Stanley G. Burgett

Genome of the Bacterium Streptococcus pneumoniae Strain R6

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

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.

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.

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

Abstract The complete nucleotide sequence of RNAα 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 (αa) 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 as polypeptide also has limited homology with the 58K (βb) protein encoded by BSMV RNAβ and includes a consensus sequence found in mononucleotide-binding polypeptides.

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.

Enzymatic degradation by the sera of various animal species of penicillium chrysogenum mycophage double-stranded RNA

Abstract The enzymatic degradation of dsRNA by the sera of a number of animal species was studied using a quantitative polyacrylamide gel assay procedure. Species ranking of activity correlate well with known biological effects of dsRNA. Human serum showed unusually high activity, and a comparison was made between it and bovine ribonuclease A using penicillium chrysogenum mycophage double-stranded RNA and yeast sRNA substrates.