Project description:Expression of nifH in 28 surface water samples collected during fall 2007 from six stations in the vicinity of the Cape Verde Islands (north-east Atlantic) was examined using reverse transcription-polymerase chain reaction (RT-PCR)-based clone libraries and quantitative RT-PCR (RT-qPCR) analysis of seven diazotrophic phylotypes. Biological nitrogen fixation (BNF) rates and nutrient concentrations were determined for these stations, which were selected based on a range in surface chlorophyll concentrations to target a gradient of primary productivity. BNF rates greater than 6?nmolN?l(-1)?h(-1) were measured at two of the near-shore stations where high concentrations of Fe and PO(4)(3-) were also measured. Six hundred and five nifH transcripts were amplified by RT-PCR, of which 76% are described by six operational taxonomic units, including Trichodesmium and the uncultivated UCYN-A, and four non-cyanobacterial diazotrophs that clustered with uncultivated Proteobacteria. Although all five cyanobacterial phylotypes quantified in RT-qPCR assays were detected at different stations in this study, UCYN-A contributed most significantly to the pool of nifH transcripts in both coastal and oligotrophic waters. A comparison of results from RT-PCR clone libraries and RT-qPCR indicated that a ?-proteobacterial phylotype was preferentially amplified in clone libraries, which underscores the need to use caution interpreting clone-library-based nifH studies, especially when considering the importance of uncultivated proteobacterial diazotrophs.
Project description:We isolated two nondefective bovine rotavirus mutants (A5-10 and A5-16 clones) which have nonsense mutations in the early portion of the open reading frame of the NSP1 gene. In the NSP1 gene (1,587 bases long) of A5-10, a nonsense codon is present at nucleotides 153 to 155 just upstream of the coding region (nucleotides 156 to 230) of a cysteine-rich Zn finger motif. A5-16 gene 5 (1,087 bases long) was found to have a large deletion of 500 bases corresponding to nucleotides 142 to 641 of a parent A5-10 NSP1 gene and to have a nonsense codon at nucleotides 183 to 185, which resulted from the deletion. Expression of gene 5-specific NSP1 could not be detected in MA-104 cells infected with the A5-10 or A5-16 clone or in an in vitro translation system using the plasmids with gene 5 cDNA from A5-10 or A5-16. Nevertheless, both A5-10 and A5-16 replicated well in cultured cells, although the plaque size of A5-16 was extremely small.