Gene amplification and analysis最新文献

A plasmid, pJL6, was constructed that contains a unique Cla I site 12 codons beyond the bacteriophage lambda cII gene initiation codon, as well as an adjacent unique Hind III site. These sites allowed us to fuse the sequences from the avian myelocytomatosis virus (MC29) v-myc gene, the avian myeloblastosis virus (AMV) v-myb gene, and the Harvey murine sarcoma virus (Ha-MuSV) v-ras gene to the amino-terminal portion of the cII gene. Transcription of the hybrid genes is controlled from the lambda PL promoter. When this promoter is derepressed, E. coli cells harboring the chimeric plasmid produce levels of fusion proteins that amount to over 5% of total cellular protein. Antibodies raised by the cII-myc fusion protein form an immunoprecipitate with the MC29 gene product, P110gag-myc. The cII-ras fusion protein is precipitated by monoclonal antibodies directed toward the Ha-MSV p21ras, binds GDP, and is capable of autophosphorylation.

This chapter describes a gene expression system of E. coli that contains a portable Shine-Dalgarno region. Transcription of this system is directed by a hybrid promoter derived from trp and lac-UV5 promoter sequences, which is followed by a region that encodes the portable Shine-Dalgarno region. We used a series of synthetic portable Shine-Dalgarno regions to vary the length (from 4 to 13 bases) of the Shine-Dalgarno by increasing the number of bases on the mRNA that are complementary to the 3' end of 16s rRNA. Increase in the Shine-Dalgarno region to 8 or 13 bases decreased the translation efficiency of the chimeric leukocyte interferon messenger by 40%. In another series of portable Shine-Dalgarno regions, we varied the four bases that follow the Shine-Dalgarno region. The presence of four A residues or four T residues in this region resulted in the highest translation efficiency. The presence of four C residues reduced translation efficiency by 50%, compared with A or T residues. The presence of four G residues after the Shine-Dalgarno region lowered translation efficiency by 75%, compared with A or T residues.

We have presented the results of our studies of the expression of the CYC genes from plasmids. All our data indicate that the levels of expression and the regulation of expression are very similar for the plasmid-borne genes and the chromosomal genes when care is taken to construct the appropriate plasmids. The usefulness of these plasmids has been demonstrated: mutations affecting regulatory sites adjacent to genes of interest have been constructed [such as the Xho I deletion and inversion in the YCpCYC1(2.4) plasmid] and selected [as in the case of the IS1 insertion into the YCpCYC7(2) plasmid], and these mutations have led us to some tentative conclusions about the location and nature of the regulatory sites of these genes. Furthermore, transformation with plasmids containing modified genes or fusions has permitted isolation of genomic regulatory mutants, as in the selection of lac+ suppressors of the lac- CYC1 1/x inversion carried on the YCpCYC1(2.4) 1/x plasmid. Although we cannot rule out the possibility that use of plasmids might cause us to miss a class of regulatory effects that can be propagated only along a chromosomal structure, we believe that the regulatory effects that we do observe can be more quickly and completely defined by working with plasmids. If any regulatory effects occur only on chromosomes, they can be studied more easily once the basic regulatory phenomena have been analyzed. The regulatory regions of the CYC1, CYC7, and TR2 genes that we have crudely mapped so far all exert their effects 100-300 bp away from the putative transcriptional starting sites. How the information in these regions is transmitted along the DNA is an intriguing question. We are engaged in a mutational analysis of these sites to locate them more precisely, to map second-site mutations that moderate the effects of the original mutations, to obtain genomic mutations that define the genes whose products interact with these sites, and to test combinations of genomic and plasmid mutations to define the sites with which regulatory elements interact. This approach should aid our understanding of the spatial relationships between yeast regulatory sites and transcriptional signals. Ultimately, obtaining mutations in regulatory genes, such as the mutations described here for the anaerobic regulation of TR2, will allow the cloning of these genes by complementation. This will lead to the isolation of the protein encoded and ultimately to an approach to the molecular mechanism of regulation through study of protein-DNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)