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S Minchin

Publications and source records attributed to S Minchin.

13 recordsLinked to original sources
PubMed2000

DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study.

We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the -10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the -10 and -35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions -15 and -14. Promoter activity is greatest when the 'non-template' strand carries T and G at positions -15 and -14, respectively. Promoter activity can be further enhanced by a second T and G at positions -17 and -16, respectively, immediately upstream of the first 'TG motif'. Our results show that the base sequence of the DNA segment upstream of the -10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of 'TG motifs' upstream of the promoters' -10 elements. We conclude that correctly placed 'TG motifs' are found at over 20% of E.coli promoters.

Base Sequence↗
PubMed1994

Thermal energy requirement for strand separation during transcription initiation: the effect of supercoiling and extended protein DNA contacts.

We have studied the role of extended protein DNA contacts and DNA topology on the ability of Escherichia coli RNA polymerase to form open complexes at several related promoters. The -35 region of several Escherichia coli promoters do not have homology with the consensus sequence, but still drive activator independent transcription initiation. This is due to the presence of a TG motif upstream from the -10 hexamer creating an 'extended -10' promoter. We have previously shown that two 'extended -10' promoters, galP1 and pBla, can form open complexes at lower temperatures than the galP1 derivative, galPcon6, which has a consensus -35 hexamer. Here we report further investigations into the mechanism of open complex formation by RNA polymerase, in particular the thermal energy requirement. A single base pair change in galPcon6 creating an 'extended -10' sequence, results in a 20 degrees C reduction in the temperature requirement for open complex formation. The DNA topology has also been shown to effect the thermal energy requirement for strand separation. Promoters carried on supercoiled plasmids form open complexes at lower temperatures than when present on linear DNA templates. We have also shown that in vivo, RNA polymerase can form open complexes at lower temperatures than those observed for linear templates in vitro, but requires slightly higher temperatures than supercoiled templates in vitro, however the promoter hierachy remains the same.

Base Composition↗
PubMed1994

Assaults by patients on psychiatric residents at three training sites.

OBJECTIVE: This study attempted to determine how often psychiatric residents are exposed to violence, the types of violence they encounter, and what institutional changes might increase their safety. METHODS: Safety conditions at two private general hospitals and one state hospital that served as training sites for a psychiatric residency program were assessed through a survey of psychiatric residents and site visits to the hospitals. The survey asked residents to quantify violent incidents occurring in the emergency rooms, wards, and clinics at each site. The site visits focused on safety issues related to staff training, physical layout, staffing patterns, current policies, and compliance with policies. RESULTS: All 47 residents in the training program responded to the survey. None reported serious injury, although as many as 56 percent had been physically assaulted on the wards of one hospital, and 54 percent of residents had encountered a weapon in one emergency room. Almost all residents had been verbally threatened or had witnessed violence to others. A paradoxical finding of the survey was that the residents felt safest in the hospital that had the highest rate of violence. The site visits revealed that deficiencies in the safety procedures were allowing weapons to be brought into patient care areas. CONCLUSIONS: Psychiatric residents are often exposed to dangerous situations, although serious injury is rare. Residents' beliefs about their level of safety seem to be influenced more by how competent they perceive the staff to be than by the frequency of violence. The findings from the site visits pointed to two steps to increase safety: creating a weapon-free environment by searching all patients and finding ways to improve compliance with existing safety measures.

Data Collection↗
PubMed1993

The Escherichia coli cysG promoter belongs to the 'extended -10' class of bacterial promoters.

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5'-TGN-3' motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the 'extended -10' class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

Base Sequence↗
PubMed1993

Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus -35 region sequences.

Methylation-interference assays have been used to identify guanine residues that make important contacts with RNA polymerase during open-complex formation at two related Escherichia coli promoters. Methylation of lower-strand G-31 at a gal consensus promoter completely prevents complex formation, while modification of upper-strand G-33 has no detectable effect. At galP1, which lacks a consensus -35 region, modification of lower-strand G-33 and upper-strand G-14 reduces, but does not prevent, complex formation. G-33 is the only guanine residue in the -35 region of galP1 where modification interferes with open-complex formation. Since this guanine residue is not protected in open complexes, we conclude that its modification causes alteration of, or interference with, a transient contact during the transcription initiation pathway.

Base Composition↗
PubMed1991

Different thermal energy requirement for open complex formation by Escherichia coli RNA polymerase at two related promoters.

We have studied the effect of temperature on transcription initiation in vitro at two related promoters ga/Pcon and ga/P1, which have the same nucleotide sequence around the -10 region and transcription start site, but differ in upstream sequences. One of the promoters, ga/Pcon, carries the consensus -35 hexamer, 5'TTGACA 3', whilst ga/P1 contains a block of 'distortable' upstream sequences that allow promoter function in the absence of a -35 region consensus sequence. RNA polymerase can form complexes with both promoters at a range of temperatures. However, the thermal energy requirement for open complex formation differs: open complexes can form at ga/P1 at low temperatures, whereas ga/Pcon requires higher temperatures. The thermal energy requirement for transcription from preformed open complexes is the same for both promoters.

Base Sequence↗
PubMed1990

Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.

The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures.

Amino Acid Sequence↗
PubMed1990

Unwinding of duplex DNA during transcription initiation at the Escherichia coli galactose operon overlapping promoters.

We have used potassium permanganate as a probe to detect DNA duplex unwinding in vitro, in open complexes between E. coli RNA polymerase and DNa fragments carrying the E. coli galactose operon regulatory region. This zone contains 3 overlapping promoters which specify transcription initiation at 3 distinct startpoints. We have used mutant gal derivatives carrying different single point mutations, each of which allows initiation from only one of the 3 start sites. This has allowed us to compare duplex unwinding in open complexes at the 3 different promoters, and to show that the extent of the unwinding is similar in each case. Further, the pattern of DNA modification by potassium permanganate suggests a model for discrimination between the upper and lower strands. Finally, we show that DNA modification by potassium permanganate at the gal promoters is the same in vivo as in vitro.

Base Sequence↗
PubMed1989

Forebrain structure in infantile autism.

Researchers implicate central nervous system dysfunction in infantile autism, but postmortem examinations and in vivo brain imaging studies have produced conflicting results concerning the neuronal systems involved. Magnetic resonance imaging--a new modality of in vivo brain imaging--was used to investigate the cerebral and thalamic structure of 105 autistic patients. Compared with the control group, there was an overall difference in the forebrain morphology of the autistic subjects due to subtle but statistically significant differences in the anterior ventricular horns, lateral ventricles, and the right lenticular nucleus. These results, when considered with previous studies of cerebral structure, suggest that there are subtle alterations in the forebrain of autistic patients.

Adolescent↗
PubMed1988

Morphological evidence for brainstem involvement in infantile autism.

Previous neurophysiological and neuroanatomic studies suggest brainstem dysfunction in infantile autism. Therefore, we investigated the brainstem structure of autistic patients by planimetric analysis of midsagittal magnetic resonance imaging scans. We found the entire brainstem and one component--the pons--to be statistically significantly smaller in the autistic group when compared with medical controls. We also noted no correlation between brainstem size and age in the autistic group--a correlation that was found in the control group. These data present morphological evidence of brainstem involvement in the infantile autism syndrome.

Adolescent↗
PubMed1987

Cerebellar structure in autism.

Several recent reports suggest cerebellar abnormalities in patients with autism. To further investigate the posterior fossa in vivo, we analyzed axial (transverse) and coronal magnetic resonance imaging (MRI) scans of autistic patients. The MRI scans were measured at life-size by planimetry. Axial MRI scans of "high level" autistic patients were compared with control scans; there were no differences in the fourth ventricles, vermes, cerebella, and cerebellar-pontine "complexes" between the groups. Coronal MRI scans were also studied. In the coronal scans, the cerebella of autistic patients were proportionally smaller and the fourth ventricles proportionally larger. This suggests that there are morphologic changes in cerebella of autistic children; such alteration may best be viewed in the MRI coronal plane.

Adolescent↗
PubMed1987

Midsagittal magnetic resonance imaging of autism.

Since recent reports suggest structural brain abnormalities in autistic patients, we analysed magnetic resonance imaging (MRI) scans of autistic children. Planimetric measurements were done on midsagittal MRI scans, produced with a 0.5 T superconducting magnet. Scans of 13 'high-level' autistic subjects were compared with 35 control MRI scans, read as anatomically normal by a neuroradiologist. Corpus callosal, fourth ventricular, cerebellar, cerebral, and cranial areas were measured. The fourth ventricle was found to be significantly larger in the autistic group. No other areas in the midsagittal scans differed statistically between groups. Results suggest that structures defining the fourth ventricle are anatomically altered in autistic patients.

Adolescent↗