Isolation and expression of a novel chick G-protein cDNA coding for a G alpha i3 protein with a G alpha 0 N-terminus.
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ABSTRACT:
SUBMITTER: Kilbourne EJ
PROVIDER: S-EPMC1137829 | biostudies-other | 1994 Jan
REPOSITORIES: biostudies-other
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Project description:Three distinct G-protein alpha o-subtypes, i.e. alpha o1, alpha o2 and a newly observed 'alpha o3', are present in membranes of mammalian brain, each appearing as two isoforms on SDS/PAGE. Only alpha o1 and alpha o2 appear to be substrates for pertussis toxin (PTX) when membranes or partially purified proteins are examined. In order to elucidate the apparent PTX-resistance of the third alpha o-subtype, we purified alpha o3 from porcine and bovine brain membranes. During the purification procedures, alpha o3 occurred in its dissociated monomeric form and, together with beta gamma-complexes, as a heterotrimer. In a first attempt, we used purified G-protein alpha i/alpha o-mixtures to obtain a final separation of alpha o3. By using f.p.l.c. anion-exchange chromatography on a Mono Q column, complete separation of alpha i1 and alpha o2 was achieved. Partial resolution of alpha o1, alpha i2 and alpha o3 was observed; alpha o3 was eluted between alpha o1 and alpha i2. If alpha o-subunits free from alpha i contaminants were loaded on to the Mono Q column, all three alpha o-subtypes were resolved. The identity of the third subtype as an alpha o-subtype was confirmed by sequence analysis of tryptic fragments. All three alpha o-subtypes bound GTP[S]. Purified alpha o3 was ADP-ribosylated when subjected to PTX treatment in the presence of beta gamma-subunits, and on SDS/PAGE the mobility of alpha o3 was similar to that of ADP-ribosylated alpha o1. On the basis of results obtained with subtype-specific antibodies, the third alpha o-subtype is immunologically more related to alpha o1 than to alpha o2. Purified alpha o3 failed to reconstitute carbachol-mediated inhibition of Ca2+ current in PTX-pretreated SH-SY5Y-cells, whereas alpha o1 and alpha o2 did successfully restore this effect. We conclude that the novel alpha o3 forms differs from alpha o1 and alpha o2 in its primary structure and may be involved in signal-transduction pathways other than those described for alpha o1 and alpha o2.
| S-EPMC1138174 | biostudies-other
Project description:We have identified and sequenced a cDNA clone coding for Trichomonas vaginalis alpha-actinin. Analysis of the obtained sequence revealed that the 2,857-nucleotide-long cDNA contained an open reading frame encoding 849 amino acids which showed consistent homology with alpha-actinins of different species. Such homology was particularly significant in regions which have been reported to represent the actin-binding and Ca2+-binding domains in other alpha-actinins. The deduced protein was also characterized by the presence of a divergent central region thought to play a role in its high immunogenicity. A study of protein localization performed by immunofluorescence revealed that the protein is diffusely distributed throughout the T. vaginalis cytoplasm when the cell is pear shaped. When parasites adhere and transform into the amoeboid morphology, the protein is located only in areas close to the cytoplasmic membrane and colocalizes with actin. Concomitantly with transformation into the amoeboid morphology, alpha-actinin mRNA expression is upregulated.
| S-EPMC108609 | biostudies-literature
Project description:We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA indicates that it encodes a protein of the 425 amino acids [tentatively named adipose differentiation-related protein (ADRP)] that does not have any similarity with sequences contained in the GenBank DNA and Protein Identification Resource protein data bases. Immunoblot of 1246 cell extracts with an antibody raised against the expressed ADRP shows that the 1246 cells contain a 50-kDa protein, the production of which increases as the cells differentiate. Localization of ADRP in 1246 cells indicates that ADRP is absent from nuclear and cytosolic fractions and is found as a membrane-associated protein. These results demonstrate that adipocyte differentiation is accompanied by early expression of a mRNA encoding a membrane-associated adipose differentiation related protein that is adipose tissue specific in vivo.
| S-EPMC49813 | biostudies-other
Project description:DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression. We previously demonstrated that DNA functions as an allosteric modulator of estrogen receptor alpha (ERalpha) conformation, alters the recruitment of regulatory proteins, and influences estrogen-responsive gene expression and reasoned that it would be useful to develop a method of isolating proteins associated with the DNA-bound ERalpha using full-length receptor and endogenously-expressed nuclear proteins.We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound ERalpha. Purified ERalpha and HeLa nuclear extracts were combined with oligos containing ERalpha binding sites and fractionated on agarose gels. The protein-DNA complexes were isolated and mass spectrometry analysis was used to identify proteins associated with the DNA-bound receptor. Rather than simply identifying individual proteins that interact with ERalpha, we identified interconnected networks of proteins with a variety of enzymatic and catalytic activities that interact not only with ERalpha, but also with each other. Characterization of a number of these proteins has demonstrated that, in addition to their previously identified functions, they also influence ERalpha activity and expression of estrogen-responsive genes.The agarose gel fractionation method we have developed would be useful in identifying proteins that interact with DNA-bound transcription factors and should be easily adapted for use with a variety of cultured cell lines, DNA sequences, and transcription factors.
| S-EPMC2585101 | biostudies-literature
Project description:A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.
| S-EPMC1148163 | biostudies-other
Project description:BackgroundInhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA.MethodsThe subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends (RACE) RT-PCR from adult rhesus monkey testis RNA.ResultsBoth the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0), which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1). Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha-variant 2 is of relatively low abundance and its biological function has not yet been ascertained.ConclusionThe data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action.
| S-EPMC526212 | biostudies-literature
Project description:A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.
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Project description:BackgroundCynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant.ResultsA gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content.ConclusionThe data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters.
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Project description:The isolation of genes from a given genomic region can be a rate-limiting step in the discovery of disease genes. We describe an approach to the isolation of cDNAs that have sequences in common with large genomic clones such as bacterial artificial chromosomes. We applied this method to loci both amplified and deleted in cancer, illustrating its usage in the identification of both oncogenes and tumor suppressor genes, respectively. The method, called rapid isolation of cDNAs by hybridization (RICH), depends on solution hybridization, enzymatic modification, and amplification/selection of sequences present in both cDNA populations and the genomic clones. The method should facilitate the development of transcription maps for large genomic clones, possibly even yeast artificial chromosomes.
| S-EPMC19911 | biostudies-literature
Project description:The Gs alpha guanine nucleotide-binding signal transduction protein is part of a heterotrimeric complex that is involved in the stimulation of adenylate cyclase upon activation of membrane receptors. We report the characterization of 16 Gs alpha cDNA clones isolated from the human adult retina and fetal eye libraries. Molecular heterogeneity in the 5'-region defines four novel Gs alpha cDNA species which are generated either by alternate splicing or by using alternative promoter. The novel exons upstream of exon 2 interrupt the highly conserved 'region A' in the Gs alpha polypeptide. Non-AUG codons in the novel 5'-exon can initiate translation of these Gs alpha species in vitro. Reverse transcription of total RNA coupled with polymerase chain reaction (RTPCR) using specific primers and in situ hybridization to mRNA in baboon tissue sections with a specific oligonucleotide probe show a high level of expression of these species in retina and brain but not in liver. Differential expression of alternatively spliced Gs alpha species suggests novel signal transducing pathways.
| S-EPMC328715 | biostudies-other