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E M Klenova

Publications and source records attributed to E M Klenova.

20 records · Page 2Linked to original sources
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[Specific interaction of a nuclear protein factor from the CTF/NF-I family with 2 different promoter regions of the rat tyrosine aminotransferase gene].

Using gel-retardation and DNase I footprinting assays, we have analysed sequence-specific DNA-protein interactions within proximal promoter fragment (from -2 to -210 bp relative to the transcription start) of the rat tyrosine aminotransferase (TAT) gene. Two distinct DNase I protection regions flanked at either boundary by sites of DNase I hypersensitivity were observed with the rat-liver nuclear extracts. The internal sequence of the region I non-coding strand, (-155)TGGGCCACCTTCCAAT(-170), is highly homologous to the NF-I consensus sequence TGG(N)6-7TGCCAA and also shares a CCAAT-box; the region II noncoding strand sequence includes asymmetrically positioned (-37)AGCCAAT(-43) recognition motif. Since there have been a number of reports about multiple DNA-binding factors that recognize CCAAT homologies, both regions were likely to interact with either a single or distinct factors. Here we show that both region I and II of the TAT gene promoter are binding to the same factor related to the human CTF/NF-I. The evidence for that is based on competition experiments using the DNA fragment containing a synthetic consensus sequence for the NF-I recognition site and on the indistinguishable chromatographic properties of the activity specifically binding to each of three DNA fragments containing NF-I consensus, region I and region II sequences.

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PubMedDate not supplied

[Nuclear factor A5 from the rat liver that requires a metal for specific interaction in vitro with distal element of the tyrosine aminotransferase gene promoter].

To detect nuclear protein factors which might account for a tissue-specific and inducible expression of the rat tyrosine aminotransferase (TAT) gene promoter, extracts from rat liver and spleen nuclei have been fractionated by heparin-sepharose chromatography and the fractions assayed for sequence-specific binding to the distal TAT gene promoter element (sequence between -313 and -210). Gel retardation experiments carried out in the presence or absence of Mg2+, Ca2+, or Zn2+ ions showed that there are at least two nuclear factors (A3 and A4) binding to the distal promoter element only in the presence of the chelator (20 mM EDTA). Incubation of the protein fractions with Zn2+ or Ca2+ instead of commonly used Mg2+allowed: (i) to avoid 3 2P-DNA-probe degradation by "contaminating" endogenous nucleases; and (ii) to detect another sequence-specific nuclear factor, A5. No other specific binding activities were found in the rat-liver nuclear fractions tested under these conditions. As the metal ions became inaccessible to chelation in excess of EDTA and EGTA when protein factor A5 was complexed to DNA we assumed that factor A5 is metalloprotein which requires Zn or Ca to maintain a structure of its DNA-binding domain. To identify the polypeptide possessing this domain, a protein gel blotting procedure was employed. By incubating gel blots with the 3 2P-DNA-probe in the buffer containing Zn2+, specific binding to the only polypeptide with approximate Mr 30 kDa was clearly revealed. Both gel retardation and gel blotting assays consistently showed that nuclear factor A5 is present in the liver, but not in the spleen extracts.

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