Recombination Between Guanidine-resistant and Dextran Sulfate-resistant Mutants of Type 1 Poliovirus

Mixed infection of monkey kidney cells with two mutants of the LSc2ab strain of poliovirus, one resistant to guanidine and the other resistant to both dextran sulfate and 2-(alpha-hydroxybenzyl)-benzimidazole (HBB), yielded progeny in which the number of gua(r)dex(r) particles exceeded by a factor of 7 to 10 the expected number of similar particles occurring through spontaneous mutation; recombination would explain the fairly high excess of doubly mutant particles that was obtained. Scoring of HBB resistance in 50 gua(r)dex(r) clones suggested that, during recombination, resistance to dextran sulfate is not associated with HBB resistance.     

Molecular Biology and Cell-free Synthesis of Poliovirus

Abstract. Poliovirus is a small icosahedral particle consisting of only five species of macromolecules: 60 copies each of the capsid protein VP1-4; and one copy of single-stranded RNA, approximately 7500 nt long. The genome, linked at the 5′ end to a small protein VPg and 3′ polyadenylylated, is of plus strand polarity. After receptor-mediated uptake of the virus and release of the RNA into the cytoplasm, the genome serves as mRNA, encoding only a single polypeptide, the polyprotein. The polyprotein is cleaved co-translationally into numerous polypeptides by its own, internal proteinases 2A^pro, 3C^pro and 3CD^pro. Initiation of translation is mediated by a novel genetic element, called internal ribosomal entry site (IRES). IRES elements, which are 400 nt long RNA segments located within the 5′ non-translated region of the viral genome, are common to all picornaviruses. Their function renders translation of picornavirus mRNAs cap- and 5′-independent, an observation that has upset the dogma of cap-dependent translation in eukaryotic cells. IRES elements have also been used to genetically dissect the viral genome and to construct novel expression vectors. Genome replication is not fully understood, the major conundrum being the initiation of RNA synthesis by the primer-dependent viral RNA polymerase 3D^pol, a process leading to VPg-linked RNA products. Nearly all non-structural proteins appear to be involved in initiation, the proteinases 2A^pro and 3CD^pro included. A HeLa cell-free system has been developed that, on programming with plasmid-transcribed viral RNA, will perform viral translation, protein processing, RNA replication, and assembly of capsid protein and newly made genomic RNA. The final yield is infectious poliovirus. This result has nullified the dictum that no virus can replicate in a cell-free medium.     

Molecular Weight of Poliovirus Ribonucleic Acid

Purified poliovirus single- and double-stranded ribonucleic acids (RNA) were examined by electron microscopy. The length of both molecules was found to be 2.37 mum. The uncorrected sedimentation coefficient for single-stranded RNA is 33S, as compared to 27S for the RNA of tobacco mosaic virus. It is calculated from these results that the molecular weight of the sodium form of poliovirus is 2.6 x 10(6) daltons.     

Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.     

Factors Influencing the Enhancement of the Infectivity of Poliovirus Ribonucleic Acid by Diethylaminoethyl-Dextran

The enhancement by diethylaminoethyl-dextran (DEAE-D) of the infectivity of poliovirus ribonucleic acid (RNA) for cell cultures was demonstrated by infective-center as well as by plaque assays, both in nonprimate (L) and primate cell systems (MK, HeLa, LLC-MK(2)). The sensitivity of plaque assays was greatly improved by using a tris (hydroxymethyl)aminomethane-buffered synthetic medium (basal medium Eagle) and freshly confluent cell monolayers. Enhancement of nucleic acid infectivity was directly dependent on the molecular weight of the DEAE-D. Two observations bearing on the action of DEAE-D appeared important: ribonuclease activity was reduced by DEAE-D, and cells pretreated with DEAE-D remained susceptible to infection with RNA in isotonic medium. Appreciable susceptibility of the treated cells persisted for at least 2 hr; the susceptible state could be reversed at will by an application of heparin. Enhancement of nucleic acid infectivity was independent of an effect of DEAE-D on intact virus and agar inhibitors.     

Molecular Analysis of Recombination Events in Drosophila

The locations of crossover junctions and gene conversion tracts, isolated in the rosy gene of Drosophila melanogaster, were determined using DNA sequencing and denaturing gradient gel electrophoresis. Frequent DNA sequence polymorphisms between the parental genes served as unselected genetic markers. All conversion tracts were continuous, and half of the reciprocal crossover events had conversion tracts at the crossover junction. These experiments have also identified the sequence polymorphisms responsible for altered gene expression in two naturally occurring rosy variants.     

含小插入片段重组子筛选鉴定方法之探讨

基因工程中,重组ＤＮＡ时,当外源ＤＮＡ很小并与载体相差很大时,其目的重组子的筛选鉴定工作不仅量大,而且常规鉴定方法无法采用。本文针对此类重组子的鉴定方法作了探讨,提出了三级四步筛选鉴定法,即：质粒筛选、重组鉴定、目的重组鉴定和双酶切鉴定。该法不仅有效地解决了国内普通实验条件下含小插入片段重组子的鉴定问题,大大缩小鉴定范围,而且快速省耗的鉴定方法也为ｐＧＥＸ载体系统的更广泛应用提供了新的启示。     

Binding of Rat Brain Hexokinase to Recombinant Yeast Mitochondria: Identification of Necessary Molecular Determinants

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wildtype, porinless, or expressing recombinant human porin) was studied in an effort to identifyminimal requirements for each component. A short hydrophobic N-terminal peptide ofhexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria.Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loopsaround residues 110 and 200, as determined by proteolytic-fragment identification usingantibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysisthan wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboringseveral natural or engineered porins from various sources, bind hexokinase to variable extentwith marked preference for the mammalian porin1 isoform. Genetic alteration of this isoformat the C-, but not the N-terminal, results in a significant reduction of hexokinase bindingability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme toall recombinant mitochondria, as well as to proteolytically digested organelles. Consequently,brain hexokinase association with heterologous mitochondria (yeast) in these conditions occursto an extent comparable to that with homologous (rat) mitochondria. The study, also pertinentto the topology and organization of porin in the membrane, represents a necessary first stepin the functional investigation of the physiological role of mammalian hexokinase binding tomitochondria in reconstituted heterologous recombinant systems, as models to cellularmetabolism.     

Identification of Potential Molecular Determinants of Murine Embryonic Stem Cell Differentiation by a Transposon-Based Approach

Embryonic stem cells (ESCs) are self-renewing pluripotent cells, capable of differentiating into all somatic cell types. The molecular control of self-renewal is relatively well-characterized, whereas how ESCs exit pluripotent state to differentiate is poorly understood. Here we identify two genes are required for differentiation and dozens of intergenic regions that potentially regulate ESC differentiation. We used PiggyBac (PB) transposon-based approach to randomly mutate the genome of ESCs, and generated hundreds of clones that resisted differentiation signals. Each clone was sequenced to determine genomic regions mutated by PB insertion. Intriguingly, many mutations were localized among intergenic regions and we identified two genes are required for differentiation. This study should facilitate further exploration of novel molecular determinants of embryonic stem cell differentiation.     

Identification of Recombination and Positively Selected Genes in Brucella

Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level.Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system.     

脊髓灰质炎病毒中Ⅰ9株基因组克隆及其部分结构分析

以脊髓灰质炎病毒RNA为模板,反转录合成了长链ds-cDNA。将合成的脊灰病毒中I9株ds—cDNA片段重组到质粒pAT153上,获得了约占脊灰病毒中I9基因组95％以上的cDNA克隆。对克隆的中I9cDNA部分片段作了限制性内切酶图谱分析和部分核酸的序列分析,发现脊灰病毒中I9部分核酸顺序有所改变。     

哺乳动物性别决定的分子要素

哺乳动物性别分化是由胚胎时期性腺分泌的激素控制,而在激素分泌前,ＸＸ和ＸＹ胎儿都具有牟勒氏管（Ｍüｌｅｒｉａｎｄｕｃｔ）,吴夫氏管（Ｗｏｉｆｉａｎｄｕｃｔ）两套生殖导管和未分化的性腺。牟勒氏管将来分化为输卵管,子宫等雌性生殖道,吴夫氏管则分化为输精管...     

外源ipt对叶片衰老分子调控研究进展(综述)

综述了叶片衰老的分子机理,并介绍了将外源异戊烯基转移酶（ipt,isopentenyl transferase)基因转入植物以获得抗衰老植株的研究进展。     

Molecular Determinants of a Native-State Prolyl Isomerization

Prolyl cis/trans isomerizations determine the rates of many protein-folding reactions, and they can serve as molecular switches and timers. The energy required to shift the prolyl cis/trans equilibrium during these processes originates from conformational reactions that are linked structurally and energetically with prolyl isomerization. We used the N2 domain of the gene-3-protein of phage fd to elucidate how such an energetic linkage develops in the course of folding. The Asp160-Pro161 bond at the tip of a β hairpin of N2 is cis in the crystal structure, but in fact, it exists as a mixture of conformers in folded N2. During refolding, about 10 kJ mol^− 1 of conformational energy becomes available for a 75-fold shift of the cis/trans equilibrium constant at Pro161, from 7/93 in the unfolded to 90/10 in the folded form. We combined single- and double-mixing kinetic experiments with a mutational analysis to identify the structural origin of this proline shift energy and to elucidate the molecular path for the transfer of this energy to Pro161. It originates largely, if not entirely, from the two-stranded β sheet at the base of the Pro161 hairpin. The two strands improve their stabilizing interactions when Pro161 is cis, and this stabilization is propagated to Pro161, because the connector peptides between the β strands and Pro161 are native-like folded when Pro161 is cis. In the presence of a trans-Pro161, the connector peptides are locally unfolded, and thus, Pro161 is structurally and energetically uncoupled from the β sheet. Such interrelations between local folding and prolyl isomerization and the potential modulation by prolyl isomerases might also be used to break and reestablish slow communication pathways in proteins.