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

黑腹果蝇通过选择性地使用三复制外显子4 (4α， 4β和4γ)，从一个基因产生三种不同类型的醛缩酶mrna，它们编码三种不同的同工酶，具有各自的羧基端。此外，我们还发现了一种新型mRNA（命名为alpha^<beta>）的存在，其中最后两个外显子4alpha和4beta未剪接，但这种mRNA的意义尚不清楚。在这里，一个含有α ^< β >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，在酶学性质和初级结构方面被发现为α同工酶，仅4 α序列作为羧基端存在。D.melanogaster醛缩酶基因外显子4 β的3′端有典型的poly (a)信号，而外显子4 α没有。这种情况可能将α -序列单独产生的mRNA抑制到低水平，并加强α ^< β >型mRNA的产生。正常的α - mrna只有在编码框下游外显子4 β的AATATA在RNA加工过程中被识别为poly (a)信号时才能产生。家蚕（Bombyx mori）具有醛脲酶同工酶S和f两种类型，它们在胚胎发育过程中被发现相互交换。用纯化的同工酶研究酶学性质。同工酶S对热处理、部分化学试剂和冷冻不稳定，F不稳定。S在早期卵子和出生后脂肪体等以及卵巢中特异性表达。F在新生儿和产后肌肉中检测强烈。S和F同工酶对两种底物的活性比（FBP/FIP）分别为3.0和10.0。从家蚕幼虫脂肪体cDNA文库中分离出7个克隆（未指定为S或F），并以黑胃醛缩酶cDNA作为证明。对其中一个基因进行测序，发现与黑腹霉醛缩酶基因有80%的同源性。Northern和Southern blot分析表明，S和F可能是由不同的基因编码的，这与d.m anogaster醛缩酶系统的情况不同，该系统通过单个基因产物的选择性剪接产生多个mrna。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