Wilkinson DC,Nieto MA.Detection of messenger RNA by in
situ hybridization to tissue sections and whole mounts.Methods Enzymol 1993;225:361-73.
16. Liang P,Pardee AB.Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.Science 1992;257:967-71.
17. Eickhoff H,Schuchhardt J,Ivanov I,Meier-Ewert S, O'Brien J,Malik A,et al.Tissue gene expression analysis using arrayed normalized cDNA libraries.Genome Res 2000; 10:1230-40.
18. JA Kerry, M Rohde, F Kwok. Brain pyridoxal kinase. Purification and characteriz ation. Eur J Biochem 1986; 158:581-5.
19. McCormick DB, Snell EE. Pyridoxal Phosphokinases. Ⅱ. EFFECTS OF INHIBITORS. J Biol Chem 1961; 236:2085-8.
20. F Kwok, JE Churchich. Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine. J Biol Chem 1979; 254:6489-95.
21. F Kwok, JE Churchich. The interaction of paramagnetic ions chelated to ATP with pyridoxal analogues. Fluorescence studies of pyridoxal kinase. Eur. J Biochem 1991; 199:157-62.
22. WF Wolkers, JD Gregory, JE Churchich, EH Serpersu. Arrangement of the substrates at the active site of brain pyridoxal kinase. J Biol Chem 1991; 266:20761-6.
23. Daniel Kornitzer, Aaron Ciechanover. Modes of Regulation of Ubiquitin-Mediated Protein Degradation. J Cell Physiol 2000; 182:1-11.
24. Peter Sutovsky. Ubiquitin-Dependent Proteolysis in Mammalian Spermatogenesis, Fertilization, and Sperm Quality Control: Killing Three Birds With One Stone. Microsc Res Tech 2003; 61:88-102.