A four-component system has been designed that makes it possible to prepare a double-stranded (ds) DNA fragment; one fragment end is predesigned (by the use of a class-IIS restriction enzyme and adapter-primer), and the other end corresponds to any normal restriction cut. The system is composed of the phage M13mp7 single-stranded (ss) target DNA; the Fok I restriction enzyme; an oligodeoxynucleotide adapter-primer, which permits one to introduce Fok I cuts at any specified site in the target DNA; and DNA polymerase, which converts the ss target into a ds form ready for cloning. In this system, the oligodeoxynucleotide adapter-primer serves several purposes. The 5' hairpin ds domain of the adapter-primer contains the Fok I recognition site. Its 3' ss domain selects a complementary site on the target ss DNA, hybridizes with it to form the ds cleavage site, and serves as a primer to convert the ss M13mp7 target to ds DNA.
SummaryMilk fat-coated microcapsules containingBrevibacterium linensand methionine were used to produce methanethiol, one of the volatile sulphur compounds implicated in Cheddar cheese flavour. Production of methanethiol from methionine occurred aerobically and anaerobically, but the production was 3- to 4-fold greater aerobically with most of the methanethiol being oxidized to dimethyl disulphide. About 35% of the total methanethiol was absorbed by the milk fat capsules and about 65% detected in the headspace. Levels of methanethiol began to decline at 26 °C after 24 h in milk fat-coated microcapsules. However, low temperature, such as 4–12 °C, stabilized levels of methanethiol in microcapsules over an 8-d analysis period. Optimum pH and temperature for methanethiol production were 8 and 26 °C respectively. The antioxidants butylated hydroxytoluene, butylated hydroxyanisole and ascorbic acid had negligible effects on methanethiol production.
Individually inactive N- and C-terminal fragments of the m5C-methyltransferase M.BspRI can complement each other resulting in specific, in vivo methylation of the DNA. This was shown by cloning the coding regions for N- and C-terminal parts of the enzyme in compatible plasmids and co-transforming them into E.coli cells. The enzyme could be detached at several different sites, producing either non-overlapping or partially overlapping fragments capable of complementation. Reconstitution of the active methyltransferase from inactive fragments was demonstrated in vitro, as well. Another GGCC-specific methyltransferase, M.BsuRI, showed a similar complementation phenomenon. Moreover, interspecies complementation was observed between appropriate fragments of the two closely related enzymes M.BspRI and M.BsuRI. Fragments of structurally and functionally more different methyltransferases were unable to complement each other.
The potential for microencapsulation of viable Brevibacterium linens with methionine or cysteine in milkfat to produce sulphur compounds was examined in this study. More than 80 per cent of B. linens cells were encapsulated and then numbers inside capsules increased about three-fold during 48 h at 26 degrees C under anaerobic conditions before a slow decline. Most of micro-organisms (7 x 10(7)/ml) were still viable in the capsules after 15 days. More than 90 per cent of cysteine and methionine were encapsulated and 90, 80 and 60 per cent of the encapsulated amino acids were maintained in the capsules at 4, 12 and 26 degrees C, respectively, after 24 h. Most of the cysteine was oxidized to cystine during microencapsulation but still available to micro-organisms whereas methionine remained in the reduced form. Partition coefficients of methionine and cysteine to milkfat were 0.117 and 0.275, respectively, indicating that most of these substrates would be available to cells of B. linens in the capsules.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.