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

2001 ◽  
Vol 69 (4) ◽  
pp. 2493-2501 ◽  
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.

scholarly journals Tissue-specific regulation of dipeptidyl peptidase IV expression during development

1991 ◽  
Vol 277 (2) ◽  
pp. 331-334 ◽  
Author(s):  
M Hildebrandt ◽  
W Reutter ◽  
J D Gitlin
Keyword(s):  
Lung Development ◽  
Kidney Development ◽  
Blot Hybridization ◽  
Dipeptidyl Peptidase ◽  
Transcriptional Level ◽  
Northern Blot Hybridization ◽  
Dpp Iv ◽  
Organ Specific ◽  
Specific Regulation ◽  
Lung And Kidney

The patterns of dipeptidyl peptidase (DPP) IV activity and protein amount in different rat organs during development were compared. In order to elucidate the molecular basis for these patterns, total RNA was isolated from lung and kidney at different stages of development and analysed by Northern-blot hybridization using an oligonucleotide derived from the DPP IV cDNA sequence. This oligonucleotide hybridized to two distinct mRNAs of approx. 3.2 and 4.8 kb respectively. During kidney development, the pattern for DPP IV mRNA paralleled that of DPP IV activity and protein amount, suggesting that, in kidney, the expression of DPP IV is primarily controlled at the transcriptional level. In contrast, the magnitude of DPP IV activity during lung development compared with that of DPP IV mRNA in lung suggests that post-transcriptional mechanisms are involved in regulating the expression of DPP IV in lung. Organ-specific regulation of DPP IV expression may provide a useful model for further comparative studies of transcriptional and post-transcriptional mechanisms of DPP IV expression within the same organism.

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

HortScience ◽  
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.

scholarly journals Identification of Genes Regulated by Low Temperature in Pachysandra terminalis Sieb. et Zucc Using cDNA Differential Display

HortScience ◽  
2005 ◽  
Vol 40 (7) ◽  
pp. 1995-1997
Author(s):  
Suping Zhou ◽  
Roger J. Sauve ◽  
Abdulah Abdulah
Keyword(s):  
Differential Display ◽  
Blot Hybridization ◽  
Leaf Tissue ◽  
Northern Blot Hybridization ◽  
Dot Blot ◽  
Novel Genes ◽  
Cold Tolerant ◽  
Reverse Northern ◽  
Stress Related Genes ◽  
And Control

Complementary Deoxyribonucleic Acid (cDNA) differential display and reverse Northern dot blot were used to identify genes in Pachysandra terminalis Sieb. & Zucc., a cold-tolerant plant, that are regulated by low temperatures. Rooted cuttings were obtained from stock plants that had been maintained in a greenhouse at 24 °C. These cuttings were subjected to the following cold treatments: 2 weeks at 12 °C, 48 hours at 4 °C, 48 hours at 0 °C, and 4 hours at –1 °C. Following leaf tissue analysis of treated and control plants, some stress-related genes and many novel genes were identified. Northern blot hybridization demonstrated that all novel genes were regulated by the cold treatments.

Post-transcriptional block in oxytocin gene expression within the seminiferous tubules of the bovine testis

1994 ◽  
Vol 140 (1) ◽  
pp. 63-72 ◽  
Author(s):  
H Ungefroren ◽  
M Davidoff ◽  
R Ivell
Keyword(s):  
Gene Expression ◽  
Leydig Cells ◽  
Blot Hybridization ◽  
Seminiferous Tubules ◽  
Transcriptional Level ◽  
Gene Transcript ◽  
Northern Blot Hybridization ◽  
Rnase Protection ◽  
Low Level

Abstract Northern blot hybridization showed that bovine and sheep testis, unlike testes from other mammals, contain moderate levels of an apparently normal oxytocin gene transcript. In situ hybridization localized this mRNA to within the seminiferous tubules, possibly in the Sertoli cells. Conflicting with this result, immunohistochemistry showed that both oxytocin and the syngeneic neurophysin I epitopes are both clearly restricted to the Leydig cells, being expressed here at a low level. Since illegitimate transcription from spurious start sites can lead to a lack of translation product, the integrity of the major ruminant testicular transcripts of the oxytocin gene was checked using differential hybridization, RNase protection and multiple polymerase chain reaction assays. All tests showed the transcripts to have a normal, translatable composition and to be transcribed from the conventional 5' initiation site. Therefore, the block in oxytocin gene expression within the tubules is probably due to a lesion at the post-transcriptional level. The low level peptide expression in the Leydig cells can probably be attributed to the presence of functional transcripts in these cells, which are below the level of significant detection for the in situ hybridization assay. Journal of Endocrinology (1994) 140, 63–72

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

1994 ◽  
Vol 267 (4) ◽  
pp. F546-F557 ◽  
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.

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

1990 ◽  
Vol 269 (2) ◽  
pp. 335-340 ◽  
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.

Expression ofptxRand its effect ontoxAandregAexpression during the growth cycle ofPseudomonas aeruginosastrain PAO1

1999 ◽  
Vol 45 (12) ◽  
pp. 1008-1016 ◽  
Author(s):  
Jane A Colmer ◽  
Abdul N Hamood
Keyword(s):  
Regulatory Gene ◽  
Blot Hybridization ◽  
Sufficient Conditions ◽  
Growth Cycle ◽  
Transcriptional Level ◽  
Northern Blot Hybridization ◽  
Exotoxin A ◽  
Strain Pao1 ◽  
Iron Deficient ◽  
Multiple Copies

The expression of the toxA and regA genes in Pseudomonas aeruginosa is negatively regulated by iron at the transcriptional level. We have previously described ptxR, an exotoxin A regulatory gene which appears to enhance toxA expression through regA. In this study, we have tried to determine if ptxR expression correlates with its effect on toxA and regA expression throughout the growth cycle of P. aeruginosa strain PAO1. This was done using Northern blot hybridization experiments (with toxA, regA, and ptxR probes), and ptxR transcriptional fusion studies. To avoid problems related to the presence of multiple copies of ptxR in PAO1, we have constructed a PAO1 strain (PAO1-XR) that carries only two ptxR genes in its chromosome. Our results showed that when PAO1-XR was grown in iron-limited conditions, the increase in exotoxin A activity and the accumulation of toxA mRNA appeared at about mid- to late-exponential phase. A similar increase in the accumulation of regA mRNA was detected. Both regA transcripts, T1 and T2, were enhanced in PAO1-XR. In iron-sufficient medium, neither toxA nor regA mRNA was detected at any time point in the growth cycle of PAO1-XR. In contrast, the accumulation of ptxR mRNA was detected throughout the growth cycle of PAO1-XR under both iron-deficient and iron-sufficient conditions. The presence of iron in the growth medium also had no effect on the level of β-galactosidase activity produced by a ptxR-lacZ fusion in PAO1. These results suggest that (i) the enhancement in toxA expression by ptxR correlates with the enhancement in regA expression; (ii) ptxR affects the expression of the regA P1 and P2 promoters; (iii) ptxR expression precedes its effect on toxA and regA expression; and (iv) unlike toxA and regA, the overall expression of ptxR throughout the growth cycle of PAO1 is not negatively regulated by iron.Key words: ptxR, differential expression, transcriptional regulation, regA, toxA.

Application of laser capture microdissection and differential display technique for screening of pathogenic genes involved in endometrial carcinoma

2007 ◽  
Vol 17 (6) ◽  
pp. 1224-1230 ◽  
Author(s):  
L. Wen-Xin ◽  
H. Xi-Shan
Keyword(s):  
Endometrial Carcinoma ◽  
Differential Display ◽  
Laser Capture Microdissection ◽  
Blot Hybridization ◽  
Local Alignment ◽  
Northern Blot Hybridization ◽  
Normal Endometrium ◽  
Pathogenic Genes ◽  
Differential Gene ◽  
Laser Capture

The objective is to eliminate the interference from other cell types; gene fragments involved in endometrial carcinoma (EC) are screened and cloned. Human normal endometrial glandular epithelia and EC cells were harvested with laser capture microdissection (LCM). The purification and concentration of minimal RNA were used to screen differential expressed gene fragments involved in EC by fluoro differential display polymerase chain reaction (FDD-PCR). The differential gene fragments were cloned, sequenced, and then identified by reverse Northern blot hybridization. Positive fragments were analyzed with basic local alignment search tool (BLAST). Cyclin-dependent kinase 7 (CDK7) expressions in EC and normal endometrial tissue were tested by immunohistochemical staining. Of 38 differential bands, 3 bands were of high expression in normal endometrium and 35 in EC. Six positive differential gene fragments were obtained and BLAST analysis for them suggested that L1.1 was homologous (99% identical) to the CDK7; L1.9 had a 99% homology with protein phosphatase 1 regulatory (inhibitor) subunit 12 A (PPP1R12A); L1.21 and L1.22 showed a 100% homology with cellular repressor of E1A-stimulated genes 1 (CREG); and L1.25 and L1.26 indicated more than 98% homology with solute carrier family 39 (zinc transporter), member 10 (SLC39A10). Immunohistochemistry revealed that CDK7 expression was higher in EC than in normal endometrium. We conclude that pathogenic genes involved in EC are obtained with LCM and FDD-PCR. It has been first found that CDK7, PPP1R12A, CREG, and SLC39A10 are correlative with EC from gene level. CDK7 is strongly associated with EC and can be used as potential molecular marker of EC for further studies

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

2006 ◽  
Vol 96 (5) ◽  
pp. 468-479 ◽  
Author(s):  
Yunjung Park ◽  
Xiaobang Chen ◽  
Zamir K. Punja
Keyword(s):  
Growth Rate ◽  
Amino Acid ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Wild Type ◽  
Melanin Production ◽  
Dsrna Element ◽  
C Elegans ◽  
Chalara Elegans ◽  
Latently Infected

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.

scholarly journals Expression of heparanase mRNA in bovine placenta during gestation

Reproduction ◽  
2001 ◽  
pp. 573-580 ◽  
Author(s):  
K Kizaki ◽  
H Nakano ◽  
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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