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.

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

1987 ◽  
Vol 7 (2) ◽  
pp. 586-594 ◽  
Author(s):  
M S Bartolomei ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Line ◽  
Resistance Mutation ◽  
Wild Type Mouse ◽  
Mutant Form ◽  
Wild Type ◽  
Gene Encoding ◽  
Base Pair Fragment ◽  
Alpha Amanitin

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.

scholarly journals A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

PLoS Genetics ◽  
2016 ◽  
Vol 12 (8) ◽  
pp. e1006227 ◽  
Author(s):  
Jeong H. Ahn ◽  
Andreas Rechsteiner ◽  
Susan Strome ◽  
William G. Kelly
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii

scholarly journals A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

1990 ◽  
Vol 10 (12) ◽  
pp. 6123-6131 ◽  
Author(s):  
J Archambault ◽  
K T Schappert ◽  
J D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Essential Gene ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Gene Encoding ◽  
Temperature Sensitive Mutation

RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.

scholarly journals Lethal and amanitin-resistance mutations in the Caenorhabditis elegans ama-1 and ama-2 genes.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 409-422
Author(s):  
T M Rogalski ◽  
A M Bullerjahn ◽  
D L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Resistance Mutations ◽  
Recessive Lethal ◽  
New Gene ◽  
Alpha Amanitin

Abstract Mutants of Caenorhabditis elegans resistant to alpha-amanitin have been isolated at a frequency of about 1.6 x 10(-6) after EMS mutagenesis of the wild-type strain, N2. Four new dominant resistance mutations have been studied genetically. Three are alleles of a previously identified gene, ama-1 IV, encoding the largest subunit of RNA polymerase II. The fourth mutation defines a new gene, ama-2 V. Unlike the ama-1 alleles, the ama-2 mutation exhibits a recessive-lethal phenotype. Growth and reproduction of N2 was inhibited at a concentration of 10 micrograms/ml amanitin, whereas ama-2/+ animals were inhibited at 100 micrograms/ml, and 800 micrograms/ml was required to inhibit growth of ama-1/+ larvae. We have also determined that two reference strains used for genetic mapping, dpy-11(e224)V and sma-1(e30)V, are at least four-fold more sensitive to amanitin that the wild-type strain. Using an amanitin-resistant ama-1(m118) or ama-1(m322) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. The frequency of EMS-induced lethal ama-1 mutations is approximately 1.7 x 10(-3), 1000-fold higher than the frequency of amanitin-resistance alleles. Nine of the lethal alleles are apparent null mutations, and they exhibit L1-lethal phenotypes at both 20 degrees and 25 degrees. Six alleles result in partial loss of RNA polymerase II function as determined by their sterile phenotypes at 20 degrees. All but one of these latter mutations exhibit a more severe phenotype at 25 degrees C. We have also selected seven EMS-induced revertants of three different ama-1 lethals. These revertants restore dominant resistance to amanitin. The selection for revertants also produced eight new dominant amanitin resistance alleles on the balancer chromosome, nT1.

scholarly journals The unc-22(IV) region of Caenorhabditis elegans: genetic analysis of lethal mutations.

Genetics ◽  
1988 ◽  
Vol 119 (2) ◽  
pp. 345-353
Author(s):  
D V Clark ◽  
T M Rogalski ◽  
L M Donati ◽  
D L Baillie
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Essential Gene ◽  
Group Iv ◽  
Essential Genes ◽  
Ems Mutagenesis ◽  
Lethal Mutations ◽  
Complementation Groups ◽  
Minimum Estimate ◽  
Caenorhabditis Elegans Genome

Abstract The organization of essential genes in the unc-22 region, defined by the deficiency sDf2 on linkage group IV, has been studied. Using the balancer nT1 (IV;V), which suppresses recombination over 49 map units, 294 lethal mutations on LGIV(right) and LGV(left) were recovered using EMS mutagenesis. Twenty-six of these mutations fell into the unc-22 region. Together with previously isolated lethal mutations, there is now a total of 63 lethal mutations which fall into 31 complementation groups. Mutations were positioned on the map using eight overlapping deficiencies in addition to sDf2. The lethal alleles and deficiencies in the unc-22 region were characterized with respect to their terminal phenotypes. Mapping of these lethal mutations shows that sDf2 deletes a minimum of 1.8 map units and a maximum of 2.5 map units. A minimum estimate of essential gene number for the region using a truncated Poisson calculation is 48. The data indicate a minimum estimate of approximately 3500 essential genes in the Caenorhabditis elegans genome.

scholarly journals Chromosome Integrity is Required for the Initiation of Meiotic Sex Chromosome Inactivation in Caenorhabditis elegans

2020 ◽  
Author(s):  
Yisrael Rappaport ◽  
Hanna Achache ◽  
Roni Falk ◽  
Omer Murik ◽  
Oren Ram ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sex Chromosomes ◽  
Sex Chromosome ◽  
Chromosome Inactivation ◽  
Chromatin Modifications ◽  
Meiotic Sex Chromosome Inactivation ◽  
Chromosome Integrity ◽  
Sex Chromosome Inactivation

During meiosis of heterogametic cells, such as XY meiocytes, sex chromosomes of many species undergo transcriptional silencing known as meiotic sex chromosome inactivation (MSCI). Silencing also occurs in aberrantly unsynapsed autosomal chromatin. The silencing of unsynapsed chromatin, is assumed to be the underline mechanism for MSCI. Initiation of MSCI is disrupted in meiocytes with sex chromosome-autosome translocations. Whether this is due to aberrant synapsis or the lack of sex chromosome integrity has never been determined. To address this, we used CRISPR to engineer Caenorhabditis elegans stable strains with broken X chromosomes that didn’t undergo translocations with autosomes. In early meiotic nuclei of these mutants, the X fragments lack silent chromatin modifications and instead the fragments are enriched with transcribing chromatin modifications. Moreover, the level of active RNA polymerase II staining on the X fragments in mutant nuclei is similar to that on autosomes, indicating active transcription on the X. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, X fragments that did not synapse were robustly stained with RNA polymerase II and gene expression levels were high throughout the broken X. Therefore, lack of synapsis does not trigger MSCI if sex chromosome integrity is lost. Moreover, our results suggest that a unique character of the chromatin of sex chromosomes underlies their lack of meiotic silencing due to both unsynapsed chromatin and sex chromosome mechanisms when their integrity is lost.

scholarly journals GENETIC ORGANIZATION IN CAENORHABDITIS ELEGANS: FINE-STRUCTURE ANALYSIS OF THE unc-22 GENE

Genetics ◽  
1979 ◽  
Vol 91 (1) ◽  
pp. 95-103 ◽  
Author(s):  
D G Moerman ◽  
D L Baillie
Keyword(s):  
Drosophila Melanogaster ◽  
Fine Structure ◽  
Caenorhabditis Elegans ◽  
Gene Conversion ◽  
Structure Analysis ◽  
Intragenic Recombination ◽  
Preliminary Estimate ◽  
Recessive Mutations ◽  
Fine Structure Analysis ◽  
New Alleles

ABSTRACT Fine-structure analysis of the unc-22 gene of Caenorhabditis elegarns has revealed a number of sites that are separable by recombination. Eight new ethyl methanesulfonate-induced recessive mutations of the unc-22 gene have been isolated. Using these new alleles, as well as e66, a number of separable sites have been identified and positioned relative to one another. The map distances obtained are found to be comparable to those associated with intragenic recombination in Drosophila melanogaster, indicating that genetic finestructure analysis is feasible in Caenorhabditis elegans. Evidence of possible gene conversion is presented. A preliminary estimate of the unc-22 gene size is 2.4 × 10-2 map units.

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.

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