scholarly journals Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei.

1993 ◽  
Vol 13 (6) ◽  
pp. 3734-3743 ◽  
Author(s):  
H M Chung ◽  
M G Lee ◽  
P Dietrich ◽  
J Huang ◽  
L H Van der Ploeg
Keyword(s):  
Amino Acid ◽  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Promoter Region ◽  
Gene Promoter ◽  
Amino Acid Substitutions ◽  
Pol Ii ◽  
Pol I ◽  
Alpha Amanitin ◽  
Gene Promoter Region

Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I.

Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei

1993 ◽  
Vol 13 (6) ◽  
pp. 3734-3743
Author(s):  
H M Chung ◽  
M G Lee ◽  
P Dietrich ◽  
J Huang ◽  
L H Van der Ploeg
Keyword(s):  
Amino Acid ◽  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Promoter Region ◽  
Gene Promoter ◽  
Amino Acid Substitutions ◽  
Pol Ii ◽  
Pol I ◽  
Alpha Amanitin ◽  
Gene Promoter Region

Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I.

scholarly journals An RNA polymerase II promoter in the hsp70 locus of Trypanosoma brucei.

1996 ◽  
Vol 16 (3) ◽  
pp. 1220-1230 ◽  
Author(s):  
M G Lee
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Reporter Gene ◽  
Intergenic Region ◽  
Regulation Of Gene Expression ◽  
Gene Promoter ◽  
Background Level ◽  
Coding Region ◽  
Pol Ii

To study of structure of RNA polymerase (pol) II transcription units a nd the influence of temperature on the regulation of gene expression in Trypanosoma brucei, and hsp70 intergenic region promoter was characterized. In T. brucei, the hsp70 locus contains, from 5' to 3', a cognate hsp70-related gene (gene 1) which is separated by about 6 kb of DNA from a cluster of five identical hsp70 genes (genes 2 to 6). Transcription proceeds on the entire 23-kb locus, and polycistronic transcription occurs in hsp70 genes 2 to 6. Transcription of hsp70 genes 2 to 6 is only moderately sensitive to UV irradiation, indicating that it cannot be driven by a single far-upstream promoter, which suggests that promoters could be located in the region close to the hsp70 coding region. Transient transformations demonstrated that sequences located upstream of hsp70 gene 2 and in the intergenic region between hsp70 genes 2 and 3 are able to direct transcription of the reporter gene, the chloramphenicol acetyltransferase (CAT) gene. The plasmid DNA driven by the hsp70 intergenic region promoter gave CAT activity approximately 85-fold above to background level. This is equivalent to approximately 1% of that derived from a CAT plasmid driven by the procyclic acidic repetitive protein gene promoter, which is controlled by RNA pol I. The hsp70 intergenic region promoter can drive alpha-amanitin-sensitive transcription at an internal position of the chromosome as well as an episome, suggesting that it is controlled by RNA pol II. However, this hsp70 intergenic region promoter, along with the 3' splice site and the 5' untranslated region of the hsp70 genes that controls the transcription of the reporter gene, cannot up-regulate the expression of the reporter gene during heat shock. This result is consistent with the previous observation that expression of the hsp70 genes in T. brucei is mainly controlled at the posttranscriptional level.

Murine α1(VI) Collagen Chain. Complete Amino Acid Sequence and Identification of the Gene Promoter Region

Matrix ◽  
1993 ◽  
Vol 13 (3) ◽  
pp. 223-233 ◽  
Author(s):  
Paolo Bonaldo ◽  
Stefano Piccolo ◽  
Donatella Marvulli ◽  
Dino Volpin ◽  
Valeria Marigo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Promoter Region ◽  
Gene Promoter ◽  
Complete Amino Acid Sequence ◽  
Chain Complete ◽  
Collagen Chain ◽  
Gene Promoter Region

scholarly journals RNA Polymerase I Transcribes Procyclin Genes and Variant Surface Glycoprotein Gene Expression Sites in Trypanosoma brucei

2003 ◽  
Vol 2 (3) ◽  
pp. 542-551 ◽  
Author(s):  
Arthur Günzl ◽  
Thomas Bruderer ◽  
Gabriele Laufer ◽  
Bernd Schimanski ◽  
Lan-Chun Tu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Protein C ◽  
Gene Promoter ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Pol I

ABSTRACT In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to α-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, αβ-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

scholarly journals TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

2011 ◽  
Vol 10 (7) ◽  
pp. 964-976 ◽  
Author(s):  
Tara M. Stanne ◽  
Manish Kushwaha ◽  
Matthew Wand ◽  
Jesse E. Taylor ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Histone Variants ◽  
Unicellular Eukaryote ◽  
Pol Ii ◽  
Content Type ◽  
Pol I ◽  
Switch Regions

ABSTRACTThe unicellular eukaryoteTrypanosoma bruceiis unusual in having very little transcriptional control. The bulk of theT. bruceigenome is constitutively transcribed by RNA polymerase II (Pol II) as extensive polycistronic transcription units. Exceptions to this rule include several RNA Pol I transcription units such as theVSGexpression sites (ESs), which are mono-allelically expressed. TbISWI, a member of the SWI2/SNF2 related chromatin remodeling ATPases, plays a role in repression of Pol I-transcribed ESs in both bloodstream- and procyclic-formT. brucei. We show that TbISWI binds both active and silent ESs but is depleted from the ES promoters themselves. TbISWI knockdown results in an increase inVSGtranscripts from the silentVSGESs. In addition to its role in the repression of the silent ESs, TbISWI also contributes to the downregulation of the Pol I-transcribed procyclin loci, as well as nontranscribedVSGbasic copy arrays and minichromosomes. We also show that TbISWI is enriched at a number of strand switch regions which form the boundaries between Pol II transcription units. These strand switch regions are the presumed sites of Pol II transcription initiation and termination and are enriched in modified histones and histone variants. Our results indicate that TbISWI is a versatile chromatin remodeler that regulates transcription at multiple Pol I loci and is particularly abundant at many Pol II transcription boundaries inT. brucei.

scholarly journals Increased expression of LD1 genes transcribed by RNA polymerase I in Leishmania donovani as a result of duplication into the rRNA gene locus.

1995 ◽  
Vol 15 (12) ◽  
pp. 6845-6853 ◽  
Author(s):  
M J Lodes ◽  
G Merlin ◽  
T deVos ◽  
A Ghosh ◽  
R Madhubala ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Leishmania Donovani ◽  
Gene Copy Number ◽  
Transcript Abundance ◽  
Gene Copy ◽  
Rrna Gene ◽  
Pol Ii ◽  
Pol I ◽  
Alpha Amanitin

Eukaryotic protein-coding genes are generally transcribed by RNA polymerase II (Pol II), which has a lower transcription rate than that of Pol I. We report here the duplication of two LD1 genes into the rRNA locus and their resultant transcription by Pol I. The multigenic LD1 locus is present in a 2.2-Mb chromosome in all stocks of Leishmania spp. and is also present in multicopy 200- to 450-kb linear chromosomes or multicopy circular DNAs in over 15% of stocks examined. Genomic rearrangement in Leishmania donovani LSB-51.1 resulted in duplication of a 3.9-kb segment of LD1 containing two genes (orfF and orfG) and of a 1.3-kb segment from approximately 10 kb downstream into the rRNA gene repeat region of the 1.2-Mb chromosome. Short sequences (12 or 13 bp) common to the 2.2-Mb LD1 and 1.2-Mb rRNA loci suggest that this gene conversion occurred by homologous recombination. Transcription of the duplicated genes is alpha-amanitin resistant, indicating transcription by Pol I, in contrast to the alpha-amanitin-sensitive (Pol II) transcription of the genes in the 2.2-Mb LD1 locus. This results in higher transcript abundance than expected from the gene copy number in LSB-51.1 and in elevated expression of at least the orfF gene product.

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