Transient gene expression in cassava using high-velocity microprojectiles

The bacterial gene encoding -glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter fromArabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3 ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.     

Transient reporter gene (GUS) expression in creeping bentgrass (Agrostis palustris) is affected by in vivo nucleolytic activity

Leaf and callus tissues of a creeping bentgrass cultivar (Penn A4) had high nuclease activities that degraded exogenously added plasmid DNA. When callus tissue was incubated for 24 h with heparin, spermidine, aurintricarboxylic acid or polyethylene glycol, only heparin and spermidine were effective as in vitro nuclease inhibitors, protecting exogenously added plasmid DNA from degradation. When -glucuronidase (GUS) reporter gene activity was evaluated in heparin-treated (0.6%), 14-month old callus following microprojectile bombardment, GUS activity increased 1000-fold compared to equivalent aged untreated Penn A4 callus. Similar enhancement from heparin pretreatment (0.6% or 1.2%) was not observed in 6-month old callus. This is likely due to much higher activities of nuclease in the younger callus.     

Locus-control-region-coupled betaS Antilles- and alpha2-hemoglobin genes select for high alpha2-hemoglobin expression in adult transgenic mice

Two transgenic lines of mice were produced which contained the ^S _Antilles- and ₂-hemoglobin genes trandemly coupled to the micro locus control region (LCR). The LCR^S _Antilles2-hemoglobin transgenic mice expressed high levels of ₂-hemoglobin while ^S _Antilles-hemoglobin expression was virtually undetectable. Abundant ₂-hemoglobin protein was observed in the blood of transgenic mice, while ^S _Antilles-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of -thalassemic mice. The LCR^S _Antilles2 transgenic mice demonstrate that if the LCR is coupled to the ^S _Antilles- and ₂-hemoglobin genes in tandem, only the distal ₂-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

Homologous recombination between plasmid DNA molecules in maize protoplasts

Summary The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for -glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB×26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adhl gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 by (Hindlll—PvuII) fragment of the yAdh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3 end of the pB×26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.     

Agrobacterium-mediated transient transformation of annatto (Bixa orellana) hypocotyls with the GUS reporter gene

Hypocotyls from annatto seedlings, were inoculated with Agrobacterium tumefaciens harboring a binary vector, pBI.121 or pCAMBIA2301, containing the -glucuronidase (gus) gene. Histochemical GUS assay of infected hypocotyls from two annatto varieties showed transient gus gene expression between 3 and 12 days after inoculation.These authors contributed equally to this work.)     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Calmodulin gene family in potato: developmental and touch-induced expression of the mRNA encoding a novel isoform

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca²⁺-binding area. The expression patterns of different genes were studied by northern analysis using the 3-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the -glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.     

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from –44 to –79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.