CA repeats in the 3'-untranslated region of bcl-2 mRNA mediate constitutive decay of bcl-2 mRNA

An AU-rich element (ARE) in the 3'-untranslated region (UTR) of bcl-2 mRNA has previously been shown to be responsible for destabilizing bcl-2 mRNA during apoptosis through increasing AUF1 binding. In the present study, we investigated the effect of the region upstream of the ARE on bcl-2 mRNA stability using serial deletion constructs of the 3'-UTR of bcl-2. Deletion of 30 nucleotides mostly consisting of the CA repeats, located upstream of the ARE, resulted in the stabilization of bcl-2 mRNA abundance, in the absence or presence of the ARE. The specificity of the CA repeats in terms of destabilizing bcl-2 mRNA was proven by the substituting the CA repeats with other alternative repeats of purine/pyrimidine, but this had no effect on the stability of bcl-2 mRNA. CA repeats alone, however, failed to confer instability to bcl-2 or gfp reporter mRNAs, indicating a requirement for additional sequences in the upstream region of the 3'-UTR. Serial deletion and replacement of a part of the region upstream of the CA repeats revealed that the entire 131-nucleotide upstream region is an essential prerequisite for the CA repeat-dependent destabilization of bcl-2 mRNA. Unlike the ARE, CA repeat-mediated degradation of bcl-2 mRNA was not accelerated upon apoptotic stimulus. Moreover, the upstream sequences and CA repeats are conserved among mammals. Collectively, CA repeats contribute to the constitutive decay of bcl-2 mRNA in the steady states, thereby maintaining appropriate bcl-2 levels in mammalian cells.     

Distinctive expression of Myf5 in relation to differentiation and plasticity of newt muscle cells

Regeneration in urodele amphibians such as the newt reflects the local plasticity of differentiated cells. Newt myotubes and myofibres undergo S phase re-entry and cellularisation in the limb blastema, and we have analysed the regulation of Myf5 in relation to these events. Surprisingly, Myf5 was expressed after fusion in cultured newt myotubes and in myofibers of the adult limb, in contrast to its familiar expression in myoblasts in other vertebrates. Its expression was markedly down regulated in cultured newt myotubes after S phase re-entry induced by serum stimulation, as well as by exposure to the trisubstituted purine called myoseverin which induces cellularisation. We have attempted to relate this striking difference from other vertebrates to the requirement for multinucleate urodele muscle cells to contribute to the regeneration blastema.     

Genetic structure of korean wild populations of the Medaka Oryzias latipes inferred from allozymic variation

Previous allozymic studies have revealed that Korean wild populations of Oryzias latipes have differentiated regionally, and are composed of two distinct groups, the East Korean Population and the China-West Korean Population. Recently, mitochondrial DNA (mtDNA) sequencing and restriction fragment length polymorphism (RFLP) analyses have confirmed these two groups, and shown that the distribution ranges of the two groups overlap in western Korea. In order to describe the detailed distributions of the two groups and the gene flow between them, genotypes of 13 allozymic loci were determined in 444 specimens from 96 localities in Korea. The two major groups were supported by remarkable allele frequency differences at six diagnostic loci: ACP*, AMY*, CK-A*, LDH-A*, PGM* and TF*. Individuals with the typical "eastern" genotype were mainly distributed in eastern and southern areas. In contrast, fish with the "western" genotype were predominant in the western area, and were further divided into two subgroups (the Han River and Geum River Subpopulations) by unique alleles at the ADH* locus. In the western coast, two distinct (eastern and western) genotypes were distributed in a mosaic fashion. This distribution pattern was identical to those from mtDNA analyses. Although the distribution patterns of the alleles at three loci (GPI-A*, LDH-C* and SOD*) showed introgressive conditions between the two groups, each population was nearly fixed as either the eastern or western genotype at all six diagnostic loci despite the proximity among samples. Therefore, it is suggested that some reproductive isolation mechanisms exist between the two groups in natural habitats.     

Expression of the hepatitis B surface S and preS2 antigens in tubers of <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

In an attempt to develop an edible vaccine, we transformed a recombinant hepatitis B virus (HBV) gene encoding the middle protein of HBV that contains the surface S and preS2 antigen into potato by Agrobacterium-mediated transformation. The HBV gene was under control of either the CaMV 35S promoter, the double 35S promoter with the AlMV 5 non-translated leader sequence, or the tuber-specific patatin promoter. HBV mRNA levels were higher with the 35S promoter than with the double 35S and patatin promoters; however, the levels of the S and preS2 antigen in the transformed tubers were higher with the patatin promoter than with the CaMV 35S and double promoters. The levels of preS2 antigen produced are the highest reported to date. Transgenic potato tubers were fed to mice, and the mice showed an immune response against the HBV S antigen.     

Differentially up-regulated genes in proliferating porcine neonatal pancreas cells caused by epidermal growth factor

Pancreatic duct cells are considered to be a major source for beta-cell regeneration or neogenesis. Although epidermal growth factor (EGF) is a well-known important growth factor for pancreas development, the control of pancreatic duct cell growth and differentiation by EGF is poorly understood. In this study, we focused on identifying the genes that were differentially up-regulated in response to EGF stimulation using monolayer cultured porcine neonatal pancreas cells. Cells were obtained from 1 to 3 day old pigs, dispersed and cultured for 8 days. Monolayer cultured porcine pancreas cells were comprised of duct cells and some endocrine and mesenchymal cells (75.2 +/- 15.1, 19.6 +/- 4.9, and 9.5 +/- 3.1%, respectively). After 16 h in serum free media, cells were treated with 100 microg/L EGF for 24 h. Differentially expressed genes were screened by subtractive hybridization. (3)H-thymidine uptake was significantly increased by EGF with time (untreated vs. 24 h treated, untreated vs. 48 h treated: 305.5 +/- 3.5 cpm vs. 380.3 +/- 17.3 cpm (P < 0.05), 309.2 +/- 4.51 vs. 929 +/- 9.19 cpm, (P < 0.005), respectively). Three hundred and fifty cDNA clones were obtained by subtractive hybridization and the inserts were confirmed in 161 colonies and then sequenced. Finally, we found increased mRNA expression of five unknown and five known genes, including cytochrome c oxidase subunit I (COI), cyclooxygenase-2 (COX-2), matrix metalloproteinase-13 (MMP-13), Wiskott-Aldrich syndrome protein interacting protein (WASPIP), and hyaluronan synthase-2 (HAS-2). We confirmed the up-regulation of these genes by Northern blot and semi-quantitative RT-PCR at various time points. The present findings opened new targets for the research on the mechanisms of pancreatic duct cell proliferation by EGF.     

Identification, evolution, and regulation of expression of Guinea pig trappin with an unusually long transglutaminase substrate domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.