Differential incorporation of biotinylated nucleotides by terminal deoxynucleotidyl transferase.
Explore the source record for details and available documents.
Base Sequence↗
Biomedical subjects
J L Flickinger
Publications and source records attributed to J L Flickinger.
Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.
A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.
Bacteriophages↗
Evidence that Lactobacillus casei insertion element ISL1 has a narrow host range.
The 1.3-kilobase-pair insertion element ISL1, originally isolated from Lactobacillus casei S-1, was found to have an extremely restricted host range. By DNA-DNA hybrizations performed with Southern transfers by using a cloned internal fragment of ISL1 as a molecular probe, it was found that only 3 of 19 L. casei strains examined contained sequences that hybridized to the ISL1 probe. In two of these, the hybridizing sequences were found on lactose plasmids. No homologous sequences were detected in a survey of 14 other Lactobacillus strains (9 species) and 15 strains of other bacteria (8 genera, 12 species).
Blotting, Southern↗
Protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum.
The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens alpha-amylase gene (amyEBamP) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody.
Bacillus↗