The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms

Abscisic acid (ABA) induces the expression of a battery of genes in mediating plant responses to environmental stresses. Here we report one of the early ABA-inducible genes in barley (Hordeum vulgare L.), HVA22, which shares little homology with other ABA-responsive genes such as LEA (late embryogenesis-abundant) and RAB (responsive to ABA) genes. In grains, the expression of HVA22 gene appears to be correlated with the dormancy status. The level of HVA22 mRNA increases during grain development, and declines to an undetectable level within 12 h after imbibition of non-dormant grains. In contrast, the HVA22 mRNA level remains high in dormant grains even after five days of imbibition. Treatment of dormant grains with gibberellin (GA) effectively breaks dormancy with a concomitant decline of the level of HVA22 mRNA. The expression of HVA22 appears to be tissue-specific with the level of its mRNA readily detectable in aleurone layers and embryos, yet undetectable in the starchy endosperm. The expression of HVA22 in vegetative tissues can be induced by ABA and environmental stresses, such as cold and drought. Apparent homologues of this barley gene are found in phylogenetically divergent eukaryotic organisms, including cereals, Arabidopsis, Caenorhabitis elegans, man, mouse and yeast, but not in any prokaryotes. Interestingly, similar to barley HVA22, the yeast homologue is also stress-inducible. These observations suggest that the HVA22 and its homologues encode a highly conserved stress-inducible protein which may play an important role in protecting cells from damage under stress conditions in many eukaryotic organisms.     

Patterns of selection: stress resistance and energy storage in density-dependent populations of Drosophila melanogaster

Populations of Drosophila melanogaster subjected to extreme larval (CU) or adult (UC) densities for multiple generations were assayed for a variety of life history characters. When reared under either crowded or uncrowded larval conditions, populations which had been selected to tolerate the limitation of resources imposed by extreme larval (CU) crowding, exhibited greater starvation resistance and lipid content than did populations which do not routinely undergo larval density-dependent regulation. Previous studies have shown that the CU populations do not show a correlated increase in longevity; as has been generally observed for these characteristics in age-structured populations of D. melanogaster. This suggests that density-dependent natural selection may not always shape life histories of the same characteristic in the same direction that age-specific selection does.     

Mechanistic studies on the inhibitory action of dietary dibenzoylmethane, a beta-diketone analogue of curcumin, on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis

Dietary factors play important roles in the carcinogenic process. The results of epidemiological data and some laboratory animal studies indicate that certain naturally occurring and synthetic components are able to block the carcinogenic process and inhibit the development of certain cancers. Dibenzoylmethane (DBM), a curcumin-related beta-diketone analogue has been reported to exhibit a remarkable inhibitory effect on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in Sencar mice. The present study investigated the possible mechanisms of inhibitory action of DBM on DMBA-induced mammary tumorigenesis in mice. The summarized results indicate that: (1) in in-vitro studies. DBM inhibited DMBA metabolism and the formation of DMBA-DNA adducts in a dose-dependent manner; (2) in the assay of competitive binding to estrogen receptors with [3H]-estradiol in vitro, DBM showed weak binding affinity; (3) in vivo, feeding of 1% DBM in the diet of immature Sencar mice for 4 -5 weeks decreased the uterine and parametrial fat pad weights, and lowered the serum estrogen and triglyceride levels. This study provides insight into the mechanisms involved in the inhibitory action of DBM in mouse mammary tumorigenesis.     

Importance of homodimerization for the in vivo function of yeast RNA triphosphatase

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.     

Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced. The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R. (1996) Genes Dev. 10, 2769-2781). On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements. GAL80 promoter region nucleosomes contain no intrinsically bent DNA.     

Cloning, expression, and complementation test of the RNA lariat debranching enzyme cDNA from mouse

The RNA lariat debranching enzyme of mouse (mDBR1) was cloned by screening a NIH/3T3 cDNA library. The sequence of full-length mDBR1 cDNA contained a single 515 amino acid open reading frame of 58 kDa protein. Comparison of the amino acid sequence of mDBR1 to other DBR proteins showed 40%, 44%, 43%, 42%, and 80% identity to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human debranching enzymes, respectively. The mDBR1 cDNA was shown to be functional in an interspecies specific complementation experiment, and an in vitro debranching enzyme assay. Mouse DBR1 could complement the intron accumulation phenotype of a S. cerevisiae dbrl null mutant strain. However, the level of complementation depended on the copy number of the mDBR1 cDNA. The integration of the mDBR1 cDNA in the chromosome of S. pombe also complemented both intron accumulation and slow growth phenotypes of the S. pombe dbr1 knock-out mutant strain.     

Involvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells

Age-related tissue Zn deficiency may contribute to neuronal and glial cell death by apoptosis in Alzheimer's dementia. To investigate this, we studied the effects of increasing or decreasing the levels of intracellular labile Zn on apoptosis of human neuroblastoma BE(2)-C cells in vitro. BE(2)-C cells were primed for 18 h with butyrate (1 mM) before addition of staurosporine (1 microM), an effector enzyme of apoptosis, for a further 3 h to induce DEVD-caspase activity. An increase in intracellular Zn using Zn ionophore pyrithione suppressed DEVD-caspase activity, while a decrease in intracellular Zn induced by Zn chelator TPEN mimicked staurosporine by activating DEVD-caspase in butyrate-primed cells. The distribution of intracellular Zn in the cells was demonstrated with the UV-excitable Zn-specific fluorophore Zinquin. Confocal images showed distinct cytoplasmic and cytoskeletal fluorescence. We propose that Zn decreases the level of apoptosis in neuronal cells exposed to toxins, possibly by stabilizing their cytoskeleton.