Promoter strength and tissue specificity effects on growth of tomato plants transformed with maize sucrose-phosphate synthase.

When sucrose-phosphate synthase (SPS; EC 2.4.1.14) is expressed in tomato (Lycopersicon esculentum Mill.) from a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit (rbcS) promoter, yields are often unchanged but when SPS is expressed from a Cauliflower Mosaic Virus 35S promoter, yield is enhanced up to 80%. Two explanations for this phenomenon are (i) that expression of SPS in tissues other than leaves accounts for the increased yield or (ii) that the lower level of expression directed by the 35S promoter is more beneficial than the high level of expression directed by the rbcS promoter. To test the first hypothesis, we conducted a reciprocal graft experiment, which showed that root SPS activity did not substantially affect growth. To test the second hypothesis, we conducted a field trial using a backcrossed, segregating, population of SPS-transformed plants derived from 35S and rbcS lines. The optimal dose of SPS activity for growth was approximately twice that of the wild type regardless of which promoter was used. The effect of SPS on growth was the result of a shift in partitioning of carbon among starch, sucrose, and ionic compounds (primarily amino acids), rather than of an increase in net photosynthesis. Excessive SPS activity resulted in a decreased rate of amino acid synthesis, which could explain the non-linear response of plant growth to the level of SPS expression.

Translational arrest in hypoxic potato tubers is correlated with the aberrant association of elongation factor EF-1 alpha with polysomes.

Translation elongation factor EF-1 alpha became stably associated with potato tuber polysomes at the onset of hypoxia, coincident with a sharp rise in lactate and decrease in tissue pH. This aberrant association of EF-1 alpha with polysomes also occurred when aerobic tuber extracts were acidified in vitro. Upon resumption of protein synthesis, an increase in the steady-state levels of EF-1 alpha, and expression of an EF-1 alpha/GUS transgene was observed. These results indicate that translational arrest results from to the failure of EF-1 alpha to dissociate from ribosomes during the elongation cycle, and that restoration of protein synthesis is coordinated with expression of EF-1 alpha.

Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit [alpha] upon Wounding in Potato Tubers.

Potato (Solanum tuberosum) tubers exhibit an increase in translational activity in response to mechanical wounding. The response is biphasic, with an initial stimulation apparent within the first 2 h after wounding and a second increase occurring 12 to 24 h after wounding. Increased activity is apparent by measurement of protein synthesis both in vivo and in vitro using a cell-free extract. Accumulation of the translational elongation factor 1 subunit [alpha] (EF-1[alpha]) parallels translational activity. Changes in the steady-state level of EF-1[alpha] mRNA, and expression of a chimeric EF-1[alpha] promoter/[beta]-glucuronidase construct in transgenic potato tubers, indicate that the gene encoding EF-1[alpha] is transcribed during both periods of translational stimulation. These results indicate that stimulation of translational activity is coordinated with increased expression and accumulation of translation factors.

Expression of Escherichia coli glycogen synthase in the tubers of transgenic potatoes (Solanum tuberosum) results in a highly branched starch.

A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen synthase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.

Demonstration of a Senescence Component in the Regulation of the Mannopine Synthase Promoter.

Regulation of the mannopine synthase (mas) promoter during senescence in leaves and flowers of tobacco (Nicotiana tabacum) plants was investigated. In plants transformed with a mas 5[prime]-[beta]-glucuronidase (GUS)-mas 3[prime] transcriptional fusion, we observed that following the onset of senescence in either intact or excised leaves of the transgenic lines, GUS activity increased significantly, whereas in excised leaves in which the senescence process was inhibited, GUS activity increased only marginally. During flower petal senescence in the transgenic tobacco, GUS activity increased approximately 6-fold over preanthesis- and anthesis-stage flowers.

The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants.

A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.

Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants.

Cyclodextrins (CDs) are cyclic oligosaccharides containing six (alpha), seven (beta), or eight (gamma) glucose molecules, respectively. The cyclodextrin glycosyltransferases (CGT), which produce CDs from starch, are found only in bacteria and are used in batch fermentors with hydrolyzed starch to produce CDs commercially. Using a CGT gene from Klebsiella, we attempted to engineer the tubers of developing potatoes to produce these novel, high-value carbohydrates. A chimeric gene, consisting of (1) the patatin promoter for tuber-specific expression, (2) the small subunit of ribulose bisphosphate carboxylase (SSU) transit peptide for plastid targeting, (3) the CGT structural gene from Klebsiella and (4) the nopaline synthase 3' region, was introduced into potatoes. Both alpha and beta CDs were produced in tubers of transgenic potatoes at levels corresponding to 0.001-0.01% of the starch being converted to CDs.

Developmental analysis of elongation factor-1 alpha expression in transgenic tobacco.

The developmental regulation of the translational elongation factor EF-1 alpha has been analyzed in tobacco. A gene fusion was constructed consisting of the 5' and 3' regions of the tomato genomic clone LeEF-A from the EF-1 alpha gene family and the beta-glucuronidase coding region. Analysis of the transgenic plants containing this chimeric gene demonstrated that the tomato LeEF-A flanking sequences were sufficient to confer expression patterns similar to those of the endogenous tobacco EF-1 alpha gene. The patterns of beta-glucuronidase activity in this system indicated that during plant growth and development EF-1 alpha is regulated with increased expression corresponding to regions of high protein synthesis, including meristems, rapidly growing tissues, and developing gametophytes. In addition, EF-1 alpha expression responds rapidly to changes in growth patterns induced by hormone treatment. Our results are in agreement with studies in animals indicating that EF-1 alpha expression may be rate limiting for protein synthesis and demonstrate that the analysis of EF-1 alpha is of value for studying interrelationships between protein synthesis and developmental control.

A rapid screen for the detection of specific DNA sequences in plants.

We have developed a simple and quick method ("wick blot") for detecting the presence of specific DNA sequences in plants, using radiolabeled DNA probes. The method requires only small amounts of tissue, about 15-25 mg. More than a hundred samples per day can be easily extracted and blotted. It works well on various species and tissues, including leaves, embryos, and callus. The method is ideally suited for screening large numbers of putative transformants, especially populations that have not been screened by prior selection.

Structure and expression of elongation factor 1 alpha in tomato.

A full-length cDNA clone, LeEF-1, has been isolated from tomato for the alpha subunit of elongation factor 1 (EF-1 alpha), a polypeptide which plays a central role in protein synthesis. The 448 amino acid protein encoded by this cDNA appears highly homologous to other EF-1 alpha s having a high degree of similarity (75-78%) to EF1 alpha previously described from both lower eukaryotes and animals. Southern analysis indicated that EF-1 alpha belongs to a small multigene family of 4-8 members in tomato. The pattern of expression of EF-1 alpha mRNA in various tomato tissues was analyzed by Northern analysis, in vitro translation and in situ hybridization. EF-1 alpha mRNA is an abundant species and higher levels of mRNA were found in developing tissues such as young leaves and green fruit compared to the mRNA levels observed in older tissues. The increased levels of EF-1 alpha mRNA therefore appear to correlate with higher levels of protein synthesis in developing tissues.

Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide.

Ribulose bisphosphate carboxylase small subunit protein is synthesized in the cytoplasm as a precursor and transported into the chloroplast where the amino-terminal portion, the transit peptide, is removed proteolytically. To obtain chloroplast delivery of the 43-kDa 5-enolpyruvyl 3-phosphoshikimate (EPSP) synthase of Salmonella typhimurium, we constructed fusion proteins between the bacterial EPSP synthase and the ribulose bisphosphate carboxylase small subunit. A fusion protein consisting of the transit peptide fused to the EPSP synthase was not transported in vitro or in vivo into chloroplasts. A second fusion protein consisting of the transit peptide and 24 amino acids of the mature small subunit fused to the EPSP synthase was transported both in vitro and in vivo into chloroplasts. It was processed into two polypeptides of 46 and 47 kDa, respectively. This heterogeneity in processing was not caused by the presence of the aroA start codon, since its removal resulted in the same pattern. Substituting 24 different amino acids for the 24 amino acids of the mature small subunit resulted in a fusion protein that was not transported into the chloroplast. It was concluded that a portion of the mature small subunit was needed for efficient chloroplast delivery.

Isolation of tobacco SSU genes: characterization of a transcriptionally active pseudogene.

Genomic clones containing three genes for the small subunit (SSU) of ribulose bisphosphate carboxylase were isolated from tobacco. Detailed analysis was performed on two of these clones to give a clearer picture of this multigene family in tobacco. This analysis demonstrated that one of the clones contained a pseudogene that was unusual in that it was transcriptionally active. This is the first transcriptionally active pseudogene that has been reported in plants. In addition, another clone was found to contain coding sequences which are 100% homologous to a previously-cloned tobacco SSU gene (Mazur, B.J. and Chiu, C-F. [1985] Nuc. Acids Res. 13, 2372-2386), indicating that gene duplication and/or gene conversion may have played a role in the evolution of the tobacco SSU family.

Transcription of cauliflower mosaic virus integrated into plant genomes.

A full-length copy of cauliflower mosaic virus (CaMV) was introduced into the T DNA of the Agrobacterium Ti plasmid and integrated into plant genomes. Northern analysis of turnip galls revealed two transcripts derived from the integrated CaMV. A 1900-nucleotide (1.9 kb) transcript arises from the region VI area of the genome and is the same size as the region VI transcript found in CaMV-infected leaves. A larger 7.5-kb transcript initiates at or near the other known CaMV promoter, which in infected cells directs synthesis of a full-length transcript, and terminates in adjacent T-DNA sequences. Comparison of the two transcripts in galls from seven plant species showed wide variation of both the ratios of the two transcripts within one species and of their levels between species.

DNA sequences necessary for transcription of the rabbit beta-globin gene in vivo.

The DNA sequences required for the expression of the rabbit-beta-globin gene in vivo have been examined. A variety of mutant rabbit beta-globin gene templates were linked to a simian virus 40-plasmid recombinant and introduced into HeLa cells; in these conditions the rabbit beta-globin gene is expressed from its own promoter. Comparison of the level of beta-globin transcripts in a variety of deletion mutants shows that for efficient transcription, both the ATA or Goldberg-Hogness box, and a region between 100 and 58 base pairs in front of the site at which transcription is initiated, are required. Deletion of either of these regions results in a decrease in the level of beta-globin transcripts by an order of magnitude; deletion of the ATA box causes an additional loss in the specificity of the site of initiation of RNA synthesis. The DNA sequences downstream from the ATA box, including the natural beta-globin mRNA cap site, are dispensable for transcription in vivo.

Structure and expression of a cloned beta o thalassaemic globin gene.

We have cloned the single beta-globin gene from an Italian patient who is a double heterozygote for beta o/delta beta o thalassaemia. RNA isolated from nucleated red cells from this patient can be translated in vitro to give detectable levels of A gamma- G gamma and alpha-globin, but no beta-globin. S1-mapping transcription studies show that beta-globin mRNA is present at about 1-3% of the level of alpha- and gamma-globin mRNA. In addition, the expression of this gene has been studied by reversed genetics. SV40-plasmid-beta o-globin gene recombinants have been transfected into Hela cells and analysed for beta-globin mRNA. In contrast to the results obtained with mRNA isolated directly from the blood of this patient, in the transfected Hela cells the same level of beta-globin mRNA is seen for the beta o thalassaemic globin gene and for a normal beta-globin gene. To elucidate the nature of the lesion, the entire DNA sequence of the beta-globin gene of this patient has been determined. The sequence shows that this gene contains a termination codon at position 39 (CAG - greater than UAG). Otherwise, there is a remarkable conservation of the entire DNA sequence.

Localization of DNA sequences necessary for transcription of the rabbit beta-globin gene in vitro.

We have studied the transcription in vitro of the rabbit beta-globin gene in the HeLa cell system. Using cloned fragments of this gene, we obtained specific transcripts with 5' ends at the "cap" site. The analysis of the transcription of a large number of deletion mutants has shown that the cap site and the conserved CCAAT box at 75 nucleotides upstream from the cap site are not required for specific in vitro transcription. Sequences localized within the region from 34 to 20 nucleotides upstream from the 5' end of the cap site, however, are required for in vitro transcription. Since the conserved "Goldberg-Hogness" box is localized in this region, we conclude that this is the major requirement for specific transcription in vitro. Finally, in at least eight cases, deletions localized to the 3' side of the Goldberg-Hogness box shift the transcription-initiation site downstream to a position approximately 30 nucleotides from the Goldberg-Hogness box. This is consistent with the idea that RNA polymerase II is bound at the Goldberg-Hogness box and initiates transcription 30 nucleotides downstream from this site. There is apparently little sequence specificity in the selection of the site of initiation.

Analysis of binding interactions between histone core complex and simian virus 40 DNA. A comparison of acetylated versus non-acetylated histone core complexes.

The interactions of acetylated and non-acetylated core complex histones with simian virus 40 (SV40) DNA 1 have been analyzed. A modified filter-binding assay utilizing micrococcal nuclease, which allows quantification of histone octamer binding to DNA has been developed. Using this assay it was determined that both non-acetylated core complex histones ad core complex histones acetylated with acetyl adenylate to levels existing in vivo bind cooperatively to SV40 DNA 1. Although both interactions are cooperative, the magnitude of the cooperativity parameter, omega, is significantly less in the acetylated case. This difference in cooperativity is in contrast to the nearly identical intrinsic association constant, K, observed in both cases.