dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics

PCR-based detection of single nucleotide polymorphisms is a powerful tool for the plant geneticist. Cleaved amplified polymorphic sequence analysis is the most widely used approach for the detection of single nucleotide polymorphisms. However, this technique is limited to mutations which create or disrupt a restriction enzyme recognition site. This paper presents a modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation. This technique is useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes. In addition, a computer program has been developed that facilitates the design of these PCR primers.     

Calcium-binding proteins and development

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intra-cellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.© Kluwer Academic Publishers     

Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation

To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies.     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.     

Phenotypic analysis and molecular cloning of discolored-1 (dsc1), a maize gene required for early kernel development

Recessive mutations in the maize dsc1 locus prevent normal kernel development. Solidification of the endosperm in homozygous dsc1– mutant kernels was undetectable 12 days after pollination, at which time the tissue was apparently completely solidified in wild-type kernels. At later times endosperm did solidify in homozygous dsc1– mutant kernels, but there was a marked reduction in the volume of the tissue. Embryo growth in homozygous dsc1– kernels was delayed compared to wild-type kernels, but proceeded to an apparently normal stage 1 in which the scutellum, coleoptile, and shoot apex were clearly defined. Embryo growth then ceased and the embryonic tissues degraded. Late in kernel development no tissue distinctions were obvious in dsc1– mutant embryos. Immature mutant embryos germinated when transplanted from kernels to tissue culture medium prior to embryonic degeneration, but only coleoptile proliferation was observed. The dsc1 gene was isolated by transposon tagging. Analysis of the two different dsc1– mutations confirmed that transposon insertion into the cloned genomic locus was responsible for the observed phenotype. Dsc1 mRNA was detected specifically in kernels 5–7 days after pollination. These data indicate Dsc1 function is required for progression of embryo development beyond a specific stage, and also is required for endosperm development.     

Use of Landmarks in Three Species of Food-storing Corvids

The use of landmarks by Clark's nutcrackers (Nucifraga columbiana), pinyon jays (Gymnorbinus cyanocepbalus), and Western scrub jays (Apbelocoma coerulescens) was investigated in three experiments. In the first experiment, birds were trained to find a hidden food reward beneath wood chips near a plastic landmark located 20 cm from one edge of a stationary tray. During unrewarded test trials, all species displaced their searching in response to movement of the landmark in both parallel and perpendicular directions; however, searching was displaced significantly more in the parallel direction. In a second experiment, the landmark was removed with the tray stationary or shifted. When the tray was shifted, all species responded by searching relative to the shifted tray. This indicates that the global (room) cues were not being used as salient landmarks. In the third experiment, the tray was placed in different positions for each trial, making global cues unreliable. As in experiment 1, all species displaced their searching in response to parallel and perpendicular movement of the landmark, with movement being similar in both directions. These results indicate no species differences on this landmark-use task.     

Characterization of dengue virus complex-specific neutralizing epitopes on envelope protein domain III of dengue 2 virus

The surface of the mature dengue virus (DENV) particle is covered with 180 envelope (E) proteins arranged as homodimers that lie relatively flat on the virion surface. Each monomer consists of three domains (ED1, ED2, and ED3), of which ED3 contains the critical neutralization determinant(s). In this study, a large panel of DENV-2 recombinant ED3 mutant proteins was used to physically and biologically map the epitopes of five DENV complex-specific monoclonal antibodies (MAbs). All five MAbs recognized a single antigenic site that includes residues K310, I312, P332, L389, and W391. The DENV complex antigenic site was located on an upper lateral surface of ED3 that was distinct but overlapped with a previously described DENV-2 type-specific antigenic site on ED3. The DENV complex-specific MAbs required significantly higher occupancy levels of available ED3 binding sites on the virion, compared to DENV-2 type-specific MAbs, in order to neutralize virus infectivity. Additionally, there was a great deal of variability in the neutralization efficacy of the DENV complex-specific MAbs with representative strains of the four DENVs. Overall, the differences in physical binding and potency of neutralization observed between DENV complex- and type-specific MAbs in this study demonstrate the critical role of the DENV type-specific antibodies in the neutralization of virus infectivity.