Isolation of a protein fraction that binds preferentially to chicken middle repetitive DNA

Sanzo, Michael A.; Stevens, Bryn; Tsai, Ming Jer; O’Malley, Bert W.

doi:10.1021/bi00321a034

Cited by 17 publications

(12 citation statements)

References 38 publications

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“…Fig. 5a illustrates schematically the longer emu clones (clones [12][13][14], and their partial DNA sequence is presented in Fig. 5b, again in comparison with the crane and chicken CR1.…”

Section: Resultsmentioning

confidence: 99%

“…The region between 284 and 292 matches a consensus silencer and is included in a 172-bp restriction fragment upstream of the chicken lysozyme gene with demonstrable effects on transcription: it exerts a strong repressing effect on weak promoters but has a weak effect on strong transcription units (14). The region between 424 and 439 is included in a somewhat larger domain that has been shown to bind a nuclear protein of unknown identity (13). (22).…”

Section: Discussionmentioning

confidence: 99%

“…Additional functional properties have been ascribed to CR1. Chromatographic columns containing CR1 DNA were used to isolate specific nuclear proteins from chicken oviduct tissue, and the fractionated proteins were found to bind at a 3' region of the CR1 sequence (13). Second, in dissecting the upstream region of the chicken lysozyme gene, Baniahamad et al (14) used a reporter gene to define a silencer element in the central portion of a CR1 member found here.…”

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confidence: 99%

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Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

Chen¹,

Ritzel²,

Lin³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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CR1 is a short interspersed repetitive DNA element originally identified in the domestic chicken (Galus gallus). However, unlike virtually all other such sequences described to date, CR1 is not confined to one or a few closely related species. It is probably a ubiquitous component of the avian genome, having been detected in representatives of nine orders encompassing a wide spectrum of the class Aves. This identification was made possible by using the polymerase chain reaction (PCR), which revealed interspecific similarities not detected by conventional Southern analysis. DNA sequence comparisons between a CR1 element isolated from a sarus crane (Grus antigone) and those isolated from an emu (Dromaius novaehollandiae) showed that two short highly conserved regions are present. These are included within two regions previously characterized in the CR1 units of domestic fowl. One of these behaves as a transcriptional silencer and the other is a binding site for a nuclear protein. Our observations suggest that CR1 has evolved under functional constraints and that interspersed repetitive sequences as a class may constitute a more significant component of the eukaryotic genome than is generally acknowledged.

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“…Fig. 5a illustrates schematically the longer emu clones (clones [12][13][14], and their partial DNA sequence is presented in Fig. 5b, again in comparison with the crane and chicken CR1.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

Chen¹,

Ritzel²,

Lin³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…The hypothesis of their implication in the regulation of gene expression was partially confirmed by Sanzo et al (1984), who showed that a particularly well-conserved CR1 segment of 39 bp has an affinity for nuclear proteins.…”

Section: Introductionmentioning

confidence: 93%

Cytogenetic repartition of chicken CR1 sequences evidenced by PRINS in Galliformes and some other birds

et al. 2005

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Chicken repeat 1 (CR1) belongs to the non-long repeat class of retrotransposons. Nearly 100000 repeats interspersed in the chicken genome are subdivided into at least six distinct subfamilies, each 300 bp long and all sharing substantial sequence similarity. CR1-like elements were found in genomes from invertebrates to mammals, suggesting their importance for genome structure and/or function. Moreover, numerous data support the hypothesis of their implication in regulation of gene expression. So, the chromosomal distribution of these CR1 sequences in vertebrates is of great interest to improve our knowledge about the genome structure, function and evolution. A comparison of the cytogenetic distribution of CR1 sequences was performed by PRINS using consensus chicken primers on the chromosomes of chicken and species of several bird orders: Galliformes, Anseriformes, Passeriformes and Falconiformes. The study revealed that CR1 repeats are spread over nearly all chicken chromosomes with a higher density on the macrochromosomes and in particular with hot spots on subtelomeric regions of chromosome 1, 2, 3q, 4q, 5q. Their distribution on the macrochromosomes forms a kind of banding pattern, which was not systematically matched with R- or G-banding. This banding pattern appears to be conserved on the chromosomes of the Galliformes studied, irrespective of their karyotypes, rearranged or not. CR1 primers also show similar signals on the chromosomes of birds phylogenetically more distant (Anseriformes, Passeriformes and Falconiformes). This fact confirms the importance of these sequences at the large scale of bird evolution and in the chromosomal structure. The location of CR1 sequences, and in particular of the hot spots, mainly within the richest CG areas are in conformity with the data on an epigenetic role of these highly conserved sequences.

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“…These include nitrocellulose filter-binding assays (1)(2)(3)(4), detection of discrete DNase-hypersensitive sites in chromatin (5-7), exonuclease III digestion of chromatin (8), affinity chromatography (9,10), and in vitro transcription (11)(12)(13)(14)(15)(16)(17)(18)(19). A limitation of these techniques is that they identify the site of protein binding within the DNA but do not identify the specific proteins involved.…”

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confidence: 99%

Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene.

Miskimins¹,

Roberts²,

McClelland³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

270

118

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We describe a procedure for detecting highaffinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.DNA sequences required for the regulation of eukaryotic gene expression have now been identified, largely through in vitro mutagenesis and subsequent analysis of the transcriptional activity of the mutagenized genes. What remains undetermined in most of these cases is the identity of specific transcription factors that interact with these DNA sequences to direct gene expression. The interaction of specific proteins with DNA regulatory sequences probably represents a fundamental process in controlling gene activity, and thus, the overall complement of sequence-specific proteins in the cell nucleus may largely determine the growth or differentiation state of the cell.The existence of site-specific DNA-protein interactions in eukaryotes has been demonstrated by several approaches. These include nitrocellulose filter-binding assays (1-4), detection of discrete DNase-hypersensitive sites in chromatin (5-7), exonuclease III digestion of chromatin (8), affinity chromatography (9, 10), and in vitro transcription (11)(12)(13)(14)(15)(16)(17)(18)(19). A limitation of these techniques is that they identify the site of protein binding within the DNA but do not identify the specific proteins involved. Only after extensive purification procedures can information be obtained about the nature of the proteins involved. Bowen et al. (20) have introduced a procedure that allows detection of DNA-binding proteins by blotting electrophoretically separated proteins on nitrocellulose. This procedure, however, has not proven to be effective for directly identifying site-specific DNA-binding proteins without partial purification (21-23) or heat-inactivation (3) of crude nuclear extracts. We describe here a procedure that uses protein blotting and that requires no prior purification steps, which has the potential for directly identifying and studying the regulation of proteins that interact with a target DNA sequence. We have used this procedure to identify a group of proteins that appear to bind with specificity and high affinity to the promoter region of the gene encoding the transferrin receptor. MATERIALS AND METHODSPreparation of Nuclear Extract. Cells were grown, at 370C in a 10% CO2 atmosphere in roller bottles, in Dulbecco's modified Eagle's medium (GIBCO) containing 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 ,ug/ml), Confluent cultur...

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Isolation of a protein fraction that binds preferentially to chicken middle repetitive DNA

Cited by 17 publications

References 38 publications

Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

Cytogenetic repartition of chicken CR1 sequences evidenced by PRINS in Galliformes and some other birds

Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene.

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