Demonstration of Z-d(5BrCGAT5BrCG) and B-d(CGCGATCGCG) form crystal structures in DNA-cobalt hexammine complexes by Kr 647.1 nm excitation of Raman spectra.

Cobalt hexammine [Co(NH3)6(3+)] is an efficient DNA complexing agent which significantly perturbs nucleic acid secondary structure. We have employed red excitation (647.1 nm) from a krypton laser to obtain Raman spectra of the highly colored complexes formed between cobalt hexammine and crystals of the DNA oligomers, d(5BrCGAT5BrCG) and d(CGCGATCGCG), both of which incorporate out-of-alternation pyrimidine/purine sequences. The Co(NH3)6(3+) complex of d(5BrCGAT5BrCG) exhibits a typical Z-form Raman signature, similar to that reported previously for the alternating d(CGCGCG) sequence. Comparison of the Raman bands of d(5BrCGAT5BrCG) with those of other oligonucleotide and polynucleotide structures suggests that C3'-endo/syn and C3'-endo/anti thymidines may exhibit distinctive nucleoside conformation markers, and tentative assignments are proposed. The Raman markers for C2'-endo/anti adenosine in this Z-DNA are consistent with those reported previously for B-DNA crystals containing C2'-endo/anti dA. Raman bands of the cobalt hexammine complex of d(CGCGATCGCG) are those of B-DNA, but with significant differences from the previously characterized B-DNA dodecamer, d(CGCAAATTTGCG). The observed differences suggest an unusual deoxyguanosine conformer, possibly related to a previously characterized structural intermediate in the B-->Z transition. The present results show that crystallization of d(CGCGATCGCG) in the presence of cobalt hexammine is not alone sufficient to induce the left-handed Z-DNA conformation. This investigation represents the first application of off-resonance Raman spectroscopy for characterization of highly chromophoric DNA and illustrates the feasibility of the Raman method for investigating other structurally perturbed states of DNA-cobalt hexammine complexes.     

Fission yeast with DNA polymerase delta temperature-sensitive alleles exhibits cell division cycle phenotype.

DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

Genetic and biochemical analysis of an endonuclease encoded by the IncN plasmid pKM101.

The IncN plasmid pKM101 nuc gene encodes a periplasmically localized endonuclease. DNA sequence analysis indicates that this gene encodes a hydrophilic protein of about 19.5 kDa containing a hydrophobic signal sequence. nuc is homologous to a partially sequenced open reading frame adjacent to the sog gene of the plasmid CollB-P9, a plasmid known to encode an endonuclease similar to that of pKM101. A partially sequenced tra gene directly upstream of nuc is homologous to the virB11 gene of Agrobacterium tumefaciens. We have partially purified the pKM101 nuclease by osmotic shock and cation exchange chromatography, and used this enzyme preparation to sequence the protein's amino terminus. The first 13 amino acids of the mature protein match amino acids 23 to 35 of the predicted sequence, indicating that the protein is proteolytically processed to a molecular mass of approximately 17 kDa, probably during export to the periplasmic space. The enzyme was able to attack many sites along an end labelled duplex DNA substrate, but showed clearly preferred cleavage sites, and may cleave preferentially at purine-rich regions.     

蛋白质中二硫键附近肽段识别规律研究

本文对蛋白质中二硫键附近的残基进行了计算机统计分析,结果发现平行和反平行残基间存在着特异的配对规律。这种残基间的相互作用或识别,可能与蛋白质折叠过程中正确地形成二硫键有关。该结果有助于蛋白质工程设计。     

白粒济宁3号小麦品种的选育

用快中子照射红粒济宁3号小麦干种子,后代获得白粒植株,经选育而成白粒济宁3号。白粒济宁3号与红粒济宁3号比,除籽粒颜色差别外,籽粒蛋白含量显著高于红粒济宁3号,而落黄性、籽粒饱满度、千粒重、产量均略低于红粒济宁3号,该品种累计种植面积10余万亩。     

激光诱变小麦后代材料籽粒贮藏蛋白编码基因变异的研究

本研究采用SDS—PAGE分析了冀麦七号、冀麦18和冀早15三个品种激光后代籽粒贮藏蛋白的变异情况。结果表明,高分子量麦谷蛋白和醇溶蛋白亚基变异较小,其控制位点为Glu—1和Gl—3或Gli—1;而低分子量部分变异较大,其控制位点多在Glu—3、Gli—2和Gli—3。这些变异具有一定的随机性。有关激光诱变对小麦面粉品质的影响有待进一步研究。     

肺癌患者胸水游离氨基酸分析

本文对11例肺癌患者胸水13种游离氨基酸作了分析,并与28例正常人血浆游离氨基酸水平作了对照,结果表明:肺癌患者胸水的必需及非必需氨基酸普遍高于正常人血浆游离氨基酸,但其胸水谷氨酰胺水平则明显低于正常人血浆水平。     

大鼠肝癌中α_2-巨球蛋白等几种分泌性蛋白基因表达的变化

用Northern blot方法对二乙基亚硝胺所诱发的大鼠肝癌中内源性蛋白酶抑制因子α_2-巨球蛋白(α_2-M)、非特异性免疫抑制剂α_1-酸性糖蛋白(α_1-AGP)及雄性激素正调控的α-2u球蛋白(α-2u)三种分泌性蛋白基因表达情况进行了分析。结果表明在大部分(14/16)肝癌样品中α_2-M RNA水平显著降低;而α_1-AGP RNA水平显著高于正常对照水平;α-2u RNA水平明显下降,但在某些雄性大鼠肝癌样品中该基因却有一定程度的表达。这些结果说明,一些肿瘤宿主血浆中α_2-M水平的显著下降及α_1-AGP水平的明显升高分别是由于基因表达活性的下降及升高所致。α-2u基因表达的异常提示,在癌变过程中机体的内分泌功能发生了某些变化。