Biomedical subjects

C M Thomas

Publications and source records attributed to C M Thomas.

At least 109 records · Page 6Linked to original sources

PubMed1995

Purification of KorA protein from broad host range plasmid RK2: definition of a hierarchy of KorA operators.

The KorA protein of promiscuous plasmid RK2 is encoded in the central control operon, which coordinates expression of genes for replication, transfer and partitioning. KorA is known to repress transcription from seven promoters on the plasmid genome but at the trfA promoter, for the vegetative replication genes, it also causes derepression of trbAp, the first promoter for the operon whose genes are required primarily for mating pair formation prior to conjugative transfer. We have overproduced and purified KorA (101 amino acid residues). Crosslinking indicates that it exists largely as a dimer in solution. Western blotting with specific antibodies suggests that there are approximately 4000 monomers per cell in exponential phase and about 600 in stationary phase. Footprinting confirmed the expected location of the KorA operator, and indicated that KorA and RNA polymerase can bind simultaneously in promoter regions. Gel retardation experiments with DNA fragments carrying each of the seven KorA operators revealed that there is a hierarchy of binding affinities. Highest affinity (Kapp = 12 to 20 nM) occurs with operators containing the 12 bp inverted repeat GTTTAGCTAAAC (klaAp, korAp and trfAp), while lower affinities (Kapp = 136 to 272 nM) occur with less perfect repeats (klcA, kleA, kleC, kfrA). In addition, specific DNA sequences flanking the 12 bp are necessary for the characteristic type I KorA binding affinity. This hierarchy may be important in providing a graded response in expression of the operons controlled by KorA as its concentration varies, as for example on transition from exponential to stationary phase.

Amino Acid Sequence↗

PubMed1995

Conformational dynamics of a mobile loop in the NAD(H)-binding subunit of proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli.

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Ths) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Ths in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Ths specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the one-dimensional 1H-NMR spectra of Ths before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Ths (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Ths with NAD+ provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Ths. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

Alanine Dehydrogenase↗

PubMed1995

Properties of the soluble polypeptide of the proton-translocating transhydrogenase from Rhodospirillum rubrum obtained by expression in Escherichia coli.

Transhydrogenase, which catalyses the reduction of NADP+ by NADH coupled to proton translocation across a membrane, may be unique in the photosynthetic bacterium Rhodospirillum rubrum. Unlike the homologous enzyme from animal mitochondria and other bacterial sources, it has a water-soluble polypeptide, which exists as a dimer (Ths), that can be reversibly dissociated from the membrane component [Williams, R., Cotton, N. P. J., Thomas, C. M. & Jackson, J. B. (1994) Microbiology, 140, 1595-1604]. We have expressed the gene for Ths in cells of Escherichia coli under control of the tac promoter and a strong ribosome binding site. The protein, purified by column chromatography, fully reconstituted transhydrogenation activity to everted membrane vesicles of Rhs. rubrum that had been washed to remove Ths. The purified expressed protein was prepared in quantities over 100-fold greater than were obtained from wild-type Rhs. rubrum. The fluorescence spectrum of purified expressed Ths had an intense and unusually short wavelength emission maximum at 310 nm with shoulders at 298 and 322 nm. Time-resolved measurements indicated that the fluorescence decay was almost monoexponential with a lifetime of 5.2 ns. On denaturation with 4 M guanidine hydrochloride, the emission band shifted to 352 nm and decreased in intensity. In the native protein, the fluorophore was relatively inaccessible to quenching solutes, such as iodide ions and acrylamide. It is concluded that the fluorescence emission arises mainly from the single tryptophan residue of Ths (Trp72), which is locked into a rigid conformation and is located in highly non-polar environment. The 310-nm fluorescence of Ths was quenched by NADH, maximally to 46%. The apparent binding constant was 18 microM. The fluorescence of Ths-bound NADH was enhanced relative to the nucleotide in free solution and its emission maximum was shifted to a shorter wavelength (440 nm). These data support previous indications that the NADH binding site is located in domain I of proton-translocating transhydrogenase. Excitation of Ths at 280 nm did not lead to sensitized emission at 440 nm from bound NADH. This indicates that the quenching of fluorescence of Ths by NADH does not result from resonance energy transfer from Trp72 to the bound nucleotide. NAD+, NADP+ and NADPH had little effect on the protein fluorescence. The kinetics of quenching of Ths fluorescence by NADH were examined after mixing in a stopped-flow device. The 'on' rate constant for nucleotide binding was approximately 8 x 10(6) M-1 s-1 and the 'off' constant approximately 150 s-1.

Amino Acid Sequence↗

PubMed1995

Neural tube defects and elevated homocysteine levels in amniotic fluid.

OBJECTIVE: Our purpose was to study maternal blood and amniotic fluid concentrations of homocysteine and relevant vitamins in relation to neural tube defects. STUDY DESIGN: Concentrations of total homocysteine, folate, and vitamins B12 and B6 were measured in maternal blood and amniotic fluid of 27 women carrying a fetus with a neural tube defect and 31 control women carrying a healthy fetus. RESULTS: The mean total homocysteine concentration in amniotic fluid of the study group was significantly higher than that of the control group. The mean concentrations of total homocysteine in blood and the vitamins folate, B12, and B6 in, respectively, blood and amniotic fluid were not significantly different between the groups. The mean concentrations of homocysteine and vitamin B6 were significantly lower in amniotic fluid than in blood in both groups, whereas vitamin B12 in amniotic fluid was higher than in blood. CONCLUSION: These results support the hypothesis that at least the cause of a subset of neural tube defects could reside in a primary or secondary maternal or fetal derangement of homocysteine metabolism.

Adult↗

PubMed1995

Haematological and biochemical profile of uncomplicated pregnancy in nulliparous women; a longitudinal study.

BACKGROUND: Most laboratory parameters change during pregnancy. A serial study of a large number of routine haematological and biochemical blood parameters and biochemical urine parameters was conducted in a group of 66 healthy nulliparous pregnant women, who had an uncomplicated pregnancy. METHODS: Blood samples and 24-h urine samples were obtained at four weeks intervals during pregnancy and at 1 (1P) and 6 (6P) weeks after delivery. RESULTS: During pregnancy, haemoglobin concentration, haematocrit and erythrocyte count were lower, mean cell volume was not different, and mean cell haemoglobin and mean cell haemoglobin concentration were enhanced. The platelet count during pregnancy was not different from the level at 6P but increased 60% at 1P. Serum ferritin decreased 50% whereas plasma fibrinogen increased 100%. Serum creatinine (-28%), uric acid (-35%) and urea (-40%) concentrations were reduced during pregnancy. The serum concentrations of sodium (-4 mmol/l) and potassium (-0.2 mmol/l) were lower, but serum chloride was unaltered. Serum protein and albumen concentrations declined by 7.8 and 9.4 g/l respectively. The serum concentrations of bilirubin, ALAT, ASAT and gamma-GT remained unaltered. Serum LDH was 30% above normal non-pregnant values at 1P. The heat-stable alkaline phosphatase level increased in the third trimester. Heat-stable and heat-labile fractions were both elevated at 1P. The serum osmolality was 9 mosmol/kg lower and urine volume was about 25% higher during pregnancy. The creatinine excretion was unaltered but creatinine clearance increased by 25%. CONCLUSIONS: The concentrations of most components change during pregnancy. The interpretation of results of laboratory tests in pregnant women should be made with caution.

Adult↗

PubMed1995

Urinary prostaglandin and prostaglandin metabolite excretion in patients with essential hypertension or hypertension with renal artery stenosis.

BACKGROUND: Recent studies have reported elevated prostaglandin levels in patients with renal artery stenosis and hypertension. To investigate whether a distinction between essential hypertension and hypertension with renal artery stenosis is possible by measuring eicosanoid excretion, we studied the excretion of these compounds in patients with essential hypertension and in hypertensives with concomitant renal artery stenosis. METHODS: The 24-h urinary excretion of metabolites of prostaglandin I2, prostaglandin E2 and metabolites of thromboxane A2 was sampled under standardised conditions, in 15 patients with essential hypertension and in 15 patients with unilateral renal artery stenosis with hypertension. Also clinical and biochemical characteristics of the subjects were analysed. RESULTS: The patients with renal artery stenosis had significantly lower excretion of prostaglandin I2 than did the essential hypertensive patients. However, the overlap in the values was large, thus not allowing a diagnostic differentiation according to urinary prostaglandin I2 levels. The excretion of prostaglandin E2 and of metabolites of thromboxane A2 showed no significant differences among the groups. CONCLUSIONS: Measurement of urinary prostaglandin or prostaglandin metabolite excretion did not contribute to the non-invasive detection of the presence of a renal artery stenosis in the patients in this study.

Adult↗

PubMed1995

Identification of amplified restriction fragment polymorphism (AFLP) markers tightly linked to the tomato Cf-9 gene for resistance to Cladosporium fulvum.

Using the technique of amplified restriction fragment polymorphism (AFLP) analysis, and bulked segregant pools from F2 progeny of the cross Lycopersicon esculentum (Cf9) x L. pennellii, approximately 42,000 AFLP loci for tight linkage to the tomato Cf-9 gene for resistance to Cladosporium fulvum have been screened. Analysis of F2 recombinants identified three markers which co-segregated with Cf-9. The Cf-9 gene has recently been isolated by transposon tagging using the maize transposon Dissociation (Ds). Analysis of plasmid clones containing Cf-9 shows that two of these markers are located on opposite sides of the gene separated by 15.5 kbp of intervening DNA. AFLP analysis provides a rapid and efficient technique for detecting large numbers of DNA markers and should expedite plant gene isolation by positional cloning and the construction of high-density molecular linkage maps of plant genomes.

Base Sequence↗

PubMed1995

Evaluation of remote stethoscopy for pediatric telecardiology.

OBJECTIVE: To investigate the interobserver reliability and diagnostic validity of a commercial electronic stethoscope for pediatric telecardiology. MATERIALS AND METHODS: Pairs of blinded pediatric cardiologists made independent diagnoses, recommendations concerning follow-up echocardiography, and specific judgments regarding heart sounds, murmurs, and congenital heart disease using an electronic (ES) or an acoustic (AS) stethoscope on 78 pediatric cardiology outpatients and at a distance of 450 km (280 miles) with 38 telemedicine cardiology outpatients. The kappa statistic (K) indexed the instruments' interexaminer reliabilities. The validity of ES was measured by K for ES versus AS and by the percentage of cases where the findings for ES and AS differed sufficiently to suggest an important ES screening error. RESULTS: For heart disease, AS, ES, and tele-ES reliabilities were satisfactory (K = 0.80, 0.67, and 0.80, respectively), as were AS agreement with hands-on ES (K = 0.65) and with tele-ES (K = 0.64). The AS and ES reliabilities and ES/AS agreement were also satisfactory for systolic regurgitant and diastolic pulmonic murmurs (K = 0.63-0.78) but were unsatisfactory for evaluable heart sounds and other murmurs (K = 0.16-0.60). The ES yielded clinically important disagreements with AS in 5.4% of the clinic cases and 10.5% of the telemedicine cases (P = 0.67). In determining the need for additional work-up (echocardiography) or follow-up appointments, hands-on ES and tele-ES had a combined accuracy of 92%, with a sensitivity of 88% and a specificity of 97%. CONCLUSIONS: Hands-on ES provided reliable and valid screening for congenital heart disease. Tele-ES was highly reliable but had reduced diagnostic validity. Examiner blinding, bandwidth limitations, and artificial restrictions on the remote assistant may have contributed to this reduced performance. As these factors are easily correctable, we regard the ES as a highly promising tool for pediatric telecardiology.

Child↗

PubMed1995

Dietary sodium restriction in the prophylaxis of hypertensive disorders of pregnancy: effects on the intake of other nutrients.

Dietary sodium restriction is used in the Netherlands in the prophylaxis of preeclampsia. To study the effects of long-term sodium restriction on the intake of other nutrients and the outcome of pregnancy, 68 healthy nulliparous pregnant women were randomly assigned to either a low-sodium diet (20 mmol/24 h) or an unrestricted diet. The diet was consumed between week 14 of gestation and delivery. The dietary intakes of energy, fat, protein, carbohydrate, sodium, potassium, and calcium were estimated with the dietary-history technique. A low-sodium diet reduced the intake of protein (by approximately 15 g/24 h), fat (by 20 g/24 h), and calcium (by 350 mg/24 h) and tended to decrease the energy intake (by approximately 0.7 MJ/24 h). The intakes of carbohydrate and potassium did not differ between the groups. The maternal weight gain was less in the low-sodium group (6.0 +/- 3.7 compared with 11.7 +/- 4.7 kg). Mean birth weight was not significantly different (3.2 +/- 0.5 compared with 3.4 +/- 0.5 kg).

Adult↗

PubMed1995

Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato.

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.

Base Sequence↗

PubMed1995

Distribution of unlinked transpositions of a Ds element from a T-DNA locus on tomato chromosome 4.

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments.

Base Sequence↗

PubMed1995

Humoral anti-OV-TL 3 response after the intravenous administration of radiolabelled Fab' or F(ab')2 fragments in ovarian cancer patients.

The human anti-mouse antibody (HAMA) response was determined in the serum of patients suspected of having ovarian cancer who underwent radioimmunoscintigraphy with either 99Tcm-OV-TL 3 Fab' (n = 20) or 111In-DTPA-OV-TL 3 F(ab')2 (n = 73). Blood samples were collected prior to and at several time points post-intravenous injection. The detection of HAMA was performed with an in-house OV-TL 3 F(ab')2-based sandwich-type immunoradiometric assay (IRMA). The homologous IRMA demonstrated that 8 of 20 (40%) patients had developed HAMA responses after injection of Fab' fragments and that 14 of 73 (19%) patients had developed HAMA responses after F(ab')2 administration. The subclass of the measured HAMA was analysed in a limited number of samples, showing IgG or IgM as well as mixed responses. The kinetics of the HAMA responses varied greatly. Our study showed the relevance of the sampling time and frequency: HAMA responses can be easily underestimated with a low sampling frequency. The homologous IRMA described in this study was able to quantify the OV-TL 3-specific HAMA responses. With additional assays, the subclass of the HAMA could be further analysed. Remarkably, the fraction of HAMA responders after injection of OV-TL 3 Fab' fragments was in the same range as the proportion of HAMA responders after F(ab')2 administration.

Animals↗

PubMed1995

Plasma homocysteine and menopausal status.

The aim of the study was to measure the concentrations of plasma homocysteine in premenopausal and postmenopausal women, and to examine a possible relationship between plasma homocysteine and oestrogen status. Homocysteine metabolism was studied by a standardized oral methionine loading test, and oestrogen status was assessed by the measurement of serum 17 beta-oestradiol. Forty-six premenopausal and 26 postmenopausal healthy women without a history of vascular disease or adverse pregnancy outcome were recruited by public advertisement. The main outcome measures were the concentrations of fasting and postmethionine plasma homocysteine, and serum 17 beta-oestradiol. Fasting plasma homocysteine concentrations (mean +/- SD) were significantly higher in postmenopausal women as compared to premenopausal women (12 +/- 4 mumol L-1 and 10 +/- 3 mumol L-1, respectively) as well as postmethionine plasma homocysteine concentrations (46 +/- 16 mumol L-1 and 32 +/- 9 mumol L-1, respectively). In premenopausal women, postmethionine plasma homocysteine was negatively and significantly correlated to serum 17 beta-oestradiol (r = -0.34). It is concluded that plasma homocysteine concentrations, both fasting and after methionine loading, are significantly higher in postmenopausal women than in premenopausal women. In premenopausal women, the higher concentrations of serum 17 beta-oestradiol may account in part for the lower concentrations of postmethionine plasma homocysteine.

Adult↗

PubMed1995

Hormonal serum profiles and follicular development after intramuscular and pulsatile intravenous administration of human menopausal gonadotrophin.

A study was performed to compare, in a randomized way, the effect of pulsatile intravenous (i.v.) and intramuscular (im) human menopausal gonadotrophin (hMG) administration on hormonal serum profiles and follicular development in in vitro fertilization (IVF). Fourteen IVF patients participated in the study, aged between 20 and 40 years, with a normal endocrine profile, no hormonal medication used for at least 3 months previously, no endometriosis, both ovaries present and a normal male factor. Seven patients were treated with im hMG at a daily dose of 150 IU and seven patients with pulsatile i.v. hMG at a daily dose of 112.5 IU, in both cases in combination with buserelin. Ultrasonography was performed every other day during the stimulation phase and blood samples were collected once daily up to five times a day during the entire IVF cycle. Serum concentrations of follicle-stimulating hormone, luteinizing hormone, 17 beta-oestradiol, progesterone and human chorionic gonadotrophin were determined. There were no differences in hormonal profiles between the two groups. The numbers of retrieved oocytes, fertilization rates and mean embryo quality were identical in this study, as was follicular growth. In conclusion, in the present randomized study no differences were observed in hormonal levels or follicular development after im and pulsatile i.v. hMG treatment.

Adult↗

PubMed1995

Analytical and clinical performance of improved Abbott IMx CA 125 assay: comparison with Abbott CA 125 RIA.

We compared the improved Abbott IMx cancer antigen (CA) 125 assay (cat. no. 7A89) with the Abbott CA 125 RIA. Serum specimens were from healthy perimenopausal women (n = 124) and from patients with benign gynecologic and nongynecologic diseases (n = 124), ovarian carcinoma (n = 104), or other malignancies (n = 193). The IMx assay detected as little as 0.193 kAU/L CA 125 (AU = arbitrary Abbott unit), demonstrated up to 29% overestimation upon serum dilution, low within-assay (2.7-5.6%) and between-assay (4.8-8.2%) CVs, and no high-dose hook effect < or = 46,000 kAU/L nor influence from human anti-mouse antibodies in serum of women injected with OC 125 F(ab')2. Values by IMx were 20% lower than by RIA for healthy perimenopausal women (n = 100; IMx = 0.80 RIA - 2.5 kAU/L), and at least 50% higher for those with benign or malignant ovarian disorders at concentrations < 100 kAU/L. Receiver-operating characteristic (ROC) curve analysis of ovarian neoplasma vs perimenopausal controls indicated a gain of specificity and sensitivity with the improved IMx assay over the RIA, but ROC performance was the same with either assay if patients with benign ovarian disorders were used as controls.

Adolescent↗

PubMed1995

Comparative evaluation of the androgenicity of four low-dose, fixed-combination oral contraceptives.

Changes in endogenous androgen metabolism were compared in healthy women taking one of four low-dose modern oral contraceptives (OCs). One hundred women were randomized to (1) 35 micrograms ethinyl estradiol (EE) + 250 micrograms norgestimate (Cilest); (2) 20 micrograms EE + 150 micrograms desogestrel (Mercilon); (3) 30 micrograms EE + 150 micrograms desogestrel (Marvelon); or (4) 30 micrograms EE + 75 micrograms gestodene (Femodene). During the luteal phase of the pretreatment cycle, body weight and blood pressure were recorded, and plasma levels of the following variables were recorded: sex-hormone-binding globulin (SHBG), cortisol-binding globulin (CBG), testosterone, free testosterone, dihydrotestosterone, androstenedione, dihydroepiandrosterone sulfate (DHEAS), and hydroxyprogesterone. The free androgen index was also calculated. These variables were remeasured during the third week of OC intake and during the fourth and sixth cycles. There were no statistically significant differences in androgenic variables among the four OCs. The DHEAS concentration decreased less with the 20 micrograms EE + desogestrel formulation compared with either 30 micrograms EE + desogesterel or norgestimate-containing formulations (20% vs. 45%). Concentrations of SHBG and CBG increased significantly in all four groups (average 263 +/- 119% and 94 +/- 26%, respectively); CBG increased less in women taking 20 micrograms EE + desogestrel (about 75%) than in the other formulations (about 100%). The four modern, low-dose OCs tested had similar impacts on endogenous androgen metabolism, yielding significant decreases in testosterone, dihydrotestosterone, androstenedione, and DHEAS. All of these formulations may be beneficial in women with androgen-related syndromes such as acne and hirsutism. Large studies are under way to establish which of the third-generation OCs is the least androgenic. In vitro studies suggest that norgestimate has the least androgenic profile.

Adult↗

PubMed1994

Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging.

The tomato Cf-9 gene confers resistance to infection by races of the fungus Cladosporium fulvum that carry the avirulence gene Avr9. The Cf-9 gene was isolated by transposon tagging with the maize transposable element Dissociation. The DNA sequence of Cf-9 encodes a putative membrane-anchored extracytoplasmic glycoprotein. The predicted protein shows homology to the receptor domain of several receptor-like protein kinases in Arabidopsis, to antifungal polygalacturonase-inhibiting proteins in plants, and to other members of the leucine-rich repeat family of proteins. This structure is consistent with that of a receptor that could bind Avr9 peptide and activate plant defense.

Amino Acid Sequence↗

PubMed1994

KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer.

The trfA and trb operons, encoding genes essential for replication and conjugative transfer of broad host range plasmid RK2, are transcribed divergently. Deletion analysis presented here indicates that trfAp and trbAp are arranged as face to face promoters. The presence of the korA gene, whose product is known to repress seven operons on RK2, including the trfA operon, is shown here to stimulate trbAp. The effect of korA on trbAp is mimicked by the trfAp-1 promoter down mutation, suggesting that a reduction in the activity of trfAp is required for derepression of trbAp activity. The trfAp-1 mutation reduces RNA polymerase binding and open complex formation at trfAp but does not stimulate melting at trbAp in vitro. Therefore, the inhibition of trbAp is most probably due to forward transcription initiated at trfAp. The simultaneous inhibition/stimulation by KorA is seen even in the presence of the other repressors KorB and TrbA, which act at this region, thus providing a dominant mode of coordinating plasmid replication and transfer. This may be one of the keys to understanding how the maintenance and spread of promiscuous plasmids are balanced in different environments.

Amino Acid Sequence↗