Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains.

ABSTRACT: By using T4 DNA polymerase rather than S1 or Bal31 nuclease to clone yeast telomeres, very little telomeric DNA is lost. These clones were used to determine the DNA sequence of virtually the entire telomeric tract. Our results demonstrated that a slightly modified version, C2-3A(CA)1-6, of the consensus derived from sequence analysis of more-internal regions (J. Shampay, J. W. Szostak, and E. H. Blackburn, Nature [London] 310:154-157, 1984) extends to the very end of the chromosome. The sequence analysis also suggests that yeast telomeres consist of two domains: the proximal 120 to 150 base pairs, which appear to be protected from processes such as recombination, degradation, and elongation, and the distal portion of the telomere, which is more susceptible to these events.

SUBMITTER: Wang SS

PROVIDER: S-EPMC361005 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains.

Wang S S SS Zakian V A VA

Molecular and cellular biology 19900801 8

By using T4 DNA polymerase rather than S1 or Bal31 nuclease to clone yeast telomeres, very little telomeric DNA is lost. These clones were used to determine the DNA sequence of virtually the entire telomeric tract. Our results demonstrated that a slightly modified version, C2-3A(CA)1-6, of the consensus derived from sequence analysis of more-internal regions (J. Shampay, J. W. Szostak, and E. H. Blackburn, Nature [London] 310:154-157, 1984) extends to the very end of the chromosome. The sequence ...[more]

PMID: 2196453

Similar Datasets

Two distinct domains in Drosophila melanogaster telomeres.

Project description:Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.

| S-EPMC1382029 | biostudies-literature

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae.

Project description:A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K(+)-selective current whose attributes resemble the "leak" conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.

| S-EPMC24080 | biostudies-literature

The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity.

Project description:The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.

| S-EPMC6486559 | biostudies-literature

Mutant T4 DNA polymerase for easy cloning and mutagenesis.

Project description:The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3'-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable manner than the wild-type enzyme, and this can be used to increase the yields of colonies containing correctly modified plasmids in cloning and mutagenesis experiments, which is particularly useful when E. coli cells are of relatively low competency. Standard protocols using the mutant T4 DNA polymerase are provided for the sequence and ligation independent cloning (SLIC) method and a modified QuikChange method, where the mutant enzyme enhances the yield of correctly mutated plasmid and further suppresses parental plasmid during digestion with DpnI. Single-stranded DNA overhangs generated by the mutant T4 DNA polymerase facilitate subsequent plasmid circularization, annealing and ligation in E. coli.

| S-EPMC6343910 | biostudies-literature

The Chromatin and Transcriptional Landscape of Native Saccharomyces cerevisiae Telomeres and Subtelomeric Domains.

Project description:Saccharomyces cerevisiae telomeres have been a paradigm for studying telomere position effects on gene expression. Telomere position effect was first described in yeast by its effect on the expression of reporter genes inserted adjacent to truncated telomeres. The reporter genes showed variable silencing that depended on the Sir2/3/4 complex. Later studies examining subtelomeric reporter genes inserted at natural telomeres hinted that telomere position effects were less pervasive than previously thought. Additionally, more recent data using the sensitive technology of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq) revealed a discrete and noncontinuous pattern of coenrichment for all three Sir proteins at a few telomeres, calling the generality of these conclusions into question. Here we combined the ChIP-Seq of the Sir proteins with RNA sequencing (RNA-Seq) of messenger RNAs (mRNAs) in wild-type and in SIR2, SIR3, and SIR4 deletion mutants to characterize the chromatin and transcriptional landscape of all native S. cerevisiae telomeres at the highest achievable resolution. Most S. cerevisiae chromosomes had subtelomeric genes that were expressed, with only ∼6% of subtelomeric genes silenced in a SIR-dependent manner. In addition, we uncovered 29 genes with previously unknown cell-type-specific patterns of expression. These detailed data provided a comprehensive assessment of the chromatin and transcriptional landscape of the subtelomeric domains of a eukaryotic genome.

| S-EPMC4492376 | biostudies-literature

Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase ? in the Alternative Lengthening of Telomeres.

Project description:Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Pol?). We observe that the depletion of Pol? leads to increased ALT activity and late DNA polymerase ? (Pol?)-dependent synthesis of telomeric DNA in mitosis. We propose that Pol? fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Pol?.

| S-EPMC5406014 | biostudies-literature

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Project description:Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

| S-EPMC7956399 | biostudies-literature

Representation of cloned genomic sequences in two sequencing vectors: correlation of DNA sequence and subclone distribution.

Project description:Representation of subcloned Caenorhabditis elegans and human DNA sequences in both M13 and pUC sequencing vectors was determined in the context of large scale genomic sequencing. In many cases, regions of subclone under-representation correlated with the occurrence of repeat sequences, and in some cases the under-representation was orientation specific. Factors which affected subclone representation included the nature and complexity of the repeat sequence, as well as the length of the repeat region. In some but not all cases, notable differences between the M13 and pUC subclone distributions existed. However, in all regions lacking one type of subclone (either M13 or pUC), an alternate subclone was identified in at least one orientation. This suggests that complementary use of M13 and pUC subclones would provide the most comprehensive subclone coverage of a given genomic sequence.

| S-EPMC146865 | biostudies-other

Bacteriophage T4 long tail fiber domains.

Project description:Bacteriophage T4 initially recognizes its host cells using its long tail fibers. Long tail fibers consist of a phage-proximal and a phage-distal rod, each around 80 nm long and attached to each other at a slight angle. The phage-proximal rod is formed by a homo-trimer of gene product 34 (gp34) and is attached to the phage-distal rod by a monomer of gp35. The phage-distal rod consists of two protein trimers: a trimer of gp36, attached to gp35, although most of the phage-distal rod, including the receptor-binding domain, is formed by a trimer of gp37. In this review, we discuss what is known about the detailed structure and function of the different long tail fiber domains. Partial crystal structures of gp34 and gp37 have revealed the presence of new protein folds, some of which are present in several repeats, while others are apparently unique. Gp38, a phage chaperone protein necessary for folding of gp37, is thought to act on an α-helical coiled-coil region in gp37. Future studies should reveal the remaining structure of the long tail fibers, how they assemble into a functional unit, and how the long tail fibers trigger the infection process after successful recognition of a suitable host bacterium.

| S-EPMC5899708 | biostudies-literature

T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing.

Project description:Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.

| S-SCDT-10_1038-S44318-024-00158-6 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data