scholarly journals Expression of Mutated Paramecium Telomerase RNAs In Vivo Leads to Templating Errors That Resemble Those Made by Retroviral Reverse Transcriptase

1999 ◽  
Vol 19 (4) ◽  
pp. 2887-2894 ◽  
Author(s):  
Amanda J. Ye ◽  
W. John Haynes ◽  
Daniel P. Romero
Keyword(s):  
Reverse Transcriptase ◽  
De Novo ◽  
Telomerase Rna ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Immunodeficiency Virus ◽  
In The Wild

ABSTRACT Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutatedP. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.

scholarly journals Improving 3-methylphenol (m-cresol) production in yeast via in vivo glycosylation or methylation

2020 ◽  
Vol 20 (8) ◽  
Author(s):  
Julia Hitschler ◽  
Eckhard Boles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Wild Type ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
In Situ Extraction ◽  
Methylsalicylic Acid Synthase ◽  
In The Wild

ABSTRACT Heterologous expression of 6-methylsalicylic acid synthase (MSAS) together with 6-MSA decarboxylase enables de novo production of the platform chemical and antiseptic additive 3-methylphenol (3-MP) in the yeast Saccharomyces cerevisiae. However, toxicity of 3-MP prevents higher production levels. In this study, we evaluated in vivo detoxification strategies to overcome limitations of 3-MP production. An orcinol-O-methyltransferase from Chinese rose hybrids (OOMT2) was expressed in the 3-MP producing yeast strain to convert 3-MP to 3-methylanisole (3-MA). Together with in situ extraction by dodecane of the highly volatile 3-MA this resulted in up to 211 mg/L 3-MA (1.7 mM) accumulation. Expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera led to the synthesis of up to 533 mg/L 3-MP as glucoside (4.9 mM). Conversion of 3-MP to 3-MA and 3-MP glucoside was not complete. Finally, deletion of phosphoglucose isomerase PGI1 together with methylation or glycosylation and feeding a fructose/glucose mixture to redirect carbon fluxes resulted in strongly increased product titers, with up to 897 mg/L 3-MA/3-MP (9 mM) and 873 mg/L 3-MP/3-MP as glucoside (8.1 mM) compared to less than 313 mg/L (2.9 mM) product titers in the wild type controls. The results show that methylation or glycosylation are promising tools to overcome limitations in further enhancing the biotechnological production of 3-MP.

scholarly journals Expression of Telomerase RNA Template, but Not Telomerase Reverse Transcriptase, Is Limiting for Telomere Length Maintenance In Vivo

2004 ◽  
Vol 24 (16) ◽  
pp. 7024-7031 ◽  
Author(s):  
Y. Jeffrey Chiang ◽  
Michael T. Hemann ◽  
Karen S. Hathcock ◽  
Lino Tessarollo ◽  
Lionel Feigenbaum ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Gene Copy ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Wild Type ◽  
Telomere Elongation

ABSTRACT Telomerase consists of two essential components, the telomerase RNA template (TR) and telomerase reverse transcriptase (TERT). The haplo-insufficiency of TR was recently shown to cause one form of human dyskeratosis congenita, an inherited disease marked by abnormal telomere shortening. Consistent with this finding, we recently reported that mice heterozygous for inactivation of mouse TR exhibit a similar haplo-insufficiency and are deficient in the ability to elongate telomeres in vivo. To further assess the genetic regulation of telomerase activity, we have compared the abilities of TR-deficient and TERT-deficient mice to maintain or elongate telomeres in interspecies crosses. Homozygous TERT knockout mice had no telomerase activity and failed to maintain telomere length. In contrast, TERT+/− heterozygotes had no detectable defect in telomere elongation compared to wild-type controls, whereas TR+/− heterozygotes were deficient in telomere elongation. Levels of TERT mRNA in heterozygous mice were one-third to one-half the levels expressed in wild-type mice, similar to the reductions in telomerase RNA observed in TR heterozygotes. These findings indicate that both TR and TERT are essential for telomere maintenance and elongation but that gene copy number and transcriptional regulation of TR, but not TERT, are limiting for telomerase activity under the in vivo conditions analyzed.

scholarly journals A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium.

1996 ◽  
Vol 16 (4) ◽  
pp. 1871-1879 ◽  
Author(s):  
M McCormick-Graham ◽  
D P Romero
Keyword(s):  
Secondary Structure ◽  
Random Distribution ◽  
Single Gene ◽  
Telomerase Rna ◽  
Paramecium Tetraurelia ◽  
Dna Repeats ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Precise Nature ◽  
Rna Genes

Paramecium telomeric DNA consists largely of a random distribution of TTGGGG and TTTGGG repeats. Given the precise nature of other ciliate telomerases, it has been postulated that there are two distinct types of the Paramecium enzyme, each synthesizing perfect telomeric repeats: one with a template RNA that specifies the addition of TTTGGG and the second dictating the synthesis of TTGGGG repeats. We have cloned and sequenced telomerase RNA genes from Paramecium tetraurelia, P. primaurelia, P. multimicronucleatum, and P. caudatum. Surprisingly, a single gene encodes telomerase RNA in all four species, although an apparently nontranscribed pseudogene is also present in the genome of P. primaurelia. The overall lengths of the telomerase RNAs range between 202 and 209 nucleotides, and they can be folded into a conserved secondary structure similar to that derived for other ciliate RNAs. All Paramecium telomerase RNAs examined include a template specific for the synthesis of TTGGGG telomeric repeats, which has not been posttranscriptionally edited to account for the conventional synthesis of TTTGGG repeats. On the basis of these results, possible mechanisms for the synthesis of variable telomeric repeats by Paramecium telomerase are discussed.

scholarly journals Template Requirements for Telomerase Translocation in Kluyveromyces lactis

2004 ◽  
Vol 24 (2) ◽  
pp. 912-923 ◽  
Author(s):  
Dana H. Underwood ◽  
Robert P. Zinzen ◽  
Michael J. McEachern
Keyword(s):  
Reverse Transcriptase ◽  
Telomere Length ◽  
Kluyveromyces Lactis ◽  
Telomerase Rna ◽  
Wild Type ◽  
Telomeric Repeats

ABSTRACT Telomeres are synthesized by telomerase, a specialized reverse transcriptase, which contains a template in its intrinsic RNA component. In Kluyveromyces lactis, the repeats synthesized by the wild-type telomerase are 25 nucleotides (nt) in length and uniform in sequence. To determine the role of the 5-nt repeats defining the ends of the K. lactis telomerase RNA template in telomerase translocation, we have made mutations in and around them and observed their effects on telomere length and the sequence of newly made telomeric repeats. These template mutations typically result in telomeres that are shorter than those of wild-type cells. The mismatches between the telomerase template and the telomeric tip that occur after telomerase-mediated incorporation of the mutations are normally not removed. Instead, the mutations lead to the synthesis of aberrant repeats that range in size from 31 to 13 bp. Therefore, the specificity with which the telomeric tip aligns with the telomere is critical for the production of the uniform repeats seen in K. lactis. In addition, the region immediately 3′ of the template may play an important role in translocation of the enzyme.

scholarly journals Extrachromosomal Telomeric Circles Contribute to Rad52-, Rad50-, and Polymerase δ-Mediated Telomere-Telomere Recombination in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Chi-Ying Lin ◽  
Hsih-Hsuan Chang ◽  
Kou-Juey Wu ◽  
Shun-Fu Tseng ◽  
Chuan-Chuan Lin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reverse Transcriptase ◽  
Telomere Maintenance ◽  
Yeast Cells ◽  
Chromosome Stability ◽  
Telomerase Reverse Transcriptase ◽  
Wild Type ◽  
Telomeric Dna ◽  
Recombination Mechanism

ABSTRACT Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the telomerase reverse transcriptase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. By using an in vivo inducible Cre-loxP system to generate and trace the fate of marked telomeric DNA-containing rings, the efficiency of telomere-telomere recombination can be determined quantitatively. We show that the telomeric loci are the primary sites at which a marked telomeric ring-containing DNA is observed among wild-type and surviving cells lacking telomerase. Marked telomeric DNAs can be transferred to telomeres and form tandem arrays through Rad52-, Rad50-, and polymerase δ-mediated recombination. Moreover, increases of extrachromosomal telomeric and Y′ rings were observed in telomerase-deficient cells. These results imply that telomeres can use looped-out telomeric rings to promote telomere-telomere recombination in telomerase-deficient Saccharomyces cerevisiae.

scholarly journals Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase.

1996 ◽  
Vol 16 (1) ◽  
pp. 66-75 ◽  
Author(s):  
D Gilley ◽  
E H Blackburn
Keyword(s):  
Active Site ◽  
Tetrahymena Thermophila ◽  
Telomerase Rna ◽  
Specific Interactions ◽  
Wild Type ◽  
Specific Primer ◽  
Telomeric Dna ◽  
Template Sequence ◽  
New Evidence

The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

Dynamics of Telomeric DNA Turnover in Yeast

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 63-73
Author(s):  
Michael J McEachern ◽  
Dana Hager Underwood ◽  
Elizabeth H Blackburn
Keyword(s):  
Kluyveromyces Lactis ◽  
Mutant Gene ◽  
Dna Repeats ◽  
Wild Type ◽  
Telomeric Dna ◽  
Cell Divisions ◽  
Dna Turnover ◽  
Length Regulation ◽  
Turn Over

Abstract Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

scholarly journals MyD88-Dependent Signaling Affects the Development of Meningococcal Sepsis by Nonlipooligosaccharide Ligands

2006 ◽  
Vol 74 (6) ◽  
pp. 3538-3546 ◽  
Author(s):  
Laura Plant ◽  
Hong Wan ◽  
Ann-Beth Jonsson
Keyword(s):  
Inflammatory Responses ◽  
Innate Immune ◽  
Meningococcal Sepsis ◽  
Wild Type ◽  
Microbial Infections ◽  
In The Wild ◽  
Myeloid Differentiation Factor ◽  
Dependent Pathway ◽  
Meningococcal Strain

ABSTRACT The Toll-like receptors (TLRs) and the adaptor myeloid differentiation factor 88 (MyD88) are important in the innate immune defenses of the host to microbial infections. Meningococcal ligands signaling via TLRs control inflammatory responses, and stimulation can result in fulminant meningococcal sepsis. In this study, we show that the responses to nonlipooligosaccharide (non-LOS) ligands of meningococci are MyD88 dependent. An isogenic LOS-deficient mutant of the serogroup C meningococcal strain FAM20 caused fatal disease in wild type C57BL/6 mice that was not observed in MyD88−/− mice. Fatality correlated with high proinflammatory cytokine and C5a levels in serum, high neutrophil numbers in blood, and increased bacteremia at 24 h postinfection in the wild-type mice. Infection with the parent strain FAM20 resulted in fatality in 100% of the wild-type mice and 50% of the MyD88−/− mice. We conclude that both LOS and another neisserial ligand cause meningococcal sepsis in an in vivo mouse model and confirm that meningococcal LOS can act via both the MyD88- dependent and -independent pathways, while the non-LOS meningococcal ligand(s) acts only via the MyD88-dependent pathway.

scholarly journals De Novo Development of mtDNA Deletion Due to Decreased POLG and SSBP1 Expression in Humans

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 284
Author(s):  
Yeonmi Lee ◽  
Taeho Kim ◽  
Miju Lee ◽  
Seongjun So ◽  
Mustafa Zafer Karagozlu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
De Novo ◽  
Wild Type Allele ◽  
Wild Type ◽  
Factors Associated ◽  
Mtdna Deletions ◽  
Nuclear Factors ◽  
Mtdna Deletion ◽  
In The Wild ◽  
Mtdna Replication

Defects in the mitochondrial genome (mitochondrial DNA (mtDNA)) are associated with both congenital and acquired disorders in humans. Nuclear-encoded DNA polymerase subunit gamma (POLG) plays an important role in mtDNA replication, and proofreading and mutations in POLG have been linked with increased mtDNA deletions. SSBP1 is also a crucial gene for mtDNA replication. Here, we describe a patient diagnosed with Pearson syndrome with large mtDNA deletions that were not detected in the somatic cells of the mother. Exome sequencing was used to evaluate the nuclear factors associated with the patient and his family, which revealed a paternal POLG mutation (c.868C > T) and a maternal SSBP1 mutation (c.320G > A). The patient showed lower POLG and SSBP1 expression than his healthy brothers and the general population of a similar age. Notably, c.868C in the wild-type allele was highly methylated in the patient compared to the same site in both his healthy brothers. These results suggest that the co- deficient expression of POLG and SSBP1 genes could contribute to the development of mtDNA deletion.

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