Saccharomyces cerevisiae telomeric DNA replicates late in S phase, and telomeric genes are transcriptionally silent. Transcriptional repression of telomere-proximal genes results from silent chromatin initiating at the chromosome end, but the relationship between telomeric chromatin and DNA replication is unknown. Mutations in SIR3, a silent chromatin component, cause telomeric DNA on chromosome V to replicate much earlier because of earlier initiation of a nearby replication origin, the Y ARS. A second telomere-proximal ARS, from an X element, does not act as an origin in a wildtype strain, whereas in a sir3 cell it does. We conclude that telomeric chromatin has a Sir3-dependent inhibitory effect on DNA replication.Received October 6, 1998; revised version accepted December 1, 1998. Different regions of the eukaryotic genome are known to replicate at distinct, reproducible times within the period of S phase. This is observed in plants, insects, and vertebrates, as well as in the yeast Saccharomyces cerevisiae (Hand 1978;Fangman and Brewer 1992). In species with large genomes, extensive regions of the chromosomes replicate early in S phase, whereas other domains do not initiate replication until after the early domains have completed synthesis. Frequently, late replication of DNA is associated with its assembly into heterochromatin, which is highly condensed chromatin that often contains repeated DNA sequences such as those found at centromeres and telomeres (John 1988). This condensed chromosome replicates much later in S phase than its transcriptionally active homolog.Telomeric chromatin in S. cerevisiae has several traits typical of heterochromatin (Grunstein 1998). In particular, telomeres confer epigenetic silencing of nearby genes (position effect variegation), and they replicate late in S phase (McCarroll and Fanagman 1988;Gottschling et al. 1990). The special chromatin found near telomeres is composed of hypoacetylated core histones as well as the SIR proteins, which are required for silencing telomeric genes (Grunstein 1998). Of the SIR proteins, Sir3p is probably the key component that defines a telomeric domain of transcriptional repression. It interacts with the tails of histones H3 and H4, spreading from the telomere inward along the chromosome, and its abundance in the cell determines how far a silent domain extends from the telomere (Renauld et al. 1993;Hecht et al. 1996).In yeast, chromosome replication initiates at ARS (autonomous replicating sequences) elements . ARS elements were originally identified as sequences that permit high-efficiency transformation of plasmids in yeast by serving as origins of DNA replication. However, in their normal chromosomal context, only a subset of the ARS elements initiate DNA replication within the ∼30 min duration of S phase (Fangman and Brewer 1992). Specific chromosomal origins of S. cerevisiae, like ARS1 on chromosome IV, initiate replication relatively early in S phase. Other ARS elements initiate later in S phase, such as ARS501 on chromosome V, which re...

Quantification of Epstein-Barr virus (EBV) in peripheral blood is important for the diagnosis and management of serious EBV diseases, including posttransplant lymphoproliferative disorder. A variety of PCRbased methods are currently in use; however, there is little information on their comparability. This study assessed the relative performance of different quantitative assays. A multicenter comparative study was performed at eight sites using three panels consisting of serial dilutions of quantified EBV DNA and extracts from a total of 19 whole-blood specimens. Samples were distributed and tested blindly. Instrumentation, probe chemistries, amplification targets, and other test-related aspects varied considerably between laboratories. Each laboratory's calibration curve indicated strong evidence of a consistent log-linear relationship between viral load and cycle threshold, suggesting that intralaboratory tracking of a given patient would yield similar relative quantitative trends among the participating test sites. There was strong concordance among laboratories with respect to qualitative test results; however, marked quantitative discordance was seen. For most samples, the across-laboratory interquartile range of the reported viral load (in copies/l) was roughly 0.6 log-units, and for one sample the overall range was approximately 4.2 log-units. While intralaboratory tracking of patients may yield similar results, these data indicate a need for caution when attempting to compare clinical results obtained at different institutions and suggest the potential value to be gained by more standardized testing methodology.

We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice.The etiology of respiratory tract infections can be difficult to diagnose by health care practitioners because clinical history and symptoms are usually nonspecific for most communityacquired pathogens. Mycoplasma pneumoniae and Chlamydophila pneumoniae cause up to 22% of community-acquired pneumonias and 5 to 10% of cases of tracheobronchitis, pharyngitis, laryngitis, and sinusitis (3-4, 7, 10, 12). Historically, culture has been the gold standard for diagnosis. However, cultivation of these microorganisms can prove challenging because they are fastidious and may require weeks for growth. Serology is more convenient and sensitive than culture, but results are often delayed, and false-negative test results often occur early in the course of illness. Although not standardized, nucleic acid-based tests, such as PCR, provide fast and sensitive results. While such tests are not always available on site in many medical centers, the 24-to 48-h delay in transit time may still be acceptable given the higher diagnostic yield of PCR.Despite the obvious limitations of culture, physicians continue to order this test frequently. In recent years, ARUP Laboratories has received large numbers of requests, including more than 3,000/year for M. pneumoniae and more than 1,500/ year for C. pneumoniae culture. Studies focusing on the value of culture either have been small in scale or have used type strains or patient isolates rather than direct patient specimens (8,13). An accurate and reliable diagnosis of M. pneumoniae and C. pneumoniae is important to differentiate them from other common respiratory pathogens because their treatments differ (1). Being aware of the poor sensitivity of culture for these pathogens, we found the high numbers of test requests for M. pneumoniae and C. pneumoniae culture from respiratory tract specimens to be concerning and to require further investigation. To examine more closely the utility of culture for diagnosing respiratory syndromes, we compared its performance to those of nucleic acid testing and serology for detection of M. pneumoniae and C. pneumoniae.From 2003 to 2008, microbiology results of culture, PCR, and serology performed at ARUP Laboratories for M. pneumoniae and C. pneumoniae were retrospectively reviewed with a specific focus on respiratory specimens (e.g., nasal wash, nasopharyngeal swab, bronchoalveolar lavage, tracheal aspirate, sputum, and pleural fluid) for PCR and culture. Respiratory specimens were transported either refrigerated or frozen, except for C. pneumoniae culture, for which only refrigerated specimens were transported. Additional data were collected for M. pneumoniae culture (1995 to 2003). C. pneumoniae enzyme-linked immunosorbent assay (ELI...

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the UnitedStates. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay.PCR is now accepted as the gold standard for diagnosis of herpes simplex virus (HSV) infections in the central nervous system (CNS), exhibiting a high degree of specificity and a sensitivity superior to that of culture or DFA (3,7,10,11,17,18). While viral culture for HSV encephalitis is still routinely employed in the clinical lab, it is only capable of detecting virus in less than 5% of patients with CNS infections (10, 14). Detection of low viral titers often found in spinal fluid requires the high level of sensitivity that is provided by amplificationbased assays (13).In recent years, real-time PCR has made testing more convenient. Real-time PCR is more amenable to automation, allowing higher throughputs and decreased turnaround times. In addition, the implementation of a one-tube closed reaction greatly reduces the possibility for contamination. Besides the potential for sensitivity roughly equivalent to gel detection, the use of target-specific probes in real-time PCR offers an additional level of specificity. These probes also allow the detection of single nucleotide polymorphisms (SNPs) using melt curve analysis, permitting typing of different viral species.While real-time PCR has been widely adopted for molecular infectious disease testing, real-time technology has often been compared to traditional methods such as culture, with a predictable enhancement in sensitivity (4,6,7,12). The current literature is more limited on the comparative performance of real-time PCR with established PCR formats. There is little definitive published evidence that real-time detection using fluorescent probes is more sensitive than end detection by gel, raising the question of whether real-time PCR is necessarily more sensitive than conventional PCR with gel detection. Therefore, it is essential during the validation of any real-time assay to compare its performance to something in ad...

FARP overall performed better than DFA with the exception of Adenovirus, making the FARP an attractive alternative to laboratories looking to replace DFA with a rapid, user-friendly, multiplex molecular assay.