Biomedical subjects

M W Bevan

Publications and source records attributed to M W Bevan.

24 records · Page 2Linked to original sources

PubMed1987

GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Cloning, Molecular↗

PubMed1987

Localization of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco.

The developing cereal grain accumulates large quantities of proteins which are unique to the endosperm tissue. The DNA sequences which determine their endosperm-specific expression have not yet been identified. In the absence of a suitable transformation-regeneration system for cereals, we have investigated whether chimaeric genes consisting of low mol. wt (LMW) and high mol. wt (HMW) glutenin gene upstream sequences coupled to the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene could be specifically expressed in transgenic tobacco. The fusions, made in a Ti-derived binary vector, were introduced into tobacco via Agrobacterium tumefaciens-mediated transformation and their activity assayed. Both the LMW and HMW glutenin chimaeric genes exhibited endosperm-specific CAT activity in the transformed plants. In addition, a deletion series of the LMW glutenin sequence indicated that sequences present between 326 bp and 160 bp upstream of the transcription start point are necessary to confer endosperm-specific CAT activity.

Journal Article↗

PubMed1987

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

Acetyltransferases↗

PubMed1985

Expression of tobacco mosaic virus coat protein by a cauliflower mosaic virus promoter in plants transformed by Agrobacterium.

The 35S transcript promoter of cauliflower mosaic virus directs the synthesis of an abundant RNA in infected leaf tissue that is both a template for reverse transcription during virus replication and also a polycistronic mRNA. The 35S promoter was isolated and linked to a cDNA of the 3' end of tobacco mosaic virus that encodes the coat protein of this single-stranded RNA virus. After transformation of this gene construction into tobacco plants using a disarmed Agrobacterium binary vector, viral coat protein synthesis was detected in transformed leaf tissue. A chimaeric 35S coat protein RNA was detected in transformed callus.

Journal Article↗

PubMed1982

T-DNA of the Agrobacterium Ti and Ri plasmids.

The study of T-DNA transmission from Agrobacterium Ti or Ri plasmid into the genomes of higher plant cells has revealed much about the consequences of transformation. It is now clear that the transformed phenotype is caused by hormonal changes produced directly or indirectly by T-DNA genes. The opine synthases are enzymes encoded in T-DNA that function in the plant cell. Our level of understanding of T-DNA-encoded functions is already sufficient to reveal clear and feasible ways to exploit T-DNA as a gene vector. What remains to challenge the crown gall investigator are many questions of fundamental importance: What is the mechanism of the seemingly illegitimate recombination between T-DNA and plant DNA, and is this process catalyzed by bacterial or host plant enzymes, or both? Do T-DNA genes encode enzymes that catalyze biosynthesis of auxin- and cytokinin-active substances? What gene in T-DNA confers immunity to A. tumefaciens, and what is its mode of action? Does T-DNA insert into random or specific sites in the host plant genome? Did T-DNA derive from plant genetic information or has prokaryotic DNA arrived at functional eukaryotic gene structure by convergent evolution? Although there is keen interest in T-DNA as a vector for genetic engineering, it holds equal interest as a unique interface between the biology of prokaryotes and eukaryotes.

Arginine↗

PubMed1982

Multiple transcripts of T-DNA detected in nopaline crown gall tumors.

Crown gall plant tumors contain neoplastic cells transformed by incorporation of a foreign DNA element, T-DNA, derived from a large tumor-inducing plasmid in the inciting Agrobacterium strain. T-DNA is covalently joined to the nuclear DNA of the tumor cell, and RNA transcripts from T-DNA are present in polyadenylated form on polysomes. This paper presents a detailed analysis of those parts of T-DNA transcribed in a nopaline-type tobacco teratoma, BT37, whose T-DNA has been mapped and cloned. Northern blots of polyA+ RNA were probed with 21 different nick-translated T-DNA fragments, and at least 13 well-defined transcripts were visualized.

Arginine↗