Sequence of human galanin and its inhibition of glucose-stimulated insulin secretion from RIN cells.

Human progalanin cDNA was cloned with polymerase chain reaction techniques. The cDNA sequence predicts that the human form of galanin has a substitution of the glycine residue found at position 30 in other species and thus is likely to retain this residue during posttranslational processing and not be amidated at the COOH terminus. Furthermore, the cDNA sequence predicts three additional amino acid substitutions compared with known galanins. Human galanin was synthesized, and its bioactivity was compared with porcine and rat galanin based on inhibition of insulin release from a glucose-responsive rat insulinoma (RIN) cell line. Human galanin inhibited glucose-stimulated insulin secretion in a dose-dependent manner in RIN cells. Human, porcine, and rat galanin exhibited similar activity with ED50 less than 1 nM.

Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus Ustilago maydis.

A cDNA library was constructed in the yeast expression vector pYcDE8 using mRNA from the phytopathogenic fungus Ustilago maydis and cDNAs capable of complementing mutations in three yeast genes, URA3, LEU2 and TPI1, were identified. Nucleotide sequence analysis indicated that the cDNA clone, which complemented the yeast ura3 mutation, carries the pyr6 gene encoding orotidine-5'-phosphate decarboxylase. The genomic copy of the pyr6 gene was isolated by hybridization with the cDNA and used to complement a pyr- mutant of U. maydis. One-step gene disruption was demonstrated by transforming U. maydis with a copy of the pyr6 gene interrupted in the coding region by a selectable marker for resistance to hygromycin B.

The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum.

Five cDNAs from the cellulolytic fungi Fusarium oxysporum that code for five distinct cellulase homologues have been cloned and sequenced. The cloning strategy exploited the hydrophobic cluster analysis-based cellulase family classification of Henrissat and Bairoch [Biochem. J. 293 (1993) 781-788] to design degenerate oligodeoxyribonucleotides (oligos) that encoded amino-acid sequences conserved in an intra-family, but not inter-family, manner among cellulases from different species. Polymerase chain reaction (PCR) experiments using F. oxysporum genomic DNA primed with these 'family-specific' oligos were used to rapidly generate PCR fragments which were in turn used to probe cDNA libraries. Two distinct cDNAs coding for cellulase C-family homologues and one cDNA each coding for homologues to the B, F and K families, were isolated in this manner. This approach is an example of the power of multiple sequence analysis to generate cross-species, homology-based probes to rapidly clone homologues in a species of interest.

Selection of functional cDNAs by complementation in yeast.

Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members. Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation. Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences. The relative abundance of the various cDNAs in the cDNA plasmid pool paralleled the abundance of the mRNAs in total poly(A)+ RNA, which ranged from approximately 0.01% to 1%. The utility of this approach to isolate rare cDNAs from higher eukaryotes is discussed.

An extensive deletion causing overproduction of yeast iso-2-cytochrome c.

CYC7-H3 is a cis-dominant regulatory mutation that causes a 20-fold overproduction of yeast iso-2-cytochrome c. The CYC7-H3 mutation is an approximately 5 kb deletion with one breakpoint located in the 5' noncoding region of the CYC7 gene, approximately 200 base from the ATG initiation codon. The deletion apparently fuses a new regulatory region to the structural portion of the CYC7 locus. The CYC7-H3 deletion encompasses the RAD23 locus, which controls UV sensitivity and the ANP1 locus, which controls osmotic sensitivity. The gene cluster CYC7-RAD23-ANP1 displays striking similarity to the gene cluster CYC1-OSM1-RAD7, which controls, respectively, iso-1-cytochrome c, osmotic sensitivity and UV sensitivity. We suggest that these gene clusters are related by an ancient transpositional event.

Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns.

A functional cDNA from Aspergillus nidulans encoding triosephosphate isomerase (TPI) was isolated by its ability to complement a tpi1 mutation in Saccharomyces cerevisiae. This cDNA was used to obtain the corresponding gene, tpiA. Alignment of the cDNA and genomic DNA nucleotide sequences indicated that tpiA contains five introns. The intron positions in the tpiA gene were compared with those in the TPI genes of human, chicken, and maize. One intron is present at an identical position in all four organisms, two other introns are located in similar positions in A. nidulans and maize, and the remaining two introns are unique to A. nidulans. These Aspergillus-specific introns are located in regions of the protein that were predicted to be interrupted by introns based on analysis of a Go plot of chicken TPI. These comparisons are discussed in relation to the evolution of introns within TPI genes.

Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene.

An Aspergillus nidulans functional cDNA encoding an alcohol dehydrogenase (ADH) was isolated by its ability to complement an adh1 mutation in Saccharomyces cerevisiae. Alignment of the cDNA and cloned genomic DNA sequences indicated that the ADH gene contains two small introns. The presence of ethanol in the growth medium was shown to result in ADH mRNA accumulation presumably due to transcriptional induction of the gene. However, ADH mRNA accumulation was at most only partially repressed by the presence of glucose. The ADH gene characterized here is designated ADH3 since it is distinct from the alcA gene which encodes ADH I and appears distinct from the gene which encodes ADH II. We demonstrated that the first intron in the A. nidulans ADH3 gene was not efficiently spliced in S. cerevisiae whereas the promoter region was utilized weakly. We also present a comparison of the primary structure of A. nidulans ADH III with the alcohol dehydrogenases of S. cerevisiae and Schizosaccharomyces pombe.

Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.

Cloning and expression of a cellular high density lipoprotein-binding protein that is up-regulated by cholesterol loading of cells.

Plasma membranes of cultured cells contain high affinity receptors for high density lipoprotein (HDL) that appear to mediate removal of excess intracellular cholesterol. Recent studies using ligand blot analysis have identified a 110-kDa membrane protein which has features predicted for an HDL receptor, in that it preferentially binds HDL apolipoproteins and undergoes up-regulation in response to cholesterol loading of cells. In this study, we isolated a cDNA clone from an expression library using an antibody raised against partially purified 110-kDa HDL-binding protein. This clone encodes a novel cell protein, designated HBP, comprised mostly of 14 imperfect tandem repeats of approximately 70 amino acids in length. Each repeat appears to contain two amphipathic helices. Expression of HBP in cultured cells was increased severalfold when cells were loaded with cholesterol, as evident by increases in both HBP mRNA and membrane-associated protein. Overexpression of HBP in mammalian cell transfectants was associated with higher HDL binding to isolated cell protein and with modest increases in HDL binding to the cell surface. Proteins identified by ligand blot analysis had lower apparent M(r) than the primary HBP gene product and varied in M(r) and in HDL binding activity between cell types, suggesting that HBP undergoes cell-specific processing. These results provide preliminary evidence that HBP is a component of a cellular pathway that facilitates removal of excess cholesterol from cells, perhaps through its interaction with HDL. However, the predicted structure of HBP does not conform to that of any known receptor, suggesting that it does not function as a classic plasma membrane receptor.