Isolation and sequence analysis of TGA1 cDNAs encoding a tomato G protein alpha subunit.

We have isolated cDNAs for a gene coding for a G protein alpha subunit from tomato (Lycopersicon esculentum, cv. VF36). This gene, named TGA1, was isolated using a cDNA of the Arabidopsis thaliana G protein alpha subunit-encoding gene, GPA1, as a DNA probe. The sequences of four cDNA clones indicate that the deduced amino acid (aa) sequence of the gene product (TG alpha 1) has 384 aa (44906 Da). The predicted TG alpha 1 protein exhibits similarity to all known G protein alpha subunits. The aa are 84.6% identical and 93% similar (identical and conservative changes) to A. thaliana GP alpha 1, and 34% identical and 59% similar to mammalian transducins. Furthermore, it has all of the consensus regions for a GTP-binding protein. Finally, hybridizations of tomato genomic DNA indicate that TGA1 is a single-copy gene.     

Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.

Full length cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rat and man have been isolated and sequenced. Many GAPDH gene-related sequences have been found in both genomes based on genomic blot hybridization analysis. Only one functional gene product is known. Results from genomic library screenings suggest that there are 300-400 copies of these sequences in the rat genome and approximately 100 in the human genome. Some of these related sequences have been shown to be processed pseudogenes. We have isolated several rat cDNA clones corresponding to these pseudogenes indicating that some pseudogenes are transcribed. Rat and human cDNAs are 89% homologous in the coding region, and 76% homologous in the first 100 base pairs of the 3'-noncoding region. Comparison of these two cDNA sequences with those of the chicken, Drosophila and yeast genes allows the analysis of the evolution of the GAPDH genes in detail.     

Occurrence of three distinct molecular species of chondroitin sulfate proteoglycan in the developing rat brain

More than 60% of brain chondroitin sulfate proteoglycans were extracted from 10-day-old rat brains by homogenization in ice-cold phosphate-buffered saline containing protease inhibitors. Although the soluble proteoglycan preparation was a mixture of chondroitin sulfate proteoglycans with a different hydrodynamic size as well as a different molecular density, each subfraction of the proteoglycans contained chondroitin sulfate side chains with virtually identical molecular weight (approximately 15,000) and chondroitin sulfate disaccharide composition (high content of 4-sulfate unit). Digestion of the purified proteoglycan preparation with protease-free chondroitinase ABC produced five core proteins with Mr = 250,000 (designated as 250K protein), 220,000 (220K), 150,000 (150K), 130,000 (130K), and 93,000 (93K). All these core proteins were obtained from chondroitin sulfate proteoglycan preparations extracted from various regions of the brain, but their composition varied among different brain regions. Analysis for amino acid composition of these core proteins and two-dimensional mapping of their proteolytic peptides revealed that three major core proteins (250K, 220K, and 150K proteins) were structurally different. These observations indicate that at least three distinct types of chondroitin sulfate proteoglycan occur in the developing rat brain.     

Cloning and tissue distribution of three murine alpha/beta hydrolase fold protein cDNAs

We have cloned 3 novel murine cDNAs encoding proteins containing an alpha/beta hydrolase fold; a catalytic domain found in a very wide range of enzymes. These proteins belong to the prosite UPF0017 uncharacterized protein family and we have named them lung alpha/beta hydrolase 1, 2, and 3 (LABH) since they were cloned from lung cDNA. All have 9 coding exons, encoding 412, 425, and 411 residue proteins respectively (46-48 kDa); LABH1 being closely related to LABH3 having 45% identity. All 3 proteins have a single predicted amino-terminus transmembrane domain. An alignment of family members from different phyla enabled the identification of the LABH1 catalytic triad as Ser211, Asp337, and His366. mRNA expression levels of LABH1 and 3 were highest in liver and LABH2 highest in testis. These findings suggest that the LABH proteins consist of a novel family of membrane bound enzymes whose function has yet to be determined.     

Characterization of G proteins in rat myometrium. A differential modulation of Gi2 alpha and Gi3 alpha during gestation

Myometrial membranes, obtained from estrogen-dominated (day 0) rat uteri, were immunoblotted with antiserum (SG1), which recognizes the alpha subunits of both Gi1 and Gi2, with antiserum (LE2) specific for Gi2 alpha, and with I3B antiserum, specific for Gi3 alpha. The data revealed the absence of detectable levels of Gi1 alpha and the simultaneous presence of Gi2 alpha and Gi3 alpha as Gi subunits in rat myometrium. The expression of Gi proteins during gestation (days 0, 12, 21) was studied with the above antibodies. No qualitative change in the nature of Gi alpha species was observed during gestation: Gi1 alpha remained undetectable, Gi2 alpha and Gi3 alpha were both present on days 12 and 21. Of significance was the increase (160%) in the amount of Gi2 alpha at midgestation (day 12) compared to days 0 and 21. A different pattern was observed with Gi3 alpha, which decreased with advancing gestation (day 0 greater than 12 greater than 21). Immunodetection of beta subunits of G proteins indicated the presence of a 35/36 kDa doublet on days 0, 12 and 21, with an increase at midgestation. The simultaneous increase in Gi2 alpha and beta subunits may provide an explanation for the previously demonstrated alteration in adenylate cyclase stimulability detected at midgestation.     

Isolation and characterization of three distinct cDNAs for the chicken c-ski gene.

Three types of c-ski cDNAs have been isolated from two different chicken cDNA libraries. Sequence comparisons suggest that the cDNAs derive from alternatively spliced mRNAs. A short stretch of sequence homology that exists between c-ski and avian leukosis virus may have played a role in viral transduction.     

Concurrent up-regulation of guanine-nucleotide-binding proteins Gi1 alpha, Gi2 alpha and Gi3 alpha in adipocytes of hypothyroid rats.

Rat white adipocytes express three distinct 'Gi-like' guanine-nucleotide-binding proteins (G-proteins) [Mitchell, Griffiths, Saggerson, Houslay, Knowler & Milligan (1989) Biochem. J. 262, 403-408]. We have previously noted elevated levels of Gi in membranes of adipocytes from hypothyroid rats [Milligan, Spiegel, Unson & Saggerson (1987) Biochem. J. 247, 223-227]. Using a series of anti-peptide antisera able to discriminate between the individual gene products we have examined levels of each Gi-like G-protein in adipocyte membranes of hypothyroid rats compared with euthyroid controls. We demonstrate that up-regulation of Gi in adipocytes of hypothyroid rats is not restricted to a single subtype of Gi but that each of Gi1 alpha, Gi2 alpha and Gi3 alpha is present at markedly higher levels compared with euthyroid animals. In contrast, levels of both the 45 and 42 kDa forms of Gs alpha were not altered substantially in the hypothyroid state.     

Isolation and sequencing of cDNAs and genomic DNAs encoding the alpha 4 chain of basement membrane collagen type IV and assignment of the gene to the distal long arm of human chromosome 2.

We cloned three overlapping cDNAs covering 2,452 base pairs encoding a new basement membrane collagen chain, alpha 4(IV), from rabbit corneal endothelial cell RNA. Nucleotide sequence analysis demonstrated that the clones encoded a triple-helical domain of 392 1/3 amino acid residues and a carboxyl non-triple-helical (NC1) domain of 231 residues. We also isolated a genomic DNA fragment for the human alpha 4(IV) chain, which contained two exons encoding from the carboxyl end of the triple-helical domain to the amino end of the NC1 domain. Identification of the clones was based on the amino acid sequence identity between the cDNA-deduced amino acid sequence and the reported amino acid sequence obtained from a fragment of the alpha 4(IV) collagen polypeptide M28+ (Butkowski, R. J., Shen, G.-Q., Wieslander, J., Michael, A. F., and Fish, A. J. (1990) J. Lab. Clin. Med. 115, 365-373). When compared with four other type IV collagen chains, the NC1 domain contained 12 cysteinyl residues in positions identical to those of the residues in those chains. The domain demonstrated 61, 70, 55, and 60% amino acid similarity with human alpha 1, human alpha 2, bovine alpha 3, and human alpha 5 chains, respectively. The human genomic DNA fragment allowed us to map the alpha 4(IV) gene (COL4A4) to the 2q35-2q37.1 region of the human genome.     

Presence of methylated adenine in GATC sequences in chromosomal DNAs from Campylobacter species.

We digested chromosomal DNAs from 12 Campylobacter strains (C. jejuni, 4 strains; C. coli, 2 strains; C. fetus subsp. fetus, 2 strains; C. hyointestinalis, 2 strains; and C. upsaliensis, 2 strains) and from 4 Helicobacter strains (H. pylori, 2 strains; and H. mustelae, 2 strains) with HindIII, SstI, BamHI, DpnI, MboI, and Sau3AI. Restriction fragments were then separated by electrophoresis in 1% agarose or 10% polyacrylamide gels. Only DNAs from three Campylobacter species (C. jejuni, C. coli, and C. upsaliensis) were digested with DpnI (an enzyme that recognizes only methylated adenine in GATC sequences). We used MboI and Sau3AI to confirm these findings.     

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.     

Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli

Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.     

Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q.

The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.     

Chemically induced hypothyroidism produces elevated amounts of the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) and the beta subunit common to all G-proteins.

Adipocytes of hypothyroid rats display an increased responsiveness to agents which function by inhibiting the production of cyclic AMP. Anti-peptide antisera which selectively recognise the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) detected a 40 kDa polypeptide in adipocyte plasma membranes of both euthyroid and hypothyroid rats. Amounts of the alpha subunit of Gi were elevated some 2-fold in the hypothyroid preparations in comparison with the euthyroid controls, when equal amounts of membrane protein of the two treatments were examined. As cells from the hypothyroid animals contained 2.7 times as much membrane protein as those from the control animals, the amounts of alpha subunit of Gi are elevated some 5.6-fold per cell in adipocytes of the hypothyroid animals compared with the euthyroid controls. Amounts of the 36 kDa beta subunit of G-proteins were also elevated in plasma membranes of adipocytes of hypothyroid animals, in this case by some 50% when compared on a protein basis. These results provide direct evidence for alterations in the amounts of the subunits of Gi caused by the hypothyroid state.