Telomeres, the chromatin structures at the ends of eukaryotic chromosomes, are essential for chromosome stability. The telomere terminates with a TG-rich 3′ overhang, which is bound by sequence-specific proteins that both protect the end and regulate the telomerase elongation process. Here, we demonstrate the presence of 3′ overhangs as long as 200 nt in asynchronously growing cells of the budding yeast Saccharomyces castellii. The 3′ overhangs show a wide distribution of 14–200 nt in length, thus resembling the distribution found in human cells. A substantially large fraction of the 3′ overhangs resides in the 70–200 nt range. Remarkably, we found an accumulation of a distinct class of 70-nt-long 3′ overhangs in the S phase of the cell cycle. Cells without a functional telomerase showed the same wide distribution of 3′ overhangs, but significantly, lacked the specific fraction of 70-nt 3′ overhangs. Hence, our data show that the highly defined 70-nt 3′ overhangs are generated by a telomerase-dependent mechanism, which is uncoupled to the mechanisms producing the bulk of the 3′ overhangs. These data provide new insights that will be helpful for deciphering the complex interplay between the specialized telomere replication machinery and the conventional DNA replication.
The sole coat protein VP2 of infectious pancreatic necrosis virus (IPNV) was isolated and purified from intact virions, propagated in CHSE-214 cells. Likewise was the full-length VP2 protein isolated and purified upon cloning and expression of the corresponding complete gene in E. coli. The two purified proteins of different synthetic origins carrying identical primary structures were utilized in an immunization program using a rabbit model. Sera obtained against both immunogens react equally well with authentic and recombinant VP2 in Western blots and ELISAs. Also, the total net binding forces as determined by avidity index (AI) calculations were high and of similar stature, exceeding 80. An IPNV infection of susceptible and permissive CHSE-214 cells could only be neutralized by IgG preparations obtained from rabbits immunized with authentic VP2. Only such antibodies were able to aggregate and sediment radiolabeled virions in glycerol gradients upon rate zonal centrifugations. The presence of sugar moieties on the authentic protein is suggested to be of pivotal importance in eliciting an immune response capable of preventing infection in cell cultures in vitro.
The possible importance of the O-linked glycosylation in virion stability and infectivity of infectious pancreatic necrosis virus (IPNV) was analysed. Enzymatic treatment with O-glycosidase of radiolabelled virions under different ionic conditions, to allow for possible alternative exposure of glycosidic enzyme cleavage sites, did not alter the specific infectivity of virions re-isolated after rate-zonal centrifugation in glycerol gradients. As an alternative method to assess the significance of carbohydrates in IPNV integrity, periodate oxidation in the presence of an aldehyde quencher was chosen. Following re-isolation of viruses, a 3-5 (10)log-unit reduction in specific infectivity was revealed and, at higher concentrations, a total disruption or virion aggregation was observed. The loss of infectivity of intact virions was not because of a lack of attachment to cells. Additionally, re-evaluation of reading values from UV-spectra of purified IPNV yielded a specific infectivity of 3 × 10(11) TCID(50)-units mg(-1) of protein and a ratio of 40 virions per TCID(50)-unit in the CHSE-214 cell system.
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