scholarly journals A Methyltransferase Targeting Assay Reveals Silencer-Telomere Interactions in Budding Yeast

2003 ◽  
Vol 23 (5) ◽  
pp. 1498-1508 ◽  
Author(s):  
Eleonore Lebrun ◽  
Geneviève Fourel ◽  
Pierre-Antoine Defossez ◽  
Eric Gilson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Subtelomeric Region ◽  
Yeast Genome ◽  
Dna Binding Domain ◽  
Bacterial Dna ◽  
Intervening Sequences ◽  
Identification Technique

ABSTRACT We have designed a modified version of the Dam identification technique and used it to probe higher-order chromatin structure in Saccharomyces cerevisiae. We fused the bacterial DNA methyltransferase Dam to the DNA-binding domain of TetR and targeted the resulting chimera to Tet operators inserted in the yeast genome at the repressed locus HML. We then monitored the methylation status of HML and other sequences by a quantitative technique combining methylation-sensitive restriction and real-time PCR. As expected, we found that TetR-Dam efficiently methylated HML in cis. More strikingly, when TetR-Dam was present at HML, we observed increased methylation in the III-L subtelomeric region but not in intervening sequences. This effect was lost when the HML silencers were inactivated by mutations. When the HM silencers and the Tet operators were transferred to a plasmid, strong methylation was clearly observed not only in the III-L subtelomeric region but also at other telomeres. These data indicate that HM silencers can specifically associate with telomeres, even those located on different chromosomes.

scholarly journals Intrachromatid Excision of Telomeric DNA as a Mechanism for Telomere Size Control in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (19) ◽  
pp. 6559-6573 ◽  
Author(s):  
Maria Bucholc ◽  
Yangsuk Park ◽  
Arthur J. Lustig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Structure ◽  
Size Control ◽  
Wild Type ◽  
Telomeric Dna ◽  
Genetic Analyses ◽  
Terminal Domain ◽  
Intervening Sequences ◽  
Restriction Sites ◽  
Strand Invasion

ABSTRACT We have previously identified a process in the yeastSaccharomyces cerevisiae that results in the contraction of elongated telomeres to wild-type length within a few generations. We have termed this process telomeric rapid deletion (TRD). In this study, we use a combination of physical and genetic assays to investigate the mechanism of TRD. First, to distinguish among several recombinational and nucleolytic pathways, we developed a novel physical assay in which HaeIII restriction sites are positioned within the telomeric tract. Specific telomeres were subsequently tested for HaeIII site movement between telomeres and for HaeIII site retention during TRD. Second, genetic analyses have demonstrated that mutations inRAD50 and MRE11 inhibit TRD. TRD, however, is independent of the Rap1p C-terminal domain, a central regulator of telomere size control. Our results provide evidence that TRD is an intrachromatid deletion process in which sequences near the extreme terminus invade end-distal sequences and excise the intervening sequences. We propose that the Mre11p-Rad50p-Xrs2p complex prepares the invading telomeric overhang for strand invasion, possibly through end processing or through alterations in chromatin structure.

scholarly journals Factors that affect the location and frequency of meiosis-induced double-strand breaks in Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 55-66 ◽  
Author(s):  
T C Wu ◽  
M Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Structure ◽  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Yeast Genome ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
A Site ◽  
Chromosomal Sequences

Abstract Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae. DSBs occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin structure in determining DSB location. We show here that the frequency of DSBs at a site is not determined simply by DNA sequence or by features of chromatin structure. An arg4-containing plasmid was inserted at several different locations in the yeast genome. Meiosis-induced DSBs occurred at similar sites in pBR322-derived portions of the construct at all insert loci, and the frequency of these breaks varied in a manner that mirrored the frequency of meiotic recombination in the arg4 portion of the insert. However, DSBs did not occur in the insert-borne arg4 gene at a site that is frequently broken at the normal ARG4 locus, even though the insert-borne arg4 gene and the normal ARG4 locus displayed similar DNase I hypersensitivity patterns. Deletions that removed active DSB sites from an insert at HIS4 restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences. We conclude that the frequency of DSB at a site can be affected by sequences several thousand nucleotides away and suggest that this is because of competition between DSB sites for locally limited factors.

scholarly journals Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

Genetics ◽  
1991 ◽  
Vol 127 (4) ◽  
pp. 681-698 ◽  
Author(s):  
A J Link ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Physical Map ◽  
Single Copy ◽  
Restriction Endonucleases ◽  
Yeast Genome ◽  
Hybridization Probes ◽  
Copy Dna ◽  
Single Copy Dna

Abstract A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

scholarly journals Ex vivo ultrasonic samples of human brain tumors in the molecular era

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Alastair J Kirby ◽  
José P Lavrador ◽  
Istvan Bodi ◽  
Francesco Vergani ◽  
Ranjeev Bhangoo ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Cellular Proliferation ◽  
Aminolevulinic Acid ◽  
Ex Vivo ◽  
Methylation Status ◽  
Protoporphyrin Ix ◽  
Intratumor Heterogeneity ◽  
Power Relationship ◽  
Surgical Biopsy ◽  
Glioma Surgery

Abstract Background Gliomas are composed of multiple clones of tumor cells. This intratumor heterogeneity contributes to the ability of gliomas to resist treatment. It is vital that gliomas are fully characterized at a molecular level when a diagnosis is made to maximize treatment effectiveness. Methods We collected ultrasonic tissue fragments during glioma surgery. Large tissue fragments were separated in the operating theater and bathed continuously in oxygenated artificial cerebrospinal fluid to keep them alive. The ex vivo tissue fragments were transferred to a laboratory and incubated in 5-aminolevulinic acid (5-ALA). 5-ALA is metabolized to Protoporphyrin IX (PpIX), which accumulates in glioma cells and makes them fluorescent. The molecular and neuropathological features of the PpIX fluorescent ultrasonic tissue fragments were studied. Results We show that PpIX fluorescence can rapidly identify tissue fragments infiltrated by glioma in the laboratory. Ultrasonic tissue fragments from the tumor core provided molecular and neuropathological information about the glioma that was comparable to the surgical biopsy. We characterized the heterogeneity within individual gliomas by studying ultrasonic tissue fragments from different parts of the tumor. We found that gliomas exhibit a power relationship between cellular proliferation and tumor infiltration. Tissue fragments that deviate from this relationship may contain foci of more malignant glioma. The methylation status of the O6-methylguanine DNA methyltransferase gene promoter varied within each glioma. Conclusions Ex vivo ultrasonic tissue fragments can be rapidly screened for glioma infiltration. They offer a viable platform to characterize heterogeneity within individual gliomas, thereby enhancing their diagnosis and treatment.

scholarly journals NIMG-37. DELAYED PSEUDOPROGRESSION IN TWO PATIENTS UNDERGOING TTFIELDS TREATMENT FOR NEWLY DIAGNOSED GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii155-ii156
Author(s):  
Allison Lowman ◽  
Sarah Hurrell ◽  
Samuel Bobholz ◽  
Jennifer Connelly ◽  
Elizabeth Cochran ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Radiographic Progression ◽  
Methylation Status ◽  
Underlying Mechanism ◽  
Recurrent Glioblastoma ◽  
Newly Diagnosed ◽  
Methylguanine Dna Methyltransferase ◽  
Specific Effects ◽  
Newly Diagnosed Glioblastoma ◽  
Associated Risk Factors

Abstract PURPOSE Tumor treatment fields (TTFields) are approved by the FDA for newly diagnosed as well as recurrent glioblastoma (GBM). TTFields have been shown to extend survival by 4.9 months in newly diagnosed GBM, and a landmark overall survival rate of 13% at 5 years. However, the specific effects remain widely unknown, which has prevented widespread clinical use of this treatment. METHODS This case study examines two glioblastoma patients, IDH-1 wildtype, MGMT unmethylated, that received TTFields (Optune) in addition to maintenance temozolomide (TMZ) following radiation (RT). Both cases were followed using standard MR imaging. Second resections were performed due to radiographic progression of contrast enhancement. RESULTS Although imaging was concerning for tumor progression, pathology showed only treatment effect, ultimately leading to the diagnosis of pseudoprogression. Both patients fell outside the normal expected timeline for chemo-radiation induced pseudoprogression. Based on the pathology, both patients resumed treatment with TMZ/TTFields. One patient expired at 25 months and one is still alive. CONCLUSIONS Pathologic confirmation was essential for guiding further treatment and allowed patients to continue treatment that was effective despite contrary indications on imaging. These findings suggest that pathological confirmation should be strongly considered in patients receiving TMZ/TTFields who develop radiographic progression, potentially with a less invasive biopsy procedure. Future studies should look to characterize the underlying mechanism of interaction between TTFields/TMZ and quantify the prevalence, associated risk factors and whether there is a genotype more susceptible. Both patients reported here had O(6)-methylguanine-DNA methyltransferase (MGMT) unmethylated GBM, and while about 60% of glioblastomas are diagnosed likewise, it is possible that MGMT methylation status plays a role in TTFields response. Better characterization of this phenomenon will improve treatment guidance, potentially reducing unnecessary surgeries and the discontinuance of successful therapies.

scholarly journals Limited role for extended maintenance temozolomide for newly diagnosed glioblastoma

Neurology ◽  
2017 ◽  
Vol 88 (15) ◽  
pp. 1422-1430 ◽  
Author(s):  
Dorothee Gramatzki ◽  
Philipp Kickingereder ◽  
Bettina Hentschel ◽  
Jörg Felsberg ◽  
Ulrich Herrlinger ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Proportional Hazards Model ◽  
Methylation Status ◽  
Residual Tumor ◽  
Cox Proportional Hazards Model ◽  
Rank Test ◽  
Karnofsky Performance Score ◽  
Newly Diagnosed ◽  
Clinical Patient ◽  
Newly Diagnosed Glioblastoma

Objective:To explore an association with survival of modifying the current standard of care for patients with newly diagnosed glioblastoma of surgery followed by radiotherapy plus concurrent and 6 cycles of maintenance temozolomide chemotherapy (TMZ/RT → TMZ) by extending TMZ beyond 6 cycles.Methods:The German Glioma Network cohort was screened for patients with newly diagnosed glioblastoma who received TMZ/RT → TMZ and completed ≥6 cycles of maintenance chemotherapy without progression. Associations of clinical patient characteristics, molecular markers, and residual tumor determined by magnetic resonance imaging after 6 cycles of TMZ with progression-free survival (PFS) and overall survival (OS) were analyzed with the log-rank test. Multivariate analyses using the Cox proportional hazards model were performed to assess associations of prolonged TMZ use with outcome.Results:Sixty-one of 142 identified patients received at least 7 maintenance TMZ cycles (median 11, range 7–20). Patients with extended maintenance TMZ treatment had better PFS (20.5 months, 95% confidence interval [CI] 17.7–23.3, vs 17.2 months, 95% CI 10.2–24.2, p = 0.035) but not OS (32.6 months, 95% CI 28.9–36.4, vs 33.2 months, 95% CI 25.3–41.0, p = 0.126). However, there was no significant association of prolonged TMZ chemotherapy with PFS (hazard ratio [HR] = 0.8, 95% CI 0.4–1.6, p = 0.559) or OS (HR = 1.6, 95% CI 0.8–3.3, p = 0.218) adjusted for age, extent of resection, Karnofsky performance score, presence of residual tumor, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status, or isocitrate dehydrogenase (IDH) mutation status.Conclusion:These data may not support the practice of prolonging maintenance TMZ chemotherapy beyond 6 cycles.Classification of evidence:This study provides Class III evidence that in patients with newly diagnosed glioblastoma, prolonged TMZ chemotherapy does not significantly increase PFS or OS.

scholarly journals Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435 ◽  
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

scholarly journals CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

Life ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 13
Author(s):  
João Rainha ◽  
Joana L. Rodrigues ◽  
Lígia R. Rodrigues
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Genome ◽  
Specific Method ◽  
Complex Processes ◽  
Biological Studies ◽  
Precise Tool ◽  
Long Time ◽  
Gene Knockouts ◽  
Heterologous Pathway ◽  
A Current

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.

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