Molecular evolution of aldolase in insect ; gene structure and isozyme generation.

昆虫醛缩酶的分子进化；

• 批准号: 05660390
• 负责人: SUGIMOTO Yasushi
• 金额: $ 1.09万
• 依托单位: Seinan-Jogakuin Junior College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660390/
• 关键词: Aldolase; Isozyme; Molecular evolution; Differential expression; Insect; Drosophila melanogaster; Bombyx mori; ショウジョウバエ; 遺伝子

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 … More 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

果蝇通过选择性地利用三重外显子4(4α、4β和4伽马)从单个基因产生三种不同类型的醛缩酶mRNAs，这些外显子编码三种不同的同工酶，具有各自的羧基末端。此外，我们还发现了一种新型的mRNA(命名为α^&lt；β&gt；)，其中最后两个外显子4α和4β保持未剪接，但该mRNA的意义尚不清楚。在此，一个包含α、β和gt；序列的cDNA克隆被插入到pINIII中，并在大肠杆菌系统中表达。该产物具有醛缩酶活性，从酶学性质和一级结构来看，该产物为同工酶α，其中4α序列为羧基末端。该基因在外显子4β的3‘端有典型的聚(A)信号，而在外显子4α没有。这种情况可能会将仅含有α序列的mrna的产生抑制到低水平的…。更多的EL，并加强阿尔法^；β；类型的mRNA的产生。只有当位于外显子4β编码框下游的AATATA在RNA加工过程中被识别为聚(A)信号时，才可能产生正常的α-信使核糖核酸。家蚕具有两种醛缩酶同工酶S和F。这两种同工酶在胚胎发育过程中被发现互换。用纯化的同工酶对其酶性质进行了研究。同工酶S对热处理、某些化学试剂和冷冻不稳定，F对热处理、某些化学试剂和冷冻不稳定。S在早期卵细胞、出生后脂肪体等组织以及卵巢中均有特异表达。F在新生儿和出生后肌肉中均有较强的表达。S同工酶和F同工酶的活性比(FBP/FIP)分别为3.0和10.0。后者与果蝇黑腹果蝇和人醛缩酶C相似。用黑腹果蝇醛缩酶基因从家蚕幼虫脂肪体文库中分离到7个克隆(非S或F)。对其中一个进行了测序，发现与黑腹葡萄球菌的醛缩酶基因有80%的同源性。Northern和Southern杂交分析表明，S和F可能是由不同的基因编码的，这与黑腹菌醛缩酶系统的情况不同，在该系统中，通过单个基因产物的选择性剪接产生多个mRNA。较少

项目成果

T.Kusakabe,K.Motoki,Y.Sugimoto et al.: "Structural and functional divergence of aldolase isozymes:I.aldolase is composed of three functionally distinct units,type-B isozyme group-specific sequences,β-barrel and connectors." Protein Engneering. 7(10). 1387

T. Kusakabe、K. Motoki、Y. Sugimoto 等人：“醛缩酶同工酶的结构和功能差异：I. 醛缩酶由三个功能不同的单元组成：B 型同工酶组特异性序列、β-桶和连接器。 “蛋白质工程。7(10)。1387

M.Yara,E.Kosegawa,Y.Sugimoto and K.Koga: "Comparison of translation product of mRNA from unfertilized and early eggs of Bombyx mori." J.Seric.Sci.Jpn.63(4). 336-340 (1994)

M.Yara、E.Kosekawa、Y.Sugimoto 和 K.Koga：“家蚕未受精卵和早期卵 mRNA 翻译产物的比较”。

S.Nagaoka,Y.Sugimoto,M.Yara and K.Koga: "Occurrence and distribution of aldolase isozymes during embryonic and post-embryonic development of the silkworm,Bombyx mori." Insect Biochem.Mol.Biol.(in press). (1995)

S.Nagaoka，Y.Sugimoto，M.Yara 和 K.Koga：“家蚕胚胎和胚胎后发育过程中醛缩酶同工酶的出现和分布。”

M.Yara,E.Kosegawa,Y.Sugimoto and K.Koga: "Comparison of translation product of mRNA from unfertilized and early eggs of Bombyx mori." J.Seric.Sci.Jpn.63(4). 336-340 ((1994))

M.Yara、E.Kosekawa、Y.Sugimoto 和 K.Koga：“家蚕未受精卵和早期卵 mRNA 翻译产物的比较”。

M.Yara,E.Kosegawa,Y.Sugimoto and K.Koga: "Comparison ot translation product of mRNA from unfertilized and early eggs of Bombyx mori." J.Seric.Sci.lpn.63(in press). (1994)

M.Yara、E.Kosekawa、Y.Sugimoto 和 K.Koga：“家蚕未受精卵和早期卵 mRNA 翻译产物的比较”。