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G Frankel

Publications and source records attributed to G Frankel.

At least 109 records · Page 6Linked to original sources
PubMed1993

Poly(GTG)5-associated profiles of Salmonella and Shigella genomic DNA.

DNA hybridization with oligonucleotide probes is a powerful technique to study population genetics and structures. The use of probes which recognize ubiquitously interspersed DNA sequences has a distinct advantage over other techniques (e.g. the analysis of patterns of restriction fragment length polymorphism) in that many independent loci can be detected simultaneously. In this communication, we investigated the use of a trinucleotide repetitive DNA oligonucleotide, poly(GTG)5, in Southern blot analysis of Salmonella serotypes and Shigella species. The strains in this study were isolated over several years from widely disparate geographic locations and can therefore be considered to represent the structure of part of the natural populations of these organisms. In most of the Salmonella serotypes, the poly(GTG)5-associated profile (GTG profile) phenotypes appeared to be clonally stable; in cases where only one isolate of a serotype was tested, the GTG profile was distinct from the others. On the other hand, when GTG profile analysis was applied to Shigella strains, each of the 12 isolates, belonging to the four Shigella species, produced a unique pattern phenotype of both the chromosome and plasmid DNA.

Autoradiography↗
PubMed1993

fliU and fliV: two flagellar genes essential for biosynthesis of Salmonella and Escherichia coli flagella.

The possible functions of two recently described flagellar genes, fliU and fliV, have been examined. Introduction of gene fliC, encoding the bacterial flagellin protein, into a number of flagellin-deficient Salmonella and Escherichia coli strains failed to complement the mutations in these strains, and the FliC flagellin was accumulated in the bacterial cytoplasm. Complementation with fliU and fliV, which map downstream of fliC, restored motility to some of the mutants which became flagellated. After inactivation of either fliU or fliV, such complementation no longer occurred and the flagellin protein accumulated in the cytoplasm, which suggested that both genes are required for the secretion of flagellin and expression of motility. Expression of these genes from high copy number plasmids resulted in the synthesis of exceptionally long flagella and in detection of the FliV protein on polyacrylamide gels.

Bacterial Proteins↗
PubMed1993

Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a.

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of Klebsiella pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK21a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-alpha-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumoniae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Mono- and bi-phasic Salmonella typhi: genetic homogeneity and distinguishing characteristics.

Several lines of evidence indicate a relatively low genetic heterogeneity in the natural Salmonella typhi population. However, some S. typhi isolates found in Indonesia express, instead of the usual fliC-d flagellin gene, a different flagellar gene fliC-j. In addition, Indonesian strains may have a second flagellar antigen fliC-z66. We have previously suggested, on the basis of the flagellar antigen constitution, that S. typhi evolved in an isolated human population in Indonesia. In order to test this hypothesis, we have gathered S. typhi isolates from around the world and tested the genetic heterogeneity among them. In general, polymorphism was greater in isolates from the Far East, as was indicated by Southern hybridizations with rDNA and fliC DNA probes. Gene fliC-j was not found in S. typhi isolates, other than those from Indonesia. However, the one-clone origin of S. typhi was indicated by a common DNA fingerprint pattern and by the occurrence, in the 5' end region of the fliC gene, of 10 scattered nucleotides that differ from the corresponding 10 nucleotides in other fliC alleles studied. These nucleotides were present in all isolates tested but did not change the amino acid sequence of the flagellin polypeptide.

Africa↗
PubMed1991

Diffuse-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico.

Diarrhea is a major cause of infantile morbidity and mortality in developing countries. A community-based, case control study was conducted in a southern Mexican Mayan village for 3 weeks during the peak diarrhea period to prospectively identify the infectious agents associated with childhood diarrheal disease. Several enteropathogens were isolated from stools of 34 of 58 cases, although none was significantly associated with diarrhea. For the 24 cases from which no enteropathogens were isolated, diffuse-adhering Escherichia coli (DAEC) strains were significantly associated with diarrheal disease (P less than .02; odds ratio = 6; 95% confidence limit, 1.08-99.0). DAEC were highly heterogeneous with respect to plasmid content and serotype. Three DNA probes designed to differentiate E. coli exhibiting localized, diffuse, or aggregative adherence were compared with results from a standard HeLa cell binding assay to assess the utility of these probes in the field. This study provides evidence for the potential pathogenic capacity of DAEC and underscores the variety of diarrheal agents operating within a community.

Bacterial Adhesion↗
PubMed1991

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Physicians↗
PubMed1990

Detection of Shigella in feces using DNA amplification.

A rapid diagnostic method employing a polymerase chain reaction procedure (PCR) was used to identify Shigella and enteroinvasive Escherichia coli. This procedure amplified a region of the invasive-associated locus (ial) from a crude DNA extract of feces. A synthetic 21-base oligonucleotide corresponding to the ial gene sequence was shown to specifically hybridize only with enteroinvasive E. coli (EIEC) strains and Shigella species. Upon PCR amplification, a 320-base pair fragment was generated in DNA extracted from feces reconstituted with EIEC or Shigella flexneri but not in DNA from 70 normal stools lacking these organisms and could be readily detected by the ial probe. For identifying Shigella and EIEC, the PCR assay was 10(5)- and 10(2)-fold more sensitive than standard biochemical tests and the macrocolony hybridization assay, respectively. These findings demonstrate a novel methodology for rapid, sensitive, and culture-independent diagnosis of diarrhea caused by these pathogens and underscores the utility of PCR in the diagnostic laboratory.

Base Sequence↗
PubMed1989

Intragenic recombination in a flagellin gene: characterization of the H1-j gene of Salmonella typhi.

Salmonella typhi, the etiologic agent of typhoid fever, typically has only a phase-1 flagellar antigen, d, but some isolates, found only in Indonesia, have antigen j instead, and may have a second flagellar antigen, z66. It appears that intragenic recombination involving a directly repeated 11 bp sequence in the H1-d flagellin gene changed the flagellar antigen to j, by deleting 261 bp in its central, antigenically determinant, part. Sequencing of the hypervariable regions of genes H1-d and H1-j, and hybridization of such genes, after amplification by the polymerase chain reaction, with oligonucleotide probes specific for the deleted segment or for the sequence produced by the recombination confirmed that all the j alleles have the postulated deletion. By applying the polymerase chain reaction to study S. typhi isolates from Jakarta, not previously tested in respect to flagellar antigen, we showed that gene H1-j was nearly as common as H1-d in these isolates.

Antigens, Bacterial↗
PubMed1989

Unique sequences in region VI of the flagellin gene of Salmonella typhi.

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.

Amino Acid Sequence↗
PubMed1989

Multi-gene amplification: simultaneous detection of three virulence genes in diarrhoeal stool.

Enterotoxigenic Escherichia coli (ETEC) and Shigella account for a substantial proportion of acute diarrhoeal illnesses among Third-World children. Rapid detection of these infectious agents in faeces followed by the prompt implementation of public health measures could help reduce their spread during the early phase of epidemics. Towards this end, three pairs of synthetic oligonucleotide primers were prepared and shown to hybridize specifically to the genes encoding the heat-stable (ST) and the heat-labile (LT) enterotoxins of ETEC and to invasion-associated loci (ial) of the large Shigella virulence plasmid. When the three primer pairs were used together in the polymerase chain reaction (PCR), the three corresponding genetic loci could be simultaneously amplified using DNA extracted directly from stool; the amplified products were readily detected by ST-, LT- and ial-specific, alkaline phosphatase-labelled oligonucleotide probes (AP probes). The performance of this system was evaluated in a Mayan community in southeastern Mexico, where diarrhoeal illnesses are a common cause of childhood morbidity and mortality. Using only simple and inexpensive laboratory equipment, multigene amplification with these primers and probes led to the identification of ETEC and/or Shigella in the stools of 20 out of 71 children with diarrhoea; the procedure could be completed in seven hours and was more sensitive than conventional diagnostic tests or DNA probes used without amplification.

Base Sequence↗
PubMed1987

Fractionation of Theiler's virus-infected BHK21 cell homogenates: isolation of virus-induced membranes.

A purified fraction containing unique membranes entrapping virions was isolated from homogenates of cells infected with the DA strain of Theiler's virus, after high-speed centrifugation through a sucrose gradient. This fraction, sedimented at 45-50% sucrose, was only found in cells infected with the DA strain but not in cells infected with the GDVII strain of Theiler's virus or in mock-infected cells. Immunogold staining of the membranes entrapping virions, using antivirus IgG antibodies, revealed that the membranes entrapping virions did not incorporate viral capsid antigens.

Animals↗
PubMed1987

Synthesis of long viral complementary DNA from 7.5 Kb poly A+ RNA templates.

The poly A+ RNA of the WW and GDVII virus isolates, belonging to the Theiler's murine encephalomyelitis virus group, were used as templates for cDNA synthesis. Since several secondary structures were present along these viral RNAs the reverse transcriptase was prematurely displaced from the RNA templates and only short cDNA molecules could be synthesized. Therefore a reliable and reproducible procedure for the synthesis of long cDNA transcripts, that can be directly used for cloning into respective plasmid or phage vectors, was developed. The precise conditions and kinetics of the several enzymatic reactions were studied. The use of methylmercury hydroxide for first strand synthesis, a correct choice of Klenow polymerase for second strand synthesis and the use of vertical gel electrophoresis in combination with zone centrifugation for removal of the excess linkers were found to be of paramount importance for the synthesis of long, up to intact, 7.5 Kb cDNA transcripts.

Animals↗
PubMed1987

Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQw1 and DQw3 alleles of the HLA-D region.

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ beta, DQ alpha, and DR beta cDNA probes. Hybridization with the DQ beta probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ alpha and DR beta probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.

Alleles↗