Construction and characterization of double mutants in nitrogenase of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Two mutants in nitrogenase of Klebsiella pneumoniae are constructed by site-directed mutagenesis and gene replacement procedure, which express the nitrogenases with Lysine and Glutamine substituting for α-Glutamine 190 and α-Histidine 194 respectively (Kp-Q α190 K and Kp-Hα194 Q). The above two substitutions are respectively introduced into a nifV mutant (expressing a citrate-containing nitrogenase) and sequentially two double mutants are obtained (Kp-Q α190 K-nifV and Kp-H α194 Q-nifV). All four mutants exhibit strict Nif phenotype under the N2-fixation condition and fail to grow diazotrophically. Altered nitrogneases are effectively depressed and the C2H2 reduction analysis shows that the double substitutions in Kp-Q α190 K-nifV abolish cell C2H2 reduction activity, but Kp-H α194 Q-nifV cells maintain a C2H2 reduction activity at 10% of that of wild type. Whole cell C2D2 reduction by all four mutants in comparison to the wild type and nifV mutant is also detected. The results show that only single α-Gln^194 substitution does not perturb the stereospecificity of protonation of C2D2. These results indicate that the α-Glutamine 190 and its combination with homocitrate are essential to the catalytic activity of nitrogenase and it is proposed that α-Glutamine 190 and its combination with homocitrate are involved in the proton and/or electron transfer to FeMoco. The nitrogenases from these double mutants will be useful in further analysis of the entry of the proton and/or electron to FeMoco and the substrate binding sites.     

Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of single nucleotide polymorphisms

A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.     

Involvement of DEG5 and DEG8 proteases in the turnover of the photosystem II reaction center D1 protein under heat stress in Arabidopsis thaliana

Deg5,deg8 and the double mutant,deg5deg8 of Arabidopsis thaliana were used to study the physiological role of the DEG proteases in the repair cycle of photosystem II (PSII) under heat stress. PSII activity in deg mutants showed increased sensitivity to heat stress, and the extent of this effect was greater in the double mutant, deg5deg8, than in the single mutants, deg5 and deg8. Degradation of the D1 protein was slower in the mutants than in the WT plants. Furthermore, the levels of other PSII reaction center proteins tested remained relatively stable in the mutant and WT plants following high-temperature treatment. Thus, our results indicate that DEG5 and DEG8 may have synergistic function in degradation of D1 protein under heat stress.     

Genetic analysis and identification of a large leaf angles （lla） mutant in rice

Mutants are essential genetic muterials to elucidation of biological functions of genes involved.Characterization and isolation of genes in mutants is one of the research tasks in functional genomic era.T-DNA insertional mutagenesis has provided an efficient way to identify genes in plant species.in which the mutated genes could be rapidly isolated once the mutant was confirmed by T-DNA inscrtion.     

ApcD is required for state transition but not involved in blue-light induced quenching in the cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

Pbycobilisomes （PBS） are able to transfer absorbed energy to photosystem Ⅰ and Ⅱ, and the distribution of light energy between two photosystems is regulated by state transitions. In this study we show that energy transfer from PBS to photosystem Ⅰ （PSI） requires ApcD. Cells were unable to perform state transitions in the absence of ApcD. The apcD mutant grows more slowly in light mainly absorbed by PBS, indicating that ApcD-dependent energy transfer to PSI is required for optimal growth under this condition. The apcD mutant showed normal blue-light induced quenching, suggesting that ApcD is not required for this process and state transitions are independent of blue-light induced quenching. Under nitrogen fixing condition, the growth rates of the wild type and the mutant were the same, indicating that energy transfer from PBS to PSI in heterocysts was not required for nitrogen fixation.     

ApcD is required for state transition but not involved in blue-light induced quenching in the cyanobacterium Anabaena sp.PCC7120

Pbycobilisomes(PBS) are able to transfer absorbed energy to photosystem I and Ⅱ,and the distribution of light energy between two photosystems is regulated by state transitions.In this study we show that energy transfer from PBS to photosystem I(PSI) requires ApcD.Cells were unable to perform state transitions in the absence of ApcD.The apcD mutant grows more slowly in light mainly absorbed by PBS,indicating that ApcD-dependent energy transfer to PSI is required for optimal growth under this condition.The apcD mutant showed normal blue-light induced quenching,suggesting that ApcD is not required for this process and state transitions are independent of blue-light induced quenching.Under nitrogen fixing condition,the growth rates of the wild type and the mutant were the same,indicating that energy transfer from PBS to PSI in heterocysts was not required for nitrogen fixation.     

Construction of DNA damage response gene pprI functiondeficient and function-complementary mutants in Deinococcus radiodurans

PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprl locus in the genome of the wild-type strain R1. The resultingpprl-deficient strain, designated YR1,was very sensitive to ionizing radiation. Meanwhile, the recombinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. radiodurans. The fragments containing complete pprl gene and 3‘-terminal deletion pprIΔ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprIΔ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. raiodurans R1, whereas YR1 pRADKpprIΔ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. radiodurans is not only useful to elucidating the relationship between domains and functions of PprI protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.     

Construction and Expression of GST and Carbon Skeleton of Amatoxins Fusion Gene

The primer, AT Forl, AT For2 and AT Back, are designed and synthesized for adding-PCR that is used to construct the fusion GST-AT gene. Depending on two-time adding-PCR amplification and the insertion of coding sequence of octapeptide carbon skeleton into the 3‘ temfinus of GST gene, the authors get the masculine recon, pGAT-1, confirmed by sequence detemfination and analysis, whose ORF is read-through. After IPTG induction and partial purification, SDS-PAGE electrophoresis is employed to detect the gene expression. The expressions of total bacterial proteins are about 21.3%, 22.5%, 19.32%, 21.73% in E. coli BL21, DH5α, JM109 and Top10 strains, respectively. Considering the quantity of induced total bacteria, the E. coli BL21 is the best one among the four recipient strains.This article supplies an academic foundation for producing biological active amatoxins.     

DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.     

Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.     

Duplications of a mutated simian virus 40 enhancer restore its activity

Enhancers are cis-acting control elements which can stimulate at a distance the activity of a variety of eukaryotic promoters. First identified as a repeated 72 base pair (bp) sequence upstream of the simian virus 40 (SV40) early gene promoter, enhancers have since been shown to be associated with numerous other viral and cellular genes. Although there are no strong homologies between the sequences of different enhancers, a number of short and degenerate consensus sequences have been identified, including the 'core' element GTGGA/TA/TA/TG and stretches of alternating purines and pyrimidines which may have the potential to form left-handed Z DNA. To study the functional significance of two alternating purine and pyrimidine sequences in the SV40 enhancer, we have introduced various combinations of point mutations into a modified SV40 enhancer which contained only one copy of the 72 bp element (W.H., Y.G., A. Nordheim and A. Rich, unpublished results); one of these combinations impaired both the activity of the enhancer and growth of SV40. We describe here the structure of 18 revertants of this mutant and suggest that in each of the 18 revertants, the defects of the original mutant have been overcome by simple tandem duplications in the enhancer region, all of which include the 'core' element.     

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.     

Activation of a transposable element in the germ line but not the soma of Caenorhabditis elegans

The genetic activity of transposable elements is tightly controlled in many species. Transposons that are relatively quiescent under certain circumstances can excise or transpose at greatly increased rates under other circumstances. For example, 'genomic shock' can activate quiescent maize transposons, 'cytotype' and tissue-specific splicing regulate Drosophila P factors, copy number controls Tn5 transposition in bacteria, and developmental timing affects the production of transposon-like intracisternal A-particles in mouse embryos. The Caenorhabditis elegans transposable element Tc1 is subject to both strain-specific and tissue-specific control. Multiple copies of Tc1 are present in the genome of all C. elegans strains collected from nature. However, these elements are genetically active in only certain isolates. For example, in C. elegans variety Bristol transposition and excision of Tc1 are undetectable, but in variety Bergerac transposition and excision are frequent. Moreover, in variety Bergerac, Tc1 is about 1,000-fold more active in somatic cells than in germ cells. We have investigated the genetic basis for the germ/soma regulation of Tc1 activity. We have isolated mutants that exhibit increased frequencies of Tc1 excision in the germ line. The frequencies of Tc1 excision in the soma are unaltered in these mutants. These mutants also exhibit high frequencies of Tc1 germ-line transposition, and this results in a mutator phenotype. Nearly all mutator-induced mutations are caused by insertion of Tc1.     

Biological Effects on Fruit Fly by N+ ion Beam Implantation

Mutation induced by low energy ion beam implantation has beenapplied widely both in plants and microbes. However, due to the vacuum limitation, such ion implantation into animals was never studied except for silkworm. In this study, Pupae of fruit fly were irradiated with different dosage N+ ions at energy 20 KeV to study the biological effect of ion beam on animal. The results showed a saddle-like curve exists between incubate rate and dosage. Damage of pupae by ion beam implantation was observed using scanning electron microscope. Some individuals with incomplete wing were obtained after implantation but no similar character was observed in their offspring. Furthermore, about 5.47% mutants with wide variation appeared in M1 generation. Therefore, ion beam implantation could be widely used for mutation breeding.     

水稻亚种间基因组水平微卫星多态性分析

从公布的水稻两个亚种（籼稻9311和粳稻Nipponbare）基因组草图序列中,搜寻了所有的完整微卫星位点,发现单碱基重复中A/T重复占大多数,二碱基重复中AT/TA、GA/TC、AG/CT重复居多,三碱基中GGC/GCC、GCG/CGC、CCG/CGG重复较多。通过相互比较,不同基序和基因区域的微卫星多态性有较大差异：内含子和基因间区中微卫星的多态性较高（分别约为45%和40%）,外显子中的微卫星多为三碱基重复,而且多态性较低（约为25%）;两个亚种具多态性的微卫星间距平均约为12.7 kb和16.4 kb。     

Frequent mtDNA mutations and its role in gastric carcinogenesis

In order to disclose the relationship between mutations of mitochondrial DNA (mtDNA) and gastric carcinogenesis, we screened the entire mtDNA sequence in 30 cases of human gastric cancer and matched normal tissues by using denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. Our data showed that high frequency (66.7%, 20/30) of mitochondrial genome mutation occurred in gastric cancer. Among these variants, 17 cases (56.7%, 17/30) were identified to be somatic mutation. High level mutant frequency was found in ND4, ND5 coding genes and D-loop control region, which was 36.7%, 26.7% and 30% respectively. Comparing with complexes Ⅲ, Ⅳ and Ⅴof the electron transport chain, we found that variants appeared to be more frequent in the subunit genes of complexⅠ. Most of mutations were base substitutions (85.4%, 41/48). Our results suggested that mutations of subunit genes encoding complexⅠ, especially ND3, ND4 and ND5 genes, might contribute to human gastric carcinogenesis.     

基于mtDNA COI基因序列的盐肤木种群遗传变异

基于mtDNA COI基因序列对贵州、湖北和四川的盐肤木(Rhus chinensis Mill.)种群进行遗传变异分析.研究共测得盐肤木9个种群50个个体的mtDNA COI基因长度为1.37kb的序列,在测得的序列中有3个核苷酸位点存在变异(占所测核苷酸序列的0.22%),T、C、A、G 4种核苷酸的组成分别为34.1%、21.8%、22.5%、21.6%,其中A+T含量(56.6%)高于G+C含量(43.4%).共检测到3个单倍型(HapA、HapB和HapC),HapA出现在所有种群中,是出现频率最高的单倍型,占所测序列的90%,该单倍型可能是祖先单倍型;HapB由安县的1个个体和竹山的3个个体共享,而HapC只存在于榕江的1个个体.分析表明盐肤木种群具低水平单倍型多样性(0.000 2)和核苷酸多样性(0.187 0),基于该基因序列的盐肤木种群间遗传多样性和遗传分化均较小.