Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12

Abstract The lacI gene has been used extensively for the recovery and analysis of mutations in bacteria with various DNA repair backgrounds and after exposure to a wide variety of mutagens. This has resulted in a large database of information on mutational mechanisms and specificity of many mutagens, as well as the effect of DNA repair background on mutagenicity. Most importantly, knowledge about the mutational sensitivity of the lacI gene is now available, yielding information about mutable nucleotides. This popularity and available knowledge resulted in the use of the lacI gene in transgenic rodents for the study of mutagenesis in mammals, where it resides in ~40 repeated copies. As the number of sequenced mutations recovered from these animals increases, we are able to analyze the sites at which mutations have been recovered in great detail and to compare the recovered sites between bacteria and transgenic animals. The nucleotides that code for the DNA-binding domain are nearly saturated with base substitutions. Even after determining the sequences of ~10,000 mutations recovered from the animals, however, new sites and new changes are still being recovered. In addition, we compare the nature of deletion mutations between bacteria and animals. Based on the nature of deletions in the animals, we conclude that each deletion occurs in a single copy of the gene.

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Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54394-5 ◽

1991 ◽

Vol 266 (35) ◽

pp. 24070-24076 ◽

Cited By ~ 2

Author(s):

A.M. Nardulli ◽

D. Lew ◽

L. Erijman ◽

D.J. Shapiro

Keyword(s):

Escherichia Coli ◽

Estrogen Receptor ◽

Dna Binding ◽

Cultured Cells ◽

Binding Domain ◽

Dna Binding Domain

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Characterization of the Elk-1 ETS DNA-binding Domain

Journal of Biological Chemistry ◽

10.1074/jbc.270.11.5805 ◽

1995 ◽

Vol 270 (11) ◽

pp. 5805-5811 ◽

Cited By ~ 32

Author(s):

Paul Shore ◽

Louise Bisset ◽

Jeremy Lakey ◽

Jonathan P. Waltho ◽

Richard Virden ◽

...

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain

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Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4297-4304.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4297-4304 ◽

Cited By ~ 1

Author(s):

D Ray ◽

R Bosselut ◽

J Ghysdael ◽

M G Mattei ◽

A Tavitian ◽

...

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Binding Domain ◽

Dna Binding Domain ◽

New Gene ◽

Carboxy Terminal Region ◽

Carboxy Terminal ◽

Hematopoietic Cell Lines

We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.1 putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-1/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19q13.3-19q13.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.

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