Brian Peter Surin

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successfulAgrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph(encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4- fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 car-rying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher s-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity com-parable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activ-ity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

A suite of novel promoters and terminators for plant biotechnology

The gene regulation signals from subterranean clover stunt virus (SCSV) were investigated for their expression in dicot plants. The SCSV genome has at least eight circular DNA molecules. Each circular DNA component contains a promoter element, a single open reading frame and a terminator. The promoters from seven of the segments were examined for their strength and tissue specificity in transgenic tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and cotton (Gossypium hirsutum L.) using a GUS reporter gene assay system. While the promoters of many of the segments were poorly expressed, promoters derived from segments 4 and 7 were shown to direct high levels of expression in various plant tissues and organs. The segment 1 promoter directs predominantly callus-specific expression and, when used to control a selectable marker gene, facilitated the transformation of all three species (tobacco, potato and cotton). From the results, a suite of plant expression vectors (pPLEX) derived from the SCSV genome were constructed and used here to produce herbicide- and insect-resistant cotton, demonstrating their utility in the expression of foreign genes in dicot crop species and their potential for use in agricultural biotechnology.

A suite of novel promoters and terminators for plant biotechnology. II. The pPLEX series for use in monocots

A suite of plant expression vectors (pPLEX), constructed from the gene regulation signals from subterranean clover stunt virus (SCSV) genome, has previously been used in dicot transformation for a variety of applications in plant biotechnology. To assess their use for the transformation of monocots, a number of modifications were made to the basic vector series and assessed in rice. In their unmodified forms, the SCSV promoters directed low levels of gene expression, however, insertion of an intron between the promoter and the transgene open reading frame (analogous to the rice actin and maize ubiquitin promoter systems) increased transgene expression 50-fold. The expression patterns from the intron-modified SCSV (segments 4 and 7) promoters were very similar to those directed by the actin or ubiquitin promoters. All promoter systems investigated directed expression that appeared to be constitutive within leaf tissue, and localised to the epidermal and vascular tissues of the root. The pPLEX vectors described here are an important counterpart to the dicot pPLEX series and have the potential to be useful in monocot research and biotechnology.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5[prime] and 3[prime] Sequences.

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5[prime] and 3[prime] regions of the Flaveria bidentis Me1 gene fused to the [beta]-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5[prime] region of Me1 determines bundle sheath specificity, whereas the 3[prime] region contains an apparent enhancer-like element that confers high-level expression in leaves. The interaction of 5[prime] and 3[prime] sequences was dependent on factors that are present in the C4 plant but not found in tobacco.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5 and 3 Sequences

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5’ and 3’ regions of the F/ayeria bidentis Mel gene fused to the P-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5‘ region of Mel determines bundle sheath specificity, whereas the 3’ region contains an apparent enhancer-like element that confers highleve1 expression in leaves. The interaction of 5‘ and 3’ sequences was dependent on factors that are present in the C4 plant but not found in tobacco.

GroEL/ES-mediated refolding of human carbonic anhydrase II: role of N-terminal helices as recognition motifs for GroEL

The presence of GroEL/ES during the refolding of human carbonic anhydrase II (pseudo-wild type) was found to increase the yield of active enzyme from 65 to 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time-course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonic anhydrase, in which N-terminal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.

Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization.

Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences.

The symmetry of Escherichia coli cpn60 (GroEL) determined by X-ray crystallography

The internal symmetries of the Escherichia coli molecular chaperone cpn60 oligomer, also called GroEL, have been examined by X-ray crystallography and self-rotation functions calculated at a resolution of 8.9 A. The oligomer ([cpn60]14) has one 7-fold symmetry axis and seven 2-fold axes that are all perpendicular to the 7-fold. The symmetry can be explained if oligomeric cpn60 is arranged as two heptamers stacked on top of each other, where the heptameric arrangement generates the 7-fold symmetry axis and the head-to-head assembly of two heptamers results in the seven 2-fold axes. This is an agreement with interpretations of electron microscopy data. However, the experimental determination of the symmetries reported here are made with an independent technique and at higher resolution. In addition self-rotation function calculations show that the symmetries observed are valid also for the internal parts of GroEL and not only for surface views. The orientations of the symmetry axes of the two independent cpn60 oligomers in the triclinic unit cell have been determined relative to the crystallographic axes. The planes formed by the 2-fold axes in the two oligomers deviate by about 2 degrees from the plane formed by the crystallographic a and c axes, while the 7-fold axes form angles of about 16 degrees with the b-axis. The two oligomers in the unit cell are arranged with their 7-fold axis parallel, but the second oligomer is rotated 26 degrees around the 7-fold axis relative to the first oligomer. Knowledge of the symmetry and orientation of the oligomers in the unit cell will be of great help in further crystallographic work.