Identification of novel sex determination loci in Japanese weedy melon.

KEY MESSAGE: Japanese weedy melon exhibits unique sex expression with interactions between previously reported sex determination genes and two novel loci. Sex expression contributes to fruit quality and yield in the Cucurbitaceae. In melon, orchestrated regulation by sex determination genes explains the mechanism of sex expression, resulting in a great variety of sexual morphologies. In this study, we examined the Japanese weedy melon UT1, which does not follow the reported model of sex expression. We conducted QTL analysis using F2 plants for flower sex on the main stem and the lateral branch and mapped "occurrence of pistil-bearing flower on the main stem" locus on Chr. 3 (Opbf3.1) and "type of pistil-bearing flower" (female or bisexual) loci on Chr. 2 (tpbf2.1) and Chr. 8 (tpbf8.1). The Opbf3.1 included the known sex determination gene CmACS11. Sequence comparison of CmACS11 between parental lines revealed three nonsynonymous SNPs. A CAPS marker developed from one of the SNPs was closely linked to the occurrence of pistil-bearing flowers on the main stem in two F2 populations with different genetic backgrounds. The UT1 allele on Opbf3.1 was dominant in F1 lines from crosses between UT1 and diverse cultivars and breeding lines. This study suggests that Opbf3.1 and tpbf8.1 may promote the development of pistil and stamen primordia by inhibiting CmWIP1 and CmACS-7 functions, respectively, making the UT1 plants hermaphrodite. The results of this study provide new insights into the molecular mechanisms of sex determination in melons and considerations for the application of femaleness in melon breeding.

New methods for reverse transfection with siRNA from a solid surface.

We describe two efficient and inexpensive methods for reverse transfection with siRNA from a solid surface. One method involves localized reverse transfection from spots on a glass slide, which is mainly useful for making "transfection microarrays" (TMAs). The other involves reverse transfection in multiple wells of microtiter plates. Conditions for cell culture, preparation of reagents, and details of reverse transfection have been determined for several lines of cells, but we focus here on experiments with HeLa cells. In particular, we evaluated the efficiency of transfection, the cytotoxic effects of reverse transfection, and the efficiency of gene "knockdown" by transfection. We also performed phenotypic screening for a functional gene, during which cell viability was evaluated in terms of fluorescence from Calcein-AM. Our methods for reverse transfection with siRNA should be powerful tools that are useful for high-throughput analysis of functional genes.

A system for analyzing the event timing profile of single cells by using a model of neurite maturation in PC12D cells.

Nerve growth factor (NGF)-induced neurite maturation in PC12D cells involves neuritogenesis and neurite outgrowth. Actions of compounds affecting the neurite maturation are sometimes invisible behind the individually variable events in nature even in the clonal population. In this study, we designed a time-lapse imaging system to determine the timing of each event in individual PC12D cells. To examine the system, we analyzed the effect of staurosporine on the neurite maturation in PC12D cells. By using the system, we obtained four event timing data sets (stimulation by NGF with and without staurosporine at the concentrations of 0.01, 0.1, and 1 microM). A permutation test of these data sets revealed that staurosporine caused an early induction of neurite outgrowth during neurite maturation in PC12D cells. These results suggest that the time-lapse imaging system to determine the timing of each event in individual cells can provide a novel insight into features of a cell mass by single-cell analysis and is expected to be utilized for profiling of compounds that can serve as candidate drugs.

Expression of a chimeric CSF1R-LTK mediates ligand-dependent neurite outgrowth.

In an earlier screening, we identified several genes for kinases that might control the extension of neurites. One of these genes encoded a leukocyte tyrosine kinase (LTK), which is a receptor tyrosine kinase whose ligands remain to be identified. To examine the possible role of this LTK in neurite outgrowth, we constructed a chimeric receptor, in which the extracellular domain of the receptor for colony-stimulating factor-1 was fused to the cytoplasmic domain of LTK, which allowed the selective activation of LTK by colony-stimulating factor-1. Our studies using this chimeric receptor suggest that activation of the tyrosine kinase activity of LTK is sufficient to promote neurite outgrowth through pathways that include reactions catalyzed by phosphatidylinositol 3-kinase and MAPK.

Highly efficient reverse transfection with siRNA in multiple wells of microtiter plates.

We have developed an efficient and inexpensive method of reverse transfection from the solid phase to suppress genes with siRNA. The method enabled the realization of (i) a high efficiency of transfection; (ii) transfection of various types of cell; (iii) a high efficiency of gene knockdown by siRNA; (iv) a low toxicity to cells; and (v) a long-term stabilization (more than 210 d) of attached transfection mixture including siRNA in multiple wells. Although array-based reverse transfection has advantages in terms of miniaturization, the method has the advantage of enabling the inclusion of various soluble factors, such as humoral factors, drugs and ligands that affect gene expression, because the liquid phase is partitioned within the individual wells of each microtiter plate. Our method of reverse transfection with siRNA in multiple wells is a powerful and high-throughput tool for the analysis of signaling pathways.