Molecular genetical and phenotypical analysis of the gerM spore germination gene of Bacillus subtilis 168.

The gerM gene, encoding a single product of 22.5 kDa, has been identified by subcloning and sequencing of DNA recovered from adjacent to a Tn917 insertion. The gene product has a potential lipoprotein signal sequence, but otherwise has no homology to known sequences. Spores of the gerM mutant were more heat sensitive than wild-type, but their dipicolinic acid content was normal. The level of cortical peptidoglycan in mutant spores is also normal but release at germination of hexosamine-containing fragments, the breakdown products of cortex degradation, is less complete than wild-type. The sporulation, resistance and germination phenotypes of the gerM mutant would be consistent with the gene product having a role, either directly or indirectly, in peptidoglycan synthesis during sporulation.

Human bradykinin B2 receptor: nucleotide sequence analysis and assignment to chromosome 14.

Functional cDNA clones for human bradykinin B2 receptor were isolated from uterus RNA by a polymerase chain reaction (PCR)-based method and by screening a human cosmid library with rat bradykinin B2 receptor probe. We isolated several overlapping clones from the cosmid library, each of which encodes the entire protein coding sequence. The human bradykinin B2 receptor gene codes for a 364-amino-acid protein with a molecular mass of 41,442 Da that is highly homologous to rat bradykinin B2 receptor cDNA (81%). The entire human cDNA sequence was cloned into an expression vector and mRNA was synthesised by in vitro transcription. Applications of bradykinin caused membrane current responses in Xenopus oocytes injected with the in vitro-synthesized mRNA. Preincubation with the potent B2 antagonist, HOE140, prevented this response. The genomic clone is intronless, and we have identified an upstream promoter region and a downstream polyadenylation signal. The human bradykinin B2 receptor gene has been mapped to chromosome 14 using PCR to specifically amplify DNA from somatic cell hybrids.

Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16.

Chromosome 21 has often been used as a model system for the development of genome mapping and cloning strategies in humans. In this report methods for systematic chromosome walking, cloning, and mapping are exemplified in the construction of a 1.5-Mb yeast artificial chromosome (YAC) contig encompassing and extending 400 kb beyond each of the genetic loci D21S13 and D21S16. Isolation of insert-terminal sequences from YACs in this contig provides a set of closely spaced physical markers. These have been used to generate a long-range genomic restriction map.

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.

Genetical and molecular studies on gerM, a new developmental locus of Bacillus subtilis.

A transposon Tn917 insertion between gerE and ilvB has identified a new developmental locus, gerM, in Bacillus subtilis. gerM96::Tn917 affects both sporulation and germination. DNA on either side of the transposon has been cloned and includes the previously cloned sdhC and gerE loci. gerE terminates 2.1 kb from the end of the transposon. The gerM96::Tn917 mutant is oligosporogenous, yielding approximately 1% of the number of wild-type heat resistant spores in liquid medium and 10% on solid medium. Six hours after the onset of sporulation alkaline phosphatase and glucose dehydrogenase levels were 90% and 7%, respectively, of those of the wild-type. At this time 50% of the mutant cells were still dividing. The occurrence of multiple polar septa and 'pygmy' cells suggested a block at stage II of sporulation. Following addition of germinants, mutant spores prepared on nutrient agar lost heat resistance normally but released slightly less dipicolinic acid than wild-type spores. They also showed only partial loss of optical density, associated with a phase-grey appearance and striations in the cortex suggesting partial degradation. Expression of the gerM gene was monitored by production of beta-galactosidase encoded by a promotorless lacZ gene fused to the gerM96::Tn917 insertion. It occurred 1.5-4 h after commencement of sporulation. Transcription was directed from a promoter on the gerE side of gerM and was unaffected by a mutation in the gerE gene.