No abstract

"Gene trapping" in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses the neor gene which lacks a polyadenylation signal and has a splice donor signal. Expression of the neor gene as fusion transcripts with the 3' end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3' rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

No abstract

For the purpose of determining the origin of horticultural evergreen azalea cultivars, this study was focused on the natural populations of azalea in Kyushu (south main island of Japan). The Kirishima mountains, the volcanic mountain mass in Kyushu, are an important centre of diversity for the Japanese evergreen azaleas. Rhododendron kiusianum Makino grows above 1000m alt., whereas R. kaempferi Planch. is distributed below 600m alt. Putative natural hybrid populations of these two species are found in the intermediate region (1000–600m alt.). These two species have been clearly distinguished by their respective morphological features. Rhododendron kiusianum has small pink-purple flowers and small elliptical leaves, whereas R. kaempferi has larger red-orange flowers with dark blotches and large oblong leaves. Interspecific hybrids show phenotypes within the range of the two species, especially with regard to flower colour and leaf shape. A morphological cline of these characteristics corresponding to altitude has been observed between these two species. PCR-RFLP analysis of chloroplast DNA detected specific bands for the two species in the 16S rDNA region when digested with HhaI restriction enzyme. Populations of interspecific hybrids were composed of individuals that had a banding pattern of either R. kiusianum or R. kaempferi. This indicates that R. kiusianum and R. kaempferi are clearly distinct species. Furthermore, natural hybrid populations consist of individuals that have one of two cpDNA. Some individuals in the populations of R. kiusianum (T-1430 and T-1030) possess the cpDNA pattern of R. kaempferi, which suggests that cytoplasmic introgression has occurred in the populations of R. kiusianum from R. kaempferi.

Naturally and artificially aged seeds of rape, Brassica napus L., produced less ethylene than freshly harvested seed during the early stage of germination. With (9); it also breaks the dormancy of seed of various species (4,9,10,18,19). Stewart and Freebaim (17) showed that heat-treated lettuce seed which became insensitive to gibberellic acid germinated well subsequent to treatment with exogenous ethylene. Ruge (15) showed earlier that ethylene enhanced the percentage germination of aged oat seed. In the present paper, the pattern of ethylene production by rape seedlings in the early stage of germination and the stimulation of the germination of aged seeds by exogenous ethylene were studied. MATERIALS AND METHODSMaterials used in the experiments were the seeds of rape, Brassica napus L., variety Chisaya-natane, harvested in 1965 and seeds were sealed in tin cans and were put with the other seed at 3 C until the experiment was started.A germination test of the seeds was conducted with three replications (100 seeds X 3) on December 10, 1969. The blotter method at 25 C was used. The first count was on the 3rd day, and the final count was on the 7th day. The number of normal seedlings, abnormal seedlings, and slowly germinating seeds was counted.For observing ethylene production by seedlings and effects of exogenous ethylene on aged seeds, all experiments were conducted in Erlenmeyer flasks equipped with rubber serum stoppers. To prevent mold growth, the flasks, stoppers, and seeds were sterilized at the start of the experiment. Flasks containing filter paper or agar media were autoclaved at 15 psi for 20 min. The rubber stoppers were immersed in 95% ethanol and dried under sterile conditions. The seeds were treated with 95% ethanol for 5 sec, rinsed with sterile water, sterilized with 0.1% sodium hypochlorite for 30 min, and washed three times with sterile water. Seedlings were germinated and grown under continuous fluorescent light in a room maintained at 25 C. Samples of ethylene given off from seedlings in a flask were withdrawn with a 1-ml syringe through the rubber stopper. The amount of ethylene was determined by gas chromatography, using an alumina column maintained at 50 C and a flame ionization detector. Each experiment was replicated three times.The first two experiments were conducted to follow the trend of ethylene production during germination. In the first trial, the seeds were maintained in a closed system in 25-ml Erlenmeyer flasks containing two layers of wet filter paper. A 1-ml gas sample was withdrawn daily for 9 days from each flask for ethylene determination. In the second trial, the seeds were transferred aseptically to 125-ml Erlenmeyer flasks containing 25 ml of 1% agar. In contrast to the first trial, a 1-ml gas sample was taken every 6 or 12 hr for ethylene analysis, after which the gaseous content of the flask was flushed with fresh ethylene-free air. This trial was conducted for 120 hr.In the third experiment, ethylene was introduced into the flask containing aged seeds t...