Paul Robert Rosteck

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.

Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa1

BACKGROUND Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

Research PaperCloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa1

BACKGROUND Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

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 BACILLUS SUBTILIS PNBA GENE ENCODING P-NITROBENZYL ESTERASE : CLONING,SEQUENCE AND HIGH-LEVEL EXPRESSION IN ESCHERICHIA COLI

p-Nitrobenzyl esters serve as protecting groups on intermediates in the manufacture of clinically important oral beta-lactam antibiotics; de-esterification of the intermediates is required for synthesis of the final product. A Bacillus subtilis PNB carboxy-esterase (PNBCE) catalyzes hydrolysis of several beta-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol. This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleotide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli. The amino acid (aa) sequence, deduced from the nucleotide sequence of the pnbA ORF, matched an experimentally determined N-terminal aa sequence of B. subtilis PNBCE and also matched an active site sequence previously identified by biochemical analyses. Specific activity of PNBCE in crude extracts was more than 90-fold greater in recombinant E. coli, as compared to B. subtilis. This increase in expression led to more than a 500-fold improvement in the efficiency of purification of PNBCE.

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.

Molecular Cloning, Expression, and Chromosomal Localization of a Human Brain-Specific Na+-Dependent Inorganic Phosphate Cotransporter

Abstract: We describe the molecular cloning of a cDNA encoding a human brain Na+‐dependent inorganic phosphate (Pi) cotransporter (hBNPI). The nucleotide and deduced amino acid sequences of hBNPI reveal a protein of 560 amino acids with six to eight putative transmembrane segments. hBNPI shares a high degree of homology with other Na+‐dependent inorganic Pi cotransporters, including those found in rat brain and human and rabbit kidney. Expression of hBNPI in COS‐1 cells results in Na+‐dependent Pi uptake. Northern blot analysis demonstrates that hBNPI mRNA is expressed predominantly in brain and most abundantly in neuron‐enriched regions such as the amygdala and hippocampus. Moderate levels of expression are also observed in glia‐enriched areas such as the corpus callosum, and low levels are observed in the substantia nigra, subthalamic nuclei, and thalamus. In situ hybridization histochemistry reveals relatively high levels of hBNPI mRNA in pyramidal neurons of the cerebral cortex and hippocampus and in granule neurons of dentate gyrus. The level of hBNPI mRNA is quite low in fetal compared with adult human brain, suggesting developmental regulation of hBNPI gene expression. Southern analyses of nine eukaryotic genomic DNAs probed under stringent conditions with hBNPI cDNA revealed that the hBNPI gene is highly conserved during vertebrate evolution and that each gene is most likely present as a single copy. Using fluorescent in situ hybridization, we localized hBNPI to the long arm of chromosome 19 (19q13) in close proximity to the late‐onset familial Alzheimers disease locus.

Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: Mechanical loading regulates osteopontin and myeloperoxidase

The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase‐polymerase chain reaction (DDRT‐PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague‐Dawley rats, approximately 8 months old, received either a sham or bending load using a four‐point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse‐transcribed into cDNA, and used in DDRT‐PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4‐fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4‐ to 10‐fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load‐induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function. J. Cell. Biochem. 68:355–365, 1998.

A physical map encompassing GP2B, EPB3, D17S183, D17S78, D17S1183, and D17S1184.

The q21 region of chromosome 17 contains the gene BRCA1, which is involved in familial early-onset breast and ovarian cancers. A physical map of a region that extends from a distal boundary of the BRCA1 region, D17S78, to GP2B has been constructed. The map consists of 30 STSs, including 2 new short tandem repeat polymorphic markers. The contig is composed of a mixture of 7 YACs, 5 P1 plasmids, and 14 cosmids and was ordered by STS-content mapping.

The human genome project: genetic and physical mapping.

The modern tools of molecular biology, recombinant DNA techniques, have given scientists the ability to isolate and study individual genes from even complex eukaryotic genomes. The availability of genes enables the study o f their structure and biologic function, and their role in normal and abnormal physiologic processes. A worldwide effort to study and understand the entire human genome is under way, which will result in information on the location of all genes, their sequences, and their complex regulation and interactions. As this knowledge becomes available, it will be rapidly applied to the practice of medicine through use in the development of diagnostic tests for genetic-based diseases and in the development of therapeutics.