Cloning of a gene for intracellular alginate lyase in a bacterium isolated from a ditch

A DNA fragment with a gene for intracellular alginate lyase in a bacterium A1 isolated from a ditch was cloned using a vector plasmid pKK223-3 and the gene was weakly expressed in Escherichia coli DH1 cells. The alginate lyase produced by E. coli DH1 cells was thought to correspond to A1-I among three kinds of alginate lyases (A1-I, A1-I-1 and A1-I-2) produced by the strain A1. Through this study, CaCl₂ was found to be a useful agent for the screening of microbial alginate lyase-producing colonies on agar plates.     

Analysis of cell variants showing differential susceptibilities to radiation- or chemical-induced neoplastic transformation: differences in their responses to growth factors

The induction of DNA synthesis in quiescent, density-arrested Balb/c 3T3 cells is known to be controlled by the sequential action of at least two functionally distinct sets of growth factors, so-called "competence factors" and "progression factors." Here we examined this induction pathway in Balb/c 3T3 A31-I variants, which showed differential susceptibilities to radiation- and chemical-induced neoplastic transformation despite their similar susceptibilities to radiation- or chemical-induced cell killing and mutagenesis. DNA synthesis was acquired only with the exposure to progression factors in a highly susceptible cell variant (A31-1-13) whereas both competence factors and progression factors were required for a less susceptible cell variant (A31-I-1). The competent state constitutively produced by an autologous mechanism in the highly transformation-susceptible A31-I-13 cells suggests the existence of an endogenous promoter that acts for the expression of the transformed phenotype in an autocrine fashion when the cells have been initiated by radiation or chemical carcinogens. The growth factor requirements acting as a determining factor for susceptibilities to transformation are discussed.     

Radiobromine labeled norcholesterol analogs. Synthesis and tissue distribution study in rats of bromine-82 labeled 6β-bromomethyl-19- norcholest-5(10)-en-3β-ol

6β-Bromomethyl-19-norcholest-5(10)-en-3β-o1 (NCL-6-Br) was synthesized directly from cholest-5-ene-3β,19-diol 19-toluene-psulfonate $\text{via}$ homoallylic rearrangement with ammonium bromide or sodium bromide in acetonitrile. This method was applied to the radiolabeling of NCL-6-Br with bromine-82. Tissue distribution of bromine-82 labeled NCL-6-Br (NCL-6-Br-82) in rats was determined. The mean percent dose per gram uptake in adrenal at 24 and 120 h was 98 and 80 %/gm, respectively, which indicated a higher adrenal uptake as compared to iodine-131 labeled 19-iodocholest-5-en-3β-o1 (CL-19-I-131), but was at a lower level than that achieved with iodine-131 labeled 6β-iodomethy 119-norcholest-S(10)-en-3β-o1 (NCL-6-I-131). The ratio of radioactivity in the adrenal-to-liver concentration was also lower than that of CL-19-I-131 or NCL-6-I 131.     

Chromosome-wide analysis of parental allele-specific chromatin and DNA methylation

To reveal the extent of domain-wide epigenetic features at imprinted gene clusters, we performed a high-resolution allele-specific chromatin analysis of over 100 megabases along the maternally or paternally duplicated distal chromosome 7 (Chr7) and Chr15 in mouse embryo fibroblasts (MEFs). We found that reciprocal allele-specific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of H3K27me3 (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H19-Igf2 domain. A paternal allele-specific gap was found along Kcnq1ot1, interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allele-specific chromatin marks at the Peg13 and Slc38a4 DMRs, Cdkn1c upstream region, and Inpp5f_v2 DMR and paternal allele-specific CTCF binding at the Peg13 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor H3K9ac and CTCF or H3K4me3 in one allele and H3K9me3 in the reciprocal allele, using a sliding-window approach, recognized with precision the parental allele specificity of known imprinted genes, H19, Igf2, Igf2as, Cdkn1c, Kcnq1ot1, and Inpp5f_v2 on Chr7 and Peg13 and Slc38a4 on Chr15. Chromatin features, therefore, can unequivocally identify genes with imprinted expression.     

自噬相关基因LC3-I的基因表达及单克隆抗体的制备

目的：通过克隆LC3．I基因,体外原核表达LC3-I蛋白后制备抗LC3单克隆抗体,作为自噬研究中的标记分子检测自噬的发生和发展过程。方法：RT．PCR方法从RAW264．7细胞基因组中克隆LC3基因,亚克隆至pQE80L原核表达载体后转化E．cobDH5a进行诱导表达,SDS—PAGE电泳及Westemblot鉴定表达蛋白。蛋白纯化后免疫BALB／c小鼠。采用淋巴细胞杂交瘤技术,制备分泌抗LC3．I杂交瘤细胞株,体内诱生腹水制备mAb,间接ELISA法测定其效价,辛酸一硫酸铵沉淀法及亲和层析法纯化mAb。结果：成功克隆了LC3一I基因,并对其在E．coilDH5a进行诱导表达,SDS-PAGE分析表明在相对分子量Mr为20×10^3有特异条带,Westernblot验证表达产物具有一定的生物学活性。建立了3株稳定分泌特异性抗LC3-ImAb的杂交瘤细胞株,诱导产生的腹水获得的抗体效价在10^5-10^7之间,结论：在E．coli中对LC3-I进行表达,并制备特异性较强抗LC3-I蛋白的单克隆抗体。为自噬研究提供了良好的标记分子,可对自噬形成和发展进行有效的检测。     

烟草磷转运蛋白基因NtPHT2;1的克隆和表达分析

植物对磷酸盐的吸收与利用主要依靠磷转运蛋白,其中PHT2家族编码的低亲和磷转运蛋白主要负责植物在正常供磷条件下磷酸盐的吸收、转运与再利用。为了探究低亲和磷转运蛋白基因NtPHT2;1在烟草转运磷酸盐中的作用和表达模式,本研究以普通烟草K326的cDNA为模板,克隆得到NtPHT2;1,对该基因进行生物信息学分析和蛋白质的亚细胞定位,并通过荧光定量PCR技术对该基因在低磷等非生物胁迫下的基因表达模式进行分析。结果表明：（1）NtPHT2;1基因的全长为1 764 bp,编码587个氨基酸。（2）亚细胞定位结果表明,NtPHT2;1蛋白定位于叶绿体上。（3）同源性比对发现,NtPHT2;1蛋白与辣椒CaPHT2;1蛋白的同源性最高达到91.00%。（4）启动子分析表明,NtPHT2;1启动子含有参与调控植物衰老、逆境胁迫相关的顺式作用元件。（5）组织表达模式分析表明,NtPHT2;1在叶片中的表达量最高,新叶中的表达量比老叶中的高;在低磷诱导条件下,该基因的表达量与正常条件相比差异不显著。（6）不同非生物胁迫下的表达模式表明,在盐胁迫和干旱胁迫下,该基因的表达量显著降低。研究认为,NtPHT2;1基因主要是负责烟株正常生长发育条件下磷酸盐的转运与利用。     

Development of biosensors for phenol determination from bacteria found in petroleum fields of West Siberia

Nine Gram-negative bacterial strains, selected from 300 strains isolated from soils of the West Siberian petroliferous basin and growing on oil and oil products, consume phenol as a single carbon and energy source. The strains were used for the development of a sensor bioreceptor. The most active 32-I strain was shown to bear a plasmid responsible for phenol degradation. The plasmid-free derivative of this strain, 32-I-1, did not grow on phenol. The possibility of creating a model biosensor for phenol based on the plasmid-containing 32-I strain is considered. The detection limit for phenol was 5 microM. The optimum conditions for the sensor operation are: pH 7.4, 35 degrees C, and operation time 30 h.     

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.     

Evolution of the ovine MHC DQA region

Southern hybridisation was used to define an apparent gene duplication event at the ovine DQA2 locus. Approximately 500 sheep from five different breeds were genotyped at their DQA1 and DQA2 loci. A subset of these were selected for further characterisation. Southern hybridisation of TaqI digested DNA revealed no DQA1 region in some sheep. It was also noted in these DQA1 null animals the DQA2 specific probe hybridised to two bands. An EcoRV-RFLP designed to distinguish copy number confirmed this duplication of the DQA2 region. The results showed that the duplication was exclusively associated with the DQA1 null haplotype and occurred only in alleles DQA2-F, -G, -I and -J. Comparison with bovine MHC genes revealed that they also contained a DQA1 null haplotype and that this haplotype was associated with a putative DQA3 gene. The potential for an ovine DQA3 locus is discussed.     

Triplication of α-globin genes is responsible for unusual α113Leu/α113His-globin chain ratios in sheep

By investigations at the DNA and protein level, it has been shown that in sheep a previously detected, presumed quantitative allele of the II alpha 113His gene, displaying a reduced efficiency (called the II alpha 113His decreases gene), is carried by a chromosome bearing three alpha-globin loci. In particular, five sheep having an alpha 113Leu/alpha 113His-chain ratio of about 13:1 (13:1 phenotype) possessed the -I alpha 113Leu-II alpha 113Leu-/-I alpha 113Leu-II alpha 113Leu-III alpha 113His decreases genotype. One sheep showing a alpha 113Leu/alpha 113His-chain ratio of about 3:1 (3:1 phenotype) had the -I alpha 113Leu-II alpha 113His-/-I alpha 113Leu-II alpha 113Leu-III alpha 113His decreases genotype, while one sheep having a chain ratio of about 6:1 (6:1 phenotype) carried the -I alpha 113Leu-II alpha 113Leu-II alpha 113His decreases-/-I alpha 113Leu-II alpha 113Leu-III alpha 113His decreases genotype. Nineteen sheep, displaying the common phenotypes, all possessed the alpha alpha/alpha alpha gene arrangement. Furthermore, the possible location of the gene with reduced efficiency and the expression of the three genes in the triple alpha-globin loci chromosome are discussed.     

Calpain 1 binding capacities of the N1-line region of titin are significantly enhanced by physiological concentrations of calcium

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.     

Influence of the genetic polymorphism in the 5'-noncoding region of the CYP1A2 gene on CYP1A2 phenotype and urinary mutagenicity in smokers

The functional significance of genetic polymorphisms on tobacco smoke-induced CYP1A2 activity was examined. The influence of three polymorphisms of the cytochrome P450 1A2 gene (CYP1A2) (-3860 G-->A (allele *1C), -2467 T-->delT (allele *1D), -163C-->A (allele *1F)), located in the 5'-noncoding promoter region of the gene, on CYP1A2 activity (measured as caffeine metabolic ratio, CMR), was studied in Caucasian current smokers (n=95). Tobacco smoke intake was calculated from the number of cigarettes/day. Also, studied was the influence of these CYP1A2 genotypes on smoking-associated urinary mutagenicity, detected in Salmonella typhimurium strain YG1024 with S9 mix, considering the urinary excretion of nicotine plus its metabolites as an internal indicator of tobacco smoke exposure. Smokers with at least one of the variant alleles CYP1A2 -3860A and -2467 delT showed a significantly increased CYP1A2 CMR (-3860 G/A versus G/G, p<0.05; -2467 delT/delT versus T/delT and T/T, p<0.01). Multiple regression analysis showed that the increase in CYP1A2 CMR (ln values) was again significantly related to the presence of CYP1A2 variants -2467delT and also to variant -163A (p<0.05), but moderately to -3860A (p=0.084). No influence of the number of cigarettes smoked per day by each subject was found. Heavy smokers (n=48, with urinary nicotine plus its metabolites>or=0.69 mg/mmol creatinine) with variant allele -2467delT or -163A had significantly increased urinary mutagenicity (p<0.01 and <0.05). CYP1A2 genetic polymorphisms are shown to influence the CYP1A2 phenotype in smokers, -2467 T-->delT having the main effect. This information is of interest for future studies assessing the possible role of tobacco smoke-inducible CYP1A2 genotypes as individual susceptibility factors in exposure to carcinogens.     

Development of Biosensors for Phenol Determination from Bacteria Found in Petroleum Fields of West Siberia

Nine gram-negative bacterial strains, selected from 300 strains isolated from soils of the West Siberian petroliferous basin and growing on oil and oil products, consume phenol as a single carbon and energy source. The strains were used for the development of a sensor bioreceptor. The most active 32-I strain was shown to bear a plasmid responsible for phenol degradation. The plasmid-free derivative of this strain, 32-I-1, did not grow on phenol. The possibility of creating a model biosensor for phenol based on the plasmid-containing 32-I strain is considered. The detection limit for phenol was 5 M. The optimum conditions for the sensor operation are: pH 7.4, 35°C, and operation time 30 h.     

Migration of vascular smooth muscle cells by phospholipase A2 via specific binding sites.

Pancreatic-type group I phospholipase A2 (PLA2-I), EC 3.1.1.4, long thought to act as a digestive enzyme, has a specific binding site in several types of tissues and cells and these sites promote PLA2-I-stimulated DNA synthesis. In this study we report a PLA2-I action on the migration of rat embryonic thoracic aorta smooth muscle cells (A7r5). A7r5 cells had a single class of PLA2-I binding site with an equilibrium binding constant (Kd) value of 1.7 nM and a maximum binding capacity (Bmax) of 40,000 sites/cell. The migration activity of PLA2-I for A7r5 cells was examined using modified Boyden chambers. PLA2-I stimulated the migration dose-dependently, and the ED50 value was about 1 nM, which was almost the same as the Kd value for PLA2-I binding. Checkerboard analysis showed that the response of A7r5 cells to PLA2-I was chemokinetic, but not chemotactic. These findings reveal a new aspect of PLA2-I in the modulation of vascular function.     

Choice of method for identifying germplasm with superior alleles

Summary An accurate and efficient method of screening the many germplasm sources available for their ability to improve elite, adapted germplasm is needed. The superiority measure (SX) of a population (P) was defined as the product of the frequency and relative superiority of the alleles in P that are more favorable than the best in an elite, adapted reference single cross I1×I2. A computer simulation was done to determine the correlations between various screening methods and the SX. The genetic model used included multiple alleles, no linkage, two types of non-epistatic gene action (additive and complete dominance) and two types of epistatic gene action (complementary and duplicate). Genetic variances in the populations and a statistic proposed by Dudley (SD={[P x I1-I1] [I1 xI2-I2]- [P x I2-I2]-[P x I2-I2] [I1 x I2-I1]}/{2[I1-I2]{) were inconsistently correlated with the SX over all types of gene action on the basis of rank correlations. The testcross to the single cross (TC[SC]=P x [I1 x I2]) and the upper bound on the SX (UBND=minimum [P x I1-I1, P x I2-I2]) were both consistently highly genetically correlated with the SX. In the set of populations simulated, there were positive correlations between products of allelic frequencies and effects at different classes of loci. The UBND usually had a higher rank correlation coefficient with the SX than did the TC(SC). The differences between their correlation coefficients were often insignificant. Although the TC(SC) gives no indication as to which inbred the population is more closely related, its ease of use and expected lowers standard error compared with the UBND indicate that it would be an appropriate choice of screening method for identifying superior populations in the sense defined.Joint contribution: USDA-ARS and Journal Paper No. J-12421 of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA 50011. Project No. 2778. Part of a dissertation submitted by senior author in partial fulfillment of PhD requirements