RESUMO
A procedure to produce an exact chromosomal replica of an insertion or insertion-deletion mutation produced in vitro in a plasmid with a ColE 1 origin of replication is presented. This procedure uses a previously described property of recD mutations (Biek DP, Cohen SN. J. Bacteriol. 1986;167:594-603) and is limited by (1) the compatibility of the new mutation with recD; and (2) the presence of some Escherichia coli DNA flanking the mutation.
Assuntos
Cromossomos Bacterianos/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Colicinas/genética , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Plasmídeos , Origem de Replicação , Deleção de SequênciaRESUMO
The primary structures of 11 proteins of ADP-glucose pyrophosphorylase are aligned and compared for relationships among them. These comparisons indicate that many domains are retained in the proteins from both the enteric bacteria and the proteins from angiosperm plants. The proteins from angiosperm plants show two main groups, with one of the main groups demonstrating two subgroups. The two main groups of angiosperm plant proteins are based upon the two subunits of the enzyme, whereas the subgroups of the large subunit group are based upon the tissue in which the particular gene had been expressed. Additionally, the small subunit group shows a slight but distinct division into a grouping based upon whether the protein is from a monocot or dicot source. Previous structure-function studies with the Escherichia coli enzyme have identified regions of the primary structure associated with the substrate binding site, the allosteric activator binding site, and the allosteric inhibitor binding site. There is conservation of the primary structure of the polypeptides for the substrate binding site and the allosteric activator binding site. The nucleotide sequences of the coding regions of the genes of 11 of these proteins are compared for relationships among them. This analysis indicates that the protein for the small subunit has been subject to greater selective pressure to retain a particular primary structure. Also, the coding region of the precursor gene for the small subunit diverged from the coding region of the precursor gene for the large subunits slightly prior to the divergence of the two coding regions of the genes for the two tissue-specific large subunit genes.
Assuntos
Nucleotidiltransferases/genética , Sítio Alostérico , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Evolução Biológica , Genes , Glucose-1-Fosfato Adenililtransferase , Dados de Sequência Molecular , Proteínas de Plantas/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
A report of a program which performs the initial computational reduction of the raw data from a cytotoxicity assay and outputs the reduced data as an image of the arrangement of the assay(s) upon the microwell plate. The program accepts the raw data either as manual or diskette file input.
Assuntos
Sistemas de Gerenciamento de Base de Dados/normas , Algoritmos , Computadores/normas , Testes Imunológicos de Citotoxicidade , HumanosRESUMO
Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to the Escherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.
Assuntos
Nucleotidiltransferases/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Glucose-1-Fosfato Adenililtransferase , Substâncias Macromoleculares , Dados de Sequência Molecular , Plantas/enzimologia , Plantas/genética , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/enzimologiaAssuntos
Frutanos/biossíntese , Nucleotidiltransferases/genética , Plantas/genética , Amido/biossíntese , Sequência de Aminoácidos , Escherichia coli/enzimologia , Escherichia coli/genética , Genes de Plantas , Glucose-1-Fosfato Adenililtransferase , Dados de Sequência Molecular , Plantas/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.
Assuntos
Regulação da Expressão Gênica , Genes ras , Leucemia Experimental/genética , Leucemia de Células T/genética , Proteínas de Membrana/genética , Vírus da Leucemia Murina de Moloney/genética , Proteínas Proto-Oncogênicas/genética , Alelos , Animais , Sequência de Bases , Linhagem Celular , Transformação Celular Neoplásica , Clonagem Molecular , Elementos de DNA Transponíveis , Rearranjo Gênico , Humanos , Immunoblotting , Leucemia Experimental/microbiologia , Leucemia de Células T/microbiologia , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas p21(ras) , RNA Viral/genética , Mapeamento por Restrição , TransfecçãoRESUMO
The xth gene of Escherichia coli K-12, which encodes exonuclease III, has been sequenced. Exonuclease III from a cloned copy of the E. coli K-12 gene has been purified and characterized. The molecular weight (30,921), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are in excellent agreement with those properties determined for the purified enzyme. The xth promoter was mapped by primer extension of in vivo transcripts. Inspection of the nucleotide sequence reveals that a region of dyad symmetry which could form a hairpin stem-loop structure in RNA characteristic of a rho-dependent terminator lies immediately downstream from the xth gene.
