Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea

Summary pSE211 fromSaccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination betweenattP andattB, the plasmid and chromosomal attachment sites. Integration depends on the presence ofint, an open reading frame (ORF) that lies adjacent toattP and encodes the putative integrase. Immediately upstream ofint liesxis (formerly calledorf2) which encodes a basic protein that is thought to exhibit DNA binding.xis andint were cloned in various combinations in pUC18 and expressed constitutively inEscherichia coli from thelac promoter.attP andattB were cloned inStreptomyces orE. coli plasmids containing kanamycin resistance (Km^R) or chloramphenicol resistance (Cm^R) markers. Stable Km^R Cm^R cointegrates formed byattP ×attB orattP ×attP recombination (integration) were obtained inE. coli hosts that expressedint. Co-integrates were not found in hosts expressingint+xis. Excision (intraplasmidatt site recombination) was examined by constructing plasmids carryingattL andattR or twoattP sites separating Cm^R from Km^R and by following segregation of the markers in various hosts. BothattL ×attR andattP ×attP excision depended on bothxis andint inE. coli. pSE211att site integration and excision were not affected by a deletion inhimA, the gene encoding a subunit of integration host factor.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

This paper describes a rapid method of constructing homologous recombinant baculovirus inE. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (Cm ^R ) to replaceorf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence oforf135, and the rest 20 bp was homologous to the each end sequence ofCm ^R . By using these primers, a linear fragment containing the completeCm ^R gene between 40 bp of homologous arms oforf135 was generated by PCR with the plasmid pKD3 which containsCm ^R as the template. By transforming the linear fragment into theE. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed inE. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.     

Identification of surface proteins ofSpiroplasma citri with protease and antibody probes

Spiroplasma citri, a helical, wall-less prokaryote, is an insect-borne phytopathogen. Though proteins having domains on the surface ofS. citri cells may be important in pathogenicity or transmissibility, only one surface protein, spiralin (29 KDa), has previously been identified. Intact cells of strain BR3 were treated with chymotrypsin, proteinase K, or trypsin, and the surviving proteins were analyzed by SDS-PAGE. Seven proteins, in addition to spiralin, were degraded, indicative of surface exposure of those polypeptides. Surface immunoprecipitation (SIP) was used to test accessibility of the proteins to anti-S. citri membrane serum, another indication of surface exposure. With unlabeled cells, five such proteins were identified. Four of these have sizes that correspond to those seen with protease treatments. When¹²⁵I surfacelabeled spiroplasmas were used for SIP, twelve surface proteins were detected, eight of which correspond to bands identified by the other methods. A protein of 89 KDa in strain BR3 was not universally detected in otherS. citri strains and spiroplasma species.     

Protoplast fusion inStreptomyces avermitilis

Summary The power of protoplast fusion as a generally applicable method for obtaining genetic recombination is demonstrated by the recombination of genes involved in avermectin biosynthesis. A backcross ofStreptomyces avermitilis strain MA6202, an improved mutant that had lost the ability to carry out the methylation of the C-5 hydroxyl of the avermectin molecule, with the original soil isolate MA4680 resulted in the recovery of at least one unambiguous recombinant class despite the instability of rifampicin resistance, one of two markers initially used for recombinant selection. Such intrinsic instability is frequently encountered in streptomycete genetics, and this result delineates the utility of protoplast fusion as a genetic tool. Other difficulties addressed include recovery of complementary recombinant classes, differences in recombination frequency due to colony density on regeneration medium, and alteration in plating efficiency on diagnostic media following protoplasting and regeneration. The results of a cross between a nicotinamide auxotroph MRG1003 and a lysine auxotroph MRG 1004 are included to aid in the elucidation of these problems as well as to support the finding of homologous recombination inS. avermitilis.     

The process of population increase and patterns of resource utilization of two spider mites,Oligonychus ununguis (Jacobi) andPanonychus citri (McGregor), under experimental conditions (Acari: Tetranychidae)

Summary Pattern of population growth and characteristics of habitat utilization and of migration by two species of spider mites were studied under experimental conditions. The population growth ofOligonychus ununguis (Jacobi) on a chestnut occurred only on a single mite-release leaf over a long period, and few individuals moved away. Most of the 2nd progeny generation females of this species emigrated from the mite-release leaf as well as the sapling by means of ballooning threads. During this growth period, population density on the mite-release leaf levelled off, whereas that on the sapling increased. In contrast, the foundress ofPanonychus citri (McGregor) on citrus actively moved over several neighbouring leaves, and until the 2nd progeny generation females emerged, individuals were distributed over all the sapling leaves by means of walking. Emigration from the sapling was not observed until the 2nd progeny females emerged, and after that the mites emigrated by means of ballooning threads. The population density ofP. citri on the sapling levelled off and was rather decreased on the mite-release leaf at the time of mite emigration. Comparing the changing pattern of the relative degree of aggregation (m ^*/m) measured in two different units between these two species, the pattern ofm ^*/m in 1 cm² on the mite release leaf inO. ununguis resembled that of the unit of leaves on the sapling inP. citri. This result as well as behavioural observations indicate that migration ofO. ununguis is the movement from leaf to leaf and that ofP. citri from sapling to sapling. It is, therefore, concluded that the boundary of the microhabitat is a single leaf forO. ununguis but sapling or foliage forP. citri. This work was presented in Annual Meeting of Jap. Soc. Appl. Ent. Zool., 1981 in Okayama.     

Comparative mapping reveals partial conservation of synteny at the apomixis locus in<Emphasis Type="Italic"> Paspalum</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

In plants, gametophytic apomixis is a form of asexual reproduction that leads to the formation of seed-derived offspring that are genetically identical to the mother plant. A common set of RFLP markers, including five rice anchor markers previously shown to be linked to apomixis in Paspalum simplex, were used to detect linkage with apomixis in P. notatum and P. malacophyllum. A comparative map of the region around the apomixis locus was constructed for the three Paspalum species, and compared to the rice map. The locus that controls apomixis in P. simplex was almost completely conserved in the closely related species P. malacophyllum, whereas it was only partially represented in the distantly related species P. notatum. Although strong synteny of markers was noted between this locus and a portion of rice chromosome 12 in both P. simplex and P. malacophyllum, the same locus in P. notatum was localized to a hybrid chromosome which carries markers that map to rice chromosomes 2 and 12. All three Paspalum species showed recombination suppression at the apomixis locus; in the case of P. notatum, this might be due to a heterozygosity for a translocation that most probably negatively interferes with chromosomal pairing near the locus. A common set of markers that show linkage with apomixis in all three Paspalum species define a portion of the apomixis-controlling locus that is likely to contain genes critical for apomictic reproduction.Communicated by R. Hagemann     

Multiplication and morphology of Spiroplasma citri in the leafhopper Euscelis plebejus

Spiroplasma citri multiplied in all Euscelis plebejus leaf hoppers injected and sometimes reached titres of over 1 × 10⁷ colony forming units per insect. Spiroplasmas could be isolated from the haemolymph at all times although helices were only apparent for a few days after injection. The salivary glands of injected insects contained membrane bound pockets densely packed with mycoplasma-like bodies. These bodies were frequently infected with virus-like particles similar to those found in cultures of S. citri. Spiroplasmas had little effect on the longevity of the leafhoppers.     

Biochemical diversity and genetic distance in two species of the genusSaguinus

A comparative study of 20 blood genetic systems was performed on three populations of genusSaguinus: S. fuscicollis weddelli, S. midas niger, andS. midas midas. Some markers are useful for the characterization of the two species.ADA ⁵, ADA⁶, CA2³, CA2⁴, andES2 ² occur only inS. fuscicollis whileADA ⁴is fixed inS. midas. S. midas midas showed heterozygosity value comparable to those previously obtained for the genusAlouatta. Estimates of genetic distance betweenS. fuscicollis andS. midas species of about 14% are in accordance with those referred to in the literature for interspecific differences. Genetic distances between subspecies ofSaguinus midas were nearly 3%, which is within the range for subspecies.     

柑橘内生真菌的分离鉴定及其发酵产物对柑橘溃疡病菌的抑制活性

本研究从柑橘抗病品种的健康植株不同组织中分离纯化和鉴定内生真菌,并测定其发酵产物对柑橘溃疡病菌的抑制活性,以明确柑橘抗病品种中内生真菌的组成及其产抗柑橘溃疡病菌活性代谢产物的潜力,为柑橘溃疡病抗菌剂的开发奠定基础。该研究通过组织培养法分离内生真菌,采用形态学和分子生物学方法对其进行鉴定; 基于前期的拮抗预试验结果,选取代表性菌株进行发酵培养,通过乙酸乙酯浸提、真空抽滤、旋转蒸发浓缩制备粗提物; 采用带毒平板涂布法测定不同菌株发酵产物乙酸乙酯提取物对柑橘溃疡病菌的抑制活性。结果表明:(1)共分离得到72株内生真菌,归为2门(Ascomycota、Basidiomycota)、14个属,其中优势属为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、链格孢属(Alternaria)和镰刀菌属(Fusarium)。(2)不同柑橘品种中内生真菌多样性指数为温州蜜柑(桂林)>沙糖桔(桂林)>沙糖桔(梧州)。(3)不同组织中内生真菌多样性变化因地理位置差异而有所不同,采自桂林的温州蜜柑和沙糖桔均为叶片中的内生真菌的多样性高于枝条,而采自梧州的沙糖桔为叶片中的多样性低于枝条,并且采自梧州的柑橘样品与采自桂林的柑橘样品中的内生真菌相似性低。(4)测定了30株内生真菌乙酸乙酯提取物对柑橘溃疡病菌的抑制活性,其中29株菌株表现出不同程度抑制活性。不同柑橘品种中的优势属的MIC介于0.312 5～10 mg·mL^-1之间,特有属的MIC介于0.156～5 mg·mL^-1,共有属镰刀菌属的MIC介于0.312 5～2.5 mg·mL^-1之间。研究结果表明柑橘抗病品种中内生真菌具有丰富多样性,并且其发酵提取物普遍对柑橘溃疡病菌具有抑制作用。特有属抑菌活性总体优于优势属,共有属镰刀菌属在不同柑橘抗病品种中均具有显著抑菌效果。     

The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis

Summary The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon, Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.Deceased, 7/89. O. Torres and R. Korman contributed equally to this work     

An estimate of the physical distance between two linked markers inHaemophilus influenzae

Using DNA clones, the physical distance between the linked genesnov andstr inHaemophilus influenzae was estimated. Although none of the cloned inserts contained both the markers, pJ1-8Str^R 13 (insert of 18·7 kb) includedstr gene at one end and part ofnov gene at the other end of the insert. By EcoRI restriction analysis and by Southern hybridization, the distance between the two EcoRI sites, cutting at which inactivates the two genes, was estimated to be 17·7 kb. A single continuous EcoRI fragment (containing 4EcoRI sites within it) carrying both the genes intact would need to be 20·4 kb in size. These estimates were confirmed independently using different clones ofnov ^r andstr ^r alleles as probes for hybridization with BamHI-digested chromosomal DNA.     

Isolation ofSpiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis

This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction, which consisted essentially of extended sheets of membranes, exhibited membrane-boundpara-nitrophenylphosphatase specific activity 1.5-fold over that of the whole-cell lysate. Only traces of soluble NADH oxidase were present in the membrane preparation. The latter fraction appeared homogeneous upon sorbitol density gradient centrifugation and banded at an equilibrium density of 1.107 g/ml. The plasma membrane proteins were then analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 40 different proteins were detected in the membrane preparations. By comparison with the patterns obtained for whole-cell extracts and cytoplasmic fractions, a protein map ofS. citri could be established in which membrane and cytoplasmic proteins were identified.     

A genetic linkage map of the diplosporous chromosomal region in<Emphasis Type="Italic"> Taraxacum officinale</Emphasis> (common dandelion; Asteraceae)

In this study, we mapped the diplosporous chromosomal region in Taraxacum officinale, by using amplified fragment length polymorphism technology (AFLP) in 73 plants from a segregating population. Taraxacum serves as a model system to investigate the genetics, ecology, and evolution of apomixis. The genus includes sexual diploid as well as apomictic polyploid, mostly triploid, plants. Apomictic Taraxacum is diplosporous, parthenogenetic, and has autonomous endosperm formation. Previous studies have indicated that these three apomixis elements are controlled by more than one locus in Taraxacum and that diplospory inherits as a dominant, monogenic trait (Ddd; DIP). A bulked segregant analysis provided 34 AFLP markers that were linked to DIP and were, together with two microsatellite markers, used for mapping the trait. The map length was 18.6 cM and markers were found on both sides of DIP, corresponding to 5.9 and 12.7 cM, respectively. None of the markers completely co-segregated with DIP. Eight markers were selected for PCR-based marker development, of which two were successfully converted. In contrast to all other mapping studies of apomeiosis to date, our results showed no evidence for suppression of recombination around the DIP locus in Taraxacum. No obvious evidence for sequence divergence between the DIP and non-DIP homologous loci was found, and no hemizygosity at the DIP locus was detected. These results may indicate that apomixis is relatively recent in Taraxacum.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination,DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

Hybridisation,genomic constitution and generic delimitation inElymus s. l. (Poaceae: Triticeae)

Intergeneric crosses were made between representatives of the genomically-defined generaElymus, Agropyron, Elytrigia, Pseudoroegneria, andThinopyrum. The genomic constitution ofElytrigia repens, the type species ofElytrigia, is shown to be SSH, a genomic combination otherwise found only inElymus. The S genome ofPseudoroegneria has almost always a dominant influence on the morphology of the taxa of which it is a component.Wang (1989) showed that the J genome inThinopyrum and the S genome have considerable homoeology, with a mean c-value of 0.35 in diploid SJ hybrids. A genetic coherence from S to SJ^e, J^e, J^eJ^b, and J^b can be expected, agreeing with the continuous morphologic variation pattern observed. Because of the absence of morphological discontinuities between the taxa,Pseudoroegneria (S),Elymus (SH, SY, sometimes with additional genomes),Elytrigia (SSH, SSHX), andThinopyrum (SJ, SJJ, J) are best treated as a single genus,Elymus, following the generic concept ofMelderis in Flora Europaea and Flora of Turkey. The basic genomic constituents ofElymus will then be the S and/or J genomes.Agropyron, with diploids, tetraploids, and hexaploids based on the P genome is morphologically distinct from other genera inTriticeae. In a few species ofElymus andPseudoroegneria, a P genome is an additional constituent. In these cases the P genome has a negligible morphological influence. Therefore, it seems reasonable to maintainAgropyron as a separate genus.     

Use of plasmid pULB113 (RP4::Mini-Mu) to construct a genomic map ofAeromonas hydrophila

Plasmid pULB113 (RP4::Mini-Mu) promoted homologous gene transfer inAeromonas hydrophila; transfer of chromosomal markers occurred at frequencies of between 10^–3 and 10^–4 per donor cell regardless of the marker selected; this indicated chromosome transfer from multiple origins. With a variety of amino acid biosynthetic markers, a single circular map of this bacterium was constructed.     

High frequency FP2 donor of Pseudomonas aeruginosa PAO

Summary After NG mutagenesis an FP2 donor was isolated which exhibited an enhanced conjugational capacity for chromosomal genes. The recombination frequency was increased by two orders of magnitude as compared to the parenal strain. In plate matings recombinants arose at a frequency up to 5×10^-1 per donor cell. Late markers also recombined efficiently.An Hfr state of the donor strain was supported by (i) the high recombination frequency, (ii) the incompatibility reaction with plasmid pRO271 (=FP2::Tn401) and (iii) the clearcut transfer kinetics in interrupted matings, even for a late marker.     

Phytohemagglutinin-enhanced hybrid colony formation by cells from aged mice: Expression of th 4mod-1 mouse locus in human-mouse hybrids

Summary Cells from a continuous human line and freshly isolated cells from old adult mice heterozygous at theMod-1 locus were fused in the presence of polyethylene glycol (PEG). The production of hybrid cells, as a function of PEG concentration in the presence and absence of phytohemagglutining (PHA), was measured by cell survival and proliferation on selective medium. The incorporation of PHA into the fusion mixture allowed cell fusion to take place at nontoxic concentrations of PEG. PHA increased the frequency of cell fusion and increased the production of viable hybrid cells from 138- to over 2800-fold depending on cell type. The results suggest that the procedure may have broad application in promoting the fusion of cells sensitive to PEG. Clones were analyzed for isozymes of malic enzyme and glucose-6-phosphate dehydrogenase. The expression of the gene encoding X-linked mouse glucose-6-phosphate dehydrogenase confirmed that the cells were hybrids. These cells lost other mouse isozymes rapidly. In those clones in which the mouse malic enzyme gene was expressed, the product ofMod-1 ^α was detected significantly more frequently than that ofMod-1 ^b.