Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants.

Telomerase is a ribonucleoprotein complex that adds telomeric DNA repeats to the ends of most eukaryotic chromosomes. The reverse transcriptase subunit of telomerase (TERT) differs from retroviral reverse transcriptases in having a long basic amino-terminal extension. We made a large library containing random mutations in the amino terminus of the EST2 gene, which encodes the Saccharomyces cerevisiae TERT, and selected functional alleles by their ability to rescue senescence of telomerase-negative cells. Through analysis of 265 mutations, the amino terminus of Est2p was found to contain at least four essential regions. This domain structure was verified by a combination of deletion and alanine-block mutations. Mutations within two essential domains of the protein reduced RNA binding, suggesting that the amino terminus of Est2p makes important contacts with the intrinsic RNA component of telomerase. A mutant close to the amino terminus retained RNA binding and in vitro enzymatic activity but was defective in vivo, suggesting a role in interaction with other macromolecular components of telomerase.

CLB5-dependent activation of late replication origins in S. cerevisiae.

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

Replication profile of Saccharomyces cerevisiae chromosome VI.

BACKGROUND: An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae. RESULTS: Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized. Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population. The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes--the telomeres replicate late in S phase, while the central region of the chromosome replicates early. CONCLUSIONS: As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.

Multiple determinants controlling activation of yeast replication origins late in S phase.

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

Optics for the mathaphobic--prism mania.

When combining like values, add and take the sign of those numbers. When combining values that are not alike, subtract and take the sign of the higher number. Draw the numbers on an optical cross or number line to see what the numbers mean. Don't panic. Prism can be a blessing to the patient with diplopia.

Analysis of the temporal program of replication initiation in yeast chromosomes.

The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase. In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an internal position on chromosome XIV. Four origins, spanning approximately 140 kb, initiate replication in the second half of S phase. At least two of these internal origins maintain their late replication time on circular plasmids. Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation. Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these 'late' elements. We report here the development of two new assays for determining replication time. The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork. The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination. This non-replicating fragment acts as an internal control for copy number within and between samples. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA. With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating regions in the genome, and to begin to analyze the effect that altering the temporal program has on chromosome function.

Emulsion stability in a total nutrient admixture for total parenteral nutrition.

A total parenteral nutrition solution containing lipid was tested up to 28 days at 4 degrees C followed by an additional 2 days at 22 degrees C (room temperature) for stability of the emulsion. The total nutrient admixture (TNA) contained 1000 ml 10% FreAmine, 1000 ml 50% dextrose, 500 ml 10% Soyacal, electrolytes, vitamins and trace elements. Stability was determined by direct observation, light and electron microscopy, Coulter counter, pH, osmolality, and fatty acid profile. Samples were tested when fresh at 0 hours, after 2 days at 22 degrees C, 14 days at 4 degrees followed by 2 days at 22 degrees C, and 28 days at 4 degrees C followed by 1 and 2 days at 22 degrees C. Light microscopy measured particles greater than 2 microns in diameter; 99% of these particles were less than 6 microns, with no increase with time. Electron microscopy found that lipid particle size increased slightly up to 30 days, at which time the mean diameter was 0.36 +/- 0.01 micron. Coulter counter studies found that lipid droplet diameter increased while at 22 degrees C; however, on day 30, 99% of the particles were less than 1.97 microns. Mean pH and osmolality were 6.35 +/- 0.04 and 1880 +/- 14.5 mOsm/kg, respectively, with no change over time. Analysis of fatty acids by gas chromatography showed that fatty acid profiles and amounts of triglyceride, phospholipid and total lipid did not change. Thus, the emulsion in the TNA was stable for 28 days refrigeration, followed by 2 days at room temperature.

Physical stability of a total nutrient admixture for total parenteral nutrition.

The stability of a total nutrient admixture (TNA) has been postulated to be less than 7 days in refrigerated storage. When a TNA destabilizes, lipid particles coalesce and enlarge. Liposomes larger than 6 microns can obstruct pulmonary capillaries. A TNA containing 1500 ml of 7% Vamin, 1000 ml of 50% dextrose and 500 ml of 10% Intralipid, including the usual electrolytes, minerals and vitamins, was studied. Liposome size was measured in the original Intralipid and the TNA at intervals up to 14 days at 4 degrees C followed by 2 days at 22 degrees C. There was a small increase in liposome size up to 16 days. However, the number of particles larger than 6 microns was insignificant (by light microscopy, 3.9 +/- 2.4 [+/- SD] per 20 high-power fields; by Coulter counter, 99.8% smaller than 1.9 microns, with 0% larger than 6 microns; and by electron microscopy, 100% smaller than 2.0 microns). The osmolality and pH of the TNA were 1472 +/- 31 mOsm/kg and 5.5 +/- 0.1 respectively (mean +/- SD), with no significant change during the study times. The authors concluded that this TNA remains physically stable when refrigerated for 14 days and at room temperature for a further 2 days.