RESUMO
Variation within genes has important implications for all biological traits. We identified 3899 single nucleotide polymorphisms (SNPs) that were present within 313 genes from 82 unrelated individuals of diverse ancestry, and we organized the SNPs into 4304 different haplotypes. Each gene had several variable SNPs and haplotypes that were present in all populations, as well as a number that were population-specific. Pairs of SNPs exhibited variability in the degree of linkage disequilibrium that was a function of their location within a gene, distance from each other, population distribution, and population frequency. Haplotypes generally had more information content (heterozygosity) than did individual SNPs. Our analysis of the pattern of variation strongly supports the recent expansion of the human population.
Assuntos
Variação Genética , Haplótipos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Povo Asiático/genética , População Negra/genética , Fosfatos de Dinucleosídeos/genética , Evolução Molecular , Feminino , Heterozigoto , Hispânico ou Latino/genética , Humanos , Masculino , Mutação , Pan troglodytes/genética , População Branca/genética , Cromossomo X/genéticaRESUMO
Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.
Assuntos
Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Proteínas de Bactérias/genética , Metabolismo Energético , Genes Bacterianos , Helicobacter pylori/genética , HumanosRESUMO
Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.
Assuntos
Genoma Bacteriano , Helicobacter pylori/genética , Úlcera Duodenal/microbiologia , Regulação Bacteriana da Expressão Gênica , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Scu-PA was cleaved by thrombin after arginine-156 to yield a two-chain molecule with low amidolytic activity and resistance to cleavage by plasmin. 125I-fibrin-labeled clots were dissolved in vitro by thrombin-cut scu-PA, but only at concentrations 10- to 50-fold greater than that needed for scu-PA. Three hours of incubation produced 100, 80, and 31% lysis with 100, 50, and 25 micrograms/ml thrombin-cut scu-PA. Thrombin-cut scu-PA, scu-PA, and tcu-PA yielded linear dose responses in the rabbit jugular venous thrombosis model. The dose required to reach 40% lysis was 2 mg/kg for scu-PA, 3 mg/kg for tcu-PA, and 4 mg/kg for thrombin-cut scu-PA. No significant consumption of fibrinogen or alpha 2-antiplasmin levels was observed with thrombin-cut scu-PA while the level of fibrinogen and alpha 2-antiplasmin decreased to about 50 and 40%, respectively, with scu-PA and to less than 10% of baseline with tcu-PA. Thus, while less potent than scu-PA, thrombin-cut scu-PA appears to be a more fibrin-specific thrombolytic agent than scu-PA.
Assuntos
Fibrinolíticos/metabolismo , Ativadores de Plasminogênio/metabolismo , Trombina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Amidas/metabolismo , Sequência de Aminoácidos , Animais , Arginina , Eletroforese em Gel de Poliacrilamida , Técnicas In Vitro , Veias Jugulares , Dados de Sequência Molecular , Coelhos , Trombose/tratamento farmacológicoRESUMO
Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-alpha) and fibroblast (HuIFN-beta) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-alpha clone, 201, has a nucleotide sequence different from published HuIFN-alpha sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-alpha in Escherichia coli.
Assuntos
Vetores Genéticos , Interferon Tipo I/genética , Sequência de Bases , Clonagem Molecular , Colífagos/genética , DNA de Cadeia Simples/genética , DNA Viral/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Interferon Tipo I/biossíntese , Hibridização de Ácido NucleicoRESUMO
A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the Mr-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.
Assuntos
Quimosina/genética , Clonagem Molecular , DNA , Genes , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Colífagos/genética , Enzimas de Restrição do DNA , Escherichia coli/genética , Hibridização de Ácido Nucleico , Plasmídeos , Poli A/genética , Biossíntese de Proteínas , RNA/genética , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The nucleotide sequence A-A-T-T was inserted into the intergenic region of the f1 genome at a site cleaved by Hae III (cleavage sequence (G-G-C-C). The resultant viable phage mutant (R199) contains a single site sensitive to the restriction endonuclease EcoRI (cleavage sequence G-A-A-T-T-C). This phage is sensitive to EcoRI restriction and modification in vivo and in vitro. Its potential for use as a cloning vector has been tested by construction in vitro of an f1/pBR322 chimeric phage. The four bases inserted into wild-type f1 to generate the R199 mutant came from a small restriction fragment obtained by digesting plasmid pBR322 with EcoRI and HindIII. The use of this linker prepared from a biological substrate is an example of a technique for constructing restriction enzyme sites in vitro. It is presented as an alternative to the use of synthetic linkers and should be generally applicable.
Assuntos
Colífagos/metabolismo , DNA Bacteriano/metabolismo , DNA Viral/metabolismo , Escherichia coli/metabolismo , Genes Virais , Sequência de Bases , Enzimas de Restrição do DNA , DNA Recombinante , Mutação , PlasmídeosRESUMO
We have characterized the 200-nucleotide-long insertion found in f1 after segregation of a chimeric phage containing the genomes of f1 and pSC101 [Ohsumi, M., Vovis, G.F. & Zinder, N.D. (1978) Virology 89, 438--449]. The insertion in this novel f1 species, called f1', is derived from pSC101 and has the potential to form an extended base-paired secondary structure, as determined by nucleotide sequence analysis. A five-nucleotide direct repeat, derived from f1 sequences, is present in f1'. The 200 additional nucleotides that are inserted into the DNA sequence coding for the carboxy terminus of f1 gene IV protein have generated a novel carboxy terminus for the f1' gene IV protein. In vitro transcription--translation studies demonstrate that a read-through protein can be expressed, as predicted from the f1' nucleotide sequence results. This 200-nucleotide-long sequence appears to be a transposable element found within pSC101 and is similar in sequence to the inverted repeat found in Tn3. Restriction enzyme analysis of the chimeric phage DNA, coupled with the nucleotide sequencing results, allows us to predict a structure for the genomic organization of this chimera.
Assuntos
Colífagos/genética , Plasmídeos , Sequência de Bases , DNA de Cadeia Simples/genética , DNA Viral/genética , Genes Virais , Recombinação Genética , Translocação GenéticaRESUMO
Four different isolates of Fusobacterium nucleatum (A,C,D and E) contain restriction endonucleases of differing specificity. Whilst many of the endonucleases are isochizomers of known enzymes, two novel activities are Fnu DII which recognizes and cleaves the sequence 5'-CGCT-3'/3'-GCGC-5' AND Fnu EI which recognizes and cleaves the sequence 5'-GATC-3'/3'-CTAG-5' irrespective of the extent of methylation of the adenine residues.
Assuntos
Enzimas de Restrição do DNA/metabolismo , Fusobacterium/enzimologia , Sequência de Bases , Enzimas de Restrição do DNA/isolamento & purificação , Peso Molecular , Oligodesoxirribonucleotídeos/análise , Especificidade por SubstratoAssuntos
Colífagos/genética , DNA Recombinante , DNA de Cadeia Simples/genética , DNA Viral/genética , Escherichia coli/genética , Plasmídeos , Colífagos/análise , DNA Recombinante/análise , DNA Viral/análise , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Tetraciclina/farmacologia , Transdução Genética , Transformação GenéticaRESUMO
Replicative form DNA of bacteriophage f1 was found to be sensitive in vitro to restriction by endonuclease R-EcoRII if the DNA was isolated from an Escherichia coli strain deficient in cytosine methylase activity. A similar observation was previously made with DNA from the closely related bacteriophage fd (S. Schlagman, S. Hattman, M. S. May, and L. Berger, submitted for publication). The two DNA fragments produced by the endo R-EcoRII digestion of f1 DNA were localized on the f1 cleavage map and their genetic content was determined. The polypeptides synthesized in a "coupled" transcription-translation system under the direction of each RII fragment were examined. The results of such experiments allow the ordering of genes V and VII and indicate the location of a RNA promotor for gene VIII.
Assuntos
Colífagos/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Viral/metabolismo , Endonucleases/metabolismo , Genes , Óperon , Mapeamento Cromossômico , Cromossomos/análise , DNA Viral/análise , Mutação , Ensaio de Placa Viral , Proteínas Virais/biossínteseAssuntos
Mapeamento Cromossômico , Colífagos , Escherichia coli , Cromatografia por Troca Iônica , Enzimas de Restrição do DNA/isolamento & purificação , DNA de Cadeia Simples , DNA Viral/análise , Eletroforese em Gel de Poliacrilamida , Haemophilus/enzimologia , Haemophilus influenzae/enzimologia , Mutação , Hibridização de Ácido Nucleico , Transformação GenéticaRESUMO
The restriction endonuclease from E. coli B is both an endonuclease and a DNA methylase. Both activities either require or are stimulated by Mg(+2), adenosine triphosphate, and S-adenosyl-L-methionine. The particular activity which the enzyme exhibits depends upon the nature of the SB sites, the genetic sites that identify substrate DNA. Enzymatic treatment of DNA that has an unmodified, wild-type SB site results in either rapid restriction of the DNA or very slow methylation of the SB site. On the other hand, a hybrid SB site (modified), which protects the DNA molecule from restriction, results in rapid methylation of that SB site.