Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells.

Cloning and expression in a stable mammalian cell line co-transfected with a glutamate transporter (RGT cells) were used as tools for studying the functions and pharmacological properties of group III metabotropic glutamate receptors (mGluRs). Complementary DNAs (cDNAs) encoding the human mGluR4, human mGluR7, and human mGluR8 were isolated from human cerebellum, fetal brain or retinal cDNA libraries. The human mGluR4, mGluR7 and mGluR8 receptors were 912, 915 and 908 amino acid residues long and share 67-70% amino acid similarity with each other and 42-45% similarity with the members of mGluR subgroups I and II. The human mGluR4 and mGluR7 had amino acid identity of 96% and 99.5% with rat mGluR4 and 7, respectively, whereas the human mGluR8 has 98.8% amino acid identity with the mouse mGluR8. The nucleotide and amino acid sequences in the coding region of human mGluR4 and mGluR7 were found to be identical to the previously published sequences by Flor et al. and Makoff et al. Following stable expression in RGT cells, highly significant inhibitions of forskolin stimulation of cAMP production by group III agonists were found for each receptor. The relative potencies of the group III agonist L-AP4 varied greatly between the group III clones, being mGluR8>mGluR4 >> mGluR7. The reported group II mGluR agonist L-CCG-I was a highly potent mGluR8 agonist (EC50=0.35 microM), with significant agonist activities at both mGluR4 (EC50=3.7 microM) and mGluR7 (EC50=47 microM). The antagonist potency of the purported group III mGluR antagonist MPPG also varied among the receptors being human mGluR8 >> mGluR4 = mGluR7. The expression and second messenger coupling of human group III mGluRs expressed in the RGT cell line are useful to clearly define the subtype selectivities of mGluR ligands.

The role of neuropeptide Y in the antiobesity action of the obese gene product.

Recently Zhang et al. cloned a gene that is expressed only in adipose tissue of the mouse. The obese phenotype of the ob/ob mouse is linked to a mutation in the obese gene that results in expression of a truncated inactive protein. Human and rat homologues for this gene are known. Previous experiments predict such a hormone to have a hypothalamic target. Hypothalamic neuropeptide Y stimulates food intake, decreases thermogenesis, and increases plasma insulin and corticosterone levels making it a potential target. Here we express the obese protein in Escherichia coli and find that it suppresses food intake and decreases body weight dramatically when administered to normal and ob/ob mice but not db/db (diabetic) mice, which are thought to lack the appropriate receptor. High-affinity binding was detected in the rat hypothalamus. One mechanism by which this protein regulated food intake and metabolism was inhibition of neuropeptide-Y synthesis and release.

Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli.

Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli. A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors. E. coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0-22% of total cell protein. This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa. Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp-IGFII in E. coli. Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.

Method for selection of transposable DNA and characterization of a new insertion sequence, IS493, from Streptomyces lividans.

A method to select for transposable elements from Streptomyces spp. by using insertional inactivation of a repressor gene that functions in Escherichia coli was developed. Plasmid pCZA126, which can replicate in Streptomyces spp. or E. coli, contains a gene coding for the lambda cI857 repressor and a gene, under repressor control, coding for apramycin resistance. E. coli cells containing the plasmid are apramycin sensitive but become apramycin resistant if the cI857 repressor gene is disrupted. Plasmids propagated in Streptomyces spp. can be screened for transposable elements that have disrupted the cI857 gene by transforming E. coli cells to apramycin resistance. This method was used to isolate a new 1.6-kilobase insertion sequence, IS493, from Streptomyces lividans CT2. IS493 duplicated host DNA at the target site, had inverted repeats at its ends, and contained two tandem open reading frames on each strand. IS493 was present in three copies in the same genomic locations in several S. lividans strains. Two of the copies appeared to be present in regions of similar DNA context that extended at least 11.5 kilobases. Several other Streptomyces spp. did not appear to contain copies of IS493.

Nucleotide sequence of barley stripe mosaic virus RNA alpha: RNA alpha encodes a single polypeptide with homology to corresponding proteins from other viruses.

The complete nucleotide sequence of RNA alpha from the Type strain of barley stripe mosaic virus has been determined. The RNA is 3768 nucleotides long and contains a single open reading frame which codes for a polypeptide of 1139 amino acids (mw 129,634). The open reading frame is flanked by a 5'-terminal sequence of 91 nucleotides and a 3'-nontranslated region composed of a short poly(A) tract followed by a 238-nucleotide tRNA-like structure. The amino acid sequence of the polypeptide (alpha a) encoded by the open reading frame has homology with the TMV 126K protein and with related polypeptides from other viruses. The carboxy-terminal portion of the alpha a polypeptide also has limited homology with the 58K (beta b) protein encoded by BSMV RNA beta and includes a consensus sequence found in mononucleotide-binding polypeptides.

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.

Cloning and nucleotide sequence of a carbomycin-resistance gene from Streptomyces thermotolerans.

Two plasmids (pOJ158 and pOJ159) containing DNA fragments from the carbomycin(Cb)-producing strain Streptomyces thermotolerans were identified in Streptomyces griseofuscus based on their ability to confer resistance to Cb. The Cb-resistance determinants on pOJ158 and pOJ159 were designated carA and carB, respectively. In S. griseofuscus, pOJ159 also confers resistance to spiramycin, rosaramicin, lincomycin, and vernamycin B, but not to tylosin; in Streptomyces lividans, pOJ159 additionally confers resistance to erythromycin and oleandomycin. The carB gene was localized on pOJ159 to a 1.25-kb region whose nucleotide sequence was determined. The sequence has a G + C content of 68% and contains the coding sequence for carB and portions of the 5' and 3' untranslated regions. A comparison of the amino acid sequence of the protein encoded by carB (as deduced from the nucleotide sequence) with the deduced amino acid sequence of the RNA methylase from Streptomyces erythraeus (encoded by ermE) revealed extensive homology, suggesting that carB also encodes an RNA methylase. The region 5' to the coding sequence does not contain a small ORF or regions of complementarity that are commonly associated with translationally regulated macrolide-lincosamide-streptogramin B resistance genes. The 3' untranslated region contains an inverted repeat sequence that potentially can form a stable RNA stem-loop structure with a calculated delta G of -70 kcal.

Hygromycin B resistance as dominant selectable marker in yeast.

Saccharomyces cerevisiae is normally sensitive to the drug hygromycin B; a hygromycin B concentration of 200 µg/ml in agar plates is sufficient to completely inhibit growth. We constructed yeast-E. coli bifunctional plasmids which confer hygromycin B resistance to Saccharomyces cerevisiae. Promoters and amino terminal coding regions of a heat shock gene, a heat shock cognate gene, and the phosphoglycerate kinase gene from yeast were fused to a bacterial hygromycin B resistance gene. In all three cases, yeast cells containing plasmids with the hybrid hygromycin B resistance gene were resistant to high levels of the drug. Yeast cells containing these plasmids can also be directly selected after transformation by using hygromycin B. The intact bacterial hygromycin B resistance gene and the kanamycin resistance gene from Tn903 were also tested in yeast for their ability to confer resistance to hygromycin B and G418. The intact bacterial genes were not effective in conferring drug resistance to yeast cells.

Analysis of a bacterial hygromycin B resistance gene by transcriptional and translational fusions and by DNA sequencing.

We have characterized hygromycin B and apramycin resistance genes from an E. coli plasmid. We have localized the coding and control regions of these genes by deletion of DNA fragments from plasmids containing the genes. It was found that polypeptides with apparent molecular weights of 33,000 and 31,500 daltons are encoded by the apramycin resistance gene and polypeptides with apparent molecular weights of 42,500 and 41,500 daltons are encoded by the hygromycin B resistance gene. DNA sequence analysis identified a typical promoter sequence upstream of the genes. Deletion of this promoter eliminated both resistance phenotypes, and hygromycin B resistance could be restored by substitution of a promoter from a foreign gene. The region known to be necessary for hygromycin B resistance contained an open reading frame large enough to encode the hygromycin B resistance gene product. This open reading frame was fused with the amino terminus of beta-galactosidase. This hybrid gene conferred hygromycin resistance to E. coli, and expression of resistance was under IPTG control.