Undermethylation at the 5′ end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture

Ott, Marie‐Odile; Sperling, Linda; Cassio, Doris; Levilliers, Jacqueline; Sala‐Trepat, José M.; Weiss, Mary C.

doi:10.1016/0092-8674(82)90287-2

Cited by 121 publications

(50 citation statements)

References 33 publications

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“…Since DNA methylation correlates in many cases with transcriptional inactivity (6,38,53,54), these findings are in accord with the idea that 5' regulatory regions of housekeeping genes may be refractory to methylation and maintained in a state of readiness in all cell types.…”

Section: Discussionsupporting

confidence: 76%

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Mitchell¹,

Carothers²,

Han³

et al. 1986

Mol. Cell. Biol.

164

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Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occwrs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G+C content and lack of DNA methylation. This region also includes a 214.base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312: [409][410][411][412][413] 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.The mammalian dihydrofolate reductase gene product (DHFR) is one of a group of S-phase-responsive enzymes important for DNA replication. Like other enzymes involved in DNA synthesis, DHFR activity is greater in proliferating cells than in quiescent cells (34). DHFR catalyzes the NADPH-dependent reduction of folate to dihydrofolate and then to tetrahydrofolate. Reduced folates are essential cofactors in the biosynthesis of glycine, purine nucleotides, and thymidylic acid. The de novo synthesis of the DNA precursor thymidylic acid during S phase is the major tetrahydrofolate-consuming reaction, and the cellular requirement for DHFR is highest at this time (34). However, even in rapidly proliferating cells, sufficient DHFR catalytic activity can be maintained by as few as 10 to 20 copies of mRNA per cell (32).Genes coding for low-abundance mRNAs represent the major portion of RNA polymerase II-specific genes that are expressed in any given cell type. Our understanding of the regulation of such genes is only just beginning. Some of the proteins encoded by low-copy mRNAs are tissue-specific enzymes and regulatory proteins involved in metabolic activities unique to a particular cellular phenotype. Others are proteins expressed to some extent in all cells and are commonly known as housekeeping gene products. The dihydrofolate reductase (dhfr) gene is referred to as a house-* Corresponding author. keeping gene because DHFR plays a role in several fundamental biosynthetic reactions, but the ternlinology is not meant to indicate that dhfr gene expression is uniform in all cells or at all times in a given cell. Housekeeping genes do show physiological and tissue-specific variations in expression. The development of cell lines with amplified dhfr genes has greatly facilitated the cloning and characterization of several...

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Section: Discussionsupporting

confidence: 76%

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Mitchell¹,

Carothers²,

Han³

et al. 1986

Mol. Cell. Biol.

164

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“…However, there is a further exception to this generalization; melanoma cell lines express a major histocompatibility gene only when a particular MspI site is methylated [18]. There are also examples of hypermethylated genes being transcribed [19,20], and of other hypomethylated genes being inactive [21,22].…”

Section: Resultsmentioning

confidence: 99%

Changes in DNA methylation are associated with loss of insecticide resistance in the peach‐potato aphid Myzus persicae (Sulz.)

Field¹,

Devonshire²,

ffrench‐Constant³

et al. 1989

FEBS Letters

103

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Insecticide resistance in peach-potato aphids arises from the overproduction of one of two very closely related esterases (E4 or FE4) that detoxify insecticidal esters, and this is associated with amplification of E4-related DNA sequences. Some highly resistant aphid clones can spontaneously lose their elevated esterase and hence their resistant phenotyp¢. We report here that such aphids also lose their elevated E4 mRNA whilst retaining their amplified genomic sequences. We have also shown that the amplified E4-related sequences are highly methylated at Mspl sites in all resistant aphid clones examined, but not in those that have lost resistance.

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“…Clones with high and low CE and/or growth rates were selected from the RH-L4 line for further analyses and main- 6.0. * Colonies were harvested individually from the agar under a microscope and expanded in liquid RPMI-1640 medium with 10-15% FCS.…”

Section: Ce and Growth Rate Of 5-azactreated Subclonesmentioning

confidence: 99%

Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate.

Olsson¹,

Due²,

Diamant³

1985

The Journal of Cell Biology

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Liquid medium cultures of three human cell lines (B-lymphoma, myeloma, and squamous lung carcinoma) with population-doubling times (PDT) and cloning efficiencies (CE) in the range of 32-43 h and 0.01-5.6%, respectively, were exposed to 5-azacytidine (5-azaC) for 3 d. The doses used (1-3/zM) were found to be nontoxic as measured by cell growth in liquid and semisolid agar medium and to be nonmutagenic as measured by the rate of generation of ouabain-and 6-thioguanine-resistant cell variants. After 5-azaC treatment, cell samples were subsequently harvested every day and assayed for their CE in semisolid agar medium. For each cell line, 30 to 42 individual clones were harvested at the day of maximal CE and expanded in liquid culture medium. PDT and CE were determined for each subclone about every 6 wk for 12 too. The majority of the subclones had unaltered PDT and CE compared to the original lines. However, several clones had profoundly changed proliferative activity with PDT on ~12-14 h and/or CE 5 to >50%. Some of the clones with altered growth properties reverted to PDT and/or CE values of untreated clones. However, a few clones of each line had stable alterations with PDT on 12-14 h and CE 5 to >50%; these clones were all significantly hypomethylated. It is concluded that the human gene repertoire does contain genes that appropriately activated can result in growth properties with very short PDT and high CE (and comparable to animal cell lines), and that this activation may be obtained by 5-azaC treatment. It is conceivable that the procedure here described to alter growth properties of human cell lines may be applied to experimental situations, where alterations of cell growth properties are desired.Many reports in recent years on regulation of both cellular 0-6) and viral~ genes (7-11) have provided experimental support to the hypothesis (12-15) that 5-methylcytosine (mSCyt) t may be important in the regulation of gene expression. Moreover, a number of experiments have shown that exposure of cells to 5-azacytidine (5-azaC) may result in hypomethylation of DNA and cause expression of otherwise silent genes of both cellular and viral origin (for review, cf. references 16, 17) to the extent that it mimics mutation induced genetic alterations, e.g., reversion frequency of thyAbbreviations used in this paper." 5-azaC, 5-azacytidine; CE, cloning efficiency; mSCyt, 5-methylcytosine; Oua r, ouabin resistant; PDT, population-doubling time; RPMI/FCS, RPMI-1640 medium SUlSplemerited with 0.3% L-glutamine and 10-15% fetal calf serum; 6-TG ~, 6-thioguanine resistant. midine kinase negative cells to thymidine kinase positive cells (18).The growth properties of malignant tumor cell lines are at least in part reflected in the growth rate and CE. However, it has hitherto not been possible by exogenous factors to alter profoundly these biological features, despite numerous attempts that mainly have been based on alterations in the growth medium conditions such as addition of growth factors like insulin and transferr...

show abstract

Undermethylation at the 5′ end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture

Cited by 121 publications

References 33 publications

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Changes in DNA methylation are associated with loss of insecticide resistance in the peach‐potato aphid Myzus persicae (Sulz.)

Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate.

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