The molecular cloning and characterization of potential chick DM-GRASP homologs in zebrafish and mouse

A full-length zebrafish cDNA clone and a partial mouse cDNA clone similar to chick DM-GRASPwere isolated and analyzed. The nucleotide sequence of the full-length zebrafish clone shares 54% identity, and predicts 39% amino acid identity, with chick DM-GRASP. The partial mouse clone shares 76% nucleotide identity, and predicts 76% amino acid identity, with chick DM-GRASP. The predicted proteins encoded by both of these clones exhibit conserved structural domains that are characteristic of the chick protein. These features may identify them as a distinct subfamily within the immunoglobulin superfamily of cell adhesion molecules. Express of the zebrafish DM-GRASP protein is similar to chick DM-GRASP and is principally restricted to a small subset of developing sensory and motor neurons during axonogenesis. Zebrafish DM-GRASP expression was temporally regulated and limited to specific axon domains. This regional expression correlated with fasciculated axon domains. These results suggest that the zebrafish and mouse cDNA clones represent the respective fish and mammalian homologs of thick DM-GRASP. The highly selective expression of zebrafish DM-GRASP suggests that it is involved in the selective fasciculation and guidance of axons along their normal pathways. 1994 John Wiley & Sons, Inc.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

A differentially expressed murine RNA encoding a protein with similarities to two types of nucleic acid binding motifs

Using differential screening, a murine cDNA, termed X16, was isolated corresponding to an mRNA which is more strongly expressed in pre-B cell lines relative to mature B-cell lines. The complete coding sequence of the mRNA predicts a 19kD protein with two domains connected by a proline-rich spacer. The N-terminal domain of about 90 amino acids encodes an RNA binding motif including the ribonucleoprotein consensus octapeptide found in one class of RNA-binding proteins and highly conserved from yeast to man. Within the very basic C-terminal domain of about 60 amino acids, several copies of two different peptides are found which are also present in several proteins which bind DNA or RNA. The expression of X16 is not limited to the lymphoid lineage. In adult mice, although the strongest expression was seen in thymus, mRNA was also found in testis, brain, spleen, and very low in heart. X16 mRNA was not detected in liver and kidney. In tissue culture, the expression of X16 mRNA can be induced by serum. The conserved protein motifs and expression pattern suggest that X16 could be involved in RNA processing correlating with cellular proliferation.     

草鱼CNBP的分子特征及其进化分析

从草鱼(Ctenopharyngodon idella)肝肾cDNA文库中克隆到细胞核酸结合蛋白基因CNBP的完整开放阅读框序列.分析表明草鱼CNBP由163个氨基酸残基组成,含有7个保守CCHC型锌指结构、核定位信号区和RGG框,与其他鱼类的同源性很高.与人及其他脊椎动物的相比,草鱼细胞核酸结合蛋白在第3个锌指中的第5个氨基酸残基由Gly变成His,另外在第1锌指和第2锌指结构间,缺失6～14个氨基酸残基.虽然如此,适应性进化分析显示细胞核酸结合蛋白没有经历正达尔文选择(ω≤1),即这种结构的差异还不足以产生新的功能.这表明CNBP处于中性进化中.     

Sequence of carbonic anhydrase II cDNA from chick retina

Sequences of three cDNA clones for carbonic anhydrase II (CA-II) from chick retina are presented. The longest cDNA clone encodes all but the first three amino acids of CA-II, and the encoded sequence generally agrees with published fragments of CA-II sequence from chick red blood cells. It is 70% identical to human CA-II; the active-site residues are conserved, but the chick protein has six extra cysteines. There is a long 3'-untranslated region which contains a second open reading frame, but this is not conserved. There appears to be a single CA-II gene in the chick. Some anomalies in cDNA synthesis and in Bal31 deletion are noted.     

Molecular Cloning and Expression of cDNA Encoding the Cysteine Proteinase Inhibitor from Upland Cotton

A cDNA encoding a novel cysteine proteinase inhibitor (CPI) was isolated from a gland mutant Xiangmian-18 of upland cotton during the pigments gland forming stage. The cDNA comprises 378 bp and encodes 125 amino acid residues with molecular mass of 13.8 kDa. It contains the conserved motif of cysteine protease inhibitors and belongs to the cystatin superfamily (Gln-Val-Val-Ala-Gly). The deduced amino acid sequences of the domains are highly similar to the normal upland cotton (96.8%). SDS-PAGE and western hybridization analysis showed that the expressed recombinant protein was recombinant CPI. The inhibitory activity of recombinant CPI was 46 u/μg which was measured by inhibiting the protease activity of papain. RT-PCR results indicated that the expression level of developing gland stage was higher than that of undeveloped gland stage.     

Nucleotide sequence of rat calretinin cDNA

A rat calretinin cDNA clone was selected by antibody screening of a λgt11 brain library. The sequence revealed remarkable nucleotide and amino acid homology with human calretinin (91.1% and 98.5%, respectively), with only four amino acid differences. A high degree of homology with chick calretinin was also observed (79.8% and 86.6%, respectively), with 36 amino acid differences.

Although the role of this central nervous system protein has not been well characterized, the evolutionarily conserved calcium binding domains and connecting regions, in addition to the limited local changes observed between rat and chick primary structure, lead us to believe that calretinin interacts with other highly conserved constituents of brain cells. This calretinin cDNA clone provides a new probe for the analysis of a specific subset of neurons in the central nervous system. The probe will allow a more detailed analysis of calretinin regulation in the brain and will be useful for screening genomic libraries for the complete chromosomal gene. (GenBank accession No. X66974.)     

Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGP Pc 2) and the other from suspension cultures of Nicotiana alata (AGP Na 2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGP Pc 2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGP Na 2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.     

家蚕磷酸吡哆醛激酶cDNA的克隆和酶活表征

吡哆醛激酶 (EC 2.7.1.35) 在 ATP 和 Zn2 的存在下,催化吡哆醛的磷酸化反应生成磷酸吡哆醛 (PLP)。在生物体内许多酶促反应中,PLP 是一种重要的辅酶因子。家蚕和哺乳动物一样,需依赖食物中的维生素 B6前体来合成 PLP。文章描述了利用家蚕基因组数据库序列信息及使用 PCR 方法,克隆出编码家蚕吡哆醛激酶的 cDNA (GenBank 登录号:DQ452397)。克隆到的 cDNA 含有一个 894 bp 的完整可读框,编码一条分子量为 33.1 kDa,含 298 个氨基酸残基的蛋白质。序列比对显示此蛋白质序列与人类吡哆醛激酶蛋白序列具有 48.6%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但其氨基酸残基数比哺乳动物和植物克隆到的吡哆醛激酶残基数均少 10 多个残基。多序列比对结果显示,吡哆醛激酶中几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换为其他种类氨基酸残基。采用 T7 启动子和 T7 聚合酶表达系统对克隆到的 cDNA 进行了原核表达并对表达粗提产物进行了酶活检测。实验结果显示表达得到的可溶性蛋白产物占其总蛋白量为 10%,细胞粗提物具有活力为 30 nmol/min/mg 的吡哆醛激酶活性,结果证实了克隆到的 cDNA 编码家蚕中的吡哆醛激酶。     

Molecular characterization of oat seed globulins

We have isolated full-length cDNA clones that encode oat (Avena sativa) seed storage globulin mRNAs from a cDNA library in the expression vector lambda gtll. The longest of these clones, pOG2, has an 1840-base pair insert that encodes a complete precursor subunit with a signal peptide of 24 amino acids followed by an acidic polypeptide of 293 amino acids and a basic polypeptide of 201 amino acids. Near the C terminus of the acidic polypeptide are four repeats of a highly conserved, glutamine-rich octapeptide. Other oat globulin cDNA clones contain five of these repeats. Nucleotide sequence comparisons between these clones indicate that the genes encoding these proteins are highly conserved. We estimate there to be 7 to 10 genes for the oat globulin per haploid genome. Comparisons of amino acid sequences show that the oat globulin is 30 to 40% homologous with storage globulins of legumes and about 70% homologous with the rice seed storage globulin (glutelin).     

甘蔗天冬氨酰半醛脱氢酶基因的电子克隆与分析

应用电子克隆技术,以水稻EF576477序列为探针,获得了甘蔗天冬氨酰半醛脱氢酶基因（aspartate．semialdehydedehy—drogenase,ASADH）的一条cDNA全长序列,命名为ScASADH。采用生物信息学方法,对该基因编码蛋白从氨基酸组成、理化性质、亚细胞定位、跨膜结构域、疏水性／亲水性、高级结构及功能域等方面进行预测和分析。结果表明：该基因全长1711bp,包含一个1128bp的开放阅读框,编码375个氨基酸,该基因编码蛋白定位于细胞核,为可溶性蛋白,存在信号肽,二级结构原件多为无规卷曲,含有多个保守功能域,主要功能为翻译。电子表达分析结果显示,该基因在甘蔗根尖、幼苗、花序、叶片和茎中组成型表达,其中在茎中的表达量比其他组织类型中表达量高。该基因的表达受葡萄糖杆菌和赤腐病菌的调控。     

A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites.

To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR.     

甘蔗细胞色素C基因的电子克隆与分析

以甘蔗类似细胞色素C的EST序列CF576943.1为探针,通过电子克隆技术获得了甘蔗细胞色素C基因（Cytochrome C,Cyt C）的一条cDNA全长序列,命名为ScCyt C.用生物信息学方法对该基因氨基酸序列与组成、亚细胞定位、跨膜区与信号肽、疏水性/亲水性、蛋白质二、三级结构以及功能等进行分析与预测.结果表明：Cyt C基因全长1 073 bp,编码112个氨基酸,该基因位于细胞质,为非分泌型蛋白,无规卷曲为主要二级结构原件,含有1个保守功能域,主要功能为翻译并且在不同植物中具有高度保守性.电子表达分析结果显示,该基因在甘蔗各个组织均有表达,其中在茎和根中的表达量比其他组织类型中表达量高,此外,该基因的表达可能受到低温的调控.为甘蔗细胞色素C基因的结构及其功能的研究奠定了一定的基础.