Biomedical subjects

C D Collier

Publications and source records attributed to C D Collier.

4 recordsLinked to original sources

PubMed1998

Modulation of TAT gene induction by glucocorticoids involves a neutralizing sequence.

Recent studies have indicated that two elements in addition to the glucocorticoid response element (GRE) are involved in the induction of the endogenous TAT gene in FuS-5 rat hepatoma cells. The first is the 21 bp glucocorticoid modulatory element (GME) at -3648 bp, which causes reporter constructs to display both a left shift in the dose-response curve for glucocorticoids and increased percentages of agonist activity for antiglucocorticoids. The second is a negative element at -3340 to -3050 that blocks the action of the GME. This last observation raised the question of how GME activity can be expressed in Fu5-5 cells in the intact TAT gene that contains both the GME and the negative element. The present study identifies a third element, a "neutralizing" sequence, that restores the activity of the GME even when otherwise inactivated by the negative element. This neutralizing sequence was located within the region surrounding the GREs of the TAT gene but is separate from the GREs. The activity of the individual GME and negative elements was found to depend upon spacing. However, in combination with the natural GRE, the native TAT gene spacing of the GME and negative elements was able to reproduce the activity of the intact gene. Thus, a total of three additional elements (an activator, a negative element, and a neutralizer) appear to cooperate with the GREs in glucocorticoid induction of the TAT gene in Fu5-5 cells. While such a grouping of elements may be novel among steroid regulated genes, it is a not uncommon occurrence for the transcriptional control of other genes.

Animals↗

PubMed1996

A negative tyrosine aminotransferase gene element that blocks glucocorticoid modulatory element-regulated modulation of glucocorticoid-induced gene expression.

Tyrosine aminotransferase (TAT) is the prototypic steroid-inducible gene. Recently, we have found that the modulation of TAT induction properties is reproduced by a novel cis-acting TAT gene element, the glucocorticoid modulatory element (GME). This GME lies about 1 kb upstream of the glucocorticoid response elements (GREs) of the TAT gene and binds a heterooligomer of two recently defined proteins. We now report the existence of an additional TAT gene element between the GME and the GREs that blocks the action of the GME and thus prevents the left shift in the glucocorticoid dose-response curve caused by the GME. This negative element has the properties of a silencer because its activity is relatively position- and orientation-independent. The interaction appears to be stoichiometric in that the effects of a single negative element can be overcome by a second GME. This negative element also has an intrinsic inhibitory activity in the absence of the GME. The majority of the negative element activity could be elicited by a 56-bp sequence between -3105 and -3050 bp of the TAT gene. Multiple, clustered mutations of this sequence reduced, but did not eliminate, the negative activity. Further efforts to restrict the negative element were unsuccessful, suggesting that multiple sequences are required for full activity. High affinity, sequence-specific binding of a trans-acting factor(s) was observed in gel shift assays. This binding was half-maximally competed by a 4.4-fold excess of nonradioactive probe and was very stable once formed (delta H [symbol: see text] dissoc. = 32 kcal/mol), suggesting that low concentrations of a high affinity binding protein(s) exist in nuclear extracts. Further support for this conclusion came from the observation that cotransfection of a plasmid containing multiple copies of the 56-bp negative element was able to relieve the negation of GME activity in a GME-56-bp-GRE reporter construct. These data directly support the role of a trans-acting factor(s) in binding to the 56-bp negative element and blocking GME activity. Collectively, these data suggest that glucocorticoid induction of TAT gene expression is subject to multiple levels of control by several new cis-acting elements and thus is much more complex than previously appreciated.

Base Sequence↗

PubMed1990

The Escherichia coli K-12 metJ193 allele contains a point mutation which alters the hydrophobic pocket responsible for in vitro binding of S-adenosylmethionine: effects on cell growth and induction of met regulon expression.

The metJ193 allele encodes one of two identified temperature-sensitive Escherichia coli K-12 met repressors. The nucleotide sequence of the metJ193 allele was determined. The point mutation was a T to A transversion at base 170 of the metJ193 open reading frame and resulted in the substitution of leucine by glutamine at the 56th amino acid residue of the MetJ193 protein. The mutational lesion altered the hydrophobic pocket responsible for in vitro binding of the corepressor S-adenosylmethionine by wild-type MetJ. MetJ193 protein formed at the permissive temperature (28 degrees C) allowed slow derepression of met regulon expression when cultures were shifted to the nonpermissive temperature (34 degrees C). When 28 degrees C cultures of strains bearing two metJ193 alleles were transferred from methionine-containing medium to minimal medium, derepression of met regulon expression did not occur quickly enough to avoid a lag in growth due to the methionine deprivation. The inability of the MetJ193 protein to easily accomplish transition between apo- and active-repressor conformations was also demonstrated by using a maxicell system to study expression of a plasmid-borne copy of the E. coli metF transcription unit. These results confirm the importance of the leucine 56 residue for the structure and function in vivo of the wild-type MetJ protein.

Alleles↗

PubMed1989

Characterization of two mutant metJ proteins with reduced, temperature-dependent capacity to regulate Escherichia coli K-12 met regulon elements.

At 28 degrees C, but not at 34 or 42 degrees C, strains with the metJ193 allele repressed chromosomal met genes but not a plasmid-borne met promoter. Increasing the metJ193 gene dosage to two copies resulted in overrepression of chromosomal and plasmid-borne met promoters at 28 degrees C. Suppressing the metJ185 amber mutation with supF (tRNATyr) produced the MetJ185F protein. Strains producing MetJ185F repressed chromosomal met promoters but not a plasmid-borne met promoter at 42 degrees C. These are the first known defective MetJ proteins with documented temperature-dependent function.

Bacterial Proteins↗