Gene-sized pieces produced by digestion of linear duplex DNA with mung bean nuclease

Kroeker, Warren D.; Kowalski, David

doi:10.1021/bi00609a010

Cited by 48 publications

(25 citation statements)

References 44 publications

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“…We found that all three nucleases also specifically cleave at the inverted repeat junction of the Tee2 circle (data not shown). These three nucleases have similar but different sensitivities (Gray et al 1975;Shenk et al 1975;Wiegand et al 1975;Kroeker and Kowalski 1978). All three act as single-stranded nucleases and as endonucleases at gaps, heteroduplex regions, or regions of unusual DNA structure (non-B DNA).…”

Section: Nuclease Sensitivity Of Circular Forms Of Tecmentioning

confidence: 99%

Elimination of Tec elements involves a novel excision process.

Jaraczewski¹,

Jahn²

1993

Genes Dev.

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Approximately 60,000 transposon-like elements of the Tecl and Tec2 families excise en masse from the micronuclear genome during formation of a macronucleus in Euplotes crassus. The circular product has been shown previously to contain the element inverted repeats joined head to head. To elucidate the mechanism of Tec excision, we have further characterized the circular products. DNA sequence analysis of cloned inverted repeat junctions and of a population of supercoiled Tec circles shows that the inverted repeat junctions consist of both copies of the target site duplication surrounding 10 additional bases. The 10 bp differs for each junction. We demonstrate that the circles are highly sensitive to Sl, mung bean and Bal 31 nucleases, and the site of sensitivity maps to the junction. Alkaline gel electrophoresis indicates that the junction does not contain a nick or gap; thus, a likely explanation for the nuclease sensitivity is the existence of a heteroduplex DNA structure at the junction. On the basis of these results, we present a model of Tec excision and discuss the relationship of Tec excision to IES elimination and chromosome fragmentation in E. crassus.

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Section: Nuclease Sensitivity Of Circular Forms Of Tecmentioning

confidence: 99%

Elimination of Tec elements involves a novel excision process.

Jaraczewski¹,

Jahn²

1993

Genes Dev.

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“…1). Unidirectional nested deletions from the 5Ј end of the p9Ka gene were produced using a time course of digestion with exonuclease III as described previously (2), removing the resulting singlestranded overhangs with mung bean nuclease (16). Following circularization of the plasmid DNA by ligation and transformation into E. coli cells, recombinant plasmids from E. coli colonies representing different time points were sequenced to precisely identify the extent of deletion of the upstream region.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Down-regulation of the Metastasis-inducing S100A4 (p9Ka) in Benign but Not in Malignant Rat Mammary Epithelial Cells by GC-factor

Chen

Davies

Rudland

et al. 1997

Journal of Biological Chemistry

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The S100-related calcium-binding protein S100A4 (p9Ka) is expressed at a low level in rat mammary epithelial cells from normal mammary gland and benign mammary tumors. In transgenic mice, expressed rat S100A4 transgenes co-operate with the activated c-erbB-2 oncogene, neu, to form metastatic mammary tumors. Elevated levels of S100A4 (p9Ka) in cultured benign rat or mouse mammary epithelial cells are associated with the induction of metastatic capability. A cis-acting sequence related to the consensus recognition sequence of GC-factor, 1,300 base pairs upstream of the start site of transcription of the rat S100A4 gene, acts as a cis-acting inhibitor of transcription of the S100A4 (p9Ka) gene in a low S100A4 (p9Ka)-expressing benign rat mammary epithelial cell line, but not in highly expressing rat mammary epithelial cell lines. There is an inverse relationship between the level of S100A4 (p9Ka) mRNA and the level of GC-factor mRNA in a range of rat mammary cell lines. The results suggest a novel mechanism for regulating the expression of the mRNA encoding an S100 protein.S100A4 (p9Ka) is a member of the S100 family of calciumbinding proteins first identified in a cultured myoepitheliallike cell line, Rama 29 (1). The S100A4 (p9Ka) gene has been isolated and its nucleotide sequence determined (2). Elevation of the level of S100A4 (p9Ka) in benign, non-metastatic rat (3) and mouse (4) cells induces metastatic capability in some of the cells. Although mice containing multiple copies of a rat (5) or mouse (6) S100A4 transgene show no observable phenotype, mice which contain cells that express both the rat S100A4 transgenes and the activated neu oncogene, or mouse S100A4 transgenes in a genetic background that yields primary mammary tumors, succumb to metastatic mammary tumors (6, 7), strongly suggesting that overexpression of S100A4 in mammary tumors can yield the metastatic phenotype. Some rat and mouse cultured mammary epithelial cell lines of high metastatic potential contain elevated levels of S100A4, when compared with their non-metastatic counterparts (8, 9).

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“…A Hinfl genomic clone fragment starting with 5' flanking sequence and ending within exon 1 was 5'-end-labeled with [y-32P]ATP. This DNA was cut with Dra II to generate a labeled 380-base-pair (bp) flanking/exon 1 Dra II-Hinfi/fragment for S1 nuclease protection (17 :20) IgG was applied for 1.5 hr at room temperature for monoclonal or polyclonal reaction, respectively. After PBS/Triton washes, 1:100 peroxidase-anti-peroxidase complex (either mouse monoclonal or rabbit antiperoxidase) was applied for an additional 1.5 hr at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Regulation of a bifunctional mRNA results in synthesis of secreted and nuclear probasin.

Spence

Sheppard

Davie

et al. 1989

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

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Probasin, a rat prostatic protein, is statistically related to members of a protein family that includes serum, cellular, and nuclear proteins. In vivo, probasin appears both in the secretions and in the nucleus of prostatic epithelial cells. Using primer extension and S1 nuclease protection assays we detected only one probasin mRNA. Thus, the localization of this protein to two separate cellular regions must be encoded by this one mRNA. Furthermore, in vitro translation of synthetic probasin mRNA demonstrated that a protein containing a signal peptide and a protein lacking a signal peptide were synthesized by initiation at different AUG codons. These data are consistent with a mechanism of translational regulation of a eukaryotic bifunctional mRNA.

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Gene-sized pieces produced by digestion of linear duplex DNA with mung bean nuclease

Cited by 48 publications

References 44 publications

Elimination of Tec elements involves a novel excision process.

Elimination of Tec elements involves a novel excision process.

Transcriptional Down-regulation of the Metastasis-inducing S100A4 (p9Ka) in Benign but Not in Malignant Rat Mammary Epithelial Cells by GC-factor

Regulation of a bifunctional mRNA results in synthesis of secreted and nuclear probasin.

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