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A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells 总被引:1,自引:1,他引:0 下载免费PDF全文
We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (105) for any target sequence for use in human cells. In this system, target sequences are fused to the 5′ end of the lacZ reporter gene and expressed in yeast. Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype. We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors. GSCs specific for c-myc were shown to regulate expression of both a c-myc–lacZ fusion gene and the endogenous c-myc gene in human cells. 相似文献
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Hongcheng Wang Haiyang Wang Meng Wang Lei Zhang Ren Wang Yanzhen Mei Weilan Shao 《World journal of microbiology & biotechnology》2014,30(6):1809-1817
Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation. 相似文献
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《Gene Analysis Techniques》1986,3(3):53-57
Vectors were constructed that allow the expression of large quantities of fused proteins in Escherichia coli. The plasmids carry the E. coli lac UV5 promotor and different portions of the coding sequence of the E. coli lacZ gene. The truncated lacZ genes are flanked by a polylinker region either at their 5′ end or their 3′ end, which has several unique restriction sites. Thus, coding sequences of any prokaryotic and eukaryotic gene can be ligated in frame to the truncated lacZ genes. These vectors were used for high-level production of herpes simplex virus type 1 envelope glycoprotein D antigens. 相似文献
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Norbert Perrimon Elizabeth Noll Kimberly McCall Andrea Brand 《Genesis (New York, N.Y. : 2000)》1991,12(3):238-252
To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of β-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development. 相似文献
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The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli -galactosidase gene (lacZ) in transiently-transformed predatory mite larvae. A construct containing the hsp70 promoter upstream of the D. melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus. LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence. Expression from the hsp70 promoter was strong and heat shock-dependent in both species. The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae. Furthermore, the lacZ gene is a useful gene for analysis of expression in both species. Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications. 相似文献
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Nathalie Bonnefoy Jane Copsey Michael J. Hynes Mervl A. Davis 《Molecular genetics and genomics : MGG》1995,246(2):223-227
The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and derepressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5′ region of amdS were cloned in front of the CYCI-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5′ sequences under carbon-limiting conditions. 相似文献
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Sowmya Raghavan Pradeep K. Burma Samir K. Brahmachari 《Journal of molecular evolution》1997,45(5):485-498
The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on
the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs
are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts
occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream
of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open
reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs
in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure
when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present
outside coding regions.
Received: 14 November 1996 / Accepted: 7 May 1997 相似文献
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Summary A 3.7 kb DNA fragment of yeast chromosome IV has been sequenced that contains the SFA gene which, when present on a multi-copy plasmid in Saccharomyces cerevisiae, confers hyper-resistance to formaldehyde. The open reading frame of SFA is 1158 by in size and encodes a polypeptide of 386 amino acids. The predicted protein shows strong homologies to several mammalian alcohol dehydrogenases and contains a sequence characteristic of binding sites for NAD. Overexpression of the SFA gene leads to enhanced consumption of formaldehyde, which is most probably the reason for the observed hyper-resistance phenotype. In sfa:LEU2 disruption mutants, sensitivity to formaldehyde is correlated with reduced degradation of the chemical. The SFA gene shares an 868 by divergent promoter with UGX2 a gene of yet unknown function. Promoter deletion studies with a SFA promoter-lacZ gene fusion construct revealed negative interference on expression of SFA by upstream sequences. The upstream region between positons – 145 and – 172 is totally or partially responsible for control of inducibility of SFA by chemicals such as formaldehyde (FA), ethanol and methyl methanesulphonate. The 41 kDa SFA-encoded protein was purified from a hyper-resistant transformant; it oxidizes long-chain alcohols and, in the presence of glutathione, is able to oxidize FA. SFA is predicted to code for a long-chain alcohol dehydrogenase (glutathione-dependent formaldehyde dehydrogenase) of the yeast S. cerevisiae. 相似文献
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Ekaterina Y Steshina Michael S Carr Elena A Glick Aleksey Yevtodiyenko Oliver K Appelbe Jennifer V Schmidt 《BMC genetics》2006,7(1):1-21