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Query: EC:2.4.2.8 (
hypoxanthine-guanine phosphoribosyltransferase
)
2,527
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
We have characterized a DNA-protein interaction within a sequence element distal from the site of transcription initiation within the mouse housekeeping gene (
HPRT
) promoter region. This interaction occurs within a 35-base pair regulatory element which confers cell type-specific gene transcription, designated as the
HPRT
cis-acting regulatory element (HCRE). Competition analysis by gel mobility shift electrophoresis indicates that this DNA-protein interaction is novel and not related to many transcription factors previously reported. Cell cycle synchronization experiments and gel mobility shift assays have demonstrated that within the HCRE a specific DNA-protein complex responds to G1 activation of the cell cycle. Experiments to purify specific DNA-binding proteins that interact with the HCRE has resulted in the purification of one sequence-specific DNA-binding protein of approximately 66 kDa. To determine the putative DNA-binding sequence, footprinting analysis has mapped the protection from DNase I hydrolysis which confers a core sequence of GTCTGGGT using both affinity purified protein and crude nuclear extract. This DNA motif represents a novel protein-binding sequence. Interestingly, data base searches have identified the same or homologous sequences of this DNA motif in additional genes, potentially related to cellular growth and proliferation. This consensus was most notable within a region 5' upstream of the
ornithine decarboxylase
gene. The unique cell type-specific regulation of the
HPRT
gene in the intestinal mucosa is not completely understood at this time but because of the relationship of
ornithine decarboxylase
expression to cell proliferation and more specifically, to mucosal cell renewal in the intestine, the function of DNA-protein interactions within the consensus sequence may prove analogous. This may account for the cell type-specific and cell-cycle responsive gene regulation previously demonstrated with
HPRT
. Identification of one sequence-specific DNA-binding protein within the HCRE suggest that this protein contributes to the trans-activation of specific genes during the immediate-early response of the cell cycle.
...
PMID:Characterization of DNA-protein interactions within a distal regulatory element upstream of a mammalian housekeeping gene promoter. 155 10
The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/
guanine phosphoribosyltransferase
(
HPRT
;
hypoxanthine phosphoribosyltransferase
; IMP:pyrophosphate phosphoribosyltransferase,
EC 2.4.2.8
), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the
HPRT
of Schistosoma mansoni and the
ornithine decarboxylase
(ODC;
L-ornithine carboxy-lyase
,
EC 4.1.1.17
) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal
HPRT
was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active
HPRT
has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of
HPRT
and ODC.
...
PMID:High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase. 200 85
