Development of a heat shock inducible gfp transgenic zebrafish line by using the zebrafish hsp27 promoter

In the present study, a zebrafish hsp27 promoter was isolated and used to develop heat shock inducible gfp transgenic zebrafish. The endogenous hsp27 mRNAs were constitutively expressed from 4 hpf and increased in several regions of brain, heart and somites in early embryogenesis until 24 hpf. Subsequently, the expression was reduced significantly but maintained in the heart and ears. Heat shock induced hsp27 mRNAs in the blastoderm from 6 hpf and later in somites, branchial arches and several regions of brain. Similarly in hsp27-gfp transgenic zebrafish, constitutive GFP expression was observed from 11 hpf. GFP expression was mainly in the skin cells and increased to the peak level at 7 dpf, followed by a reduction. The constitutive GFP expression in the heart was initiated from 50 hpf and maintained even in the adult fish. After heat shock, GFP expression was mainly induced in the muscle in addition to a mild increase in the skin and heart. The early stages of the embryos were more sensitive than late stages as the time required for induced GFP expression in the muscle is shorter. Thus, the hsp27-gfp transgenic line generally recapitulates the expression pattern and heat shock inducibility of endogenous hsp27 RNAs. We also tested the potential of using the hsp27-gfp transgenic zebrafish embryos for heavy metal induction and demonstrated the inducibility of GFP expression by arsenic; this pattern of induction was also supported by examination of endogenous hsp27 mRNA.     

Analysis of an ABA-responsive rice gene promoter in transgenic tobacco

We have analyzed in transgenic tobacco the expression of a chimeric gene containing 5 sequences of the rice rab-16B gene fused to the -glucuronidase (GUS) reporter gene. This construct, a translational fusion (–482 to +184) including 14 amino acids of the RAB-16B protein, is expressed only in zygotic and pollen-derived embryos. In zygotic embryos, GUS activity begins to accumulate 10 days after flowering (daf), and increases until seed maturation at 25 daf. Immunological measurements of endogenous abscisic acid (ABA) accumulation in these seeds showed a close parallel between hormone levels and GUS activity. However, GUS activity could not be reproducibly induced by treatment of immature embryos with ABA (10 M). Neither GUS activity nor GUS mRNA could be detected in leaves of transgenic tobacco even after ABA treatment. In contrast, GUS activity could be induced to high levels in pollen-derived embryos by treatment with ABA. Our results show that 482 bp of 5 sequences of the rice rab-16B promoter can confer in transgenic tobacco developmentally regulated expression in embryos but not ABA-responsive expression in vegetative tissues.     

Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: localization and possible functional implications

We isolated and sequenced Ha hsp 17.9, a DNA complementary (cDNA) of dry-seed stored mRNA that encodes a low-molecular-weight heat-shock protein (LMW HSP). Sequence analysis identified Ha hsp17.9, and the previously reported Ha hsp17.6, as cDNAs encoding proteins (HSP17.6 and HSP17.9) which belong to different families of cytoplasmic LMW HSPs. Using specific antibodies we observed differential expression of both proteins during zygotic embryogenesis under controlled environment, and a remarkable persistence of these LMW HSPs during germination. Immuno-blot analysis of HSP17.9 proteins in two-dimensional gels revealed that the polypeptides expressed in embryos were indistinguishable from LMW HSPs expressed in vegetative tissues in response to water deficit; but they appeared different from homologeous proteins expressed in response to thermal-stress. Tissue-print immunolocalization experiments showed that HSP17.9 and HSP17.6 were homogeneously distributed in every tissue of desiccation-tolerant dry seeds and young seedlings under non-stress conditions. These results demonstrate developmental regulation of specific, cytoplasmic, plant LMW HSPs, suggesting also their involvement in water-stress tolerance.     

Analysis of Metal-Regulated Metallothionein and Heat Shock Gene Expression in HeLa-Derived Cadmium-Resistant Cells

The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd²⁺and Zn²⁺exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd²⁺-exposed H454 cells at levels comparable to the ones present in Cd²⁺-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes.