Protein Interactions at the Transferrin Receptor Gene Promoter

Miskimins, W. Keith; Roberts, M P; McClelland, Alan; Ruddle, Frank H.

doi:10.1101/sqb.1986.051.01.085

Cited by 4 publications

(2 citation statements)

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“…The NL2‐252 clone sequence (EMBL accession number Z22395), a 237 bp segment, was completely identical to the 5′ part of the TFRC gene [10, 19]. This gene has been shown previously to be important for growth/differentiation and probably is involved in the development of leukemia [20, 21].…”

Section: Resultsmentioning

confidence: 95%

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors

et al. 1997

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Section: Resultsmentioning

confidence: 95%

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors

et al. 1997

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“…Cell-type-specific transcription is presumably conferred by the interaction of selective proteins with certain of these sequences. Genetic methods, such as cell fusion or transfection experiments, provide an indirect approach in the study of trans-acting factors, while in vitro transcription assays [l], DNA protection 121, gel retardation [3,41 and Western blotting [5] methods have yielded direct information on the interaction between sitespecific binding proteins and DNA regulatory sequences. A complete characterisation of these factors, however, necessarily depends on their purification.…”

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confidence: 99%

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

1987

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We describe a method for the purification of proteins binding to specific DNA sites based on the strong interaction between streptavidin and biotin. We tested the efficiency of this method using the Escherichia coli lactose operon operator-repressor system. dUTP coupled to biotin is incorporated into a DNA fragment containing the lactose operator. A crude E. coli extract is first incubated with the biotinylated fragment and the reaction mixture is filtered on a streptavidin-agarose column. Proteins retained on the column are either eluted alone by high salt or isopropyl p-D-thiogalactoside, or as a complex with the DNA site by enzymatic digestion of the DNA. We thus obtained a 3400-fold enrichment of the repressor complexed to the operator in one step. The method is simple and makes use of commercially available reagents. The large concentration of biotin-binding sites of the streptavidin-agarose matrix (0.1 pmol/ml packed gel) provides a very high capacity for the concentration and purification of large amounts of proteins. The advantage of this method for the detection and purification of other DNA-binding proteins is discussed.Gene transcription is regulated by both cis and transacting factors. Regulatory sequences located either near the transcription start site, such as promoters, or variably located such as enhancers, have been identified. Cell-type-specific transcription is presumably conferred by the interaction of selective proteins with certain of these sequences. Genetic methods, such as cell fusion or transfection experiments, provide an indirect approach in the study of trans-acting factors, while in vitro transcription assays [l], DNA protection 121, gel retardation [3, 41 and Western blotting [5] methods have yielded direct information on the interaction between sitespecific binding proteins and DNA regulatory sequences. A complete characterisation of these factors, however, necessarily depends on their purification. Since they are generally present at very low cellular concentration, powerful methods such as affinity chromatography using the target DNA must be employed.Several authors have recently reported different methods to obtain a DNA site matrix. Rosenfeld and Kelly [6] coupled a plasmid bearing multiple recognition sites to cellulose. Alternatively, Kadonaga and Tjian [7] used a polymerised synthetic oligonucleotide. Levens and Howley [8] fixed a / Igalactosidase -lac-repressor fusion protein to anti-(pga1actosidase)-Sepharose. The DNA sequence of interest was ligated to the lactose operator sequence and thus retained on the column. Kasher et al.[9] used biotin-cellulose and a biotinylated DNA fragment to obtain a DNA matrix via streptavidin bridges.To various degrees all these methods suffer from difficulties due to competition between non-specific DNAbinding proteins and the protein of interest for binding to the target DNA, and to inactivation of the purified protein. We present here a novel method designed to minimize these problems, uniting individual advantages of the preceding met...

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Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

Lieberman¹,

Lin²,

Yeh³

et al. 1988

Mol. Cell. Biol.

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We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from Go to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in Go arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serumstimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed.Thymidine kinase (TK; EC 2.7.1.21) participates in the salvage pathway for pyrimidine nucleotide biosynthesis, catalyzing the phosphorylation of thymidine (TdR) to form thymidine 5'-monophosphate. Two major versions of the enzyme have been characterized in mammalian cells; one is found in the cytoplasm, and the other is found in the mitochondria (8, 27). They are encoded by genes located on different autosomes (15,30,37,63), and although they catalyze the same basic phosphorylation reaction, the biochemical characteristics of each enzyme and of each reaction are unique (27). The cytosolic form of TK is of special interest, since its activity is regulated in association with several cellular metabolic activities and events. Enzyme activity is high in cells infected with certain oncogenic viruses (for a review, see reference 29), in many rapidly proliferating tumor cell populations (14), and in cells during passage through the S phase of the cell cycle (3, 35). In contrast, nonproliferating cells, including those that have terminally differentiated (42), and also cells that are not in the S phase (3, 35), express little or no TK activity.We previously described the cloning and phys...

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Protein Interactions at the Transferrin Receptor Gene Promoter

Cited by 4 publications

References 0 publications

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

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