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Targeting transgene expression to specific cells and tissues is desirable for a number of reasons (Gittins et al., 2000) not least of which is the requirement to reduce perceived risks with GM technology. In fruit crops, where invariably the product is consumed fresh, this assumes unparalleled importance. To this end, the ability of several heterologous and homologous gene promoters to drive expression of the βglucuronidase (gusA) marker gene in the vegetative tissues of transgenic apple (Malus pumila, Mill.) have and are being tested. These promoters originally drove expression in leaves (Rubisco, small subunit (SSU), RBCS3C from tomato, SRS1 from soybean), in vascular tissue (rolC, and CoYMV) and in root tissue (extA from Brassica). Homologous promoters from apple were cloned for expression in fruit. Transgenic lines were produced by Agrobacterium-mediated transformation and the levels of gusA activity in the vegetative tissues of young plants were compared with those using the CaMV 35S promoter to drive expression of the same gene. Quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with the chromogenic substrate X-Gluc. Light-regulation and patterns of expression are recorded in various vegetative tissues of apple.

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of β-glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.

Within the 5′ flanking regions of two ripening-related genes from apple (Malus pumila), two short repeat sequences were identified and named Ars1 andArs2 (Apple repetitivesequences). Both elements were present in the promoter of the Md-ACS1-1 ACC synthase gene and Ars2was also found in the promoter of a gene homologous to the ABG1 β-galactosidase gene. Database searches revealed other examples of each element in apple genomic sequences and a partial Ars2element in a sequence from Asian pear (Pyrus pyrifolia). DNA dot blot analysis demonstrated that both elements are present in high numbers in the apple genome. Ars1 is thought to represent a MITEminiature inverted-repeat transposable element) related to the MITE Alien, while the identity ofArs2, though it is undoubtedly a transposable element, remains undetermined. DNA dot blot analysis and PCR amplification using primers designed from Ars1and Ars2 showed that elements similar to Ars1 and Ars2 are present across the Rosaceae. The amplification patterns obtained using Ars1- and Ars2-specific primers with genomic DNA from different cultivars or mapping populations suggest that these elements could be used for finger printing and segregation analysis in Rosaceae. Evidence from DNA database searches suggests that they may be applicable to genetic studies in a wide range of other organisms.

The genomic framework that enables corals to adjust to unfavourable conditions is crucial for coral reef survival in a rapidly changing climate. We have explored the striking intraspecific variability in the expression of coral pigments from the green fluorescent protein (GFP) family to elucidate the genomic basis for the plasticity of stress responses among reef corals. We show that multicopy genes can greatly increase the dynamic range over which corals can modulate transcript levels in response to the light environment. Using the red fluorescent protein amilFP597 in the coral Acropora millepora as a model, we demonstrate that its expression increases with light intensity, but both the minimal and maximal gene transcript levels vary markedly among colour morphs. The pigment concentration in the tissue of different morphs is strongly correlated with the number of gene copies with a particular promoter type. These findings indicate that colour polymorphism in reef corals can be caused by the environmentally regulated expression of multicopy genes. High‐level expression of amilFP597 is correlated with reduced photodamage of zooxanthellae under acute light stress, supporting a photoprotective function of this pigment. The cluster of light‐regulated pigment genes can enable corals to invest either in expensive high‐level pigmentation, offering benefits under light stress, or to rely on low tissue pigment concentrations and use the conserved resources for other purposes, which is preferable in less light‐exposed environments. The genomic framework described here allows corals to pursue different strategies to succeed in habitats with highly variable light stress levels. In summary, our results suggest that the intraspecific plasticity of reef corals’ stress responses is larger than previously thought.

In order to study metabolic pathways and utilize them for biotechnology, it is frequently necessary to clone gene clusters that can be tens of kilobases in size. Traditional library construction, followed by screening and sub-cloning, is time consuming and costly. PCR amplification and assembly is more precise, quicker and cheaper, but the DNA polymerases used have an inherent error rate and incorrect fragment pairing can occur. Total gene synthesis can be rapid, but again sequence verification is necessary.

DNA fragments containing the 5' promoter regions of the Pisum sativum sadA and sadC genes were amplified from genomic DNA, cloned and sequenced. These sequences contain a number of conserved cis-acting elements, which are potentially involved in stress-induced transcription of the sad genes. To determine whether any of the identified elements are active in binding nuclear factors in vitro, 11 60-bp overlapping (by 30 bp) DNA probe fragments covering the proximal sadC promoter sequence (360 bp) were used in electrophoretic mobility shift assays with competition. Binding activities were compared in nuclear extracts from control, UV-B-stressed and wounded pea leaves. The pattern of DNA binding was almost identical with all three extracts, with one 30-bp region being the predominant site for factor binding. Using overlapping sub-fragments of this region, the majority of the specific binding could be attributed to the novel 11-bp GC-rich sequence GTGGCGCCCAC. An almost identical sequence is conserved in the sadA promoter. This motif has features in common with a number of recognised cis-elements, which suggests a possible binding site for factors which play a role in regulating sad gene transcription.

The Brassica napus extensin A gene is highly expressed in root tissue of oilseed rape. In an attempt to identify an effective root-specific promoter for biotechnological applications, we have examined the ability of the −940 extA promoter to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill cv. Greensleeves). Transgenic apple lines were produced by Agrobacterium tumefaciens-mediated transformation and GUS activity was analysed both quantitatively and qualitatively. The extA promoter was active in all tissues of young plants in all 15 clones examined. However Southern blot data suggested that only a proportion of the population contained the entire promoter and that others had suffered deletions of unknown length. This may have contributed to the variation seen in the quantitative and qualitative expression of GUS. Specific GUS activity was highest in the stems where it approached, and in some clones, exceeded that using the constitutive CaMV 35S promoter. Histochemical analysis confirmed that GUS was localised to tissues involved in structural support of the stem. Staining was particularly intense at nodal junctions where high tensile stress is exerted on the tissues. Maturing phloem tissues showed localisation of expression to the phloem parenchyma cells and phloem fibres. Transverse sections of the root revealed staining of primary procambial tissues including the young endodermis but no staining was seen in the cortex. Although the −940 extA promoter is clearly not root-specific in apple, it is likely to have useful biotechnological applications in tree species.

The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif. A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a. at the C-terminus, and a much larger 728 a.a. sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E. coli and purified. The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis. The 91 a.a. protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a. protein has very low ( approximately microM) affinity for the same sequence. The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a. sequence C-terminal to the zinc finger. Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein. These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA. We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91).

It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) small- subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max (h.) Mers.), to drive expression of the b-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approxi- mately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple.

A copper resistance gene cluster (6 genes, ∼8.2kb) was isolated from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery (RR). Following integration of a narrow-host-range plasmid vector adjacent to the target region in the Synechocystis genome (pSYSX), DNA was isolated from transformed cells and the plasmid plus flanking sequence circularized by recombineering to precisely clone the gene cluster. Complementation of a copper-sensitive Escherichia coli mutant demonstrated the functionality of the pcopM gene encoding a copper-binding protein. RR provides a novel alternative method for cloning large DNA fragments from species that can be transformed by homologous recombination.