Detection of potential suberinase-encoding genes in Streptomyces scabiei strains and other actinobacteria

2013 ◽  
Vol 59 (5) ◽  
pp. 294-303 ◽  
Author(s):  
Doaa Komeil ◽  
Anne-Marie Simao-Beaunoir ◽  
Carole Beaulieu
Keyword(s):  
Esterase Activity ◽  
Common Scab ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Open Reading Frames ◽  
Streptomyces Scabiei ◽  
Polymerase Chain ◽  
Encoding Genes ◽  
Reading Frames ◽  
Important Disease

Streptomyces scabiei causes common scab, an economically important disease of potato tubers. Some authors have previously suggested that S. scabiei penetration into host plant tissue is facilitated by secretion of esterase enzymes degrading suberin, a lipidic biopolymer of the potato periderm. In the present study, S. scabiei EF-35 showed high esterase activity in suberin-containing media. This strain also exhibited esterase activity in the presence of other biopolymers, such as lignin, cutin, or xylan, but at a much lower level. In an attempt to identify the esterases involved in suberin degradation, translated open reading frames of S. scabiei 87-22 were examined for the presence of protein sequences corresponding to extracellular esterases of S. scabiei FL1 and of the fungus Coprinopsis cinerea VTT D-041011, which have previously been shown to be produced in the presence of suberin. Two putative extracellular suberinase genes, estA and sub1, were identified. The presence of these genes in several actinobacteria was investigated by Southern blot hybridization, and both genes were found in most common-scab-inducing strains. Moreover, reverse transcription – polymerase chain reaction performed with S. scabiei EF-35 showed that estA was expressed in the presence of various biopolymers, including suberin, whereas the sub1 gene appeared to be specifically expressed in the presence of suberin and cutin.

scholarly journals Spinach curly top virus: A Newly Described Curtovirus Species from Southwest Texas with Incongruent Gene Phylogenies

2004 ◽  
Vol 94 (7) ◽  
pp. 772-779 ◽  
Author(s):  
Surendranath Baliji ◽  
Mark C. Black ◽  
Roy French ◽  
Drake C. Stenger ◽  
Garry Sunter
Keyword(s):  
Tandem Repeats ◽  
Infectious Clone ◽  
Blot Hybridization ◽  
Systemic Infection ◽  
Southern Blot Hybridization ◽  
Complete Sequence ◽  
Open Reading Frames ◽  
Viral Dna ◽  
Severe Stunting ◽  
Reading Frames

A curtovirus associated with a disease of spinach was isolated in southwest Texas during 1996. Disease symptoms included severe stunting and chlorosis, with younger leaves curled, distorted, and dwarfed. Viral DNA was purified and an infectious clone obtained. Agroinoculation using a construct bearing full-length tandem repeats of the cloned viral genome resulted in systemic infection of species in six of seven plant families tested, indicating that the virus has a wide host range. Symptoms produced in spinach agroinoculated with cloned viral DNA were similar to those observed in the field. Viral single-stranded and double-stranded DNA forms typical of curtovirus infection were detected in host plants by Southern blot hybridization. The complete sequence of the infectious clone comprised 2,925 nucleotides, with seven open reading frames encoding proteins homologous to those of other curtoviruses. Complete genome comparisons revealed that the spinach curtovirus shared 64.2 to 83.9% nucleotide sequence identity relative to four previously characterized curtovirus species: Beet curly top virus, Beet severe curly top virus, Beet mild curly top virus, and Horseradish curly top virus. Phylogenetic analysis of individual open reading frames indicated that the evolutionary history of the three virion-sense genes was different from that of the four complementary-sense genes, suggesting that recombination among curtoviruses may have occurred. Collectively, these results indicate that the spinach curtovirus characterized here represents a newly described species of the genus Curtovirus, for which we propose the name Spinach curly top virus.

scholarly journals Identification of a New Enamovirus Associated with Citrus Vein Enation Disease by Deep Sequencing of Small RNAs

2013 ◽  
Vol 103 (10) ◽  
pp. 1077-1086 ◽  
Author(s):  
Mari Carmen Vives ◽  
Karelia Velázquez ◽  
José Antonio Pina ◽  
Pedro Moreno ◽  
José Guerri ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Blot Hybridization ◽  
Proximal Region ◽  
Open Reading Frames ◽  
Dot Blot ◽  
Certification Programs ◽  
Polymerase Chain ◽  
Pea Enation Mosaic Virus ◽  
Reading Frames ◽  
Biased Distribution

To identify the causal agent of citrus vein enation disease, we examined by deep sequencing (Solexa-Illumina) the small RNA (sRNA) fraction from infected and healthy Etrog citron plants. Our results showed that virus-derived sRNAs (vsRNAs): (i) represent about 14.21% of the total sRNA population, (ii) are predominantly of 21 and 24 nucleotides with a biased distribution of their 5′ nucleotide and with a clear prevalence of those of (+) polarity, and (iii) derive from all the viral genome, although a prominent hotspot is present at a 5′-proximal region. Contigs assembled from vsRNAs showed similarity with luteovirus sequences, particularly with Pea enation mosaic virus, the type member of the genus Enamovirus. The genomic RNA (gRNA) sequence of a new virus, provisionally named Citrus vein enation virus (CVEV), was completed and characterized. The CVEV gRNA was found to be single-stranded, positive-sense, with a size of 5,983 nucleotides and five open reading frames. Phylogenetic comparisons based on amino acid signatures of the RNA polymerase and the coat protein clearly classifies CVEV within the genus Enamovirus. Dot-blot hybridization and reverse transcription-polymerase chain reaction tests were developed to detect CVEV in plants affected by vein enation disease. CVEV detection by these methods has already been adopted for use in the Spanish citrus quarantine, sanitation, and certification programs.

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.

scholarly journals Absence of the t(2;5) in Hodgkin's disease [see comments]

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2845-2847 ◽  
Author(s):  
LM Weiss ◽  
JR Lopategui ◽  
LH Sun ◽  
OW Kamel ◽  
CH Koo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Hodgkin's Disease ◽  
Hodgkin’S Disease ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Large Cell ◽  
Common Finding ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Hodgkin's Lymphomas

The cytogenetics of Hodgkin's disease (HD) is poorly understood. However, a t(2;5) is a common finding in CD30+ anaplastic large cell lymphoma (ALCL), a neoplasm thought by some to be closely related to HD. Recently, the t(2;5) has been cloned and found to represent fusion of the NPM gene with the ALK gene. Using Southern blot hybridization, one group has reported finding rearrangements of NPM in a proportion of cases of both ALCL and HD. In the current study, we used a highly sensitive reverse transcriptase-polymerase chain reaction methodology to analyze 34 cases of HD for the t(2;5). We were unable to find polymerase chain reaction evidence for the t(2;5) in any of the cases of HD, a result significantly different from our previous study of CD30+ non-Hodgkin's lymphomas (P < .02) including ALCL (P < .04), using identical methods. Our results do not support the hypothesis that the t(2;5) represents a common chromosomal abnormality for both HD and ALCL.

Inactivation of the Streptococcus mutans fxpC Gene Confers Resistance to Xylitol, a Caries-preventive Natural Carbohydrate Sweetener

2002 ◽  
Vol 81 (6) ◽  
pp. 380-386 ◽  
Author(s):  
H. Benchabane ◽  
L.-A. Lortie ◽  
N.D. Buckley ◽  
L. Trahan ◽  
M. Frenette
Keyword(s):  
Streptococcus Mutans ◽  
Northern Blot Analysis ◽  
Regulatory Protein ◽  
Open Reading Frames ◽  
Phosphotransferase System ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Reading Frames

Xylitol is transported by Streptococcus mutans via a constitutive phosphoenolpyruvate:fructose phosphotransferase system (PTS) composed of a IIABC protein. Spontaneous xylitol-resistant strains are depleted in constitutive fructose-PTS activity, exhibit additional phenotypes, and are associated with the caries-preventive properties of xylitol. Polymerase chain-reactions and chromosome walking were used to clone the fxp operon that codes for the constitutive fructose/xylitol-PTS. The operon contained three open reading frames: fxpA, which coded for a putative regulatory protein of the deoxyribose repressor (DeoR) family, fxpB, which coded for a 1-phosphofructokinase, and fxpC, which coded for a IIABC protein of the fructose-PTS family. Northern blot analysis revealed that these genes were co-transcribed into a 4.4-kb mRNA even in the absence of fructose. Inactivation of the fxpC gene conferred resistance to xylitol, confirming its function. The fxp operon is also present in the genomes of other xylitol-sensitive streptococci, which could explain their sensitivity to xylitol.

scholarly journals Cloning of two Bombyx homologues of the Drosophila rosy gene and their relationship to larval translucent skin colour mutants

1998 ◽  
Vol 71 (1) ◽  
pp. 11-19 ◽  
Author(s):  
YUJI YASUKOCHI ◽  
TOSHIO KANDA ◽  
TOSHIKI TAMURA
Keyword(s):  
Tissue Specificity ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Striking Similarity ◽  
Wild Type ◽  
Rt Pcr ◽  
Significant Difference ◽  
Phage Dna ◽  
Polymerase Chain ◽  
Reading Frames

To clone the Bombyx xanthine dehydrogenase (XDH) gene as a dominant marker for silkworm transgenesis, we performed nested reverse transcriptase–polymerase chain reaction (RT-PCR) using embryonic mRNA and primers designed from the conserved region of Drosophila and rat XDH genes. Sequencing of amplified 180 bp fragments showed that two different sequences were present in the fragments. Since both possessed striking similarity to XDH genes of other organisms, we considered these to be portions of silkworm XDH genes and designated them BmXDH1 and BmXDH2. Subsequently we cloned separately the entire region of the two cDNAs by PCR using phage DNA of an embryonic cDNA library and sequenced them. The two cDNAs were around 4 kb in size and possessed complete open reading frames. The deduced amino acid sequences of the two BmXDHs were very similar to each other and to those of other organisms. The expression pattern of wild-type larvae basically followed the tissue specificity of the enzyme and no significant difference was observed between the two XDH genes. The expression of both genes was detected in the XDH-deficient mutants, oq and og, but non-synonymous substitutions were specifically detected in the BmXDH1 of the oq mutant. In addition, a length polymorphism of the second intron of the BmXDH1 co-segregated with the oq translucent phenotype, suggesting that deficiency in BmXDH1 is the cause of the oq translucent phenotype.

The elusive factor Xa receptor: failure to detect transcripts that correspond to the published sequence of EPR-1

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 145-148 ◽  
Author(s):  
Guido J. R. Zaman ◽  
Edward M. Conway
Keyword(s):  
Expressed Sequence Tags ◽  
Messenger Rna ◽  
Blot Hybridization ◽  
Factor Xa ◽  
Southern Blot Hybridization ◽  
Cellular Responses ◽  
Cellular Effects ◽  
Strong Homology ◽  
Polymerase Chain ◽  
Expressed Sequence

The coagulation protease factor Xa induces cellular responses implicated in cardiovascular and inflammatory disease. Effector-cell protease receptor 1 (EPR-1) is a functionally characterized receptor of factor Xa, and the EPR-1complementary DNA (cDNA) was published. Remarkably, the cDNA encoding an inhibitor of apoptosis, survivin, is reportedly identical to that ofEPR-1 except for a few nucleotide differences and its orientation opposite to EPR-1. To isolate the EPR-1cDNA and gene, we surveyed gene databases for expressed sequence tags (ESTs) that could be derived from EPR-1. All ESTs with strong homology to EPR-1/survivin were derived from survivinand could not encode EPR-1. By polymerase chain reaction and Southern blot hybridization, EPR-1 was not detectable in the human or murine genome, but survivin was. Our data suggest that EPR-1 is either highly cell-specific or the published EPR-1 cDNA includes sequences from clones derived from survivin messenger RNA. The means by which factor Xa mediates its cellular effects requires further evaluation.

scholarly journals Vibrio vulnificus Has the Transmembrane Transcription Activator ToxRS Stimulating the Expression of the Hemolysin GenevvhA

2000 ◽  
Vol 182 (12) ◽  
pp. 3405-3415 ◽  
Author(s):  
Shee Eun Lee ◽  
Sung Heui Shin ◽  
Soo Young Kim ◽  
Young Ran Kim ◽  
Dong Hyeon Shin ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Wild Type ◽  
Transcription Activator ◽  
Reading Frame ◽  
Type Gene ◽  
Virulence Regulator

ABSTRACT In an attempt to dissect the virulence regulatory mechanism inVibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS(toxRS Vc) homologs in V. vulnificus. By comparing the sequences of toxRS ofV. cholerae and V. parahaemolyticus(toxRS Vp), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kbBglII-HindIII fragment and a 1.2-kbHindIII fragment containing two complete open reading frames and one partial open reading frame attributable totoxR Vv, toxS Vv, andhtpG Vv were cloned. ToxRVv shared 55.0 and 63.0% sequence homology with ToxRVc and ToxRVp, respectively. ToxSVv was 71.5 and 65.7% homologous to ToxSVc and ToxSVp, respectively. The amino acid sequences of ToxRSVv showed transmembrane and activity domains similar to those observed in ToxRSVc and ToxRSVp. Western blot analysis proved the expression of ToxRVv in V. vulnificus. ToxRSVv enhanced, in an Escherichia coli background, the expression of the V. vulnificushemolysin gene (vvhA) fivefold. ToxRSVv also activated the ToxRVc-regulated ctx promoter incorporated into an E. coli chromosome. AtoxR Vv null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. ThetoxR Vv mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxRVv may regulate the virulence expression of V. vulnificus.

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