1994 Fiscal Year Final Research Report Summary
Molecular evolution of aldolase in insect ; gene structure and isozyme generation.
| Project/Area Number |
05660390
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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 |
Applied molecular and cellular biology
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| Research Institution | Seinan-Jogakuin Junior College |
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Principal Investigator |
SUGIMOTO Yasushi Seinan-Jogakuin Junior College, Department of Food and Nutrition, Associate Professor, 食物栄養科, 助教授 (10100736)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Aldolase / Isozyme / Molecular evolution / Differential expression / Insect / Drosophila melanogaster / Bombyx mori |
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| Research Abstract |
Drosophila melanogaster generates three differnt types of aldolase mRNAs from a single gene by selective usage of the triplicate exons 4 (4alpha, 4beta and 4gamma) , which encode three different isozymes having respective carboxyl termini. Moreover, we have found the presence of a novel-type mRNA (named alpha^<beta>) in which the two final exons 4alpha and 4beta were retained unspliced, but the significance of this mRNA was obscure. Here, a cDNA clone containing the alpha^<beta> sequence was inserted into pINIII and expressed in an Escherichia coli system. The product, which exhibited aldolase activity, was found to be isozyme alpha in term of the enzymological properties and the primary structure with the 4alpha sequence alone being present as the carboxyl terminus. The D.melanogaster aldolase gene has a typical poly (A) signal at the 3' end in the exon 4beta but not in the exon 4alpha. This situation probably restrains the production of mRNA with the alpha-sequence alone to a low lev
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el and enforces the alpha^<beta>-type mRNA production. Normal alpha-mRNA might be produced only when AATATA,residing downstream of the coding frame in the exon 4beta, is recognized as a poly (A) signal during RNA processing.
The silkworm, Bombyx mori, has the two types of aldolase isozymes S and F.These were found to interchange during embryonic devel-opment. Enzymatic properties were investigated using purified iso-zymes. Isozyme S was stable while F was unstable toward thermal teratment, some chemical reagents and freezing. S was expressed specifically in the eggs at early stages and in the postnatal fat bodies etc.as well as in the ovaries. F was detected strongly in the neonates and in the postnatal muscles. The S and F isozymes exhibited the values of 3.0 and 10.0, respectively, for the activity ratios vs.the two substrates (FBP/FIP). The latter value was similar to those of Drosophila melanogaster aldolase and human aldolase C.Seven clones (not specified for S or F) were isolated from the fat body cDNA library of B.mori larva using a D.melano-gaster aldolase cDNA as a prove. One of them was sequenced and found to have 80% homology to D.melanogaster aldolase gene. Northern and Southern blot analyzes indicated that S and F are presumably coded for by distinct genes, the situation different from that of the D.melanogaster aldolase system where plural mRNAs are produced by alternative splicing of a single gene product. Less
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