Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis

Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase delta (Pol delta) with 5.-flap cutting by FEN1(RAD27) endonuclease. Excessive strand displacement is normally prevented by the 3.-exonuclease activity of Pol delta. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol delta) or PIF1 helicase genes can suppress lethality or growth defects of rad27Delta pol3-D520V mutants (defective for FEN1(RAD27) and the 3.-exonuclease of Pol delta) that produce long flaps and of dna2Delta mutants that are defective in cutting long flaps. On the contrary, pol32Delta or pif1Delta caused lethality of rad27Delta exo1Delta double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1(RAD27) and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol delta is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1(RAD27) during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol delta to FEN1(RAD27). Finally, RNA-DNA hybrids are more readily displaced by Pol delta than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.     

An Assessment of the Potential for Selenium to Impair Reproduction in Bull Trout, Salvelinus confluentus, from an Area of Active Coal Mining

Selenium (Se) is an essential nutrient, but in higher concentrations can reduce recruitment in fish populations by increasing rates of deformities during early development. Recent work has identified elevated levels of Se in water and biota collected downstream from coal mining activity in Alberta's northeast slopes region. We also recently identified increased incidence of terata and edema in rainbow trout and brook trout with elevated tissue Se from this area. However, there is currently no information regarding the potential for Se to contribute to declining stocks of bull trout, a species of concern in the area. The present study provides an assessment of the potential for Se to contribute to low recruitment in bull trout downstream from coal mining activity. Non-destructive muscle biopsy sampling and a sensitive atomic fluorescence analysis technique are used to determine Se. Results indicate that most bull trout (>90%) captured immediately downstream from coal mining activity in the region have concentrations of Se that would be expected to impair recruitment. Additional work is required to determine the extent of Se's contribution to low recruitment in bull trout.     

Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.     

CLADISTICS OF THE BRYOPSIDALES: A PRELIMINARY ANALYSIS

The Bryopsidales contains some of the most species rich and ecologically dominant algae in tropical ecosystems. However, the evolutionary relationships among the 29 genera and several hundred species of this order remain poorly resolved. Because of a lack of known reproductive characters for many taxa, evolutionary hypotheses grouped genera by similarities in morphological characters. To apply standard cladistical analyses to further our understanding of this group, this study presents the first comprehensive compilation of reported morphological, reproductive, and subcellular characteristics for genera in the Bryopsidales. Computer-assisted cladistical analyses ultimately identified phylogenetically informative and uninformative characters. Although the topology of the trees generated in this study is expected to change as additional data are added to this matrix, many traditional groupings and recent groupings based on molecular data were supported.     

Short communication. Identification of developmentally-specific markers in germinating and haustorial stages of Striga hermonthica (Del.) Benth. seedlings

Developmentally-specific markers have been identified in the germinating and haustorial stages of Striga hermonthica seedlings. Four water-soluble proteins, preferentially expressed in germinated seedlings, were microsequenced. An haustorial-specific cDNA clone was isolated by differential screening. Tissue specificity for this clone was assessed by Northern blot hybridization analysis.Key words: Striga hermonthica (Del.) Benth, microsequencing, lipid transfer protein, superoxide dismutase.      

Cryptosporidium parvum Mixed Genotypes Detected by PCR-Restriction Fragment Length Polymorphism Analysis

Combinations of 10 Cryptosporidium parvum oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.     

Bicarbonate transport proteins.

Bicarbonate is not freely permeable to membranes. Yet, bicarbonate must be moved across membranes, as part of CO2 metabolism and to regulate cell pH. Mammalian cells ubiquitously express bicarbonate transport proteins to facilitate the transmembrane bicarbonate flux. These bicarbonate transporters, which function by different transport mechanisms, together catalyse transmembrane bicarbonate movement. Recent advances have allowed the identification of several new bicarbonate transporter genes. Bicarbonate transporters cluster into two separate families: (i) the anion exachanger (AE) family of Cl-/HCO3- exchangers is related in sequence to the NBC family of Na+/HCO3- cotransporters and the Na(+)-dependent Cl/HCO3- exchangers and (ii) some members of the SLC26a family of sulfate transporters will also transport bicarbonate but are not related in sequence to the AE/NBC family of transporters. This review summarizes our understanding of the mammalian bicarbonate transporter superfamily.