Studies on Abscisic Acid

Methyl α-ionylideneacetates were oxidized with selenium dioxide to a mixture of methyl 3′-keto-β-ionylideneacetates and a small amount of methyl 4′-keto-α-ionylidene-acetates followed by treatment with active manganese dioxide. By a similar oxidation methyl 3′-keto-β-ionylideneacetates were prepared from methyl β-ionylidene acetates. Methyl 4′-keto-α-ionylideneacetates were obtained by oxidation of methyl α-ionylideneacetates with tert-butyl chromate. Dehydrobromination of methyl bromoionylideneacetate, obtained by bromination of methyl 2-trans-α-ionylideneacetate with N-bromosuccinimide, gave a mixture of methyl 2-trans-dehydro-β-ionylideneacetate and methyl 2-cis-dehydro-β-ionylideneacetate. The growth inhibitory activities of these sesquiterpene carboxylic acids and keto esters on rice seedlings were tested.     

The WD repeat protein FAN regulates lysosome size independent from abnormal downregulation/membrane recruitment of protein kinase C

FAN (factor associated with neutral sphingomyelinase [N-SMase] activation) exhibits striking structural homologies to Lyst (lysosomal trafficking regulator), a BEACH protein whose inactivation causes formation of giant lysosomes/Chediak-Higashi syndrome. Here, we show that cells lacking FAN show a statistically significant increase in lysosome size (although less pronounced as Lyst), pointing to previously unrecognized functions of FAN in regulation of the lysosomal compartment. Since FAN regulates activation of N-SMase in complex with receptor for activated C-kinase (RACK)1, a scaffolding protein that recruits and stabilizes activated protein kinase C (PKC) isotypes at cellular membranes, and since an abnormal (calpain-mediated) downregulation/membrane recruitment of PKC has been linked to the defects observed in Lyst-deficient cells, we assessed whether PKC is also of relevance in FAN signaling. Our results demonstrate that activation of PKC is not required for regulation of N-SMase by FAN/RACK1. Conversely, activation of PKC and recruitment/stabilization by RACK1 occurs uniformly in the presence or absence of FAN (and equally, Lyst). Furthermore, regulation of lysosome size by FAN is not coupled to an abnormal downregulation/membrane recruitment of PKC by calpain. Identical results were obtained for Lyst, questioning the previously reported relevance of PKC for formation of giant lysosomes and in Chediak-Higashi syndrome. In summary, FAN mediates activation of N-SMase as well as regulation of lysosome size by signaling pathways that operate independent from activation/membrane recruitment of PKC.     

Construction of a BAC Contig for a 3 cM Biologically Significant Region of Mouse Chromosome 1

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.     

Cytogenetic mapping of five YAC clones to human chromosome region 2q31 --> q32.1 in relation to the FRA2G common fragile site

In this work, five YAC clones have been mapped by fluorescent in situ hybridization (FISH) to human chromosome region 2q31 q32.1 and ordered in relation to each other and to the FRA2G common fragile site. YAC clones that span the fragile site have been identified. Moreover a deleted HOXD 13 gene has been identified on the 942D2 YAC.     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

Mapping candidate hotspots of meiotic recombination in segments of human DNA cloned in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The hotspots of meiotic recombination in the human genome can be localized by genetic techniques. The resolution of these techniques is in the range of kilobases and depends on the density of the physical markers identifying allelic variants of the chromosomal loci. We thought it would be interesting to localize these sites with higher resolution. Assuming that some human chromosomal sites conserve their propensity for recombination when cloned in yeast, we localized the hotspots of recombination in several yeast artificial chromosomes (YACs) carrying human DNA. A number of potential recombination hotspots could be identified in the clones studied. Among them there are two classes of sites that are particularly recombination prone also in human meiotic cells: sites associated with CpG islands and sites located in the vicinity of long minisatellite sequences.Communicated by G. P. Georgiev     

A Physical Map of Arabidopsis thaliana Chromosome 3 Represented by Two Contigs of CIC YAC, P1, TAC and BAC Clones

We have constructed a physical map of Arabidopsis thaliana chromosome3 by ordering the clones from CIC YAC, P1, TAC and BAC librariesusing the sequences of a variety of genetic and EST markersand terminal sequences of clones. The markers used were 112DNA markers, 145 YAC end sequences, and 156 end sequences ofP1, TAC and BAC clones. The entire genome of chromosome 3, exceptfor the centromeric and telomeric regions, was covered by twolarge contigs, 13.6 Mb and 9.2 Mb long. This physical map willfacilitate map-based cloning experiments as well as genome sequencingof chromosome 3. The map and end sequence information are availableon the KAOS (Kazusa Arabidopsis data Opening Site) web siteat http://www.kazusa.or.jp/arabi/.     

用万能引物PCR法从YAC中分离制备荧光原位杂交探针的研究

报道了一种从微量且未知碱基顺序的YAC DNA中制备FISH探针的新方法——万能引物PCR(UP PCR),即把复性后的UP-Linker连接于PFGE分离后经Alu Ⅰ酶切的YAC DNA上,再用UP对其进行PCR扩增和标记。由于Alu Ⅰ的广谱性和UP与Linker长度不同等,使该法能根据PCR效率调节扩增产物的广泛性向特异性的转变,且产物能覆盖YAC嵌入DNA的全长。此法制备的人21号染色体FISH探针,特异性强,杂交信号明亮,21号染色体的检出率高。该法稳定简便。     

Localization of <Emphasis Type="Italic">pms3</Emphasis>, a gene for photoperiod-sensitive genic male sterility,to a 28.4-kb DNA fragment

Photoperiod-sensitive genic male-sterile (PSGMS) rice, in which pollen fertility is regulated by day-length, originally arose as a natural mutant in the rice cultivar Nongken 58 (Oryza sativa ssp. japonica). Previous studies identified pms3 on chromosome 12 as the locus of the original PSGMS mutation. In this study we have assigned the pms3 locus to a 28.4-kb DNA fragment by genetic and physical mapping. A cross between Nongken 58S (PSGMS line) and DH80 was used to produce an F₂ population of about 7000 plants, from which 892 highly sterile individuals were obtained for recombination analysis. By analyzing recombination events in the sterile individuals using a total of 157 RFLP probes from a BAC contig covering the pms3 region, the pms3 locus was localized to a sub-region of less than 1.7 cM. Further analysis of recombination events using 49 additional probes isolated from this sub-region identified markers flanking the pms3 region on each side; these markers are only 28.4-kb apart. Sequence analysis of this fragment predicted the presence of five ORFs, found high homology with two ESTs in public databases, and detected three SNPs between the mutant and the wild-type parents, which may be helpful for identifying a candidate gene for pms3.