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.     

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.     

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.     

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.     

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.     

Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila

Summary We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the -amanitinresistant allele, RpII215 ^C4 , includes all sequences required to produce amanitin-resistant transformants.     

Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit

A fragment of the Drosophila melanogaster RpIIC4 locus, which encodes the RNA polymerase II subunit that determines amanitin sensitivity, was inserted into a bacterial plasmid cloning vehicle useful for over-production of hybrid proteins. Two plasmid constructions encoded hybrid proteins that reacted with antibodies against D. melanogaster RNA polymerase II. Use of subunit-specific antibodies indicated that these hybrid proteins displayed antigenic determinants unique to the largest polypeptide (215 kDa) of the enzyme. This RpII locus, the site at which mutations to amanitin-resistance occur, must therefore encode the largest polymerase II subunit.     

Modifiers of bx1 alter the distribution of Ubx proteins in haltere imaginal discs of Drosophila.

The bithorax (bx) mutations in the Ultrabithorax (Ubx) gene of Drosophila melanogaster cause homeotic transformations of anterior third thoracic structures (T3a) toward anterior second thoracic structures (T2a) in the adult fly. A corresponding loss of Ubx protein expression in T3a of bx imaginal discs has been observed (White and Wilcox, 1985). We describe two genetic loci which modify the bx-induced transformation. A locus which we map very close to the pink peach (pp) gene suppresses the bx1 phenotype. In contrast, mutations in the suppressor of sable (su(s)) gene enhance the bx1 phenotype. A correlation was observed between patterns of Ubx protein expression and the phenotypic transformations observed.     

Developmental Genetic Analysis of Contrabithorax Mutations in Drosophila Melanogaster

A developmental analysis of the Contrabithorax (Cbx) alleles offers the opportunity to examine the role of the Ultrabithorax (Ubx) gene in controlling haltere, as alternative to wing, morphogenesis in Drosophila. Several Cbx alleles are known with different spatial specificity in their wing toward haltere homeotic transformation. The molecular data on these mutations, however, does not readily explain differences among mutant phenotypes. In this work, we have analyzed the "apogenetic" mosaic spots of transformation in their adult phenotype, in mitotic recombination clones and in the spatial distribution of Ubx proteins in imaginal discs. The results suggest that the phenotypes emerge from early clonality in some Cbx alleles, and from cell-cell interactions leading to recruitment of cells to Ubx gene expression in others. We have found, in addition, mutual interactions between haltere and wing territories in pattern and dorsoventral symmetries, suggesting short distance influences, "accommodation," during cell proliferation of the anlage. These findings are considered in an attempt to explain allele specificity in molecular and developmental terms.     

Transvection in the Drosophila Ultrabithorax Gene: A Cbx(1) Mutant Allele Induces Ectopic Expression of a Normal Allele in Trans

In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one.     

The role of Ultrabithorax in the patterning of adult thoracic muscles in Drosophila melanogaster

Mutations in the homeotic gene, Ultrabithorax (Ubx), result in the transformation of the third thoracic (T3) segment into the second thoracic (T2) segment. Although it has been well established that these mutations have striking effects on adult epidermal structures in T3, the effect of these mutations on the adult musculature has been controversial. In this study, a series of Ubx regulatory mutations, anterobithorax, bithorax, postbithorax, and bithoraxoid, as well as combinations of these alleles were used to reevaluate the role of Ubx in the patterning of the T3 musculature. Homeotic indirect and direct flight muscles (IFMs and DFMs) were identified in the transformed T3 segment of all alleles and allelic combinations with the exception of postbithorax. We critically evaluated the pattern and amount of these muscles and found that while the amount and/or quality of homeotic IFMs increased, the amount of homeotic DFMs did not vary significantly as the severity of the ectodermal transformation increased. Because Ubx is not expressed in the adult mesoderm of T3, these results suggest that inductive cues play a major role in the patterning of adult thoracic muscles. We provide a model that illustrates the central role of inductive cues in generating the final adult muscle pattern in the thorax.     

Effect of Abx, Bx and Pbx Mutations on Expression of Homeotic Genes in Drosophila Larvae

We have examined the patterns of expression of the homeotic gene Ubx in imaginal discs of Drosophila larvae carrying mutations in the abx, bx and pbx regulatory domains. In haltere discs, all five bx insertion mutations examined led to a general reduction in Ubx expression in the anterior compartment; for a given allele, the strength of the adult cuticle phenotype correlated with the degree of Ubx reduction. Deletions mapping near or overlapping the sites of bx insertions, including three abx alleles and the bx34e-prv(bx-prv) allele, showed greatly reduced Ubx expression in parts of the anterior compartment of the haltere disc; however, anterior patches of strong Ubx expression often remained, in highly variable patterns. As expected, the pbx1 mutation led to reduced Ubx expression in the posterior compartment of the haltere disc; surprisingly, pbx1 also led to altered expression of the en protein near the compartment border in the central region of the disc. In the metathoracic leg, all the bx alleles caused extreme reduction in Ubx expression in the anterior regions, with no allele-specific differences. In contrast, abx and bx-prv alleles resulted in patchy anterior reductions in third leg discs. In the larval central nervous system, abx but not bx alleles affected Ubx expression; the bx-prv deletion gave a wild-type phenotype, but it could not fully complement abx mutations. In the posterior wing disc, the bx-prv allele, and to a much lesser extent the bx34e chromosome from which it arose, led to ectopic expression of Ubx. Unlike other grain-of-function mutations in the BX-C, this phenotype appeared to be partially recessive to wild type. Finally, we asked whether the ppx transformation, which results from early lack of Ubx+ function in the mesothorax and is seen in abx animals, is due to ectopic Scr expression. Some mesothoracic leg and wing discs from abx2 larvae displayed ectopic expression of Scr, which was variable in extent but always confined to the posterior compartment.     

Negative regulation of dorsoventral signaling by the homeotic gene Ultrabithorax during haltere development in Drosophila.

Growth and patterning during Drosophila wing development are mediated by signaling from its dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ultrabithorax (Ubx) to mediate development of a pair of tiny balancing organs, the halteres. Here we show that expression of Ubx in the haltere D/V boundary down-regulates its D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx(-) clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhanced the homeotic transformation of Ubx(-) cells in the capitellum but also caused homeotic transformation of even Ubx(+) cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles of Ubx during maintenance of cell fate and patterning in the halteres.     

Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.