The bacteriophage straight phi29 portal motor can package DNA against a large internal force

As part of the viral infection cycle, viruses must package their newly replicated genomes for delivery to other host cells. Bacteriophage straight phi29 packages its 6.6-microm long, double-stranded DNA into a 42 x 54 nm capsid by means of a portal complex that hydrolyses ATP. This process is remarkable because entropic, electrostatic and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. Here we use optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force-generating motor. This motor can work against loads of up to 57 pN on average, making it one of the strongest molecular motors reported to date. Movements of over 5 microm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Notably, the packaging rate decreases as the prohead is filled, indicating that an internal force builds up to approximately 50 pN owing to DNA confinement. Our data suggest that this force may be available for initiating the ejection of the DNA from the capsid during infection.     

Recognition sequence of bacteriophage phi X174 gene A protein--an initiator of DNA replication

Gene A protein, the initiator protein of bacteriophage phi X174 DNA replication, cleaves synthetic single-stranded oligodeoxyribonucleotides at the same site as the corresponding sequence at the phi X origin. The results identify the recognition sequence within the decamer CAACTTGATA which is cleaved next to the G residue. Further requirements for cleavage of double-stranded DNA by the gene A protein are supercoiling and an A + T-rich domain adjacent to the recognition sequence.     

'Z-RNA'--a left-handed RNA double helix

In contrast to double-stranded DNA, there has so far been little evidence that double-stranded RNA can undergo major conformational transitions. We have investigated the conformation in different conditions of the double-stranded RNA molecule poly(G-C).poly(G-C), by NMR, circular dichroism and absorbance spectroscopy. We report here evidence obtained by these different spectroscopic techniques that poly(G-C).poly(G-C) undergoes a transition from the A-form to a left-handed Z-form in conditions of high ionic strength and at temperatures above 35 degrees C. This conformational transition may be of relevance to the biological situations in which double-stranded RNA occurs, such as in ribosomes and in some viruses.     

Evolvability of an RNA virus is determined by its mutational neighbourhood

The ubiquity of mechanisms that generate genetic variation has spurred arguments that evolvability, the ability to generate adaptive variation, has itself evolved in response to natural selection. The high mutation rate of RNA viruses is postulated to be an adaptation for evolvability, but the paradox is that whereas some RNA viruses evolve at high rates, others are highly stable. Here we show that evolvability in the RNA bacteriophage phi6 is also determined by the accessibility of advantageous genotypes within the mutational neighbourhood (the set of mutants one or a few mutational steps away). We found that two phi6 populations that were derived from a single ancestral phage repeatedly evolved at different rates and toward different fitness maxima. Fitness measurements of individual phages showed that the fitness distribution of mutants differed between the two populations. Whereas population A, which evolved toward a higher maximum, had a distribution that contained many advantageous mutants, population B, which evolved toward a lower maximum, had a distribution that contained only deleterious mutants. We interpret these distributions to measure the fitness effects of genotypes that are mutationally available to the two populations. Thus, the evolvability of phi6 is constrained by the distribution of its mutational neighbours, despite the fact that this phage has the characteristic high mutation rate of RNA viruses.     

Filamentous phage integration requires the host recombinases XerC and XerD

Many bacteriophages and animal viruses integrate their genomes into the chromosomal DNA of their hosts as a method of promoting vertical transmission. Phages that integrate in a site-specific fashion encode an integrase enzyme that catalyses recombination between the phage and host genomes. CTX phi is a filamentous bacteriophage that contains the genes encoding cholera toxin, the principal virulence factor of the diarrhoea-causing Gram-negative bacterium Vibrio cholerae. CTX phi integrates into the V. cholerae genome in a site-specific manner; however, the approximately 6.9-kilobase (kb) CTX phi genome does not encode any protein with significant homology to known recombinases. Here we report that XerC and XerD, two chromosome-encoded recombinases that ordinarily function to resolve chromosome dimers at the dif recombination site, are essential for CTX phi integration into the V. cholerae genome. The CTX phi integration site was found to overlap with the dif site of the larger of the two V. cholerae chromosomes. Examination of sequences of the integration sites of other filamentous phages indicates that the XerCD recombinases also mediate the integration of these phage genomes at dif-like sites in various bacterial species.     

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%.     

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with approximately 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.     

Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3'-->5' exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a 'right hand' shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an 'open fingers' state in which the active site samples nucleotides, and a 'closed' state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of approximately 34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.     

Translocation step size and mechanism of the RecBC DNA helicase

DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.     

古菌病毒

古菌病毒的研究起步较晚,但发展很快。近年来,人们已经从热泉等特殊环境分离得到了数十种古菌病毒。来自热泉的古菌病毒表现出了极为独特、多样的形态学和基因组学特征,据此目前已经或正在建议建立7个病毒新科。已知的古菌病毒均属于双链DNA病毒,极端嗜热古菌病毒中绝大多数基因的功能尚不清楚。古菌病毒的研究不仅极大地丰富了人们对自然界病毒多样性的认识,还为探索病毒的起源与进化提供了重要启示。本文对古菌病毒的主要类群及特点作了简单介绍,并进一步探讨了病毒的起源与进化。     

DNA场效应生物传感器的研制

利用亲和素作为连接臂将生物素修饰的单链DNA固定在传感器的栅区表面上，制成DNA场效应管生物传感器，研究DNA场效应管的稳定性和特异性，并测定该传感器对不同浓度DNA互补链检测的效应值．     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.     

钢与高强混凝土连续组合梁连接件的布置方式

采用ANSYS通用有限元软件,针对钢与高强混凝土连续组合梁本身结构及受力性能的复杂性,考虑材料非线性,提出了一个对钢与高强混凝土连续组合梁从加载到破坏的全过程非线性分析模式,对钢与高强混凝土连续组合梁连接件设计进行分析.研究连接件的布置方式对连续组合梁的影响,完善钢与高强混凝土连续组合梁中抗剪连接件的设计理论.     

Virus-specific effects of interferon in embryonal carcinoma cells

Embryonal carcinoma (EC) cells are susceptible to infection by a variety of viruses, but do not become resistant to infection by Semliki Forest virus or vesicular stomatitis virus (VSV) on treatment with interferon. These observations have led to the conclusion that interferon does not induce an antiviral state in EC cells. We report here, however, that EC cells treated with interferon become resistant to infection by two picornaviruses and two ts mutants of VSV, whereas they remain sensitive to wild-type VSV, Sindbis and influenza virus infectin. These results suggest that a partial antiviral state is induced in EC cells by interferon and that the induced antiviral protein(s) interferes with the replication of specific viruses. A significant common feature of these viruses is their replication through structures containing double-stranded RNA (dsRNA).     

滚环DNA扩增的原理、应用和展望

滚环DNA扩增(Rolling circle DNA amplification,RCA)是一种等温信号扩增方法,其线性扩增倍数为10^5,指数化扩增能力大于10^9,产生的扩增产物连接在固相支持物(如玻片、微孔板等)表面的DNA引物或抗体上(适宜作生物芯片研究)。RCA是一种适合在芯片上(on-chip)进行信号扩增的新技术,它既能提供研究分析的敏感性和特异性,又能保持立体分析的多元性。RCA亦是一种痕量的分子检测方法,可用于极其微量的生物大分子和生物标志的检测与研究。,     

Uncoupling of initiation site cleavage from subsequent headful cleavages in bacteriophage T1 DNA packaging

The packaging of intracellular DNA into heads is a key feature in the morphogenesis of bacteriophage particles. In many phages a performed empty head precursor, the prohead, is filled with DNA from a concatemeric substrate consisting of tandemly repeated genome lengths. The addition of outer shell proteins completes head formation. The DNA molecules released from particles of the coliphage T1 exist as three major permutations of nucleotide sequence. Such limited permutation can be explained by the modification of Streisinger's 'headful' mechanism proposed for phage P22. DNA packaging is initiated at a specific site (the pac site) on the concatemeric precursor. While this site is cleaved, subsequent cleavages (headful cleavages) are dependent only on head-filling and are not defined in terms of nucleotide sequence. Headfuls of DNA, consisting of slightly more than a genome length, are packaged in three successive cycles of head-filling to produce the permuted and terminally redundant molecules characteristic of T1 DNA. To elucidate the regulation of this process, we have studied the DNA metabolism of T1 head mutants. We describe here the properties of a mutant in gene 13.3 which is defective for headful cleavage but remains proficient in pac site cleavage. The observation in this mutant that concatemers are degraded to unit-length molecules by repeated pac site cleavage suggests a model of headful packaging in which pac site initiation and processive head-filling compete for the DNA substrate.