Transgenes 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.

RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation.

Transgenic Nicotiana tabacum cv. Burley 49 plants were generated that express the 5' untranslated region of the tobacco etch potyvirus (TEV) genome ligated to a mutated version of the TEV coat protein gene sequence that rendered it untranslatable. Eight different transgenic plant lines were analyzed for transgene expression and for resistance to TEV. Three different responses were noted when the transgenic plant lines were inoculated with TEV: 1) some were highly resistant, and no virus replication occurred; 2) some were susceptible but able to recover from systemic TEV infection; and 3) some were susceptible to TEV infection. Plant tissue displaying the recovery phenotype was analyzed for virus replication and transgene expression. Recovered tissue could not be infected with TEV and had steady-state transgene RNA levels which were five- to eightfold lower than those of unchallenged transgenic plant tissue. Nuclear runoff assays suggested a post-transcriptional reduction in specific RNA levels. The highly resistant and recovery phenotypes associated with TEV challenge inoculation and the reduction of steady-state RNA levels in recovered transgenic leaf tissue may be manifestations of a common mechanism.

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).

Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays.

Potyviruses express their genetic information from a genome length RNA as a single polyprotein, which is post-translationally processed by at least two different viral-encoded proteolytic activities. Since regulation of the expression of individual genes is not likely to occur at the transcriptional level, we sought to determine if post-translational regulation of gene expression was possible via differential proteolytic processing. Modulating the rate of cleavage at different gene product junctions could be a potential mechanism to regulate the kinetics of formation of specific gene products. We have examined the proteolytic processing of two tobacco etch virus (TEV) cleavage sites using a cell-free system in which synthetic polyprotein precursors were processed by the TEV 49-kDa proteinase. The amino acid sequences at these two sites contained both conserved and variable positions. The cleavage reaction at the TEV 50/71-kDa junction was characterized as "slow" (T1/2 = approximately 27 min) while the cleavage reaction at the 58/30-kDa junction was judged "fast" (T1/2 = approximately 5 min). Similar cleavage reactions were observed whether the cleavage site was in its natural polyprotein context or placed in a foreign position. The slow and fast cleavage sites could be interconverted by changing the nonconserved amino acid positions. The data suggest that the heptapeptide sequence proximal to a TEV cleavage site determines not only cleavage at a particular junction but influences the cleavage reaction profile in cell-free studies.

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.

Substrate recognition by the NIa 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.

Molecular genetic analysis of a plant virus polyprotein cleavage site: a model.

The RNA genome of tobacco etch virus (TEV) is expressed as a polyprotein which is co- and post-translationally processed by viral encoded proteinases. The TEV 49,000 dalton (49-kDa) proteinase cleaves the polyprotein at five positions each defined by the seven amino acid consensus sequence, (formula; see text) One of the cleavage sites, the 58-kDa nuclear inclusion/30-kDa capsid protein junction was altered by site-directed mutagenesis and the effects of these alterations on cleavage were determined. Polyprotein precursors were synthesized by translation of T7 polymerase-derived transcripts and processed in a cell-free system using TEV nuclear inclusion bodies as a source of 49-kDa proteolytic activity. A wild-type cleavage site and 61 substrates containing site-directed amino acid replacements at the nonconserved P7, P5, P4, P2, and P'2 positions were examined. Amino acid replacements flanking the putative TEV cleavage sequence at the P7 and P'2 positions had minimal effects on cleavage. Amino acid substitutions at positions P5, P4, and P2 resulted in substrates which were processed by the 49-kDa TEV proteinase, albeit generally at reduced rates. No substitution at any of these five positions resulted in total elimination of cleavage. A model is presented which proposes different roles for conserved and variable positions in the TEV heptapeptide cleavage sequence.

Cleavage profiles of tobacco etch virus (TEV)-derived substrates mediated by precursor and processed forms of the TEV NIa proteinase.

Nucleotide sequences coding for proteins containing the tobacco etch virus (TEV) NIa proteinase were generated by polymerase chain reaction amplification and/or site-directed mutagenesis. These coding regions contained sequences for the proteinase alone or as part of higher Mr precursors. Following transcription and translation of these sequences in a cell-free system, the various polyproteins, all containing an active small nuclear inclusion protein (NIa) proteinase, were used to process a TEV substrate series. Most substrates were processed in a similar fashion by all proteolytic forms. However, one substrate which contained the TEV 50K/71K protein junction was differently processed by several of the polyproteins containing NIa proteinase. Substrates which previously had no identified TEV NIa proteinase cleavage sites also were tested and were not cleaved by any of the proteinase-containing polyprotein forms.

Molecular genetic analyses of the soybean mosaic virus NIa proteinase.

Recombinant DNA molecules containing cDNA to a soybean mosaic virus (SMV) RNA genome were constructed and partial nucleotide sequences determined for two cDNA inserts, pSMV-34 and pSMV-35. Comparison of the predicted amino acid sequence encoded by the pSMV-34 cDNA insert to other potyvirus protein sequences revealed extensive homology with the region of the genome encoding the NIa proteinase, with conservation of the amino acids proposed to form the catalytic triad of the active site. Cell-free transcription and translation of the cloned cDNA sequence containing the NIa open reading frame and flanking sequences revealed that NIa proteinase sequences, which were expressed as part of a high Mr precursor, were able to undergo proteolytic processing. Alteration of the codon for one of the putative active site residues by site-directed mutagenesis eliminated processing and resulted in the accumulation of a high Mr precursor. Based on predicted amino acid sequences at five putative cleavage sites within the SMV polyprotein, a consensus SMV NI a proteinase cleavage sequence of Glu/Asn-Xaa-Val-Xaa-Xaa-Gln decreases Gly/Ser was proposed. The SMV NIa proteinase and its putative cleavage sites maintained motifs found in other potyviruses.

Biochemical and mutational analysis of a plant virus polyprotein cleavage site.

The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346,000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus-encoded proteinase at five different sites between the dipeptides Gln-Ser or Gln-Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence...Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly.... We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site-directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell-free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.

Identification of an aspartate transfer RNA gene in maize mitochondrial DNA.

A gene for a transfer RNA (tRNA) specific for aspartic acid was identified in maize mitochondrial DNA. The nucleotide sequence and predicted secondary structure of this tRNA more closely resemble eubacterial and chloroplast aspartate tRNA genes than other mitochondrial aspartate tRNA genes. This gene is located on a 3,123 base pair EcoRI DNA fragment that also contains an elongator methionine tRNA gene. These two tRNA genes are separated by 726 nucleotides and are located on opposite strands of DNA.

A second proteinase encoded by a plant potyvirus genome.

The RNA genome of tobacco etch virus (TEV) encodes a large polyprotein precursor that is processed to mature proteins by virus-specific proteinases. Cleavage sites located within the carboxyl-terminal two-thirds of the polyprotein are processed by a TEV-encoded 49 kd proteinase, while the enzyme(s) responsible for cleaving the remaining sites has not been found. In this study, a second TEV-encoded proteinase has been identified based on cell-free expression of defined RNA transcripts. The boundaries of this proteinase have been delineated by deletion analysis and site-directed mutagenesis. The proteolytically active domain has been localized to the carboxyl-terminal half of the 56 kd aphid-transmission helper component. A cleavage site that is recognized by this proteinase has been identified in the polyprotein adjacent to the carboxyl-terminus of the enzyme, and the proteinase appears to cleave by an autocatalytic mechanism. Proteolysis in vitro occurs between a Gly-Gly dipeptide as determined by radiochemical sequencing at the amino-terminus of the proteolytic product.

Autocatalytic activity of the tobacco etch virus NIa proteinase in viral and foreign protein sequences.

The small nuclear inclusion (NIa) protein of the tobacco etch virus (TEV) is synthesized initially as part of a genome-derived high M(r) precursor. The NIa protein releases itself from this genome-derived precursor by self-cleavage, or an autocatalytic processing event. Cleavage between specific glutamine-glycine dipeptides at the N and C termini generates the 430 amino acid or 49,000 M(r) (49K) NIa protein. The requirements of this autocatalytic release, or cis cleavage, were examined by constructing gene cassettes encoding the TEV NIa protein which could be ligated into particular locations in cDNA of the TEV genome and also into foreign gene DNA sequences. Using cell-free transcription and translation systems, polyproteins containing TEV NIa sequences were synthesized and assayed for (i) autocatalysis and (ii) the ability of a functional NIa proteinase, purified from plant tissue, to cleave in bimolecular or trans reactions various artificial polyproteins which contained an inactive form of the NIa proteinase. The NIa self-cleavage events required an active proteinase sequence and a consensus TEV cleavage site sequence at the N and C termini. These results were consistent for NIa protein sequences placed at a foreign TEV cleavage site or in unrelated proteins. Differences were noted in the trans cleavage of these sites.

Safety and efficacy of sedation in children using a structured sedation program.

OBJECTIVE: The purpose of this study was to evaluate the safety and efficacy of sedating pediatric outpatients in a radiology department using a structured sedation program modeled after the guidelines of the American Academy of Pediatrics. MATERIALS AND METHODS: Medical records of 6006 pediatric outpatients who underwent sedation for imaging studies under the guidelines of a structured sedation program were retrospectively reviewed for safety and efficacy of sedation, complications related to sedation, and rate of successful sedation. RESULTS: No cases of cardiovascular or respiratory arrest or fatalities occurred. Four children who required overnight hospitalization because of CNS depression had no sequelae. Forty-eight patients experienced transient respiratory depression that proved responsive to supplemental oxygen. Delayed complications were found in 29 children, none of whom required hospitalization. The rate of sedation failure was approximately 1%. CONCLUSION: Sedation of children can be done in a safe and highly efficacious manner in a hospital radiology department using a structured sedation program modeled after the guidelines of the American Academy of Pediatrics.

Identification of two methionine transfer RNA genes in the maize mitochondrial genome.

Two methionine transfer RNA (tRNA) genes were identified in the maize mitochondrial genome by nucleotide sequence analysis. One tRNA gene was similar in nucleotide sequence and secondary structure to the initiator methionine tRNA genes of eubacteria and higher plant chloroplast genomes. This tRNA gene also had extensive nucleotide homology (99%) with an initiator methionine tRNA gene described for the wheat mitochondrial genome. The other methionine tRNA gene sequence was distinct and more closely resembled an elongator methionine tRNA.

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.

Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs.

Haploid leaf tissue of tobacco cultivars K326 and K149 was transformed with several transgenes containing cDNA of the potato virus Y (PVY) coat protein (CP) open reading frame (ORF). The various transgenes containing the PVY CP ORF sequence produced (1) the expected mRNA and CP product, (2) an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense RNA that was untranslatable as a result of the incorrect orientation of the PVY CP ORF behind the transcriptional promoter. Homozygous doubled haploid (DH) (diploid) plants were generated, and selfed progeny from these plants were examined. Resistance was virus specific, functioning only against PVY. An inverse correlation between transgene-derived PVY transcript steady state levels and resistance was generally noted with lines expressing the untranslatable sense version of the PVY CP ORF. A collection of DH lines, derived from a single transformation event of a common haploid plant and isogenic for the PVY transgenes expressing untranslatable sense RNA, displayed different levels of PVY resistance. Lines with actively transcribed, methylated transgene sequences had low steady state levels of transgene transcript and a virus-resistant phenotype. These results are discussed within the context of sense suppression in plants.

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.

Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase.

An improved method for the production, cleavage, and purification of fusion proteins and peptides is described. The unique aspect of this method is dependent on the use of a proteinase from tobacco etch virus (TEV). The proteinase used is a recombinant TEV proteinase produced with a polyhistidine tract positioned at the amino terminus. The proteinase recognizes a specific, extended cleavage site sequence. The peptide or protein of interest is purified as a fusion protein with a TEV proteinase cleavage site sequence located between it and an affinity carrier portion of the fusion. Incubation with the recombinant TEV proteinase mediates release of the peptide or protein of interest. Use of the recombinant TEV proteinase to cleave fusion proteins is an improvement over use of other proteinases for several reasons, including its high degree of specificity, its insensitivity to many proteinase inhibitors generally used in protein purification, and the ready separation of both the affinity tag and the proteinase from the cleaved product of interest.

Genetic and biochemical dissection of transgenic RNA-mediated virus resistance.

RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines.