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W Cannon

Publications and source records attributed to W Cannon.

At least 37 records · Page 2Linked to original sources
PubMed1992

Central domain of the positive control protein NifA and its role in transcriptional activation.

The positive control protein NifA of Klebsiella pneumoniae activates transcription by RNA polymerase containing sigma 54 by catalysing open promoter complex formation. We show that the integrity of the putative ATP-binding pocket in the central domain of NifA is necessary for the positive control function of NifA, but is not required for DNA-binding or recognition of NifA by NifL. The inactive mutant NifA proteins are trans dominant to wild-type NifA and are unable to catalyse formation of open promoter complexes irrespective of whether a closed promoter complex at the nifH promoter has preformed. Formation of the closed complex results in a DNA structural distortion adjacent to the DNA region melted in the open promoter complex. This distortion lies at the leading edge of the E sigma 54 footprint. Although unable to catalyse open complex formation, some mutant NifAs altered the chemical reactivity of the distorted base-pair indicating that they retain the ability to recognize the closed promoter complex. The activation phenotype of partially active NifA molecules was sensitive to promoter sequences known to influence closed complex formation, indicating differences in (1) the susceptibility of the closed complexes towards activation and (2) their requirements for NifA during activation.

Amino Acid Sequence↗
PubMed1992

Activator-independent formation of a closed complex between sigma 54-holoenzyme and nifH and nifU promoters of Klebsiella pneumoniae.

The alternative sigma factor sigma 54 is required for transcription of nitrogen fixation genes in Klebsiella pneumoniae and other diazotrophs. The nif genes, and other E sigma 54-dependent genes whose products are necessary for a wide range of processes, are postively regulated. A unifying model that is well supported by studies on nif and other nitrogen-regulated (ntr) genes includes the central tenet that sigma 54 confers upon core RNA polymerase the ability to recognize and bind specific promoter sequences, but not the ability to isomerize to the open complex without assistance from the appropriate activator protein. Direct physical evidence for formation of an activator-independent complex between E sigma 54 and the NifA-dependent K. pneumoniae nifH and nifU promoters has, to date, been lacking. Using purified components we have now demonstrated formation of the closed complex at these promoters, indicating that it is an intermediate along the pathway to open complex formation. The closed complex was not detected when conserved features of the promoter were altered by mutation, nor was its stability increased when integration host factor protein was bound adjacent to the E sigma 54 recognition sequence.

Base Sequence↗
PubMed1991

Organization and function of binding sites for the transcriptional activator NifA in the Klebsiella pneumoniae nifE and nifU promoters.

The interaction of the Klebsiella pneumoniae NifA protein, a sigma 54-dependent activator, with the nifE and nifU promoters was analysed. At these promoters NifA established contacts in addition to those predicted by the minimal formulation NifA binding site (5'-TGT-N10-ACA). The positions of the contacts indicate that bound NifA molecules could assemble to form an oligomer. At both promoters contacts with NifA are made predominantly on one face of the DNA helix, and all contacts appear necessary for full activation by NifA. The close contacts made by NifA appear to be made by the DNA-binding domain of NifA. This domain shows specific DNA-binding activity in vitro. The binding of NifA to one site in the nifU promoter depends upon occupancy of additional upstream sequences by NifA. At the nifE promoter NifA binds adjacent to an integration host factor (IHF) binding site, but in contrast to results obtained with the nifU promoter IHF does not diminish nifE promoter occupancy by NifA. The IHF requirement for efficient in vivo activation of the nifU promoter by NifA was greater than that of the nifE promoter. Accordingly, the affinity of IHF for the nifU promoter is higher than for the nifE promoter. Amongst promoters utilizing the sigma 54 holoenzyme, the nifE promoter appears somewhat atypical in having the activator bound at around position -74 rather than the usual 100 base-pairs or more upstream from the transcription start site.

Bacterial Proteins↗
PubMed1990

The influence of the Klebsiella pneumoniae regulatory gene nifL upon the transcriptional activator protein NifA.

The influence of the Klebsiella pneumoniae nifL gene product upon the interaction of the transcriptional activator protein NifA with the nifH promoter has been examined using in vivo dimethylsulphate 'footprinting'. Binding of NifA to the upstream activator sequence (UAS) of the nifH promoter in the presence of the NifL protein was observed under nitrogen-limiting growth conditions. Growth in the presence of NH4+ or addition of NH4+ to nitrogen-limited cells diminished the interaction of NifA with the UAS when NifL was present. Repression of nif transcription by NifL may therefore involve an interaction between NifL and NifA which reduces the affinity of NifA for the UAS.

Ammonia↗
PubMed1989

Mutations in the RNA polymerase recognition sequence of the Klebsiella pneumoniae nifH promoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences.

Positive control of the wild-type Klebsiella pneumoniae nifH promoter by the NifA protein requires that NifA is bound at the upstream activator sequence (UAS). By introducing base substitutions at -15 to -17 in the RNA polymerase recognition sequence of the nifH promoter, positive control by a form of NifA unable to bind to the UAS was greatly increased when compared to the wild-type promoter. Transcriptional activation still required the rpoN encoded sigma factor and was initiated at the same nucleotide as in the wild-type promoter. Mutations at -15 to -17 suppressed the requirement that the UAS should be located on the correct face of the DNA helix with respect to the RNA polymerase recognition sequence in order that titration of NifA and efficient activation occur. This result supports the suggestion that upstream bound NifA interacts with the RNA polymerase-RpoN complex. To examine the minimal carboxy terminal sequences required for the positive control function of NifA a series of carboxy terminal deletions were constructed. Efficient positive control at a UAS-independent promoter was only observed in deletions which did not extend beyond the proposed boundary separating the carboxy terminal NifA DNA-binding domain from its central domain.

Amino Acid Sequence↗
PubMed1988

The DNA-binding domain of the transcriptional activator protein NifA resides in its carboxy terminus, recognises the upstream activator sequences of nif promoters and can be separated from the positive control function of NifA.

The positive control protein NifA activates transcription of nitrogen fixation promoters in Klebsiella pneumoniae. NifA is believed to bind to specific sites, the upstream activator sequences (UAS's), of the nif promoters which it activates. We have now shown by mutation of the carboxy terminus of NifA that this is the DNA-binding domain and that the DNA-binding and positive activator functions of NifA can be separated. Mutational analysis of the nifH UAS and in vivo methylation protection analysis of the interaction of NifA with the nifH promoter demonstrates that the UAS is recognised by the carboxy terminus of NifA. The UAS's of K. pneumoniae nif promoters are also required for activation by the Rhizobium meliloti NifA indicating that this activator also possesses DNA-binding activity.

Amino Acid Sequence↗
PubMed1987

Mutational analysis of upstream sequences required for transcriptional activation of the Klebsiella pneumoniae nifH promoter.

Upstream sequences of the Klebsiella pneumoniae nifH promoter were mutagenised and activation of the mutated promoters by the nif-specific transcriptional activator protein NifA examined in vivo. Of the sixteen mutations analysed, only those within the nifH upstream activator sequence (UAS), characterised by a TGT-N10-ACA motif, influenced nifH promoter activity. Mutations altering the two-fold rotational symmetry of the UAS or the spacing between the TGT and ACA motifs reduced promoter activity, consistent with the UAS functioning as a NifA binding site. The bases flanking the TGT-ACA motif of the UAS also appear to influence activation by NifA. Substituting the nifH UAS with a binding site for the transcriptional activator NtrC resulted in improved NtrC-dependent activation of the nifH promoter demonstrating that the activator specificity of the nifH promoter is dependent upon the presence of the appropriate upstream sequences to which the activator binds.

Base Sequence↗
PubMed1987

Frameshifts close to the Klebsiella pneumoniae nifH promoter prevent multicopy inhibition by hybrid nifH plasmids.

Certain multicopy plasmids bearing promoter sequences of nitrogen fixation (nif) genes inhibit expression of chromosomal genes in Nif+ Klebsiella pneumoniae, hence leading to a Nif- phenotype. This 'multicopy inhibition' has been attributed to the titration of the nif-specific activator protein NifA by the plasmid-borne promoter sequences. We now report that multicopy inhibition by nifH translational fusions is sensitive to frameshifts close to the nifH promoter. Transcriptional nifH fusion plasmids in which translation terminated near the nifH promoter were transcriptionally active and showed multicopy inhibition; introduction of a transcription terminator after the nifH coding sequence in these plasmids prevented their multicopy inhibition. Therefore it seems likely that premature termination of transcription prevents multicopy inhibition by the nifH promoter.

Cloning, Molecular↗
PubMed1987

Positional requirements for the function of nif-specific upstream activator sequences.

The upstream activator sequence (UAS) found in Klebsiella pneumoniae nif promoters and required for the activation of transcription by nifA, is absent from the nifF-nifL intergenic region, but is present downstream from the nifLA transcription start at +59. To determine whether nif upstream activator sequences can function in a 3' position, the nifH UAS was cloned downstream from the NifH transcription start, but no activation of transcription by nifA dependent upon the UAS in its 3' location could be detected. A mild repressive effect was detectable when the nifH UAS was placed downstream of the nifH promoter, but not when the cat promoter was substituted for the nifLA promoter upstream from the motif at +59 described above. However, deletion analysis showed that the UAS motif located downstream of the nifLA promoter has a role in transcription from the nifF promoter, although it is situated at position -263 with respect to the nifF transcription start, about 100 bp further upstream than previously described occurrences of the activator sequence.

Genes, Bacterial↗
PubMed1987

Transcriptional activation of the Klebsiella pneumoniae nitrogenase promoter may involve DNA loop formation.

Transcriptional activation of nitrogen fixation genes by NifA in Klebsiella pneumoniae requires an upstream NifA binding site. We now report that the introduction of half turns of the DNA helix into the DNA separating the upstream NifA binding site from the downstream promoter element of the nifH promoter decreases NifA-mediated activation to a greater extent than does the introduction of full helical turns. Reducing the spacing between the upstream and downstream elements of the nifH promoter also results in a promoter down phenotype. Introduction of a tight protein-binding site, the lac operator, between the upstream and downstream promoter elements did not render activation of the nifH promoter sensitive to occupancy of this site by the lac repressor. These findings indicate that NifA-mediated activation of transcription requires that NifA is bound upstream, and to the correct face of the DNA helix, in order to interact with downstream transcription factors. This implies that the interaction is brought about by the formation of a DNA loop between upstream and downstream promoter elements rather than by NifA sliding downstream.

DNA Restriction Enzymes↗
PubMed1982

Regression of pituitary microadenoma during and following bromocriptine therapy: persistent defect in prolactin regulation before and throughout pregnancy.

During 5 years of clinical, endocrinologic, and radiologic observations in a woman with a microprolactinoma treated medically with bromocriptine for 29 months, serial hypothalamic-pituitary studies revealed a defect in lactotrope function after prolactin (PRL) concentrations and ovulation were restored to normal. This defect persisted throughout a spontaneously conceived pregnancy in which the PRL, estradiol, and progesterone levels were subnormal, while, the dehydroepiandrosterone sulfate levels were normal and estriol concentrations were elevated. Levels of the beta subunit of human chorionic gonadotropin (hCG) were close to and slightly above the normal ranges. These observations are consistent with a role for PRL, interacting with hCG, in the control of estrogen and progesterone secretion by the fetoplacental unit. Lactation was initiated and maintained post partum. Pituitary function and PrL responses to suckling suggested improved lactotrope function 22 to 25 months after withdrawal of bromocriptine. The impaired lactotrope function, therefore, did not preclude normal implantation, pregnancy maintenance, onset of parturition, fetal development, and lactation.

Adenoma↗
PubMed1982

Prolactin, estradiol, and thyroid hormones in umbilical cord blood of neonates with and without hyaline membrane disease: a study of 405 neonates from midpregnancy to term.

Concentrations of prolactin (PRL), estradiol (E2), thyroxine (T4), triiodothyronine (T3), and reverse T3 in umbilical cord blood were measured by radioimmunoassay in neonates with (n = 60) and in a control group without (n = 345) hyaline membrane disease. Mean levels of all hormones assayed were not significantly different between the two groups at various stages of gestation. In the control group, gestational age correlated positively with PRL and inversely with reverse T3, whereas birth weight correlated positively with PRL, T4, and T3, but inversely with reverse T3 levels. Thus, larger, more mature neonates tended to have higher cord levels of PRL, T4, T3 and lower concentrations of reverse T3. The data also suggest that, in the premature neonate, various obstetric complications and exposure in utero to beta-mimetic drugs and glucocorticoids may be important determinants of the concentrations of E2 and thyroid hormone in cord blood.

Birth Weight↗
PubMed1981

Regulation of ovarian follicular and luteal function during treatment with exogenous gonadotropins in anovulatory infertility.

The dosage, duration of treatment, and plasma hormone levels were analyzed statistically between and within groups of treatment cycles with (n = 46) and without (n = 10) ovulation. A significant difference was observed in the dosage of human menopausal gonadotropins (hMG) over various days of treatment, but not in the mean dosage of hMG and human chorionic gonadotropin (hCG) administered per cycle. Follicle-stimulating hormone (FSH):luteinizing hormone (LH) ratios, prolactin (PRL) levels, and the magnitude and the duration of the estradiol response were greater in the ovulatory cycles. Additionally, in the ovulatory cycles, the dose of hMG correlated with the plasma levels of estradiol, FSH, and LH, while in the anovulatory cycles, hMG dosage correlated only with the LH concentrations. After administration of hCG, the mean plasma concentrations of its beta subunit peaked within 1 day and remained detectable for up to 10 days thereafter. In the ovulatory cycles, the mean progesterone level was maximal 6 days following hCG administration. In these cycles, luteal phase progesterone levels correlated positively with the preovulatory estradiol and inversely with concentrations of the beta subunit of hCG. The data demonstrate that, in contrast to anovulatory follicles, ovulatory follicles were exposed to a relative "dominance" of FSH over LH, with higher concentrations of estradiol and PRL for several days before hCG was administered. Apart from hMG dosage, the endogenous discharge of LH appeared to be an important determinant of the ovarian response. A single 10,000 IU dose of hCG was adequate for inducing ovulation and maintaining luteal function.

Adult↗
PubMed1981

Molar pregnancy in British Columbia: estimated incidence and postevacuation regression patterns of the beta subunit of human chorionic gonadotropin.

A total of 171 cases of molar pregnancy were followed by serial radioimmunoassay of the beta subunit of human chorionic gonadotropin (hCG-beta) over a 4-year period. The incidence over the study interval was 1:1,202 pregnancies in the province. In 120 women with intact uteri and measurable plasma levels of hCG-beta at the initiation of follow-up, the hormone remained detectable for up to 219 days (between 31 and 32 weeks) following evacuation, with 50% of patients remaining positive at 63 days (between 8 and 9 weeks). In five women undergoing hysterectomy, the hormone remained detectable for up to 112 days (between 15 and 16 weeks) postoperatively. In 92% of the cases, hCG-beta regressed to negative without chemotherapy. The prolonged presence of hCG in plasma after evacuation of the mole, regardless of the presence or absence of the uterus, points to persistence of molar tissue and its gradual rejection from uterine and extrauterine sites as a common phenomenon in the natural history of the uncomplicated postmolar course of this disease.

Adolescent↗
PubMed1981

Human chorionic gonadotropin, estradiol, progesterone, prolactin, and B-scan ultrasound monitoring of complications in early pregnancy.

In a prospective study of women in the first trimester who were at risk for recurrent abortion, data from 20 pregnancies with normal outcomes were contrasted with those of 9 pregnancies that aborted spontaneously between 7 and 12 weeks. In pregnancies in which an embryo was present before abortion, the mean concentrations of human chorionic gonadotropin beta-subunit (hCG-beta) were low with a steady decline in progesterone levels, whereas estradiol (E2) and prolactin concentrations approximated those in normal pregnancies. With growth disorganization, mean hCG-beta, E2, and progesterone levels were significantly lower than normal. In 87.5% of embryopathic pregnancies, conclusive evidence of this disorder was obtained with ultrasound monitoring. Subnormal levels of hCG-beta, E2, and progesterone were observed in 88.9, 100, and 57.1%, respectively. The karyotype was abnormal in 80% of embryopathic pregnancies successfully cultured. The normal production of E2 appeared dependent on the presence of the embryo in the first trimester of pregnancy. Endocrine failure of the trophoblast and/or the corpus luteum in embryopathic pregnancy is suggested by diminished hCG-beta and progesterone concentrations. Serial ultrasound scanning and hormone assay, particularly of hCG-beta and E2, may be useful in assessing pregnancy at risk for recurrent abortion. However, because subnormal hormone levels may occur relatively late in the clinical course of embryopathic gestation, ultrasound scanning may yield more useful results as compared with hormone assay. Further studies are required to define more clearly the mechanisms of endocrine failure in embryopathic pregnancy.

Abortion, Spontaneous↗
PubMed1980

A possible role for prolactin in the control of human chorionic gonadotropin and estrogen secretion by the fetoplacental unit.

The effects of prolactin (PRL) on fetoplacental function were studied by measuring the beta subunit of human chorionic gonadotropin (hCG-beta), estradiol (E2), progesterone (P), and PRL concentrations throughout seven pregnancies in a control group (N = 6 women) and in three pregnant women with prolactinomas, who were receiving bromocriptine. In one of the latter, estriol (E3) was also assayed. Long-term suppression of PRL was associated with augmentation of hCG-beta at the first-treimester peak and in late pregnancy. Concomitant augmentation of E3 (in late gestation) and possibly E2, but not P, levels was also observed. This effect on hCG appeared dependent on PRL rather than the dopaminergic effect of bromocriptine. Short-term drug induced alterations in PRL (over 3 hours) during early pregnancy did not result in significant changes in hCG-beta or steroid concentrations. In each control pregnancy, a significant negative correlation (p less than 0.05) was observed between hCG-beta and PRL, while a significant positive correlation between the latter and E2 concentrations in these women was also demonstrated. Apart from its effect on lactation, osmoregulation, and gonadal and adrenal function, a further role for PRL during reproduction appears to be in the control of hCG and estrogen secretion in the fetoplacental unit.

Adult↗
PubMed1979

Plasma prolactin, human chorionic gonadotropin, estradiol, testosterone, and progesterone in the ovarian hyperstimulation syndrome.

Plasma prolactin, estradiol, progesterone, and testosterone, but not HCG-beta levels, were higher in a patient who developed the ovarian hyperstimulation syndrome while undergoing ovulation induction with human gonadotropins than in two other women who also became pregnant after similar treatment without complications. These results suggest that hyperprolactinema, in association with elevated ovarian steroid levels, may be factors in the pathogenesis of this disorder.

Blood Proteins↗