Stringent control in the archaeal genus Sulfolobus.

Six Archaea belonging to the phylum Euryarchaeota were previously analyzed with respect to stringent control. Only one of the strains studied was shown to possess Bacteria-like stringent control over stable RNA accumulation; ppGpp and pppGpp production was totally lacking in all Archaea analyzed. To broaden our knowledge of stringent control in the Archaea, we examined here the accumulation of stable RNA and the production of ppGpp and pppGpp under amino acid starvation in three species of the genus Sulfolobus belonging to the Crenarchaeota, an archaeal phylum distant from the Euryarchaeota. In these species the accumulation of sRNA was arrested when aminoacylation of tRNA was inhibited by pseudomonic acid. Furthermore, stringent control of stable RNA accumulation was relaxed by some protein synthesis inhibitors that do not interfere with aminoacylation of tRNA, a feature typical of bacterial stringent control. Neither ppGpp nor pppGpp could be detected during growth or under amino acid starvation, and the intracellular GTP levels did not decrease in the course of the stringent response. These results show that: (1) stringency is widespread in wild-type thermoacidophilic archaea; (2) in the crenarchaeal species analyzed here SC depends on the deaminoacylation of tRNA; (3) in the strains analyzed ppGpp is not produced during normal growth nor during the stringent reaction; it is therefore not an effector either of SC over sRNA synthesis or of growth control. (p)ppGpp appears to be completely absent from the Archaea and thus constitutes an additional feature that distinguishes the Bacteria from the Archaea.

Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig.

A de novo SAC was constructed by making use of YAC technology and a humanized yeast strain. The construct (about 310 kb) contained pig centromeric DNA and the Neo gene. The construct was introduced into a pig cell line by yeast-mammalian cell fusion and G418 resistant clones were obtained. One clone was characterized by FISH and shown to contain an episomally located microchromosome containing YAC, Neo and pig centromere sequences. FISH analysis over time showed that the SAC was mitotically stable for at least 34 generations in the absence of selection. The size of the SAC was determined by confocal microscopy of the SAC and shown to be approximately 7 Mb, which is about 25-fold greater than the size of the original YAC. From its behavior in pulsed field gel electrophoresis, FISH analysis of stretched DNA fibers, and its appearance under scanning confocal microscopy, it was concluded that the SAC is a circularized and multimerized derivative of the original YAC. Possible applications as vectors for animal transgenesis are discussed.