Biomedical subjects

Lynn C Thomason

Publications and source records attributed to Lynn C Thomason.

4 recordsLinked to original sources

PubMed2004

In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides.

We demonstrate that the bacteriophage lambda Red functions efficiently recombine linear DNA or single-strand oligonucleotides (ss-oligos) into bacteriophage lambda to create specific changes in the viral genome. Point mutations, deletions, and gene replacements have been created. While recombineering with oligonucleotides, we encountered other mutations accompanying the desired point mutational change. DNA sequence analysis suggests that these unwanted mutations are mainly frameshift deletions introduced during oligonucleotide synthesis.

Bacteriophage lambda↗

PubMed2004

Identification of the Escherichia coli K-12 ybhE gene as pgl, encoding 6-phosphogluconolactonase.

We report identification of the Escherichia coli ybhE gene as the pgl gene that encodes 6-phosphogluconolactonase. A tentative identification was first made based on the known approximate location of the pgl gene and the similarity of the presumptive ybhE-encoded protein sequence to a known Pgl enzyme. To test this notion, the ybhE gene was deleted and replaced with a drug marker. Like previously characterized pgl mutants, the ybhE deletion mutant had a Blu- phenotype (dark-blue staining with iodine due to accumulation of starch after growth on minimal maltose) and demonstrated impaired growth on minimal glucose medium when combined with a pgi mutation. Biochemical assay of crude extracts for 6-phosphogluconolactonase enzymatic activity showed that ybhE encodes this activity. The ybhE gene was transferred from the E. coli chromosome to an expression vector. This ybhE clone complemented both the precise deletion of the ybhE gene and a larger deletion, pglDelta8, for the Blu- phenotype and for phosphogluconolactonase activity, confirming that ybhE is the pgl gene. A newly observed phenotype of pgl strains is a lowered frequency of appearance of Bgl+ mutants that can utilize the beta-glucoside salicin. This is likely due to poor growth of Bgl+ pgl strains on salicin due to the accumulation of 6-phosphogluconolactone.

Carboxylic Ester Hydrolases↗

PubMed2002

Genetic engineering using homologous recombination.

In the past few years, in vivo technologies have emerged that, due to their efficiency and simplicity, may one day replace standard genetic engineering techniques. Constructs can be made on plasmids or directly on the Escherichia coli chromosome from PCR products or synthetic oligonucleotides by homologous recombination. This is possible because bacteriophage-encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 50 base pairs. This technology, termed recombineering, is providing new ways to modify genes and segments of the chromosome. This review describes not only recombineering and its applications, but also summarizes homologous recombination in E. coli and early uses of homologous recombination to modify the bacterial chromosome. Finally, based on the premise that phage-mediated recombination functions act at replication forks, specific molecular models are proposed.

Bacteriophage lambda↗

PubMed2002

Gene products encoded in the ninR region of phage lambda participate in Red-mediated recombination.

BACKGROUND: The ninR region of phage lambda contains two recombination genes, orf (ninB) and rap (ninG), that were previously shown to have roles when the RecF and RecBCD recombination pathways of E. coli, respectively, operate on phage lambda. RESULTS: When lambda DNA replication is blocked, recombination is focused at the termini of the virion chromosome. Deletion of the ninR region of lambda decreases the sharpness of the focusing without diminishing the overall rate of recombination. The phenotype is accounted for in large part by the deletion of rap and of orf. Mutation of the recJ gene of the host partially suppresses the Rap- phenotype. CONCLUSION: ninR functions Orf and Rap participate in Red recombination, the primary pathway operating when wild-type lambda grows lytically in rec+ cells. The ability of recJ mutation to suppress the Rap- phenotype indicates that RecJ exonuclease can participate in Red-mediated recombination, at least in the absence of Rap function. A model is presented for Red-mediated RecA-dependent recombination that includes these newly identified participants.

Bacterial Proteins↗