Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.Abbreviations GUS ß-glucuronidase (EC 3.2.1.31) - NPT-II neomycin phosphotransferase II (EC 2.7.1.95) - EDTA ethylene-diamine-tetracetic acid     

pIDDLE6: A system for ligation and expression-cloning in E. coli

Summary The pIDDLE6 (for PCR-based, in-frame, directional DNA ligation and expression) system utilises novel ligation procedures to clone and overexpress almost any coding sequence. Inserts are cloned either by blunt ligation in the presence of PmeI or by ligation between dissimilar SfiI sites. A chimeric protein with both N- and C-terminal tags is produced, allowing purification by via starch-or nickel-affinity under native or denaturing conditions. The tags can be removed independently. The the vector and ligation procedures have been used successfully as described.     

A nucleic acid sequence-based amplification (NASBA) system for Listeria monocytogenes and simple method for detection of amplimers

A NASBA system amplifying specific sequences of the Listeria monocytogenes hlyA gene and an immunoenzymatic assay for the detection of amplimers was developed. The immunoenzymatic assay utilized a simple microtiter plate format and an anti-RNA:DNA hybrid antibody for the detection of NASBA product (predominantly RNA) hybridized to an immobilized DNA probe. This highly sensitive isothermal amplification and detection system was reactive with genomic DNA from various L. monocytogenes isolates but not with other Listeria or non-Listeria species.     

Plasmid dna transformation of Lactobacillus strains by electropermeabilization

Summary Two thermophilic strains of Lactobacillus were transformed by electroporation; L.fermentum with a maximum of frequency of 1×10⁵/ug of plasmid vector pPSC₂₀DNA and 1.4×10³/ug pSA₃DNA. L.helveticus showed a very low frequency of transformation, from 9 to 26 transformants/ug DNA in all the experiments carried out with both the vectors. While L.fermentum transformants were very stable, in L.helveticus the acquired plasmid was lost after 30–50 generations.     

Mos1 transposition as a tool to engineer the Caenorhabditis elegans genome by homologous recombination

Gene knockouts and knock-ins have emerged as powerful tools to study gene function in model organisms. The construction of such engineered alleles requires that homologous recombination between a transgenic fragment carrying the modifications desired in the genome and the locus to engineer occurs at high frequencies. Homologous recombination frequency is significantly increased in the vicinity of a DNA double-strand break. Based on this observation, a new generation of transgene-instructed genome engineering protocols was developed. Here, we present MosTIC (for “Mos1 excision-induced transgene-instructed gene conversion”), a new technique that provides a means to engineer the Caenorhabditis elegans genome. MosTIC is initiated by the mobilization of Mos1, a Drosophila transposon experimentally introduced in C. elegans. During MosTIC, a Mos1 insertion localized in the genomic region to engineer is mobilized after germline expression of the Mos transposase. Mos1 excision generates a DNA double-strand break, which is repaired by homologous recombination using a transgenic repair template. This results in the transfer of information from the transgene into the genome. Depending on the method used to trigger Mos1 excision, two alternative MosTIC protocols are available, which are presented here in detail. This technique can be used for a wide range of applications, such as structure-function analysis, protein localization and purification, genetic screens or generation of single copy transgenes at a defined locus in the genome.     

Genotypic and phenotypic correlationships among some strains ofLactobacillus helveticus

Summary Evidence for the presence of extrachromosomal elements inLactobacillus helveticus ATCC 15009 and the absence of plasmid DNA in two other strains ofL. helveticus is reported. These three strains did not show any difference in regard to lactose metabolism, proteolytic activity, and antibiotic resistance or in N-acetyl-D-glucosamine fermentation. The only difference found is a higher resistance to arsenate forL. helveticus ATCC 15009, suggesting linkage of this resistance to plasmids present in this strain.     

MICA: desktop software for comprehensive searching of DNA databases

Background  

Molecular biologists work with DNA databases that often include entire genomes. A common requirement is to search a DNA database to find exact matches for a nondegenerate or partially degenerate query. The software programs available for such purposes are normally designed to run on remote servers, but an appealing alternative is to work with DNA databases stored on local computers. We describe a desktop software program termed MICA (K-Mer Indexing with Compact Arrays) that allows large DNA databases to be searched efficiently using very little memory.     

Plasmid-encoded ropiness production inLactobacillus casei SSP.casei

Summary Genetic determinants of the Muc⁺ character were investigated in two ropy strains,Lactobacillus delbrueckii ssp.bulgaricus 201 andL. casei ssp.casei NCIB 4114, which secrete a large amount of slime in culture media. Plasmid DNA analysis revealed the presence of two plasmids (4.5 and 2.3 Mdal) inL. casei ssp.casei, whileL. delbrueckii ssp.bulgaricus was plasmid free, suggesting a chromosomal location of Muc⁺ character in this strain. Curing experiments carried out onL. casei ssp.casei NCIB 4114 indicated a correlation between the Muc⁺ phenotype and the 4.5 Mdal plasmid.     

Development of the SapI/AarI Incision Mediated Plasmid Editing Method

Plasmid engineering and molecular cloning is a virtually ubiquitous tool in biology. Although various methods have been developed for ligating DNA molecules or targeted mutagenesis of plasmids, each has its limitations. Many of the commonly used laboratory strategies are inefficient, while commercially available kits are quite costly and often specialized for highly specific circumstances. Here, we describe the SapI/AarI incision mediated plasmid editing (SIMPLE) method, which allows users to perform site-directed mutagenesis, deletions, and even short insertions into any plasmid in a single PCR reaction, using just one restriction enzyme. In addition, the SIMPLE method can be adapted to insert any sized DNA fragment into a vector using a two-step PCR approach, and can be used to ligate any number of DNA fragments with non-compatible ends in the specific order desired. The SIMPLE method provides researches an efficient and powerful tool with a broad range of applications for molecular cloning.     

Pool sizes of the deoxynucleoside triphosphates in the sea urchin egg and developing embryo.

The four deoxynucleoside triphosphate pools in unfertilized eggs of L. pictus and S. purpuratus were measured and found to be very large, ranging from 10^?3 to 10^?2 pmoles per egg. The high levels of the individual dNTP pools are sufficient for one to eight rounds of DNA synthesis. During the first division cycle these pools fluctuate with the highest levels being attained prior to DNA synthesis. The pools then decrease just preceding or during the S period. There is a large reduction in the total cellular dNTP in later stages of development when DNA synthesis is reduced relative to the cleavage stages.     

A Dynamic Equation for Plant Interaction and Application to Yield-density-time Relations

A model for plant interaction is developed based on a definitionof space in terms of actual and potential amount of growth factorsabsorbed per unit of time. The resulting equation is a second-orderdifferential equation which is solved by dynamic simulation.Five data sets on yield-density relations are used to demonstratethe model's excellent predictive power. Competition model, plant interaction, yield-density relations, Richards function, subterranean clover (Trifolium subterraneum L.), Wimmera ryegrass (Lolium loliaceum Hand-Mazz.), lettuce (Lactuca sativa L.), maize (Zea mays L.), lucerne (Medicago sativa L     

Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production

Summary Candida shehatae ATCC 22984 and Pichia stipitis CBS 5776 were tested for ethanol production from xylose, glucose-xylose mixtures, and aspen wood total hydrolysates. Adaptation of these yeasts to wood hydrolysate solutions by recycling resulted in improved substrate utilization and ethanol production. Compared to the non-adapted cultures, recycled C. shehatae and P. stipitis in aspen hydrolysate increased g ethanol/g sugar consumed from 0.39 and 0.41 to 0.45 and 0.47; while ethanol production from a 70:30 glucose-xylose solution (total sugars 140 g/L) was 45 g/L in 24 h and 60 g/L in 72 h with the adapted yeasts compared to 15 g/L and 28 g/L in the same times with the parent strains.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Transcriptional role in DNA replication: degradation of RNA primer during DNA synthesis

DNA synthesis catalyzed in vitro by E. coli DNA polymeraseI in the presence of single stranded fd DNA or poly (dT) as template is stimulated by RNA primers. When poly(dT) fully or partially saturated with polyriboadenylic acid strands is used as template - primer, DNA synthesis proceeds with concomitant degradation of the ribostrands to 5′-adenosine monophosphate. The fragment of DNA polymerase lacking the 5′→3′ exonuclease shows comparable RNA primer dependency but reduced efficiency for the degradation of the RNA primer from the 5′-end.     

A parenchymatic medium solidifier for plant in vitro culture

A new material for the solidification of liquid culture media was prepared from plant parenchyma tissues by mechanical subdivision, solute extration and dessication from ethanol. It is suitable for in vitro culture and propagation of callus as well as shoot tip cultures. The following plant materials have been grown by means of the new medium solidifier: shoot cultures of Betula pendula Roth, Gerbera jamesonii H. Bolus ex Hook and Floribunda rose "Triumph", callus tissues of Daucus carota L. and Chenopodium album L. The new solidifying material has special advantages over agar for application in the rooting phase of in vitro propagation.Abbrevations PMS parenchymatic medium solidifier - MS Murashige and Scoog's medium - IAA Indole-3-acetic acid - B biotin - K kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - ch caseine hydrolysate     

Agrobacterium — and microprojectile — mediated viral DNA delivery into barley microspore-derived cultures

Summary Anther cultures of barley (Hordeum vulgare L. var. Igri) were used as targets for Agrobacterium-mediated DNA transfer and direct DNA uptake by particle bombardment. A wheat dwarf virus construct which can replicate to a high copy number in cereal cells provided a sensitive marker for successful DNA delivery. Although DNA delivery was achieved using both procedures, particle bombardment gave more reproducible and higher levels of infection. The ability to deliver DNA into cereal cells which have a high regeneration capacity may provide a route for stable transformation.Abbreviations WDV Wheat dwarf virus - Gus -glucuronidase - ^mA N⁶-methyladenine     

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1,2, and 5 positions of the α3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNTTT-. a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12mol/L1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the     

In situ detection of early replication phases of a gemini virus in legume protoplasts

Protoplasts of Phaseolus vulgaris L. (Top Crop), infected with bean golden mosaic virus, were isolated and fixed by various methods for in situ hybridization. An iodine-125 labeled probe was made from the replicative form of the virus. The localization and quantitation was done by autoradiography. Cell wall removal lowered the background and allowed a more accurate analysis. RNase was used to eliminate the possibility of hybrids to RNA. The evidence suggests a sequence of virus movements starting from rough endoplasm reticulum, moving to the nuclear membrane, and finally with the highest concentration inside the nucleus.Abbreviations BGMV bean golden mosaic virus - rfBGMV or rfDNA replicative double-stranded DNA virus - ssDNA single-stranded virus     

Stable ploidy levels in long-term callus cultures of loblolly pine

Ploidy levels were calculated for callus cultures of loblolly pine (Pinus taeda L.), based on nuclear DNA content measured by Feulgen cytophotometry. The nuclear DNA content of initial stem explants showed a predominant 2C condition with less 3C and 4C, in ratios approximating those expected from diploid cells as they replicate DNA in the mitotic cell cycle. Cells with higher ploidy were produced during callus initiation, as indicated by a sharp reduction in the 2C population and a concomitant increase in higher DNA levels up to 8C. A gradual decrease in the higher ploidy levels occurred in subsequent subculture intervals, so that by 18 weeks the diploid nuclear DNA distribution was again observed, with complete elimination of DNA levels greater than 4C. Established callus cultures derived from stem or embryo explants and cultured on three different nutrient media for 48–76 weeks also showed the diploid nuclear DNA distribution with no indication of polyploid cells.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - BL Brown and Lawrence's medium - BLG modified BL medium - LM Litvay's medium Paper No. 11952 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh NC 27695-7643, USA     

Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

Background  

In mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function.