Ronald R. D. Croy

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

SummaryMaturing 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 legumintype 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.

Characterisation of the storage protein subunits synthesised in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.) : II. Vicilin.

Evidence is presented to show that legumin, the major storage protein in Pisum, is synthesised in vitro by the wheat germ and reticulocyte lysate systems, from polyribosomes and mRNA isolated from developing pea seeds. While legumin isolated from mature pea seeds consists of 40,000 and 20,000 MW subunits, the in vitro legumin is synthesised as a 60,000 MW precursor consisting of covalently linked 40,000 and 20,000 MW subunits. The implications of these findings are discussed in relationship to studies with other systems.

A gene from pea (Pisum sativum L.) with homology to metallothionein genes

While searching for ‘organ‐specific’ genes in pea (Pisum sativum L.) we have isolated a gene (designated PsMT A) which has an ORF encoding a predicted protein with some similarity to metallothioneins (MTs). The PsMT A transcript is abundant in roots which have not been exposed to elevated concentrations of trace metals.

Prokaryotic Metallothionein Gene Characterization and Expression: Chromosome Crawling by Ligation-Mediated PCR

A strategy is described for the characterization of a novel gene employing the polymerase chain reaction, inosine-containing oligonucleotide primers, and ligation mediated, or anchored polymerase chain reaction (APCR). The same primers, designed from the known protein sequence, are used to amplify the coding region of the gene and subsequently to ‘chromosome crawl’ by APCR. This strategy was applied to the characterization of a prokaryotic metallothionein gene, designated smtA, from the cyanobacterium Synechococcus PCC 6301 ( ═ Anacystis nidulans). The abundance of smtA transcripts was examined in extracts from cells exposed to heat shock and elevated concentrations of cadmium, zinc and copper ions. There was no detectable change in smtA transcript abundance following exposure to heat shock, while exposure to all three metal ions led to an increase in abundance. A smtA homologue was also identified in Synechococcus PCC 7942 ( ═ Anacystis nidulans R2).

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.

Transformation of Brassica napus L. (oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes - a comparison.

Abstract We have produced transgenic oilseed rape plants by two different transformation strategies. The first method utilised an A. rhizogenes binary system comprising of p Ri1855 and p Bin19 plasmids, to induce hairy root proliferation at the cotyledonary nodes. Clonal hairy root explants selected on kanamycin were then used to regenerate whole transgenic rape plants which though exhibiting the hairy root phenotype to varying degrees, were fertile. These plants were outcrossed with “wild type” rape lines and the F1 progeny analysed to show segregation of hairy root and antibiotic resistance genes. In the second method, an A. tumefaciens binary system was used to transform inflorescence stalks of different varieties of rape, using modifications of existing protocols which resulted in more efficient transformation. These modifications included the use of acetosyringone, the use of N. plumbaginifolia feeder cells, the inclusion of Seaplaque agarose in the media, and the postponement of kanamycin selection in the shooting medium for 2–3 weeks. Regenerated shoots were rooted and whole rape plants produced. Putative transgenic plants were subjected to phenotypic and Southern analyses to confirm the presence or absence of the introduced genes. The methods are effective with both Spring and Winter varieties of rape. Details of the two methods are presented and the advantages and disadvantages of each method are discussed.

The synthesis and structure of pea storage proteins

The seeds of pea, Pisum sativum L., like those of other legumes, accumulate proteins in their cotyledons during development. These proteins are mainly globulins, soluble in aqueous salt solutions at neutral pH; in keeping with their physiological storage role they are multimeric proteins readily rendered insoluble for deposition, and have high proportions of amide amino acids. They are degraded on germination of the seed to provide nutrients for the growing plant. The two major seed proteins in pea are legumin, a hexameric molecule (Mr ⋍ 380,000) where each monomer (Mr ⋍ 60,000) contains an “acidic”; (Mr ⋍ 40,000) and a “basic”; (Mr ⋍ 20,000) subunit linked by a disulphide bond, and vicilin, a trimeric molecule (Mr ⋍ 150,000) where each monomer (Mr ⋍ 50,000) may contain breaks in its polypeptide chain, giving rise to a variety of smaller subunits. Both these proteins are synthesised on polyribosomes bound to the endoplasmic reticulum in cotyledon cells and are transported from there to membrane‐enclosed p...

Gene copy number and levels of expression in transgenic plants of a seed specific gene

Abstract Two variants of the seed specific pea legA gene encoding legumin, the major storage protein of pea seeds were transferred to Nicotiana plumbaginifolia plants using the Agrobacterium tumefaciens binary vector Bin 19. A wide variation was observed both in the gene copy number of the introduced genes and in the level of pea legumin synthesised in the seeds of several individual transgenic N. plumbaginifolia plants. The number of gene copies of the introduced legA genes could not be correlated with expression levels in the same plants, suggesting that the variation in expression resulted from other factors, such as chromosomal position effects of the integrated genes in the Nicotiana genome.

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.

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

SummaryA 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 1300 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.