T. Dawn Parks

Transgene and gene suppression: telling us something new?

Transgenes provide unique opportunities to assess the relationship between genotype and phenotype in an organism. In most cases, introduction and subsequent expression of a transgene will increase (with a sense RNA) or decrease (with an antisense RNA) the steady-state level of a specific gene product. However, a number of surprising observations have been made in the course of many transgenic studies. We develop a hypothesis that suggests that many examples of endogenous gene suppression by either antisense or sense transcripts are mediated by the same cellular mechanism.

Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase

The 49-kDa proteinase of tobacco etch virus (TEV) cleaves the polyprotein derived from the TEV genomic RNA at five locations. Molecular genetic and biochemical analyses of the 49-kDa TEV proteinase were performed to test its homology to the cellular trypsin-like serine proteases. A cDNA fragment, containing the TEV 49-kDa proteinase gene and flanking sequences, was expressed in a cell-free transcription/translation system and resulted in the formation of a polyprotein precursor that underwent rapid self-processing. Site-directed mutagenesis was used to test the effect of altering individual 49-kDa amino acid residues on proteolysis. The data suggest that the catalytic triad of the TEV 49-kDa proteinase could be composed of the His234, Asp269, and Cys339. These findings are consistent with the hypothesis that the TEV 49-kDa proteinase is structurally similar to the trypsin-like family of serine proteinases with the substitution of Cys339 as the active site nucleophile. A structural model of the TEV 49-kDa proteinase proposes other virus-specific differences in the vicinity of the active site triad and substrate-binding pocket. The structure may explain the observed negligible effect of most cellular proteinase inhibitors on the activity of this viral proteinase.

Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L.

Treatment of tall and dwarf (3β-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA4, reduced the shoot contents of C19-GAs, including GA1, and increased the concentration of the C20-GA, GA19. In shoots of the slender (la crys) mutant, the content of C19-GAs was lower and GA19 content was higher than in those of the tall line. Metabolism of GA19 and GA20 in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment. The results suggest feedback regulation of the 20-oxidation and 3β-hydroxylation reactions. Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase cDNA from pea. The cDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences. After expression of this cDNA clone in Escherichia coli, the product oxidized GA12 to GA15, GA24 and the C19-GA, GA9, which was the major product. The 13-hydroxylated substrate GA53 was similarly oxidized, but less effectively than GA12, giving mainly GA44 with low yields of GA19 and GA20. Ps074 hybridized to polyadenylated RNA from expanding shoots of pea. Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treatment with GA3, suggesting that there is feed-back regulation of GA 20-oxidase gene expression.

Post-translational processing of the tobacco etch virus 49-kDa small nuclear inclusion polyprotein: Identification of an internal cleavage site and delimitation of VPg and proteinase domains

The 49,000-dalton (49-kDa) small nuclear inclusion (NI) protein of tobacco etch virus (TEV) has two distinct functions associated with it. An N-terminal segment is covalently attached to the genomic length RNA and likely involved in RNA replication, while the C-terminal half is associated with a proteolytic activity critical for genome expression. The junction delineating these two proteins has not been identified. We have analyzed naturally occurring cleavage products of TEV NI proteins and have identified a possible internal cleavage site between Glu and Gly residues at TEV 49-kDa NI protein amino acids 189-190. Similar 49-kDa-derived products are formed in cell-free translation studies in minor amounts upon the addition of excess amounts of NI protein. Cleavage of the 49-kDa (430 amino acids) protein is predicted to result in the formation of two products, 21-kDa (189 amino acids) and 27 kDa (241 amino acids) in size. Complementary DNA encoding the 27-kDa C-terminal portion of the 49-kDa protein gene was cloned into various DNA sequences. This allowed us to express the 27-kDa protein alone or as part of higher molecular weight polyproteins containing flanking TEV or foreign protein sequences. This 27-kDa amino acid sequence had a proteolytic activity similar to the 49-kDa-associated activity.

Isolation and nucleotide sequence of a novel cDNA clone encoding the major form of pathogenesis-related protein R

Pathogenesis-related (PR) proteins are acid-soluble, protease-resistant proteins which accumulate in the intercellular spaces of many plants as a result of the hypersensitive reaction to a pathogen [10]. The accumulat ion of PR proteins has also been observed in leaves of plants treated with certain chemical inducers such as acetylsalicylic acid and polyacrylic acid [4, 11]. The presence of PR proteins correlates with the onset o f a broad-range systemic resistance [3, 10] which suggests that the PR proteins may have a role in the establishment or maintenence of the resistant state. The predominant PR proteins expressed after TMV infection in tobacco are PRla, lb, lc, -2, -N, -O, -P, -Q, -R and -S [7, 10]. In an effort to isolate cDNA clones for PR proteins, Bol and colleagues at the University of Leiden constructed a cDNA library from TMV-infected tobacco leaves (Nicotiana tabacurn cv Samsun NN) and cDNA clones were isolated from this library that were induced upon TMV infection [2, 5, 6]. One group of these cDNA clones was shown to encode a protein with 65% identity to the sweet-tasting protein, thaumat in [1]. Later it was found that the thaumatin-like protein has about 60% identity to a maize t rypsin/a-amylase inhibitor [9]. Recently, the thaumatin-like protein has been identified as the tobacco pathogenesis-related protein, PR-R [8]. PR-R exists as two isoforms in tobacco, a major and a minor form which are expressed at a ratio of approximately 3:2 [8]. Cornelissen et al. described the isolation and sequence of the minor form of PR-R [1]. Here we present the sequence of a clone isolated from a cDNA library generated from RNA isolated from leaves of TMV-infected N. tabacum cv. Xanthi-nc which encodes the major form of PR-R. The two cDNA clones of PR-R share 95% identity at the nucleic acid level and 98% identity at the level of the encoded amino acid sequence. When 11 cDNA clones were randomly isolated and partially sequenced it was found that 8 code for the major form of PR-R and 3 code for the minor form of PR-R. Our deduced amino acid sequence for the major and minor forms of PR-R agrees with the protein sequence data from Pierpoint et al. [8] at every position except the asparagine residue at position 26 of the major form. There is no obvious reason for this one discrepancy. The DNA sequence for the minor form of PR-R does encode an asparagine at position 26 ([1], our unpublished results); however, the DNA sequence analysis resulting from sequencing both strands of two independent cDNA clones confirms that there is indeed a G at base 166 which would code for an aspartate.

Isolation of the genomic clone for pathogenesis-related protein 1a from Nicotiana tabacum cv. Xanthi-nc

We describe the isolation of the chromosomal gene for pathogenesis-related protein 1a from Nicotiana tabacum. A 2 kb fragment containing the PR-1a gene as well as 5′ and 3′ flanking DNA has been sequenced and the transcriptional start site has been determined by primer extension and S1 nuclease mapping. 80% of the protein sequence from purified PR-1a and 20% of the sequence of purified PR-1b has also been determined and used to verify the nomenclature established for the PR-1 cDNAs.

Substrate recognition by the Nla proteinase of two potyviruses involves multiple domains: Characterization using genetically engineered hybrid proteinase molecules

The proteolytic activity associated with the small nuclear inclusion protein (NIa proteinase) of tobacco etch virus (TEV), a potyvirus, catalyzes several cleavages at sites within the polyprotein derived from the TEV RNA genome. The homologous proteinase of tobacco vein mottling virus (TVMV), a closely related potyvirus, cleaves at similar, yet distinct, recognition sites. We examined these proteinases, in a cell-free cleavage system, in an attempt to define the biochemical basis of substrate specificity. Each proteinase was specific for its own cleavage site sequence in cell-free trans processing reactions, and no processing of the heterologous cleavage site was evident. Domains of the proteinase which were important in determining this substrate specificity were identified by generating hybrid proteinase genes containing both TEV and TVMV NIa proteinase coding sequences. Using site-directed mutagenesis and standard recombinant DNA techniques, plasmids were constructed which contained coding sequences for hybrid TEV-TVMV proteinases. These plasmids were expressed and tested in a cell-free environment for their ability to cleave both TEV and TVMV substrates. The data suggest that the carboxy-terminal 150 amino acids of the NIa protein contain the necessary information to specifically recognize a particular cleavage site sequence, and that specificity determinants are contained in at least three interactive subdomains within this region.

Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5′ end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease.

Application of TEV Protease in Protein Production.

In many cases, the analysis of a specific protein is impeded by the inability to purify large amounts of it from a native source. Proteins of interest may be present in minute quantities and/or purification may be plagued with technical problems. Recombinant DNA methodologies have enabled researchers to circumvent some of these limitations by producing and purifying large quantities of protein in a nonnative system. Various systems and strategies have been successfully employed, depending on the specific protein of interest and the desired use of the final end product (antibody production, crystallography studies etc.). This chapter reviews some common methods for the production of recombinant fusion proteins and specifically describes a versatrle method for the removal of affinity tags from recombinant fusions using a highly purified proteinase with an unparalleled degree of specificity. This proteinase, from the genome of tobacco etch virus (TEV), demonstrates specific proteolytic activity under a wide range of parameters (salt, temperature, pH), making it an excellent choice for cleavage of fusion proteins (1,2).