ompared the expression of MMPs in these oocytes and cumulus cell throughout oocytes maturated. In an attempt to investigate the effect of MMP activation and inhibitors in total protein of cumulus cell and, oocytes during oocytes maturation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), TIMPs (TIMP-2 and TIMP-3), as well as their expression profiles (Real-time PCR, Gelatin Zymography and ELISA). Our results that the bovine oocytes MMP-2 and MMP-9 level was significantly associated with the rate of maturity of oocytes (P<0.05). In cumulus cell, MMP-2 was highly expressed in all stages of the oocyte’s maturation. The final oocytes maturation exhibited strong gelatinase activity. There was no significant correlation between cumulus cell MMP-9 and the maturation rate of oocytes. However, for the oocyte cytoplasm MMP-9 expression was significant correlation to the maturation oocytes. There was no significant correlation between cumulonimbus cells MMP-9 and oocyte maturation rates; however, for oocyte cytoplasm, MMP-9 expression was significantly correlated with mature oocyte. However, the TIMP-1 and TIMP-2 protein expression patterns are not correlated with the maturation rate of the oocyte. Our results suggest that MMP different expression pattern may regulate the morphological remodeling of oocyte's in the cumulus cell. Further, the MMP-2 expression has a strong relation with a higher maturation rate of the oocyte.

Oyster mushroom is one of mushrooms that are cultivated and consumed a lot in Korea. P. ostreatus 'ASI 2504(Suhan)' is a preferable cultivar to mushroom farmers because it has a dark pileus and a thick stipe. But it is very sensitive to environmental conditions, so farmers demand an alternative cultivar of ‘Suhan’ continuously. To develop a new cultivar, parental strains ‘ASI 2504(Suhan)’ and ‘ASI 0665(Heuktari)’ were selected from P. ostreatus’s various collected strains according to morphological characteristics. P. ostreatus ‘Soltari’ was developed by the method of Di-Mono crossing between dikaryotic strain ‘Suhan’ and monokaryotic strain derived from ‘Heuktari’. Analysis of the mitochondrial genetic characteristics was performed for primary selection in 100 crossed strains. The mitochondrial DNA profile of ‘Soltari’ was same as that of ‘Heuktari’, when mitochondrial DNA primer MtPo1 was used. And a nuclear DNA profile of ‘Soltari’ was similar as those of the parental strains, ‘Suhan’ and ‘Heuktari’, when RAPD(Random Amplified Polymorphic DNA) primer URP 1, 3 were used. The optimum temperature for mycelial growth was 30°C for ‘Soltari’. ‘Soltari’ was appropriate for middle high temperature to grow, especially 13~18°C. Fruiting body production per bottle (1,100mL) was about 158.6g. When compared to the control strain ‘Suhan’, the stipe’s length and thickness of ‘Soltari’ were similar to those of ’Suhan’. But the pileus diameter of ‘Soltari’ was a little shorter than that of ‘Suhan’, the former was 42.72mm, while the latter was 51.33mm. And the pileus thickness of ‘Soltari’ and ‘Suhan’ were 18.18mm and 25.46mm, respectively. ‘Soltari’ was more resistant at high CO2 concentration than ‘Suhan’ and the color of pileus of ‘Soltari’ was kept dark gray at high temperature. Therefore, it is suggested that this new cultivar ‘Soltari’ be an alternative of ‘Suhan’ and contribute to energy saving effect in oyster mushroom farms.

The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, and known to play pivotal functions for the synthesis of proteins involved in cell proliferation and cell cycle control. Its nuclear localization has an important implication for the eIF5A functions in nucleus, but the evidence of the nuclear translocation is still in controversy. This study is aimed to elucidate the nuclear localization of eIF5A in the epithelial cells of oral leukoplakia by the immunohistochemistry using trypsin digestion to remove their cytoplasms. The keratinocytes of the acanthotic and basal cell layers in oral leukoplakia showed the complete removal of their cytoplasmic components, but the nuclei of those cell layers were remained on the microsection. The immunostainings using both polyclonal and monoclonal antibody against eIF5A showed the strong positive reaction in the nuclei remained after trypsin digestion. And the immunostaining was more intensely expressed in the nuclei of the basal and suprabasal keratinocytes than in the nuclei of the upper spinous keratinocytes. These data directly indicate the post-translationally modified eIF5A is abundantly localized in the nuclear matrix components including nucleoli, which are resistant to the trypsin digestion. It is also presumed that the nuclear eIF5A localized at the trypsin resistant nuclear matrix, i.e., histone and r ibosomal proteins, may be closely relevant to the control of mRNA production or to the nuclear-cytoplamic trafficking for mRNA transportation.

본 연구는 융합 전 수핵란의 활성화 처리 유무와 활성화 처리 시간이 소 체세포 유래 핵이식란의 체외 발육능에 미치는 영향을 검토하기 위해 수행하였다. 소 체외성숙란의 탈핵 후 체세포 핵을 이식하고 일부는 전기 융합 후 활성화를 유기하였고(pre-AC), 일부는 먼저 활성화 처리 후 융합을 실시(post-AC)하였다. 난자의 활성화는 Ca2+-ionophore(A23187)로 처리 후 DMAP로 4시간 배양하는 방법으로 유기하였다. 핵이식란은 CR1aa액에서 9일간 배양하여 발육율을 검토하였으며, 활성화 후 30분～2.5시간에 고정하여 confocal microscope 하에서 핵형 변화를 검사하였다. 배반포기까지 발육율은 post-AC구(20.6%)가 pre-AC(15.3%)보다 다소 높게 나타났다. 또한 활성화 처리를 하여 핵이식란의 배 발달을 비교한 결과 post-AC구가 더 늦은 배 발달 속도를 나타내었다. Post-AC구를 활성화 후 30분, 2시간, 4시간으로 나누어 융합하여 발육율을 검토한 결과 발육율에 차이가 없었다. 본 연구의 결과는 수핵란의 활성화 시간에 따라 핵이식란의 발육 및 핵형이 영향을 받을 수 있음을 시사한다.

본 연구는 한우 난포란이 체외성숙된 환경의 변화를 단백질 측면으로부터 검토하기 위하여 체외성숙 배지와 세포질내 단백질 변화와 종류를 검토하였다. 그 결과 배지 내의 단백질 발현량은 배양 4.5시간째까지 감소하였고, 배양 13.5시간째까지는 변화가 없었다. 그러나 배양 13.5~18시간 사이에 증가한 후 배양 18시간 이후에 다시 감소하는 경향이었다. 세포질내의 단백질 발현량은 배양 4.5시간째까지 증가하였고 배양 9시간째까지 급격히 감소하였다. 배양 9시간째부터 18시간째 까지는 단백질 발현량이 유사한 경향이었으나 배양 18시간째부터 24시간째까지 다시 증가하였다. 한편 체외성숙한 배지와 세포질을 2차원 전기영동하여 각각 298개 및 35개의 단백질 spot을 확인하였고, 그 중 배지에서는 28개, 세포질에서는 5개의 spot이 유의적인 변화를 확인하였다. 이들 spot 대한MALDI-TOP분석으로 배지와 세포질에서 각각 8개 및 1개의 단백질을 동정하였다. 그 종류는 aldose reductase, alpha enolase, apolipoprotein A-1 precursor, 43M1a collectin precursor, heat shock 27kDa protein, plasminogen activator inhibitor-1 precursor, thrombospondin 1 transitional endoplasmic reticulum ATPase 및 β-tubulin이었다.

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated Gphase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

Radish (Raphanus sativus L.) is a widely-consumed root vegetable that is grown worldwide. To utilize the radish genetic resources for breeding research, we collected radish germplasms and evaluated their morphological and genetical characteristics. Here, phylogenetic relationship of 288 accessions were analyzed using 16 SSR markers and classified cytoplasm male sterility (CMS) types using cpDNA-based molecular markers. To create a collection of 288 accessions, 188 and 73 accessions were selected from RDA-Genebank (Korea) and NIAS-Genebank (Japan), respectively, after generation advancement for the accessions with low uniformity. In addition, 27 elite lines currently used for commercial radish breeding programs were included. In the result of phylogenetic analysis, 288 accessions were clustered into 5 major groups corresponding to the morphological traits and origins at the similarity coefficient value of 0.51. Analysis of CMS types revealed that majority of accessions were determined as DBRMF1 and DBRMF2 mitotypes, 15 accessions to Ogura and 4 accessions to DCGMS mitotypes. Further genetic analysis for radish germplasm will be valuable in assisting radish f1 hybrid breeding.

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared to seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.

Cytoplasmic male sterility caused by DCGMS (Dongbu cytoplasmic and genic male-sterility) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome 3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group 2 co-segregated with male fertility in an F2 population. Using radish ESTs positioned in linkage group 2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely-linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome 3. Synteny between the radish and Arabidopsis genomes in the 985-kbp interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome 3.