RESUMO
Phage infections still represent a serious risk to the dairy industry, in which Streptococcus thermophilus is used in starter cultures for the manufacture of yogurt and cheese. The goal of the present study was to analyze the biodiversity of the virulent S. thermophilus phage population in one Argentinean cheese plant. Ten distinct S. thermophilus phages were isolated from cheese whey samples collected in a 2-mo survey. They were then characterized by their morphology, host range, and restriction patterns. These phages were also classified within the 2 main groups of S. thermophilus phages (cos- and pac-type) using a newly adapted multiplex PCR method. Six phages were classified as cos-type phages, whereas the 4 others belonged to the pac-type group. This study illustrates the phage diversity that can be found in one factory that rotates several cultures of S. thermophilus. Limiting the number of starter cultures is likely to reduce phage biodiversity within a fermentation facility.
Assuntos
Queijo/microbiologia , Queijo/virologia , Variação Genética , Fagos de Streptococcus/genética , Streptococcus thermophilus/virologia , Argentina , Proteínas do Capsídeo/genética , Primers do DNA/química , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese em Gel de Campo Pulsado , Indústria de Processamento de Alimentos/normas , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência , Fagos de Streptococcus/classificação , Fagos de Streptococcus/isolamento & purificação , Streptococcus thermophilus/ultraestruturaRESUMO
Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with contractions of D4Z4 repeat on 4q35. It displays a remarkable inter- and intra-familial clinical variability ranging from severe phenotype to asymptomatic carriers. Mosaicism for the contracted FSHD-sized allele is a recurrent finding, but only DNA from lymphocytes had been studied. It is currently not known if mosaicism is unequally distributed between different tissues and if muscle is relatively spared for the presence of the disease allele in mosaic asymptomatic carriers of a disease allele. Here we compare DNA extracted from peripheral blood lymphocytes (PBL), fibroblasts and muscle from a mosaic asymptomatic female carrier and mother of a FSHD patient. PFGE analysis showed a complex allelic segregation: two independent mitotic rearrangement episodes occurred, resulting in mosaicism for a contracted D4Z4 repeat on 4q35 in the mother and mosaicism for an expanded D4Z4 repeat on 10q26 in the affected daughter. The results show that the proportion of mosaicism in PBL and muscle were comparable, while in fibroblasts there was some variation in the mosaicism, which might be caused by culturing artefacts. This finding supports the hypothesis that a mitotic contraction of D4Z4 is an early embryonic event and indicates that the degree of mosaicism in PBL is representative for that of muscle.
Assuntos
Mosaicismo , Distrofia Muscular Facioescapuloumeral/patologia , Sequências Repetitivas de Ácido Nucleico/genética , Alelos , Cromossomos Humanos Par 4 , DNA/genética , DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonuclease HindIII/metabolismo , Eletroforese em Gel de Campo Pulsado , Saúde da Família , Feminino , Fibroblastos/metabolismo , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Músculos/metabolismo , Distrofia Muscular Facioescapuloumeral/sangue , Distrofia Muscular Facioescapuloumeral/genética , LinhagemRESUMO
The nucleotide sequence of a 4,539 bp fragment of Bacillus stearothermophilus V mediating tellurite resistance in Escherichia coli was determined. Four ORFs of more than 150 amino acids encoding polypeptides of 244, 258, 308, and 421 residues were found in the restriction fragment. E. coli cells harboring a recombinant plasmid containing the Ter determinant express, when challenged with tellurite, a 32 kDa protein with an amino terminal sequence identical to the ten first residues of the 308 ORF. This ORF shows great similarity with the cysteine synthase gene (cysK) of a number of organisms. Recombinant clones carrying the active cysK gene have minimal inhibitory concentrations to K2TeO3 that were tenfold higher than those determined for the host strain or that of clones carrying ORFs 244, 258, and 421. Introduction of the B. stearothermophilus V cysK gene into a cysK strain of Salmonella typhimurium LT2 resulted in complementation of the mutation as well as transfer of tellurite resistance.
Assuntos
Cisteína Sintase/genética , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Geobacillus stearothermophilus/enzimologia , Telúrio/farmacologia , Clonagem Molecular , Cisteína Sintase/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Geobacillus stearothermophilus/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Plasmídeos/genética , Salmonella typhimurium , Análise de Sequência de DNARESUMO
The evolutionary relationships of five Atlantic Aeglidae species (Aegla neuquensis affinis, A. humahuaca, A. jujuyana, A. platensis, and A. uruguayana) were studied by (i) satellite DNA analysis using a restriction enzyme digestion and hybridization pattern approach and (ii) genome screening by using randomly amplified polymorphic DNA (RAPD) typing. The identical restriction patterns and intense interspecific hybridization patterns obtained in this study strongly suggest a recent cladogenetic event for the Aeglidae. The species-specific amplification products which were detected using RAPD markers allowed species characterization. A total of 49 amplification products were used to construct trees by cluster analysis. The new scheme agrees in part with previous proposals based on biogeography and morphology. We considered that the subdivision northwestern-platensis species was probably due to the rising of the Andes, which started in the Middle Miocene. Divergence due to altitude is suggested by the different altitudinal distribution of three northwestern species along the same river. The possible role of selection by ecological factor/s was observed at the population level in A. jujuyana, which has a wider altitudinal range distribution. RAPD markers revealed a high level of intraspecific diversity and important genetic flow among populations. However, a few markers showed significant differences in frequency or H between the lowermost population and the other populations, located in a different biogeographical region. The differences were not in relation to geographical distance, and we interpreted them as being due to selection. Repetitive sequences constitute an important reservoir of genetic variation, and these results show their usefulness in testing and proposing evolutionary hypothesis in crabs. These sequences seem to have played an important role in aeglid evolution. Ecological factors related to altitude have probably influenced macro- and microevolutionary processes, at least in northwestern species.
Assuntos
Evolução Biológica , Crustáceos/genética , DNA/química , Sequências Repetitivas de Ácido Nucleico , Animais , Argentina , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonuclease HindIII/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Hibridização de Ácido Nucleico , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
The pachytene ZW pair of the chicken has been studied with a novel combination of fluorescent in situ hybridization and silver staining of the synaptonemal complexes, and with electron microscopic in situ hybridization. Probes for the EcoRI and the XhoI repeat sequences were used for light microscopy and probes for XhoI for electron microscopy. XhoI repeats are pericentromeric and correspond to 27.3% of the W axis length. EcoRI repeats form three distinct domains: domain I covers the distal 19.4% of the late-synapsing arm, and domains II and III cover respectively 21.7% and 8.5% of the W axis. The early-synapsing end containing the recombination nodule is free from any of both signals. This non-hybridizing region accounts for 25.9% of the W axis. It is suggested that this region is composed of a proximal region containing other repeat types and a terminal region which is the recombining or pseudoautosomal region. The successful combination of silver staining and fluorescent in situ hybridization will be generally useful for high-resolution localization of DNA sequences in meiotic chromosomes.
Assuntos
Galinhas/genética , Meiose , Sequências Repetitivas de Ácido Nucleico/genética , Cromossomos Sexuais/genética , Animais , Sequência de Bases , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Feminino , Hibridização in Situ Fluorescente , Masculino , Microscopia Eletrônica , Modelos Genéticos , Dados de Sequência Molecular , Cromossomos Sexuais/ultraestrutura , Coloração pela PrataRESUMO
A method is described that facilitates the cloning of large synthetic genes in plasmid vectors such as pUC and pGEM. This protocol uses unpurified synthetic oligonucleotides of moderate length (ca. 50-90 bp) to construct larger DNA molecules by a reiterative, directional cloning procedure. Open reading frames are maintained by the flexible use of six-base pair blunt-end restriction sites that are not present in the DNA sequence of the plasmid cloning vector. Rapid production and analysis of plasmid intermediates are achieved by standard recombinant DNA cloning methods. The use of an anchored sticky-end restriction site and a variable blunt-end restriction site in each step of the cloning scheme gives specific orientation to each oligonucleotide fragment and results in high cloning efficiencies. The flexibility of the method allows for the construction of a gene of any size. Very large synthetic genes can be made by generating the intermediate gene fragments in parallel vectors and simply cloning the fragments in frame to produce the final construct.
Assuntos
Clonagem Molecular/métodos , DNA/biossíntese , Oligonucleotídeos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , DNA/química , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonuclease HindIII/metabolismo , Eletroporação , Escherichia coli/genética , Vetores Genéticos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase , Precursores de Proteínas/química , Precursores de Proteínas/genética , Xenopus laevisRESUMO
We describe a novel gross deletion of the factor VIII gene in 5 related patients with severe hemophilia A. The deletion extends from intron 15 to at least 8.5 kb beyond the 3' end of the gene (at least 95 kb of extension), and is associated with variable levels of FVIII inhibitor in 4 of the patients. The carrier detection in the family was based on the abnormal restriction pattern of the partially deleted gene.
Assuntos
Deleção Cromossômica , Fator VIII/genética , Hemofilia A/genética , Adolescente , Adulto , Southern Blotting , Criança , Pré-Escolar , DNA/genética , DNA/metabolismo , Desoxirribonuclease BamHI/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Éxons , Fator VIII/antagonistas & inibidores , Humanos , Hibridização de Ácido NucleicoRESUMO
Staphylococcus aureus isolates (n = 1201) from 20 centers in Europe, the USA and Brazil were evaluated for the presence of epidemiologic markers. Plasmid typing and restriction endonuclease analysis of plasmid DNA confirmed the presence of an apparently identical plasmid in 13% of clinical isolates. The plasmid was recovered from all 20 hospitals studied, with an overall frequency of greater than 10% on each of the three continents. Since relatively few staphylococcal plasmids may be shared by epidemiologically unrelated strains, there are inherent limitations to this otherwise useful technique. Additionally, these data demonstrate the importance of including unrelated strains of Staphylococcus aureus from the local region as controls when molecular typing methods are performed.