Sequence of a cDNA encoding mouse F1F0-ATP synthase g subunit.

A pregnant-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as a full-length cDNA encoding the F1F0-ATP synthase g subunit. Comparison of the deduced amino acid sequences of mouse ATP synthase g subunit with those of bovine species showed 86% identity. The high levels of ATP synthase g subunit mRNA were detected in heart and uterine tissues.     

葡萄白藜芦醇合酶基因的克隆及序列分析

目的:从葡萄中克隆白藜芦醇合酶基因vrs1并对其序列进行生物信息学分析.方法:利用葡萄总RNA为模板,采用RT - PCR技术克隆白藜芦醇合酶基因vrs1并亚克隆进T- Vector.利用生物信息学工具对其核酸和蛋白序列进行分析.结果:测序结果显示其cDNA序列全长为1 257bp,含有一个1 179bp的开放阅读框.生物信息学分析表明葡萄白藜芦醇合酶基因编码392个氨基酸,分子量为42.9kDa,理论等电点为5.97,具有芪合酶家族固有的氨基酸保守结构域,二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成.结论:该基因的克隆、生物信息学分析为进一步研究其功能奠定了基础.     

桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

Human mitochondrial ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling factor 6

Coupling factor 6 (F6) is a component of mitochondrial ATP synthase which is required for the interactions of the catalytic and proton-translocating segments. A human fetal muscle cDNA clone encoding this protein was isolated by screening a lambda gt10 library with oligodeoxyribonucleotide probes. The 497-bp F6 cDNA included a 96-bp segment that delineated a presequence of 32 amino acids (aa) in the precursor protein, and 140 bp of 3'-untranslated sequence. The remainder of the cDNA sequence coded for a mature human F6 protein of 76 aa. The deduced primary aa sequence showed 81% homology to that of bovine F6, differing in 14 aa. Almost all of these aa substitutions were conservative and comparison of the hydropathy profiles revealed a similar pattern.     

Isolation and sequence determination of cDNA encoding P2 protein of human peripheral myelin.

A full length cDNA of P2 protein of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 2150 base pairs (bp) in length and contains a 393 bp open reading frame encoding a polypeptide of 131 residues. The deduced amino acid sequence is highly homologous to P2 protein from other species.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin.

A full length cDNA of the major structural protein of peripheral myelin (P0 protein) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs (bp) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

大豆异黄酮合酶基因的克隆及序列分析

目的:大豆异黄酮是多酚类混合物,有防治肿瘤发生,提高机体免疫力等多种保健功能。异黄酮合酶(isoflavone synthase,IFS)是合成异黄酮的关键酶。本文为了利用异黄酮的特有生物学功能,从大豆中克隆了该基因。方法:采用PCR扩增从大豆[Glycine max(Linn.)Merr.]总RNA中分离了异黄酮合酶基因,并将其克隆到pUCm-T载体并测序。结果:得到全长1583bp的片段。以期用于构建诱导表达基因敲除系统,并用于无性繁殖植物的无标记基因转化。结论:序列分析表明,异黄酮合酶基因(IFS1)含1583个核苷酸,与已报道的序列比较,核苷酸的同源性为92%。     

Isolation and sequence determination of cDNA encoding PMP-22 (PAS-II/SR13/Gas-3) of human peripheral myelin.

A full length cDNA of PMP-22 (PAS-II/SR13/Gas-3) of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 1823 base pairs (bp) in length and contains a 480 bp open reading frame encoding a polypeptide of 160 residues. The deduced amino acid sequence is highly homologous to PMP-22 from bovine (PAS-II), rat (SR13) and mouse (Gas-3).     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析

海藻糖-6-磷酸合成酶(Trehalose-6-phosphate synthse, TPS)是植物海藻糖合成途径的关键酶, 在旱生卷柏等复苏植物对逆境胁迫应答中起重要作用。文章以我国特有旱生植物垫状卷柏(Selaginella pulvinata)为材料, 采用同源扩增与RACE技术相结合的方法克隆了海藻糖-6-磷酸合成酶基因SpTPS1, cDNA全长3 223 bp, 包括一个2 790 bp的开放阅读框, 推导的氨基酸序列与模式物种的海藻糖-6-磷酸合成酶具有较高的序列相似性, 催化活性中心保守位点基本一致。酵母功能互补实验证明, 用SpTPS1基因开放阅读框转化的海藻糖合成酶基因突变(tps1△)酵母菌株, 可恢复在以葡萄糖作为唯一碳源培养基上的生长, 说明垫状卷柏海藻糖-6-磷酸合成酶基因SpTPS1的编码蛋白具有生物活性, 可应用于植物抗逆性的转基因改良。     

cDNA sequence of the long mRNA for human glutamine synthase.

Screening a human liver cDNA library in lambda ZAP revealed several clones for the mRNA of glutamine synthase. The longest clone was completely sequenced and consists of a 109 bp 5' untranslated region, a 1119 bp protein coding region, a 1498 bp 3' untranslated region and a poly(A) tract of 12 bp.     

Bovine liver cDNA clones encoding a precursor of the alpha-subunit of the mitochondrial ATP synthase complex

cDNA clones encoding a precursor of the alpha-subunit of the mitochondrial ATP synthase complex have been isolated from a bovine liver cDNA library using the alpha-subunit gene from Saccharomyces cerevisiae as a probe. Analyses of the nucleotide sequence of these cDNA clones reveal that the bovine liver ATP synthase alpha-subunit is initially synthesized as a precursor with an aminoterminal extension 43 amino acids in length. This aminoterminal presequence contains seven basic residues, no acidic residues, and seven polar uncharged serine and threonine residues. A single mRNA species, approximately 1.85 kb in length, was detected for the ATP synthase alpha-subunit precursor in both bovine liver and heart.