Cloning, sequencing, and characterization of CYP1A1 cDNA from leaping mullet (Liza Saliens) liver and implications for the potential functions of its conserved amino acids

A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a lambdaZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69-96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method.     

Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.     

小麦PAL基因的克隆及赤霉菌诱导下的表达分析

利用苯丙氨酸解氨酶(PAL,phenylalanine ammonia-lyase)基因保守区域从小麦抗赤霉病材料苏麦3号中克隆获得4个PAL基因,分别命名为Ta PAL1、Ta PAL2、Ta PAL3、Ta PAL4。4个基因的开放阅读框(ORF,open reading frame)长度分别为2142 bp、2016 bp、2118 bp和2139 bp,分别编码714个、672个、706个和713个氨基酸。基因序列比对发现其相似性达到88.35%,所编码的氨基酸相似性为91.92%,氨基酸序列分析表明4个基因都包含HAL-PAL结构域及PAL结构域。通过接种禾谷镰刀菌,利用荧光定量PCR对PAL基因进行表达分析发现,4个PAL基因全部为上调表达,其中Ta PAL2、Ta PAL3和Ta PAL4最为明显。PAL基因的上调表达,说明PAL基因在小麦抵抗赤霉病菌侵染的机制中可能起着重要作用。     

The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase

Recently we reported the sequence of a cDNA clone (pUOX-1), isolated from a lambda gt11 cDNA library, which encoded for rat liver urate oxidase (EC 1.7.3.3), but this clone lacked the nucleotide sequences encoding the N-terminal region for this enzyme. Using the cDNA insert from the pUOX-1 clone as a probe, we have now isolated a full length cDNA clone, pUOX-2, from a lambda gt10 library by plaque hybridization. Nucleotide sequence analysis of the pUOX-2 clone showed that it has 1379 base pairs with an open reading frame coding for 303 amino acid residues corresponding to a molecular mass of 34,931 daltons. In addition to the open reading frame the pUOX-2 contains 439 bp of 3'-untranslated and 41 bp of 5'-untranslated sequences. The consensus polyadenylation signal AATAAA precedes a stretch of poly(A)+ residues at the 3' end.     

鳜肌酸激酶M-CK cDNA的克隆与组织表达分析

利用RT–PCR和cDNA末端快速扩增法（RACE）克隆了鳜（Siniperca chuatsi）肌酸激酶（creatine kinase,CK）cDNA序列,并分析了该基因的结构特征和系统关系。鳜CK cDNA序列全长1586 bp,包括5′端非翻译区92 bp,3′端非翻译区348 bp和开放阅读框（ORF）1 146 bp,共编码381个氨基酸。鳜CK具有脊椎动物CK共有的保守结构域和肌型肌酸激酶（M-CK）同工酶的特异识别位点;氨基酸序列与M-CK型的相似度最高,而与脑型肌酸激酶（B-CK）和线粒体型肌酸激酶（Mi-CKs）的相似度较低;在CK系统关系树中鳜CK与M-CK群聚类。这些均表明,鳜CK属脊椎动物M-CK型。RT-PCR分析表明,鳜M-CK在成体不同组织中的表达量不同,其中,在皮肤、卵巢、肾脏、胃、肌肉和心脏中表达较强;而在眼和脑、肝胰脏中表达较弱。     

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.