Abstract
A plasmid was constructed that allows the selection in vivo of gene fusions between the Escherichia coli beta-galactosidase gene and the yeast (Saccharomyces cerevisiae) URA3 gene. A large yeast DNA fragment containing the URA3 gene was placed upstream of an amino-terminally deleted version of the lacZ gene. The plasmid vehicle contains sequences that allow selection and maintenance of the plasmid in both yeast and E. coli. Selection for Lac+ in E. coli yielded numerous deletions that fused the lacZ gene to the URA3 gene and flanking yeast sequences, to the bacterial tetracycline-resistance gene from the parent plasmid pBR322, and to the yeast 2-micrometer plasmid DNA. Some of these fusion plasmids produced beta-galactosidase activity when introduced into yeast. One of the fusions to the URA3 gene itself has been shown to place the expression of beta-galactosidase activity under uracil regulation in yeasts.
Full text
PDF![2460](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/89ca/319366/4fd1a657f83a/pnas00655-0503.png)
![2461](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/89ca/319366/c5280c87b139/pnas00655-0504.png)
![2462](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/89ca/319366/c41b471939f1/pnas00655-0505.png)
![2463](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/89ca/319366/8408d720bb51/pnas00655-0506.png)
![2464](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/89ca/319366/d4dbd113ab29/pnas00655-0507.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bach M. L., Lacroute F., Botstein D. Evidence for transcriptional regulation of orotidine-5'-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jan;76(1):386–390. doi: 10.1073/pnas.76.1.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [DOI] [PubMed] [Google Scholar]
- Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Brickman E., Silhavy T. J., Bassford P. J., Jr, Shuman H. A., Beckwith J. R. Sites within gene lacZ of Escherichia coli for formation of active hybrid beta-galactosidase molecules. J Bacteriol. 1979 Jul;139(1):13–18. doi: 10.1128/jb.139.1.13-18.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Broach J. R., Atkins J. F., McGill C., Chow L. Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2mu circle. Cell. 1979 Apr;16(4):827–839. doi: 10.1016/0092-8674(79)90098-9. [DOI] [PubMed] [Google Scholar]
- Broach J. R., Hicks J. B. Replication and recombination functions associated with the yeast plasmid, 2 mu circle. Cell. 1980 Sep;21(2):501–508. doi: 10.1016/0092-8674(80)90487-0. [DOI] [PubMed] [Google Scholar]
- Cameron J. R., Philippsen P., Davis R. W. Analysis of chromosomal integration and deletions of yeast plasmids. Nucleic Acids Res. 1977;4(5):1429–1448. doi: 10.1093/nar/4.5.1429. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
- Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J. Regulation of the regulatory gene for the arabinose pathway, araC. J Mol Biol. 1976 Jul 5;104(3):557–566. doi: 10.1016/0022-2836(76)90120-0. [DOI] [PubMed] [Google Scholar]
- Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
- Chevallier M. R., Bloch J. C., Lacroute F. Transcriptional and translational expression of a chimeric bacterial-yeast plasmid in yeasts. Gene. 1980 Oct;11(1-2):11–19. doi: 10.1016/0378-1119(80)90082-7. [DOI] [PubMed] [Google Scholar]
- Chou J., Lemaux P. G., Casadaban M. J., Cohen S. N. Transposition protein of Tn3: identification and characterisation of an essential repressor-controlled gene product. Nature. 1979 Dec 20;282(5741):801–806. doi: 10.1038/282801a0. [DOI] [PubMed] [Google Scholar]
- Clarke L., Carbon J. Isolation of the centromere-linked CDC10 gene by complementation in yeast. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2173–2177. doi: 10.1073/pnas.77.4.2173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franklin N. C. Genetic fusions for operon analysis. Annu Rev Genet. 1978;12:193–221. doi: 10.1146/annurev.ge.12.120178.001205. [DOI] [PubMed] [Google Scholar]
- Guarente L., Ptashne M. Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2199–2203. doi: 10.1073/pnas.78.4.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jund R., Lacroute F. Regulation of orotidylic acid pyrophosphorylase in Saccharomyces cerevisiae. J Bacteriol. 1972 Jan;109(1):196–202. doi: 10.1128/jb.109.1.196-202.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kenyon C. J., Walker G. C. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A. 1980 May;77(5):2819–2823. doi: 10.1073/pnas.77.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Komeda Y., Iino T. Regulation of expression of the flagellin gene (hag) in Escherichia coli K-12: analysis of hag-lac gene fusions. J Bacteriol. 1979 Sep;139(3):721–729. doi: 10.1128/jb.139.3.721-729.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lacroute F. Regulation of pyrimidine biosynthesis in Saccharomyces cerevisiae. J Bacteriol. 1968 Mar;95(3):824–832. doi: 10.1128/jb.95.3.824-832.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lapeyre J. N., Beattie W. G., Dugaiczyk A., Vizard D., Becker F. F. EcoRI-generated reiterated components of the rat genome. I. Sequence of two (92 and 93 bp) related DNA fragments. Gene. 1980 Sep;10(4):339–346. doi: 10.1016/0378-1119(80)90154-7. [DOI] [PubMed] [Google Scholar]
- Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
- Nasmyth K. A., Reed S. I. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2119–2123. doi: 10.1073/pnas.77.4.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shanabruch W. G., Walker G. C. Localization of the plasmid (pKM101) gene(s) involved in recA+lexA+-dependent mutagenesis. Mol Gen Genet. 1980;179(2):289–297. doi: 10.1007/BF00425456. [DOI] [PubMed] [Google Scholar]
- Shuman H. A., Silhavy T. J., Beckwith J. R. Labeling of proteins with beta-galactosidase by gene fusion. Identification of a cytoplasmic membrane component of the Escherichia coli maltose transport system. J Biol Chem. 1980 Jan 10;255(1):168–174. [PubMed] [Google Scholar]
- Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]