RESUMO
A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.
Assuntos
Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Lactococcus lactis/genética , Bacteriófagos/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Ribossômico/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Marcadores Genéticos , Mutagênese Insercional , Hibridização de Ácido Nucleico , Óperon/genética , Mapeamento por Restrição , Especificidade da EspécieRESUMO
The chromosome structure of lactic acid bacteria has been investigated only recently. The development of pulsed-field gel electrophoresis (PFGE) combined with other DNA-based techniques enables whole-genome analysis of any bacterium, and has allowed rapid progress to be made in the knowledge of the lactic acid bacteria genome. Lactic acid bacteria possess one of the smallest eubacterial chromosomes. Depending on the species, the genome sizes range from 1.1 to 2.6 Mb. Combined physical and genetic maps of several species are already available or close to being achieved. Knowledge of the genomic structure of these organisms will serve as a basis for future genetic studies. Macrorestriction fingerprinting by PFGE is already one of the major tools for strain differentiation, identification of individual strains, and the detection of strain lineages. The genome data resulting from these studies will be of general application strain improvement.
Assuntos
Mapeamento Cromossômico , Genoma Bacteriano , Lactobacillus/genética , Lactococcus lactis/genética , Streptococcaceae/genética , Cromossomos Bacterianos , Ácido Láctico , Mapeamento Físico do CromossomoRESUMO
A combined physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403 was determined. We constructed a restriction map for the NotI, ApaI, and SmaI enzymes. The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments. The strain IL1403 chromosome was found to be circular and 2,420 kb in size. A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L. lactis and various other bacteria. Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome. Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped. At least seven copies of IS1076 were present and were located on 50% of the chromosome. In contrast, no copy of ISS1RS was detected. Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction. A comparison of the physical maps of L. lactis subsp. lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters. In contrast, despite major restriction pattern dissimilarities between L. lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains.
Assuntos
Cromossomos Bacterianos , Genes Bacterianos/genética , Lactococcus lactis/genética , Lactococcus/genética , Mapeamento por Restrição , Clonagem Molecular , Elementos de DNA Transponíveis/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Variação Genética , Mutagênese Insercional , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico/genética , Especificidade da EspécieRESUMO
Tools for the genetic and physical analysis of the Lactococcus lactis subsp. lactis genome were developed. Plasmid pRC1 does not replicate in Gram+ bacteria; it contains unique ApaI, NotI and SmaI restriction sites and an erythromycin-resistance (ErR) encoding gene, ermAM, functional in L. lactis subsp. lactis. When a chromosomal L. lactis subsp. lactis DNA fragment was cloned into this vector, the resulting plasmid became integrated, after transformation, into the bacterial chromosome by homologous recombination in a Campbell-like manner. The integration lead to the generation of new rare restriction sites near to the host fragment. This procedure allows precise mapping of cloned genes onto the chromosomal restriction map. The mapping of the his operon of L. lactis subsp. lactis provides an illustration. The cloning into pRC1 of an IS element able to transpose into the chromosome of the target cell, gave rise to an integration plasmid able to insert randomly rare restriction sites onto the bacterial chromosome. The L. lactis IS element, ISS1RS, was cloned into pRC1, yielding pRL1. Pulsed-field gel electrophoresis analysis of ErR clones obtained after transformation with pRL1, showed that this plasmid was stably integrated at a number of different sites in the L. lactis subsp. lactis chromosome, via transposition. Plasmids pRC1 and pRL1 can greatly facilitate the construction of the physical and genetic map of the chromosome of lactococcal strains.
Assuntos
Lactococcus/genética , Plasmídeos , Mapeamento por Restrição , Clonagem Molecular , Elementos de DNA Transponíveis , Eletroforese em Gel de Campo Pulsado , Técnicas Genéticas , Hibridização de Ácido Nucleico , ÓperonRESUMO
Pretreatment of cells of Lactococcus lactis subsp. lactis with low levels of N-methyl-N'-nitro-N-nitrosoguanidine does not reduce the cytotoxic and mutagenic effects caused by high concentration of this agent. This observation indicates that there is no efficient inducible error-free repair system for alkylation damage similar to the 'adaptive response' described in detail for Escherichia coli.
Assuntos
Reparo do DNA , Lactococcus lactis/efeitos dos fármacos , Metilnitronitrosoguanidina/toxicidade , Mutagênicos , Resposta SOS em Genética , Alquilantes/toxicidade , Dano ao DNA , MutaçãoRESUMO
About two clinical cases with maculopathy resulting from irradiation with so low dosage as to have been previously considered harmless in most of the literature. The authors suggest that early photocoagulation laser treatment as a mean of preserving a good visual prognosis for these eyes.
Assuntos
Macula Lutea , Lesões por Radiação/etiologia , Doenças Retinianas/etiologia , Adolescente , Adulto , Transplante de Medula Óssea , Feminino , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/cirurgia , Masculino , Irradiação Corporal Total/efeitos adversosRESUMO
The lethal and mutagenic effects of various mutagens on three strains of Streptococcus lactis were investigated. Lethality studies demonstrated that S. lactis was relatively sensitive to UV irradiation, methyl methanesulphonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and, to a lesser extent, to ethyl methanesulphonate (EMS). A spontaneous derivative Lac-, which has lost a 37-Md plasmid, was slightly more resistant and much less mutable than the wild-type after UV irradiation. Although the three strains were strongly mutated by EMS for the genetic marker assayed (Rifr), an increase in the mutation frequency was also observed after MMS and MNNG treatments.
Assuntos
Alquilantes/toxicidade , Lactococcus lactis/genética , Mutação , Raios Ultravioleta/efeitos adversos , DNA Bacteriano/análise , Eletroforese em Gel de Ágar , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/efeitos da radiação , PlasmídeosRESUMO
Six cases of intra-ocular metallic foreign bodies located into the retino-choroidal wall are presented. A surgical treatment was performed, and included a vitrectomy, a foreign body extraction with intraocular forceps, a primary or a secondary scleral buckling for the peripheral wound, and a retino-choroidectomy for one of the posteriorly located foreign bodies. In all the cases, we observed a cicatricial retraction of the retino-choroidal wound. When the wound was peripheral, the retina detached in the cases without buckling and it was necessary to do a secondary scleral buckling procedure. When the wound was located at the posterior pole, the retina remained flat in one case, with a 6 mm metallic body, probably because of the relaxing retinotomy performed to extract the foreign body. We think that it is better to perform a primary scleral buckle of the peripheral wounds. The various aspects of the treatment are discussed.