scholarly journals Identification of a Locus Involved in Systemic Dissemination of Yersinia enterocolitica

2001 ◽  
Vol 69 (10) ◽  
pp. 6201-6208 ◽  
Author(s):  
Kristin M. Nelson ◽  
Glenn M. Young ◽  
Virginia L. Miller
Keyword(s):  
Mouse Model ◽  
Yersinia Enterocolitica ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Transcriptional Regulators ◽  
Host Cells ◽  
Systemic Dissemination ◽  
Southern Blot Hybridization Analysis ◽  
Lysr Family

ABSTRACT A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by Yersinia enterocolitica (G. M. Young and V. L. Miller, Mol. Microbiol. 25:319–328, 1997). An insertion in hre20, now designated rscR, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated inrscR, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in rscR was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal rscR gene and carrying rscR on a plasmid under the control of the inducible araBAD promoter was mutagenized with an mTn5Km-2 transposon containing a promoterless lacZY. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of Serratia marcescens, and a locus (designated rscBAC) with similarity to thehmwABC locus of Haemophilus influenzae. ThehmwABC locus promotes adherence of H. influenzae to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320–3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875–2879, 1993). A strain containing a deletion mutant of rscA, the hmwA homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the rscR mutant. This suggests that the phenotype of an rscR mutant is due to the loss of RscA.

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii

1983 ◽  
Vol 3 (6) ◽  
pp. 1070-1076
Author(s):  
S M Landfear ◽  
D McMahon-Pratt ◽  
D F Wirth
Keyword(s):  
Tandem Repeat ◽  
Blot Hybridization ◽  
Protozoan Parasite ◽  
Southern Blot Hybridization ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Southern Blot Hybridization Analysis ◽  
Leishmania Enriettii

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

scholarly journals Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

1983 ◽  
Vol 3 (6) ◽  
pp. 1070-1076 ◽  
Author(s):  
S M Landfear ◽  
D McMahon-Pratt ◽  
D F Wirth
Keyword(s):  
Tandem Repeat ◽  
Blot Hybridization ◽  
Protozoan Parasite ◽  
Southern Blot Hybridization ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Southern Blot Hybridization Analysis ◽  
Leishmania Enriettii

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

Characterization of the pTFI91 -family replicon of Thiobacillus ferrooxidans plasmids

1995 ◽  
Vol 41 (4-5) ◽  
pp. 354-365 ◽  
Author(s):  
Leena Chakravarty ◽  
Thomas J. Zupancic ◽  
Beth Baker ◽  
Joseph D. Kittle ◽  
Ilona J. Fry ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Thiobacillus Ferrooxidans ◽  
Blot Hybridization ◽  
Marker Gene ◽  
Southern Blot Hybridization ◽  
Structural Features ◽  
Broad Host Range ◽  
Origin Of Replication ◽  
E Coli ◽  
Southern Blot Hybridization Analysis

Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those of T. ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5α cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I in E. coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation of T. ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either in E. coli or in P. putida. Since pTFI91 contains the same origin of replication as other plasmids in several other T. ferrooxidans strains, this replicon may be commonly distributed in T. ferrooxidans.Key words: nucleotide sequence, origin of replication, plasmid DNA, replicon, Thiobacillus ferrooxidans.

scholarly journals Characterization of the Yersinia enterocolitica Type III Secretion ATPase YscN and Its Regulator, YscL

2006 ◽  
Vol 188 (10) ◽  
pp. 3525-3534 ◽  
Author(s):  
Bill Blaylock ◽  
Kelly E. Riordan ◽  
Dominique M. Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Atp Hydrolysis ◽  
Biochemical Properties ◽  
Host Cells ◽  
Gram Negative Bacteria ◽  
Type Iii ◽  
Bacterial Proteins

ABSTRACT Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.

scholarly journals Conjugation of Hydroxyethyl Starch to Desferrioxamine (DFO) Modulates the Dual Role of DFO in Yersinia enterocolitica Infection

2000 ◽  
Vol 7 (3) ◽  
pp. 457-462 ◽  
Author(s):  
Sören Schubert ◽  
Ingo B. Autenrieth
Keyword(s):  
Yersinia Enterocolitica ◽  
Hydroxyethyl Starch ◽  
Growth Promotion ◽  
Iron Chelator ◽  
Dual Role ◽  
Host Cells ◽  
Immunosuppressive Action

ABSTRACT The iron chelator desferrioxamine (DFO) B is widely used in the therapy of patients with iron overload. As a side effect, DFO may favor the occurrence of fulminant Yersinia infections. Previous work from our laboratory showed that this might be due to a dual role of DFO: growth promotion of the pathogen and immunosuppression of the host. In this study, we sought to determine whether conjugation of DFO to hydroxyethyl starch (HES-DFO) may prevent exacerbation ofYersinia infection in mice. We found HES-DFO to promote neither growth of Yersinia enterocolitica nor mitogen-induced T-cell proliferation and gamma interferon production by T cells in vitro. Nevertheless, in vivo HES-DFO promoted growth ofY. enterocolitica possibly due to cleavage of HES and release of DFO. The pretreatment of mice with DFO resulted in death of all mice 2 to 5 days after application of a normally sublethal inoculum of Y. enterocolitica, while none of the mice pretreated with HES-DFO died within the first 7 days postinfection. However, some of the HES-DFO-treated mice died 8 to 14 days postinfection. Thus, due to the delayed in vivo effect HES-DFO failed to triggerYersinia-induced septic shock, which accounts for early mortality in DFO-associated septicemia. Moreover, our data suggest that DFO needs to be taken up by host cells in order to exert its immunosuppressive action. These results strongly suggest that HES-DFO might be a favorable drug with fewer side effects than DFO in terms of DFO-promoted fulminant infections.

Leptospira interrogans serovar sejroe infection in a group of laboratory dogs

1995 ◽  
Vol 29 (3) ◽  
pp. 300-306 ◽  
Author(s):  
E. Scanziani ◽  
L. Crippa ◽  
Anna M. Giusti ◽  
M. Luini ◽  
Maria L. Pacciarini ◽  
...  
Keyword(s):  
Interstitial Nephritis ◽  
Urine Analysis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Leptospira Interrogans ◽  
Normal Ranges ◽  
Southern Blot Hybridization Analysis ◽  
Antibody Titres ◽  
Renal Infection ◽  
Endonuclease Digestion

Interstitial nephritis was seen histologically in 19 (59%) out of 32 pure-breed beagle dogs (16 males and 16 females) subjected to standard safety tests. In these animals no clinical abnormalities were observed and all the tested parameters (haematology, biochemistry and urine analysis) were within the normal ranges. Leptospiral antibody titres ranging from 1:100 to 1:6400, against a serovar ( hardio) belonging to the Sejroe serogroup, were detected by the microscopic agglutination test (MAT) in the serum of the 19 dogs with interstitial nephritis. All animals without renal lesions were seronegative. Leptospiral antigen was detected immunohistochemically in the kidneys of 4 dogs; leptospires were detected in Warthin-Starry stained sections of one dog. Leptospires were isolated from the kidneys of 3 of the 4 dogs examined by bacterial culture. The isolated strains were typed as serovar sejroe by restriction endonuclease digestion and Southern blot hybridization analysis of their DNA. It was concluded that Leptospira interrogans serovar sejroe, was responsible for an asymptomatic chronic renal infection which was widespread in this group of laboratory dogs.

scholarly journals Genetic Transformation of Garlic (Allium sativum L.) by Particle Bombardment

HortScience ◽  
2004 ◽  
Vol 39 (6) ◽  
pp. 1208-1211 ◽  
Author(s):  
Alejandrina Robledo-Paz ◽  
José Luis Cabrera-Ponce ◽  
Víctor Manuel Villalobos-Arámbula ◽  
Luis Herrera-Estrella ◽  
Alba Estela Jofre-Garfias
Keyword(s):  
Transgenic Plants ◽  
Plasmid Dna ◽  
Allium Sativum ◽  
Microprojectile Bombardment ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Hygromycin B ◽  
Gus Histochemical Assay ◽  
Embryogenic Calluses ◽  
Southern Blot Hybridization Analysis

Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Embryogenic calluses, derived from garlic cultivar `GT96-1', were bombarded with plasmid DNA containing genes coding for hygromycin phosphotransferase and β-glucuronidase. Putatively transformed calluses were identified in the bombarded tissue after 4 months of selection on 20 mg·L-1 hygromycin B. The transgenic nature of the selected material was demonstrated by GUS histochemical assay and Southern blot hybridization analysis, and twenty transgenic plants were regenerated.

scholarly journals Molecular defect of a phosphoglycerate kinase variant (PGK-Matsue) associated with hemolytic anemia: Leu----Pro substitution caused by T/A- ---C/G transition in exon 3

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1348-1352
Author(s):  
M Maeda ◽  
A Yoshida
Keyword(s):  
Hemolytic Anemia ◽  
Blot Hybridization ◽  
Phosphoglycerate Kinase ◽  
Southern Blot Hybridization ◽  
Enzyme Deficiency ◽  
Nucleotide Sequencing ◽  
Molecular Defect ◽  
Coding Regions ◽  
Variant Gene

We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2- terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.

scholarly journals Southern blot-hybridization analysis of HBV DNA in patients with HBsAg seropositive chronic liver diseases.

Kanzo ◽  
1985 ◽  
Vol 26 (5) ◽  
pp. 565-571 ◽  
Author(s):  
Osamu YOKOSUKA ◽  
Masao OMATA ◽  
Fumio IMAZEKI ◽  
Yoshimi ITO ◽  
Junko MORI ◽  
...  
Keyword(s):  
Southern Blot ◽  
Liver Diseases ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Hbv Dna ◽  
Chronic Liver ◽  
Hybridization Analysis ◽  
Chronic Liver Diseases ◽  
Southern Blot Hybridization Analysis

scholarly journals Protein C deficiency Hong Kong 1 and 2: hereditary protein C deficiency caused by two mutant alleles, a 5-nucleotide deletion and a missense mutation

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 126-133 ◽  
Author(s):  
Y Sugahara ◽  
O Miura ◽  
P Yuen ◽  
N Aoki
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Catalytic Domain ◽  
Stop Codon ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Expression Vectors ◽  
Protein C Deficiency ◽  
Mutant Proteins ◽  
Southern Blot Hybridization Analysis

Abstract We characterized a mutant protein C gene from an individual with no detectable protein C antigen in blood plasma. Southern blot hybridization analysis with human protein C cDNA demonstrated neither gross deletion nor rearrangement of the gene. Sequencing all the exons and exon-intron boundaries of the gene except the 3′ noncoding region showed two mutant alleles. The one, derived from the mother, represents a deletion of 5 nucleotides (nt) (CCCGC) in the end of exon VI (mutation I), predicted to result in the generation of a new stop codon due to a reading frameshift and the premature termination of translation. The other, derived from the father, represents a point mutation (G to A) in exon IX (mutation II), resulting in an amino acid substitution, Gly-376(GGC) to Asp(GAC), in the catalytic domain of the protein. Allele-specific oligonucleotide probe hybridization confirmed the presence of the two mutations. Mutation I would result in a truncated polypeptide of 169 amino acid residues that lacks the heavy chain. Mutation II gives rise to an alteration of a highly conserved amino acid, Gly-376. These data indicate that this patient is a compound heterozygote of the two mutant alleles, each one inherited from each parent. Transient expression assays using COS-7 cells transfected with mutated protein C expression vectors suggested that each of the two mutations leads to the protein C deficiency by causing an impairment of secretion of the respective mutant proteins.

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