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

果蝇（Drosophilamelanogaster）通过选择性地使用一式三份的外显子4（4 α、4 β和4 γ）从单个基因产生三种不同类型的醛缩酶mRNA，所述外显子4编码三种具有各自羧基末端的不同同工酶。此外，我们还发现了一种新型mRNA（命名为α ^<beta>），其中最后两个外显子4 α和4 β未被剪接，但这种mRNA的意义尚不清楚。在此，将含有α序列的cDNA克隆<beta>插入pIN III中，并在大肠杆菌系统中表达。该产物表现出醛缩酶活性，被认为是同工酶α的酶学性质和一级结构与4 α序列单独存在作为羧基末端。黑腹果蝇醛缩酶基因在外显子4 β的3'端具有典型的poly（A）信号，但在外显子4 α中没有。这种情况可能会将单独使用α序列的mRNA的产生抑制到较低水平。 ...更多信息 el并促进α ^<beta>-型mRNA的产生。正常的α-mRNA只有当位于外显子4 β编码框下游的AATATA在RNA加工过程中被识别为poly（A）信号时才能产生。酶的性质进行了研究，使用纯化的同工酶。同工酶S对热处理、某些化学试剂和冷冻不稳定，而F对热处理、某些化学试剂和冷冻不稳定。S在早期阶段的卵子和出生后的脂肪体以及卵巢中特异性表达。etc.as在新生儿和出生后的肌肉中检测到强烈的F。S和F同工酶的活性比值分别为3.0和10.0。后者的值与果蝇和人的醛缩酶C的值相似。以一个果蝇醛缩酶cDNA作为证据，从B.mori幼虫脂肪体cDNA文库中分离到7个克隆（不指定S或F）。对其中一个克隆进行了测序，发现与果蝇醛缩酶基因有80%的同源性。北方和南方印迹分析表明，S和F可能是由不同的基因编码的，这种情况与黑腹果蝇醛缩酶系统不同，在该系统中，多个mRNA是通过单个基因产物的选择性剪接产生的。少

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