Gotlib R W, Bishop D F, Wang A M, Zeidner K M, Ioannou Y A, Adler D A, Disteche C M, Desnick R J
Department of Human Genetics, Mount Sinai School of Medicine, New York 10029, USA.
Biochem Mol Med. 1996 Apr;57(2):139-48. doi: 10.1006/bmme.1996.0020.
The full-length cDNA and genomic sequences encoding mouse alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22), a lysosomal galactohydrolase, were isolated and characterized. The cDNA's open reading frame encoded 419 amino acids and had 82% nucleotide (nt) and 78% amino acid identity with the human sequence, although the carboxy terminus of the mouse alpha-Gal A polypeptide was 10 amino acids shorter. The functional integrity of the mouse cDNA was demonstrated by transient expression in COS-1 cells. Northern analysis revealed two mRNA species of about 1.6 and 3.4 kb due to alternative polyadenylation signals. The entire 14.4-kb mouse genomic sequence was determined; each of its seven exons was interrupted by intronic sequence at the identical positions as the exons in the human gene. The mouse 5' flanking region (250 nt) had one Sp1, site, five CAAT boxes, and no TATA box and had 67% identity with the human promoter region. The gene contained 18 complete or partial Alu-repetitive elements (13 type 1 and 5 type 2 repeats), and three putative functional AATAAA consensus polyadenylation signals were identified 72, 1668, and 1682 nt after the TAA termination codon. Use of the 72-nt site and the 1866 and/or 1682 sites were consistent with the shorter and longer transcripts. The availability of the full-length cDNA and genomic sequence encoding mouse alpha-Gal A should facilitate structure/function studies of this lysosomal glycosidase and the construction of alpha-Gal A-deficient mice by targeted gene disruption.
编码小鼠α-半乳糖苷酶A（α-Gal A；EC 3.2.1.22）（一种溶酶体半乳糖水解酶）的全长cDNA和基因组序列被分离并进行了表征。该cDNA的开放阅读框编码419个氨基酸，与人类序列具有82%的核苷酸（nt）和78%的氨基酸同一性，不过小鼠α-Gal A多肽的羧基末端短10个氨基酸。通过在COS-1细胞中的瞬时表达证明了小鼠cDNA的功能完整性。Northern分析显示由于可变聚腺苷酸化信号存在约1.6和3.4 kb的两种mRNA。确定了整个14.4 kb的小鼠基因组序列；其七个外显子中的每一个都在与人类基因中外显子相同的位置被内含子序列打断。小鼠5'侧翼区域（250 nt）有一个Sp1位点、五个CAAT框且没有TATA框，与人类启动子区域具有67%的同一性。该基因包含18个完整或部分的Alu重复元件（13个1型和5个2型重复），并且在TAA终止密码子后72、1668和1682 nt处鉴定出三个推定的功能性AATAAA共有聚腺苷酸化信号。使用72 nt位点以及1866和/或1682位点与较短和较长的转录本一致。编码小鼠α-Gal A的全长cDNA和基因组序列的可得性应有助于对这种溶酶体糖苷酶进行结构/功能研究以及通过靶向基因破坏构建α-Gal A缺陷小鼠。
