First Genome Data from Uncultured Upland Soil Cluster Alpha Methanotrophs Provide Further Evidence for a Close Phylogenetic Relationship to Methylocapsa acidiphila B2 and for High-Affinity Methanotrophy Involving Particulate Methane Monooxygenase

ABSTRACT Members of upland soil cluster alpha (USCα) are assumed to be methanotrophic bacteria (MB) adapted to the trace level of atmospheric methane. So far, these MB have eluded all cultivation attempts. While the 16S rRNA phylogeny of USCα members is still not known, phylogenies constructed for the active-site polypeptide (encoded by pmoA) of particulate methane monooxygenase (pMMO) placed USCα next to the alphaproteobacterial Methylocapsa acidiphila B2. To assess whether the pmoA tree reflects the evolutionary identity of USCα, a 42-kb genomic contig of a USCα representative was obtained from acidic forest soil by screening a metagenomic fosmid library of 250,000 clones using pmoA-targeted PCR. For comparison, a 101-kb genomic contig from M. acidiphila was analyzed, including the pmo operon. The following three lines of evidence confirmed a close phylogenetic relationship between USCα and M. acidiphila: (i) tetranucleotide frequency patterns of 5-kb genomic subfragments, (ii) annotation and comparative analysis of the genomic fragments against all completely sequenced genomes available in public domain databases, and (iii) three single gene phylogenies constructed using the deduced amino acid sequences of a putative prephenate dehydratase, a staphylococcal-like nuclease, and a putative zinc metalloprotease. A comparative analysis of the pmo operons of USCα and M. acidiphila corroborated previous reports that both the pmo operon structure and the predicted secondary structure of deduced pMMO are highly conserved among all MB.

Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster

Assigning functional significance to completed genome sequences is one of the next challenges in biological science. Conventional genetic tools such as deficiency chromosomes help assign essential complementation groups to their corresponding genes. We describe an F2 genetic screen to identify lethal mutations within cytogenetic region 61D-61F of the third chromosome of Drosophila melanogaster. One hundred sixteen mutations were identified by their failure to complement both Df(3L)bab-PG and Df(3L)3C7. These alleles were assigned to 14 complementation groups and 9 deficiency intervals. Complementation groups were ordered using existing deficiencies, as well as new deficiencies generated in this study. With the aid of the genomic sequence, genetic and physical maps in the region were correlated by use of PCR to localize the breakpoints of deficiencies within a 268-kb genomic contig (GenBank accession No. AC005847). Six essential complementation groups were assigned to specific genes, including genes encoding a porphobilinogen deaminase and a Sac1-like protein.Key words: Drosophila, functional genomics, porphobilinogen deaminase, synaptojanin.