Molecular and Biological Characterization of a Mitovirus in Chalara elegans (Thielaviopsis basicola)

A 2.8-kb double-stranded RNA (dsRNA) element in strain BK18 of Chalara elegans originally isolated from cotton soil in California was characterized by obtaining a full-length cDNA sequence (2,896 nucleotides in length) from a series of overlapping clones. Sequence analysis revealed the presence of one large open reading frame (ORF I) using the mitochondrial genetic code, with 20 to 34% amino acid identity to the ORF I of other previously reported fungal mitochondrial RNA viruses. The ORF I encoded a putative protein of 705 amino acids and contained the conserved motif characteristic of RNA-dependent RNA polymerases. Purification of mitochondria from strain BK18 confirmed the co-localization of this dsRNA, and northern blot hybridization with a strand-specific probe revealed the (+) single-stranded nature. This Chalara elegans mitovirus (CeMV) is designated as a new member of the genus Mitovirus of the family Narnaviridae. Using dsRNA-specific primers, the ORF I region (positions 427 to 2544) was obtained from an additional 2.8-kb dsRNA element in strain HA2 originating from carrot roots in the Netherlands. Both ORFs had 98% homology at the nucleotide and amino acid levels. CeMV was also found to be present in five additional strains of C. elegans from different geographic locations worldwide, and a 97 to 100% nucleotide sequence identity was observed within a 300-bp region of ORF I in these strains. To determine the biological effects of CeMV on C. elegans, attempts to cure strain BK18 of the dsRNA were made. Sequential transfers of mycelium at 35 to 37°C yielded a colony which lacked the 2.8-kb dsRNA when visualized on agarose gels and also in northern blot hybridization analysis. However, reverse transcription-polymerase chain reaction with specific primer sets revealed a band, indicating that dsRNA replication had been significantly repressed (latent). The wild type and latently infected strains were compared for colony morphology, growth rate, melanin production, various enzymatic assays (polyphenoloxidase, laccase, tyrosinase, and esterase), and virulence on carrot roots. Colony morphology on V8 agar was comparable between the two strains, while growth rate, melanin production, and virulence were enhanced in the latently infected strain. There were no detectable differences in enzymatic activity. Transmission electron microscopy of hyphae of the wild type and latently infected strains revealed differences in the number and size of the mitochondria, which were enhanced in the latently infected strain. Our results show that CeMV is a new member of the genus Mitovirus with some disruptive effects on its fungal host and is present in C. elegans strains from different locations worldwide.

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Identification of Stress-Responsive Genes in Streptococcus mutans by Differential Display Reverse Transcription-PCR

Infection and Immunity ◽

10.1128/iai.69.4.2493-2501.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2493-2501 ◽

Cited By ~ 51

Author(s):

Jean-San Chia ◽

Ya-Yu Lee ◽

Peng-Tu Huang ◽

Jen-Yang Chen

Keyword(s):

Amino Acid ◽

Environmental Stress ◽

Streptococcus Mutans ◽

Differential Display ◽

Acid Treatment ◽

Blot Hybridization ◽

Stress Protein ◽

Transcriptional Level ◽

Northern Blot Hybridization ◽

High Osmolarity

ABSTRACT Streptococcus mutans, which causes dental caries in the human oral cavity and occasionally causes infective endocarditis in the heart, withstands adverse environmental stress through diverse alterations in protein synthesis. Differential gene expression in response to environmental stress was analyzed by RNA fingerprinting using arbitrarily primed PCR with a panel of 11mer primers designed for differential display in Enterobacteriaceae. Dot and Northern blot hybridization confirmed that the transcription of several genes was up- or down-regulated following exposure to acid shock from pH 7.5 to 5.5. RNA of a gene designated AP-185 (acid-stress protein) was induced specifically by acid treatment, while RNA of GSP-781 (general-stress protein) was up-regulated significantly when bacteria were exposed to high osmolarity and temperature, as well as low pH. The deduced amino acid sequence of AP-185 shares homology (78% identity) with branched-chain amino acid aminotransferase. Cloning and sequence analysis of GSP-781 revealed a potential secreted protein of a molecular mass of about 43 kDa and with a pI predicted to be 5.5. Transcriptional levels of another gene, designated AR-186 (acid-repressed protein), which encodes putative aconitase, were repressed by acid treatment but were enhanced by plasma or serum components. Analogous results were identified in icd andcitZ genes, and repression of these genes, along with AR-186, was also observed when they were exposed to high osmolarity and temperature. These results indicate that differential regulation of specific genes at the transcriptional level is triggered by different stress and that genes responsible for glutamate biosynthesis in the citrate pathway are coordinately regulated during the stress response of S. mutans.

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Influence of double-stranded RNAs on growth, sporulation, pathogenicity, and survival of Chalara elegans

Canadian Journal of Botany ◽

10.1139/b95-109 ◽

1995 ◽

Vol 73 (7) ◽

pp. 1001-1009 ◽

Cited By ~ 9

Author(s):

Zamir K. Punja

Keyword(s):

Plant Pathogen ◽

Dry Weight ◽

Complete Loss ◽

Thielaviopsis Basicola ◽

Wild Type ◽

C Elegans ◽

Chalara Elegans ◽

In The Wild ◽

Rna Fragments ◽

Type Strains

Three strains of Chalara elegans from diverse geographical areas that contained multiple (4 or 5) double-stranded RNA fragments were compared with spontaneously derived cultures from these strains that were either partially cured or completely free of dsRNA. In the wild-type strains, presence of the dsRNAs was found to significantly enhance phialospore production and pigmentation of colonies, whereas radial growth and mycelial dry weight accumulation were reduced. The rate and overall percentage of phialospore germination on 1% Noble water agar were also significantly reduced by the presence of the dsRNAs. In two partially cured strains (only one 2.8-kb fragment remaining), pathogenicity to various plant tissues was significantly enhanced when compared with the wild-type strains containing multiple dsRNA. However, survival in field soil was enhanced in one strain and reduced in the other. In the completely cured strain, the loss of multiple dsRNA fragments was associated with enhanced growth, reduced phialospore production, and a complete loss of pathogenicity and capability for survival in soil. These results indicate that the effects of dsRNAs in C. elegans vary with the strain. In general, the presence of multiple dsRNAs in this fungus enhanced sporulation, altered colony morphology, and reduced growth and pathogenicity. However, since the complete loss of dsRNA was found to eliminate pathogenicity and reduce survival, it suggests that some dsRNA fragments in C. elegans may confer an advantage to this soil-borne facultative plant pathogen. Key words: black root rot, soil-borne plant pathogen, Thielaviopsis basicola.

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348 SENESCENCE OF DAYLILY (HEMEROCALLIS) IS ASSOCIATED WITH EXPRESSION OF A MADS-BOX GENE

HortScience ◽

10.21273/hortsci.29.5.480e ◽

1994 ◽

Vol 29 (5) ◽

pp. 480e-480

Author(s):

Nathan E. Lange ◽

Michael S. Reid ◽

Victoriano Valpuesta ◽

Consuelo Guerrero ◽

Miguel A. Botella

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Amino Acid ◽

Blot Hybridization ◽

Flower Senescence ◽

Northern Blot Hybridization ◽

Mads Box ◽

Homologous Genes ◽

Mads Box Gene ◽

Commercially Important

As in many commercially important flowers, especially the monocotyledonous geophytes, senescence of the ephemeral daylily flower (Hemerocallis) does not involve ethylene. By differentially screening a cDNA library constructed from mRNA extracted from daylily petals in the earliest stages of senescence, clones were isolated whose transcription is up-regulated coordinately with the onset of senescence. One of these clones, sen12, was found to be a transcription factor. The deduced amino acid sequence of sen12 contains a MADS-box and an associated K-box similar to transcription factors suggested to control floral morphogenesis in a variety of different species. Northern blot hybridization showed sen12 to be highly upregulated before and during visible flower senescence. The expression of homologous genes during senescence of other flowers will be reported.

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Cloning and Inactivation of a Branched-Chain-Amino-Acid Aminotransferase Gene from Staphylococcus carnosus and Characterization of the Enzyme

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.4007-4014.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 4007-4014 ◽

Cited By ~ 31

Author(s):

Søren M. Madsen ◽

Hans Christian Beck ◽

Peter Ravn ◽

Astrid Vrang ◽

Anne Maria Hansen ◽

...

Keyword(s):

Growth Rate ◽

Amino Acid ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Biomass Yield ◽

Degradation Products ◽

Wild Type ◽

Staphylococcus Carnosus ◽

Branched Chain

ABSTRACT Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the corresponding enzyme in Staphylococcus aureus and that the ilvE gene was constitutively expressed as a monocistronic transcript. To study the influence of ilvE on BCAA catabolism, we constructed an ilvE deletion mutant by gene replacement. The IlvE protein from S. carnosus was shown mainly to catalyze the transamination of isoleucine, valine, leucine, and, to some extent, methionine using pyridoxal 5′-phosphate as a coenzyme. The ilvE mutant degraded less than 5% of the BCAAs, while the wild-type strain degraded 75 to 95%. Furthermore, the mutant strain produced approximately 100-fold less of the methyl-branched carboxy acids, 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which derived from the BCAA catabolism, clearly emphasizing the role of IlvE in aroma formation. In contrast to previous reports, we found that IlvE was the only enzyme that catalyzed the deamination of BCAAs in S. carnosus. The ilvE mutant strain showed remarkably lower growth rate and biomass yield compared to those of the wild-type strain when grown in rich medium. Normal growth rate and biomass yield were restored by addition of the three BCAA-derived α-keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.

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Rat kidney glutamyl aminopeptidase (aminopeptidase A): molecular identity and cellular localization

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.4.f546 ◽

1994 ◽

Vol 267 (4) ◽

pp. F546-F557 ◽

Cited By ~ 8

Author(s):

L. Song ◽

M. Ye ◽

M. Troyanovskaya ◽

E. Wilk ◽

S. Wilk ◽

...

Keyword(s):

Amino Acid ◽

Angiotensin Ii ◽

Cellular Localization ◽

Mesangial Cells ◽

Rat Kidney ◽

Blot Hybridization ◽

Northern Blot Hybridization ◽

Glomerular Mesangial Cells ◽

Partial Cdna ◽

Aminopeptidase A

Glutamyl aminopeptidase [aminopeptidase A (EAP), EC 3.4.11.7] is an ectoenzyme that selectively hydrolyzes acidic amino acid residues from the amino terminus of oligopeptides. EAP activity is highest within the kidney and small intestine. The murine pre-B cell BP-1/6C3 and the human kidney glycoprotein gp160 differentiation antigens have been reported to have biochemical properties indistinguishable from EAP. It is not known, however, if rat kidney EAP is a homologue of these antigens or molecularly distinct. Using the reverse transcription-polymerase chain reaction method with oligonucleotide primers based on the BP-1/6C3 nucleotide sequence, we isolated a 450-bp partial cDNA from rat kidney poly(A)+ RNA. The partial cDNA encoded a predicted protein that was 92% and 86% identical to the murine BP-1/6C3 and human gp160 antigens, respectively; the amino acid sequence within the zinc-binding domain was completely conserved. Purification of EAP from rat kidney and microsequence analysis of a tryptic digest peptide fragment (18-mer) indicated that the fragment was highly similar to a region within the BP-1/6C3 and gp160 proteins. Northern blot hybridization and immunoblot analyses were also consistent with labeling of products the same size as reported for the BP-1/6C3 and gp160 antigens. There was a good correlation between the cellular distribution of EAP mRNA and EAP immunoreactivity, with proximal tubules and glomerular mesangial cells having the highest densities. These results indicate that rat kidney EAP is a species homologue of the murine BP-1/6C3 and human gp160 antigens. Furthermore, on the basis of its cellular localization, rat kidney EAP is likely to be involved in degradation of oligopeptides within the glomerulus and the glomerular filtrate. Since cells that express EAP also express receptors for angiotensin II, an intrarenal vasoactive hormone that is a substrate for EAP, these results further suggest that EAP may play a role in modulating the activity of intrarenal angiotensin II.

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Sequence of cDNA for rat cystathionine γ-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes

Biochemical Journal ◽

10.1042/bj2690335 ◽

1990 ◽

Vol 269 (2) ◽

pp. 335-340 ◽

Cited By ~ 98

Author(s):

P F Erickson ◽

I H Maxwell ◽

L J Su ◽

M Baumann ◽

L M Glode

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Blot Hybridization ◽

Cdna Clones ◽

Sequence Information ◽

Northern Blot Hybridization ◽

Monospecific Antiserum ◽

Positive Hybridization ◽

Amino Acid Sequence Information

A cDNA clone for cystathionine gamma-lyase was isolated from a rat cDNA library in lambda gt11 by screening with a monospecific antiserum. The identity of this clone, containing 600 bp proximal to the 3′-end of the gene, was confirmed by positive hybridization selection. Northern-blot hybridization showed the expected higher abundance of the corresponding mRNA in liver than in brain. Two further cDNA clones from a plasmid pcD library were isolated by colony hybridization with the first clone and were found to contain inserts of 1600 and 1850 bp. One of these was confirmed as encoding cystathionine gamma-lyase by hybridization with two independent pools of oligodeoxynucleotides corresponding to partial amino acid sequence information for cystathionine gamma-lyase. The other clone (estimated to represent all but 8% of the 5′-end of the mRNA) was sequenced and its deduced amino acid sequence showed similarity to those of the Escherichia coli enzymes cystathionine beta-lyase and cystathionine gamma-synthase throughout its length, especially to that of the latter.

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Morphological and molecular characterization of Chalara elegans (Thielaviopsis basicola), cause of black root rot on diverse plant species

Canadian Journal of Botany ◽

10.1139/b99-164 ◽

2000 ◽

Vol 77 (12) ◽

pp. 1801-1812 ◽

Cited By ~ 3

Author(s):

Zamir K Punja ◽

Li-Juan Sun

Keyword(s):

British Columbia ◽

Random Amplified Polymorphic Dna ◽

Similarity Index ◽

Thielaviopsis Basicola ◽

Wild Type ◽

C Elegans ◽

Chalara Elegans ◽

Average Similarity ◽

High Degree ◽

Diverse Plant Species

The extent of variation in colony morphology and chlamydospore size, septation, and pigmentation was studied in 50 isolates of Chalara elegans Nag Raj et Kendrick (syn. Thielaviopsis basicola (Berk. et Br.) Ferr.) originating from 12 different geographic areas and substrates. In addition, the extent of genetic variation among these isolates was determined using random amplified polymorphic DNA (RAPD) analysis. Five general morphological groups could be distinguished among the isolates, two of which were aberrant phenotypes (albino and mycelial) that were derived upon continuous subculture of some wild-type isolates in the laboratory. The isolates with the most variation in phenotype originated from British Columbia and California. Six primers (10-mers) were used to generate 90 bands in RAPD-PCR, of which 75 were polymorphic. A high degree of diversity was apparent within C. elegans, and some banding patterns generated by specific primers were unique to certain isolates, thereby generating fingerprints. Distinct groups (clusters) were obtained following UPGMA analysis and, generally, these were composed of isolates from similar geographic regions or hosts. However, isolates from some areas, for example, British Columbia, were also found to belong to different clusters. There was generally a good relationship between groups assigned on the basis of morphology and those derived from cluster analysis, that is, isolates within a cluster tended to have similar morphology. In a few isolates, the aberrant phenotypes (albino and mycelial) could be distinguished using RAPDs from the wild type by the absence of 1 or 2 bands, indicating that changes in the nucleotide sequence had occurred, possibly through mutation. The average similarity index among all 50 isolates of C. elegans was 87%. An outgroup species (Chalara thielaviodes) had a similarity value of 40%.

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Expression of heparanase mRNA in bovine placenta during gestation

Reproduction ◽

10.1530/rep.0.1210573 ◽

2001 ◽

pp. 573-580 ◽

Cited By ~ 31

Author(s):

K Kizaki ◽

H Nakano ◽

T Takahashi ◽

K Imai ◽

...

Keyword(s):

Amino Acid ◽

Blot Hybridization ◽

Amino Acid Sequences ◽

Pcr Analysis ◽

Fetal Membrane ◽

Northern Blot Hybridization ◽

Amino Acid Residues ◽

Placental Development ◽

Bovine Placenta ◽

And Function

This study reports the identification and sequence of a partial cDNA for bovine heparanase and the expression of its mRNA in the placenta during gestation. The 364 amino acid residues deduced from the 1092 bp cDNA fragment share 81.9% and 80.5% identity with amino acid sequences of human and rat heparanase, respectively. Northern blot hybridization showed that two mRNAs (2.0 and 3.5 kb) are strongly expressed in placenta, and weakly expressed in the kidney, lung, spleen and non-pregnant uterus. In the placenta, these transcripts were detected in the cotyledon at all stages of gestation examined, and in the intercotyledonary fetal membrane and caruncle on day 60, day 120 and day 260. Quantitative real-time RT-PCR analysis showed very low expression of heparanase mRNA in the conceptus before implantation (day 17), but high expression in the cotyledon-containing fetal membrane (days 27-34) after implantation. Furthermore, heparanase mRNA was detected in the cotyledon, intercotyledonary fetal membrane and caruncle after days 60-64 of gestation. However, no significant expression of heparanase mRNA was observed in intercaruncular endometrium at all stages of gestation examined. These results demonstrate that heparanase mRNA is expressed in the placentome, indicating that heparanase may play a role in implantation, and in placental development and function.

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An extracellular aminopeptidase encoded by the ywaD gene plays an important role in supplying nitrogen nutrition for the growth of Bacillus subtilis 168

Canadian Journal of Microbiology ◽

10.1139/cjm-2016-0602 ◽

2017 ◽

Vol 63 (6) ◽

pp. 516-524

Author(s):

Zhongmei Liu ◽

Xinxing Gao ◽

Li Zhou ◽

Wenjing Cui ◽

Yaping Tian ◽

...

Keyword(s):

Growth Rate ◽

Bacillus Subtilis ◽

Amino Acid ◽

Culture Media ◽

Nitrogen Nutrition ◽

Soybean Protein ◽

Physiological Role ◽

Wild Type ◽

Bacillus Subtilis 168 ◽

Culture Supernatants

To investigate the physiological role of an extracellular aminopeptidase (BSAP168) encoded by the ywaD gene in Bacillus subtilis 168, we constructed the ywaD-deletion mutant (BS-AP-K). Compared with that of the wild-type strain, the maximum growth rate of BS-AP-K was reduced by 28% when grown in soybean protein medium at 37 °C, but not in Luria–Bertani medium. The impaired growth rate was more marked at higher temperature and could be compensated by supplementation of amino acid to the culture media. Further studies showed that in regards to the amino acid compositions and peptide distribution in the culture supernatants, there was an obvious difference between the culture supernatants of wild-type and BS-AP-K strains. In addition, another mutant strain (BS-AP-R) was constructed by replacing ywaD with ywaD-ΔPA to evaluate the effect of a protease-associated domain in BSAP168 on growth. All these findings indicated that BSAP168 played an important role in supplying the amino acids required for growth.

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Triose phosphate isomerase of Stellaria longipes (Caryophyllaceae)

Genome ◽

10.1139/g94-019 ◽

1994 ◽

Vol 37 (1) ◽

pp. 148-156 ◽

Cited By ~ 7

Author(s):

Xing-Hai Zhang ◽

C. C. Chinnappa

Keyword(s):

Amino Acid ◽

Blot Hybridization ◽

Triose Phosphate Isomerase ◽

Amino Acid Sequences ◽

Ecological Effect ◽

Northern Blot Hybridization ◽

Specific Expression ◽

Stellaria Longipes ◽

Triose Phosphate ◽

Low Degree

A cDNA encoding triose phosphate isomerase (TPI) of Stellaria longipes has been isolated and sequenced. The TPI of S. longipes exhibits high similarity to the TPIs from other species at both the nucleotide and amino acid levels. Southern blot analysis showed that the S. longipes genome contained multiple TPI-like nucleotide sequences and only a low degree of polymorphism was observed among genotypes of different habitats. The levels of TPI mRNA, detected by hybridization to the TPI cDNA, appeared similar between the genotypes. However, different genotypes showed variations in the total TPI activity, reflecting the possible ecological effect on the TPI gene expression. Northern blot hybridization to the cDNA also indicated a higher level of TPI mRNA in leaves than in roots, suggesting tissue-specific expression of TPI gene(s). Phylogenetic analysis of the TPI amino acid sequences from 16 taxa suggested that the TPI from S. longipes was more closely related to cytosolic TPIs from other plants than it was to TPIs from prokaryotes.Key words: triose phosphate isomerase, Stellaria longipes, genotype, ecotypes, molecular phylogeny.

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