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At least 667 records · Page 37Linked to original sources
PubMed2001

Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1.

The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family. Among the proteic killer genes described so far, hig is unique in that the toxin gene (higB) exists upstream of the antidote gene (higA). There are two promoters in the hig locus, Phig and PhigA, and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins. In this study, we purified HigA protein by means of GST fusion. The electrophoretic mobility shift assay using the purified protein revealed that HigA specifically bound to the Phig region, but not to PhigA. The HigA-binding sequence was determined by DNase I footprinting assay to be a 56-bp sequence that completely covered the -35 and -10 boxes of Phig. The presence of two inverted repeats in the binding sequence and the identification of a dimer form of HigA by cross-linking experiment suggested that the protein bound to the Phig region as a dimer. HigB was purified as a GST fusion protein as well, though it was achieved only in the presence of HigA. HigA and GST-HigB formed a highly stable complex where the two proteins were present in an equimolar ratio.

Amino Acid Sequence↗
PubMed1991

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

Base Sequence↗
PubMed1983

Plasmid transformation in Bacillus subtilis. Effects of the insertion of Bacillus subtilis rRNA genes into plasmids.

Two HindIII generated DNA fragments of 3.0 and 2.3 Kb derived from rRNA genes of B. subtilis were cloned in E. coli with pBR322. The 3.0 Kb fragment could be subcloned in B. subtilis using pC194. However, only the multimeric, but not the monomeric derivatives of this hybrid plasmid were active in transformation of B. subtilis cells. The 2.3 Kb fragment could neither be subcloned in pC194 nor in pPL603, using both cell and protoplast transformation. We attribute the nonclonability of the 2.3 Kb fragment in B. subtilis to the presence of strong promoter activity in this fragment. Direct proof for the presence of a strong promoter, which is apparently responsible for the transcription of the rRNA gene, was obtained in experiments with B. subtilis and E. coli promoter search plasmids.

Bacillus subtilis↗
PubMed1984

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer.

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.

Bacillus thuringiensis↗
PubMed2001

Periodic solutions in a model of competition between plasmid-bearing and plasmid-free organisms in a chemostat with an inhibitor.

We obtain necessary and sufficient conditions on the existence of a unique positive equilibrium point and a set of sufficient conditions on the existence of periodic solutions for a 3-dimensional system which arises from a model of competition between plasmid-bearing and plasmid-free organisms in a chemostat with an inhibitor. Our results improve the corresponding results obtained by Hsu, Luo, and Waltman [1].

Anti-Bacterial Agents↗
PubMed1984

A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1.

Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product. The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E. coli oriC consensus sequence. Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number. par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.

Autoradiography↗
PubMed1980

Plasmids controlled exclusion of the K2 killer double-stranded RNA plasmid of yeast.

Saccharomyces strains of two types (K1+R1+ and K2+R2+) kill each other and K-R--sensitive strains by secreting protein toxins. K1 killer strains carry a 1.25 X 10(6) dalton double-stranded RNA plasmid, [KIL-k1], while K2 killers have a 1.0 X 10(6) dalton double-stranded RNA plasmid, [KIL-k2]. Mating [KIL-k1] haploids with [KIL-k2] haploids yields only [KIL-k1] diploids, that is, [KIL-k1] excludes [KIL-k2]. [EXL], a new non-Mendelian genetic element from a nonkiller strain, excludes [KIL-k2] but does not exclude [KIL-k1]. A second new non-Mendelian genetic element, called [NEX], when present prevents [EXL] from excluding [KIL-k2]. [NEX] does not prevent [KIL-k1] or [KIL-s1] (a suppressive mutant of [KIL-k1]) from excluding [KIL-k2]. A chromosomal gene, called MKT1, is needed for maintenance of [KIL-k2] if [NEX] is present. In the absence of [NEX], [KIL-k2] does not need MKT1. [KIL-k1] does not need MKT1 even if [NEX] is present. [EXL] replication depends on at least the products of MAK1, MAK3, MAK10 and PET18. [NEX] replication depends on MAK3 but is independent of MAK4, MAK6, MAK27 and MKT1.

Crosses, Genetic↗
PubMed1983

The origin of replication of plasmid p15A and comparative studies on the nucleotide sequences around the origin of related plasmids.

Replication of Escherichia coli plasmid p15A was examined by use of a cell extract or a mixture of three purified E. coli enzymes: RNA polymerase; RNAase H; and DNA polymerase I. In each system, replication initiates at any of three consecutive nucleotides located at a unique site. Primer transcription starts 508 bp upstream of the replication origin. The region between 294 and 524 bp upstream of the origin determines the incompatibility property. This region specifies an RNA (RNA I) of about 105 nucleotides that is involved in regulation of primer formation. We compare the nucleotide sequences around the origins of related plasmids p15A, ColE1, pBR322, RSF1030 and CloDF13, and discuss the significance of possible RNA secondary structures in primer formation.

Base Sequence↗
PubMed1984

A molecular characterization of H. influenzae plasmid pVe445: a comparison with N. gonorrhoeae plasmids pNG10 and pNG18.

Haemophilus influenzae plasmid pVe445 (4.6 MDa) has been compared with Neisseria gonorrhoeae plasmids pNG18 (3.3 MDa) and pNG10 (4.6 MDa) by heteroduplex and restriction enzyme analysis. This study demonstrates that pVe445 differs from pNG18 only that it carries an additional 1.3-MDa DNA fragment between coordinates 0.9 and 2.2. Heteroduplex studies demonstrated that pNG10 and pVe445 are indistinguishable whereas restriction endonuclease analysis indicated minor differences in the distribution of AluI sites.

DNA Restriction Enzymes↗
PubMed1988

Map location on Agrobacterium root-inducing plasmids of homologies with the virulence region of tumor-inducing plasmids.

Southern-type hybridizations were carried out in order to identify sequence homologies with the pTi vir loci, on an agropine-type plasmid (pRiHRI) and a mannopine-type plasmid (pRi8196) of Agrobacterium rhizogenes. The localization of the sequences hybridizing with subcloned fragments containing vir A, B, G, C, and D from pTiAch5 indicated a similar linear organization of the pTi vir loci and their homologies on pRiHRI and pRi8196, though no homology was detected on both pRi with a 1.1-kb internal fragment of virD. No homology was detected either with the vir E locus on pRiHRI vir region, nor with the virF locus on both pRi vir regions. As on nopaline pTiC58, fragments bearing the homologies with virC and virG are closer together on both pRi than on octopine pTiAch5. A preliminary functional map of the pRiHRI vir region is deduced from this study.

DNA Restriction Enzymes↗
PubMed1990

The sequences of genes bordering oriT in the enterotoxin plasmid P307: comparison with the sequences of plasmids F and R1.

The nucleotide sequences of the enterotoxin plasmid P307 transfer genes traM, finP, traJ, traY, and gene 19 were determined. Gene 19 is highly conserved; its product is very similar to that coded by the F and R1 plasmids. The TraM protein is similar in P307 and in F; the R1 sequence shows differences in the 40 N-terminal amino acids. The traJ product is very different in P307, F, and R1. The traY gene from P307, which in F is almost twice as long, is similar in size to that from R1. The finP RNA shows a high degree of homology with that from R1 and F, except for the two loop regions where base changes were observed. The genes coding for proteins, except traY, could be expressed in minicell- and T7 promoter-driven expression systems, whereas traJ and gene 19 could be expressed only in the latter system.

Amino Acid Sequence↗
PubMed1991

Insertion of Tn916 into Bacillus pumilus plasmid pMGD302 and evidence for plasmid transfer by conjugation.

As part of an effort to develop systems for genetic analysis of strains of Bacillus pumilus which are being used as a microbial hay preservative, we introduced the conjugative Enterococcus faecalis transposon Tn916 into B. pumilus ATCC 1 and two naturally occurring hay isolates of B. pumilus. B. pumilus transconjugants resistant to tetracycline were detected at a frequency of approximately 6.5 x 10(-7) per recipient after filter mating with E. faecalis CG110. Southern hybridization confirmed the insertion of Tn916 into several different sites in the B. pumilus chromosome. Transfer of Tn916 also was observed between strains of B. pumilus in filter matings, and one donor strain transferred tetracycline resistance to recipients in broth matings at high frequency (up to 3.4 x 10(-5) per recipient). Transfer from this donor strain in broth matings was DNase-resistant and was not mediated by culture filtrates. Transconjugants from these broth matings contained derivatives of a cryptic plasmid (pMGD302, approx 60 kb) from the donor strain with Tn916 inserted at various sites. The plasmids containing Tn916 insertions transferred to a B. pumilus recipient strain at frequencies of approx 5 x 10(-6) per recipient. This evidence suggests that pMGD302 can transfer by a process resembling conjugation between strains of B. pumilus.

Bacillus↗
PubMed1988

The origin of conjugative IncP plasmid transfer: interaction with plasmid-encoded products and the nucleotide sequence at the relaxation site.

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication we isolated and dissected the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. Essential features of oriT are conserved: symmetric sequence repeats, the nic site and a pair of potential promoter sites that allow for divergent transcription of two tra operons. The relaxation nick and the end of a 19 bp inverted repeat are interspaced by eight basepairs. The 5'-terminal nucleotide at the nick is modified by an alkali-resistant residue and the 3'-nucleotide is accessible to extension by DNA polymerase I. Transfer gene products essential for the formation of the initiation complex (relaxosome) of conjugative DNA synthesis map adjacent to oriT. Two of these products, TraJ and TraK confer specificity to their homologous oriT exclusively. Proteins TraJ and TraK are the only components of the RP4 and R751 transfer machinery which cannot be interchanged. TraJ and at least two additional plasmid-encoded products are necessary for specific relaxation. The purified TraJ protein of RP4 possesses oriT-binding ability. The recognition sequence contains a palindromic sequence located within the right arm of the 19 bp inverted repeat. The TraJ binding site and the nic site are located on one side of the DNA double helix. We presume that this nucleoprotein structure is the initial complex in the pathway to the assembly of functional relaxosomes.

Bacterial Proteins↗
PubMed1994

Transpogenes: the transposition-like integration of short sequence DNA into the yeast 2 micron plasmid creates the STB locus and plasmid-size polymorphism.

The type-2 2 mu plasmid of industrial yeast strains exhibits extensive size polymorphism in the STB (plasmid stability) locus and IR (inverted repeat)-right region. Comparative DNA sequence analyses of STB alleles identified a 38-bp sequence flanked by a 25-bp direct repeat as the underlying structural motif. Variable unequal recombination within the direct repeat accounted for the observed polymorphism of STB alleles. IR-right polymorphism was observed to result from tandem duplication of a 22-bp sequence flanked by a 9-bp direct repeat. The flanking direct repeats marked both loci as originating from the transposition-like integration of short DNA fragments. We call these structures transpogenes and note that these are hybrid structures of host and foreign DNA which can evolve into functional loci.

Base Sequence↗
PubMed1989

Application of an asymmetrical flow field-flow fractionation channel to the separation and characterization of proteins, plasmids, plasmid fragments, polysaccharides and unicellular algae.

The asymmetrical flow field-flow fractionation channel has been improved by introduction of a different sample loading method, downstream central injection, which reduces sample relaxation and focusing time considerably and allows the concentrations of material in the channel, thereby enabling the loading of very large sample volumes. The performance of the channel was demonstrated by the separation of a protein from its dimer, retention of nucleic acids, i.e., plasmids and enzymatically cleaved plasmids, hyaluronate and unicellular algae.

Chemical Fractionation↗