Prokaryotes commonly present outer cell wall structures composed of a crystalline array of proteinaceous subunits, known as surface layers (S-layers). The ORF encoding the S-layer protein (SlpA) of ...Lactobacillus brevis was cloned into Lactococcus lactis under the transcriptional control of the xylose-inducible expression system (XIES). SlpA was secreted into the extracellular medium, as determined by immunoblotting, and assays on the kinetics of SlpA production revealed that repression of the system with glucose did not require the depletion of xylose from the medium that allows transitory ORF expression. The successful use of XIES to express S-layer proteins in the versatile and generally recognized as safe species L. lactis opens new possibilities for an efficient production and isolation of SlpA S-layer protein for its various applications in biotechnology and importantly as an antigen-carrying vehicle.
Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by ...Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.
The genomic diversity of Argentine Tospoviruses from different geographical areas, and from several distinct crops was analysed here. For each isolate, RT-PCR, cloning and sequencing of a 450 nt ...fragment of the N gene were performed. Comparisons of RNA and predicted amino acid sequence identity and similarity were made. A partial sequence of the N gene was able to classify our local isolates within three Tospovirus species previously described (Tomato spotted wilt virus, TSWV; tomato chlorotic spot virus, TCSV and groundnut ringspot virus, GRSV). With the sequence data currently available, we performed a cladistic phylogenetic analysis which gave a possible genealogy among members of the Tospovirus genus.
Citrus tristeza closterovirus (CTV), is a phloem-limited virus
transmitted by aphids in a semipersistent manner. The genome of CTV is
composed of a ssRNA with two capsid proteins: CP, covering about ...95% of
the particle length, and a diverged coat protein (dCP), present only in
one end of the particle, forming a rattlesnake structure. dCP is the
product of p27 gene for which it is also postulated a function in the
transmissibility by aphid vectors. Hybridization analysis showed a p27
gene region, which exhibits different patterns with two probes derived
from two biological distinct CTV isolates. In an attempt to screen
whether that gene region differs in mild and severe strains, six CTV
isolates belonging to different biogroups were compared for variations
in their p27 gene by analysis of single-strand conformation
polymorphism (SSCP). The p27 gene was reverse transcribed and amplified
by PCR and thirty clones of each isolate were obtained. From each
clone, two fragments of the gene were amplified by PCR: fragment (a),
459 bp long, and fragment (b), 281 bp long. Sequence variations in both
gene fragments were studied by SSCP analysis. A variety of SSCP
patterns was obtained from each isolate, being isolates belonging to
the groups II-IV and III those with the higher and lower number of
them. Moreover, SSCP analysis provided a rapid procedure to screen the
genetic heterogeneity of the viral isolates reducing considerably the
amount of nucleic acid sequenciation necessary to gain that knowledge.
Citrus tristeza closterovirus (CTV), is a phloem-limited virus transmitted by aphids in a semipersistent manner. The genome of CTV is composed of a ssRNA with two capsid proteins: CP, covering about ...95% of the particle length, and a diverged coat protein (dCP), present only in one end of the particle, forming a rattlesnake structure. dCP is the product of p27 gene for which it is also postulated a function in the transmissibility by aphid vectors. Hybridization analysis showed a p27 gene region, which exhibits different patterns with two probes derived from two biological distinct CTV isolates. In an attempt to screen whether that gene region differs in mild and severe strains, six CTV isolates belonging to different biogroups were compared for variations in their p27 gene by analysis of single-strand conformation polymorphism (SSCP). The p27 gene was reverse transcribed and amplified by PCR and thirty clones of each isolate were obtained. From each clone, two fragments of the gene were amplified by PCR: fragment (a), 459 bp long, and fragment (b), 281 bp long. Sequence variations in both gene fragments were studied by SSCP analysis. A variety of SSCP patterns was obtained from each isolate, being isolates belonging to the groups II-IV and III those with the higher and lower number of them. Moreover, SSCP analysis provided a rapid procedure to screen the genetic heterogeneity of the viral isolates reducing considerably the amount of nucleic acid sequenciation necessary to gain that knowledge.
Citrus tristeza closterovirus (CTV), is a phloem-limited virus transmitted by aphids in a semipersistent manner. The genome of CTV is composed of a ssRNA with two capsid proteins: CP, covering about ...95% of the particle length, and a diverged coat protein (dCP), present only in one end of the particle, forming a rattlesnake structure. dCP is the product of p27 gene for which it is also postulated a function in the transmissibility by aphid vectors. Hybridization analysis showed a p27 gene region, which exhibits different patterns with two probes derived from two biological distinct CTV isolates. In an attempt to screen whether that gene region differs in mild and severe strains, six CTV isolates belonging to different biogroups were compared for variations in their p27 gene by analysis of single-strand conformation polymorphism (SSCP). The p27 gene was reverse transcribed and amplified by PCR and thirty clones of each isolate were obtained. From each clone, two fragments of the gene were amplified by PCR: fragment (a), 459 bp long, and fragment (b), 281 bp long. Sequence variations in both gene fragments were studied by SSCP analysis. A variety of SSCP patterns was obtained from each isolate, being isolates belonging to the groups II-IV and III those with the higher and lower number of them. Moreover, SSCP analysis provided a rapid procedure to screen the genetic heterogeneity of the viral isolates reducing considerably the amount of nucleic acid sequenciation necessary to gain that knowledge.
Interspecies repetitive DNA homology was studied in akodont rodents related at generic and suprageneric levels. The homology was determined by taking the species Akodon molinae as the reference ...species. The 3H-DNA/DNA hybridization on filters showed a closer relationship between A. molinae and A. azarae, A. dolores and A. mollis than between A. molinae and Bolomys obscurus. These data agree with the taxonomical ranking of the species. The quantity and quality of the hybrid DNAs were measured by investigating their thermal stabilities and subsequent comparison to the results obtained on the reference species. These data indicate high similitude between the repetitive DNA of A. dolores and A. molinae. Increasing differences were shown to occur in the repetitive DNA of A. mollis, B. obscurus and A. azarae, respectively. Since these results coincide with the G-banding homologies and differ slightly from the taxonomical rank, it is speculated that the divergency between the DNA of A. molinae and A. azarae is the result of a differential process of DNA amplification which is not related to the phylogenetical distance separating the two species.
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