New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacA gene sequences encoding an enzymatically active carboxy-terminal portion of β-galactosidase

Shapira, Stuart K.; Chou, Joany; Richaud, F; Casadaban, Malcolm J.

doi:10.1016/0378-1119(83)90169-5

Cited by 424 publications

(157 citation statements)

References 18 publications

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“…Since all known proteins of the BglG family are involved in the regulation of sugar utilization and since the expression of most of these proteins responds to the presence of specific sugars, the effects of various sugars on the expression of bglR were examined. A bglR::lacZ fusion was constructed by inserting a 1,277-nucleotide EcoRV-RsaI fragment of the trpA-bglR region of the IL1403 chromosome, made blunt by treatment with T4 polymerase, at the SmaI site of plasmid pMC1871 (37). A PstI cassette carrying bglR::lacZ was then inserted at the unique PstI site of pE194 (21), which does not replicate in L. IL3677 which carries a bglR::lacZ chromosomal fusion was grown in M17 medium containing the indicated sugars (1%) to mid-log phase, and the ,-galactosidase specific activity was measured.…”

Section: Methodsmentioning

confidence: 99%

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

1994

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The bgl operon of Escherichia coli is involved in the utilization of P-glucoside sugars (25,36). This operon is cryptic in wild-type strains but can be rendered functional by a variety of spontaneous mutations (30,31,35). When functional, this operon is inducible by 3-glucosides. This control is exerted through transcription antitermination mediated by the BglG protein (3,22,25,33,34). Transcription initiates constitutively at the bgl promoter, but in the absence of 3-glucosides, most transcripts terminate at a p-independent terminator located in the leader region, immediately upstream of the bglG gene. In the presence of P-glucosides, the BglG protein binds to a specific sequence of the mRNA, overlapping part of the transcription terminator, thus preventing RNA polymerase from terminating. The binding of BglG is modulated by its phosphorylation by EIIBgi, an enzyme of the phosphoenolpyruvate:p-glucoside phosphotransferase system, which is involved in uptake and phosphorylation of P-glucosides (36). In the absence of 0-glucosides, P-EIIBgl phosphorylates BglG, which thus becomes unable to bind to RNA and act as an antiterminator (3, 4). If 3-glucosides are present, they are phosphorylated by P-EII'9' and P-BglG is dephosphorylated. The protein can thus bind to RNA, which results in transcription antitermination.Based on a strong conservation of the amino acid sequences of the regulatory proteins and of their putative RNA systems. These systems include the control of the Bacillus subtilis levansucrase sacB gene by SacY (7, 13), of the B. subtilis sacPA operon by SacT (6, 14, 39), of B. subtilis 13-glucan utilization by licT (40), of Lactobacillus casei lactose utilization (1), and of the Erwinia chrysanthemi P-glucoside phosphotransferase gene arbF by ArbG (15).In this article, we describe the Lactococcus lactis bglR gene, whose product shares homology with the BglG family of antiterminators. bglR is preceded by a transcription terminator partially overlapped by the conserved target sequence of the BglG antiterminators. Constitutive expression of bglR in E. coli increased the expression of a bglG::lacZ transcriptional fusion. The fact that the expression of BglG is autoregulated in E. coli suggests that BglG and BglR are functionally related. In L.lactis, the expression of bglR is positively controlled by P-glucosides and its disruption results in a deficiency in P-glucoside utilization. Taken together, these results indicate that BglR is a lactococcal counterpart of the E. coli BglG regulator of 3-glucoside utilization. MATERIALS AND METHODSBacterial strains and media. L. lactis subsp. lactis IL1403 (11) and derivatives, E. coli TG1 (18) and MA152 (24), and B. subtilis MT119 (41) were grown as previously described (8). Growth on various carbohydrates in a chemically defined broth (28,29,38), supplemented with the appropriate sugar at 1% final concentration, was monitored. The medium was inoculated with 1% of a culture grown overnight and then washed twice.DNA manipulations. Plasmids and chromosomal DNAs were e...

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Section: Methodsmentioning

confidence: 99%

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

1994

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“…Plasmids were transformed into yeast cells using lithium acetate (26) and plated on medium lacking uracil. Plasmid pR116 carries an ADE5-lacZ fusion and was constructed by first introducing a 3.0-kb BamHI fragment carrying the E. coli lacZ gene (27) into the BamHI site located in the ADE5,7 gene of plasmid pYEADE5,7(5.2R) (23) to produce plasmid pR111. The ϳ8-kb SalI to BspEI fragment containing the ADE5-lacZ fusion from pR111 was subcloned into the SalI and XmaI sites of a modified form of pRS316, lacking the ␣-fragment of lacZ, to produce pR116.…”

Section: ј-Cgtcagatctgccccgnnngtagtgac-3јmentioning

confidence: 99%

“…PCR reactions were performed as described above using the labeled oligonucleotides as primers and plasmids containing the appropriate ADE5,7 sequences as templates to amplify the region between Ϫ211 and Ϫ145 (relative to the ADE5,7 start codon). The templates were pR224, carrying the wild-type ADE5,7 fragment, or selected plasmids carrying mutated ADE5,7 promoter fragments, constructs numbered 5,7,10,11,13,14,19,20,27,29,34,35,39, and 43 as listed in Table IV. After PCR, a portion of each sample was separated by electrophoresis, and concentrations of the PCR products were estimated by ethidium bromide staining in comparison with known concentrations of duplex oligonucleotides of approximately the same length.…”

Section: ј-Cgtcagatctgccccgnnngtagtgac-3јmentioning

confidence: 99%

The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

Rolfes

Zhang

Hinnebusch

1997

Journal of Biological Chemistry

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Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves downregulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.

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“…The yolk protein component of pCR2 extends to the NcoI site in yp2 ( + 105 of yp2), which is fused to the Sinai site of a modified E. coli lacZ gene [pMLB1034(&AvaI) ~ Shapira et al 1983] by a linker (5'-GCCATGCGGTCGACCGGGGATCCC-3'; underlining indicates the NcoI and SmaI sites remaining in pCR2). The 3' end of the lacZ gene region was terminated at the Bali site by insertion of a 620-bp HpaI-HindIII fragment from the D. melanogaster al-tubulin gene (Theurkauf et al 1986) that contains the oL1 polyadenylation site in the appropriate ori- entation.…”

Section: Construction Of P-element Plasmidsmentioning

confidence: 99%

Ovarian follicle cell enhancers from the Drosophila yolk protein genes: different segments of one enhancer have different cell-type specificities that interact to give normal expression.

Logan

Wensink²

1990

Genes Dev.

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This paper examines ovarian transcription of the divergently oriented yolk protein genes 1 and 2 (ypl and yp2) of Drosophila melanogaster. We report germ line transformation results demonstrating that ypl and yp2 are transcribed in the same subpopulations of ovarian follicle cells. Our results show that this expression pattern is directed by two enhancers: ovarian enhancer 1, located between the genes, and ovarian enhancer 2, located within the first exon of yp2. Analysis of the expression pattern resulting from alterations in ovarian enhancer 1 demonstrates that different segments of this enhancer have different positive or negative effects on the cell-type specificity of transcription.

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New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacA gene sequences encoding an enzymatically active carboxy-terminal portion of β-galactosidase

Cited by 424 publications

References 18 publications

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

Ovarian follicle cell enhancers from the Drosophila yolk protein genes: different segments of one enhancer have different cell-type specificities that interact to give normal expression.

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