Crystallization and preliminary crystallographic studies of the decadeoxyoligonucleotide d(CpGpTpApCpGpTpApCpG)

Crystals of the self-complementary decadeoxyoligonucleotide d(CpGpTpApCpGpTpApCpG) have been grown from a solution containing [Co(NH3)6]Cl3 and spermine. The amber-colored crystals are hexagonal and belong to the space group P6(5) (or P6(1] with unit cell parameters a = 17.93 A, c = 43.41 A. Precession photography and molecular packing considerations indicate that the unit cell consists of a 12 nucleotide duplex. The asymmetric unit, therefore, is a disordered duplex dimer in which each pyrimidine-purine base-pair is occupied 60% of the time by a C . G pair and 40% of the time by a T . A pair. The above considerations and preliminary structure analysis reveal that this alternating pyrimidine-purine oligomer assumes a Z-DNA conformation.     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

A nuclear specific glycoprotein representative of a unique pattern of glycosylation

Whole rat liver nuclei were reacted with UDP-[14C]galactose in the presence of bovine beta(1----4) galactosyltransferase. The reaction mixture was electrophoresed on a reducing sodium dodecyl sulfate-polyacrylamide gel. Autoradiograms of the gel demonstrated a major labeled broad band migrating with an apparent molecular weight of 65,000-66,000. A number of other less prominently labeled bands were also present. The labeled 65,000-66,000 band when cut from the gel and subjected to alkaline reduction while in the gel matrix exclusively yielded a 14C-labeled disaccharide that co-migrated with a [14C]Gal-GlcNAcol standard in descending paper chromatography. Treatment of this disaccharide with beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) from Aspergillus niger removed all the [14C]galactose label. Treatment of the labeled 65,000-66,000 polypeptide with Endoglycosidase F, however, did not remove the [14C]galactose label. Western transfer blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels performed with horseradish peroxidase-labeled succinyl wheat germ agglutinin, a lectin specific for GlcNAc, on unlabeled nuclei revealed a dominant band at 63,000-64,000. Subjecting 14C-labeled nuclei to this procedure resulted in a shift of the major horseradish peroxidase-labeled succinyl wheat germ agglutinin band to 65,000-66,000. The shifted band was coincident with the [14C]galactose band as visualized on an autoradiogram. A survey of other rat tissue nuclei revealed the same spectrum of [14C]galactose acceptor proteins with a dominant 65,000-66,000 galactose-labeled band.     

Isolation of mutants of the obligate methanotroph Methylomonas albus defective in growth on methane

A strain of Methylomonas albus BG8WM, a type 1 obligate methanotroph, which had been maintained for 2 ycars by serial passage on solid medium containing methanol as a carbon source was found to mutate at a frequency of 10^-5-10^-6 to resistance to dichloromethane (DCM^R), the parental strain BG8 did not give rise to DCM^R colonies. DCM^R strains were no longer capable of growth on methane as a carbon cource and exhibited greatly reduced or undetectable methane mono-oxygenase activity. The mutants fell into three groups on the basis of SDS-PAGE analysis of the polypeptide profiles of the particulate fraction of cell extracts. One or more of four polypeptides of Mr 70,000, 50,000, 25,000 and 23,000 were implicated as being components of the methane mono-oxygenase. Spontaneous reversion to growth on methane and sensitivity to dichloromethane occurred at a frequency of about 10^-8. The loss of methane mono-oxygenase activity was not associated with loss of the resident 55 kb plasmid.Abbreviations DCM^R dichloromethane-resistant - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - NMS nitrate minimal salts medium     

Predator detection and avoidance by starlings under differing scenarios of predation risk

Practically all animals must find food while avoiding predators.An individual's perception of predation risk may depend on manyfactors, such as distance to refuge and group size, but it isunclear whether individuals respond to different factors ina similar manner. We tested whether flocks of foraging starlingsresponded in the same way to an increased perception of predationrisk by assessing three factors: (1) neighbor distances, (2)habitat obstruction, and (3) recent exposure to a predator.We found that in all three scenarios of increased risk, starlingsreduced their interscan intervals (food-searching bouts), whichincreased the frequency of their vigilance periods. We thenexamined how one of these factors, habitat obstruction, affectedescape speed by simulating an attack with a model predator.Starlings were slower to respond in visually obstructed habitats(long grass swards) and slower when they had their head downin obstructed habitats than when they had their head down inopen habitats. In addition, reaction times were quicker whenstarlings could employ their peripheral fields of vision. Ourresults demonstrate that different sources of increased riskcan generate similar behavioral responses within a species.The degree of visibility in the physical and social environmentaffects both the actual and perceived risk of predation.     

N-糖蛋白去糖基化酶（PNGase）的研究进展

N-糖蛋白去糖基化酶（PNGase）是一种广泛存在于真菌、植物、哺乳动物中的去糖基化酶，可以水解N-糖蛋白或 N-糖肽上天冬酰胺与寡糖链连接的化学键，并释放出完整的N-寡糖。PNGase在生物体内参与蛋白质降解、器官发育、个体生长等过程。人PNGase基因功能缺陷会导致先天性去糖基化障碍，小鼠PNGase缺陷会导致胚胎致死性，线虫PNGase缺陷使其寿命下降。本文对PNGase在不同物种的分布、蛋白质结构、酶学功能及生物学功能进行阐述，为PNGase的生理病理功能及致病机制的基础研究提供思路，为PNGase作为糖生物学工具酶或药物开发的创新应用研究奠定基础。