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At least 19 recordsLinked to original sources
PubMed1979

Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids.

Hybrid plasmids were constructed from fragments of F'ara episomes formed by the restriction endonuclease EcoRI and a linear form of the plasmid ColE1 created by cleavage with EcoRI. Hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted. E. coli K12 host strains were constructed which contained different deletions of the ara region. The hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid. The initial differential rates of synthesis of L-arabinose isomerase, the product of the araA gene, were determined for the Ara+, plasmid containing strains. These studies demonstrated that strains containing delta(araOIBA)718 produce elevated levels of araC protein, suggesting the araC promoter has been altered by this deletion. Evidence is also presented which suggests that araC protein activates the ara-BAD operon to higher levels when it is present in cis rather than trans. Amplification of the products of the cloned genes is observed when compared to haploid levels in some cases.

Arabinose↗
PubMed1977

Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids.

Replication of the multicopy mini-R1 plasmid, Rsc11, is dependent on host replication functions dna A, B, C, E and G but independent of polA1. Chloramphenicol immediately stops its replication. A stable relaxation complex is not formed. Composite plasmids were constructed with Rsc11 and other small replicons like pSC101, ColE1 and mini-ColE1. In all combinations the amount of hybrid plasmid DNA in the cell never exceeds the amount of Rsc11 DNA itself. This leads to varying copy numbers of the hybrid plasmids depending on the size of the second plasmid. Replication of the composite plasmids proceeds probably always under the control of the Rsc11 part although the second replicon is still functional. The composite plasmids are incompatible with both the parent replicons.

Chloramphenicol↗
PubMed1977

E. coli phosphofructokinase synthesized in vitro from a ColE1 hybrid plasmid.

A hybrid plasmid, pLC 16-4, from the ColE1-DNA (E. coli) bank of Clarke and Carbon (1976) carrying pfkA was used to program an in vitro protein synthesis system from E. coli. Phosphofructokinase was the main product, as determined by enzyme assay, immunoprecipitation and gel electrophoresis. The enzyme was synthesized in vitro without added cAMP at a rate (enzyme/genome/h) ca. 30% the in vivo value, a higher efficiency than usually found in cell free systems. The plasmic molecular weight is ca. 16.10(6) daltons.

Coliphages↗
PubMed1976

[Transmissible hybrid plasmid RP4-ColE1].

Hybrid plasmid RP4-ColE1 was obtained by joining DNA plasmids RP4 and ColE1, each of which possessed only one site of restriction for EcoR1. These plasmid molecules were restricted by endonuclease EcoR1 and then treated by ligase. The hybrid plasmid retained the property of transmissibility typical for drug factor resistance RP4. Non-transmissible mutant of the hybrid plasmid selected by the character of the resistance of Escherichia coli C600 (RP4-ColE1) to the phage PRR1 is used for subsequent investigations.

Colicins↗
PubMed1977

Construction and restriction endonuclease mapping of hybrid plasmids containing Saccharomyces cerevisiae ribosomal DNA.

Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.

DNA↗
PubMed1979

[Hybrid plasmid pSD1 containing the immunity region of bacteriophage lambda].

Hybrid plasmid pSD1 carrying the immunity region of the coliphage lambda and bio operon have been obtained by means of studying the efficiency of transcription DNA fragments in the plasmid RSF2124. The molecular weight of this plasmid is 17.2 Md. The growth inhibition of phage lambdavir has been observed in cells carrying the new hybrid plasma. The properties of the plasmid pSD1 and probable reasons of the growth inhibition of phage lambdavir are discussed. The hybrid plasmid pSD2 carrying genes R, A and J of phage lambda has been constructed on the basis of the plasmid RSF2124. There are cohesive ends in this plasmid which make possible its packing in the phage lambda head. Hybrid plasmid pSD3 carrying genes P and Q of phage lambda has also been constructed.

Bacteriophage lambda↗
PubMed1976

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method.

The hybrid plasmid PBETAG, consisting of plasmid PMB9 DNA with an insert of rabbit globin DNA (about 600 base pairs) flanked by poly(dA) poly(dT) regions (Maniatis, T., Kee, S.G., Efstratiadis, A. and Kafatos, F.C. (1976) Cell 8, 163-182), was cleaved into two fragments by endonuclease S1 under conditions of partial denaturation. Only the smaller fragment (575 base pairs) contained globin-specific sequences, showing that excision had occurred in the A-T-rich regions. This method of cleavage provides a useful procedure for assessing the length of inserts in hybrid plasmids prepared by the poly(dA)-POLY(DT) tail method, and allows the preparative recovery of the insert.

Animals↗
PubMed1977

Properties of hybrid plasmids, consisting of parts of the mini-R1 factor Rsc11 and ColE1.

In vitro joining of the two small multicopy plasmids Rsc11 and ColE1 by a poly dAdT linker resulted in hybrid plasmids, which determine resistance to ampicillin and immunity to colicin E1. Isolation of the plasmid DNA from single colonies revealed that a variety of hybrid plasmids was formed. Cleavage of these plasmids with restriction endonucleases HinII, HindIII, EcoRI, SmaI and BamI and hybridization with ColE1 demonstrated that they contain different parts of the parent plasmids, Rsc11 and ColE1. Their copy number in the cell is between 6 and 15 per chromosome depending on the plasmid. None of these plasmids can replicate in polA mutants. Replication continues in the presence of chloramphenicol. This suggests that replication can only occur from the ColE1 origin and that the replication function of the Rsc11 part is lost. The hybrid plasmids are compatible with Rsc11 but not with ColE1. The comparison of the physical maps of these Rsc11--ColE1 hybrids with their functions allows a partial determination of the location of ampicillin resistance, replication and incompatibility on the Rsc11 genome.

Ampicillin↗
PubMed1977

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

A convenient procedure for the isolation of specific EcoRI-fragments of E. coli genome and their amplification on Km-resistance plasmid victor CK delta11 is described. The hybrid molecules were constructued in vitro using EcoRI-digestion, followed by ligation. Then appropriated E. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK delta11-arg+, CK delta11-his+, CK delta11-thr+ and CK delta11-leu+ containing loci of E. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of one EcoRI-fragment of initial plasmid CK delta11 and one respective specific portion of E. coli genome.

Arginine↗
PubMedDate not supplied

[Construction and molecular cloning of hybrid plasmids containing specific fragments of Escherichia coli DNA].

In the paper a convenient procedure for the isolation of specific Eco RI-fragments of E. coli genome and their amplification on Km-resistance plasmid vector CKdelta11 is described. Plasmid CKdelta11 contains Col E1 replicon and has only one Eco RI site. The hybrid molecules were constructed in vitro using Eco RI-digestion followed by ligation. Then appropriated E. coli strain (polyauxotrophic strain E. coli K12 AB 2463) was transformed with ligated DNA mixture and hybrid plasmids, containing arg, leu, his and thr chromosomal markers were selected by molecular cloning and isolated from the obtained E. coli clones. The hybrid plasmids have two Eco RI sites and consist of one Eco RI-fragment of initial plasmid CKdelta11 and one Eco RI-fragment of El coli DNA. The method described allows to isolate and amplify on hybrid plasmids DNA fragments, containing any selectable genes or genes adjacent to the selectable ones.

Chemical Phenomena↗
PubMed1978

Deletion plasmids from transformants of Pseudomonas aeruginosa trp cells with the RSF1010-trp hybrid plasmid and high levels of enzyme activity from the gene on the plasmid.

A RSF1010-trp hybrid plasmid which contained the tryptophan operon of Escherichia coli was introduced into Pseudomonas aeruginosa trp cells by transformation. From the Trp+ transformants several deletion plasmids were obtained, and their physical maps with restriction endonucleases were constructed. P. aeruginosa trp cells with these plasmids showed at first more than 100 times higher levels of tryptophan synthetase beta activity over that of the control P. aeruginosa wild-type cells, but these levels were drastically decreased by 1 week of successive transfers of cultures. This decrease in enzyme activity was found to be due to the change on the plasmids but not to the host cells. The production of E. coli tryptophan synthetase beta enzyme in P. aeruginosa cells was proved by immunological test.

Anthranilate Synthase↗
PubMed1979

Hybrid plasmids complement a putP mutation in Escherichia coli K12.

Clarke and Carbon have prepared a colony bank of 2000 Escherichia coli strains each containing a random segment of the Escherichia coli chromosome inserted into the EcoR1 restriction site of the plasmid ColE1. We have screened the colony bank by conjugation and have identified three strains bearing hybrid plasmids that complement a defective putP gene. Each of these strains shows increased L-proline uptake activity in comparison with the unmodified host or with the host bearing noncomplementing hybrid plasmids. However, CS520, the DNA source strain employed in constructing the hybrid plasmids, is a putP mutant. Since Escherichia coli possesses two L-proline porters, a variety of possible complementation mechanisms are discussed.

Escherichia coli↗
PubMed1979

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA).poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid. Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such at thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec- recipient strain of B. subtilis was used.

Bacillus subtilis↗
PubMed1975

Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome.

Using a poly(dA-dT) "connector" method, a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle containing one full-length molecule of poly(dT)-tailed DNA from E1 colicinogenic factor (Col E1) fragmented by EcoRI endonuclease annealed to any one of a collection of poly(dA)-tailed linear DNA fragments of the entire E. coli genome. This annealed, but unligated, hybrid DNA was used to transform several different auxotrophic mutants of E. coli, and by direct selection, bacterial clones were isolated which contained specific hybrid plasmids. In this manner, bacterial strains containing Col E1 hybrid plasmids carrying the entire tryptophan operon or the arabinsoe and leucine operons were isolated. The methods described should allow the molecular cloning of any portion of the E. coli genome by selection from a pool of DNA molecules containing at least several hundred different hybrids representing the entire bacterial genome.

Arabinose↗
PubMed1979

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid.

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.

Alleles↗
PubMed1978

Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein.

The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes.

Base Sequence↗