Isolation and characterization of microsatellite markers in black muntjac (Muntiacus crinifrons)

The black muntjac (Muntiacus crinifrons) is a vulnerable species found in the mountains of eastern China, about which little is known. Here we develop 11 polymorphic microsatellite loci from a (CA)(n) -enrichment library for the animal. In the analyses of 25 individuals sampled, unbiased expected heterozygosity levels varied from 0.686 to 0.876 and the number of alleles per locus varied from five to nine. Results that 11 microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as parentage, mating system and population genetic structure of M. crinifrons.     

Genomic instability is associated with natural life span variation in Saccharomyces cerevisiae

Increasing genomic instability is associated with aging in eukaryotes, but the connection between genomic instability and natural variation in life span is unknown. We have quantified chronological life span and loss-of-heterozygosity (LOH) in 11 natural isolates of Saccharomyces cerevisiae. We show that genomic instability increases and mitotic asymmetry breaks down during chronological aging. The age-dependent increase of genomic instability generally lags behind the drop of viability and this delay accounts for approximately 50% of the observed natural variation of replicative life span in these yeast isolates. We conclude that the abilities of yeast strains to tolerate genomic instability co-vary with their replicative life spans. To the best of our knowledge, this is the first quantitative evidence that demonstrates a link between genomic instability and natural variation in life span.     

Salt bridges in the hyperthermophilic protein Ssh10b are resilient to temperature increases

A double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in the hyperthermophilic protein Ssh10b. The coupling free energy were 2.4 +/- 0.4 kJ/mol at 298 K and 2.2 +/- 0.4 kJ/mol at 353 K for Glu-54/Arg-57, and 6.0 +/- 0.2 kJ/mol at 298 K and 5.9 +/- 0.6 kJ/mol at 353 K for Glu-36/Lys-68. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures. The reason for the difference in coupling free energy between salt bridges Glu-54/Arg-57 and Glu-36/Lys-68 is discussed. Comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the energy contribution of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.     

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.     

Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10-30% lower than that of UGPase-2, depending on direction of the reaction, whereas its K(m) values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03-0.14 mM vs. 0.07-0.36 mM, respectively). Both proteins were "true" UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.