We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome 4 and 5, respectively, and BAC clones from the KBrH library, we studied conservation and microsynteny corresponding to the Arabidopsis regions in B. rapa ssp. pekinensis. The BAC contigs assembled according to the Arabidopsis homoeologues revealed triplication and rearrangements in the Chinese cabbage. In general, collinearity of genes in the paralogous segments was maintained, but gene contents were highly variable with interstitial losses. We also used representative BAC clones, from the assembled contigs, as probes and hybridized them on mitotic (metaphase) and/or meiotic (leptotene/pachytene/metaphase I) chromosomes of Chinese cabbage using bicolor fluorescence in situ hybridization. The hybridization pattern physically identified the paralogous segments of the Arabidopsis homoeologues on B. rapa ssp. pekinensis chromosomes. The homoeologous segments corresponding to chromosome 4 of Arabidopsis were located on chromosomes 2, 8 and 7, whereas those of chromosome 5 were present on chromosomes 6, 1 and 4 of B. rapa ssp. pekinensis.
We estimated the genome size of Korean ginseng (Panax ginseng C.A. Meyer), a medicinal herb, constructed a HindIII BAC library, and analyzed BAC-end sequences to provide an initial characterization of the library. The 1C nuclear DNA content of Korean ginseng was estimated to be 3.33 pg (3.12 x 10(3) Mb). The BAC library consists of 106,368 clones with an average size of 98.61 kb, amounting to 3.34 genome equivalents. Sequencing of 2167 BAC clones generated 2492 BAC-end sequences with an average length of 400 bp. Analysis using BLAST and motif searches revealed that 10.2%, 20.9% and 3.8% of the BAC-end sequences contained protein-coding regions, transposable elements and microsatellites, respectively. A comparison of the functional categories represented by the protein-coding regions found in BAC-end sequences with those of Arabidopsis revealed that proteins pertaining to energy metabolism, subcellular localization, cofactor requirement and transport facilitation were more highly represented in the P. ginseng sample. In addition, a sequence encoding a glucosyltransferase-like protein implicated in the ginsenoside biosynthesis pathway was also found. The majority of the transposable element sequences found belonged to the gypsy type (67.6%), followed by copia (11.7%) and LINE (8.0%) retrotransposons, whereas DNA transposons accounted for only 2.1% of the total in our sequence sample. Higher levels of transposable elements than protein-coding regions suggest that mobile elements have played an important role in the evolution of the genome of Korean ginseng, and contributed significantly to its complexity. We also identified 103 microsatellites with 3-38 repeats in their motifs. The BAC library and BAC-end sequences will serve as a useful resource for physical mapping, positional cloning and genome sequencing of P. ginseng.
Several factors influence the limited application of assisted reproductive technologies (ART) in the canine species. Most problems arise because of the complex nature of reproductive physiology of the dog. For example, dogs are monoestrus, generally exhibiting oestrus only once every 6 month to 1 year. In the canine species, there has been little research on the ART because of difficulties associated with anatomy and reproductive physiology. Because in vitro maturation of canine oocytes has been particularly difficult, in vivo matured oocytes have been used in somatic cell nuclear transfer. However, the number of oocytes that can be obtained using this approach is limited, with ∼6 to 10 good oocytes being obtained per collection. The present study was undertaken to evaluate the effects of different dosages of eCG on folliculogenesis in the dog and to determine the number of oocytes that might be obtained after ovulation. The experimental design involved 3 groups that were treated with different dosages of eCG at the early stage of proestrus; Group A was a nontreatment (control) group, Group B received 200-IU eCG SC injections every day, and Group C was injected with 500 IU of eCG every 2 days until reaching 2 to 3 ng mL–1 serum progesterone concentration, respectively. Dogs in Groups B and C received a 1 000-IU hCG SC injection when progesterone concentrations reached 2 to 3 ng mL–1. The serum progesterone concentration was examined with a Radioimmunoassay Kit (Diagnostic Systems Laboratories Inc., Webster, TX, USA). The day of ovulation was considered as the day when serum progesterone concentration reached 4.0 to 7.2 ng mL–1. Approximately 70 to 76 h after ovulation, the dogs were subjected to the oocyte collection procedure. One-way ANOVA followed by Duncan’s multiple range tests was performed. The significance level was <0.05. In total, 446 oocytes were recovered from 60 bitches, with an average of 6.4 oocytes/dog in Group A (from 49 bitches), 16.6 oocytes/dog in Group B (from 5 bitches), and 8.5 oocytes/dog in Group C (from 6 bitches). The oocytes collection rate (number of oocytes per dog) in Group B was higher (P < 0.05) than those in the other 2 groups. In conclusion, the results showed that eCG treatment with hCG in early-proestrus-stage bitches can result in a greater number of recovered in vivo matured oocytes. This technology could become a useful research tool for canine cloning and ART.
The amniotic fluids contain mesenchymal stem cells and can be readily available for tissue engineering. Recently, regenerative treatments such as tissue engineering, cell therapy, and transplantation have shown potential in clinical trials of degenerative diseases. Physiologically, disease presentation and clinical responses in the dog are much more similar to that in the human compared with other traditional mammalian models. In addition, several researchers have demonstrated Canis familiaris is a suitable model for human diseases. The aim of the present study was to investigate whether canine amniotic fluid (cAF)-derived mesenchymal stem cells (MSC) can differentiate into neural precursor cells in vitro by neural induction reagent. The conditions of differentiation of MSC into neural cells were DMEM and N2-supplement, dibutyryl cyclic adenosine monophosphate, and butylated hydroxyanisole. During neural precursor differentiation, cAF-MSC can progressively acquire neuron-like morphology. Expressions of neuron cell-specific markers were examined before and after in vitro induction of differentiation. Changes in mRNA levels of specific genes were quantified by RT-PCR. The mRNA levels of NEFL (730%), GFAP (350%), β-tubuline 3 (2900%), and NSE (960%) were significantly increased after induction. The value of change in mRNA levels before and after induction was evaluated with the Image J program. In addition, the nestin, β-tubuline 3, and tyrosine hydroxylase protein expressions were confirmed by immunocytochemistry assay following the induction of differentiation, compared with the noninduction. In conclusion, this study demonstrated that cAF-MSC have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegeneration diseases including Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.
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