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Biomedical subjects

S J Austin

Publications and source records attributed to S J Austin.

At least 19 recordsLinked to original sources
PubMed1992

Biochemical activities of the parA partition protein of the P1 plasmid.

The unit-copy P1 plasmid depends for stability on a plasmid-encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition-critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP-dependent, site-specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The role of the ATP-dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.

Adenosine Triphosphatases↗
PubMed1992

Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids.

The maintenance of partition-defective (Par-) mini-P1 and mini-F plasmids was studied in topA strains of Escherichia coli, which are defective in topoisomerase I activity. The partition defects were substantially but not completely suppressed in broth-grown cultures. This suppression was not due to a large increase in copy number. However, the absolute number of copies of Par- mini-P1 plasmids per average dividing cell is sufficiently high to account for the modest stability observed if a random distribution of the copies to daughter cells is assumed. The similar number of Par- plasmid copies in wild-type cells are distributed in a considerably worse-than-random fashion. Thus, it is unnecessary to propose, as was suggested previously, that an active, par-independent pathway operates in topA strains to ensure proper segregation of the plasmids to daughter cells. Rather, it seems likely that the lack of topoisomerase I activity aids the random distribution of the partition-defective plasmids, perhaps by facilitating their separation after replication. The results of studies carried out at reduced growth rates were consistent with this view; when topA cells containing Par- mini-P1 plasmids were cultured in minimal medium, in which the copy number of the plasmids per average cell is sharply reduced, very little suppression of the partition defect was observed.

DNA Topoisomerases, Type I↗
PubMed1991

Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

Bacterial Proteins↗
PubMed1991

Localized 1H NMR spectroscopy in Canavan's disease: a report of two cases.

Two children with Canavan's Disease, an autosomal recessive leukodystrophy, were studied by localized 1H spectroscopy. The N-acetylaspartate (NAA) signal intensity was high relative to other metabolite signals, and the signal intensity from choline-containing compounds was low. These findings are discussed in relation to a possible role for NAA in normal myelination.

Aspartic Acid↗
PubMed1991

Approaches to editing, assignment and interpretation of proton spectra.

Clinical 1H spectroscopy of the brain is complemented by parallel analyses of biopsy specimens and by studies of animal models of disease. 1H spectroscopy has been carried out on perchloric acid extracts of biopsy specimens from patients with intracranial tumours. The data suggest that clinical spectroscopy may be useful in the identification and grading of these tumours. In addition, the spectra from extracts derived from normal white matter add weight to the possibility that acetyl-containing compounds other than N-acetylaspartate may make a significant contribution to the signal at 2.0 ppm in vivo. Edited 1H spectra of brain metabolites in rats with acute liver failure demonstrate an elevation of glutamine and of lactate, suggesting a role for 1H spectroscopy in clinical investigations of metabolic encephalopathies. However, the observation and resolution of signals from glutamate and glutamine is more difficult at the lower fields that are available for clinical spectroscopy. Finally, some studies of patients with inborn errors of metabolism are described. It is shown that in a disorder of oxidative metabolism, brain lactate can be detected without the need for complex spectral editing techniques. Investigations of the metabolic abnormalities associated with Canavan's disease have shed further light on a possible role for N-acetylaspartate.

Animals↗
PubMed1990

Specificity switching of the P1 plasmid centromere-like site.

The P1 plasmid partition site acts like a centromere, promoting accurate segregation of copies to daughter cells. A 34 bp segment is essential for partition and binds the plasmid ParB protein. Additional sequences act as specificity elements that direct the choice of copies for partition. They include a second ParB binding site and a site for the host integration host factor protein. Sites lacking one or more of these additional elements are switched to a different specificity. Defined mutants were scored for partition specificity and protein binding. The results suggest that the wild-type site is folded in a specific DNA-protein complex. Disruption of the complex leads to an open configuration which, while still active in partition, has altered recognition specificity.

Base Sequence↗
PubMed1990

Oncogene alterations in in vitro transformed rat tracheal epithelial cells.

10 derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 nontumorigenic cell line transformed in vitro by treatment with 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for oncogene alterations. No abnormalities of Ha-ras or Ki-ras were seen that were suggestive of amplification, rearrangement or the presence of RFLPs. Analysis of specific-point mutations in Ha-ras using Pst I digestion (codon 12, GGA to GCA) or Ha-ras and Ki-ras using Xba I (codon 61, CAA to CTA) were negative. In one cell line derived by DMBA treatment, changes in the c-myc restriction digest pattern were seen after incubation with Bam HI and Hind III. Northern analysis revealed consistent differences between normal and transformed cells when probed with Ha-ras; c-myc expression was of low intensity, and the expression of Ki-ras could not be detected. Transfection of RTE cell DNAs into NIH/3T3 cells did not result in the appearance of morphologic transformants. The studies suggest that Ha-ras or Ki-ras codon 61 A to T transversions (CAA to CTA) are not associated with the immortal/tumorigenic phenotype in RTE cells transformed by DMBA or TPA, and are in contrast to results reported in some other biological systems.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

A single DnaA box is sufficient for initiation from the P1 plasmid origin.

The P1 plasmid replication origin requires the host DnaA protein for function. Two DnaA-binding boxes lie in tandem within the previously defined minimal origin, constituting its left boundary. Three more boxes lie 200 base pairs to the right of these, in the leader region for the P1 repA gene. We show that either set alone is active for origin function. One of the two origin boxes is relatively inactive. Constructs with just one of the five boxes are active for specific origin function as long as the box conforms exactly to the published consensus. This single consensus box is functional when placed either to the left or right of the core origin sequences. The flexibility shown by this system suggests that the boxes play a role different from those in the host oriC origin, where the number and position of boxes are critical.

Bacterial Proteins↗
PubMed1989

Absence of mutations in codon 61 of the Ha-ras oncogene in epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene.

Epithelial cells of the respiratory tract of rats were transformed in vitro by 7,12-dimethylbenz(a)anthracene (DMBA) which has been reported to cause A----T transversion mutations of the second position of Ha-ras in codon 61 in several biological models. In this study Ha-ras exon 2 was amplified by the polymerase chain reaction (PCR) and then sequenced directly. In 10 transformed cell lines, of which 5 are known to be tumorigenic, no mutations in codon 61 were found. The results suggest that Ha-ras codon 61 mutations are not associated with cell transformation initiated with DMBA in this particular cell transformation system. These data imply that other genes (oncogenes) are responsible for transformation of these cells. The results are discussed in relation to observations in various transformation systems in vivo and in vitro.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Consistent oncogene methylation changes in epithelial cells chemically transformed in vitro.

We have examined the restriction digest patterns of CCGG sequences in Kiras, Ha-ras, and c-myc oncogenes in rat tracheal epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene/12-O-tetradecanoylphorbol-13-acetate (TPA), or TPA alone. Oncogenes c-myc and Ha-ras in transformed cell lines, compared to normal rat tracheal epithelial cells and untreated primary cultures, had altered Hpa II restriction patterns as demonstrated by hybridizing bands of different molecular weight, or loss of bands. Ki-ras was hypermethylated in all cell derivations, including normal cells. These molecular alterations have not previously been reported for epithelial cells transformed in vitro by polycyclic hydrocarbons and tumor promoters, and suggest common mechanisms of action for agents with diverse molecular targets.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Plasmid-partition functions of the P7 prophage.

The sequences responsible for the proper partition of the P7 plasmid prophage to daughter cells lie within a discrete block of non-similarity between P7 and its close relative P1. The DNA sequence of the P7 region was determined. A segment with near identity to the replication (rep) region of P1 is followed by sequences (P7 par) that are clearly related to but very divergent from the P1 partition region. Subcloning was used to define the ends of the functional P7 partition region. It begins with a transcription promoter followed by two large open reading frames, parA and parB, that overlap by a single base and are read in the same direction. The genes direct the synthesis of two proteins, P7 ParA and ParB, with apparent Mr of 44,000 and 37,000. Specific frameshift mutations were introduced into the two genes. Each mutation blocked plasmid partition and both were complemented when the P7 ParA and ParB proteins were supplied in trans. The amino acid sequences of the P7 proteins show strong similarities to the P1 ParA and ParB proteins. However, the DNA sequences of the P7 and P1 open reading frames are remarkably divergent, largely caused by variability at the third positions in the codons. Interspecific complementation tests showed that the P7 proteins are unable to complement P1 parA or parB mutants, and the P1 proteins fail to complement the P7 mutations. Downstream from the P7 parB open reading frame is a sequence that conserves 27 of the 34 base-pairs of the P1 partition site parS. Unlike the P1 parS site, the P7 equivalent does not contain as extensive an inverted repeat. The heptamer sequences that define ParB binding sites within P1 parS are represented in P7 but differ from it by one base. A related sequence that coincides with the secondary ParB binding site within the P1 incB sequences is present nearby. Other sequences within the P7 incB region are rather different from their P1 counterparts. The basis for the major differences in specificity of the P1 and P7 par components is discussed. Comparison of the P1 and P7 sequences, and the nature of the junctions between similar and different sequences, suggest that the phages could have evolved by the pickup of divergent cassettes by recombination.

Amino Acid Sequence↗
PubMed1989

Protein-DNA interactions in regulation of P1 plasmid replication.

The P1 RepA protein appears to play three roles in P1 plasmid replication: acting at the origin both as a specific initiator and as a repressor of transcription, and interacting with the copy-control locus incA to bring about a negative control of initiation. We have used the DNase I footprinting technique to show that RepA binds specifically to repeat units of a 19-base-pair consensus sequence present in both the origin and incA control regions. RNA polymerase was shown to bind to two specific regions within the origin repeats. One of these constitutes the known promoter sequence for the repA gene. We show evidence that the polymerase can be efficiently displaced from the promoter by subsequent RepA binding, thus providing a direct mechanism for RepA autoregulation. Under the conditions used, there were no obvious differences in the affinities of individual repeat sequences for the purified protein.

Bacterial Proteins↗
PubMed1988

Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor.

The P1 plasmid partition system is responsible for segregation of daughter plasmids during division of the Escherichia coli host cell. The P1-encoded elements consist of two essential proteins, ParA and ParB, and the cis-acting incB region. The incB region determines partition-mediated incompatibility and contains the centromere-like site parS. We have isolated and purified the two proteins. ParB binds specifically to the incB region in vitro. DNase I footprinting assays place a strong binding site over the 35-bp parS sequence previously shown to be sufficient for partition when the Par proteins are supplied in trans. A weaker site lies within the incB region in sequences that are important for specifying incompatibility, but are not essential for partition. Gel band retardation assays show that a host factor binds specifically to the incB sequence. The factor strongly stimulates binding of ParB. Cutting the region at a site between the two ParB binding sites yields two fragments that can bind ParB but not host factor. Thus, information for host-factor binding lies in the region determining the specificity of plasmid incompatibility. The roles of parB and the host factor in partition and the specificity of plasmid incompatibility are discussed.

Bacterial Proteins↗
PubMed1988

Plasmid partition.

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DNA Replication↗