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S D Ehrlich

Publications and source records attributed to S D Ehrlich.

At least 199 records · Page 11Linked to original sources
PubMed1985

Saccharomyces cerevisiae ARS on a plasmid from Staphylococcus aureus.

Staphylococcus aureus plasmid pC194 carries three sequences closely related to a consensus sequence defined previously by analysis of different genetic elements which replicate autonomously in yeast Saccharomyces cerevisiae. Two of these enable the plasmid to replicate in yeast, the third does not. A new consensus sequence A/T T T T A T R T T T, 1 bp shorter than the previous one, can be deduced from our results. Replacement of the T with G at the position 9 of the sequence abolishes its activity. The presence of the two active sequences on pC194 genome can be explained by the A + T-rich base composition of the plasmid.

Base Sequence↗
PubMed1985

Stable gene amplification in the chromosome of Bacillus subtilis.

We constructed five different structures, consisting of a genetic marker flanked by directly repeated sequences 2-4 kb long, in the Bacillus subtilis chromosome. When a selective pressure was applied amplification of the marker and one of the repeats was observed in all cases. Amplification was not detected with two markers which were not flanked by the repeated sequences. The maximum amplification level observed with the different structures varied between 5 and 50. The size of the most amplified structure corresponded to 7.5% of the chromosome. Amplification was stable upon growth of cells under non-selective conditions. Each copy of an amplified gene was expressed with equal efficiency. These results indicate that chromosomal gene amplification may be useful for constructing genetically engineered B. subtilis strains.

Bacillus subtilis↗
PubMed1985

Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements.

Linear DNA molecules composed of a central region nonhomologous with the Bacillus subtilis chromosome and two flanking regions homologous with the chromosome can integrate into the chromosome, provided that the homologous regions have the same relative orientation. The resulting chromosome can be maintained in a haploid or in a merodiploid cell together with a parental chromosome. This can most easily be explained by supposing that the integration occurs by crossing over at each homologous region and that a part of the chromosome between these regions is deleted and replaced by the central nonhomologous region of the integrating molecule. If no essential genes were replaced during that process a haploid cell would be obtained; if essential genes were replaced a merodiploid cell would be obtained. The use of appropriate linear molecules therefore should allow the induction of deletions, extending from a given chromosomal site in a predetermined direction, and defined duplications in the B. subtilis chromosome.

Bacillus subtilis↗
PubMed1984

Recombination efficiency is a quadratic function of the length of homology during plasmid transformation of Bacillus subtilis protoplasts and Escherichia coli competent cells.

Recombination efficiency between transforming and resident plasmids was 100 times higher in Bacillus subtilis protoplasts than in Escherichia coli competent cells. In both systems it varied with the square of the length of the sequences common to two plasmids within the studied range 0.96-8.8 kb in B. subtilis, 0.72-3.9 kb in E. coli. The observed exponential dependence may be characteristic of recombination occurring within a relatively short interval bordered by relatively long heterologous regions.

Bacillus subtilis↗
PubMed1984

Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage.

Escherichia coli plasmids pBR313 and pBR322 were transduced by phage M13 with low efficiency (10(-8) transductants/phage). Hybrid plasmids pHV12 or pHV33, composed of Staphylococcus aureus plasmid pC194 and pBR313 or pBR322, respectively, were transduced much more efficiently (10(-4) transductants/phage). Inactivation of either of the two zones necessary for pC194 replication, one coding for a protein, the other not, reduced the transforming efficiency of hybrids to the level of pBR322. Activity of the pC194 replication region was not necessary for the formation of chimeras between M13 and the transduced plasmid in the donor cells, but rather for the establishment of the plasmid in the recipient cells.

Amino Acid Sequence↗
PubMed1984

Genes involved in transitory recombination between phage M13 and plasmid pHV33.

Plasmid pHV33 and phage M13 combine in Escherichia coli cells to form a chimera, which decombines to regenerate two parental genomes. Combination can occur via two genetic pathways, one defined by the recBC genes, the other by recA, recF and possibly recL genes. Decombination can also occur via two pathways, one defined again by the recBC genes, the other by a gene not identified, but active only in the absence of the recL gene product.

Bacterial Proteins↗
PubMed1983

Transitory recombination between plasmid pHV33 and phage M13.

Plasmid pHV33 and phage M13 which have no homology exceeding 13 bp, combine in Escherichia coli cells. The chimeric genome is encapsidated in phage proteins and injected into a recipient cell, where it decombines to regenerate the two parental genomes. We call this combination-decombination process 'transitory recombination'.

Chimera↗
PubMed1983

Intermolecular recombination during transformation of Bacillus subtilis competent cells by monomeric and dimeric plasmids.

Bacillus subtilis competent cells harboring plasmid pUB110 were transformed by plasmids unable to replicate in this host but carrying segments of pUB110, 260 to 4500 bp long. Recombinants between the incoming and the resident plasmids were found in the transformed cells. Transforming efficiency of the incoming plasmids depended strongly on their molecular form and the length of their region homologous with the resident plasmid. It increased with the fourth to fifth power of that length for monomers having at least 900 bp of homology. Activity of monomers having less than 900 bp homology was too low to be measured in our experiments. Transforming efficiency of dimers was much greater than that of monomers, and varied with the square of the length of the homologous region. These results indicate that dimeric and monomeric plasmid molecules are processed differently during transformation of B. subtilis competent cells.

Bacillus subtilis↗
PubMed1983

Inversion in the lactose region of Escherichia coli K-12: inversion termini map within IS3 elements alpha 3 beta 3 and beta 5 alpha 5.

In this work, the previously described inversion in the lactose region of the Escherichia coli K-12 chromosome (D. J. Savić, J. Bacteriol. 140:311-319, 1979) is analyzed in greater detail. The results presented indicate that the inversion most likely occurred by a homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements.

Chromosome Inversion↗
PubMed1982

Plasmids from Staphylococcus aureus replicate in yeast Saccharomyces cerevisiae.

It is known that some plasmids, such as RP4, can replicate in many Gram-negative bacteria. Certain small Staphylococcus aureus plasmids have an even broader host range, being able to replicate in not only phylogenetically distant Gram-positive bacteria such as Bacillus subtilis or Streptococcus pneumoniae, but also in the Gram-negative bacterium Escherichia coli. Here we have examined whether these plasmids can also replicate in a lower eukaryote, the yeast Saccharomyces cerevisiae. For this purpose we constructed hybrids between a S. aureus plasmid pC194 and an E. coli plasmid YIp5, which carries a ura-3 gene easy to select for in yeast but cannot replicate in this host. We found that the hybrids transformed yeast with high efficiency (as did hybrids between YIp5 and three other S. aureus plasmids); were maintained extrachromosomally in yeast; and were not modified during residence in yeast. We conclude from this evidence that S. aureus plasmids can replicate in yeast, which raises the questions of whether the replication signals used by prokaryotes and eukaryotes are similar, and how far up the phylogenetic tree the organisms still able to be hosts to S. aureus plasmids may be.

Base Sequence↗
PubMed1982

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.

Bacillus subtilis↗
PubMed1982

Recombination between short direct repeats in a recA host.

Spontaneous deletion derivatives of plasmid pHV14, a recombinant of pBR322, have been isolated in the recA strain HB101. About 2.5 kilobases (Kb) of DNA has been lost in each of the four plasmids described including, in two cases the region involved in control of copy number and the initiation of replication. Sequence analysis of each deletion junction has revealed that in 3 out of 4 cases the deletion event occurred by recombination between short direct repeats of as little as 7 base pairs (bp).

Adenosine Triphosphatases↗
PubMed1982

Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning.

The plasmid pHV32, which replicates in Escherichia coli but not in Bacillus subtilis, transformed B. subtilis-competent cells efficiently when linked in vitro to EcoRI B. subtilis DNA segments. The transformed clones carried pHV32 inserted in their chromosomes, and often displayed a mutant phenotype. One of the transformed clones carried pHV32 inserted close to the thyB gene. We cleaved the DNA extracted from this clone with BglII restriction endonuclease, for which no sites exist on pHV32, ligated the released segments and used them to transform E. coli selecting for pHV32-carried genetic markers. The transformants harbored a hybrid plasmid which carried the B. subtilis thyB gene. Circular molecules composed of pHV32 joined to B. subtilis DNA inserted into the chromosome by a Campbell-like recombination event. Linear molecules, in which pHV32 was flanked by two non-adjacent DNA segments, underwent a double cross-over recombination with the chromosome. In this case the chromosomal sequences between the non-adjacent segments were deleted, and replaced by pHV32 sequences.

Bacillus subtilis↗
PubMed1980

DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors.

Random segments of Bacillus amyloliquefaciens and yeast Saccharomyces cerevisiae DNA were used to determine two parameters pertinent to cloning in Bacillus subtilis, the yield of hybrids and the mean size of cloned segments. 10(3) to 10(4) hybrids/micrograms of DNA segments were obtained. Hybrids represented 11--18% of transformants. Mean m. wt. of cloned DNA segments was about 1 x 10(6), substantially lower than 3 x 10(6) found for donor DNAs after digestion with restriction endonucleases. We have cloned a B. amyloliquefaciens DNA segment which complemented a deficiency in B. subtilis hisH and E. coli hisC genes, which encode imidazolylacetolphosphate aminotransferase. The cloning efficiency for this gene was 10 transformed hosts/micrograms of donor DNA. Several B. subtilis insertional-inactivation cloning vectors were examined. One, pHV41, allows inactivation of the kanamycin-resistance (KmR) gene by insertion into its unique Bg/II site. In two other vectors, pHV11 and pHV23, insertion in their unique Kpn site inactivates the tetracycline-resistance (TcR) gene. pHV23 replicates both in E. coli and B. subtilis, and carries unique sites for seven restriction endonucleases (BamHI, EcoRI, HpaI, KpnI, PstI, SalI, XbaI). This makes it one of the most versatile B. subtilis cloning vectors yet described.

Bacillus↗