ABSTRACT
Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation of cloned inserts can be determined. Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.
Subject(s)
Cloning, Molecular , DNA/genetics , Base Sequence , DNA Primers , Escherichia coli/genetics , Genetic Vectors , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
A hybrid gene containing the 5' sequence of a hamster histone H3 gene and the coding sequence of the bacterial neomycin-resistance gene (neo) was constructed. Upon transfection into the hamster fibroblast cell line K12, the hybrid gene exhibited cell cycle-dependent regulation, as evidenced by the maximal accumulation of the neo transcripts during synthesis of DNA in the cell cycle. In addition, cells arrested in the prereplicative phase, as a consequence of the K12 temperature-sensitive mutation, produced significantly less neo messenger RNA.
Subject(s)
Cell Cycle , Gene Expression Regulation , Histones/genetics , Animals , Cricetinae , DNA Replication , DNA, Recombinant , Drug Resistance , Neomycin/pharmacology , Poly A/genetics , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
Using two cDNA clones which encode hamster genes specifically induced by glucose starvation, we demonstrated that an 8- and 30-fold increase, respectively, in the transcription rates of these genes was coordinately effected by calcium ionophore A23187 treatment, resulting in a similar increase in the steady-state levels of their mRNAs. This response was observed within several hours of ionophore treatment in several mammalian cell types and appeared to be specifically mediated by A23187 but not by other ionophores in general. To define the regulatory sequence which mediates this Ca2+-induced response, we showed by gene transfection techniques that the 5' flanking sequence of a rat glucose-regulated gene contained the region for induction by A23187. The system reported here offers attractive features for the study of specific gene regulation by Ca2+.