Calcium-dependent priming of DNA synthesis in isolated rat liver nuclei

Burgoyne, Leigh A.; Waqar, M. Anwar; Atkinson, M. R.

doi:10.1016/0006-291x(70)90786-2

Cited by 143 publications

(37 citation statements)

References 14 publications

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“…Subconfluent monolayers from two 150-mm-diameter dishes were harvested by scraping and washed twice in cold phosphate-buffered saline. Nuclei were prepared (8) and stored at -95°C in 2x transcription buffer (16). Transcription in the thawed nuclei was carried out essentially as described previously (16,37 mutants (DUl, -2, -3, -6, -9, -12, -14, and -15).…”

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Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

et al. 1989

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Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.In an effort to identify and understand those aspects of gene structure that play a role in gene expression in mammalian cells, we have been carrying out a detailed mutational analysis of the dihydrofolate reductase (dhfir) gene in Chinese hamster ovary (CHO) cells. dlifr exhibits many typical characteristics of mammalian housekeeping genes: it is 25 kilobase pairs (kbp) in size, contains five variously sized introns, and has a promoter region with no TATA box but with a very high G+C content and an Spl binding site (10,22,40,53). The basic strategy has been to isolate mutants that are deficient in DHFR enzyme activity; such mutants can be readily selected from a CHO cell line that is hemizygous at this locus (58, 59). To focus on lesions that may affect transcription or RNA processing, mutants can be screened for alterations that have affected dimfi-mRNA, either qualitatively or quantitatively.We have previously described spontaneous point mutations in th...

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confidence: 99%

Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

et al. 1989

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“…Muscle cell squash preparations were from adult fly thoraces. All whole tissue squashes were prepared in buffer A as originally described by Burgoyne et al (1971;see also Paddy et al,, 1990) supplemented with 0.1% Triton X-100 and 3.7 % formaldehyde. Adult fly heads were dissected manually, prepared for cryosectioning, and cryosections cut as previously described for whole third instar larvae (Smith and Fisher, 1984).…”

Section: Lmmunocytochemistrymentioning

confidence: 99%

Developmental regulation of Drosophila DNA topoisomerase II.

Whalen

McConnell

Fisher

1991

The Journal of Cell Biology

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Abstract. Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo H) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M.

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“…Particularly in the case of micrococcal nuclease which requires Ca' + , the activity of the endogenous enzyme may have been a source of serious error, especially where the specificities of the introduced enzyme were being investigated. Nuclear deoxyribonucleases have been implicated in the replication of DNA [5], in the regulation of actin filament formation [6] and developmental gene rearrangement and diminution [7]. The role most likely to be played by the Xenopus Ca' + -DNase is in programmed cell destruction.…”

Section: Discussionmentioning

confidence: 99%