Gene expression studies on developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene

Previous studies have identified two tissue- and cell-specific, yet functionally redundant, sucrose synthase (SuSy) genes, Sh1 and Sus1, which encode biochemically similar isozymes, SH1 and SUS1 (previously referred to as SS1 and SS2, respectively). Here we report evidence for a third SuSy gene in maize, Sus3, which is more similar to dicot than to monocot SuSys. RNA and/or protein blot analyses on developing kernels and other tissues show evidence of expression of Sus3, although at the lowest steady-state levels of the three SuSy gene products and without a unique pattern of tissue specificity. Immunoblots of sh1sus1-1 embryos that are either lacking or deficient for the embryo-specific SUS1 protein have shown a protein band which we attribute to the Sus3 gene, and may contribute to the residual enzyme activity seen in embryos of the double mutant. We also studied developing seeds of the double mutant sh1sus1-1, which is missing 99.5% of SuSy enzyme activity, for evidence of co-regulation of several genes of sugar metabolism. We found a significant reduction in the steady-state levels of Miniature-1 encoded cell wall invertase2, and Sucrose transporter (Sut) mRNAs in the double mutant, relative to the lineage-related sh1Sus1 and sh1Sus1 kernels. Down-regulation of the Mn1 gene was also reflected in significant reductions in cell wall invertase activity. Co-regulatory changes were not seen in the expression of Sucrose phosphate synthase, UDP-glucose pyrophosphorylase, and ADP-glucose pyrophosphorylase.     

Oxalate decarboxylase from Collybia velutipes. Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato

Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mechanism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5'-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548-23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants expressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotiorum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.     

Packing-induced conformational and functional changes in the subunits of alpha -crystallin

The heteroaggregate alpha-crystallin and homoaggregates of its subunits, alphaA- and alphaB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of alpha-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulation in vivo have not been elucidated. The subunits of alpha-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the alpha-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observed in vivo. We have investigated the time-dependent structural and functional changes during the course of heteroaggregate formation by the exchange of subunits between homoaggregates of alphaA- and alphaB-crystallins. Native isoelectric focusing was used to follow the time course of subunit exchange. Circular dichroism revealed large tertiary structural alterations in the subunits upon subunit exchange and packing into heteroaggregates, indicating specific homologous and heterologous interactions between the subunits. Subunit exchange also resulted in quaternary structural changes as demonstrated by gel filtration chromatography. Interestingly, we found time-dependent changes in chaperone-like activity against the dithiothreitol-induced aggregation of insulin, which correlated with subunit exchange and the resulting tertiary and quaternary structural changes. Heteroaggregates of varying subunit composition, as observed during eye lens epithelial cell differentiation, generated by subunit exchange displayed differential chaperone-like activity. It was possible to alter chaperone-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit exchange and the resulting structural and functional changes observed could constitute a mechanism of regulation of chaperone-like activity of alpha-crystallin (and possibly other mammalian sHSPs) in vivo.     

An enigmatic peptide ligation reaction: protease-catalyzed oligomerization of a native protein segment in neat aqueous solution

We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free alpha-carboxyl and alpha-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 degrees C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase."     

Checkpoint-dependent activation of mutagenic repair in Saccharomyces cerevisiae pol3-01 mutants

The Saccharomyces cerevisiae DNA polymerase delta proofreading exonuclease-defective mutation pol3-01 is known to cause high rates of accumulating mutations. The pol3-01 mutant was found to have abnormal cell cycle progression due to activation of the S phase checkpoint. Inactivation of the S phase checkpoint suppressed both the pol3-01 cell cycle progression defect and mutator phenotype, indicating that the pol3-01 mutator phenotype was dependent on the S phase damage checkpoint pathway. Epistasis analysis suggested that a portion of the pol3-01 mutator phenotype involves members of the RAD6 epistasis group that function in both error-free and error-prone repair. These results indicate that activation of a checkpoint in response to certain types of replicative defects can result in the accumulation of mutations.     

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.