scholarly journals The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila.

1989 ◽  
Vol 9 (2) ◽  
pp. 452-460 ◽  
Author(s):  
P C Yaeger ◽  
E Orias ◽  
W L Shaiu ◽  
D D Larson ◽  
E H Blackburn
Keyword(s):  
Inbred Strain ◽  
Southern Blot Analysis ◽  
Tetrahymena Thermophila ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Wild Type ◽  
Recombinational Event ◽  
Somatic Nucleus ◽  
Somatic Recombination ◽  
General Method

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.

scholarly journals The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila

1989 ◽  
Vol 9 (2) ◽  
pp. 452-460
Author(s):  
P C Yaeger ◽  
E Orias ◽  
W L Shaiu ◽  
D D Larson ◽  
E H Blackburn
Keyword(s):  
Inbred Strain ◽  
Southern Blot Analysis ◽  
Tetrahymena Thermophila ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Wild Type ◽  
Recombinational Event ◽  
Somatic Nucleus ◽  
Somatic Recombination ◽  
General Method

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.

scholarly journals Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter.

1995 ◽  
Vol 15 (6) ◽  
pp. 3372-3381 ◽  
Author(s):  
W J Pan ◽  
R C Gallagher ◽  
E H Blackburn
Keyword(s):  
Rna Polymerase ◽  
Tetrahymena Thermophila ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Wild Type ◽  
Independent Evidence ◽  
Origin Region ◽  
Origin Function ◽  
Polymerase I ◽  
Wild Type Promoter

In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer. The replication of this rDNA minichromosome is under both cell cycle and copy number control. We addressed the effect on origin function of transcription through this origin region. A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome. Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J. Pan and E. H. Blackburn, Nucleic Acids Res, in press). We present direct evidence that the +G mutation inactivates this rRNA promoter. It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript. Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter. Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance. Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region. We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila

1989 ◽  
Vol 9 (3) ◽  
pp. 1092-1099
Author(s):  
M C Yao ◽  
C H Yao
Keyword(s):  
Developmental Stages ◽  
Transformation Efficiency ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Rrna Genes ◽  
Somatic Nucleus ◽  
Dna Breakage ◽  
Cis Acting ◽  
Palindromic Structure ◽  
Rdna Amplification

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.

scholarly journals Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.

1989 ◽  
Vol 9 (3) ◽  
pp. 1092-1099 ◽  
Author(s):  
M C Yao ◽  
C H Yao
Keyword(s):  
Developmental Stages ◽  
Transformation Efficiency ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Rrna Genes ◽  
Somatic Nucleus ◽  
Dna Breakage ◽  
Cis Acting ◽  
Palindromic Structure ◽  
Rdna Amplification

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.

REGULATION OF RIBOSOMAL RNA GENE MULTIPLICITY IN DROSOPHILA MELANOGASTER

Genetics ◽  
1973 ◽  
Vol 73 (1) ◽  
pp. 57-71
Author(s):  
Kenneth D Tartof
Keyword(s):  
Drosophila Melanogaster ◽  
Ribosomal Rna ◽  
Homologous Chromosome ◽  
Germ Line ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Wild Type ◽  
Wild Type Level ◽  
New Genotype ◽  
Successive Generations

ABSTRACT The ribosomal RNA (rRNA) genes of Drosophila melanogaster can undergo a disproportionate replication of their number. This occurs when the cluster of rRNA genes (rDNA) of one chromosome is maintained with a homologous chromosome that is completely or partially deficient in its rDNA. Under appropriate genetic conditions, it appears that disproportionate rDNA replication can be generated at the level of both somatic and germ line cells. In the latter case, mutants partially deficient for rDNA can increase their rRNA gene number to the wild type level and transmit this new genotype to successive generations.

scholarly journals Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila.

1990 ◽  
Vol 10 (5) ◽  
pp. 2070-2080 ◽  
Author(s):  
G L Yu ◽  
E H Blackburn
Keyword(s):  
Replication Origin ◽  
Tetrahymena Thermophila ◽  
Recipient Cell ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Present Evidence ◽  
Cell Divisions ◽  
Inverted Orientation ◽  
Replication Control ◽  
Origin Region

The macronuclear rRNA genes (rDNA) in the ciliate Tetrahymena thermophila are normally palindromic linear replicons, containing two copies of the replication origin region in inverted orientation. A circular plasmid containing a single Tetrahymena rRNA gene (one half palindrome) joined to a tandem repeat of a 1.9-kilobase (kb) rDNA segment encompassing the rDNA replication origin and known replication control elements was used to transform Tetrahymena macronuclei by microinjection. This plasmid was shown previously to have a replication advantage over the rDNA allele of the recipient cell strain (G.-L. Yu and E. H. Blackburn, Proc. Natl. Acad. Sci. USA 86:8487-8491, 1990). During vegetative cell divisions, the circular and palindromic rDNAs were rapidly replaced by novel, successively longer linear rDNAs that eventually contained up to 30 tandem 1.9-kb repeats, resulting from homologous but unequal crossovers between the 1.9-kb repeats. We present evidence to show that increasing the number of copies of the replication control regions increases the replicative advantage of the rDNA, the first such situation for a cellular nuclear replicon in a eucaryote.

Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila

1993 ◽  
Vol 13 (8) ◽  
pp. 4814-4825
Author(s):  
R Sweeney ◽  
L Chen ◽  
M C Yao
Keyword(s):  
Tertiary Structure ◽  
Tetrahymena Thermophila ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Variable Regions ◽  
Small Subunit Rrna Gene

Tetrahymena thermophila is an ideal organism with which to study functional aspects of the rRNAs in vivo since the somatic rRNA genes of T. thermophila can be totally replaced by cloned copies introduced via microinjection. In this study, we made small insertions into seven sites within the small subunit rRNA gene and observed their phenotypic effects on transformed cells. Two mutated genes coding for rRNA (rDNAs), both of which bear insertions in highly conserved sequences, failed to transform and are therefore believed to produce nonfunctional rRNAs. Three other altered rDNAs produce functional rRNAs that can substitute for most or all of the cellular rRNA. Two of these bear insertions in highly variable regions, and, surprisingly, the other has an insertion in a region that is well conserved for both sequence and secondary structure among eucaryotes. In addition, two other insertions appear to destabilize rRNAs that contain them. Our findings make predictions concerning the positions of some of these sites within the tertiary structure of the small ribosomal subunit and thus serve as an in vivo test of the existing tertiary structure models for the small subunit rRNA. Our results are in good agreement with expectations based on sequence comparison and in vitro work.

Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila

1990 ◽  
Vol 10 (5) ◽  
pp. 2070-2080
Author(s):  
G L Yu ◽  
E H Blackburn
Keyword(s):  
Replication Origin ◽  
Tetrahymena Thermophila ◽  
Recipient Cell ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Present Evidence ◽  
Cell Divisions ◽  
Inverted Orientation ◽  
Replication Control ◽  
Origin Region

The macronuclear rRNA genes (rDNA) in the ciliate Tetrahymena thermophila are normally palindromic linear replicons, containing two copies of the replication origin region in inverted orientation. A circular plasmid containing a single Tetrahymena rRNA gene (one half palindrome) joined to a tandem repeat of a 1.9-kilobase (kb) rDNA segment encompassing the rDNA replication origin and known replication control elements was used to transform Tetrahymena macronuclei by microinjection. This plasmid was shown previously to have a replication advantage over the rDNA allele of the recipient cell strain (G.-L. Yu and E. H. Blackburn, Proc. Natl. Acad. Sci. USA 86:8487-8491, 1990). During vegetative cell divisions, the circular and palindromic rDNAs were rapidly replaced by novel, successively longer linear rDNAs that eventually contained up to 30 tandem 1.9-kb repeats, resulting from homologous but unequal crossovers between the 1.9-kb repeats. We present evidence to show that increasing the number of copies of the replication control regions increases the replicative advantage of the rDNA, the first such situation for a cellular nuclear replicon in a eucaryote.

scholarly journals Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila.

1993 ◽  
Vol 13 (8) ◽  
pp. 4814-4825 ◽  
Author(s):  
R Sweeney ◽  
L Chen ◽  
M C Yao
Keyword(s):  
Tertiary Structure ◽  
Tetrahymena Thermophila ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Variable Regions ◽  
Small Subunit Rrna Gene

Tetrahymena thermophila is an ideal organism with which to study functional aspects of the rRNAs in vivo since the somatic rRNA genes of T. thermophila can be totally replaced by cloned copies introduced via microinjection. In this study, we made small insertions into seven sites within the small subunit rRNA gene and observed their phenotypic effects on transformed cells. Two mutated genes coding for rRNA (rDNAs), both of which bear insertions in highly conserved sequences, failed to transform and are therefore believed to produce nonfunctional rRNAs. Three other altered rDNAs produce functional rRNAs that can substitute for most or all of the cellular rRNA. Two of these bear insertions in highly variable regions, and, surprisingly, the other has an insertion in a region that is well conserved for both sequence and secondary structure among eucaryotes. In addition, two other insertions appear to destabilize rRNAs that contain them. Our findings make predictions concerning the positions of some of these sites within the tertiary structure of the small ribosomal subunit and thus serve as an in vivo test of the existing tertiary structure models for the small subunit rRNA. Our results are in good agreement with expectations based on sequence comparison and in vitro work.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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