Mechanisms of Mutagenesis by a Bulky DNA Lesion at the Guanine N7 Position

The RpII215 locus encodes the large subunit of RNA polymerase II (polII). Three of 22 RpII215 alleles cause a synergistic enhancement of the mutant phenotype elicited by mutations in the Ultrabithorax (Ubx) locus. We have recovered and analyzed three new mutations that suppress this enhancement. All three mutations map to the RpII215 locus. In addition to suppressing the Ubx enhancement of other RpII215 alleles, two of the new mutations, JH1 and WJK2, themselves enhance Ubx. RpII215 alleles can be placed into three classes based on their ability to enhance Ubx. Class I alleles, including Ubl, C4, C11, JH1, and WJK2, enhance Ubx when heterozygous with class II alleles, which include wild-type RpII215. Class III alleles, which include amorphic alleles, do not enhance Ubx. The third new mutation, WJK1, is a conditional amorphic allele, which behaves like a class III allele at 29 degrees but like a class II allele at 19 degrees. Another mutant phenotype is caused by certain RpII215 alleles, including all class I alleles. This phenotype is a synergistic enhancement of a mutant phenotype elicited by mutations at the Delta (Dl) locus. Unlike the enhancement of Ubx, the enhancement of Dl is not dependent upon antagonistic interactions between different classes of RpII215 alleles.     

Clonal analysis of two mutations in the large subunit of RNA polymerase II of Drosophila

Summary Two mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215 ^ts(ts), only displays a conditional recessive lethality, while the other, RpII215 ^Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/- tissue in gynandromorphs are morphologically normal. Cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215 ⁺, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/- cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interaction described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term negative heterosis to describe the aforementioned interactions.We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells. Mutant somatic cells (either Ubl/- or ts/-, the latter shifted to restrictive temperature) reduce cell proliferation, but otherwise do not appear to disrupt cell differentiation. However, mutant germ cells often differentiate into morphologically abnormal oocytes.     

Genetic and molecular variation in the RpII215 region of Drosophila melanogaster

Summary The RpII215 region of the X chromosome of Drosophila melanogaster was investigated to identify genetic functions and correlate these with the known molecular organization of the region. Five genetic loci were identified in a subregion that is reported to transcribe nine or more messages. One locus is nod, which causes meiotic abnormalities, and three other loci are recessive lethal mutations whose developmental lesions are unknown. The fifth and most mutable of the loci is RpII215, which encodes the 215,000 dalton subunit of RNA polymerase II. Mutant effects of RpII215 alleles include: temperature-dependent (heat and cold) survival, altered sensitivity to -amanitin, male sterility, maternal effects and epistatic enhancement of mutant effects of other loci.     

Regulation of RNA polymerase II activity in α-amanitin-resistant CHO hybrid cells

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to α-amanitin with mutant cells containing RNA polymerase II activity resistant to α-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing α-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to α-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the α-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975).A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in α-amanitin exhibited a 2–3 fold increase in the activity of the α-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the α-amanitin-inactivated polymerase II of wild-type sensitivity by ³H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of ³H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.     

Evidence for variation in the number of functional gene copies at the AmaR locus in chinese hamster cell lines

The hypothesis of functional hemizygosity has been examined for the α-amanitin resistant (Ama^R, a codominant marker) locus in a series of Chinese hamster cell lines. Ama^R mutants were obtained from different cell lines, e.g., CHO, CHW, M3-1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards α-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to α-amanitin inhibition, only about 50% of the activity is highly resistant in Ama^R mutants of CHW and M3-1 cell lines. The remaining activity in the latter cell lines shows α-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by α-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.     

Reverse Genetics of Drosophila RNA Polymerase II: Identification and Characterization of Rpii140, the Genomic Locus for the Second-Largest Subunit

We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus.     

Functional Studies of the Carboxy-Terminal Repeat Domain of Drosophila RNA Polymerase II in Vivo

To understand the in vivo function of the unique and conserved carboxy-terminal repeat domain (CTD) of RNA polymerase II largest subunit (RpII215), we have studied RNA polymerase II biosynthesis, activity and genetic function in Drosophila RpII215 mutants that possessed all (C4), half (W81) or none (IIt) of the CTD repeats. We have discovered that steady-state mRNA levels from transgenes encoding a fully truncated, CTD-less subunit (IIt) are essentially equal to wild-type levels, whereas the levels of the CTD-less subunit itself and the amount of polymerase harboring it (Pol IIT) are significantly lower than wild type. In contrast, for the half-CTD mutant (W81), steady-state mRNA levels are somewhat lower than for wild type or IIt, while W81 subunit and polymerase amounts are much less than wild type. Finally, we have tested genetically the ability of CTD mutants to complement (rescue) partially functional RpII215 alleles and have found that IIt fails to complement whereas W81 complements partially to completely. These results suggest that removal of the entire CTD renders polymerase completely defective in vivo, whereas eliminating half of the CTD results in a polymerase with significant in vivo activity.     

α-Amanitin-sensitive DNA-dependent RNA polymerase I from cherry salmon (Oncorhynchus masou) liver nuclei

DNA-dependent RNA polymerases I and II were purified approximately 3900- and 13300 fold, respectively, from a sonicated nuclear extract of the cherry salmon liver by column chromatographies on DEAE-Sephadex, heparin-Sepharose and DNA-cellulose. The RNA polymerases were examined with respect to template-specificity, the effects of Mn²⁺, Mg²⁺ and ammonium sulfate, α-amanitin sensitivity. Results showed that the RNA polymerase I differed from other eukaryotic RNA polymerase I in α-amanitin sensitivity.     

Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe

A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4. To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpb1 gene, which encodes RpII LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures. First, we examined the effects of the mutation on haploid yeast cells. The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed. Tetrad analysis suggested that these phenotypes were associated with the mutation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These observations demonstrate that the S. pomberpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4.     

The RNA polymerase II of an α-amanitin-resistant Chinese hamster ovary cell line

Amal, an α-amanitin-resistant mutant of the Chinese hamster ovary cell line, contains an RNA polymerase activity which elutes from DEAE-Sephadex at a salt concentration characteristic of an RNA polymerase II, but which is not sensitive to α-amanitin at levels where the polymerase II of wild-type cells is strongly inhibited. This result suggests that Amal owes its amanitin-resistant phenotype to a mutation affecting one of its genes for RNA polymerase II. To test this hypothesis, we purified the enzyme from Amal and then compared its properties with those of the wild-type enzyme. The mutant enzyme is indeed a polymerase II, and is over 600 times less sensitive to α-amanitin and more thermolabile than the wild-type enzyme.