Application of ferric sludge to immobilize leachable mercury in soils and concrete.

A Hg-contaminated site in B.C. Province, Canada was caused by the previous operation of Hg-cell in chlor-alkali process for over 25 years. The soils and groundwater at the site are highly contaminated with mercury. An analysis of groundwater at the site has shown that most of the mercury is bonded with humic and fulvic acids (HFA) in colloidal form. The Hg-HFA colloids can be completely removed from the groundwater with ferric chloride treatment under optimized process conditions to form ferric sludge (FS), which is rendered non-leachable by standard TCLP (Toxicity Characteristic Leaching Procedure) test. The effluent discharged from a clarifier has achieved mercury levels of < 0.5 microkg l(-1). The studies of mercury adsorption characteristics of FS show it has low mercury leachability by TCLP, and great mercury adsorption capability. This feature is the basis for the application of FS to immobilization of leachable Hg-contaminants in solid wastes. Full-scale stabilization tests of Hg-contaminated soil have been carried out, and the time-based stability of the treated soil has been monitored by TCLP over a period of 60 days. All the results have shown a small variation in TCLP mercury levels within a range of 10-40 microg l(-1). Based on these results and with the approval of the B.C. Ministry of the Environment, 1850 tons of Hg-contaminated soils and 260 tons of Hg-contaminated concrete fines have been treated, stabilized with FS, and disposed in a non-hazardous waste disposal site.

A homologue of the 65 kDa regulatory subunit of protein phosphatase 2A in early pea (Pisum sativum L.) embryos.

A partial cDNA, isolated from an early developing pea (Pisum sativum L.) embryo library, was found to encode a plant homologue of the regulatory subunit (PR65) of protein phosphatase 2A (PP2A). Comparison of the deduced amino acid sequence with a human PR65 sequence showed that the regulatory subunit of PP2A has been highly conserved during evolution. Southern analysis demonstrated that in pea and rape the catalytic and regulatory subunits of PP2A are encoded by multigene families. The levels of the transcripts encoding each subunit are developmentally regulated during pea embryogenesis and expression of the regulatory subunit is not solely restricted to the embryo.

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo.

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequences on two cyanogen bromide fragments were determined. An oligonucleotide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3-5-day old seedling hypocotyls, in lambda ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539-1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7-15 per haploid genome).

Insect pest control by copying nature using genetically engineered crops.

A 'copy Nature' strategy for insect pest control is presented which aims to be relatively sustainable and environmentally friendly. Many higher plant genes encoding insecticidal proteins exist in Nature which can be expressed in transgenic plants in a tissue or development-specific manner, or in response to environmental stimuli. These genes can either be expressed singly or in combination so as to enhance host resistance to insect pests. The results so far, which have been obtained mainly in the growth-room, are discussed in both a scientific and applied context. The feasibility of this technology, either as a partial substitution technology for synthetic chemicals or as a component in IPM systems, now needs to be evaluated in the farmer's field. If proven there, its long-term use may depend on promoting agronomic and farm management which minimizes the build-up of resistance in insect populations.

A complete sequence of the rice sucrose synthase-1 (RSs1) gene.

Using a fragment of the maize sucrose synthase gene Sh-1 as probe, the rice genome was shown to contain at least three genes encoding sucrose synthase. One of these genes was isolated from a genomic library, and its full sequence, including 1.7 kb of 5' flanking sequence and 0.9 kb of 3' flanking sequence, is reported. The new rice gene, designated RSs1, is highly homologous to maize Sh-1 (approx. 94% identity in derived amino acid sequence), and contains an identical intron-exon structure (16 exons and 15 introns). Both RSs1 and maize Sh-1 show similar sequence homologies to a second rice sucrose synthase gene described recently (designated RSs2, Yu et al. (1992) Plant Mol Biol 18: 139-142), although both the rice genes predict an extra 6 amino acids at the C-terminus of the protein when compared to the maize gene. The RSs1 5' flanking sequence contains a number of promoter-like sequences, including putative protein-binding regions similar to maize zein genes.

Partial sequences of 18S ribosomal RNA of two genera from each of six flowering plant families.

Partial nucleotide sequences of 18S ribosomal RNA from two genera of each of six families of flowering plants were analysed using parsimony programmes. The results are discussed with reference to their usefulness in plant phylogenetic studies.

Pea lectin is correctly processed, stable and active in leaves of transgenic potato plants.

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating alpha and beta subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.

The extensin gene family in oilseed rape (Brassica napus L.): characterisation of sequences of representative members of the family.

A family of cross-hybridising cDNA clones has been isolated from a cDNA library produced with poly(A)+ RNA from the roots of oilseed-rape (Brassica napus L.). The clones were selected as abundantly expressed in root by differential screening of the root cDNA library with cDNA probes prepared from root, green leaf, etiolated leaf and developing seed. mRNA species corresponding to the selected abundant clones were expressed in roots at levels of at least 400 times those in other organs, as shown by Northern blot analysis and RNase protection assays. Complete nucleotide sequence determination of the cDNA clones showed that they encoded proteins homologous to carrot extensin and were the products of at least three different genes. An extensin gene, designated extA, was obtained from an oilseed rape (B. napus L.) genomic library screened with a cDNA species encoding a protein expressed abundantly in roots. The gene is a member of a multigene family, consisting of about 3 members per haploid genome with strong homology to the probe, and a further 20 or so members with weaker homology. The isolated gene, although not identical to the cDNA probe, was also found to be specifically expressed in roots, and was transcribed into a mRNA species approximately 1,300 nucleotides in size. A single transcription start was identified by S1 mapping. The complete nucleotide sequence of the extA gene and its flanking regions has been determined and shown to encode a protein homologous to carrot and tomato extensins.

A cDNA encoding an S-locus specific glycoprotein from Brassica oleracea plants containing the S5 self-incompatibility allele.

A cDNA sequence homologous to the Brassica self-incompatibility locus specific glycoprotein (SLSG) sequence was isolated from stigmas of B. oleracea plants homozygous for the S5 allele. The nucleotide sequence of this cDNA was obtained and compared with the S6 allelic form of the SLSG. Evidence is presented which indicates that this sequence does not specify the self-incompatibility response of pollen.

Protein and cDNA sequences of Bowman-Birk protease inhibitors from the cowpea (Vigna unguiculata Walp.).

The protein and gene sequences of the cowpea Bowman-Birk type trypsin inhibitor which confers enhanced insect resistance to transgenic tobacco plants, and of cowpea trypsin/chymotrypsin inhibitors are presented. There are regions of high conservation and high divergence within the 5' leader, mature protein and 3' non-coding regions of the Bowman-Birk inhibitors and in the genes which encode them in different members of this family within the Leguminosae. The practical implications of this finding for studies on the evolution of plants and the utilization of these genes for enhancing insect resistance is discussed.

Use of cowpea trypsin inhibitor (CpTI) to protect plants against insect predation.

Transgenic tobacco plants expressing a cowpea trypsin inhibitor gene have enhanced levels of insect resistance to a variety of insect pests. Furthermore, insect bioassay has shown the cowpea trypsin inhibitor to have anti-metabolic activity to insect pests of the orders Lepidoptera, Coleoptera and Orthoptera. The advantages and disadvantages of this approach to developing insect resistant crops is discussed in relationship to other methods, including conventional plant breeding and chemical control. Eventually it is hoped that African farmers will benefit from this industrially sponsored research.

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco.

Maturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumin-type genes of several legume species. Constructs containing -833 and -1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions -97 and -549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions -549 and -1203 bp appear to function as enhancer-like elements, to increase expression.

Transcriptional and posttranscriptional regulation of seed storage-protein gene expression in pea (Pisum sativum L.).

At least three classes of legumin, encoded by the gene families legA, legJ and legS, and a lectin, encoded by a single gene, accumulate in the developing cotyledons of Pisum sativum L. Transcription rates for the genes encoding these proteins were measured in nuclei isolated from cotyledons at 12 and 16 days after flowering (DAF). The steady-state levels of the corresponding mRNA species were also measured in absolute terms throughout cotyledon development, from 8-9 to 28 DAF. When transcription rates and steady-state mRNA levels of the different gene families are compared, there is little correlation. This indicates a posttranscriptional regulation of the level of expression of these storage proteins in the developing cotyledons. Expression of the legumin genes is known to be seed-specific, whereas expression of the lectin gene is found in both seed and root. When transcription rates were measured in leaf nuclei the levels of legumin and lectin transcripts detected approached background levels, indicating that these genes are either inactive or transcribed at very low levels in leaves; however, the rate of transcription of the chlorophyll a/b-binding protein gene was high. This points to transcriptional control as the major factor in the organ-specificity of legumin and lectin expression.

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.

Tissue-specific expression of a pea legumin gene in seeds of Nicotiana plumbaginifolia.

A 3.4-kilobase genomic DNA fragment from Pisum sativum L. containing the LegA gene, which encodes a major legumin storage protein, was transferred to Nicotiana plumbaginifolia using an Agrobacterium tumefaciens strain containing the Bin 19 binary vector system. Northern hybridisation analysis of legA-transformed plants demonstrated that legumin-specific RNA was present in developing seeds but not in developing leaves. Legumin protein was immunologically detected in the mature seeds of legA-transformed plants, and was present as the correct-size protein composed of disulphide-bonded polypeptides. It is concluded that the transferred pea genomic fragment contains all the information necessary for seed-specific expression of the legA gene, and for correct processing of the primary transcript and the precursor legumin protein.

Nuclease sensitivity of storage-protein genes in isolated nuclei of pea seeds.

The chromatin structure of legumin genes in nuclei isolated from leaves and cytyledons of pea (Pisum sativum L.) was investigated. Digestions with micrococcal nuclease (EC 3.1.31.1.) showed that the nucleosomal repeat length of total chromatin (171±25 base pairs) was similar in nuclei from both tissues. The sensitivity to pancreatic deoxyribonuclease (DNase I; EC 3.1.21.1.) of the legumin genes in cotyledon nuclei was greater than that in leaf nuclei; this increase in sensitivity correlated with transcriptional activity of the genes. No DNase-I-hypersensitive sites were detected in these genes in either tissue.

Regulation of storage-protein synthesis in pea (Pisum sativum L.) cotyledons under conditions of sulphur deficiency.

The effects of sulphur deficiency on the expression of storage-protein genes in developing pea (Pisum sativum) cotyledons were studied. Legumin-gene transcription was decreased by S-deficiency, but not to the same extent as the decrease in the level of legumin mRNA. Vicilin-gene transcription was not significantly affected. Control of gene expression may thus occur during transcription and/or post-transcriptional events.

The 5'-flanking regions of three pea legumin genes: comparison of the DNA sequences.

Approximately 1200 nucleotides of sequence data from the promoter and 5'-flanking regions of each of three pea (Pisum sativum L.) legumin genes (legA, legB and legC) are presented. The promoter regions of all three genes were found to be identical including the "TATA box", and "CAAT box', and sequences showing homology to the SV40 enhancers. The legA sequence begins to diverge from the others about 300bp from the start codon, whereas the other two genes remain identical for another 550bp. The regions of partial homology exhibit deletions or insertions and some short, comparatively well conserved sequences. The significance of these features is discussed in terms of evolutionary mechanisms and their possible functional roles. The legC gene contains a region that may potentially form either of two mutually exclusive stem-loop structures, one of which has a stem 42bp long, which suggests that it could be fairly stable. We suggest that a mechanism of switching between such alternative structures may play some role in gene control or may represent the insertion of a transposable element.