Hayami K, Noumi T, Inoue H, Sun-Wada G, Yoshimizu T, Kanazawa H
Department of Biotechnology, Faculty of Engineering Sciences, Okayama University, Okayama, Japan.
Gene. 2001 Aug 8;273(2):199-206. doi: 10.1016/s0378-1119(01)00590-x.
We cloned the genomic genes encoding the murine 16 kDa subunit (proteolipid, PL16) of vacuolar H(+)-ATPase (V-ATPase) and determined their nucleotide sequences. At least three independent genes were found in the murine genome. One gene consisted of three exons and was largely identical in sequence to that of PL16 cDNA reported previously (Hanada et al., Biochem. Biophys. Res. Commun. 176 (1991) 1062). In the 5'-flanking region of this gene, several possible transcriptional cis-elements were found. TATA and CAAT sequences were not found, which is characteristic for promoters of house-keeping genes. The other two genes identified did not contain introns. One of these genes had an open reading frame that potentially encoded PL16 but contained six amino acid substitutions and a frame-shift mutation that would result in a truncated protein unable to participate in V-ATPase activity. The other gene had the same sequence in the reading frame as that in the cDNA. However, this gene contained a polyA sequence at the same position where polyA is normally added to mRNA. The gene also had 15 bp repetitive sequences near the transcription initiation site and next to the polyA sequence. These observations suggest that this gene may have been generated by the insertion of reverse-transcribed double-stranded cDNA, as is usually observed for pseudogenes. In conclusion, there is a single functional PL16 gene and two pseudogenes in the murine genome. It is unlikely that PL16 isoforms contribute to variation in V-ATPase function.
我们克隆了编码鼠液泡H(+)-ATP酶（V-ATP酶）16 kDa亚基（蛋白脂质，PL16）的基因组基因，并测定了它们的核苷酸序列。在鼠基因组中发现了至少三个独立的基因。一个基因由三个外显子组成，其序列与先前报道的PL16 cDNA的序列基本相同（Hanada等人，《生物化学与生物物理研究通讯》176 (1991) 1062）。在该基因的5'侧翼区域，发现了几个可能的转录顺式元件。未发现TATA和CAAT序列，这是管家基因启动子的特征。鉴定出的另外两个基因不含内含子。其中一个基因有一个开放阅读框，可能编码PL16，但包含六个氨基酸取代和一个移码突变，这将导致产生一个截短的蛋白质，无法参与V-ATP酶活性。另一个基因的阅读框序列与cDNA中的相同。然而，该基因在通常添加polyA到mRNA的相同位置含有一个polyA序列。该基因在转录起始位点附近和polyA序列旁边也有15 bp的重复序列。这些观察结果表明，该基因可能是由逆转录双链cDNA插入产生的，这在假基因中通常可以观察到。总之，在鼠基因组中有一个功能性的PL16基因和两个假基因。PL16同工型不太可能导致V-ATP酶功能的变异。
