Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

[Concordance between phenotypical features and PCR-REA for the identification of Malassezia spp]. / Concordancia entre características fenotípicas y PCR-REA en la identificación de especies de Malassezia.

The genus Malassezia has been recently revised and nowadays includes 11 species that cannot always be differentiated from each other by physiological and morphological tests. This study was aimed to evaluate the correlation between a molecular method and conventional phenotypic features in the identification of Malassezia spp. To achieve this aim, 92 Argentinean clinical strains isolated between 2001 and 2005 were analyzed along with three reference strains (Malassezia furfur CBS 7019, Malassezia sympodialis CBS 7222 and Malassezia slooffiae CBS 7956). By using PCR and restriction enzyme analysis with three different DNA endonucleases (PCR-REA), the molecular method consistently identified all three reference strains and all 92 clinical isolates as follows: 63 M. sympodialis, 18 M. furfur, 10 Malassezia globosa and one Malassezia obtusa. Phenotypic studies undentified 85 clinical isolates and two of the reference strains (total agreement > 91%). In particular for M. sympodialis, M. furfur and M. globosa, the species more frequently involved in human pathology, the agreement ranged between 84 and 96%. This result suggests that phenotypic studies are suitable for the presumptive identification of important Malassezia species in the clinical medical mycology laboratories where molecular methodologies are not available.

Association of a distinctive strain of Epstein-Barr virus with gastric cancer.

Epstein-Barr virus (EBV) has been linked to gastric carcinoma (GC) with worldwide geographical variations attributable to types and variants of EBV. Here, we compare EBV strains between EBVaGC and healthy donors in Latin America, a high frequency area for EBVaGC. Tumor samples from 73 EBVaGC cases and throat washings from 329 healthy adults were examined for types 1 and 2 EBV and polymorphism at BamHI-F and BamHI-W1/I1 boundary regions and XhoI restriction site in LMP1 gene. Type 1 and prototype F of BamHI- F polymorphism accounted 59 (81%) and 69 (95%) of EBVaGC cases and 257 (78%) and 267 (81%) of healthy donors, respectively. Types I and "i" of BamHI W1/I1 polymorphism accounted 2 (3%) and 62 (85%) of EBVaGC and 85 (26%) and 170 (52%) of healthy donors, respectively (p<0.001). XhoI+ and - polymorphism accounted 60 (82%) and 4 (5%) of EBVaGC and 142 (43%) and 92 (28%) of healthy donors, respectively (p<0.001). Cosegregation analysis demonstrated that most of the 62 type "i" EBVaGC cases harbor XhoI+ strain (81%). However, among 143 type "i" healthy adults, both XhoI polymorphism were present in relatively similar frequencies (XhoI+ 58% and XhoI- 42%) (OR 9.0; 95% CI 1.2-69). Our findings are against to the proposed hypothesis that EBV strains are geographically but not disease-restricted. We conclude that most of the EBVaGC cases harbor a distinctive EBV strain (type "i"/XhoI +), but in healthy donors, this strain was as common as other strains. This finding is contrary to the proposed hypothesis that EBV strains are geographically but not disease-restricted and identified a healthy population group that share the same strain that predominate in EBVaGC cases.

Isolation of equine herpesvirus-2 from the lung of an aborted fetus.

This study describes the isolation of equine herpesvirus-2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti-EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause-effect relationship was established between the EHV-2 isolate and the abortion.

Isolation and identification of Adenovirus in hospitalized children, under five years, with acute respiratory disease, in Havana, Cuba

Nine Adenovirus (Ad) strains isolated in Cuba, from 128 nasopharingeal swab specimens of children below five years old, with acute respiratory diseases, during 1996 and 1997, were studied by restriction enzyme analysis of genomic DNA with two endonucleases BamH I and Sma I. All different fragment patterns were compared with the respective prototypes. The identified adenoviruses were Ad 1 (n=4), Ad 2 (n=1) and Ad 6 (n=4). Males were more frequently infected than females. The analysis of the occurrence of these Adenovirus strains of subgenus C revealed that Ad 1 and Ad 6 were the predominant serotypes in 1996 and in 1997, respectively

Isolation and identification of adenovirus in hospitalized children, under five years, with acute respiratory disease, in Havana, Cuba.

Nine Adenovirus (Ad) strains isolated in Cuba, from 128 nasopharingeal swab specimens of children below five years old, with acute respiratory diseases, during 1996 and 1997, were studied by restriction enzyme analysis of genomic DNA with two endonucleases BamH I and Sma I. All different fragment patterns were compared with the respective prototypes. The identified adenoviruses were Ad 1 (n=4), Ad 2 (n=1) and Ad 6 (n=4). Males were more frequently infected than females. The analysis of the occurrence of these Adenovirus strains of subgenus C revealed that Ad 1 and Ad 6 were the predominant serotypes in 1996 and in 1997, respectively.

Intrachromosomal localization of aberration breakpoints induced by neutrons and gamma rays in Chinese hamster ovary cells.

Chromosome breakpoints induced by neutrons or gamma rays in Chinese hamster ovary cells were mapped to Giemsa-light or Giemsa-dark bands or to band junctions. Radiation-induced breakpoints were found to be distributed nonrandomly according to chromosome or band length. More than 60% of the breakpoints were localized in G-light bands. A group of 13 bands which corresponded to only 7% of the total chromosome length contained 22% of the breakpoints produced by neutrons and 14% of those induced by gamma rays. Seven of these 13 bands are also preferentially damaged by AluI, BamHI and DNase I as reported previously. The results indicate that chromatin and nuclear structure may play a role in the distribution of breakpoints produced by ionizing radiation and endonucleases.

Diversity of genomic electropherotypes of naturally occurring equine herpesvirus 1 isolates in Argentina.

The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, BamHI and BglII, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype.

Diversity of genomic electropherotypes of naturally occurring equine herpesvirus 1 isolates in Argentina

The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, Bam HI and Bgl II, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype

Genome composition in Venezuelan spiny-rats of the genus Proechimys(Rodentia, Echimyidae). I. Genome size, C-heterochromatin and repetitive DNAs in situ hybridization patterns.

The genome sizes of the Venezuelan spiny-rats Proechimys guairae guairae (2n = 48) and P. trinitatis (2n = 62) were evaluated and proved to be 12.5 +/- 0.5 pg and 12.6 +/- 0.3 pg respectively, the highest so far recorded among mammals; also the C-heterochromatin (32.7%, Coefficient of Variation [CV] 3.8 and 35.8%, CV 4.4) and GC (44.2%, CV 2.7 and 43.6%, CV 2.9) contents are very high. Highly repetitive (rep) DNAs were isolated from restriction enzyme digested genomic DNAs of both species. The intra- and inter-specific chromosomal allocations of these rep DNAs were analyzed by direct and cross-hybridizations. Results show that the two genomes harbour several rep DNA families which show both species-specificity and interspecific relatedness in their in situ hybridization patterns. The rep DNA families show an equilocal distribution at both the pericentromeric areas of all chromosomes and in the whole arms of two pairs of the uniarmed group, suggesting co-evolution of the rep DNAs. P. g. guairae BamHI digested DNA, when cloned and sequenced, proved to consist of a long "composite" unit (1,239 bp) containing two copies of each of 75-bp and 110-bp internal subrepeats. Karyotype restructuring between P. g. guairae and P. trinitatis, mainly due to Robertsonian changes, was accompanied by slight intra- and intergenomic movements of the putative satellite DNA families within stable genome sizes and C-heterochromatin contents. We discuss the findings obtained in Proechimys in the light of those regarding the kangaroo rat, the pocket gopher and the house mouse; they support the idea that karyotype restructuring could be the expression of molecular driven events of rep DNA amplification and homogenisation through non-homologous chromosomes.

Localization of chromosome breakpoints induced by DNase I in Chinese hamster ovary (CHO) cells.

DNase I was electroporated into S-phase CHO cells and induced chromosome breakpoints were localized in G-banded metaphases. More than 75% of breakpoints mapped to Giemsa-light bands, 18% to Giemsa-dark bands and about 7% to band junctions. Chromosome breakpoint clusters produced by DNase I colocalized with chromosome breakpoints induced by the restriction endonucleases AluI and BamHI in the G1- and S-phases of the cell cycle in CHO cells. Digestion of metaphase spreads with AluI, BamHI and DNase I produced G-bands, indicating that G-light bands are more sensitive to endonuclease action. The possible role of nuclease-sensitive sites in active chromatin as selective targets for the induction of chromosome breakpoints by these endonucleases is discussed.

Intrachromosomal localization of breakpoints induced by the restriction endonucleases AluI and BamHI in Chinese hamster ovary cells treated in S phase of the cell cycle.

The restriction endonucleases (REs) AluI and BamHI were electroporated into Chinese hamster ovary (CHO) cells during S phase of the cell cycle and breakpoints in G-banded metaphases were mapped to Giemsa-light or -dark bands or to band junctions. The majority of AluI- and BamHI-induced breakpoints were located in Giemsa-light bands. Both REs induced similar distributions of breakpoint clusters. The localization pattern of S phase-induced breakpoints in CHO cells is similar to the pattern of G1-induced breakpoints reported earlier. These data show that breakpoint localization for both REs is independent of the cell cycle stage (G1 or S) in which aberrations are induced and give further support to the hypothesis that nuclease hypersensitive regions (NHRs) associated with active genes play an important role in the distribution of breakpoints.

Localization of chromosome breakpoints induced by AluI and BamHI in Chinese hamster ovary cells treated in the G1 phase of the cell cycle.

Intact Chinese hamster ovary cells were exposed to the restriction endonucleases (REs) AluI or BamHI. In metaphase spreads from these cells, 300 breakpoints per RE were localized in G-banded chromosome type aberrations (dicentrics, translocations, rings, terminal and interstitial deletions). The majority of breakpoints induced by both REs were localized in G-light bands and showed a similar distribution of breakpoint clusters. RE digestion of metaphase spreads with AluI induced C-banding, and with BamHI G-banding. The data indicate that nuclease sensitive sites associated with active genes are mainly responsible for the distribution of breakpoints.

The genotype of Aujeszky's disease viruses isolated in Argentina.

Genomes of four Argentine isolates of Aujeszky's disease virus (ADV) (Rio Cuarto/79, Mercedes, Chanar Ladeado-7 and Chanar Ladeado-15) from pigs were characterized and compared with four ADV strains obtained from U.S.A. (Indiana-S), Sweden (Sweden 66), France (Alfort) and Japan (Yamagata-S81) by restriction endonuclease (RE) analysis. Although three Argentine isolates were classified into type I of BamHI cleavage pattern, one isolate, Mercedes, belonged to type II, according to the classification by Herrmann et al. [6]. Since this type II virus was first isolated in 1981, no outbreak of ADV infection by this type has so far been reported in Argentina. This may imply that the immediate measures by total slaughter of pigs in the farm led successful eradication of the type II ADV infection in Argentina. This report is the first epidemiological study using RE analysis on ADV strains in this country.

Location and rapid analysis of the intragenic BamHI polymorphic site of the factor IX gene.

We report the precise location and a polymerase chain reaction (PCR)-based method for the analysis of the intragenic BamHI polymorphism of the factor IX (FIX) gene. After screening DNA samples from the Brazilian Black population by a selective amplification of a segment of the FIX gene containing entire exon 2, intron 2, and exon 3 followed by digestion of PCR products with BamHI, we were able to identify individuals presenting the polymorphic BamHI site. By DNA sequencing of selected samples, the dimorphic base was located at nucleotide number 6575 (intron 2). The PCR method outlined here allows rapid and easy analysis of this polymorphism. Its application may be particularly useful for carrier detection and prenatal diagnosis of hemophilia B in the Black population, thus far the only one that has been shown to be polymorphic for this site. Because of its apparent restrictive pattern it may also be used in combination with other markers for estimates of racial admixture in mixed populations in which the Black population is present (as is the case for most countries in the American continent).

Location and rapid analysis of the intragenic BamHI polymorphic site of the factor IX gene

We report the precise location and a polymerase chain reaction (PCR)-based method for the analysis of the intragenic BamHI polymorphism of the factor IX (FIX) gene. After screening DNA samples from the Brazilian Black population by a selective amplification of a segment of the FIX gene containing entire exon 2, intron 2, and exon 3 followed by digestion of PCR products with BamHI, we were able to identify individuals presenting the polymorphic BamHI site. By DNA sequencing of selected samples, the dimorphic base was located at nucleotide number 6575 (intron 2). The PCR method outlined here allows rapid and easy analysis of this polymorphism. Its application may be particularly useful for carrier detection and prenatal diagnosis of hemophilia B in the Black population, thus far the only one that has been shown to be polymorphic for this site. Because of its apparent restrictive pattern it may also be used in combination with other markers for estimates of racial admixture in mixed populations in which the Black population is present (as is the case for most countries in the American continent).

Restriction site patterns in the ribosomal DNA of camelidae.

The restriction map of rDNA from South American camelids and the Bactrian camel was analyzed by digestion of high-molecular-weight DNA with endonucleases EcoRI,BamHI and the two combined followed by Southern blot hybridization with probes for the 18S and 28S rDNA sequences. We scored a total of 17 restriction sites, six of which were mapped conserved in all the species. The other eleven corresponded to spacer regions and revealed variations between these taxa. The study showed that the two groups differ in the length of the internal transcribed spacer. Also they showed the existence of two regions of fast evolution on the opposite termini of the external spacer. A restriction site present at low frequency in the non-transcribed spacer of guanaco and llama was the only difference encountered within the South American group.

[Cleft lip and palate in the Chilean population: association with BamH1 polymorphism of the transforming growth factor alpha (TGFA) gene]. / Fisura labiopalatina en población chilena: asociación con polimorfismo BamH1 del gen factor transformante del crecimiento alfa (TGFA).

In recent studies we have demonstrated that the model that better explains the genetic etiology of non syndromic cleft lip/palate (CL/P) in the Chilean population is one that postulates the existence of a major dominant autosomic locus with low penetrance, without discarding the possible influence of polygenes. Similar conclusions have been communicated by others authors in different populations. Thus, investigations have been initiated to seek possible associations between candidate genes and restriction length polymorphisms (RFLP's), specifically between Transforming Growth Factor Alpha (TGFA) gene RFLP's and CL/P, in caucasian populations. Results thus far obtained have been inconclusive. Therefore, the aim of this work was to study this association in the Chilean population, that is ethnically different. The gene and phenotype frequencies of the TGFA gene BamH1 polymorphism in CL/P probands (n = 21) and controls (n = 16) were determined. No significant differences were detected in the frequencies of the A1 and A2 alleles of the TGFA gene between probands and controls. These results do not support an association between the cleft palate phenotype and TGFA RFLP.

A novel deletion of FVIII gene associated with variable levels of FVIII inhibitor.

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.

Demethylated CD8 gene in CD4+ T cells suggests that CD4+ cells develop from CD8+ precursors.

Mature T cells and medullary thymocytes bear either the CD4 or CD8 differentiation antigen. Precursor cells in the thymus express neither CD4 nor CD8 (CD4-8-), but most cortical thymocytes are CD4+8+. Whether CD4+ and CD8+ mature T cells arise directly from CD4-8- precursors or from a CD4+8+ intermediate remains unresolved. In this study, methylation of the CD8 gene in murine T cells and thymocytes was examined. There was progressive demethylation of the CD8 gene in the thymus during the transition from CD4-8- to CD4+8+. A similar pattern of demethylation of the CD8 gene was seen in CD4+ mature T cells, suggesting previous expression of CD8 in the CD4+ lineage.