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1.
Virus Res ; 104(1): 11-6, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15177887

ABSTRACT

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.


Subject(s)
Endogenous Retroviruses/genetics , Enhancer Elements, Genetic/physiology , Promoter Regions, Genetic/physiology , Terminal Repeat Sequences/physiology , Animals , CHO Cells , Cricetinae , Genes, Reporter , Humans , Luciferases/genetics , Organ Specificity , Tumor Cells, Cultured
2.
J Chromatogr A ; 976(1-2): 95-101, 2002 Nov 08.
Article in English | MEDLINE | ID: mdl-12462600

ABSTRACT

A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.


Subject(s)
Chromatography, Affinity/methods , Endogenous Retroviruses/genetics , Repetitive Sequences, Nucleic Acid , Base Sequence , DNA Primers , Electrophoresis, Polyacrylamide Gel , Humans , Protein Binding , Species Specificity
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