No abstract

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.

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.

We have isolated a brain-specific cDNA that encodes a Na+-dependent inorganic phosphate (Po) Subtractive Cloning. Subtractive hybridization was used to identify genes differentially expressed after NMDA exposure (12 hr) of cerebellar granule cells. Rat cerebellar granule cells (P8) were cultured and exposed to NMDA as described (6). Poly(A)+ RNA was isolated by using oligo(dT)-cellulose columns (9) from NMDA-treated (12 hr) (tester) and control (untreated) cerebellar granule cells (driver). The tester mRNA (1 I.g) was used to generate[32P]dCTP-labeled first-strand cDNA (6000 Ci/mmol; NEN; 1 Ci = 37 GBq). Driver mRNA (10 pg) was photobiotinylated with a 300 W Sylvania clear bulb and was used to hybridize to tester cDNAs. Subtractive hybridization was done by using subtractor 1, as described by the manufacturer (Invitrogen). After hybridization, common sequences were removed by adding streptavidin and extracting with phenol/chloroform. Resulting subtracted cDNA was used to screen a Zap II cDNA library constructed from NMDA-treated cerebellar granule cells. Positive clones were rescued, replated individually, and rescreened with labeled, single-stranded cDNAs derived from driver and tester mRNAs. Those cDNAs that were detected only by tester probes, and were not detected by driver probes, were isolated for further analysis. Plasmids were excised from A phage, as described by the manufacturer (Stratagene), digested with EcoRI, and electrophoresed in a 1% agarose gel before DNA blotting. cDNA inserts that were specifically detected by tester probes were sequenced.DNA Sequencing and Sequence Analysis. The nucleotide sequence of the BNPI cDNA clone was determined for both strands. Sequence reactions were done by using doublestranded DNA templates, sequence-specific oligonucleotide primers, fluorescently labeled dideoxynucleotide terminators (Applied Biosystems) and Ampli-Taq polymerase in cyclesequencing reactions modified as described (10). Individual sequences were assembled with an Applied Biosystems Abbreviations: NMDA, N-methyl-D-aspartate; BNPI, brain Na+-dependent inorganic phosphate cotransporter I. tPresent address: