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

2009 ◽  
Vol 54 (No. 10) ◽  
pp. 473-482 ◽  
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 <I>qnr</I> and <I>qepA</I> genes in 660 <I>Escherichia coli</I> 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 <I>qnrS</I> gene, but not <I>qnrA, qnrB, </I> and <I>qepA</I> were detected in 12/360 pig isolates (3.33%) and in 6/300 chicken isolates (2%). Southern blot hybridization analysis indicated that <I>qnrS</I> was located on plasmids ranging in size from 50–165 kb. Eleven of the 18 <I>qnrS</I> 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 <I>gyrA</I> and <I>parC</I>. Only two <I>qnrS</I>-positive isolates carried the <I>aac(6’)-Ib-cr</I>variant that mediates FQ acetylation. For the high percentage resistance of cephalosporins, the<I> bla</I><sub>CTX-M</sub> gene was also examined in <I>qnrS</I>-positive isolates. The <I>bla</I><sub>CTX-M</sub> gene was detected in fifteen isolates (15/18, 83.3%) of which 12 isolates were <I>bla</I><sub>CTX-M-1</sub> and three isolates were <I>bl</I><sub>CTX-M-15</sub>. This study demonstrated a close linkage between the <I>qnrS</I> gene and <I>bla</I><sub>CTX-M-1</sub>, suggesting CTX-M and Qnr-based mechanisms might be co-emerging in <I>E. coli</I> strains isolated from healthy chickens and pigs under selective pressure of quinolone and cephalosporine administration.

scholarly journals Length polymorphisms in human proline-rich protein genes generated by intragenic unequal crossing over.

Genetics ◽  
1988 ◽  
Vol 120 (1) ◽  
pp. 267-278 ◽  
Author(s):  
K M Lyons ◽  
J H Stein ◽  
O Smithies
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Length Variation ◽  
Length Polymorphisms ◽  
Dna Strands ◽  
Southern Blot Hybridization Analysis ◽  
Genomic Dnas ◽  
Proline Rich Protein

Abstract Southern blot hybridization analysis of genomic DNAs from 44 unrelated individuals revealed extensive insertion/deletion polymorphisms within the BstNI-type loci (PRB1, PRB2, PRB3 and PRB4) of the human proline-rich protein (PRP) multigene family. Ten length variants were cloned, including alleles at each of the four PRB loci, and in every case the region of length difference was localized to the tandemly repetitious third exon. DNA sequences covering the region of length variation were determined for seven of the alleles. The data indicate (1) that the PRB loci can be divided into two subtypes, PRB1 plus PRB2, and PRB3 plus PRB4, and (2) that the length differences result from different numbers of tandem repeats in the third exons. Variant chromosomes were also identified with different numbers of PRP loci resulting from homologous but unequal exchange between the PRB1 and PRB2 loci. The overall data are compatible with the observed length variants having been generated via homologous but unequal intragenic exchange. The results also indicate that these crossover events are sensitive to the amount of homology shared between the interacting DNA strands. Allelic length variants have arisen independently at least 20 times at the PRB loci, but only one has been detected at a PRH locus. Comparison of the detailed structures of the repetitious regions in PRB and PRH loci shows that the repeats in PRB genes are very similar to each other in sequence and in length. The PRH genes contain fewer repeats, which differ considerably in their individual lengths. These differences suggest that the larger number of length variants in PRB genes is related to their greater ease of homologous but unequal pairing compared to PRH genes.

scholarly journals A Tn7-Like Transposon Is Present in theglmUS Region of the Obligately Chemoautolithotrophic Bacterium Thiobacillus ferrooxidans

1998 ◽  
Vol 180 (11) ◽  
pp. 3007-3012 ◽  
Author(s):  
Joseph C. Oppon ◽  
Robert J. Sarnovsky ◽  
Nancy L. Craig ◽  
Douglas E. Rawlings
Keyword(s):  
Thiobacillus Ferrooxidans ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Open Reading Frames ◽  
Ecological Niches ◽  
E Coli ◽  
Repeat Sequences ◽  
Genes Encoding ◽  
Reading Frames ◽  
Different Parts

ABSTRACT The region downstream of the Thiobacillus ferrooxidansATCC 33020 atp operon was examined, and the genes encodingN-acetylglucosamine-1-uridyltransferase (glmU) and glucosamine synthetase (glmS) were found. ThisatpEFHAGDC-glmUS gene order is identical to that ofEscherichia coli. The T. ferrooxidans glmS gene was shown to complement E. coli glmS mutants for growth on minimal medium lacking glucosamine. A Tn7-like transposon, Tn5468, was found inserted into the region immediately downstream of the glmS gene in a manner similar to the site-specific insertion of transposon Tn7 within the termination region of the E. coli glmS gene. Tn5468 was sequenced, and Tn7-like terminal repeat sequences as well as several open reading frames which are related to the Tn7 transposition genes tnsA,tnsB, tnsC, and tnsD were found. Tn5468 is the closest relative of Tn7 to have been characterized to date. Southern blot hybridization indicated that a similar or identical transposon was present in three T. ferrooxidans strains isolated from different parts of the world but not in two Thiobacillus thiooxidans strains or aLeptospirillum ferrooxidans strain. Since T. ferrooxidans is an obligately acidophilic autotroph and E. coli is a heterotroph, ancestors of the Tn7-like transposons must have been active in a variety of physiologically different bacteria so that their descendants are now found in bacteria that occupy very different ecological niches.

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.

Presence of Human Herpesvirus 8 DNA Sequences in Renal Transplantation-Associated Pleural Kaposi Sarcoma

1999 ◽  
Vol 123 (12) ◽  
pp. 1269-1273
Author(s):  
J. Javier Gómez-Román ◽  
J. Gonzalo Ocejo-Vinyals ◽  
Pablo Sánchez-Velasco ◽  
Francisco Leyva-Cobián ◽  
J. Fernando Val-Bernal
Keyword(s):  
Kidney Transplantation ◽  
Pleural Effusion ◽  
Dna Sequences ◽  
Blot Hybridization ◽  
Human Herpesvirus ◽  
Kaposi Sarcoma ◽  
Southern Blot Hybridization ◽  
Pulmonary Involvement ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8

Abstract Objective.—To describe one case of symptomatic skin and pleural Kaposi sarcoma (KS) associated with kidney transplantation. Diagnosis was supported by morphologic study and human herpesvirus 8 (HHV-8) detection in both tissues. Pulmonary involvement was not present. Design.—The presence of HHV-8 DNA sequences was proved using polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization. Setting.—Human herpesvirus 8 is found in most KS from patients with and without the acquired immunodeficiency syndrome. Clinically significant pulmonary infiltration by KS is diagnosed uncommonly antemortem, and pleural disease is exceptional. Patient.—A 49-year-old man who had renal transplant with immunosuppressive therapy (tacrolimus and prednisone) and developed a cutaneous KS. A pleural effusion appeared without pulmonary involvement. Both lesions disappeared when immunosuppressive drugs were suspended. Later, the pleural effusion and the cutaneous lesions reappeared. Pleural biopsy specimens showed KS infiltration. Outcome.—The patient refused treatment and was lost to follow-up. Results.—The skin and pleural biopsies showed a proliferation of spindle-shaped cells positive for CD34. The HHV-8 sequences were detected by nested PCR. No amplification was detected in uninvolved skin from the patient or in peripheral blood mononuclear cells from 10 healthy individuals used as controls. The Southern blot hybridization confirmed these results. Conclusions.—To our knowledge, this is the first report of HHV-8 in symptomatic pleural KS, which was probably associated with immunosuppression after kidney transplantation. The demonstration of HHV-8 DNA in biopsy material in the appropriate cells could be diagnostic when the morphologic setting is consistent with KS.

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.

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 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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