Molecular analyses of a repetitive DNA sequence in wheat (Triticum aestivum L.).

A repetitive sequence designated WE35 was isolated from wheat genomic DNA. This sequence consists of a 320-bp repeat unit and represents approximately 0.002% of the total wheat DNA. It is unidirectionally distributed either continuously or discretely in the genome. Ladder-like banding patterns were observed in Southern blots when the wheat genomic DNA was restricted with endonuclease enzymes EcoRI, HincII, NciI, and NdeI, which is characteristic for tandemly organized sequences. Two DNA fragments in p451 were frequently associated with the WE35 repetitive unit in a majority of lambda wheat genomic clones. A 475-bp fragment homologous to the 5'-end long terminal repeat (LTR) of cereal retroelements was also found in some lambda wheat genomic clones containing the repetitive unit. Physical mapping by fluorescence in situ hybridization (FISH) indicated that one pair of wheat chromosomes could be specifically detected with the WE35 positive probe p551. WE35 can be considered a chromosome-specific repetitive sequence. This repetitive unit could be used as a molecular marker for genetic, phylogenetic, and evolutionary studies in the tribe Triticeae.     

Identification of sequences necessary for packaging DNA into lambda phage heads

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.     

New approach for discrimination of Vibrio vulnificus by real-time PCR before and after gamma-irradiation

The effects of gamma-irradiation on the destruction of Vibrio vulnificus by real-time PCR were studied. gamma-irradiation was found to result in extensive reduction in the molecular size of DNA. Irradiation of viable cells (1 x 10(6) CFU/ml) at 1.08 KGy resulted in 100% destruction determined by plate counts, with most of the DNA from the irradiated cells having a bp-length of less than 1000. The use of a pair of primers to amplify a 1000-bp sequence of DNA from cells exposed to 1.08 KGy failed to yield amplification. In contrast, primers designed to amplify sequences of 700, 300, and 70-bp yielded amplification with C(t) values resulting in 13.4, 27.6, and 45.4% detection of genomic targets. When viable cells of V. vulnificus were exposed to 1.08, 3.0, and 5.0 kGy, the average molecular size of genomic DNA visualized in an agarose gel decreased with increasing dose, corresponding to an increased probability of amplification with primers targeting sequences of decreasing size.     

Size-dependent DNA mobility in cytoplasm and nucleus

The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (D(w)) decreased from 53 x 10(-8) to 0.81 x 10(-8) cm(2)/s for sizes of 21-6000 bp; D(w) was related empirically to DNA size: D(w) = 4.9 x 10(-6) cm(2)/s.[bp size](-0.72). DNA diffusion coefficients in cytoplasm (D(cyto)) were lower than D(w) and depended strongly on DNA size. D(cyto)/D(w) decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was <0.01 for >2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.     

Replication defective RP4 plasmids recovered after chromosomal integration

pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication. Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen. This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA. It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon. The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations. The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division.     

A simple method for direct automated sequencing of PCR fragments.

A simple and rapid method for direct sequencing of PCR-generated fragments has been developed for use on Applied Biosystems 373A Automated DNA Sequencer utilizing the DyeDeoxy terminator chemistry. Standard PCR conditions are used to generate a DNA fragment, which is subsequently gel-purified to remove excess primers and unwanted PCR products. The sequencing reactions are carried out in a thermal cycler using the purified product as template DNA and the Dye-Deoxy terminators. The sequence of 500-bp region in the bacteriophage lambda genome and a 320-bp fragment of the human genomic erythropoietin gene were sequenced with greater than 99% accuracy using this method.     

基于多重PCR的DNA Marker制备方法

报道了一种简便的制备分子量大小为100-1000bp DNA marker的方法,其原理是以一段特异的DNA片段为模板,设计PCR引物,采用多重PCR的方法一次扩增100-1000bp系列条带,酚/氯仿抽提,乙醇沉淀,即可得到条带清晰的DNA marker。     

Electrotransfection of Escherichia coli with λgt10 DNA

We have used electroporation to introduce lambda gt10 DNA into E. coli C600 (Electrotransfection). We obtained approximately 10(7) pfu (plaque forming units) per micrograms of lambda gt10 DNA. This frequency is 100-fold higher than the maximum reported for classical calcium chloride-induced transfection. We have also compared electrotransfection with in vitro packaging in cloning experiments using relatively small amounts (less than or equal to 10 ng) of DNA. Our results slow that electrotransfection can generate approximately 1000-fold more plaques than in vitro packaging at these concentrations.     

Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme. We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). We further show that lambda terminase adopts a heterogeneous mixture of higher-order structures, with an average molecular mass of 528(+/-34) kDa. Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. Furthermore, the ATPase activity of the higher-order species is approximately 1000-fold greater than that of the heterotrimer. These data suggest that IHF bending of the duplex at the cos site in viral DNA promotes the assembly of the heterotrimer into a biologically active, higher-order packaging motor. We propose that a single, higher-order hetero-oligomer of gpA and gpNu1 functions throughout lambda development.     

The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda DNA.

cosN is the site at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. Genetic and molecular studies show that cosN is recognized specifically by terminase and that effects of cosN mutations on lambda DNA packaging and cosN cleavage are well correlated. Mutations affecting a particular base-pair of cosN are unusual in being lethal in spite of causing only a moderate defect in cosN cleavage and DNA packaging. The particular base-pair is the rightmost duplex base-pair in mature chromosomes, at position 48,502 in the numbering system of Daniels et al; herein called position - 1. A G.C to T.A transversion mutation at position - 1, called cosN - 1T, reduces the particle yield of lambda fivefold, and the particles formed are not infectious. lambda cosN - 1T particles have wild-type morphology, and contain chromosomes that have normal cohesive ends. The chromosomes of lambda cosN - 1T particles, like the chromosomes of lambda + particles, are associated with the tail. lambda cosN - 1T particles, in spite of being normal structurally, are defective in injection of DNA into a host cell. Only approximately 25% of lambda cosN - 1T particles are able to eject DNA from the capsid in contrast to 100% for lambda +. Furthermore, for the 25% that do eject, there is a further injection defect because the ejected lambda cosN - 1T chromosomes fail to cyclize, in contrast to the efficient cyclization found for wild-type chromosomes following injection. The cosN - 1T mutation has no effect on Ca2+ mediated transformation by lambda DNA, indicating that the effect of the mutation on DNA fate is specific to the process of DNA injection. Models in which specific DNA : protein interactions necessary for DNA injection, and involving the rightmost base-pair of the lambda chromosome, are considered.     

SCAR markers for discriminating species of two genera of medicinal plants, Liriope and Ophiopogon

The development of DNA markers that can closely discriminate between Liriope and Ophiopogon species is vital for efficient and accurate identification of these species, and to ensure the quality, safety, and efficacy of medicines made from these plants. We developed species-specific molecular markers for these two genera. Forty RAPD primers were tested to detect polymorphism; species-specific RAPD bands were gel-purified, cloned, and sequenced. Primers for sequence-characterized amplified regions (SCARs) were then designed, based on nucleotide sequences of specific RAPD primers. SCAR markers SA06 and SB05, specific to Ophiopogon japonicus, amplified 460- and 553-bp DNA fragments, respectively. The marker SA12 amplified a 485-bp fragment specific to Liriope platyphylla. This is the first report of a species-specific SCAR marker for this group. These markers will be useful for rapid identification of closely related Liriope and Ophiopogon species.     

Analysis of DNA markers specific for G-genome of wheat

A RAPD marker specific for the G genome of wheat was identified. The corresponding 1171-bp DNA sequence was cloned and analyzed. Screening of the database did not reveal any homologies with the known plant DNA sequences. Using the primers specific to the flanking regions of the marker sequence, PCR analysis of the polyploid wheat species and the diploid species of the section Sitopsis was carried out. In addition, using the cloned sequence as a molecular hybridization probe, RFLP analysis of the genomic DNA of these species was performed.     

Maize transformation using xylose isomerase gene as a selection marker]

The xylA gene, encoding xylose isomerase, was cloned as a 1342-bp BamHI/SacI fragment from the E. coli. As a selection marker, the xylA gene was fused between the enhanced CaMV 35S promoter (E35S) and terminator (35St) in pBAC413 (Fig.2). pBAC413 was constructed to prevent the expression of sbeIIb in maize. PDS1000/He was used to bombard maize calli, which were induced to form by the elite inbred lines. The selection was carried out on the media containing concentrations of xylose from 0 to 100%. The results showed that the media containing 50% to 100% D-xylose were better, but differed with the genotype of maize (Tables 1 and 2). Successful integration of xylA gene into the maize genome was confirmed by DNA dot blotting, PCR and PCR-Southern hybridization (Figs.4 to 6). A method was established in which transformed maize cells were successively screened on a medium containing xylose instead of antibiotic and herbicide for bio-safety.     

Development of a PCR marker for rapid identification of the Bt-10 gene for common bunt resistance in wheat.

In western Canada, the Bt-10 resistance gene in wheat (Triticum aestivum) is effective against all the known races of common bunt caused by Tilletia tritici and T laevis. The genotypes of 199 F2 plants, originated from a cross between BW553 containing Bt-10 and the susceptible spring wheat cultivar 'Neepawa,' were established in greenhouse and field inoculation studies. A ratio of 1:2:1 resistant : heterozygous : susceptible was observed for bunt reaction, indicating that Bt-10 was expressed in a partially dominant fashion. A polymorphic DNA fragment, amplified using RAPD, and previously shown to be linked to Bt-10 was sequenced and SCAR (sequence characterized amplified region) primers devised. However, SCAR primers failed to amplify the polymorphic fragment. Restriction of PCR products with DraI revealed a polymorphic fragment of 490 bp resulting from a single base pair difference between lines possessing Bt-10 and those lacking the gene. As per the base pair difference, FSD and RSA primers were designed to generate a 275-bp polymorphic DNA fragment. Both 275- and 490-bp polymorphic fragments were present in all of the 22 cultivars known to carry Bt-10, and absent in all 16 cultivars lacking Bt-10. A 3:1 ratio was observed for presence: absence of the 275-bp marker in the F2 population. Using Southern analysis, the 490-bp fragment was effective in differentiating homozygous resistant plants from those heterozygous for Bt-10, based on its presence and the hybridization signal strength. A 1:2:1 resistant : heterozygous : susceptible ratio was also observed for the molecular marker and corresponded to 88% of the phenotypes deduced from the original F2 population. The molecular marker was estimated to be between 1.1 cM and 6.5 cM away from the Bt-10 resistance gene, based on the segregation analysis. Segregation analyses of Bt-10 and the 275-bp marker, evaluated in three different Canada Prairie Spring (CPS) wheat populations, demonstrated a segregation ratio of 3:1 for the molecular marker in two of the populations. These results demonstrated that the PCR marker system using the FSD and RSA primer pair permitted a rapid and reliable identification of individual lines carrying the Bt-10 gene for resistance to common bunt.     

Retroviral integration: in vitro host site selection by avian integrase.

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.     

Analysis of the DNA Marker Specific to the Wheat G Genome

A RAPD marker specific for the G genome of wheat was identified. The corresponding 1171-bp DNA sequence was cloned and analyzed. Screening of the database did not reveal any homologies with the known plant DNA sequences. Using the primers specific to the flanking regions of the marker sequence, PCR analysis of the polyploid wheat species and the diploid species of the section Sitopsis was carried out. In addition, using the cloned sequence as a molecular hybridization probe, RFLP analysis of the genomic DNA of these species was performed.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

The 9-bp deletion in region V of mitochondrial DNA: evidence of mutation recurrence

A deletion of one of the two copies of a 9-bp direct repeat sequence (CCCCCTCTA) in region V of mitochondrial DNA has previously been used as a polymorphic anthropological marker for people of east Asian origin, and to a lesser extent, in Oceanian and African populations. We report the presence of the 9-bp deletion in homoplasmy in skeletal muscle fibers and lymphocytes of a Spanish Caucasian individual. Other mitochondrial DNA polymorphisms associated with the 9-bp deletion characteristic of other populations were not present. Our results suggest that the 9-bp deletion probably originated independently in the maternal lineage of the propositus, and that it can thus be described as a recurrent mutation.     

Linear nicking endonuclease-mediated strand-displacement DNA amplification

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly.