Structural analysis of transcripts for the protein L-isoaspartyl methyltransferase reveals multiple transcription initiation sites and a distinct pattern of expression in mouse testis: identification of a 5'-flanking sequence with promoter activity.
Structural analysis of transcripts for the protein L-isoaspartyl methyltransferase reveals multiple transcription initiation sites and a distinct pattern of expression in mouse testis: identification of a 5'-flanking sequence with promoter activity.
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对 L-异天冬氨酰甲基转移酶蛋白转录物的结构分析揭示了小鼠睾丸中的多个转录起始位点和独特的表达模式：鉴定出具有启动子活性的 5 侧翼序列。
DOI：
10.1006/abbi.1994.1341
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发表时间：
1994
影响因子：
3.9
通讯作者：
O'Connor,CM
中科院分区：
文献类型：
--
作者：
Galus,A;Lagos,A;Romanik,EA;O'Connor,CM
编码蛋白质L-异戊酰基-（D-戊酰基）甲基转移酶（蛋白质羧基甲基转移酶，PCMT）的基因在细菌和真核细胞中广泛表达。以小鼠PCMT基因第一外显子为探针，设计了一种反义探针[E. A.罗马尼克角L.拉迪诺，8岁。C. D′Ardenne和C. M. O′康纳（1992）基因118，217-222]被用于核糖核酸酶保护测定，以鉴定小鼠睾丸、脑和肝组织中PCMT转录的起始位点。两个主要的起始位点，155-157个核苷酸（nt）和119个核苷酸上游的ATG起始密码子，除了几个次要的网站，在所有组织中被确定。用连接介导的5′-快速扩增cDNA末端（RACE）方法确定睾丸RNA起始位点的位置。这些起始位点位于407 bp基因组序列的3′端，该基因组序列足以在NIH/3 T3细胞的瞬时转染试验中驱动萤火虫荧光素酶基因的表达。407 bp的序列类似于管家基因启动子，其高G + C含量，缺乏TATA盒和转录因子Sp1和ETF的多个潜在结合位点的存在。PCMT转录物C端编码序列和3′-非翻译区的选择性剪接产生三类不同的mRNA，这些mRNA是用3′-RACE从睾丸poly（A）+RNA中克隆的。转录本剪接在外显子7中终止密码子下游38 nt或上游7 nt处产生编码PCMT同工酶的mRNA，在其C末端分别具有-RWK或-RDEL。睾丸中的主要转录本，这是没有检测到体细胞组织中的北方印迹，这可能是特定的生殖细胞，不剪接外显子7内，也编码-RWK同工酶。
The gene encoding the protein L-isoaspartyl-(D-aspartyl) methyltransferase (protein carboxyl methyltransferase, PCMT) is widely expressed in bacteria and eucaryotic cells. An antisense probe encompassing the first exon of the murinePCMTgene [E. A. Romanik, C. L. Ladino, 8. C. D′Ardenne, and C. M. O′Connor (1992)Gene118, 217-222] was used in ribonuclease protection assays to identify the initiation sites forPCMTtranscription in mouse testis, brain, and liver tissues. Two major initiation sites, 155-157 nucleotides (nt) and 119 nt upstream from the ATG initiation codon, were identified in all tissues in addition to several minor sites. The locations of the initiation sites in testicular RNA were confirmed using ligation-mediated 5′-rapidamplification ofcDNAends (RACE). These initiation sites are situated at the 3′-end of a 407-bp genomic sequence which is sufficient to drive the expression of a firefly luciferase gene in transient transfection assays with NIH/3T3 cells. The 407-bp sequence resembles a housekeeping gene promoter in its high G + C content, lack of a TATA box and the presence of multiple potential binding sites for the transcription factors Sp1 and ETF. Alternative splicing in the C-terminal encoding sequence and in the 3′-untranslated regions ofPCMTtranscripts generates three distinct classes of mRNAs which were cloned from testicular poly(A)+RNA using 3′-RACE. Transcript splicing either 38 nt downstream or 7 nt upstream from the termination codon in exon 7 produces mRNAs encoding PCMT isozymes with -RWK or -RDEL, respectively, at their C-termini. The predominant transcript in testis, which is not detected in somatic tissues by Northern blotting and which may be specific to germ cells, is not spliced within exon 7 and also encodes the -RWK isozyme.