Polymerase chain reaction-restriction fragment length polymorphism assays to distinguish Liriomyza huidobrensis (Diptera: Agromyzidae) from associated species on lettuce cropping systems in Italy

The pea leafminer, Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), is a serious insect pest infesting open field lettuce plantings in northern Italy. In these cropping systems, it coexists with several other agromyzid species that have negligible economic importance on open field vegetables. The rapid detection of L. huidobrensis is crucial for effective management strategies, but the identification of agromyzids to species can be very difficult at adult as well at immature stages. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay is proposed to separate L. huidobrensis from Liriomyza bryoniae (Kaltenbach), Liriomyza trifolii (Burgess), and Chromatomyia horticola (Goureau), which usually occur in the same lettuce plantings. An approximately 1,031-bp region of the mitochondrial genome encompassing the 3' region of cytochrome oxidase I, the whole leucine tRNA, and all of the cytochrome oxidase II was amplified by PCR and digested using the enzymes PvuII and SnaBI separately. Both endonucleases cut the amplicons of L. huidobrensis in two fragments, whereas the original band was not cleaved in the other analyzed species. The presence of Dacnusa spp. DNA does not bias the assay, because the PCR conditions and the primer set here described do not amplify any tract of this endoparasitic wasp genome.     

Molecular evidence of cryptic species within the Liriomyza huidobrensis (Diptera: Agromyzidae)

Phylogenetic relationships among populations of the polyphagous pea leafminer, Liriomyza huidobrensis (Blanchard), were investigated using DNA sequence data. Maximum parsimony analysis of 941 bp of mitochondrial cytochrome oxidase I and II genes showed that L. huidobrensis contains two well-defined monophyletic groups, one composed of specimens from California and Hawaii and one composed of specimens from South and Central America together with populations that have been recently introduced into other parts of the world. The differentiation between the two clades within L. huidobrensis is equivalent to that seen between other agromyzid species, suggesting that L. huidobrensis as currently defined contains two cryptic species. This finding is consistent with field observations of differences in pest status and insecticide resistance between L. huidobrensis populations. Until additional studies are complete, no changes in L. huidobrensis taxonomy are proposed. However, researchers and quarantine officials may wish to consider the findings of the current study in designing research, pest management, and quarantine programs for L. huidobrensis.     

Identification of EcoRV fragments spanning the N alpha-tubulin gene of Physarum

The N alpha-tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross-hybridised with another alpha-tubulin gene. Probes derived from either 5'- or 3'-flanking regions were gene-specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5'-fragment) and 6.2 kb (3'-fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5'-end) and 5.5 kb (3'-end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.     

Establishment of a physical and genetic map for bacteriophage PRD1

DNA was isolated from the lipid-containing bacteriophage PRD1 and subjected to restriction endonuclease analysis. The total genome size is 14.7 kb. PRD1 DNA was resistant to cutting by fifteen restriction endonucleases with six base specificity. HaeII made thirty-seven cuts in the DNA, MboI made one cut, and MnlI made six cuts. DNA that was not treated with protease yielded two fewer fragments when treated with HaeII. Evidence is presented to indicate that the PRD1 DNA has protein at the ends of the DNA. The thirty-eight HaeII fragments were ordered using the ladder technique of Smith and Birnstiel (1976) on MboI and MnlI fragments of the genome. Clones of HaeII partial digests of PRD1 DNA in pBR322 were analyzed by HaeII digestion and were then assigned to specific regions of the genome by their HaeII fragment composition. A comparison of the marker rescue characteristics of the cloned DNA with the overall restriction fragment map generated a physical map of the genome. Some genes that have not been mapped because of a lack of mutants or leakiness at restrictive conditions were mapped by studying the in vitro protein synthesis of restriction endonuclease fragments.     

Distinct mouse DNA sequences enable establishment and persistence of plasmid DNA polymers in mouse cells.

Distinct elements isolated from mouse genomic DNA confer on plasmid DNA the ability to persist at high copy numbers in mouse L fibroblasts (1). Field inversion gel electrophoresis demonstrated that - in contrast to our previous assumption - the persisting plasmid DNA does not exist extrachromosomally but as clusters of tandem repeats integrated into genomic DNA. Digestion with restriction endonucleases that do not cut within the plasmid DNA results in fragments of 50-300 kb in length indicating reiteration of 10-50 plasmid DNA molecules. Restriction with several enzymes that cut once or twice within the plasmid sequences lead to fragment(s) indicative for head-to-tail tandem repeats. In situ hybridization revealed signals for a long homogeneously staining region (HSR) in one or two chromosomes per cell nucleus. Possibilities how these elements could act in the establishment and/or maintenance of the head-to-tail polymers of plasmid DNA in mouse cells are discussed.     

Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).     

Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo

The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.     

Phylogenetic relationships of sorghum taxa inferred from mitochondrial DNA restriction fragment analysis.

To elucidate the evolutionary history and affinity of sorghum species, 41 sorghum taxa were analyzed using variability in mitochondrial DNA. Analysis of species relationships at the molecular level can provide additional data to supplement the existing classification based on morphological characters and may also furnish unexpected but useful information. Total DNA extracted from each of the sorghum accessions was digested with each of five restriction enzymes, BamHI, HindIII, EcoRI, EcoRV, and XbaI, and probed with five mitochondrial DNAs cloned from Sorghumbicolor. A total of 180 restriction fragments was detected by the 25 probe-enzyme combinations. Forty-three fragment bands were phylogenetically informative. Multiple correspondence analysis was performed to visualize associations among the accessions and suggested that section Eusorghum species may be divided into four groups, with Sorghumlaxiflorum (section Heterosorghum) and Sorghumnitidum (section Parasorghum) appearing as outliers. A phylogenetic tree was assembled from mitochondrial restriction fragment data. The taxa analyzed formed three major groups comprising section Heterosorghum (group I), section Parasorghum (group II), and all accessions in section Eusorghum (group III). Group III is further divided into four groups: (i) two sweet sorghums and shattercane; (ii) Sorghumhalepense, Sorghummiliaceum, Sorghumhewisonii, Sorghumaethiopicum, Sorghumverticilliflorum, and S. bicolor, including Sorghumsudanense (sudangrass), the Chinese Kaoliangs, and a number of commercial sorghum inbreds from the U.S.A.; (iii) Sorghumpropinquum; and (iv) Sorghumarundinaceum, Sorghumniloticum, Sorghumalmum, Sorghumcontroversum, and the Chinese material C-401 and 5-27. Results indicate that the analysis of fragmented mitochondrial DNA was diagnostic and useful in sorghum phylogenetic and taxonomic research at the species, subspecies, and race levels, and can complement results from those analyses using nuclear ribosomal DNA and chloroplast DNA that effectively distinguish taxa at species and genus levels. Key words : Sorghum, mitochondrial DNA, phylogeny, restriction fragment.     

重大潜在入侵害虫番茄潜叶蛾的SS-CoI快速检测技术

【背景】番茄潜叶蛾是源自南美洲的一种最具毁灭性的世界性入侵害虫,其适生区范围广,且与其他潜叶类昆虫难以准确、快速识别。【方法】以田间常见的其他6种潜叶类害虫为对照,采用基于mtDNACOl基因的种特异性SS—COI方法,研究番茄潜叶蛾快速分子检测技术。【结果】种特异性检测结果显示,SS-COl引物只对番茄潜叶蛾mtDNA具有扩增能力,对美洲斑潜蝇、南美斑潜蝇、三叶草斑潜蝇、葱斑潜蝇、豌豆潜叶蝇、桃潜叶蛾等其他潜叶类害虫不具有扩增效果。该引物不仅对成虫和单粒卵具有很好的扩增效能,对单根触角、头部、胸部、腹部以及翅和足等成虫残体亦具有同样的扩增能力,其最低检出阈值为312．5Pg·μL^-1。【结论与意义】该方法在有效防范番茄潜叶蛾侵入我国,保障农业生产安全和生态环境安全中意义重大。     

Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes.

The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined.     

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.     

Application of molecular techniques to distinguish Liriomyza trifolii from L. sativae (Diptera: Agromyzidae) on tomato cultivation in Japan

A molecular method is applied for differentiating the morphologically related leafminers Liriomyza trifolli and L. sativae on tomato cultivation. The method requires multiplex polymerase chain reaction (PCR) amplification of a region of mitochondrial cytochrome oxidase DNA using multiprimer sets. The method divides the mitochondrial fragment of L. trifolli into two fragments and L. sativae into three fragments. It is faster, less costly, and easier than random amplified polymorphic DNA-PCR, PCR-restriction fragment-length polymorphism, and DNA sequencing and more sensitive than the enzyme electrophoresis method, which are other ways to differentiate these two species. We applied the method to samples from populations of another place, sex, and stage and obtained the same results.     

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Complex Satellite DNA of Tropaeolum majus L.: Partial Characterization of Isolated and of Cloned Restriction Fragments

Satellite DNA (satDNA) was purified by repeated neutral CsCl,ethidium bromide/CsCl and actinomycin D/CsCl density centrifugationfrom total nuclear DNA of Tropaeolum majus. Digestion patternsof satDNA and main band DNA as generated with 22 different restrictionenzymes were partially characterized and hybridized by the Southernblot technique with Hpa II-generated satDNA fragments and aribosomal DNA fragment. Some restriction fragments were sequencedby the chemical method, either directly, or after cloning, andpossible secondary structures were determined. The results showthat the satellite is of high complexity and evidently composedof different, interspersed repeats, and is also interspersedwith ribosomal sequences. Some sequences of the satellite aredispersed throughout the Tropaeolum genome, and even found inthe chloroplast genome. Most of the restriction enzymes cutthe satDNA into many fragments of lengths between 14 bp and3000 bp. Hpa II cut the satellite DNA into at least 63 differentsized fragments (more than is known from any other plant satDNA).Sal I cut the satDNA into a single fragment of 1040 bp in size,which probably represented the superrepeat unit. Sequence analysisof satDNA restriction fragments and of cloned fragments isolatedat different times, indicated a rapid diversification in vivoand in vitro. Computer modeling of most likely secondary structuressuggest the occurrence of palindromic loops (cruciform structures).These could be the basis of rapid diversification in being sitesof preferred intragenomic recombination and rearrangement. Cloning, palindromic sequences, restriction analysis, satellite DNA complexity, Tropaeolum majus L, nasturtium     

Paternal leakage of mitochondrial DNA inPinus

Summary We studied mitochondrial DNA restriction fragment length polymorphism in 11 parents and 125 seedlings of 23 controlled matings within and between jack pine (Pinus banksiana Lamb.) and lodgepole pine (P. contorta Dougl.). A potential mitochondrial distinction between these two conifers was evident in the parental samples. Only maternal mitochondrial restriction fragments were observed in a majority of the seedlings, which is consistent with results from angiosperms and other members of the genusPinus L. However, we detected exclusively paternal mitochondrial DNA in six of the seedlings. These unusual seedlings were not attributable to heteroplasmy or contamination of the experimental material, indicating that mitochondrial inheritance was not strictly maternal. Paternal mitochondrial leakage inPinus may permit novel insights into the transmission genetics and evolution of organellar polymorphisms.     

美洲斑潜蝇SS-PCR检测技术研究

【背景】美洲斑潜蝇是一种严重威胁瓜果蔬菜、烟草、棉花等经济作物和花卉生产的入侵性害虫。由于潜叶蝇类害虫体型较小、生活方式隐蔽、形态相似,本文针对其难以快速准确地进行形态鉴别的问题,以美洲斑潜蝇为研究对象,以菜田常见的4种潜叶蝇类害虫为参照,采用种特异性PCR方法（species-specific PCR,SS-PCR）,研究其快速分子检测鉴定技术。【方法】调用GenBank中一段936bp的美洲斑潜蝇线粒体DNA（mtDNA）细胞色素氧化酶亚基Ⅰ基因（COⅠ）的序列（Gen-Bank登录号为EU219613）,并根据此基因片段的碱基序列设计引物1对,其扩增片段大小为294bp。【结果】种特异性检验结果显示,该引物只对美洲斑潜蝇的COⅠ基因具有扩增能力,对其他种类如南美斑潜蝇、三叶斑潜蝇、葱斑潜蝇、豌豆潜叶蝇等没有扩增能力。该引物不仅对成虫具有良好的扩增效果,对蛹、幼虫以及单粒卵也具有同样的扩增效果,其最低检出阈值为1/3840头成虫。【结论与意义】SS-PCR技术体系可用于美洲斑潜蝇的鉴定识别与检测监测,对阻止其进一步扩散蔓延具有重要意义。     

Electron microscopy and biochemical analysis of mouse metaphase chromosomes after digestion with restriction endonucleases

Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.     

Translation in Escherichia coli mini-cells containing hamster mitochondrial DNA-Co1E1 - Ampr recombinant plasmids.

Hamster mitochondrial DNA is cleaved into two fragments (4.2 and 11.4 kilobase pairs of DNA (kb)) by the restriction enzyme, Eco RI. Recombinant DNA molecules formed in vitro between an Escherichia coli plasmid, Co1E1 - Ampr, and Eco RI-digested hamster mitochondrial DNA were transformed into E. coli K12. The translation products of the parent plasmid, Co1E1 - Ampr, and recombinant plasmid DNAs containing (i) the 4.2 kb mitochondrial DNA fragment and (ii) the 11.4 kb fragment were characterized on sodium dodecyl sulfate-polyacrylamide gels using bacterial mini-cell lysates. The Co1E1 - Ampr plasmid specifies at least six polypeptides whose structural genes comprise 56% of the plasmid DNA. Insertion of hamster mitochondrial DNA at the Eco RI site of the plasmid alters the relative rate of synthesis of these six polypeptides and induces the occurrence of a new band on sodium dodecyl sulfate-polyacrylamide gels which is probably not specified by the inserted mitochondrial DNA sequences.