Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2-3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica.

This study represents the first ß-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one ß-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. ß-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke ß-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.

Globin Gene Expression: Role of Transcription Factors

La dérégulation de l'expression génétique est une base pathophysiologique de plusieurs maladies. On a utilisé le locus du gène β-globine humain comme modèle pour élucider le mécanisme de régulation de la transcription génétique et évaluer son expression génétique durant l'érythropoïèse. La famille des protéines 'E' est composée de facteurs de transcription qui possèdent plusieurs sites de liaison au sein de locus du gène β-globine, suggérant leur rôle potentiel dans la régulation de l’expression de ces gènes. Nous avons montré que les facteurs HEB, E2A et ETO2 interagissent d’une manière significative avec la région contrôle du Locus (LCR) et avec les promoteurs des gènes de la famille β-globine. Le recrutement de ces facteurs au locus est modifié lors de l'érythropoïèse dans les cellules souches hematopoitiques et les cellules erythroides de souris transgéniques pour le locus de la β-globine humain, ainsi que dans les cellules progénitrices hématopoïétiques humaines. De plus par cette étude, nous démontrons pour la première fois que le gène β-globine humain est dans une chromatine active et qu’il interagit avec des facteurs de transcriptions de type suppresseurs dans les cellules progénitrices lymphoïdes (voie de différentiation alternative). Cette étude a aussi été faite dans des souris ayant une génétique mutante caractérisée par l'absence des facteurs de transcription E2A ou HEB.

Emergence of Klebsiella pneumoniae carrying the novel extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and the class 1 integron-associated bla(GES-7) in Brazil

A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-7) was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere.

The frequency of 844ins68 mutation in the cystathionine beta-synthase gene is not increased in patients with venous thrombosis

Background and Objectives. A frequent mutation in the cystathionine beta-synthase (CBS) gene (844ins68, a 68-bp insertion in the coding region of exon 8) was recently discovered. In the present study we Investigated this mutation as a candidate risk factor for venous thrombosis.Design and Methods. The prevalence of the 844ins68 CBS mutation was determined in 101 patients with objectively diagnosed deep venous thrombosis and in 101 healthy controls matched for age, sex and race. PCR amplification of a DNA fragment containing exon 8 of the CBS gene was employed to determine the genotypes, Additionally, Bsrl restriction enzyme digestion of the PCR products was performed in all samples from carriers of the insertion, to test for concurrent presence of a second mutation (T833C) in the CBS gene.Results. The insertion was found in 21 out of 101 patients (20.8%; allele frequency 0.109) and in 20 out of 101 controls (19.8%; allele frequency 0.114), yielding a relative risk for venous thrombosis related to the 844ins68 CBS mutation close to 1.0. In addition, the T833C GBS mutation was detected in all alleles carrying the 844ins68 CBS insertion, confirming the co-inheritance of the two mutations.Interpretation and Conclusions. Our findings do not support the hypothesis that the 844ins68 mutation in the CBS gene is a genetic risk factor for venous thrombosis. (C)1998, Ferrata Storti Foundation.

Molecular investigation of zoonotic genotypes of Giardia intestinalis isolates in humans, dogs and cats, sheep, goats and cattle in Araçatuba (Sãoo Paulo State, Brazil) by the analysis of beta-giardin gene fragments

In the period from July 2009 to October 2010, fecal samples from 61 animals and 154 humans from the municipality of Aracatuba (São Paulo State, Brazil) were studied. Fecal samples from animals were collected in the Municipal Animal Shelter and the Veterinary Hospital of the Universidade Estadual Paulista. Human fecal specimens were collected in playschools in the outskirts of the city by the private network of clinical analysis laboratories of the municipal. Diagnosis was done by optical microscopy using the Faust and Hoffmann, Pons and Janer techniques. The genotypes of Giardia intestinalis were characterized by PCR-RFLP and confirmed by sequencing the ß-giardin gene. Human specimens were positive in 25.3% (39/154) of the cases with 26.8% (36/134) of the specimens from children and 15% (3/20) from adults being positive. The frequency of G. intestinalis among the animals was 23.0% (14/61). A total of 32 isolates of G. intestinalis obtained from human feces and six from dogs and cats were characteristic of the A genotype (AI and AII/AIII). The results of this study in respect to frequency of giardiasis are similar to reported in most studies in Brazil. The prevalence observed in animal populations conforms to worldwide infection rates. G. intestinalis genotypes considered zoonotic were detected in both pets and humans from the city of Aractuba, suggesting a possible zoonotic transmission of the parasite in the northwestern region of São Paulo State. The absence of these genotypes in farm animals may imply that they are not involved in the chain of transmission to humans in this region.

Complete Sequence of Broad-Host-Range Plasmid pRIO-5 Harboring the Extended-Spectrum-beta-Lactamase Gene bla(BES-1)

Broad-host-range plasmid pRIO-5, harboring the extended-spectrum beta-lactamase bla(BES-1) gene in Serratia marcescens, was fully sequenced. Analysis of the 12,957-bp sequence of this IncP6-type plasmid revealed that the bla(BES-1) gene was associated with two copies of the insertion sequence IS26. The promoter responsible for the bla(BES-1) expression was hybrid, made of a - 35 box located inside the inverted repeat of IS26 and a - 10 box inside a remnant of an insertion sequence.

β-Globin polymorphisms in amerindian populations from the brazilian amazon

Objectives: This investigation was performed to examine genetic variation at the beta-globin locus in a sample of 30 healthy individuals from native populations in South America. The patterns of haplotypic variation were compared with those of previous studies including samples for various worldwide populations in an attempt to make inferences about the occupation of the Americas from a deeper temporal perspective than is typically available with haploid markers. Methods: A 2.67-kb segment containing the beta-globin gene and its flanking regions was examined for genetic variation in a sample of 60 chromosomes from native populations in South America. The fragment was PCR-amplified and directly sequenced. To determine linkage relationships in compound heterozygotes, we used the amplification refractory mutation system. In addition, we assessed genetic variability and differentiation among populations, and we performed tests of selective neutrality. These analyses were performed for Brazilian Amerindian group and other worldwide populations previously studied. Results: Eleven polymorphic sites were found in the studied fragment, which distinguished eight different haplotypes, three recombinants haplotypes (present as single copies) and five previously described haplotypes, including some of those most highly differentiated. Genetic variation found in the pooled sample is substantial. Conclusions: Although only five known haplotypes are observed in Amazonia, some of these are highly divergent, resulting in patterns of molecular polymorphism equal to or higher than those from other world regions. Am. J. Hum. Biol. 2012. (C) 2012 Wiley Periodicals, Inc.

Short communication: duplication in the 5'-flanking region of the beta-lactoglobulin gene is linked to the BLG A allele

beta-Lactoglobulin (beta-LG) is the major whey protein in the milk of cows and other ruminants. It is well established that the predominant genetic variants beta-LG A and B are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants. However, the genetic basis for the differentially expressed BLG A and B alleles is still elusive. In this study additional genetic variation further upstream in the 5'-flanking region of the BLG gene was identified, including 6 single nucleotide substitutions, a single nucleotide deletion, and a 7-bp duplication. Comparison of DNA sequences showed that the investigated 5'-flanking region is highly conserved between ruminants, and the duplication g.-1885_-1879dupCTCTCGC and the substitution g.-1888A>G are only found in the BLG A and D alleles in cattle. The cytosine at position g.-1957 and the thymines at positions g.-2008 and g.-2049 are only found in BLG B alleles of cattle. It is suggested that the described genetic variability contributes to the differential allelic expression of the BLG gene.

Characterization of the beta-lactamase gene from {\it Enterococcus faecalis\/} strain HH22

The plasmid-encoded, constitutively produced $\beta$-lactamase gene from Enterococcus faecalis strain HH22 was genetically characterized. A restriction endonuclease map of the 5.1 kb EcoRI fragment encoding the enterococcal $\beta$-lactamase was prepared and compared with the restriction map of a cloned staphylococcal $\beta$-lactamase gene (from the naturally-occurring staphylococcal $\beta$-lactamase plasmid pI258). Comparison and hybridization studies showed that there were identical restriction sites in the region of the $\beta$-lactamase structural gene but not in the region surrounding this gene. Also the enterococcal $\beta$-lactamase plasmid did not encode resistance to mercury or cadmium which is encoded by the small, transducible staphylococcal $\beta$-lactamase plasmids. The nucleotide sequence of the enterococcal gene was shown to be identical to the published sequences of three of four staphylococcal type A $\beta$-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred-forty nucleotides upstream of the $\beta$-lactamase start codon were also determined for the inducible staphylococcal $\beta$-lactamase gene on pI258; this sequence was identical to that of the constitutively expressed enterococcal gene indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The gene for the enterococcal $\beta$-lactamase and an inducible staphylococcal $\beta$-lactamase were each cloned into a shuttle vector and then transformed into enterococcal and staphylococcal recipients. The major difference between the two host backgrounds was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene but no qualitative difference was seen between the two genera. Also a difference in the level of resistance to ampicillin was seen between the two backgrounds with the cloned enzymes by MIC and time-kill studies. The location of the enzyme was found to be host dependent since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell bound enzyme in the enterococcus. Based on the identity of the enterococcal $\beta$-lactamase and several staphylococcal $\beta$-lactamases, these data suggest recent spread of $\beta$-lactamase to enterococci and also suggest loss of a functional repressor. ^

A duplication in the canine beta-galactosidase gene GLB1 causes exon skipping and GM1-gangliosidosis in Alaskan huskies

GM(1)-gangliosidosis is a lysosomal storage disease that is inherited as an autosomal recessive disorder, predominantly caused by structural defects in the beta-galactosidase gene (GLB1). The molecular cause of GM(1)-gangliosidosis in Alaskan huskies was investigated and a novel 19-bp duplication in exon 15 of the GLB1 gene was identified. The duplication comprised positions +1688-+1706 of the GLB1 cDNA. It partially disrupted a potential exon splicing enhancer (ESE), leading to exon skipping in a fraction of the transcripts. Thus, the mutation caused the expression of two different mRNAs from the mutant allele. One transcript contained the complete exon 15 with the 19-bp duplication, while the other transcript lacked exon 15. In the transcript containing exon 15 with the 19-bp duplication a premature termination codon (PTC) appeared, but due to its localization in the last exon of canine GLB1, nonsense-mediated RNA decay (NMD) did not occur. As a consequence of these molecular events two different truncated GLB1 proteins are predicted to be expressed from the mutant GLB1 allele. In heterozygous carrier animals the wild-type allele produces sufficient amounts of the active enzyme to prevent clinical signs of disease. In affected homozygous dogs no functional GLB1 is synthesized and G(M1)-gangliosidosis occurs.

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.

The molecular basis for beta-lactamase gene expression in B. anthracis, B. cereus and B. thuringiensis

The susceptibility of most Bacillus anthracis strains to β-lactam antibiotics is intriguing considering that the B. anthracis genome harbors two β-lactamase genes, bla1 and bla2, and closely-related species, Bacillus cereus and Bacillus thuringiensis, typically produce β-lactamases. This work demonstrates that B. anthracis bla expression is affected by two genes, sigP and rsp, predicted to encode an extracytoplasmic function sigma factor and an antisigma factor, respectively. Deletion of the sigP/rsp locus abolished bla expression in a penicillin-resistant clinical isolate and had no effect on bla expression in a prototypical penicillin-susceptible strain. Complementation with sigP/rsp from the penicillin-resistant strain, but not the penicillin-susceptible strain, conferred β-lactamase activity upon both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsp in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsp homologues are required for inducible penicillin resistance in those species. Expression of the B. cereus or B. thuringiensis sigP and rsp genes in a B. anthracis sigP/rsp-null mutant confers resistance to β-lactam antibiotics, suggesting that while B. anthracis contains the genes necessary for sensing β-lactam antibiotics, the B. anthracis sigP/rsp gene products are insufficient for bla induction. ^ Because alternative sigma factors recognize unique promoter sequence, direct targets can be elucidated by comparing transcriptional profiling results with an in silico search using the sigma factor binding sequence. Potential σP -10 and -35 promoter elements were identified upstream from bla1 bla2 and sigP. Results obtained from searching the B. anthracis genome with the conserved sequences were evaluated against transcriptional profiling results comparing B. anthracis 32 and an isogenic sigP/rsp -null strain. Results from these analyses indicate that while the absence of the sigP gene significantly affects the transcript levels of 16 genes, only bla1, bla2 and sigP are directly regulated by σP. The genomes of B. cereus and B. thuringiensis strains were also analyzed for the potential σP binding elements. The sequence was located upstream from the sigP and bla genes, and previously unidentified genes predicted to encode a penicillin-binding protein (PBP) and a D-alanyl-D-alanine carboxypeptidase, indicating that the σ P regulon in these species responds to cell-wall stress caused by β-lactam antibiotics. ^ β-lactam antibiotics prevent attachment of new peptidoglycan to the cell wall by blocking the active site of PBPs. A B. cereus and B. thuringiensis pbp-encoding gene located near bla1 contains a potential σP recognition sequence upstream from the annotated translational start. Deletion of this gene abolished β-lactam resistance in both strains. Mutations in the active site of the PBP were detrimental to β-lactam resistance in B. cereus, but not B. thuringiensis, indicating that the transpeptidase activity is only important in B. cereus. I also found that transcript levels of the PBP-encoding gene are not significantly affected by the presence of β-lactam antibiotic. Based on these data I hypothesize that the gene product acts a sensor of β-lactam antibiotic. ^