Development of microsatellite markers for Rhodiola rosea

Rhodiola rosea L. is an important adaptogen medicinal plant. In this study two new microsatellite markers were developed. The assessment of the genetic diversity of R. rosea has recently started with molecular markers, but only a few species-specific microsatellite markers have been published so far. However the small number of markers allows only a limited insight into the genetic variability of the species therefore the aim of our work was to develop new microsatellite markers for R. rosea with a microsatellite enrichment library technique. Genomic DNA was cleaved with an endonuclease enzyme followed by adaptor ligation and PCR amplification. DNA fragments that contained microsatellites were first isolated using a biotin-streptavidin linkage based magnetic selection and then cloned into plasmids. Out of forty-three sequenced clones three contained  microsatellites, in these cases primers were designed for the amplification of the microsatellite repeats. The newly developed primer pairs were tested on individuals from distant R. rosea populations and the variability of the amplified fragments was estimated by fragment-length analysis. The locus RhpB14a was found to be monomorphic while RhpB14b and RhpB13 were polymorphic. As a result of the present study, two novel variable microsatellite loci were identified in the genome of R. rosea

Telomere length regulation: coupling DNA end processing to feedback regulation of telomerase

The conventional DNA polymerase machinery is unable to fully replicate the ends of linear chromosomes. To surmount this problem, nearly all eukaryotes use the telomerase enzyme, a specialized reverse transcriptase that utizes its own RNA template to add short TG-rich repeats to chromosome ends, thus reversing their gradual erosion occurring at each round of replication. This unique, non-DNA templated mode of telomere replication requires a regulatory mechanism to ensure that telomerase acts at telomeres whose TG tracts are too short, but not at those with long tracts, thus maintaining the protective TG repeat cap at an appropriate average length. The prevailing notion in the field is that telomere length regulation is brought about through a negative feedback mechanism that counts TG repeat-bound protein complexes to generate a signal that regulates telomerase action. This review summarizes experiments leading up to this model and then focuses on more recent experiments, primarily from yeast, that begin to suggest how this counting mechanism might work. The emerging picture is that of a complex interplay between the conventional DNA replication machinery, DNA damage response factors, and a specialized set of proteins that help to recruit and regulate the telomerase enzyme

Development of microsatellite markers for Rhodiola rosea

Rhodiola rosea L. is an important adaptogen medicinal plant. In this study two new microsatellite markers were developed. The assessment of the genetic diversity of R. rosea has recently started with molecular markers, but only a few species-specific microsatellite markers have been published so far. However the small number of markers allows only a limited insight into the genetic variability of the species therefore the aim of our work was to develop new microsatellite markers for R. rosea with a microsatellite enrichment library technique. Genomic DNA was cleaved with an endonuclease enzyme followed by adaptor ligation and PCR amplification. DNA fragments that contained microsatellites were first isolated using a biotin-streptavidin linkage based magnetic selection and then cloned into plasmids. Out of forty-three sequenced clones three contained  microsatellites, in these cases primers were designed for the amplification of the microsatellite repeats. The newly developed primer pairs were tested on individuals from distant R. rosea populations and the variability of the amplified fragments was estimated by fragment-length analysis. The locus RhpB14a was found to be monomorphic while RhpB14b and RhpB13 were polymorphic. As a result of the present study, two novel variable microsatellite loci were identified in the genome of R. rosea

Compensatory Paracrine Mechanisms That Define The Urothelial Response to Injury in Partial Bladder Outlet Obstruction

Diseases and conditions affecting the lower urinary tract are a leading cause of dysfunctional sexual health, incontinence, infection, and kidney failure. The growth, differentiation, and repair of the bladder's epithelial lining are regulated, in part, by fibroblast growth factor (FGF)-7 and -10 via a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the receptor for FGF-7 and -10 within the transitional epithelium (urothelium). The FGF-7 gene is located at the 15q15-q21.1 locus on chromosome 15 and four exons generate a 3.852-kb mRNA. Five duplicated FGF-7 gene sequences that localized to chromosome 9 were predicted not to generate functional protein products, thus validating the use of FGF-7-null mice as an experimental model. Recombinant FGF-7 and -10 induced proliferation of human urothelial cells in vitro and transitional epithelium of wild-type and FGF-7-null mice in vivo.To determine the extent that induction of urothelial cell proliferation during the bladder response to injury is dependent on FGF-7, an animal model of partial bladder outlet obstruction was developed. Unbiased stereology was used to measure the percentage of proliferating urothelial cells between obstructed groups of wild-type and FGF-7-null mice. The stereological analysis indicated that a statistical significant difference did not exist between the two groups, suggesting that FGF-7 is not essential for urothelial cell proliferation in response to partial outlet obstruction. In contrast, a significant increase in FGF-10 expression was observed in the obstructed FGF-7-null group, indicating that the compensatory pathway that functions in this model results in urothelial repair

Novel Phosphorylation Sites in the S. cerevisiae Cdc13 Protein Reveal New Targets for Telomere Length Regulation

The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics. [Image: see text

Localization of telomeres and telomere-associated proteins in telomerase-negative Saccharomyces cerevisiae

Cells lacking telomerase cannot maintain their telomeres and undergo a telomere erosion phase leading to senescence and crisis in which most cells become nonviable. On rare occasions survivors emerge from these cultures that maintain their telomeres in alternative ways. The movement of five marked telomeres in Saccharomyces cerevisiae was followed in wild-type cells and through erosion, senescence/crisis and eventual survival in telomerase-negative (est2::HYG) yeast cells. It was found that during erosion, movements of telomeres in est2::HYG cells were indistinguishable from wild-type telomere movements. At senescence/crisis, however, most cells were in G2 arrest and the nucleus and telomeres traversed back and forth across the bud neck, presumably until cell death. Type I survivors, using subtelomeric Y′ amplification for telomere maintenance, continued to show this aberrant telomere movement. However, Type II survivors, maintaining telomeres by a sudden elongation of the telomere repeats, became indistinguishable from wild-type cells, consistent with growth properties of the two types of survivors. When telomere-associated proteins Sir2p, Sir3p and Rap1p were tagged, the same general trend was seen—Type I survivors retained the senescence/crisis state of protein localization, while Type II survivors were restored to wild type