Recombination Between a Thermosensitive Kanamycin Resistance Factor and a Nonthermosensitive Multiple-Drug Resistance Factor

The thermosensitive kanamycin (KM) resistance factor, R(KM)(t), and a nonthermosensitive multiple-drug resistance factor, R(100), were simultaneously introduced into Escherichia coli and Salmonella typhimurium. The temperature sensitivity of both R factors remained unchanged as long as they replicated independently. Under certain conditions, however, a new thermosensitive R factor harboring resistance markers for kanamycin, streptomycin (SM), and sulfanilamide (SA) was obtained by recombination between the R(KM)(t) and R(100) factors. R factors carrying resistance markers for KM and SA, or for SM and SA, were obtained from the recombinant R(KM SA SM)(t) by spontaneous segregation. Though the R(100) factor has been known as an fi(+) (positive for F-mediated fertility inhibition of its host) type and it does not restrict any coexisting phages, the thermosensitive recombinants of R(100) with R(KM)(t) and their segregants were found to be fi(-) and to restrict the replication of all T-even phages, as does the R(KM)(t) factor. Double infection immunity was not observed between the R(KM)(t) and R(100) factors.     

Round of Replication Mutant of a Drug Resistance Factor

A derivative of the R factor NR1 (called R12) has been isolated which undergoes an increased number of rounds of replication each division cycle in Proteus mirabilis, Escherichia coli, and Salmonella typhimurium. The alteration resulting in the increased number of copies (round of replication mutation) is associated with the transfer factor component of the R factor. R12 has the same drug resistance pattern as NR1, is the same size as shown by sedimentation in a sucrose gradient and electron microscopy (63 × 10⁶ daltons), and has the same partial denaturation map. The level of the R factor gene product chloramphenicol acetyltransferase has been examined in P. mirabilis and was found to be consistent with gene dosage effects. The plasmid to chromosomal deoxyribonucleic acid ratio of NR1 increases several fold after entry into stationary phase, whereas this ratio for R12 remains approximately constant. Individual copies of R12 are selected at random for replication from a multicopy plasmid pool. A smaller percentage of R12 copies replicate during amino acid starvation than has previously been found for NR1 in similar experiments.     

几种白菜类蔬菜卡那霉素抗性的研究

标记基因的利用是筛选和鉴定基因转化的细胞、组织和转基因植株的有效方法。针对目前遗传转化中常用的卡那霉素(Km)抗性基因,对几种白菜类蔬菜的卡那霉素抗性作了较系统的探索,发现5.0 mg/L Km可完全抑制大白菜、小白菜及菜心子叶的再生;幼苗Km抗性测验表明各品种均表现出随着Km浓度的提高,子叶黄化率升高、根茎变短、鲜重减轻的趋势。白菜类蔬菜的卡那霉素抗性检测为遗传转化和阳性后代筛选奠定了基础。     

Host Cell Growth in the Presence of the Thermosensitive Drug Resistance Factor, Rts1

We have confirmed and extended the observation of Terawaki et al. that the R factor, Rts1, alters the growth of its host at 42 C. In all media tested there was a period during which total cell numbers increased linearly, while viable counts remained constant. During this period the rate of precursor incorporation per cell particle into deoxyribonucleic acid, ribonucleic acid, and protein declined steadily. These patterns were a consequence of the accumulation of increasing numbers of cells which had lost colony-forming ability. A temperature shiftdown experiment showed that the colony formers could, after a lag, go on to divide normally, whereas most of the noncolony formers could not undergo even a limited number of divisions after shiftdown. The number of normal divisions which occurred after shiftup of Rts1 cells to 42 C was medium dependent. In rich medium there were, on the average, two or three doublings; in glucose medium, one; and in glycerol medium, only a fraction of a doubling. Even in glucose medium, however, no increase in viable counts was observed during growth at 42 C if the cells were first starved for glucose for 1 h at 42 C. A temperature shiftdown from 42 C to 27 C during glucose starvation reversed the effect of starvation at 42 C alone. These results are consistent with the hypothesis that the thermosensitive Rts1 component(s) responsible for the host effects is present at permissive temperature, but can undergo a reversible temperature-induced alteration which then interferes with some essential host function. The detrimental effects of this R factor on its host were also reflected in a heightened sensitivity to kanamycin and actinomycin D at 42 C. Electron microscope observations revealed changes in the appearance of the cell membrane. Membranous invaginations were noted at discrete sites in the cell.     

Negative Regulation of Systemic Acquired Resistance by Replication Factor C Subunit3 in Arabidopsis

Systemic acquired resistance (SAR) is a plant immune response induced by local necrotizing pathogen infections. Expression of SAR in Arabidopsis (Arabidopsis thaliana) plants correlates with accumulation of salicylic acid (SA) and up-regulation of Pathogenesis-Related (PR) genes. SA is an essential and sufficient signal for SAR. In a genetic screen to search for negative regulators of PR gene expression and SAR, we found a new mutant that is hypersensitive to SA and exhibits enhanced induction of PR genes and resistance against the virulent oomycete Hyaloperonospora arabidopsidis Noco2. The enhanced pathogen resistance in the mutant is Nonexpressor of PR genes1 independent. The mutant gene was identified by map-based cloning, and it encodes a protein with high homology to Replication Factor C Subunit3 (RFC3) of yeast and other eukaryotes; thus, the mutant was named rfc3-1. rfc3-1 mutant plants are smaller than wild-type plants and have narrower leaves and petals. On the epidermis of true leaves, there are fewer cells in rfc3-1 compared with the wild type. Cell production rate is reduced in rfc3-1 mutant roots, indicating that the mutated RFC3 slows down cell proliferation. As Replication Factor C is involved in replication-coupled chromatin assembly, our data suggest that chromatin assembly and remodeling may play important roles in the negative control of PR gene expression and SAR.     

Direct Gene Transfer in Psophocarpus tetragonolobus Resistance to Kanamycin

Epicotyl-derived protoplasts of Psophocarpus tetragonolobuswere isolated and regenerated to plants. These protoplasts weretransformed to kanamycin resistance following uptake of plasmid(pABDl or pHP23) DNA in combination with PEG treatment. Protoplast-derivedtransformed colonies were selected on kanamycin (75 mg l^–1).The transformed calli expressed NPT II activity and also exhibitedthe presence of the plasmid gene integrated into the plant genome.However, none of the transformed clones showed regenerationof shoot buds. Psophocarpus tetragonolobus, winged bean, naked DNA transformation, protoplast culture, regenerated plants     

Molecular Nature of the Deoxyribonucleic Acid of a Thermosensitive R Factor, Rts1

The deoxyribonucleic acid of the thermosensitive R factor, Rts1, has been examined by the technique of sedimentation in alkaline sucrose, electron microscopy, and radiation target size. All these methods yielded a molecular weight of approximately 120 million for Rts1 deoxyribonucleic acid in Escherichia coli. Sedimentation analysis revealed that Rts1 deoxyribonucleic acid in Proteus mirabilis was also 120 million daltons. Rts1 did not segregate into E. coli minicells under the conditions where another smaller non-thermosensitive R factor could.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

Replication Control and Switch-Off Function as Observed with a Mini-F Factor Plasmid

Mini-F is a fragment of the F plasmid, consisting of 9,000 base pairs, which carries all of the genes and sites required for replicon maintenance and control. Its copy number is one to two per chromosome. This plasmid is joined to ColE1, whose copy number is 16 to 20. Under normal circumstances the composite plasmid replication exhibited ColE1 characteristics, maintaining a high copy number. However, when ColE1 replication was inhibited by deoxyribonucleic acid polymerase I inactivation, its replication exhibited mini-F characteristics, maintaining a low copy number. These observations are in complete agreement with those of Timmis et al. (Proc. Natl. Acad. Sci. U.S.A. 71:4556-4560, 1974), who examined the behavior of a recombinant plasmid formed between pSC101 and ColE1. The transition from high to low copy number allowed us to examine the control system acting in cells carrying plasmids exhibiting intermediate copy numbers. The initiation of the mini-F replication system as represented by deoxyribonucleic acid synthesis of the composite plasmid was completely blocked when there were multiple copies of mini-F in a cell. It was not restored until the copy number was lowered to one to two, after which replication was first detected. ppF, a mini-F replicon packaged in a phage λ head behaved similarly: its replication was completely shut off when the resident mini-F genome copy number was high and was inhibited partially when the resident mini-F genome copy number was low. These experiments clearly demonstrate that there is a switch-off mechanism acting on deoxyribonucleic acid synthesis (initiation) in a cell carrying mini-F, and its intensity is related to the plasmid copy number. This result supports the “inhibitor dilution model” proposed by Pritchard et al. (Symp. Soc. Gen. Microbiol. 19:263-297, 1969). The nature of the hypothetical inhibitor is discussed.     

Pattern of Replication of a Colicin Factor During the Cell Cycle of Escherichia coli

The ColBM, trp, lac episome was transferred to a lacZ derivative of Escherichia coli B/r, and the manner of replication of this colicinogenic factor was followed through the cell cycle. The results suggest a pattern of replication not connected with any particular stage in the cell cycle.     

Role and Regulation Mechanism of Kanamycin Acetyltransferase in Kanamycin Biosynthesis

The formation of kanamycin (KM) and KM-acetyltransferase (KAT) in washed cell suspensions of a strain of Streptomyces kanamyceticus was induced by 4-O-(6-aminoglycosyl)-2-deoxystreptamine, i.e. 6–AG–DOS, but repressed by 6-O-(3-aminoglycosyl)-DOS and KM, i.e. 3–AG–DOS and 6′–AG–DOS–3″–AG. The “feedback” inhibition of the formation of KM and KAT by KM was relieved by addition of 3–AG in the presence of 6–AG–DOS. Acetylation of 6–AG–DOS was supposed to be prerequisite for binding 3–AG. The activity of KAT in a cell-free extract was inhibited by inorganic phosphate, ADP, phosphoenolpyruvate, oxalacetate and malate. Citrate and oleate were available to the acetylation reaction as a substitute for acetate. Sclerin stimulated both formation and activity of KAT throughout the experiments mentioned above.     

Structure-Function Analysis of DipA,a Francisella tularensis Virulence Factor Required for Intracellular Replication

Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella , indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host–pathogen interface to influence the intracellular fate of this pathogen.     

Replication of the R Factor Rts1 in Proteus mirabilis

The replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density-gradient centrifugation. The proportion of Rts1 deoxyribonucleic acid (DNA) relative to the host chromosomal DNA (% R-DNA) was 7% in both exponential and stationary growth phases in Penassay Broth and supplemented M9 minimal medium at 30 C. The chromosomal DNA content per cell varied over a threefold range in the different growth media. In agreement with previous genetic observations, the replication of Rts1 was found to be temperature-sensitive and Rts1 DNA was diluted from the cells during exponential growth at 42 C. (14)N-(15)N medium transfer experiments have shown that individual copies of Rts1 are selected at random for replication during the duplication of the multicopy episome pool.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP^Sc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). Stable variations in PrP^Sc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP^Sc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP^Sc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP^Sc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP^Sc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP^Sc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP^Sc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.     

拟南芥复制因子C亚基3在黄化苗下胚轴伸长生长中的作用

拟南芥遮光培养2．5d时,rfc3-1突变体黄化幼苗的下胚轴平均长度约比野生型植株黄化幼苗的下胚轴长27．5％。观察表明,相对于野生型复制因子C亚基3（replication factor C3,AtRFC3）基因突变体的下胚轴表皮细胞,特别是上部靠近子叶部分的表皮细胞,单细胞长度变长。将野生型RFC3基因转染到rfc3-1后,突变体恢复野生型表型,进一步说明RFC3在黄化苗的下胚轴伸长生长中有作用。