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...

It has been shown that 5-azacytidine (5-Aza-Cyd) can reactivate genes on the inactive human X chromosome. It is assumed that the 5-Aza-Cyd acts by causing demethylation of the DNA at specific sites, but this cannot be demonstrated directly without a cloned probe. Instead, we have utilized the technique of DNA-mediated transformation to show that the 5-Aza-Cyd-induced reactivation occurs at the DNA level. DNAs from various mouse-human or hamster-human hybrid cell lines, deficient for mouse or hamster hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8) and varying in whether they contained either an active or inactive human X chromosome, were used in transformation of HPRT-cells. DNA from the active human X chromosome-containing cell lines yielded HPRT' transformants, whereas DNA from the inactive X chromosome-containing cell lines did not. The inactive X chromosomal DNA was able to transform thymidine Idnase-deficient mouse cells, indicating that the DNA solution was normal. These results confirm that inactivation of the X chromosome involves a DNA modification. Furthermore, DNAs from three cell lines with a 5-Aza-Cyd-reactivated X chromosome also transform HPRT-cells, demonstrating that the 5-Aza-Cyd has altered the DNA structure and supporting the idea that methylation plays a role in X chromosome inactivation. Dosage compensation for most ofthe X chromosome is achieved in mammalian females early in development when one of the X chromosomes undergoes a condensation and ceases most transcriptional activity (1, 2). In eutherian mammals, either the maternal or paternal X chromosome may be inactivated in somatic cells, but once established, the pattern of inactivation remains the same for each cell and its descendants. It was originally proposed that the entire X chromosome is inactivated, but there is now evidence that the steroid sulfatase (STS; sterolsulfate sulfohydrolase, EC 3.1.6.2) locus and the Xg locus to which it is linked escape inactivation (3-6).Liskay and Evans (7) have shown that DNA from the inactive X chromosome will not function in DNA-mediated transformation of the hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) gene, suggesting that the inactive X chromosome has been modified at the DNA level. One possible DNA modification that has been proposed as a mechanism for X chromosome inactivation is methylation ofcytosine residues (8-10). Recently, a number of reports have appeared correlating hypomethylation ofspecific sites with gene expression (for reviews see refs. 11 and 12). 5-Azacytidine (5-Aza-Cyd) is an analog of cytidine known to inhibit methylation of DNA (13). It has been used in a number of studies designed to investigate the relationship between DNA methylation and gene activity. Mohandas et al. (14) treated mouse-human hybrid cells, retaining an inactive human X chromosome, with 5-Aza-Cyd and were able to demonstrate reactivation of the human HPRT locus. Some ofthe HPRT' clones obtained also showed reactivation ofother X chromosome-linked enzymes, glucose-6-phosp...

No abstract

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 occurs 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. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 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 mechanism of X-chromosome inactivation has been investigated recently using DNA-mediated transformation of the X-linked hypoxanthine phosphoribosyl transferase (hprt) locus. Several experiments indicate that inactive X-chromosomal DNA does not function in HPRT transformation. Liskay and Evans used DNA from hamster or mouse cells which had an hprt- allele on the active X chromosome and an hprt+ allele on the inactive X chromosome. We and others used rodent-human hybrid cell lines which had an hprt+ allele on the inactive human X chromosome alone. DNA from all of these cells failed to transform HPRT- recipients. Recently, Chapman et al. have shown that inactive X-chromosome DNA from several tissues of adult female mice is strikingly inefficient in genetic transformation for the hprt gene. On the other hand, de Jonge et al., using simian virus 40 (SV40)-transformed fibroblasts from a human heterozygous for an HPRT deficiency, observed HPRT transformation regardless of whether the hprt+ allele was on the active or the inactive X chromosome of the donor cells. We have done an experiment similar to that of deJonge et al., and report here results which clearly indicate that DNA from the inactive X chromosome functions very poorly in HPRT transformation, thus supporting the original interpretation of Liskay and Evans that inactive X-chromosomal DNA is structurally modified.