Benito O. de Lumen

A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells

A soybean cDNA encoding the small subunit peptide of a cotyledon-specific 2S albumin (Gm2S-1) is thought to play a role in arresting mitosis during the DNA endoreduplication and cell expansion phase of seed development. The peptide (termed lunasin) contains the cell adhesion motif Arg-Gly-Asp (RGD) followed by eight aspartic acid residues at its C-terminal end. A chimeric gene encoding the lunasin peptide tagged with green fluorescent protein (GFP) arrested cell division, caused abnormal spindle fiber elongation, chromosomal fragmentation, and cell lysis when transiently transfected into murine embryo fibroblast, murine hepatoma, and human breast cancer cells. Deletion of the polyaspartyl end abolished the antimitotic effect. Subcellular localization of lunasin and immunobinding assay using synthetic peptides revealed the preferential adherence of lunasin to chromatin. Immunofluorescence showed that kinetochore proteins were displaced from the centromere in lunasin-transfected cells. These observations suggest that lunasin binds to the chromatin, leading to disruption of kinetochore formation and inhibition of mitosis.

Galactinol synthase (GS): increased enzyme activity and levels of mRNA due to cold and desiccation

Galactinol synthase (GS) catalyzes the first committed step in the biosynthesis of the raffinose family oligosaccharides (RFO) and could play a key regulatory role in the carbon partitioning between sucrose and RFO in the developing seed. RFO have been proposed to be osmoprotectants when plants are exposed to environmental water deficit stresses, such as cold and desiccation, and during the developmentally induced desiccation in the late maturation stage of seed development. We present the following evidences obtained from three plant species in support of these proposed roles for GS and RFO: (a) GS activity increased in kidney bean seeds upon exposure of plants to cold; (b) GS mRNA levels increased significantly upon cold exposure in the vegetative tissues of Arabidopsis thaliana, a chill resistant species, with the transcripts diminishing upon re-exposure to room temperature. A smaller increase due to cold was also observed in the siliques with the transcripts likewise disappearing upon re-exposure of the plants to room temperature; (c) we established by protein sequence comparison that a previously unidentified gene belonging to a group of ABA-independent, desiccation stress inducible genes isolated from rice with acquired chill resistance, encodes the rice homologue of the GS gene.

Identification and isolation of methionine-cysteine rich proteins in soybean seed.

We recently developed a method to identify methionine-containing proteins and quantitate their methionine contents [4]. We applied this method to soybeans and identified relatively methionine-rich proteins (MRP) among the albumins. By acidic methanol extraction of the albumins, we obtained a group of low molecular weight methionine-cysteine rich proteins (MCRP) that analyzed 4.0% methionine and 8.8% cysteine. MCRP made up 1–2% of the total protein in soybeans. Reversed-phase HPLC purification of MCRP yielded a protein peak that exhibited a single major band on denaturing polyacrylamide gel electrophoresis, had a molecular weight of 16kD and contained 6.2% methionine and 18.8% cysteine. We are cloning the gene for this protein. Increasing its level through genetic engineering could increase the methionine-cysteine content of soybeans.We recently developed a method to identify methionine-containing proteins and quantitate their methionine contents [4]. We applied this method to soybeans and identified relatively methionine-rich proteins (MRP) among the albumins. By acidic methanol extraction of the albumins, we obtained a group of low molecular weight methionine-cysteine rich proteins (MCRP) that analyzed 4.0% methionine and 8.8% cysteine. MCRP made up 1–2% of the total protein in soybeans. Reversed-phase HPLC purification of MCRP yielded a protein peak that exhibited a single major band on denaturing polyacrylamide gel electrophoresis, had a molecular weight of 16kD and contained 6.2% methionine and 18.8% cysteine. We are cloning the gene for this protein. Increasing its level through genetic engineering could increase the methionine-cysteine content of soybeans.

Cloning and expression of rice (Oryza sativa) sucrose synthase 1 (RSs1) in developing seed endosperm

Abstract A previously cloned cDNA to rice ( Oryza sativa L.) sucrose synthase 1 ( RSs1 ) was used to determine spatial expression of the gene in rice tissues and temporal expression in developing rice endosperm. RSs1 was expressed predominantly in the endosperm of milky stage rice seeds with maximum expression at 3–5 days after pollination which were 8–10-fold over leaf levels. The peak in RSs1 transcript levels preceded the peak of enzyme activity which occured 9–11 days after pollination. RSs1 transcript and activity levels were analyzed in two starch deficient mutants of rice to determine if the lesion in these mutants resides at the locus for RSs1 . No correlation between sucrose synthase activity and starch biosynthesis was seen in these mutants, although slight elevations of RSs1 transcript levels were observed.

Methionine-containing proteins in twoPhaseolus vulgaris cultivars with different methionine bioavailabilities

The full utilization of legumes as human food is limited by a deficiency of sulfur amino acids, low protein digestibility, low methionine bioavailability and the presence of anti-nutritional factors. A new cultivar ofPhaseolus vulgaris (Carioca 80) has 56.8% available methionine, compared with 29.3% availability in the parent cultivar Carioca. The total methionine content, denaturing gel electrophoretic patterns of methionine-containing proteins, and the percentage of phaseolins (the major storage proteins in Phaseolus) relative to the total protein are similar in the two cultivars. Although the digestibility of the two cultivars is similar, the increased biological value of Carioca 80 may indicate that there are differences in overall bean composition that affect protein hydrolysis and utilization. We suggest the tentative explanation that this is due to differences in the distribution of methionine in the methionine-containing proteins of the two cultivars.

Generation of anNco I restriction site for translational fusions using PCR-mediated, site-directed mutagenesis

A polymerase chain reaction-based method of site-directed mutagenesis was used to introduce anNco I restriction site on the translation start site of a tomato peroxidase gene. This quick and efficient method utilized two overlapping synthetic oligonucleotide primers containing the requisite base pair changes on the ATG translation start site and two flanking primers in PCR. The resulting DNA amplified fragments were fused together byNco I digestion at the mutated ends followed by a T4 ligation reaction. A rapid alternative method utilizing the overlapping fragments and the flanking primers in PCR can also be used for ligating the two fragments. Cloning and sequencing of the PCR-amplified fragments provided additional evidence for the presence of the site-specific mutations. Unique restriction sites upstream and downstream of the site-specific mutation allows for the easy transfer of this mutated region into the wild type peroxidase gene.

Test paper for detection of lipoxygenase.

Abstract A quick, simple, and specific test for lipoxygenase was developed. Filter paper impregnated with linoleic acid and N,N′-dimethyl-p-phenylenediamine gave a blue color as a positive test. The specificity of the test was established by: use of inhibitors of lipoxygenase and heme-catalyzed peroxidation, use of nonsubstrates, use of inactivated enzymes, proportionality of color intensity to amount of enzyme applied, and use of nonoptimum pH. Heattreated tomato samples showed decreasing color intensities with increasing temperatures. The technique should prove useful as a quality control tool in the quick detection of lipoxygenase during processing and especially in in situ localization of the enzyme in fruits and vegetables.