Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2).

The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS) was determined. Proviral DNA of ARV-2 (9737 base pairs) has long terminal repeat structures (636 base pairs) and long open reading frames encoding gag (506 codons), pol (1003 codons), and env (863 codons) genes. Two additional open reading frames were identified. Significant amino acid homology with several other retroviruses was noted in the predicted product of gag and pol, but ARV-2 was as closely related to murine and avian retroviruses as it was to human T-cell leukemia viruses (HTLV-I and HTLV-II). By means of an SV-40 vector in transfected simian cells, the cloned gag and env genes of ARV-2 were shown to express viral proteins.

High level expression of proinsulin in the yeast, Saccharomyces cerevisiae.

Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.

The specificity of pilin DNA sequences for the detection of pathogenic Neisseria.

A nucleic acid hybridization assay for the detection of the pilin gene of Neisseria gonorrhoeae has been devised. The method involves solution hybridization of pilin specific synthetic oligonucleotide probes to genomic DNA in crude cell lysates. This is followed by capture of the probe-target complex onto a microtitre dish well, signal amplification and labelling based on horseradish peroxidase conjugated to oligonucleotides. Detection is achieved with a chemiluminescent enzyme substrate. With a detection limit of about 20,000 cells, the 4-h assay is as sensitive as a radioactive dot-blot method. Over 150 strains of Neisseria gonorrhoeae collected from a variety of sources were detected with the assay. Several N. meningitidis serogroups were also found to react positively. No reactivity was observed with non-pathogenic Neisseria spp. or with other known pathogenic or normal microbial inhabitants of the human urogenital tract.

Rapid nucleic acid assay for detection of bacteria with tetM-mediated tetracycline resistance.

A novel nucleic acid assay has been developed to screen bacterial populations for the presence of the tetM structural gene. The method involves the specific hybridization of several synthetic oligonucleotides to the gene in a crude bacterial lysate solution. As few as 1.5 x 10(4) CFU can be detected with the assay.

Sequences of liver cDNAs encoding two different mouse insulin-like growth factor I precursors.

Complementary DNAs encoding mouse liver insulin-like growth factor I (IGF-I) have been isolated and sequenced. Alternative RNA splicing results in the synthesis of two types of mouse IGF-I precursor that differ in the size and sequence of the COOH-terminal peptide. The sequences of the signal peptides, IGF-I moieties and the first 16 amino acids of the COOH-terminal peptides or E-domains of the two precursors are identical. The sequence difference results from the presence in preproIGF-IB mRNA of a 52 base insertion which introduces a 17 amino acid segment into the COOH-terminal peptide of preproIGF-IB and also causes a shift in the reading frame of the mRNA. As a consequence of this insertion, the COOH-terminal 19 and 25 amino acids of mouse preproIGF-IA and -IB, respectively, are different. The sequences of mouse and human preproIGF-IA are highly conserved and possess 94% identity. In contrast, the sequences of mouse and human preproIGF-IB are quite different in the region of the COOH-terminal peptide. A comparison of the sequences of mouse and human preproIGF-IB mRNA indicates that they are generated by different molecular mechanisms.

Sequence of a placental cDNA encoding the mouse insulin-like growth factor II precursor.

cDNAs encoding the mouse insulin-like growth factor II (IGF-II) precursor have been isolated from a placental library and sequenced. Mouse prepro-IGF-II is predicted to be 180 amino acids and has 84% and 97% identity with human and rat IGF-II precursors, respectively. There are two prepro-IGF-II transcripts in mouse placenta; the major transcript is 3850 bases and the minor one is 4800 bases. Prepro-IGF-II mRNA cannot be detected in adult mouse liver, but it is readily detected in adult human liver, indicating that there are differences in the regulation of expression of the IGF-II gene in the liver of adult mice and human beings.

Evolution of CuZn superoxide dismutase and the Greek key beta-barrel structural motif.

Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron-exon boundary locations, gene duplication, and Greek key beta-barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of superoxide dismutation. The beta-strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the beta-strands and at both termini. Thus, the beta-barrel with only four invariant residues is apparently over-determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility of the Greek key beta-barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.

Sequence of a cDNA clone encoding human preproinsulin-like growth factor II.

The insulin-like growth factors (IGF) I and II are single-chain serum proteins of 70 and 67 amino acids, respectively, which are synthesized by the liver and possibly other tissues. They are probably required for normal fetal and postnatal growth and development. They also stimulate the growth of cultured cells, possibly by controlling the progression through the G1 phase of the cell cycle. In contrast to IGF-II whose concentration does not vary during postnatal development, the serum levels of IGF-I increase several-fold to adult levels during puberty. The serum concentration of IGF-I is a sensitive monitor of growth hormone levels and is decreased in individuals with growth hormone deficiency and elevated in those with growth hormone-secreting tumours. As a first step in studying the biosynthesis of these proteins and elucidating their role(s) in normal development and in tumorigenesis, we have isolated and sequenced cDNAs prepared from adult human liver mRNA which encode the precursors to IGF-I and -II. We report here the sequence of a cDNA encoding a 180-amino acid protein which is the precursor to IGF-II.

Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization.

Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini, respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover, 15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein.