Plasmid-Borne Genes Code for an Angular Dioxygenase Involved in Dibenzofuran Degradation by Terrabacter sp. Strain YK3

ABSTRACT The genes responsible for angular dioxygenation of dibenzofuran in actinomycetes were cloned by using a degenerate set of PCR primers designed by using conserved sequences of the dioxygenase alpha subunit genes. One sequence of alpha subunit genes was commonly amplified from four dibenzofuran-utilizing actinomycetes: Terrabacter sp. strains YK1 and YK3, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18. A 5.2-kb PstI fragment encoding the alpha and beta subunits of the terminal dioxygenase, ferredoxin, and ferredoxin reductase (designated dfdA1 to dfdA4, respectively) was cloned from the large circular plasmid pYK3 isolated from Terrabacter sp. strain YK3. We confirmed that transcription of the dfdA1 gene was induced by dibenzofuran in Terrabacter sp. strain YK3. Southern blot hybridization analysis revealed that this type of dioxygenase gene is distributed among diverse dibenzofuran-utilizing actinomycetes. However, genes homologous to dfdA1 were not detected in dibenzofuran utilization-deficient mutants of Terrabacter, Rhodococcus, and Microbacterium species. When the dfdA1 to dfdA4 genes were introduced into a non-dibenzofuran-degrading mutant of Rhodococcus sp. strain YK2, strain YK2-RD2, which had spontaneously lost the gene homologous to dfdA1, the ability to degrade dibenzofuran was restored. Analysis of the breakdown products indicated that DfdA has angular dioxygenase activity. This dfdA transformant degraded several aromatic compounds, indicating that the novel angular dioxygenase possesses broad substrate specificity.

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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 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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Engineering Resistance Against Cotton Leaf Curl Kokhran Virus-Burewala Strain Using CRISPR-Cas9 System in Nicotiana Benthamiana

10.21203/rs.3.rs-604666/v1 ◽

2021 ◽

Author(s):

Muhammad Hamza ◽

Muhammad Zuhaib Khan ◽

Roma Mustafa ◽

Hira Kamal ◽

Aneela Hussain ◽

...

Keyword(s):

Genome Editing ◽

Nicotiana Benthamiana ◽

Durable Resistance ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Pcr Primers ◽

Cotton Leaf ◽

A Genome ◽

Intact Virus ◽

Leaf Curl

Abstract Clustered regularly interspaced palindromic repeats (CRISPR) and associated Cas9 nuclease (CRISPR-Cas9) systems provide adaptive immunity to prokaryotes against infectious phage particles that can be engineered as a genome-editing tool. Guided by an RNA strand, the class II type II CRISPR-Cas9 system can be employed to provide resistance against plant DNA viruses. Here we describe an efficient CRISPR-Cas9 genome editing system based on simultaneous targeting of the highly conserved intergenic region (IR) of the virus that can provide resistance against Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bur) in Nicotiana benthamiana plants. The data revealed that upon infection, the transgenic plants harboring CRISPR-Cas9 and two gRNAs showed complete resistance against CLCuKoV-Bur/Cotton leaf curl Multan betasatellite (CLCuMB). All efforts failed to find the intact virus in CLCuKoV-Bur/CLCuMB challenged transgenic (OX:Cas9NB:IR) plants using either gene specific PCR primers or CLCuKoV-Bur as a probe in southern blot hybridization. Thus, our results have demonstrated an efficient CRISPR-Cas9 approach to engineer durable resistance against CLCuKoV-Bur in a model system. The implications of these findings are discussed.

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

Kanzo ◽

10.2957/kanzo.26.565 ◽

1985 ◽

Vol 26 (5) ◽

pp. 565-571 ◽

Cited By ~ 1

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

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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 ◽

10.1182/blood.v80.1.126.126 ◽

1992 ◽

Vol 80 (1) ◽

pp. 126-133 ◽

Cited By ~ 11

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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Molecular genetic demonstration of the diverse evolution of Richter's syndrome (chronic lymphocytic leukemia and subsequent large cell lymphoma)

Blood ◽

10.1182/blood.v83.5.1363.1363 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1363-1372 ◽

Cited By ~ 94

Author(s):

A Matolcsy ◽

G Inghirami ◽

DM Knowles

Keyword(s):

Tumor Suppressor ◽

Tumor Suppressor Gene ◽

Gene Rearrangement ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Large Cell ◽

Suppressor Gene ◽

Lymphocytic Leukemia ◽

Southern Blot Hybridization Analysis ◽

Richter’S Syndrome

Abstract Paired samples of chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) and the subsequent diffuse large cell lymphoma (DLL) of six cases of Richterxybs syndrome were investigated to establish the clonal relationship between the CLL/SLL and the DLL components and to define the oncogene and/or tumor-suppressor gene alterations involved in the morphologic transformation of CLL/SLL. Southern blot hybridization analysis showed identical clonal immunoglobulin (Ig) gene-rearrangement patterns in the CLL/SLL and DLL components in four cases and different Ig gene-rearrangement patterns in two cases. Polymerase chain reaction (PCR) amplification, cloning, and DNA sequencing of complementary determinant region 3 (CDR3) of the Ig-heavy chain gene of one of the two cases in which the Ig gene- rearrangement patterns were different showed nonidentical sequences in the CLL/SLL and DLL components. In the other case, monomorphic Epstein- Barr virus (EBV) genome integration was detected in the DLL but not in the CLL, suggesting that the CLL and DLL components in this case of Richter's syndrome also represent unrelated clones. Single-strand conformation polymorphism (SSCP) analysis and sequencing of exons 5 through 9 of the p53 tumor-suppressor gene showed a mutation in codon 176 of the DLL but not in the CLL/SLL component in one case where the CLL/SLL and DLL represented different clones. The p53 mutation probably played a role in the development of the lymphoma rather than morphologic transformation of the CLL/SLL in this case. SSCP analysis and sequencing also showed identical mutations in codon 282 in both the CLL/SLL and DLL components in a case where the CLL and DLL represented identical clones. Thus, this p53 gene mutation was present both before and after morphologic transformation, and therefore, probably did not play a primary role in this process. Southern blot hybridization analysis failed to show evidence of bcl-1, bcl-2, c-myc proto-oncogene or retinoblastoma (Rb) tumor-suppressor gene rearrangements in these six cases of Richter's syndrome. In conclusion, the original CLL/SLL and the subsequent DLL in Richter's syndrome may or may not be derived from identical clones, and the well-known proto-oncogenes and tumor- suppressor genes do not appear to play an obvious and consistent role in the morphologic transformation of CLL/SLL to DLL.

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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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