| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1990: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Research Abstract |
Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVRalpha, PVRbeta, PVRgamma, and PVRdelta. The predicted amino acid sequences indicate that PVRalpha and PVRdelta, corresponding to the previously described cDNA clones H20A and H20B, respectively, and integral membrane proteins while the other two molecules lack a putative transmembrane domain. Mouse cell transformants carrying PVRalpha were permissive for poliovirus infection, but those carrying PVRbeta were hardly permissive. In contrast to PVRalpha, PVRbeta was not detected on the sur
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face of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVRalpha, PVRbeta and PVRgamma were detected by polymerase chain reaction using appropriate primers in poly (A)^+RNAs of the brain, leukocyte, liver, lung and placenta of humans ; the choice of primers used did not permit detection of PVRdelta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1 - q13.2 of human chromosome 19. Nucleotide sequence analysis of possible requratory region of the receptor gene did not clarify TATA box of the gene. Furthermore position of transcriptional initiation was not detected. It is possible therefore that unique initiation mechanism for the transcription is involved in the expression of PVR gene. To know whether similar reguration occur in a different animal species, poliovirus-sensitive transgenic mice were produced by introducing the human PVR gene into the mouse genome. Expression of the receptor mRNAs in tissues of the transgenic mice was analyzed by using RNA blot hybridization and the polymerase chain reaction. The human gene is expressed in many tissues of the transgenic mice just, although strong expression was observed only in the brain and spinal cord just like as in tissues of humans. Thus the expression of the human PVR gene seems to be controlled by a similar mechanisms both in humans and mice. Less
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