Tel1ATM dictates the replication timing of short yeast telomeres

Telomerase action is temporally linked to DNA replication. Although yeast telomeres are normally late replicating, telomere shortening leads to early firing of subtelomeric DNA replication origins. We show that double‐strand breaks flanked by short telomeric arrays cause origin firing early in S phase at late‐replicating loci and that this effect on origin firing time is dependent on the Tel1ATM checkpoint kinase. The effect of Tel1ATM on telomere replication timing extends to endogenous telomeres and is stronger than that elicited by Rif1 loss. These results establish that Tel1ATM specifies not only the extent but also the timing of telomerase recruitment

Characterization of the role of SUMO in telomere length homeostasis and overhang processing at yeast telomeres

Telomeres protect the ends of the linear chromosomes by assembling a nucleoprotein complex called shelterin. This complex regulates the action of telomerase, a reverse transcriptase enzyme that adds telomeric DNA to the G-rich 3’ end of the chromosomes. CST (CTC1/Cdc13-Stn1-Ten1) complex, also associated with telomeres, counteracts telomerase mediated telomere overhang elongation by recruiting lagging strand DNA polymerases for C-Strand synthesis. SUMOylation, a post-translational modification, is known to contribute towards negative regulation of telomerase in both budding yeast and fission yeast. It has previously been shown in budding yeast that SUMOylation of Cdc13 enhances its interaction with Stn1 to restrain telomerase function. In humans, CST interacts with overhang-binding shelterin proteins TPP1/POT1, but there is no evidence whether this interaction is regulated. The work in this thesis provides a mechanistic insight into the role of SUMOylation in telomere length regulation in fission yeast. It was established in this study that fission yeast telomeric protein Tpz1, an ortholog of human TPP1, is SUMOylated at lysine 242 by SUMO E3 ligase Pli1. The mutation of this lysine residue leads to telomere elongation in a telomerase-dependent manner. Chromatin immunoprecipitation (ChIP) analysis indicated that the association of telomerase with telomeres is increased in a tpz1-K242R mutant, whereas that of Stn1 and Ten1 is greatly reduced. In addition, a SUMO-Tpz1 fusion protein showed increased affinity for Stn1, in the yeast two-hybrid assays. The assay was also used to define minimal region in Tpz1 required for its interaction with Stn1. This data indicated that SUMOylation of Tpz1 facilitates the recruitment and function of Stn1-Ten1 at telomeres, which in turn down-regulates telomerase. Collectively, this study highlight the evolutionary conservation of the regulation of (C)ST function by SUMOylation and provides further insight into the role of Tpz1 in telomerase regulation. In order to obtain further insight into the dynamics of telomerase action and overhang fill-in by C-strand synthesis I also developed and validated a PCR-based assay for precise measurement of telomere overhang i budding and fission yeast

Tpz1TPP1 SUMOylation reveals evolutionary conservation of SUMO-dependent Stn1 telomere association

Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang-binding Cdc13 is increased by Cdc13 SUMOylation. Human and fission yeast CST instead interact with overhang-binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association,and a SUMO-Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation

Persistencia de malezas gramíneas en cultivos de trigo del sudeste bonaerense

En la presente tesis se estudió la persistencia de especies poáceas en cultivos de trigo del sudeste de Buenos Aires. En dicha región, Avena fatua L. y Lolium multiflorum Lam. son las malezas poáceas más importantes, tanto por la dificultad de control como por sus efectos competitivos sobre el cultivo. A los efectos de cuantificar la persistencia de dichas especies, se estudió la composición de la comunidad de malezas en dos momentos del ciclo: preaplicación de herbicidas y precosecha. Individuos de ambas malezas fueron registrados en ambos momentos como consecuencia de “escapes” al control realizado con herbicidas, siendo A. fatua más constante que L. mutiflorum. Posteriormente, se estudiaron los procesos que definen la persistencia de ambas malezas. Los resultados obtenidos indican que el ajuste del momento de emergencia es jerárquicamente el factor más importante para explicar la persistencia de A. fatua. Se demostró que los modelos de germinación son diferentes según las semillas provengan de un lote agrícola o de una condición de no cultivo, siendo estas diferencias de naturaleza genética. Por otro lado, la variabilidad en la supervivencia a los herbicidas es el factor que mejor explica la persistencia de L. multiflorum, habiéndose documentado resistencia cruzada a los herbicidas inhibidores de la ALS, pyroxsulam, imazamox y flucarbazone, sin antecedentes previos en la región. Los índices de resistencia encontrados presentan variación con la temperatura ambiente en post-aplicación del herbicida, habiéndose registrado mayor resistencia con mayor temperatura. Además, se comprobó que los individuos resistentes presentan menor tiempo a floración que los susceptibles. Tal atributo puede significar una ventaja demográfica para dichas poblaciones. Queda así demostrada la persistencia de A. fatua y L. multiflorum durante el ciclo del cultivo más allá de las prácticas de control realizadas y la participación de dos procesos demográficos distintos (establecimiento y supervivencia) en dicha persistencia

Evaluation of thickness of palatal masticatory mucosa in posterior teeth and its relation with age and gender

Background: To evaluate the thickness of palatal masticatory mucosa in posterior teeth by transgingival probing and determine its relation with age and gender. Materials and Methods: Forty systemically healthy volunteers with age range 16–38 years were selected based on the inclusion and exclusion criteria. Eighteen measurement points were made on the cast that was transferred onto the palate using clear acrylic stent to measure thickness. Three different lines a, b, and c: 3, 8, and 12 mm, respectively, from the gingival margin were made starting at the mid-palatal aspect of the canine and ending over the palatal root of the second molar. Six points were defined on each of the lines: Ca (mid-palatal aspect of the canine), P1 (mid-palatal aspect of the first premolar), P2 (mid-palatal aspect of the second premolar), M1 (palatal root of the first molar), Mi (interproximal aspect between the first and second molar), and M2 (palatal root of the second molar). Results: Soft-tissue thickness progressively increased in sites further from the gingival margin and was thickest adjacent to mid-palatal aspect of the second premolar 12 mm away from gingival margin. Younger age group patients had thinner posterior palatal mucosa as compared to older age group patients. Males had thicker posterior palatal mucosa as compared to females, but results were statistically insignificant. Conclusion: The thickness of posterior palatal mucosa showed a varied degree of variation at different marked areas in different teeth, and the difference in the mean thickness was also associated with age and gender

Downregulation of Mitogen-Activated Protein Kinase 1 of Leishmania donovani Field Isolates Is Associated with Antimony Resistance

Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed in Escherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates