Use of mutant RNAs in studies on yeast 5S rRNA structure and function

1991 ◽  
Vol 69 (4) ◽  
pp. 217-222 ◽  
Author(s):  
Ross N. Nazar ◽  
Don I. Van Ryk ◽  
Yoon Lee ◽  
C. David Guyer
Keyword(s):  
Structural Changes ◽  
Growth Conditions ◽  
5S Rrna ◽  
Rrna Genes ◽  
Shuttle Vectors ◽  
5S Rrna Genes ◽  
And Function ◽  
Optimized Conditions ◽  
Rna Concentration

The expression of mutant yeast 5S rRNA genes in vivo is reviewed as a basis for further studies on the structure, function, and regulation of the ribosomal 5S rRNA. Specific base substitutions, insertions, or deletions can result in substantial structural changes which can be detected readily by gel electrophoresis, permitting the assay of mutant RNA synthesis and utilization. Furthermore, the use of high and low copy shuttle vectors, as well as alternate growth conditions, permits a wide adjustment of the mutant RNA concentration. Under optimized conditions more than 80% of the cell's RNA can be replaced with mutant molecules. The application of this strategy to studies on the biosynthesis and structure of the 5S rRNA are demonstrated through recently isolated mutations.Key words: site-specific mitogenesis, 5S RNA, ribosomes, yeast transformation.

scholarly journals Restricted Specificity of Xenopus TFIIIA for Transcription of Somatic 5S rRNA Genes

2004 ◽  
Vol 24 (6) ◽  
pp. 2467-2477 ◽  
Author(s):  
Romi Ghose ◽  
Mariam Malik ◽  
Paul W. Huber
Keyword(s):  
Transcription Factor ◽  
Glutamic Acid ◽  
Rna Binding ◽  
Early Stage ◽  
Nuclear Extract ◽  
5S Rrna ◽  
Rrna Genes ◽  
Wild Type ◽  
5S Rrna Genes

ABSTRACT Xenopus transcription factor IIIA (TFIIIA) is phosphorylated on serine-16 by CK2. Replacements with alanine or glutamic acid were made at this position in order to address the question of whether phosphorylation possibly influences the function of this factor. Neither substitution has an effect on the DNA or RNA binding activity of TFIIIA. The wild-type factor and the alanine variant activate transcription of somatic- and oocyte-type 5S rRNA genes in nuclear extract immunodepleted of endogenous TFIIIA. The glutamic acid variant (S16E) supports the transcription of somatic-type genes at levels comparable to those of wild-type TFIIIA; however, there is no transcription of the oocyte-type genes. This differential behavior of the phosphomimetic mutant protein is also observed in vivo when using early-stage embryos, where this mutant failed to activate transcription of the endogenous oocyte-type genes. Template exclusion assays establish that the S16E mutant binds to the oocyte-type 5S rRNA genes and recruits at least one other polymerase III transcription factor into an inactive complex. Phosphorylation of TFIIIA by CK2 may allow the factor to continue to act as a positive activator of the somatic-type genes and simultaneously as a repressor of the oocyte-type 5S rRNA genes, indicating that there is a mechanism that actively promotes repression of the oocyte-type genes at the end of oogenesis.

scholarly journals A Methylated Neurospora 5S rRNA Pseudogene Contains a Transposable Element Inactivated by Repeat-Induced Point Mutation

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1787-1797
Author(s):  
Brian S Margolin ◽  
Phillip W Garrett-Engele ◽  
Judith N Stevens ◽  
Deborah Y Fritz ◽  
Carrie Garrett-Engele ◽  
...  
Keyword(s):  
Transposable Element ◽  
Point Mutation ◽  
5S Rrna ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Related Sequence ◽  
5S Rrna Genes ◽  
And Function ◽  
Type Strains ◽  
Repeat Induced Point Mutation

Abstract In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Ψ63, was identified. We characterized the Ψ63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Ψ63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Ψ63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Ψ63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Ψ63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt.

scholarly journals RNA Polymerase III Transcription Complexes on Chromosomal 5S rRNA Genes In Vivo: TFIIIB Occupancy and Promoter Opening

2001 ◽  
Vol 21 (9) ◽  
pp. 3166-3178 ◽  
Author(s):  
G. Costanzo ◽  
S. Camier ◽  
P. Carlucci ◽  
L. Burderi ◽  
R. Negri
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Dimethyl Sulfate ◽  
Experimental System ◽  
5S Rrna ◽  
Rrna Genes ◽  
Polymerase Iii ◽  
5S Rrna Genes ◽  
Transcription Complexes

ABSTRACT Quantitative analysis of multiple-hit potassium permanganate (KMnO4) footprinting has been carried out in vivo onSaccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established.

scholarly journals Ribosomal DNA localization on Lathyrus species chromosomes by FISH

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Hoda B. M. Ali ◽  
Samira A. Osman
Keyword(s):  
Genome Mapping ◽  
Chromosome Complement ◽  
5S Rdna ◽  
Rdna Locus ◽  
5S Rrna ◽  
Rrna Genes ◽  
45S Rdna ◽  
Metaphase Chromosomes ◽  
Lathyrus Odoratus ◽  
5S Rrna Genes

Abstract Background Fluorescence In Situ Hybridization (FISH) played an essential role to locate the ribosomal RNA genes on the chromosomes that offered a new tool to study the chromosome structure and evolution in plant. The 45S and 5S rRNA genes are independent and localized at one or more loci per the chromosome complement, their positions along chromosomes offer useful markers for chromosome discriminations. In the current study FISH has been performed to locate 45S and 5S rRNA genes on the chromosomes of nine Lathyrus species belong to five different sections, all have chromosome number 2n=14, Lathyrus gorgoni Parl, Lathyrus hirsutus L., Lathyrus amphicarpos L., Lathyrus odoratus L., Lathyrus sphaericus Retz, Lathyrus incospicuus L, Lathyrus paranensis Burkart, Lathyrus nissolia L., and Lathyrus articulates L. Results The revealed loci of 45S and 5S rDNA by FISH on metaphase chromosomes of the examined species were as follow: all of the studied species have one 45S rDNA locus and one 5S rDNA locus except L. odoratus L., L. amphicarpos L. and L. sphaericus Retz L. have two loci of 5S rDNA. Three out of the nine examined species have the loci of 45S and 5S rRNA genes on the opposite arms of the same chromosome (L. nissolia L., L. amphicarpos L., and L. incospicuus L.), while L. hirsutus L. has both loci on the same chromosome arm. The other five species showed the loci of the two types of rDNA on different chromosomes. Conclusion The detected 5S and 45S rDNA loci in Lathyrus could be used as chromosomal markers to discriminate the chromosome pairs of the examined species. FISH could discriminate only one chromosome pair out of the seven pairs in three species, in L. hirsutus L., L. nissolia L. and L. incospicuus L., and two chromosome pairs in five species, in L. paranensis Burkart, L. odoratus L., L. amphicarpos L., L. gorgoni Parl. and L. articulatus L., while it could discriminate three chromosome pairs in L. sphaericus Retz. these results could contribute into the physical genome mapping of Lathyrus species and the evolution of rDNA patterns by FISH in the coming studies in future.

Microheterogeneity in Aspergillus nidulans 5S rRNA genes

1987 ◽  
Vol 11 (6-7) ◽  
pp. 571-573 ◽  
Author(s):  
S?awomir Bartoszewski ◽  
Piotr Borsuk ◽  
Izabela Kern ◽  
Ewa Bartnik
Keyword(s):  
Aspergillus Nidulans ◽  
5S Rrna ◽  
Rrna Genes ◽  
5S Rrna Genes

Characterization of 5S rRNA genes from mouse

Gene ◽  
1994 ◽  
Vol 142 (2) ◽  
pp. 291-295 ◽  
Author(s):  
Charlotte Hallenberg ◽  
Jens <SNM> Nederby Nielsen ◽  
Sune Frederiksen
Keyword(s):  
5S Rrna ◽  
Rrna Genes ◽  
5S Rrna Genes

A natural case of RIP: degeneration of the DNA sequence in an ancestral tandem duplication

1989 ◽  
Vol 9 (10) ◽  
pp. 4416-4421
Author(s):  
W S Grayburn ◽  
E U Selker
Keyword(s):  
Dna Methylation ◽  
Tandem Duplication ◽  
De Novo ◽  
5S Rrna ◽  
Rrna Genes ◽  
Structural Basis ◽  
Rrna Gene ◽  
Oak Ridge ◽  
5S Rrna Gene ◽  
5S Rrna Genes

5S rRNA genes of Neurospora crassa are generally dispersed in the genome and are unmethylated. The xi-eta region of Oak Ridge strains represents an informative exception. Most of the cytosines in this region, which consists of a diverged tandem duplication of a 0.8-kilobase-pair segment including a 5S rRNA gene, appear to be methylated (E. U. Selker and J. N. Stevens, Proc. Natl. Acad. Sci. USA 82:8114-8118, 1985). Previous work demonstrated that the xi-eta region functions as a portable signal for de novo DNA methylation (E. U. Selker and J. N. Stevens, Mol. Cell. Biol. 7:1032-1038, 1987; E. U. Selker, B. C. Jensen, and G. A. Richardson, Science 238:48-53, 1987). To identify the structural basis of this property, we have isolated and characterized an unmethylated allele of the xi-eta region from N. crassa Abbott 4. The Abbott 4 allele includes a single 5S rRNA gene, theta, which is different from all previously identified Neurospora 5S rRNA genes. Sequence analysis suggests that the xi-eta region arose from the theta region by duplication of a 794-base-pair segment followed by 267 G.C to A.T mutations in the duplicated DNA. The distribution of these mutations is not random. We propose that the RIP process of N. crassa (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987; E. U. Selker, and P. W. Garrett, Proc. Natl. Acad. Sci. USA 85:6870-6874, 1988; E. B. Cambareri, B. C. Jensen, E. Schabtach, and E. U. Selker, Science 244:1571-1575, 1989) is responsible for the numerous transition mutations and DNA methylation in the xi-eta region. A long homopurine-homopyrimidine stretch immediately following the duplicated segment is 9 base pairs longer in the Oak Ridge allele than in the Abbott 4 allele. Triplex DNA, known to occur in homopurine-homopyrimidine sequences, may have mediated the tandem duplication.

Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits

1993 ◽  
Vol 13 (5) ◽  
pp. 2835-2845
Author(s):  
M Deshmukh ◽  
Y F Tsay ◽  
A G Paulovich ◽  
J L Woolford
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Single Copy ◽  
5S Rrna ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Ribosomal Protein L1 ◽  
The Stability ◽  
And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

scholarly journals InEuglena, spliced-leader RNA (SL-RNA) and 5S rRNA genes are tandemly repeated

1992 ◽  
Vol 20 (7) ◽  
pp. 1711-1715 ◽  
Author(s):  
M. Keller ◽  
L. H. Tessier ◽  
R. L. Chan ◽  
J. H. Weil ◽  
P. lmbault
Keyword(s):  
5S Rrna ◽  
Rrna Genes ◽  
Spliced Leader ◽  
5S Rrna Genes ◽  
Spliced Leader Rna
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