Expression In Tobacco Protoplasts Research Articles

Rice aquaporin OsPIP2;2 is a water-transporting facilitator in relevance to drought-tolerant responses.

In rice (Oryza sativa), the PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family of aquaporin has 11 members, OsPIP1;1 to OsPIP1;3, and OsPIP2;1 to OsPIP2;8, which are hypothesized to facilitate the transport of H2O and other small compounds across cell membranes. To date, however, only OsPIP1;2, OsPIP2;1, and OsPIP2;4 have been demonstrated for substrate selectivity in their source plant (rice). In this study, OsPIP2;2 was characterized as the most efficient facilitator of H2O transport across cell membranes in comparison with the other 10 OsPIPs. In concomitant tests of all OsPIPs, four genes (OsPIP1;3, OsPIP2;1, OsPIP2;2, and OsPIP2;4) were induced to express in leaves of rice plants following a physiological drought stress, while OsPIP2;2 was expressed to the highest level. After de novo expression in frog oocytes and yeast cells, the four OsPIP proteins were localized to the plasma membranes in trimer and tetramer and displayed the activity to increase the membrane permeability to H2O. In comparison, OsPIP2;2 was most supportive to H2O import to oocytes and yeast cells. After de novo expression in tobacco protoplasts, OsPIP2;2 exceeded OsPIP1;3, OsPIP2;1, and OsPIP2;4 to support H2O transport across the plasma membranes. OsPIP2;2‐mediated H2O transport was accompanied by drought‐tolerant responses, including increases in concentrations of proline and polyamines, both of which are physiological markers of drought tolerance. In rice protoplasts, H2O transport and drought‐tolerant responses, which included expression of marker genes of drought tolerance pathway, were considerably enhanced by OsPIP2;2 overexpression but strongly inhibited by the gene silencing. Furthermore, OsPIP2;2 played a role in maintenance of the cell membrane integrity and effectively protected rice cells from electrolyte leakage caused by the physiological drought stress. These results suggest that OsPIP2;2 is a predominant facilitator of H2O transport in relevance to drought tolerance in the plant.

Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

BackgroundPlants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco.ResultsWe designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure.ConclusionWe have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

Intracellular sorting of the tail-anchored protein cytochrome b5 in plants: a comparative study using different isoforms from rabbit and Arabidopsis

Tail-anchored (TA) proteins are bound to membranes by a hydrophobic sequence located very close to the C-terminus, followed by a short luminal polar region. Their active domains are exposed to the cytosol. TA proteins are synthesized on free cytosolic ribosomes and are found on the surface of every subcellular compartment, where they play various roles. The basic mechanisms of sorting and targeting of TA proteins to the correct membrane are poorly characterized. In mammalian cells, the net charge of the luminal region determines the sorting to the correct target membrane, a positive charge leading to mitochondria and negative or null charge to the endoplasmic reticulum (ER). Here sorting signals of TA proteins were studied in plant cells and compared with those of mammalian proteins, using in vitro translation-translocation and in vivo expression in tobacco protoplasts or leaves. It is shown that rabbit cytochrome b5 (cyt b5) with a negative charge is faithfully sorted to the plant ER, whereas a change to a positive charge leads to chloroplast targeting (instead of to mitochondria as observed in mammalian cells). The subcellular location of two cyt b5 isoforms from Arabidopsis thaliana (At1g26340 and At5g48810, both with positive net charge) was then determined. At5g48810 is targeted to the ER, and At1g26340 to the chloroplast envelope. The results show that the plant ER, unlike the mammalian ER, can accommodate cytochromes with opposite C-terminal net charge, and plant cells have a specific and as yet uncharacterized mechanism to sort TA proteins with the same positive C-terminal charge to different membranes.

Formation of Disulfide Bridges by a Single-chain Fv Antibody in the Reducing Ectopic Environment of the Plant Cytosol

Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody fragment consists of the variable light and heavy chain domains from a mouse IgG antibody, which are connected by a flexible linker peptide. In each domain one disulfide bridge is present. The functionality of antibodies, which are normally secreted via the oxidizing environment of the endoplasmic reticulum, depends on the formation of intramolecular disulfide bridges. We demonstrate that a scFv can form intramolecular disulfide bridges and is functionally expressed in the cytosol of stably transformed plants. In addition, the formation of intermolecular disulfide bridges through a cysteine present in the linker peptide was observed. In contrast, transient expression in tobacco protoplasts resulted in a cytosolic scFv lacking disulfide bridges, which had a substantially reduced affinity for the antigen. This indicates that functionality rather than stability is determined by the presence of disulfide bridges in the in planta-expressed scFv antibody. The controversial observation of disulfide bond formation in the cytosol is discussed.

Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import.

A deletion and mutagenesis study was performed on the mitochondrial presequence of the beta-subunit of the F(1)-ATP synthase from Nicotiana plumbaginifolia linked to the green fluorescent protein (GFP). The various constructs were tested in vivo by transient expression in tobacco protoplasts. GFP distribution in transformed cells was analysed in situ by confocal microscopy, and in vitro in subcellular fractions by Western blotting. Despite its being highly conserved in different species, deletion of the C-terminal region (residues 48-54) of the presequence did not affect mitochondrial import. Deletion of the conserved residues 40-47 and the less conserved intermediate region (residues 18-39) resulted in 60% reduction in GFP import, whereas mutation of conserved residues within these regions had little effect. Further shortening of the presequence progressively reduced import, with the construct retaining the predicted N-terminal amphiphilic alpha-helix (residues 1-12) being unable to mediate mitochondrial import. However, point mutation showed that this last region plays an important role through its basic residues and amphiphilicity, but also through its hydrophobic residues. Replacing Arg4 and Arg5 by alanine residues and shifting the Arg5 and Leu6 (in order to disturb amphiphilicity) resulted in reduction of the presequence import efficiency. The most dramatic effects were seen with single or double mutations of the four Leu residues (positions 5, 6, 10 and 11), which resulted in marked reduction or abolition of GFP import, respectively. We conclude that the N-terminal helical structure of the presequence is necessary but not sufficient for efficient mitochondrial import, and that its hydrophobic residues play an essential role in in vivo mitochondrial targeting.

Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue.

Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone. Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Lüscher and Nelson, 1995). It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6(G)-fructosyl transferases. Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast, Pichia pastoris. When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST. When the cDNA was expressed in P. pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate. The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan 6(G)-fructosyl transferase activity. The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.

Characterization of a pine multigene family containing elicitor-responsive stilbene synthase genes.

Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial beta-glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.

Adenine methylation at GATC sequences regulates activity of tobacco PR-1 and PR-2 promoters in electroporated protoplasts

Three different constructs, containing promoters of genes which belong to the Pathogenesis Related family (PR-1a, PR-2d) and CaMV 35S promoter, fused with uidA gene (GUS) as a reporter, were tested individually for expression in tobacco protoplasts. DNA containing constructs were amplified in Escherichia coli K12 strains: DH5α (wild type) or GM33 and GM48 defective in methylation systems (Dam and Dam/Dcm mutants, respectively). In contrast to 35S promoter, marked differences in transient activity of both PR promoters were observed depending on the methylation state of the templates. High levels of GUS expression driven by PR-1a or PR-2d were observed only when the template was obtained from the DH5α strain. In vitro DNA methylation with Dam methylase, prior to protoplast transfection, restored high levels of activity to the investigated promoters. A gel mobility shift assay was used to check if there are any differences in binding of PR-1a promoter fragments, containing GATC or G mATC sequences, with tobacco nuclear extract. Our data suggest that N 6 methyl-deoxyadenine (6-mA), present in GATC sequences, may modulate the regulation of PR-s genes expression.

Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform.

Three genes coding for different multifunctional acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) isoenzymes from Brassica napus were isolated and divided into two major classes according to structural features in their 5' regions: class I comprises two genes with an additional coding exon of approximately 300 bp at the 5' end, and class II is represented by one gene carrying an intron of 586 bp in its 5' untranslated region. Fusion of the peptide sequence encoded by the additional first exon of a class I ACCase gene to the jellyfish Aequorea victoria green fluorescent protein (GFP) and transient expression in tobacco protoplasts targeted GFP to the chloroplasts. In contrast to the deduced primary structure of the biotin carboxylase domain encoded by the class I gene, the corresponding amino acid sequence of the class II ACCase shows higher identity with that of the Arabidopsis ACCase, both lacking a transit peptide. The Arabidopsis ACCase has been proposed to be a cytosolic isoenzyme. These observations indicate that the two classes of ACCase genes encode plastidic and cytosolic isoforms of multi-functional, eukaryotic type, respectively, and that B. napus contains at least one multi-functional ACCase besides the multi-subunit, prokaryotic type located in plastids. Southern blot analysis of genomic DNA from B. napus, Brassica rapa, and Brassica oleracea, the ancestors of amphidiploid rapeseed, using a fragment of a multi-functional ACCase gene as a probe revealed that ACCase is encoded by a multi-gene family of at least five members.

An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication.

Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.

Regulation of African cassava mosaic virus complementary-sense gene expression by N-terminal sequences of the replication-associated protein AC1

Fragments of the African cassava mosaic virus (ACMV) genome, cloned upstream of the beta-glucuronidase (GUS) reporter gene in an expression cassette, were analysed for their ability to direct complementary-sense gene expression in tobacco protoplasts by measuring GUS activity. Five arbitrary domains (A-E) have been designated that contribute to the expression of AC1 (replication-associated protein) and AC4. Consistent with earlier reports, AC1 gene expression was negatively regulated (80% reduction in activity) by its own protein product, and suppression was mimicked by truncated versions of AC1 comprising the N-terminal 57 amino acids. AC1 also suppressed AC4 gene expression to a similar extent. Nucleotide sequences responsible for suppression were mapped to domain A, a 92 bp fragment located immediately upstream of the AC1 initiation codon encompassing the consensus TATA box and transcription start point. Complementary-sense gene expression also decreased by 30-40% in the presence of AV1 (coat protein) although other DNA A-encoded proteins (AV2, AC2, AC3 and AC4) had no effect. The results are discussed in the light of recent advances concerning the initiation of viral DNA replication and the control of gene expression.

Regulatory sequences involved in the peroxidase gene expression in Arabidopsis thaliana

Organ-specific expression of two peroxidase genes (prxCa and prxEa) from Arabidopsis thaliana was studied. The prxCa gene showed non-specific expression with relatively high levels of mRNA accumulation in the roots, stems and leaves of A. thaliana. The prxEa gene, on the other hand, accumulated high levels of mRNA only in roots. Promoter fragments from each gene were fused to the coding region of β-glucuronidase (gusA) reporter gene introduced into tobacco. Promoter/gusA contructs were transferred to tobacco (Nicotiana tabacum BY-2) protoplasts by electroporation or to N. tabacum SR-1 by Agrobacterium tumefaciens-mediated leaf disk transformation. Transient expression in tobacco protoplasts showed that the 580 fragment from prxEa (Ea-580) expressed thirteen-fold and eight-fold higher GUS activity than prxCa (Ca-622) fragment and CaMV35S promoter, respectively. Tobacco plants transformed with the gusA gene, fused to the -580 deletion (Ea-580), exhibited high GUS expression in roots. The root-specific expression of GUS gene was also observed when the -281 bp deletion end point was used. Although the GUS activity in transgenic tobacco under the control of Ca-622 was low, the activity was found in all organs examined. Histochemical analyses of stem and root tissues of Ea-580 showed that the GUS gene was expressed specifically in phloem and pith parenchyma cells. For Ca-622, high level, specific expression of the gusA gene was observed in the xylem of roots. The results of this study implicate multiple cis-elements in the control of transcription from the prxEa promoter.

Transfer RNA-mediated suppression of stop codons in protoplasts and transgenic plants.

We have developed a simple, rapid and sensitive assay for tRNA gene expression in plant cells. A plant tRNA(Leu) gene was site-specifically mutated to encode each of the three anticodon sequences (CUA, UUA and UCA) that recognize, respectively, the amber, ochre and opal stop codons. The suppression activity of these genes was detected by their ability to restore transient beta-glucuronidase (GUS) expression in tobacco protoplasts electroporated with GUS genes containing premature stop codons. Protoplasts co-electroporated with the amber suppressor tRNA gene and a GUS gene containing a premature amber stop codon showed up to 20-25% of the activity found in protoplasts transfected with the functional control GUS gene. Ochre and opal suppressors presented maximum efficiencies of less than 1%. This system could be adapted to examine transcription, processing or aminoacylation of tRNAs in plant cells. In addition, phenotypically normal, fertile tobacco plants expressing a stably incorporated amber suppressor tRNA gene have been obtained. This suppressor tRNA can be used to transactivate a target gene containing a premature amber stop codon by a factor of at least several hundred-fold.

Tomato Golden Mosaic Virus Leftward Gene Expression: Autoregulation of Geminivirus Replication Protein

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two DNA components, designated DNA A and DNA B. DNA A encodes AL1, the only viral protein required for DNA replication. AL1 protein interacts specifically with sequences in the common region that is conserved between the two genome components, near sequences involved in the transcription of complementary sense genes encoding BL1 protein and the AL1 protein itseft. In the experiments described here, we replaced the AL1 and BL1 open reading frames with the β-glacuronidase (GUS) reporter gene and used the gone replacement constructs to examine AL1 and BL1 gone expression in tobacco protoplasts. We found that expression of the GUS reporter in the AL1 replacement construct was reduced to background levels when transfactions included a plasmid expressing AL1 protein from the cauliflower mosaic virus 35S promoter, indicating that AL1 gene expression is autoregulated. Surprisingly, a similar repression of BL1 gene expression by AL1 protein was not observed. Plasmids expressing the TGMV AL2 or AL3 proteins had no significant effect on AL1 or BL1 gone expression. In the course of these studies, we showed for the first time that the product of the AL3 ORF alone is sufficient to complement the replication-deficient phenotype of a TGMV AL3 mutant. The results are discussed in light of the multiple activities of AL1 protein.

Characterisation of LTR sequences involved in the protoplast specific expression of the tobacco Tnt1 retrotransposon.

The tobacco Tnt1 retrotransposon is the only plant retrotransposon that has been shown to be transcriptionally active, and its transcription is strongly induced when preparing leaf-derived protoplasts. We have analysed in this paper the LTR sequences important for Tnt1 expression in tobacco protoplasts. We show that LTR sequences upstream of the TATA box are sufficient to confer protoplast-dependent induction to a heterologous promoter. We also show that this region contains two short activator elements, and that one of these sequences, BII, interacts with protoplast-specific nuclear factors.

The combination of a novel stimulatory element in the first exon of the maize Shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold

Both exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene individually stimulate expression of reporter genes in transient gene expression experiments if present within the transcription unit. The Sh1 exon 1 mediates a 10-fold increase in activity when inserted at the 5' end of the bacterial chloramphenicol transacetylase (CAT) marker gene in both monocot and dicot protoplasts. The Sh1 intron 1 enhances chimeric gene expression in rice and maize protoplasts approximately 100-fold but inhibits CAT expression in tobacco protoplasts. In combination, the stimulatory effects of Sh1 exon 1 and intron 1 are multiplicative in monocot protoplasts resulting in a final enhancement of up to 1000-fold compared to the unmodified CAT or luciferase marker genes.

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone lambda CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on lambda CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspension-cultured cells, the chimeric gene consisting of 1.1 kb CHN50 5' upstream region fused to the coding sequence of beta-glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5' deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.

Direct gene transfer to plant protoplasts by electroporation by alternating, rectangular and exponentially decaying pulses

The efficiency of electroporation of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) protoplasts by alternating, rectangular and exponentially decaying pulses was studied by assaying transient expression of an introduced gene for chloramphenicol acetyltransferase. A simple device for electroporation by alternating current was constructed. The mains (220 V) were used as power supply and the pulse duration was controlled by the blow-out of a small fuse. Electroporation of sugar beet protoplasts by alternating current and exponentially decaying pulses resulted in 3-4 fold higher transient expression compared to rectangular pulses. Transient expression in tobacco protoplasts electroporated by exponentially decaying pulses was 30 % and 85 % higher than when electroporated by rectangular and alternating current pulses, respectively.

Intra- and intramolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase

A site-specific recombination system has many potential uses for rearranging genetic material in higher eukaryotic cells: for example, the control of gene expression by deletion or inversion of DNA segments, the clustering of transgenic constructs via site-specific integration, and the generation of chromosomal translocations. In this report, we describe a first step towards the application of a site-specific recombination system in plant cells. By use of a transient assay, we demonstrate that the bacteriophage P1 cre gene can be expressed as a functional recombinase in tobacco cells. Upon expression in tobacco protoplasts, Cre recognizes its target sites, lox, and mediates reciprocal genetic crossovers at these sites. When the lox sites are present in cis to one another, and arranged in either direct or inverted orientations, we detect Cre/ lox-specific deletion and inversion events, respectively. The placement of lox sites in trans resulted in the co-integration of the substrates by Cre-mediated intermolecular recombination. These results indicate that the Cre/ lox site-specific recombinaton system might be further developed as an additional tool for manipulating DNA in plant cells. Applications relevant to the genetic engineering of higher plants are discussed.

Biological activity of hemimethylated and single-stranded DNA after direct gene transfer into tobacco protoplasts

In this investigation we analyzed the effect of DNA methylation on gene expression in tobacco protoplasts. Methylation of the chimeric GUS gene (CaMV 35s/GUS/NOS) with the HpaII methyltransferase had no effect on transient and on stable GUS gene expression. The chimeric gene was then cloned as single-stranded DNA. In vitro second-strand DNA synthesis was performed with 5-methylcytosine, leading to methylation of every cytosine in one DNA strand. After transfection this DNA was totally inactive. Control experiments revealed that the single-stranded DNA was as active as the double-stranded form.