Foldback transposable elements in plants.

A novel transposon family was discovered in plants. This family, designated SoFT (Solanaceae Foldback Transposon), exhibit striking structural similarity to the 'foldback' class of animal transposons. SoFT elements consist of a middle segment surrounded by long terminal inverted repeats. Two of the identified SoFT elements have 'classical' foldback structure: their inverted repeats are divided into two domains. The outer domain consists of tandemly arranged subrepeats, whereas the inner domain is non-repetitive and AT-rich. The existence of foldback elements in plants as well as in animals suggests that long inverted repeat (foldback) transposons are ubiquitous among eukaryotes.

Plant farnesyltransferase can restore yeast Ras signaling and mating.

Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes.

The promoter for tomato 3-hydroxy-3-methylglutaryl coenzyme A reductase gene 2 has unusual regulatory elements that direct high-level expression.

The promoter region of tomato (Lycopersicon esculentum) 3-hydroxy-3-methylglutaryl coenzyme A reductase gene 2 (HMG2) has been analyzed using the transient expression of HMG2-luciferase fusions in red fruit pericarp. The mRNA for HMG2 accumulates to high level during fruit ripening, in a pattern that coincides with the synthesis of the carotenoid lycopene. Unlike most promoters, the region that is upstream of the HMG2 TATA element is not required for high-level expression. The 180-bp region containing the TATA element, the 5' untranslated region, and the translation start site are comparable in strength of the full-length 35S cauliflower mosaic virus promoter. Pyrimidine-rich sequences present in the 5' untranslated leader are important in regulating expression. Also, the ATG start region has been found to increase translation efficiency by a factor of 4 to 10. An alternative hairpin secondary structure has been identified surrounding the HMG2 initiator ATG, which could participate in the translational regulation of this locus. HMG2 appears to be a novel class of strong plant promoters that incorporate unusual, positive regulators of gene expression.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.

Tomato hydroxymethylglutaryl-CoA reductase is required early in fruit development but not during ripening.

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and the level of its mRNA have been determined at various stages of tomato fruit development. The HMGR reaction makes mevalonate, a necessary component in the synthesis of all isoprene containing compounds, such as sterols and carotenoids. A cDNA clone encoding the active site region of HMGR has been isolated from a tomato library derived from young-fruit mRNA. The clone hybridizes to a one- or two-copy fragment in high-stringency DNA gel blot analyses and detects an mRNA of approximately 3.0 kb. Both HMGR activity and mRNA levels are high in early stages of tomato fruit development, when rapid cell division occurs, as well as in the subsequent early stages of cellular expansion. In contrast, ripening fruit have very low levels of reductase activity and mRNA, even though large amounts of the carotenoid lycopene are synthesized during this period. Furthermore, in vivo inhibition of HMGR during early fruit stages disrupts subsequent development, whereas inhibition during later stages of fruit expansion has no apparent effect on ripening. We conclude that the pool of mevalonate responsible for the synthesis of phytosterols is synthesized primarily during the first half of tomato fruit development. In addition, the final period of fruit expansion and ripening is not dependent upon HMGR activity, but instead utilizes a preexisting pool of pathway intermediates or requires the use of salvage pathways in the cell.

A subpopulation of spinach chloroplast tRNA genes does not require upstream promoter elements for transcription.

We have identified a class of spinach plastid tRNA genes which do not require 5' upstream promoter elements for their expression in a chloroplast transcription system. The 5' DNA sequences flanking the trnR1 and trnS1 coding regions have little or no homology to previously characterized chloroplast promoter sequences. The deletion of the 5' DNA sequences from these genes to positions close to the start of the coding regions has little effect on their transcription in vitro. In addition, a synthetic DNA fragment homologous to the 5' region of trnS1 does not support the transcription of the promoter (-) trnM2 mutant 51 in a promoter/trnM2-51 fusion assay. In a dicistronic construct the wild type trnS1 gene does not support transcription of trnS1 transcription occurs immediately following the 3' end of the coding region. Both trnS1 and trnR1 compete with trnM2 for the same chloroplast RNA polymerase and/or common transcription factors.

Characterization of a Euglena gracilis chloroplast RNA polymerase specific for ribosomal RNA genes.

Euglena gracilis chloroplasts contain a 145,000-base pair chromosome that encodes genes for ribosomal, transfer, and messenger RNAs. These genes are transcribed within the organelle by chloroplast RNA polymerase activities that are specific for different classes of RNA. Two transcriptional activities have been isolated from Euglena chloroplasts. (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259, 14880-14887). One, the "soluble extract," contains enzymes active in tRNA transcription and processing. The other activity, the transcriptionally active chromosome, consisting of a chloroplast DNA-dependent RNA polymerase tightly bound to chloroplast DNA, only transcribes rRNA genes even though the entire chloroplast genome is present. We have extensively purified the transcriptionally active chromosome using high salt concentrations to dissociate loosely bound proteins. The result is a highly enriched extract containing three major polypeptides of Mr 116,000-118,000, 83,000-88,000, and 24,000-26,000 that retains complete selectivity for rDNA transcription. It is probable that one, or both, of the high molecular weight proteins are functional components of the DNA-dependent RNA polymerase. The identification and characterization of the transcriptionally active chromosome is a first step towards understanding how chloroplast rRNA synthesis is regulated.

Evidence for two RNA polymerase activities in Euglena gracilis chloroplasts.

Two types of RNA polymerase activity were isolated from Euglena gracilis chloroplasts and compared. One polymerase is tightly bound to chloroplast DNA; this complex is called the transcriptionally active chromosome (TAC) (Rushlow, K. E., Orozco, E. M., Jr., Lipper, C., and Hallick, R. B. (1980) J. Biol. Chem. 255, 3786-3792). The other activity is found in a soluble extract of Euglena chloroplasts. The soluble extract is dependent upon an exogenous DNA template for activity. The two activities can be isolated in two distinct subchloroplast fractions from a single chloroplast preparation. The soluble extract is selective for transcription of transfer RNA genes, whereas the TAC is selective for ribosomal RNA genes. TAC and the soluble extract respond differently to KCl and Mg2+. The soluble extract is sensitive to heparin, and TAC is resistant. The two activities have different temperature optima. Based on this evidence, we conclude that Euglena chloroplasts have at least two distinct RNA polymerase activities.

Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs.

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.

Selective in vitro transcription of chloroplast genes.

Transcription of Euglena gracilis chloroplast genes has been investigated by using in vitro transcription systems. A DNA-dependent RNA polymerase responsible for the transcription of rRNA genes has been isolated as a nucleoprotein complex (transcriptionally active chromosome). The RNA polymerase remains tightly bound to the chloroplast DNA template and does not initiate transcription with cloned chloroplast genes. A transcriptionally active extract has been prepared from intact Euglena chloroplasts. The soluble RNA polymerase in this extract recognizes cloned chloroplast tRNA genes and tRNA-sized products have been detected after transcription. The tRNA-sized molecules specifically hybridize to the tRNA genes in the plasmid DNA. At least five tRNA-sized products have been identified from transcription of a trnY1trnH1-trnM1-trnE1-trnW1-trnG1 cluster. Evidence is also presented that processing enzymes in the chloroplast-extract can recognize a polycistronic tRNAVal-tRNAAsn-tRNAArg precursor and process it into tRNA-sized molecules. Truncated templates have been used to demonstrate that the chloroplast tRNA genes are actively transcribed. From a comparison of 5' flanking sequences in chloroplast tRNA genes, a consensus sequence which might function as a promoter, has been identified. The properties of the RNA polymerase involved in the transcription of chloroplast rRNA genes and tRNA genes have been investigated and compared.