1990 Fiscal Year Final Research Report Summary
On the Protein Carboxyl Methylation of the Aged Protein in the Brain
| Project/Area Number |
01580183
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Yamagata University |
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Principal Investigator |
SATO Michihiko School of Med. Dept. of Molecular and Pathological Biochemistry, Associate Professor, 医学部, 助教授 (00135344)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Tadashi School of Med. Dept. of Molecular and Pathological Biochemistry, Professor, 医学部, 教授 (10004673)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Protein carboxyl methyltransferase / Aging / cDNA for protein carboxyl methyltransferase / Brain / Methylation |
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| Research Abstract |
Two cDNA clones for protein carboxyl methyltransferase were isolated from a rat brain cDNA library in lambdagt 11 with synthetic oligonucleotides as probes. The two clones differ in size, but the nucleotide sequence including the whole coding region of the shorter cDNA is completely identical with the corresponding sequence of the longer cDNA. The open reading frame encodes a polypeptide of 227 amino acid residues, with a molecular weight of 24,626. This molecular weight is comparable to those reported for other protein carboxyl methyltransferases from several animals, which were determined by gel filtration chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Using the coding region of cDNA for PCM as a probe, three transcripts of 3.4 kb, 2.0 kb, and 1.3 kb were detected in rat brain by RNA blot hybridization analysis. The shorter (1.3 kb) transcript may be a product transcribed to the atypical polyadenylation signals, ATTAAA and AAGAAA, positioned at 250 b downstream from the stop codon. However, the property of the longer (3.4 kb) one is not clear yet. Consequently, we performed Southern blot analysis of rat genomic DNA. A single band was detected, when we used 5'-terminal region of the cDNA (about 200 bp) as a probe. Judging from the results, we concluded that single gene for PCM occurred in rat genomic DNA. Following the Southern blot analysis we screened the rat genomic DNA library with the cDNA as a probe and obtained two positive clones. We found 5 exons in these two clones, but which have no 5' and 3' terminal regions of the cDNA. Thus, a new genomic DNA library, which was constructed in lambdaEMBL3 using Sau 3A partial digests of rat genomic DNA was rescreened. We have fortunately obtained several positive clones for the missing regions at the first screening. They may help us to clarify whole structure of the rat PCM gene.
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