Characterization of Insertions of IS476and Two Newly Identified Insertion Sequences, IS1478 and IS1479, in Xanthomonas campestris pv. campestris

ABSTRACT Thirty-two plasmid insertion mutants were independently isolated from two strains of Xanthomonas campestris pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS3 family member, IS476, and two new insertion sequences (IS), IS1478 and IS1479. While IS1478does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS1479 demonstrated 73% sequence homology with IS1051 in X. campestrispv. dieffenbachiae, 62% homology with IS52 inPseudomonas syringae pv. glycinea, and 60% homology with IS5 in Escherichia coli. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS1478 by itself constitutes a new subfamily of the widespread IS5 family, whereas IS1479, along with IS1051, IS52, and IS5, belongs to the IS5 subfamily of the IS5 family. All but one of the IS476 insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS476 insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS1478 and IS1479, on the other hand, were not random, and IS1478 and IS1479 each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS476, IS1478, and IS1479 are present in the genomes of all seven X. campestris pv. campestris strains tested and several X. campestrispathovars.

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Characterization of plasmid-mediated quinolone resistance by the qnrS gene in Escherichia coli isolated from healthy chickens and pigs

Veterinární Medicína ◽

10.17221/108/2009-vetmed ◽

2009 ◽

Vol 54 (No. 10) ◽

pp. 473-482 ◽

Cited By ~ 10

Author(s):

H.-C. Kuo ◽

C.-C. Chou ◽

C. Tu ◽

S.-R. Gong ◽

C.-L. Han ◽

...

Keyword(s):

Escherichia Coli ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Quinolone Resistance ◽

E Coli ◽

Sequence Variations ◽

Southern Blot Hybridization Analysis ◽

Ciprofloxacin Resistance ◽

Polymerase Chain

The prevalence of qnr and qepA genes in 660 Escherichia coli isolates was investigated in healthy animals from 30 pig farms and 30 chicken farms in Taiwan from January 2005 to February 2006 by the polymerase chain reaction. The qnrS gene, but not qnrA, qnrB, and qepA were detected in 12/360 pig isolates (3.33%) and in 6/300 chicken isolates (2%). Southern blot hybridization analysis indicated that qnrS was located on plasmids ranging in size from 50–165 kb. Eleven of the 18 qnrS positive isolates which showed a high ciprofloxacin resistance phenotype (minimum inhibitory concentration ≥ 8 mg/l) also had amino acid sequence variations in chromosomal quinolone resistance-determining regions of gyrA and parC. Only two qnrS-positive isolates carried the aac(6’)-Ib-crvariant that mediates FQ acetylation. For the high percentage resistance of cephalosporins, the blaCTX-M gene was also examined in qnrS-positive isolates. The blaCTX-M gene was detected in fifteen isolates (15/18, 83.3%) of which 12 isolates were blaCTX-M-1 and three isolates were blCTX-M-15. This study demonstrated a close linkage between the qnrS gene and blaCTX-M-1, suggesting CTX-M and Qnr-based mechanisms might be co-emerging in E. coli strains isolated from healthy chickens and pigs under selective pressure of quinolone and cephalosporine administration.

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Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii

Molecular and Cellular Biology ◽

10.1128/mcb.3.6.1070-1076.1983 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.3.6.1070 ◽

1983 ◽

Vol 3 (6) ◽

pp. 1070-1076 ◽

Cited By ~ 69

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

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Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination

Molecular and Cellular Biology ◽

10.1128/mcb.3.11.1943-1948.1983 ◽

1983 ◽

Vol 3 (11) ◽

pp. 1943-1948

Author(s):

L J Kelly ◽

R Kelly ◽

H L Ennis

Keyword(s):

Dictyostelium Discoideum ◽

Spore Germination ◽

Developmental Regulation ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

In Vitro Translation ◽

Cdna Clones ◽

Molecular Weights ◽

Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

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Characterization of two BHV-4 strains isolated in the Czech Republic

Veterinární Medicína ◽

10.17221/18/2010-vetmed ◽

2010 ◽

Vol 55 (No. 3) ◽

pp. 106-112 ◽

Cited By ~ 1

Author(s):

V. Fichtelova ◽

K. Kovarcik

Keyword(s):

Reference Strain ◽

Fragment Size ◽

Blot Hybridization ◽

Size Variation ◽

Southern Blot Hybridization ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

The Czech Republic ◽

Herpesvirus 4

This study describes the isolation of bovine herpesvirus 4 (BHV-4) from the respiratory tract of animals suffering from respiratory disease. DNA of new isolates, CH and Ni, was cleaved with BamHI, EcoRI and HindIII in restriction enzyme analysis and the fragments were identified by co-migration with the restriction profile of the reference strain Movar 33/63 cleaved with the appropriate endonuclease. Typical profiles with polyrepetitive DNA (prDNA) fragments were detected. In order to localize the size variation within the obtained digestion fragments, Southern blot hybridization was performed. Differences between the isolates CH, Ni were localized in both the prDNAs and the unique central part of the genome and were restricted to fragment size variation.

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Detection and Characterization of Molecular Amplification Products: Agarose Gel Electrophoresis, Southern Blot Hybridization, Restriction Enzyme Digest Analysis, and Enzyme-Linked Immunoassay

Advanced Techniques in Diagnostic Microbiology ◽

10.1007/0-387-32892-0_15 ◽

2007 ◽

pp. 243-263 ◽

Cited By ~ 1

Author(s):

Raymond P. Podzorski ◽

Mike Loeffelholz ◽

Randall T. Hayden

Keyword(s):

Southern Blot ◽

Restriction Enzyme ◽

Gel Electrophoresis ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Agarose Gel ◽

Agarose Gel Electrophoresis ◽

Enzyme Digest

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Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa532 ◽

2020 ◽

Author(s):

Kyriaki Xanthopoulou ◽

Julia Wille ◽

Janine Zweigner ◽

Kai Lucaßen ◽

Thorsten Wille ◽

...

Keyword(s):

Blood Culture ◽

Enterococcus Faecium ◽

Core Genome ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Vana Gene ◽

Plasmid Analysis ◽

Vancomycin Resistant ◽

Faecium Isolate

Abstract Objectives To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. Methods The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Results Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Conclusions Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

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Characterization of the pTFI91 -family replicon of Thiobacillus ferrooxidans plasmids

Canadian Journal of Microbiology ◽

10.1139/m95-048 ◽

1995 ◽

Vol 41 (4-5) ◽

pp. 354-365 ◽

Cited By ~ 6

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.

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Leptospira interrogans serovar sejroe infection in a group of laboratory dogs

Laboratory Animals ◽

10.1258/002367795781088261 ◽

1995 ◽

Vol 29 (3) ◽

pp. 300-306 ◽

Cited By ~ 14

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.

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

HortScience ◽

10.21273/hortsci.39.6.1208 ◽

2004 ◽

Vol 39 (6) ◽

pp. 1208-1211 ◽

Cited By ~ 5

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.

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