Earlier studies showed that the predominant steroid (estradiol E2, testosterone T, progesterone P4) secreted by in vitro cultured amphibian (Rana nigromaculata) ovarian follicles varied with their ...size. E2 was mainly produced by medium-sized follicles, T by intermediate-sized follicles, and P4 by the largest follicles. Experiments were carried out to ascertain whether the activities of steroidogenic enzymes changed during follicle development and were responsive to gonadotropic stimulation. Enzyme activities were measured indirectly by monitoring conversion of exogenously added substrates to products during in vitro culture of isolated follicles. Different stage follicles were cultured in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors (25-200 ng/2 ml). Amounts of E2, T, androstenedione (AD), 17 alpha-hydroxyprogesterone (17 alpha-OHP4), or P4 secreted into the medium were measured by RIA. Exogenous pregnenolone (P5) was converted to P4 by all types of follicles in a dose-dependent manner in the absence of FPH. Addition of FPH markedly enhanced medium levels of P4 in all sized follicles. Highest levels of P4 were presented in cultures containing the largest follicles. Such follicles were much less efficient than intermediate follicles in metabolizing P4 to AD or T. FPH suppressed conversion of exogenous 17 alpha-OHP4 but not androstenedione to testosterone by the largest follicles. Exogenous T was converted to E2 only by medium-sized follicles and FPH had little or no stimulating or inhibiting effect on this process in either medium- or intermediate-sized follicles.
For large DNA fragment transformation in dicots and monocots, BIBAC2 vector system was applied to Arabidopsis thaliana and Oryza sativa L. cv. Jinmi as a model plant, respectively. For Arabidopsis, ...the Th1 gene in T23L3 BAC clone whose size is about 90 kb was used as the target gene source for transformation. Because T23L3 BAC clone was originally constructed in pBelloBAC11, the target gene was reconstructed into BIBAC2. As the results of reconstruction, 476 colonies were survived in selection medium containing 40 mg/L kanamycin. In colony hybridization analysis, 24 out of 476 colonies exhibited positive signals. In the pulsed-field gel electrophoresis analysis, 11 out of 24 positive clones exhibited the band at the location of 90 kb. In Southern hybridization, positive signal band at the location of 90 kb was observed in all 11 transformants. Using these verified clones, Agrobacterium-mediated transformation was applied to Arabidopsis thaliana th1-201 mutant for genetic complementation test. Twelve thousands T$_1$ seeds were harvested, and antibiotic selection test is being analyzed to verify whether these seeds were transformed. for rice, COR356 that contains 150 kb human genomic DNA in a BIBAC2 vector was used as the target gene. As the results of transformation, 151 out of 210 co-cultivated calli were survived in selection medium containing 5 mg/L hygromycin, and 45 out of 151 survived calli were regenerated into plants. Transformation efficiency was 21.6%. Progeny test using 71 seeds is being analyzed now. These results provide the potential that large DNA fragments can be transferred into both dicots and monocot by Agrobacterium-mediate d transformation system.
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