Martiniuk F, Mehler M, Tzall S, Meredith G, Hirschhorn R
New York University Medical Center, Department of Medicine, NY 10016.
DNA Cell Biol. 1990 Mar;9(2):85-94. doi: 10.1089/dna.1990.9.85.
Acid maltase or acid alpha-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 Hoefsloot et 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 Hoefsloot et 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.
酸性麦芽糖酶或酸性α-葡萄糖苷酶（GAA）是一种溶酶体酶，可将糖原水解为葡萄糖，在II型糖原贮积病中缺乏该酶。以前，我们分离出了人GAA的部分cDNA（1.9 kb）；现在我们利用该cDNA从另外四个独立的cDNA文库中分离并确定更长cDNA的序列。引物延伸研究表明，mRNA在获得的cDNA序列的5'端大约延伸了200 bp。因此，我们分离出一个包含5' cDNA序列的基因组片段，该序列与先前的cDNA序列重叠，并在Kozak共有序列内的起始密码子处又延伸了24 bp。基因组克隆的序列显示在ATG的5'端32 bp处有一个内含子-外显子接头，表明5'前导序列被一个内含子打断。其余186 bp的5'非翻译序列在大约3 kb上游被鉴定出来。转录起始位点上游的启动子区域富含GC，并且包含与Sp1结合位点同源的区域，但没有可识别的CAAT或TATA框。综合数据得出从ATG到终止密码子的编码区核苷酸序列为2856 bp，预测有一个952个氨基酸的蛋白质。3'非翻译区包含555 bp，在3385 bp处有一个聚腺苷酸化信号，随后在聚（A）尾巴之前有16 bp。GAA编码区的这个序列在三个区域与Hoefsloot等人（1988年）报道的不同，总共改变了42个氨基酸。对其中一个区域氨基酸序列的直接测定证实了此处报道的核苷酸序列，但也与Hoefsloot等人（1988年）直接测定的氨基酸序列不一致。在另外两个区域，碱基对的变化预测了新的限制性酶切位点，这些位点在来自几个独立文库的cDNA中被鉴定出来。所有三个区域的氨基酸变化都增加了与兔-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。我们还鉴定出了人GAA在18个位点的单碱基对多态性，其中一些不是沉默突变。
