Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains.

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive alpha-helical structure of 21-33 amino acids (subdomain B). 149 amino acids at the N-terminus and 71 amino acids at the C-terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites. Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213-9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N-terminal and C-terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.     

Cloning and sequencing of a cDNA encoding the rat Bcl-2 protein

A rat cDNA encoding the Bcl-2 protein was cloned and sequenced. The primary amino-acid sequence deduced from the nucleotide sequence reveals a 236-aa protein having extensive homology with the mouse (95%), human (87%) and chicken (71%) Bcl-2 proteins.     

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.     

Isolation and oncogenic potential of a novel human src-like gene.

We have isolated cDNA molecules representing the complete coding sequence of a new human gene which is a member of the src family of oncogenes. Nucleotide sequence analysis revealed that this gene, termed slk, encoded a 537-residue protein which was 86% identical to the chicken proto-oncogene product, p60c-src, over a stretch of 191 amino acids at its carboxy terminus. In contrast, only 6% amino acid homology was observed within the amino-terminal 82 amino acid residues of these two proteins. It was possible to activate slk as a transforming gene by substituting approximately two-thirds of the slk coding sequence for an analogous region of the v-fgr onc gene present in Gardner-Rasheed feline sarcoma virus. The resulting hybrid protein molecule expressed in transformed cells demonstrated protein kinase activity with specificity for tyrosine residues.     

Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.     

Molecular cloning, expression and evolutionary analysis of the avian tyrosine kinase JAK1

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133 kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1–JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5 kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.     

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor alpha-subunits and platelet glycoprotein IIb

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated alpha- and beta-subunits. The present study was designed to compare the cDNA-derived protein sequences of the alpha-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the alpha-subunit of the FnR (FnR alpha) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR alpha-subunit (VnR alpha) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR alpha. Translation of these sequences showed that the FNR alpha, the VnR alpha, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca2+-binding domains of about 30 amino acids that have sequence similarities to other Ca2+-binding proteins. The identity among the protein sequences of the three receptor alpha-subunits ranges from 36.1% to 44.5%, with the Ca2+-binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication.     

Cloning and sequence analysis of the human liver rhodanese: comparison with the bovine and chicken enzymes

The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library. Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively. The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins. A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.     

Isolation of a cDNA clone for human antithrombin III

Antithrombin III (ATIII) is an important plasma protease inhibitor with a central role in the coagulation system. On the basis of its protein sequence, ATIII is one member of a "super family" of protease inhibitors that includes alpha 1-antitrypsin and chicken ovalbumin. An increased risk of thromboembolism is associated with inherited ATIII deficiency. To study the structure and expression of the human ATIII gene, we have isolated complementary (cDNA) clones for ATIII from human liver mRNA. ATIII cDNA clones were identified by hybridization to a mixture of synthetic oligodeoxynucleotides encoding amino acids 251-256 of the ATIII protein sequence. The largest cDNA clone (1.4 kilobases) included the coding region of ATIII mRNA from codon 10 through a 3'-untranslated region. Comparison of ATIII cDNA clones from two different sources revealed a sequence polymorphism at an internal PstI restriction site. Analysis of both total genomic DNAs and an ATIII gene cloned in a bacteriophage Charon 4A showed that the ATIII gene is present once per haploid genome and is distributed over 10-16 kilobases of DNA. Computer-assisted comparison of the cDNA sequence with those for baboon alpha 1-antitrypsin and chicken ovalbumin revealed homologies consistent with their inclusion in the protease inhibitor superfamily.     

北京鸭卵磷脂胆固醇酰基转移酶的cDNA和蛋白质的结构分析

为获得不易感动脉粥样硬化动物北京鸭卵磷脂胆固醇酰基转移酶 (LCAT)的cDNA和蛋白质序列 ,分析其结构特点 .以从北京鸭肝脏mRNA反转录获得的cDNA一链为模板 ,应用SMART RACE技术 ,获得了北京鸭LCAT的cDNA序列 ,推导出其蛋白质氨基酸序列 ,应用分子生物学软件对该蛋白的一级、二级结构进行分析和比较 .北京鸭LCATcDNA (在GenBank中的注册号为AF32 4 887)全长 195 3bp ,其中开放阅读框架 135 6bp ,编码 4 5 1个氨基酸 ,包括一个由 2 3个氨基酸构成的疏水性信号肽和一个由 4 2 8个氨基酸组成的成熟蛋白 .该成熟蛋白比人LCAT在C端多 12个氨基酸 ,其与鸡、人、家兔的同源性依次为 98%、83%和 82 % .与其它种属LCAT蛋白序列的比较结果表明 ,北京鸭LCAT蛋白质序列虽然在长度上和结构上与其它种属有一定的差异 ,但序列中与酶催化活性相关的序列均非常保守     

Cloning and characterization of zfBLP1, a Bcl-XL homologue from the zebrafish, Danio rerio

The importance of the Bcl-2 family proteins in normal vertebrate embryogenesis is being recognized; however, their regulatory mechanism is poorly understood. We report here the cloning and characterization of a novel zebrafish Bcl-2 family protein, zfBLP1. The zfBLP1 cDNA is 1942 nucleotides long, encoding a polypeptide of 238 amino acids. The primary sequence of zfBLP1 shares 50% identity to human Bcl-XL, and contains all four conserved BH domains of the Bcl-2 family proteins. Primary sequence analysis identified a consensus ER retention signal at the C-terminal end of zfBLP1. Northern blot analysis indicated that there were two major and two minor zfBLP1 mRNA species expressed during embryonic development. Among the two major mRNA species, the short one, approx. 3 kb in size, was expressed throughout embryonic development, while the long one, approx. 7 kb long, was not detectable until the gastrula stage. These results suggest that zfBLP1 is a novel Bcl-2 family protein under complicated regulations, and is likely to play an important role in zebrafish oogenesis and embryogenesis.     

Molecular cloning of a splicing variant of human RECQL helicase

A cDNA was isolated from the human heart library. This cDNA variant was produced by the deletion of 176 bases at the 5(') end of human RecQL gene, presumably by an alternative mRNA splicing. The cDNA contains two open reading frames and so may encode two isoforms of human RECQL. The first isoform is a 105 amino acid protein with the first 53 N-terminal amino acids identical to the known sequence of RECQL protein and followed by 52 amino acids introduced by in-frame premature stop codon. The second isoform is a 537 amino acid protein that has the same sequence as the published human RECQL helicase, except the first 112 amino acids at the N-terminal end were absent. The possible roles of both of these proteins are discussed.     

The primary structure of chicken ribosomal protein L5.

The nucleotide sequence of a cDNA for chicken ribosomal protein L5, which is considered to associate with 5S rRNA, was determined. The cDNA is 975 bp long. The deduced protein has 297 amino acids and has a molecular mass of 34,090 Da. A comparative analysis of the amino acid sequences of chicken L5 and its homologous proteins revealed an extremely conserved region which contains a cluster of basic amino acids.     

Novel chicken CXC and CC chemokines

Upon stimulation with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes factors with cytokine activity. To characterize these molecules, representational difference analysis with RNA of LPS-induced and uninduced HD-11 cells was performed. Two cDNA clones were isolated that code for polypeptides with structural features of chemokines. cDNA K60 codes for a novel CXC chemokine of 104 residues including a putative signal peptide of 20 amino acids at the N-terminus. It is 67% identical to the previously cloned chicken chemokine 9E3/CEF4. K60 exhibits a similar degree of sequence identity to human interleukin 8 and other related CXC chemokines (about 50%), rendering straight-forward predictions of its biological properties difficult. cDNA K203 codes for a novel CC chemokine of 89 amino acids including a putative N-terminal signal peptide of 21 residues. It is 43% identical to a previously characterized chicken protein with homology to mammalian macrophage inflammatory protein 1beta (MIP-1beta). K203 exhibits about 50% sequence identity to human MIP-1beta and other related CC chemokines.     

Structural organization and regulation of the chicken estrogen receptor

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.     

Cloning and characterization of chicken adipocyte fatty acid binding protein gene

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.     

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes.

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.