scholarly journals A Caenorhabditis elegans RNA polymerase II gene, ama-1 IV, and nearby essential genes.

Genetics ◽  
1988 ◽  
Vol 118 (1) ◽  
pp. 61-74
Author(s):  
T M Rogalski ◽  
D L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gamma Ray ◽  
Temperature Shift ◽  
Developmental Rate ◽  
Egg Laying ◽  
Essential Genes ◽  
Sensitive Period ◽  
Temperature Sensitive

Abstract The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20 degrees but are arrested as larvae at 25 degrees, and two others are fertile at 20 degrees and sterile at 25 degrees. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25 degrees is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four gamma-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development.

scholarly journals Caenorhabditis elegans mutants defective in the functioning of the motor neurons responsible for egg laying.

Genetics ◽  
1989 ◽  
Vol 121 (4) ◽  
pp. 703-721 ◽  
Author(s):  
C Desai ◽  
H R Horvitz
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptic Transmission ◽  
Motor Neurons ◽  
Egg Laying ◽  
The Other ◽  
Single Type ◽  
Sensitive Period ◽  
Temperature Sensitive ◽  
Sensitive Allele

Abstract We have isolated and characterized 45 Caenorhabditis elegans mutants presumed to be defective in the functioning of the hermaphrodite-specific neurons (HSNs). Like hermaphrodites that lack the HSN motor neurons, these mutants are egg-laying defective and do not lay eggs in response to exogenous imipramine but do lay eggs in response to exogenous serotonin. Twenty of the 45 mutations define 10 new egl genes; the other 25 mutations are alleles of five previously defined genes, four of which are known to affect the HSNs. Seven mutations in three genes cause the HSNs to die in hermaphrodites, as they normally do in males. These genes appear to be involved in the determination of the sexual phenotype of the HSNs, and one of them (egl-41) is a newly identified gene that may function generally in sex determination. Five of the 15 genes are defined only by mutations that have dominant effects on egg laying. One gene egl(n1108), is defined by a temperature-sensitive allele that has a temperature-sensitive period after HSN development is complete, suggesting that egl(n1108) may be involved in HSN synaptic transmission. Four of the genes are defined by single alleles, which suggests that other such genes remain to be discovered. Mutations in no more than 4 of the 15 genes specifically affect the HSNs, indicating that there are few genes with functions needed only in this single type of nerve cell.

scholarly journals Mutant Caenorhabditis elegans RNA polymerase II with a 20,000-fold reduced sensitivity to alpha-amanitin.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 889-898
Author(s):  
T M Rogalski ◽  
M Golomb ◽  
D L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Cell Nuclei ◽  
Spliced Leader ◽  
Triton X ◽  
Alpha Amanitin

Abstract A doubly mutant ama-1(m118m526) gene results in an RNA polymerase (Rpo) II that is unusually resistant to alpha-amanitin. Rpo II activity in isolated Caenorhabditis elegans cell nuclei is inhibited 50% by alpha-amanitin at a concentration of 150 micrograms/ml, making this enzyme 150 times more resistant to the toxin than Rpo II from the singly mutant allele, ama-1(m118), 20,000 times more resistant than the wild-type Rpo II, and about six times more resistant to amanitin than is Rpo III. It was determined that the SL1 spliced leader precursor is transcribed by Rpo II, and this transcript was used to measure Rpo II activity. The Rpo II activity is unstable in vitro, and the mutant strain has a temperature-sensitive sterile phenotype. The highly resistant double mutant was selected among four million progeny of the mutagenized ama-1(m118) parent by its ability to grow and reproduce in 200 micrograms/ml amanitin in the presence of a permeabilizing agent, Triton X-100.

scholarly journals Identification and Characterization of Genes That Interact With lin-12 in Caenorhabditis elegans

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1675-1695 ◽  
Author(s):  
Frans E Tax ◽  
James H Thomas ◽  
Edwin L Ferguson ◽  
H Robert Horvitzt
Keyword(s):  
Cell Fate ◽  
Low Frequency ◽  
Signal Transduction Pathway ◽  
Temperature Shift ◽  
Egg Laying ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Suppressor Mutations

Abstract We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-l7, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup1 7 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup1 7and lag-2, suggest that both genes act at approximately the same time as lin-12in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

scholarly journals The Essential WD Repeat Protein Swd2 Has Dual Functions in RNA Polymerase II Transcription Termination and Lysine 4 Methylation of Histone H3

2004 ◽  
Vol 24 (7) ◽  
pp. 2932-2943 ◽  
Author(s):  
Hailing Cheng ◽  
Xiaoyuan He ◽  
Claire Moore
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Modification ◽  
Histone H3 ◽  
Temperature Sensitive ◽  
Repeat Protein ◽  
Tightly Coupled ◽  
Cleavage And Polyadenylation ◽  
Wd Repeat

ABSTRACT Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3′ end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled.

Genetic exploration of interactive domains in RNA polymerase II subunits

1990 ◽  
Vol 10 (5) ◽  
pp. 1908-1914
Author(s):  
C Martin ◽  
S Okamura ◽  
R Young
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Temperature Sensitive ◽  
Suppressor Mutations ◽  
Region I ◽  
Temperature Sensitive Mutation ◽  
Allele Specific ◽  
Separate Protein

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.

scholarly journals Fine-structure genetics of ama-1, an essential gene encoding the amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 423-434
Author(s):  
A M Bullerjahn ◽  
D L Riddle
Keyword(s):  
Fine Structure ◽  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Essential Gene ◽  
Resistance Mutation ◽  
Lethal Mutation ◽  
Intragenic Recombination ◽  
Lethal Mutations ◽  
Binding Subunit

Abstract A fine-structure genetic map has been constructed for ama-1 IV, an essential gene in Caenorhabditis elegans encoding the amanitin-binding subunit of RNA polymerase II. Sixteen EMS-induced recessive-lethal mutations have been positioned in the gene by determining their intragenic recombination frequencies with m118, a mutation that confers dominant resistance to alpha-amanitin. The 16 mutants, all isolated in the ama-1(m118) background, include 13 that are early larval lethals, and three that are mid-larval lethals, at 25 degrees. Six of the mutants exhibit temperature-dependence in the severity of their phenotype. Intragenic recombination between the lethal site and the parental resistance mutation was detected by means of resistance to amanitin. Recombinants were detected at frequencies as low as 2 X 10(-6). The segregation of the closely linked flanking markers, unc-17 and unc-5, revealed whether the lethal mutation was to the left or the right of m118. By adding the distances between the extreme left and right mutations, the ama-1 gene is estimated to be 0.011 map unit long, with m118 positioned 0.004 map unit from the left-most lethal mutation. To order the lethal mutations with respect to each other, viable heteroallelic strains were constructed using the free duplication, mDp1[unc-17(e113) dpy-13(+) ama-1(+)]. The heteroallelic strains were sensitive to amanitin, and recombination events between the lethal mutations were specifically selected by means of the dominant amanitin resistance encoded on the recombinant chromosome. The segregation of outside markers revealed the left-right order of the lethal mutations. The position of mutations within the gene is nonrandom. Functional domains of the ama-1 gene indicated by the various lethal phenotypes are discussed.

Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein

2000 ◽  
Vol 113 (15) ◽  
pp. 2679-2683 ◽  
Author(s):  
K. Sugaya ◽  
M. Vigneron ◽  
P.R. Cook
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Temperature Sensitive ◽  
Mammalian Cell Lines ◽  
Eukaryotic Genes ◽  
Green Fluorescent

RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional ‘holoenzymes’ involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.

KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth

1989 ◽  
Vol 9 (6) ◽  
pp. 2341-2349
Author(s):  
C Martin ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Gene ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Temperature Dependent ◽  
Growth Properties ◽  
Cold Sensitive ◽  
Dependent Growth ◽  
Kex2 Protease

Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum

1989 ◽  
Vol 9 (8) ◽  
pp. 3543-3547
Author(s):  
T W Nilsen ◽  
J Shambaugh ◽  
J Denker ◽  
G Chubb ◽  
C Faser ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Secondary Structure ◽  
Rna Polymerase ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Ascaris Lumbricoides ◽  
Parasitic Nematode ◽  
Secondary Structures ◽  
Spliced Leader ◽  
C Elegans

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.

scholarly journals The Rpb6 Subunit of Fission Yeast RNA Polymerase II Is a Contact Target of the Transcription Elongation Factor TFIIS

2000 ◽  
Vol 20 (4) ◽  
pp. 1263-1270 ◽  
Author(s):  
Akira Ishiguro ◽  
Yasuhisa Nogi ◽  
Koji Hisatake ◽  
Masami Muramatsu ◽  
Akira Ishihama
Keyword(s):  
Rna Polymerase ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Direct Interaction ◽  
Temperature Sensitive ◽  
Single Mutation ◽  
Transcription Elongation Factor ◽  
Essential Region

ABSTRACT The Rpb6 subunit of RNA polymerase II is one of the five subunits common to three forms of eukaryotic RNA polymerase. Deletion and truncation analyses of the rpb6 gene in the fission yeastSchizosaccharomyces pombe indicated that Rpb6, consisting of 142 amino acid residues, is an essential protein for cell viability, and the essential region is located in the C-terminal half between residues 61 and 139. After random mutagenesis, a total of 14 temperature-sensitive mutants were isolated, each carrying a single (or double in three cases and triple in one) mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (6AU), which inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and temperature-sensitive phenotypes of these rpb6 mutants were suppressed by overexpression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS-RNA polymerase II complexes using a fusion form of TFIIS with glutathioneS-transferase. Moreover, the direct interaction between TFIIS and RNA polymerase II was competed by the addition of Rpb6. Taken together, the results lead us to propose that Rpb6 plays a role in the interaction between RNA polymerase II and the transcription elongation factor TFIIS.

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