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M Crabeel

Publications and source records attributed to M Crabeel.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of the DNA target site for the yeast ARGR regulatory complex, a sequence able to mediate repression or induction by arginine.

We have determined the sequences and positions of the cis elements required for proper functioning of the ARG3 promoter and proper arginine-specific control. A TATA box located 100 nucleotides upstream of the transcription start was shown to be essential for ARG3 transcription. Two sequences involved in normal arginine-mediated repression lie immediately downstream of the TATA box: an essential one (arginine box 1 [AB1]) and a secondary one (arginine box 2 [AB2]). AB1 was defined by saturation mutagenesis and is an asymmetrical sequence. A stringently required CGPu motif in AB1 is conserved in all known target sites of C6 zinc cluster DNA-binding proteins, leading us to propose that AB1 is the binding site of ARGRII, another member of the C6 family. The palindromic AB2 sequence is suggested, on the basis of published data, to be the binding site of ARGRI, possibly in heterodimerization with MCM1. AB2 and AB1 correspond respectively to the 5' and 3' halves of two adjacent similar sequences of 29 bp that appear to constitute tandem operators. Indeed, mutations increasing the similarity of the other halves with AB1 and AB2 cause hyperrepression. To mediate repression, the operator must be located close to the transcription initiation region. It remains functional if the TATA box is moved downstream of it but becomes inoperative in repression when displaced to a far-upstream position where it mediates an arginine and ARGR-dependent induction of gene expression. The ability of the ARG3 operator to act either as an operator or as an upstream activator sequence, depending on its location, and the functional organization of the anabolic and catabolic arginine genes suggest a simple model for arginine regulation in which an activator complex can turn into a repressor when able to interfere sterically with the process of transcription initiation.

Arginine↗
PubMed1992

Analysis of the DNA binding and transcriptional activation properties of the Ets1 oncoprotein.

The c-ets1 gene product (Ets1) is the prototype of a family of sequence-specific transcriptional activators which have been implicated in various developmental processes and in the response of cells to a variety of extracellular stimuli. We report here a structure-function analysis of the DNA binding and transcriptional activation properties of Ets1. The minimal region required for specific DNA binding is located at the carboxy-terminus of Ets1, a domain highly conserved in all known members of the Ets family. Transcriptional activation by Ets1 in mammalian cells requires an additional domain of 110 amino acids characterized by a high content of acidic residues and localized in the amino-terminal half of the protein. This domain also functions as a transcriptional activation domain in yeast cells when linked to the heterologous DNA binding domain of Gal4. In contrast to its conservation in Ets1 proteins across vertebrate species, this activation domain is not conserved in other members of the Ets family. These results indicate that an important level of specificity between different members of the Ets family may reside in the differential interactions of their respective activation domains with distinct general transcription factors or different associated coactivators.

Base Sequence↗
PubMed1991

Ets1, when fused to the GAL4 DNA binding domain, efficiently enhances galactose promotor dependent gene expression in yeast.

Ets1, the translation product of the c-ets1 proto-oncogene and the related Ets2 protein, act as sequence-specific transcriptional factors in transient transfection experiments in animal cells. We report here that in S. cerevisiae, expression of a lacZ test gene placed under the control of the GAL1 promoter is stimulated efficiently by a fusion protein in which the chicken Ets1 sequence starting from amino acid 37, is linked to the DNA binding domain of the yeast GAL4 transcriptional activator. This suggests that Ets1 contains one or more intrinsic transcription activation domain(s). However, the GAL4 integral of Ets1 fusion protein was unable to restore growth of a gal4 deletion mutant on galactose, implying that the fusion product cannot substitute for GAL4 enhancement on all GAL genes.

DNA-Binding Proteins↗
PubMed1991

Two c-myb proteins differing by their aminotermini exhibit different transcriptional transactivation activities (yeast/reporter-effector system).

We assayed in the yeast S. cerevisiae the transcriptional transactivation activity of the c-myb products encoded by a normal thymus cDNA and of an aminoterminally truncated version of it (minus 58 amino acids) corresponding to the cDNAs isolated from lymphoma and leukemia cells from different origins. Both proto-oncogene products were expressed under the control of the galactose inducible GAL10 promoter. The reporter system used to monitor the transactivation potential of the myb products consisted of a CYCl-lacZ gene fusion in which the UASCYC signals were replaced by one or multiple copies of the myb recognition element (mRE). As shown by Northern blot analyses and by primer extension experiments both c-myb products increase the level of beta-galactosidase transcription. Interestingly, the c-myb product corresponding to lymphoma cDNAs stimulates transcription four to five times more efficiently than does the normal thymic c-myb product.

Base Sequence↗
PubMed1990

Sequences of the genes encoding argininosuccinate synthetase in Escherichia coli and Saccharomyces cerevisiae: comparison with methanogenic archaebacteria and mammals.

The nucleotide (nt) sequences of the genes encoding argininosuccinate synthetase from Escherichia coli K-12 (argG) and Saccharomyces cerevisiae (ARG1) were determined. The deduced amino-acid sequences were compared to each other and to their counterparts in two methanogens and in mammals. Three regions are highly conserved. Two of them appear to contain possible Walker-type nt-binding sites [Walker et al., EMBO J. 1 (1982) 945-951] and are therefore candidates for ATP-binding sites. The third region shows some similarity to a short portion of the N-proximal part of the PurA enzyme which catalyses an analogous reaction.

Amino Acid Sequence↗
PubMed1990

Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferase: evolutionary relationship with ornithine aminotransferase.

Genes argD and ARG8, encoding the acetylornithine aminotransferase (ACOAT) subunit in Escherichia coli and Saccharomyces cerevisiae, respectively, have been cloned and sequenced. The deduced amino acid sequences show substantial similarity. Moreover, they resemble ornithine aminotransferase (OAT) sequences (i.e., those from yeast, rat and man); the observed similarities are statistically significant, indicating that the enzymes are homologous. However, in contrast to OATs, which appear to be substrate (i.e., ornithine)-specific, S. cerevisiae ACOAT transaminates ornithine about as efficiently as E. coli does. The evolutionary relationship between ACOATs and OATs is discussed in terms of substrate ambiguity.

Amino Acid Sequence↗
PubMed1990

Arginine-specific repression in Saccharomyces cerevisiae: kinetic data on ARG1 and ARG3 mRNA transcription and stability support a transcriptional control mechanism.

A specific repression mechanism regulates arginine biosynthesis in Saccharomyces cerevisiae. The involvement of regulatory proteins displaying DNA-binding features and the location of an operator region between the TATA box and the transcription start of the structural gene ARG3 suggest that this mechanism operates at the level of transcription. A posttranscriptional mechanism has, however, been proposed to account for the conspicuous lack of proportionality between ARG3 mRNA steady-state levels (as determined by Northern [RNA] assays; F. Messenguy and E. Dubois, Mol. Gen. Genet. 189:148-156, 1983) and the cognate enzyme activities. In this work, we have analyzed the time course of the incorporation of radioactive precursors into ARG1 and ARG3 mRNAs and the kinetics of their decay under different regulatory statuses. The results (expressed in terms of relative mRNA levels, relative transcription rates, and mRNA half-lives) give the picture expected from a purely transcriptional control. A similar analysis of expression of the gene CPA1, for which a translational regulation by arginine has been clearly demonstrated (M. Werner, A. Feller, F. Messenguy, and A. Piérard, Cell 49:805-813, 1987), indicates that this gene is also partly regulated at the transcriptional level by the ARGR repressor system. Moreover, the half-life of CPA1 mRNA is reduced twofold in the presence of excess arginine; we suggest that this could be inherent in the mechanism of translational regulation of CPA1.

Arginine↗
PubMed1988

Arginine repression of the Saccharomyces cerevisiae ARG1 gene. Comparison of the ARG1 and ARG3 control regions.

The Saccharomyces cerevisiae ARG1 gene coding for argininosuccinate synthetase has been isolated and the nucleotide sequence of both its control region and of its amino terminal end coding region determined. The startpoint of transcription was established by S1-mapping and reverse transcriptase procedures. Northern blot hybridizations showed that whereas arginine-specific repression reduced the enzyme activity fivefold, it did not reduce the steady state level of the corresponding messenger in proportion; by analogy with the coregulated ARG3 gene, this result suggests a post-transcriptional regulatory mechanism. In contrast, proportionally between enzyme activity and mRNA content was observed under conditions where general amino acid control (known to be transcriptional) was operating. Comparing the 5' untranscribed domains of ARG1 and ARG3 revealed a first region of homology between the TATA box and the transcription startpoint. In this region a 10 bp (ARG3) or 11 bp (ARG1) central box is flanked by two segments which, by mutation, have been shown to be part of the ARG operator (Crabeel et al. 1985). The repressor is assumed to bind at this primary target site prior to establishing contacts with the proximal part of the nascent mRNA molecule (Crabeel et al. 1985). By in vitro directed deletion mutagenesis we show that the central conserved box of ARG3 is not essential for arginine-specific repression to occur. Another region of homology was found in the leader part of the messenger RNA; deletion of this region causes a small reduction in ARG3 expression but also does not alter regulation. Neither of these two regions are thus part of the primary repressor target site. In addition, in terms of post-transcriptional regulation, the latter result indicates that no sequence specificity is required in the RNA recognition step.

Amino Acid Sequence↗
PubMed1979

Tandem and inverted repeats of arginine genes in Escherichia coli: structural and evolutionary considerations.

Duplications of arg genes produced in the Rec+ and in the recA genetic backgrounds are shown by heteroduplex analysis to be strictly tandem at the level of resolution of this technique. The formation of these particular rearrangements therefore does not require the inclusion of transposons or other sequences of an appreciable size in their final structure. Duplications of short segments (about 2,000 nucleotides) appear unexpectedly stable when compared with duplications of longer segments (about 10,000 nucleotides). One of the structures analyzed displays two inversely repeated argE genes rearranged into an artificial divergent operon. The bearing of this observation on the origin of bipolar operons, of "mirror-image" map symmetries and on the production of inverted repeats in general, is discussed.

Arginine↗
PubMed1979

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.

Arginine↗
PubMed1978

Studies on the control region of the bipolar argECBH operon of Escherichia coli. I. Effect of regulatory mutations and IS2 insertions.

Several mutations affecting the control or the potential of gene expression in the argECBH bipolar operon have been characterized by enzyme assays, genetic mapping, dominance tests and pulse labelled RNA determinations. None of the mutations involves DNA rearrangements detectable by heteroduplex analysis (Charlier et al., 1978). Partially constitutive transcription of both argE and argCBH has been observed in mutant L10 while constitutive argE transcription and normal argCBH control characterize mutants L9, LL13 and LL2. The control region thus appears to contain two overlapping operators, as suggested previously (Elseviers et al., 1972). Two mutants (L2, LL1) and strain 6-8 from Bretscher and Baumberg (1976) display an increase in acetylornithinase specific activity (argE product) without concommittant increased argE transcription. In addition, they exhibit a decreased argCBH transcription. It is suggested that in these organisms, argE translation and argCBH transcription may be affected by the same genetic event; this explanation is compatible with present working hypothesis for the structure of the control region. An interpretation in terms of messenger attenuation also appears possible. From the properties of two strains harbouring an IS2 insertion in the control region (Charlier et al., 1978) the following conclusion may be drawn: 1. When inserted in orientation I close to the proximal end of a silent gene IS2 appears to promote a low but detectable transcription readthrough into that gene. 2. Insertion of an IS2 element in orientation II close to a neighbouring gene is not a sufficient condition to express that gene at a high rate. The properties of the two insertions appear compatible with the structure proposed for the control region.

Acetyltransferases↗
PubMed1977

Studies on the bipolar argECBH operon of E. coli: characterization of restriction endonuclease fragments obtained from gammadargECBH transducing phages and a ColE1 argECBH plasmid.

The isolation of a new type of gamma transducing phage carrying the bipolar argECBH operon of E. coli K12 is described. The argECBH segment is inserted in the phage in a direction which is opposite from that of previously isolated argECBH-carrying phages. A colE1 argECBH plasmid has been constructed. DNA fragments resulting from digestion of these genetic elements with Eco RI and Hind III restriction enzymes have been characterized by agarose gel electrophoresis and electron microscopy, including hetero-duplex analysis. Two fragments are of special significance for studies on the control of arginine synthesis, one of length 9.8 kilobases carrying the whole argECBH region, the other of length 2 kilobases carrying most or all of the control region between argE and argC.

Arginine↗