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S J Austin

Publications and source records attributed to S J Austin.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1988

The P1 plasmid-partition system synthesizes two essential proteins from an autoregulated operon.

The P1 partition region contains two large open reading frames that encode the proteins ParA and ParB. It was previously shown that ParA is essential for partition activity. Using a novel assay, we show that ParB protein is also an absolute requirement for partition and that it is active in trans to the partitioning plasmid. Development of complementation tests for parA and parB allowed us to assign a number of partition-defective point mutants of a P1 miniplasmid to the parA and parB cistrons. Using gene fusion techniques, it was shown that parA and parB constitute an operon controlled from a promoter proximal to the start of parA. Transcription from this promoter is autoregulated by a feedback loop that is sensitive to the ParA and ParB proteins in concert. The parB gene also appears to be expressed at a low level from a second promoter at the intercistronic boundary. This results in a low level of expression and tight autoregulation for the ParA protein and slightly less stringent control for ParB synthesis.

Bacteriophages↗
PubMed1987

P1 plasmid replication requires methylated DNA.

Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.

Base Sequence↗
PubMed1987

The plasmid-maintenance functions of the P7 prophage.

The region responsible for the maintenance of the prophage of bacteriophage P7 as a stable, unit-copy plasmid was isolated in a lambda att vector which lysogenizes Escherichia coli as a stable unit-copy plasmid under the control of the P7 replication origin. The P7 plasmid-maintenance region was shown to consist of adjacent replication and partition regions capable of functioning independently. The isolated replication region could support plasmid maintenance but the resulting plasmids were highly unstable unless the partition region was also included. Stable composite plasmids were isolated containing the putative P7 partition region and the origin of replication of the unrelated plasmid F, indicating that P7 encoded an active partition mechanism. The replication regions of P7 and P1 were shown to be highly homologous but the partition regions of the two plasmids appear to be unrelated in sequence. The incompatibility determinants associated with the two replication regions showed the same specificity, whereas the partition-region incompatibility determinants were different, showing no cross-specificity.

Chromosome Deletion↗
PubMed1987

Partition site of the P1 plasmid.

We have defined a minimal partition site, parS, from the plasmid P1. It contains sufficient cis-acting information to direct accurate segregation of low-copy-number plasmids that contain it as long as the two essential P1 Par proteins are supplied in trans. The site is, at most, 34 base pairs and contains a perfect 13-base-pair inverted repeat. Site-directed mutations were made within the repeat sequence that abolished activity whether or not the symmetry of the palindrome was maintained. Partition appears to be a competitive process, as differentially marked plasmids carrying the same type of partition site are not independently segregated but are randomly distributed with respect to each other. We have studied competition between plasmids carrying various fragments encompassing the parS site. As expected, two plasmids carrying the minimal parS site compete with each other. However, a sequence that lies to the left of the minimal parS site acts as a major modulator of this competition, changing the specificity of the competitive effect completely. Thus, this adjacent sequence appears to be an important determinant of the specificity of the wild-type P1 partition system without being necessary for its efficient function.

Base Sequence↗
PubMed1985

Partition of unit-copy miniplasmids to daughter cells. III. The DNA sequence and functional organization of the P1 partition region.

The boundaries of the P1 par (plasmid partition) region of the unit-copy plasmid P1 were defined to within 2.7 X 10(3) base-pairs of DNA. The DNA sequence of the region revealed two large open reading frames that could encode proteins of Mr 44,000 and Mr 38,000. Both would be read in the same direction. The first open reading frame corresponds to the par A gene, the Mr 44,000 protein product of which was shown to be trans acting and essential for partition. The second open reading frame (parB) follows closely and may be cotranscribed with par A. The codon usage frequency for parB is consistent with its producing a protein product. The ParB protein was identified in cell extracts as a product with an apparent Mr of 45,000, suggesting that it behaves anomolously on gel electrophoresis. Following parB is the incB region, an incompatibility determinant thought to be the cis acting site that constitutes the putative attachment point on the DNA for the cellular partition apparatus. Subcloning of this site showed it to consist of a maximum of 174 base-pairs. The incB sequence is highly A + T-rich and contains a 20 base-pair inverted repeat. Another A + T-rich inverted repeat of similar size but different sequence is found between the putative parA promoter and the ribosome initiation sequence at the start of the parA open reading frame and may be involved in the autoregulation of ParA synthesis. The par region appears to contain a functional analog of the centromere of eukaryotic chromosomes. It is responsible for ensuring that newly replicated plasmids are properly distributed to daughter cells during cell division of its Escherichia coli host.

Bacterial Proteins↗
PubMed1985

Trans- and cis-acting elements for the replication of P1 miniplasmids.

Replication-deficient mutants of the unit-copy miniplasmid lambda-P1:5R were isolated after hydroxylamine mutagenesis. Complementation tests showed that the majority of these mutants are defective in the production of the repA protein product. Two of these mutants have suppressible nonsense (amber) mutations. The DNA sequence of one of these, repA103, has been determined. The lesion lies within the repA open reading frame, showing that the repA product is essential for plasmid replication. Complementation of deletion mutants of lambda-P1:5R by repA protein showed that the origin of replication lies to the left of repA and that this 300-base-pair origin region is the only portion of the DNA essential for plasmid replication if repA protein is supplied in trans. Six of the 21 hydroxylamine-induced mutants were not complemented by repA. Replication of three of these could be restored by introduction into the plasmid of a wild-type origin region, suggesting that they were origin-defective. The DNA sequence of two mutants was determined. Mutant rep-11 has a 43-base-pair deletion within the incC sequence (incC is a series of five direct repeats of a 19-base-pair sequence known to be involved in the regulation of plasmid replication). The deletion appears to have been generated by homologous recombination between two repeats. Mutant rep-30 has a single base substitution in a region just to the left of incC that destroys one of five G-A-T-C (dam methylation) sites in this region. As lambda-P1:5R is unable to establish itself as a plasmid in a methylase-defective (dam-) strain, it seems probable that methylation of the G-A-T-C sequences is important for origin function. The incC region and the sequences to its left appear to constitute an essential part of the origin of replication.

Bacteriophages↗