``break Copy'''' Duplication: A Model for Chromosome Fragment Formation in Saccharomyces Cerevisiae

Introduction of a chromosome fragmentation vector (CFV) into the budding yeast Saccharomyces cerevisiae results in a targeted homologous recombination event that yields an independently segregating chromosome fragment (CF) and an alteration in the strain's karyotype. Fragmentation with an acentric CFV directed in a centromere-proximal orientation generates a CF that contains all sequences proximal to the targeting segment and results in loss of the endogenous targeted chromosome to yield a 2N-1+CF karyotype. In contrast, fragmentation with a centric CFV directed in a centromere-distal orientation generates a CF that contains all sequences distal to the targeting segment and retention of the endogenous targeted chromosome to yield a 2N+CF karyotype. We have termed this phenomenon ``break copy' duplication. Using yeast strains in which the centromere had been transposed to a new location, it was demonstrated that the centromere inhibited break copy duplication. These data suggest that CF formation is the product of an unscheduled DNA replication event initiated by the free end of the CFV and is analogous to a ``half' double-strand break gap-repair reaction. We suggest that break copy duplication may have evolved as a mechanism for maintenance of ploidy following DNA breakage.     

Analysis and parameter resolution in highly cooperative systems

We have examined common methods of analysis of highly cooperative systems such as oxygen binding by hemoglobin and thermal denaturation. Through extensive simulation of ligand-binding data for a tetrameric macromolecule we show that careful attention must be paid to the formulation of the fitting function and to proper assessment of the number of parameters involved. We conclude that the partition function should be formulated in terms of overall reaction parameters as opposed to stepwise reaction parameters and that bias is introduced by fixing physical parameters such as extrapolated end points.     

Identification and metabolic characteristics of an apolipoprotein C-II variant isolated from a hypertriglyceridemic subject

The very low density lipoprotein (VLDL) apolipoproteins from a Type IV hypertriglyceridemic Caucasian subject (plasma TG: 645 mg/dl) and his brother (plasma TG: 328 mg/dl) were separated by isoelectric focusing gel electrophoresis (IEF) and found to contain two isoforms of apoC-II, identified by immunoblot. These corresponded to normal apoC-II-1 (isoelectric point: pI 4.88) and a variant isoform (apoC-II-v, pI 4.74). The pI of C-II-v was not altered by neuraminidase treatment, indicating that it was not sialylated. The concentration of total immunoreactive C-II in VLDL was elevated (18 mg/dl vs normal; 5.0 +/- 2 mg/dl) but similar to that in other Type IV subjects. In VLDL, which contained 90% of the plasma immunoreactive apoC-II, the ratio (by IEF) of C-II-1:C-II-v was 2:1, whereas in high density lipoproteins (HDL) the ratio was 1:1. VLDL apoB turnover was measured after the pulse injection of 125I-labeled VLDL. VLDL apoB kinetic parameters for the proband and four Type IV subjects were similar: production rate, 28 mg/kg per day versus 30 mg/kg per day; fractional catabolic rate, 1.62.day-1 versus 1.96.day-1; and pool size, 17 mg/kg versus 18 mg/kg. The decline in VLDL triglyceride (TG) after the infusion of heparin (9,000 IU over 4 h) was also similar to that observed in Type IV subjects. In VLDL, the fractional catabolic rates of apoC-II-1 and C-II-v were similar (C-II-1: 0.31.day-1, C-II-v: 0.29.day-1) whereas in HDL, although similar to each other, the rates were greater than in VLDL (C-II-1: 0.48.day-1, C-II-v: 0.44.day-1). VLDL and HDL from the proband were normal in their ability to activate bovine skim milk lipase, compared to Type IV VLDL and HDL without C-II-v. Purified apoC-II-1 and apoC-II-v activated the milk lipase to a similar extent (at 1 microgram of C-II; C-II-1: 34 units/h, C-II-v: 35 units/h). Thus, apoC-II-v is a newly recognized isoform of apoC-II-1. It remains to be determined whether this mutation plays a role in the genesis of hypertriglyceridemia.     

Identification of a telomeric fragment from the right arm of chromosome III of Aspergillus nidulans

The activities of pyruvate decarboxylase, alcohol dehydrogenase and certain glycosidases were measured for six species of yeast. Five of these yeasts could utilize one or more disaccharides aerobically, but not anaerobically, although all could use D-glucose anaerobically. That is, each of the five showed the Kluyver effect; but the sixth yeast, Saccharomyces cerevisiae, did not do so. When grown on a glycoside with which it gave the Kluyver effect, each yeast had much less pyruvate decarboxylase activity than when grown on D-glucose or another glycoside. There was no consistent corresponding lowering of activity of either alcohol dehydrogenase, or of the appropriate glycosidase. Hence, pyruvate decarboxylase may have a role in producing the Kluyver effect.     

Chorismate mutase isoenzymes from Sorghum bicolor: purification and properties

Two forms of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, have been detected in etiolated seedlings of Sorghum bicolor after DEAE-cellulose chromatography. CM-1 and CM-2 contained 44 and 56%, respectively, of the total activity measured after DEAE-cellulose chromatography. CM-1 was activated by tryptophan and inhibited by phenylalanine and tyrosine. In contrast, CM-2 was insensitive to all three aromatic amino acids. CM-1 and CM-2 were purified 1389- and 1018-fold, respectively, by anion exchange, hydrophobic, and dye matrix chromatography. The molecular weights estimated by gel filtration on Sephacryl S-200 were 56,000 for CM-1 and 48,000 for CM-2. Subunit molecular weights of the two forms were estimated by sodium dodecyl sulfate-gel electrophoresis at 36,000 and 51,000 for CM-1 and CM-2, respectively. Tryptophan was required for the stability of CM-1 at all stages of purification. Both isoenzymes were stable at 0 or -20 degrees C and had broad pH optima (6-10 for CM-1 and 7.5-9.5 for CM-2).     

Geographic/ethnic differences in human herpesvirus-6 antibody patterns.

Serum samples from healthy adults in four geographic/ethnic groups (Ghanaian Blacks, Malaysian Chinese, Malaysian Indians and United States Caucasians) were tested under code for antibodies to human herpesvirus-6 (HHV-6). The prevalence and titer of HHV-6 antibody in the healthy Ghanaians were significantly higher than in the Malaysian Chinese; United States Caucasians and Malaysian Indians had intermediate prevalence and titer of antibodies. Thus far, no specific differences in HHV-6-associated diseases have been noted between geographic/ethnic groups with these marked variations in antibody patterns.     

Freshwater crabs (Decapoda: Pseudothelphusidae) increase rates of leaf breakdown in a neotropical headwater stream

1. Freshwater crabs are the largest macroconsumers in many neotropical headwater streams, but few studies have examined their roles in ecosystem processes such as leaf litter breakdown. As omnivorous macroconsumers, freshwater crabs affect multiple trophic levels. They may directly increase leaf breakdown through fragmentation and consumption or indirectly decrease breakdown by consuming other macroinvertebrates, including shredders and detritivores.
2. In a headwater stream in Monteverde, Costa Rica, we conducted an in-stream experiment with 40 enclosures to quantify the effects of pseudothelphusid crabs on both leaf breakdown and macroinvertebrate colonisation of leaves. Half of the enclosures were randomly selected to contain two crabs (mean carapace width = 30 mm) and half were controls without crabs. We sampled mixed leaf packs from the enclosures on days 11, 19, 28, 34, and 42. We found the leaves of one species (Koanophyllon pittieri) almost completely decomposed by day 28 in both treatments (crab versus no crab). The other two leaf species (Meliosma idiopoda, Quercus brenesii) composed the remaining leaf mass at the end of the experiment.
3. At 42 days, enclosures with crabs had faster rates of leaf breakdown than those without crabs (with crabs: k = −0.020; without crabs: k = −0.016; p = 0.034). This suggests that the magnitude of direct leaf breakdown by crabs, due to fragmentation, consumption, or manipulation of leaves, was greater than any indirect effects on leaf breakdown via crab consumption of other leaf-consuming species.
4. Macroinvertebrate composition based on taxa abundances or biomasses did not significantly differ between treatments (ANOSIM; p = 0.73 and p = 0.65, respectively). Shredder and detritivore abundances and biomasses increased significantly through time (ANOVA; p ≤ 0.001), but there was no evidence of an effect of crab presence (p > 0.2), nor were there significant interactions between crab presence and time (p > 0.3).
5. This is one of the first studies to quantify the effects of pseudothelphusid freshwater crabs on leaf breakdown rates. Our results suggest that these crabs can play a significant role in detrital processing in neotropical headwater streams. This study has also demonstrated that short-term enclosure experiments are useful in measuring in-stream effects of crab activity on leaf breakdown.