The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an aL-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer aL-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of a-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of a-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.The genes for several eucaryotic RNA polymerase subunits have been cloned and sequenced (1,2,6,8,10,12,20). These studies have revealed that the two largest subunits (IHo and IIc) of RNA polymerase TI are related to the ,B' and P subunits, respectively, of Escherichia coli RNA polymerase (2,8,11,12,27). Genetic and biochemical studies of the procaryotic transcription apparatus have shown that the ,B and P' subunits, together with two a subunits, make up the catalytic core of RNA polymerase (29, 30). Conservation of P-and P'-homologous sequences among eucaryotic subunits indicates that the basic catalytic core functions provided by these subunits have been maintained through evolution.The largest subunit of eucaryotic RNA polymerase II (RPII215) contains an additional domain for which no procaryotic counterpart exists. This domain consists of a repeated amino acid block with the consensus sequence TyrSer-Pro-Thr-Ser-Pro-Ser (see Fig. 1D). This sequence is repeated 26 times in Saccharomyces cerevisiae (2, 18a), about 40 times in Drosophila melanogaster (15), and 52 times in the mouse (11) and Chinese hamster (2a). The S. cerevisiae repetitive domain is made up almost entirely of consensus repeats (2), while the Drosophila domain shows more variability from the consensus sequence (15). In the mammalian repetitive domain, some repeats adhere strongly to the consensus sequence, whereas other repeats diverge considerably (2a, 11).The evolutionary conservation of the RNA polymerase II carboxyl-terminal domain (CTD) among eucaryotic species and the fact that no similar domain is found in the equivalent subunits of RNA polymerases I...
Mammalian sex-dosage compensation is mediated by maintaining activity of only one X chromosome. The asynchronous DNA synthesis characterizing the silent human X chromosome is thought to be reversible only during ontogeny of oocytes. We have previously shown that the glucose-6-phosphate dehydrogenase (G6PD) locus (G6PD) on the allocyclic X chromosome in chorionic villi is partially expressed. We now show that in hybrids derived from a clone of chorionic villi cells (heterozygous for G6PD A) and mouse A9 cells, the loci for G6PD, hypoxanthine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase are expressed on both human X chromosomes; the human X chromosomes carrying either G6PD A or B replicate synchronously with each other and with murine chromosomes. The X chromosome with G6PD A was identified as the original late-replicating X, because methylation in the body of the HPRT gene on this chromosome remained characteristic of the inactive X chromosome. These results indicate that X-chromosome inactivation is completely reversible in cells of trophoblast origin; induction of full transcriptional activity is accompanied by acquisition of isocyclic replication, showing an intimate relationship between these processes. The molecular events responsible for this reversal may be similar to those occurring during maturation of oocytes. Chorionic vilii and derivative hybrids provide in vitro models for exploring early events that program the single active X chromosome.In mammals, the sex difference in dosage of X chromosomes is compensated by silencing all but one X chromosome in female somatic cells (1). Dosage compensation is not established simultaneously in all tissues (2) but is programmed along with other tissue-specific functions. At the time of tissue differentiation, only one X chromosome achieves potential transcriptional activity; the others become inactive, condense at interphase, and replicate asynchronously [later than the autosomes and the active X chromosome (3)]. The inactivity of the allocyclic X chromosome is rigorously maintained (4), and spontaneous reversal of the processi.e., reactivating the entire chromosome and reversing allocyclic replication-has not been observed in somatic cells. The spontaneous reactivation events that have been noted (5, 6) affect some loci but not others, and the chromosome remains late-replicating (5). On the other hand, reactivation of the entire X chromosome is believed to occur regularly during the ontogeny of oocytes. Primordial germ cells in the yolk sac of female rodents have a condensed X chromosome (7), and there is evidence from mouse embryos that glucose-6-phosphate dehydrogenase (G6PD) genes on both X chromosomes are expressed shortly after germ cells am've at the gonadal ridges, whereas only one locus was active previously (8).Although recent observations provide insights into maintenance of X chromosome dosage compensation (9-11), little is known about initiation of the process. Determining the initial steps in X chromosome differentiation-thos...
Maintenance of dosage compensation for housekeeping genes on the human X chromosome is mediated through differential methylation of clustered CpG nucleotides associated with these genes. To determine if methylation has a role in maintaining inactivity of X‐linked genes which show tissue‐specific expression, we examined the locus for blood clotting Factor IX. The analysis encompassed 91% of the HpaII and HhaI sites in the 41‐kb region that includes the presumed promoter region, 5 kb of 5′‐ and 4 kb of 3′‐flanking sequences. Although there are sex differences in methylation of the locus in leukocytes, the methylation pattern in liver, where the gene is expressed, is essentially the same for loci on the active and inactive X chromosome. The lack of differences in methylation of active and inactive genes makes it unlikely that methylation within the locus has a role in expression of the Factor IX gene. These findings, along with the absence of clustered CpG dinucleotides within the Factor IX locus, suggest that functional differences in DNA methylation related to X chromosome dosage compensation may be limited to CpG clusters. In any event, dosage compensation seems to be maintained regionally, rather than locus by locus.
The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.
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