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.

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 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.

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

scholarly journals The Pseudomonas aeruginosa Exotoxin A Regulatory Gene, ptxS: Evidence for Negative Autoregulation

1999 ◽  
Vol 181 (16) ◽  
pp. 4890-4895 ◽  
Author(s):  
Britta L. Swanson ◽  
Jane A. Colmer ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Upstream Region ◽  
Transcriptional Level ◽  
Exotoxin A ◽  
Specific Sequence ◽  
T7 Promoter ◽  
Gel Shift

ABSTRACT We have previously described a Pseudomonas aeruginosagene, ptxR, which enhances exotoxin A production at the transcriptional level. We have also described another gene,ptxS, which is transcribed divergently fromptxR and interferes with the enhancement of exotoxin A synthesis by ptxR. However, the mechanisms through whichptxR and/or ptxS are regulated is not known. In this study, we attempted (by using the DNA gel shift assay) to determine if P. aeruginosa contains a potential regulatory protein that binds specifically to the ptxR orptxS upstream region. In the initial analysis, different-sized gel shift bands were detected when a probe containing the ptxR-ptxS intergenic region was incubated with the lysate of P. aeruginosa PAO1. The strongest binding activity was detected with a smaller fragment that represents theptxS upstream region. Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream ofptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of the ptxS isogenic mutant PAO1::ptxS. However, the binding band was regenerated when a plasmid carrying ptxS intact was introduced into PAO1::ptxS. In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate of Escherichia coli in which ptxS was overexpressed from the T7 promoter. The effect of PtxS onptxS expression was examined by using aptxS-lacZ fusion plasmid. The level of β-galactosidase activity produced by PAO1::ptxS carrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid. Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site. These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within the ptxSupstream region.

Effect of static growth and different levels of environmental oxygen on toxA and ptxR expression in the Pseudomonas aeruginosa strain PAO1

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2263-2275 ◽  
Author(s):  
Jennifer M. Gaines ◽  
Nancy L. Carty ◽  
Jane A. Colmer-Hamood ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sandwich Elisa ◽  
Growth Cycle ◽  
Anaerobic Conditions ◽  
Strain Pao1 ◽  
Time Points ◽  
Iron Deficient ◽  
Deficient Medium ◽  
Enhanced Expression ◽  
Different Levels

Within certain infection sites, such as the lung of cystic fibrosis patients, Pseudomonas aeruginosa grows statically under either decreased oxygen tension or anaerobic conditions, a situation that is likely to influence the production of virulence factors. The goal of this study was to determine the effect of static growth under microaerobic (decreased oxygen) and anaerobic conditions on the expression of the P. aeruginosa exotoxin A (ETA) gene toxA and its positive regulator ptxR. Using toxA–lacZ and ptxR–lacZ fusion plasmids, the level of toxA and ptxR expression was measured throughout the growth cycle of strain PAO1, which was grown in either iron-deficient or iron-sufficient medium under four different conditions: 20 %-SH (aerobic, shaking), 20 %-ST (aerobic, static), 10 %-ST (microaerobic, static) and 0 %-ST (anaerobic, static). In iron-deficient medium, toxA expression was higher under 20 %-ST and 10 %-ST than under 20 %-SH. However, the highest level of toxA expression occurred under 0 %-ST. Analysis of ETA protein using sandwich ELISA revealed that at time points between 8 and 24 h of the growth curve, PAO1 produced higher levels of ETA under 0 %-ST than under 20 %-SH. In iron-sufficient medium, toxA expression was significantly repressed under all conditions. Additional analyses using PAO1 strains that carry lacZ fusions with the toxA regulatory genes regA and pvdS revealed that the expression of regA and pvdS is reduced rather than increased at 0 %-ST. ptxR expression under different conditions paralleled that of toxA expression, except that it was repressed by iron under 20 %-SH only. Between 6 and 24 h of growth, and under all conditions, the level of dissolved oxygen (DO) within the PAO1 cultures was sharply reduced. These results suggest that (1) the combined effect of static growth and anaerobic conditions produce a significant increase in toxA and ptxR expression in PAO1; (2) this effect appears to be unique to toxA and ptxR, since the level of regA and pvdS expression was reduced under the same conditions; (3) neither static growth nor anaerobic conditions interfere with the repression of toxA expression by iron, although static growth deregulates ptxR expression with respect to iron; and (4) the enhanced expression of toxA and ptxR is not related to the reduced levels of DO in PAO1 cultures.

Characterization of a Tn551-mutant of Staphylococcus aureus defective in the production of several exoproteins

1994 ◽  
Vol 40 (8) ◽  
pp. 677-681 ◽  
Author(s):  
Ana T. Giraudo ◽  
Claudia G. Raspanti ◽  
Aldo Calzolari ◽  
Rosa Nagel
Keyword(s):  
Staphylococcus Aureus ◽  
Regulatory Gene ◽  
Blot Hybridization ◽  
Protein A ◽  
Southern Blot Hybridization ◽  
Terminal Segment ◽  
Transcriptional Level ◽  
Extracellular Form ◽  
Single Insertion

A Tn551 insertional pleiotropic mutant defective in the production of several exoproteins was isolated from Staphylococcus aureus 196E and characterized. The pleiotropism of the mutant was due to a single insertion of the transposon as evidenced by Southern blot hybridization and by the transfer of its phenotype by transduction to S. aureus ISP479. The mutants showed diminished or null levels of α- and β-hemolysins, DNase, coagulase, and protein A in the supernatants of broth cultures. Production of proteases, lipase, staphylokinase, or enterotoxin A was not modified. The mutants did synthesize the cell-bound form of protein A and also the extracellular form of this protein coded by pRIT11, which lacks the COOH-terminal segment of the molecule. These observations suggest that the sae locus does not involve a positive regulatory gene acting at the transcriptional level. The phenotype of the mutant was different from that of other insertional mutants affecting exoprotein synthesis, such as agr, xpr, or sar. This new mutation has been designated sae (for S. aureus exoprotein expression).Key words: S. aureus, transpositional mutant, exoprotein production, pleiotropism, protein A.

scholarly journals Location of the initial cleavage sites in mouse pre-rRNA.

1983 ◽  
Vol 3 (8) ◽  
pp. 1501-1510 ◽  
Author(s):  
L H Bowman ◽  
W E Goldman ◽  
G I Goldberg ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
28S Rrna ◽  
Cleavage Sites ◽  
S1 Nuclease ◽  
5.8S Rrna ◽  
Initial Cleavage ◽  
S1 Nuclease Mapping ◽  
Mapping Techniques ◽  
Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

Location of the initial cleavage sites in mouse pre-rRNA

1983 ◽  
Vol 3 (8) ◽  
pp. 1501-1510
Author(s):  
L H Bowman ◽  
W E Goldman ◽  
G I Goldberg ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
28S Rrna ◽  
Cleavage Sites ◽  
S1 Nuclease ◽  
5.8S Rrna ◽  
Initial Cleavage ◽  
S1 Nuclease Mapping ◽  
Mapping Techniques ◽  
Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

Renal expression of osteopontin and alkaline phosphatase correlates with BUN levels in aged rats

1995 ◽  
Vol 269 (3) ◽  
pp. F398-F404
Author(s):  
C. T. Liang ◽  
J. Barnes
Keyword(s):  
Alkaline Phosphatase ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Tgf Beta ◽  
Young Rats ◽  
Beta Actin ◽  
Old Rats ◽  
Renal Expression

Renal expression of alkaline phosphatase (AP) and osteopontin (OP) in rats of different age was examined. Northern blot hybridization showed that AP mRNA was reduced moderately, whereas OP mRNA was stimulated drastically in old rats. Dot-blot quantitation analysis showed that AP mRNA decreased 30% in 24-compared with 6-mo-old rats. In contrast, OP mRNA increased 3.1- and 9.1-fold, respectively, in 12- and 24-mo-old rats. beta-Actin mRNA did not change with age. Blood urea nitrogen (BUN) increased 47 and 187% in 12- and 24-mo-old rats, respectively. Correlation analysis showed that BUN correlated negatively with AP mRNA and positively with OP mRNA. No correlation was observed with beta-actin. The expression of these markers was also examined in femurs. AP and OP mRNAs were marginally reduced in old bones. To test whether the correlation also exists in other types of renal insufficiency, we examined these parameters in young rats infused with parathyroid hormone (PTH). BUN was elevated 3.5-fold, whereas AP mRNA decreased 48%, and OP mRNA increased 15.3-fold in kidneys of PTH-treated rats. To elucidate the possible mechanisms that lead to the overexpression of OP in kidney, we examined the expression of transforming growth factor-beta 1 (TGF-beta 1) mRNA. No significant differences in TGF-beta 1 expression were observed between young and old rats and control and PTH-treated young rats. Changes in the expression of OP were also visualized by immunostaining of renal sections. Alterations in the levels of OP and AP were validated by Western blot analysis and enzyme assay of homogenate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Detection and Differentiation of Bovine Group A Rotavirus Serotypes using Polymerase Chain Reaction-Generated Probes to the VP7 Gene

1992 ◽  
Vol 4 (2) ◽  
pp. 148-158 ◽  
Author(s):  
Anil V. Parwani ◽  
Blair I. Rosen ◽  
Jorge Flores ◽  
Malcolm A. McCrae ◽  
Mario Gorziglia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Chain Reaction ◽  
Diarrhea Virus ◽  
Bovine Rotavirus ◽  
Group A ◽  
Polymerase Chain ◽  
Vp7 Gene ◽  
Hybridization Assays

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Cracker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture-propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.

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