Gines M J, Dove M J, Seligy V L
Division of Biological Sciences, National Research Council of Canada, Ottawa.
Gene. 1989 Jun 30;79(1):107-17. doi: 10.1016/0378-1119(89)90096-6.
cDNA and genomic DNA for two nearly identical genes, AmyI and AmyII, coding for the enzyme Taka-amylase A (TA-A) of the fungus Aspergillus oryzae have been cloned and characterized. These genes are apparently unlinked, differing by only 3 nucleotides (nt) out of the 2720 nt that span the coding regions. The 617-nt 5'-flanking regions differ only at nt -372 (T or A) from the putative ATG start codon and contain four sets of short, inverted repeats (IR) upstream from the putative TATAAA box at nt -100 and the transcription start point at nt -69. The coding regions consist of 499 codons disrupted by eight intervening sequences. The putative proenzymes differ by only two amino acids (aa) and consist of the 478-aa extracellular enzyme plus a 21-aa hydrophobic leader sequence. Except for the replacement site changes in codons 35 (Arg----Gln) and 151 (Phe----Leu), the identity of the two genes continues downstream for 58 nt past the TGA stop codon before diverging. Exon 9 codes for 94 of the 98 aa of the domain B of mature TA-A. Little conservation of TA-A exons was found when these exons were aligned with those of human amylase. The genes are flanked by at least 6 to 10 kb of unrelated chromosomal nucleotide sequence. The Amy genes are co-expressed, since mRNA (cDNA) specific to the 3'-UTR of both genes was recovered from mycelia grown on starch, a known inducer of TA-A biosynthesis. The 3'-UTRs of cDNAs related to AmyI are shorter (128 nt and 145 nt) than those of AmyII (179 nt and 297 nt). The AmyI specific 3'-UTR is characterized by the absence of IR sequences and the presence of a putative 'AATAAA' polyadenylation signal.
已克隆并鉴定了编码米曲霉真菌的耐热淀粉酶A（TA-A）的两个几乎相同的基因AmyI和AmyII的cDNA和基因组DNA。这些基因显然不连锁，在跨越编码区的2720个核苷酸（nt）中仅相差3个核苷酸。617 nt的5'侧翼区与推定的ATG起始密码子仅在nt -372（T或A）处不同，并且在nt -100处的推定TATAAA框和nt -69处的转录起始点上游包含四组短的反向重复序列（IR）。编码区由499个密码子组成，被八个间隔序列打断。推定的酶原仅相差两个氨基酸（aa），由478个aa的细胞外酶加上一个21个aa的疏水前导序列组成。除了密码子35（Arg→Gln）和151（Phe→Leu）的替换位点变化外，这两个基因的一致性在TGA终止密码子下游持续58 nt，然后才出现分歧。外显子9编码成熟TA-A结构域B的98个aa中的94个。当这些外显子与人淀粉酶的外显子比对时，未发现TA-A外显子有太多保守性。这些基因两侧至少有6至10 kb的不相关染色体核苷酸序列。Amy基因共表达，因为从在淀粉上生长的菌丝体中回收了两个基因3'-UTR特异的mRNA（cDNA），淀粉是TA-A生物合成的已知诱导剂。与AmyI相关的cDNA的3'-UTR比AmyII的3'-UTR短（128 nt和145 nt）（179 nt和297 nt）。AmyI特异的3'-UTR的特征是没有IR序列且存在推定的“AATAAA”聚腺苷酸化信号。
