Isaac-CACTA transposons: new genetic markers in maize and sorghum.

CACTA is an En/Spm transposon superfamily present in high copy number in plant genomes, and Isaac is a subfamily of the CACTA superfamily. The TIR sequence of the Isaac subfamily was used for the purpose of transposon display (TD) in maize and sorghum. The Isaac TD produced 50-80 amplified fragments, depending on the primer combination, and the amplification profile was highly polymorphic among maize inbred lines. Isaac TD-based phylogenetic clustering distinguished the maize inbred lines according to their lineages and was consistent with the results of phylogenetic reports derived from other marker techniques by others researchers. The Isaac TD profile proved to be highly reproducible with different brands of Taq DNA polymerases and thermocyclers. The Isaac TD was also applied to recombinant inbred lines to assess genetic segregation; we observed 40-50 recordable segregation markers, depending on the primer combination. These Isaac TD markers segregated mostly as dominant markers, although, in a few cases, non-parental bands were observed in the segregating populations. In addition, the Isaac TD was very successful in the amplification of sorghum accessions. Therefore, the Isaac TD may provide another useful protocol for genetic analysis in maize and sorghum.     

Pong-like elements in Arabidopsis and Brassica rapa: its regulation of F-box protein gene in different ecotypes of Arabidopsis thaliana

Pong elements are active class 2 transposons in rice. We found Pong-like elements in Arabidopsis thaliana and named them as AtPong (Arabidopsis thaliana Pong) elements. The AtPong elements carried 13 bp of TIRs and two ORFs in which NAM DNA binding domain and DDE catalytic domain are encoded. Ping and mPing (miniature Ping) elements are deficient Pong elements and we found AtPong derived deficient AtPing and AtmPing elements. We also found a homologous element of the AtPong element in Brassica rapa. This element was a deficient element by internal deletion, but showed high sequence similarity with the AtmPing so that it was named as BrmPing (Brassica rapa miniature Ping). The AtPong, AtPing, and AtmPing elements were present in intergenic regions except one element, AtPing1, which was present in an exon of a F-box protein gene. Among the different A. thaliana ecotypes, the AtPing1 showed polymorphisms of present and absent in the F-box protein gene. The excision of the AtPing1 restored the expression of the F-box protein gene, indicating that the expression of the F-box protein genes is regulated by the AtPing1.     

Selective inhibition of MAPKK Wis1 in the stress-activated MAPK cascade of Schizosaccharomyces pombe by novel berberine derivatives.

Intracellular molecular targets of novel berberine derivatives, HWY 289 and HWY 336, were identified by a screen of a variety of mutants in fission yeast Schizosaccharomyces pombe. HWY 289 and HWY 336 completely inhibited the proliferation of wild type as well as various mutant fission yeast cells (minimal inhibitory concentrations were 29.52 microm for HWY 289 and 11.83 microm for HWY 336), but did not affect the proliferation of Wis1 mitogen-activated protein kinase kinase (MAPKK) deletion mutants. In addition, HWY 289 with an IC(50) value of 7.3 microm or HWY 336 with IC(50) of 5.7 microm specifically inhibited in vitro kinase activities of purified Wis1, whereas either compound did not affect the activities of other kinases in the mitogen-activated protein kinase (MAPK) cascades of fission yeast. These genetic and biochemical results demonstrate the high degree of specificity of HWY 289 and HWY 336 to MAPKK Wis1 and suggest that the cytotoxicity of these compounds is not simply due to the inhibition of Wis1 kinase activity. High salt wash experiments have shown that strong noncovalent binding occurs between Wis1 and either HWY 289 or HWY 336. The preincubation of Wis1 kinase with ATP did not affect the inhibition of Wis1 by HWY 289 and HWY 336, but when Wis1 was preincubated with MBP, a protein substrate, Wis1 kinase activity was no longer inhibited. These observations demonstrate that HWY 289/HWY 336 do inhibit Wis1 kinase, not by binding to the ATP-binding site but by disturbing the binding of substrate to the kinase. Target validation of the complex of HWY 289/HWY 336 and Wis1 kinase will provide important clues for the mechanism of specific cytotoxicity of these compounds in S. pombe. On a broader aspect, it would create an initiative to further modify and develop compounds that selectively inhibit kinases and cause cytotoxicity in various MAPK cascades including those of mammals.     

Differential induction of three pathogenesis-related genes, PR10, PR1b and PR5 by the ethylene generator ethephon under light and dark in rice (Oryza sativa L.) seedlings

Ethylene has been shown to be involved in triggering pathogenesis-related (PR) gene expression mainly in dicotyledonous species; however, less attention has been devoted identifying and characterizing PR genes in rice (Oryza sativa L.), a monocot and a model of cereal crop genera. Here, we demonstrate that ethylene induces at least three important rice PR genes, the PR10, PR1 basic (PR1b), and PR5 genes in rice (cv. Nipponbare) seedling leaf, upon treatment with the ethylene generator, ethephon (ET), in a light-, time- and dose-dependent manner. Induction of these PR genes was partially blocked by cycloheximide (CHX), a eukaryotic cytoplasmic protein synthesis inhibitor, which indicates an involvement of cytoplasmic de novo protein synthesis in their induction. These results clearly indicate a dynamic role for ethylene in PR genes induction in rice.     

Genetic diversity of maize kernel starch-synthesis genes with SNAPs.

Measuring genetic diversity in populations of a crop species is very important for understanding the genetic structure of and subsequently improving the crop species by genetic manipulation. Single-nucleotide amplified polymorphisms (SNAPs) among and within maize populations of waxy, dent, and sweet corns at 25 single-nucleotide polymorphism (SNP) sites in 6 kernel starch-synthesis genes (sh2, bt2, su1, ae1, wx1, and sh1) were determined. Because of the intensive selection of some favorable alleles in starch-synthesis genes during the breeding process, and the resultant strong linkage disequilibrium (LD), the number of haplotypes in each population was far less than expected. Subsequent phenetic clustering analysis with the SNAPs indicated that the dent, waxy, and sweet corns formed distinct subclusters, except in a few incidences. LD was surveyed among SNAPs of intragenic, intergenic, and intrachromosomal SNPs in whole and subpopulations, which revealed that some SNAPs showed high LD with many other SNAPs, but some SNAPs showed low or no significant LD with others, depending on the subpopulation, indicating that these starch genes have undergone different selection in each subpopulation during the breeding process. Because the starch synthesis genes used in this study are important in maize breeding, the genetic diversity, LD, and accessions having rare SNAP alleles might be valuable in maize improvement programs.     

Retrotransposons in <Emphasis Type="Italic">Betula nana</Emphasis>, and interspecific relationships in the Betuloideae,based on inter-retrotransposon amplified polymorphism (IRAP) markers

The Betulaceae family comprises two subfamilies, Betuloideae and Corylaceae. The subfamily Betuloideae contains two genera, Alnus Mill. and Betula L. Twenty putative long terminal repeat (LTR) retrotransposons were mined from 171 scaffolds containing 5,208,995 bp of dwarf birch (Betula nana) genome sequences. Five retrotransposons were finally selected after filtering the retrotransposon canonical features and nucleotide similarities between left and right LTR sequences. Of the five retroelements, three elements were found to be Ty1/Copia retrotransposons; identity of the other two elements could not be ascertained due to sequence undetermined ‘N’ bases in the sequence database. Inter-retrotranposon amplified polymorphism (IRAP) analysis, based on the LTR sequences of the mined LTR-retrotransposons, produced 179 discernible IRAP bands among the Alnus and Betula genera. Sequence analysis revealed no size homoplasy among the homologous IRAP bands. Phylogenetic and principle coordinate analysis, based on the band sharing among the taxa, showed the species in two different genera were clearly separated. The subgenera in each genus of Alnus and Betula were also distinguishable from the IRAP profiles. In the genus Betula, the species in subgenus Betula showed mixed clustering between species. This is incongruent with the phylogeographical distribution of the species.     

A Mutation in PMP2 Causes Dominant Demyelinating Charcot-Marie-Tooth Neuropathy

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of peripheral neuropathies with diverse genetic causes. In this study, we identified p.I43N mutation in PMP2 from a family exhibiting autosomal dominant demyelinating CMT neuropathy by whole exome sequencing and characterized the clinical features. The age at onset was the first to second decades and muscle atrophy started in the distal portion of the leg. Predominant fatty replacement in the anterior and lateral compartment was similar to that in CMT1A caused by PMP22 duplication. Sural nerve biopsy showed onion bulbs and degenerating fibers with various myelin abnormalities. The relevance of PMP2 mutation as a genetic cause of dominant CMT1 was assessed using transgenic mouse models. Transgenic mice expressing wild type or mutant (p.I43N) PMP2 exhibited abnormal motor function. Electrophysiological data revealed that both mice had reduced motor nerve conduction velocities (MNCV). Electron microscopy revealed that demyelinating fibers and internodal lengths were shortened in both transgenic mice. These data imply that overexpression of wild type as well as mutant PMP2 also causes the CMT1 phenotype, which has been documented in the PMP22. This report might expand the genetic and clinical features of CMT and a further mechanism study will enhance our understanding of PMP2-associated peripheral neuropathy.     

Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species

Dual-color fluorescence in situ hybridization (FISH) analysis of three Cucurbitaceae species from different genera was conducted using 5S and 45S rDNA probes. In Benincasa hispida (Thunb.) Cogn. (2n=24), the 45S rDNA probe hybridized on two chromosomes, one in the short arm of a medium-sized metacentric chromosome and another at the satellite of a chromosome. The 5S rDNA hybridized at a site proximal to the centromere of the same short arm of the 45S rRNA gene locus that occupied almost the entire short arm. For Citrullus lanatus (Thunb.) Matsum & Nakai (2n=22), the 45S rDNA probe hybridized at sites in the short arms of two chromosomes and the 5S rDNA probe was co-localized with the 45S rRNA locus at the region proximal to the centromere in one chromosome. The 45S rRNA loci occupied almost all of the short arms in both chromosomes. In Cucurbita moschata Duch. (2n=40), the 45S rDNA probe hybridized in five chromosomes in which the 45S rRNA genes occupied almost two-thirds of the chromosomes in two large chromosomes and the entire short arm of a medium-sized chromosome. Two other loci were present in two medium-sized chromosomes, one in the proximal region in the short arm of a chromosome and another at the tip of the long arm of a chromosome. Chromosomes of B. hispida were relatively larger than those of the other two species. The karyotype of B. hispida is composed of two metacentrics and 10 submetacentrics, while that of C. lanatus is composed of seven metacentrics and four submetacentrics and that of C. moschata is composed of 18 metacentrics and two submetacentrics. Comparative chromosome evolution among the three Cucurbitaceae species was attempted using the karyotypes and the chromosomal distribution patterns of the 5S and 45S rDNAs. The results presented herein will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species, and will provide basic data for their breeding programs.