Sequence of the cDNA and 5'-flanking region for human acid alpha-glucosidase, detection of an intron in the 5' untranslated leader sequence, definition of 18-bp polymorphisms, and differences with previous cDNA and amino acid sequences.
Sequence of the cDNA and 5'-flanking region for human acid alpha-glucosidase, detection of an intron in the 5' untranslated leader sequence, definition of 18-bp polymorphisms, and differences with previous cDNA and amino acid sequences.
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人酸性α-葡萄糖苷酶的 cDNA 和 5 侧翼区域的序列、5 非翻译前导序列中内含子的检测、18 bp 多态性的定义以及与先前 cDNA 和氨基酸序列的差异。
DOI：
10.1089/dna.1990.9.85
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发表时间：
1990
影响因子：
3.1
通讯作者：
Hirschhorn,R
中科院分区：
文献类型：
--
作者：
Martiniuk,F;Mehler,M;Tzall,S;Meredith,G;Hirschhorn,R
酸性麦芽糖酶或酸性α-葡糖苷酶（GAA）是一种将糖原水解为葡萄糖的溶酶体酶，在II型糖原累积病中缺乏。以前，我们分离出的部分cDNA（1.9 kb）的人GAA，我们现在已经使用此cDNA分离和确定序列在较长的cDNA从另外四个独立的cDNA文库。引物延伸研究表明，mRNA延伸了获得的cDNA序列的约200 bp 5′。因此，我们分离了一个基因组片段，该片段含有与先前的cDNA序列重叠的5′端cDNA序列，并在Kozak共有序列内的起始密码子上额外延伸了24 bp。该基因组克隆的序列显示，在ATG的5′端有一个32 bp的内含子-外显子连接，表明5′前导序列被一个内含子中断。在上游约3 kb处发现了剩余的186 bp的5′非翻译序列。启动子区上游的起始位点的转录是GC丰富的，并含有领域的同源性Sp1结合位点，但没有可识别的CAAT或TATA盒。组合的数据给出了从ATG到终止密码子的编码区的2，856 bp的核苷酸序列，预测了952个氨基酸的蛋白质。3′非翻译区包含555 bp，在3，385 bp处有多聚腺苷酸化信号，随后是16 bp，然后是poly（A）尾。GAA编码区的该序列不同于Hoefslootet等人报道的序列。（1988）在三个区域共改变了42个氨基酸。直接测定这些区域之一的氨基酸序列证实了本文报道的核苷酸序列，但也不同意Hoefslootet等人报道的直接测定的氨基酸序列。（1988年）。在另外两个区域，碱基对的变化预测了在几个独立文库的cDNA中鉴定出的新的限制性位点。所有三个战神的氨基酸变化增加了与兔-人异麦芽糖酶的同源性。因此，我们认为我们的GAA核苷酸序列更精确。我们还确定了人类GAA的18个位点的单碱基对多态性，其中一些不是沉默的。
Acid maltase or acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose and is deficient in glycogen storage disease type II. Previously, we isolated a partial cDNA (1.9 kb) for human GAA; we have now used this cDNA to isolate and determine sequence in longer cDNAs from four additional independent cDNA libraries. Primer extension studies indicated that the mRNA extended approximately 200 bp 5′ of the cDNA sequence obtained. Therefore, we isolated a genomic fragment containing 5′ cDNA sequences that overlapped the previous cDNA sequence and extended an additional 24 bp to an initiation codon within a Kozak consensus sequence. The sequence of the genomic clone revealed an intron–exon junction 32 bp 5′ to the ATG, indicating that the 5′ leader sequence was interrupted by an intron. The remaining 186 bp of 5′ untranslated sequence was identified approximately 3 kb upstream. The promoter region upstream from the start site of transcription was GC rich and contained areas of homology to Sp1 binding sites but no identifiable CAAT or TATA box. The combined data gave a nucleotide sequence of 2,856 bp for the coding region from the ATG to a stop codon, predicting a protein of 952 amino acids. The 3′ untranslated region contained 555 bp with a polyadenylation signal at 3,385 bp followed by 16 bp prior to a poly(A) tail. This sequence of the GAA coding region differs from that reported by Hoefslootet al.(1988) in three areas that change a total of 42 amino acids. Direct determination of the amino acid sequence in one of these areas confirmed the nucleotide sequence reported here but also disagreed with the directly determined amino acid sequence reported by Hoefslootet al.(1988). At two other areas, changes in base pairs predicted new restriction sites that were identified in cDNAs from several independent libraries. The amino acid changes in all three ares increased the homology to rabbit–human isomaltase. Therefore, we believe that our nucleotide sequence for GAA is more precise. We have also identified single base-pair polymorphisms at 18 sites for human GAA, some of which are not silent.