phosphorus-radioisotopes and Chromosome-Deletion

The herpes simplex virus 1 genome was shown to encode two genes, US3 and UL13, exhibiting amino acid sequence motifs common to protein kinases. Elsewhere this laboratory reported that the prominent substrate of the US3 protein kinase is the product of the UL34 gene, an essential nonglycosylated membrane protein. In the absence of the US3 kinase, the UL34 protein remains unphosphorylated but forms a complex with four proteins that become phosphorylated uniquely when UL34 is not. To investigate the role of UL13 protein in this process, recombinant viruses lacking UL13 or both UL13 and US3 were constructed. We report that UL13 is dispensable for viral replication in cell culture and is not involved in the processing of UL34 or of associated phosphoproteins. UL13 is, however, responsible for the posttranslational processing associated with phosphorylation of infected-cell protein 22, the product of the alpha 22 gene. This gene was previously reported to play a regulatory role in selected cell lines. UL13 appears to be either a protein kinase or a phosphotransferase and its major substrate is the alpha 22 protein.

Topics: Animals; Autoradiography; Cell Line; Chromosome Deletion; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Genes, Viral; Genome, Viral; Genotype; Phenotype; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Protein Processing, Post-Translational; Restriction Mapping; Simplexvirus; Sulfur Radioisotopes; Viral Proteins

PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.

Topics: Amino Acid Sequence; Base Sequence; beta-Galactosidase; Blotting, Southern; Chromosome Deletion; Cloning, Molecular; DNA, Bacterial; Escherichia coli; Genes, Bacterial; Molecular Sequence Data; Mutagenesis, Site-Directed; Myxococcus xanthus; Oligodeoxyribonucleotides; Open Reading Frames; Phenotype; Phosphoproteins; Phosphorus Radioisotopes; Polymerase Chain Reaction; Protein Kinases; Protein Serine-Threonine Kinases; Recombinant Proteins; Restriction Mapping; Sequence Homology, Nucleic Acid; Spores, Bacterial

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.

Topics: Animals; Autoradiography; Blotting, Southern; Caenorhabditis; Chromosome Deletion; Cloning, Molecular; DNA; Genes; Mutation; Myosins; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Polymorphism, Genetic; Restriction Mapping

Topics: Chromosome Deletion; Cloning, Molecular; Escherichia coli; Genes, Bacterial; Mutation; Nucleic Acid Hybridization; Operon; Phosphorus Radioisotopes; Radioisotope Dilution Technique; RNA, Ribosomal; RNA, Ribosomal, 5S