John Wiley & Sons - 2004 - Analysis of Genes and Genomes

are diluted by diffusion through the membrane into the feeding chamber. This can result in high levels of protein expression by allowing protein synthesis to continue for long periods of up to 24 h (Martin et al., 2001).

Structural genomics promises to increase greatly our knowledge of the types of protein fold that polypeptides may adopt. The danger is that only those proteins that either are easy to crystallize, or are suitably small to be studied by NMR, will have their structures solved. Efforts will still be required to analyse biologically interesting, but structurally difficult, proteins.

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economic benefits. We will discuss some of these issues at the end of this chapter. First, however, we need to understand the differences between animal and plant cells and the mechanisms that can be employed to get foreign DNA – called a transgene – into the latter. One of the major differences between plant and animal cells is that plants retain a high degree of plasticity. That is, an isolated plant cell under appropriate culturing conditions can have the capability of regenerating an entire new plant. This ability has proved vital for the production of engineered plants since individual cells, either as cell cultures or other forms, can be manipulated and then used to reform an intact plant. Recently, a series of in planta transformation techniques have been developed in which plant embryos, seeds or even pollen have been treated to take up foreign DNA (Touraev et al., 1997), but culture and regeneration remains widely used.

11.1.1Agrobacterium Tumefaciens

Plants do not contain any naturally occurring plasmid DNA molecules. However, a bacterial plasmid, the tumour inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens, has been used extensively to introduce genes into plant cells. A. tumefaciens is responsible for crown gall disease in a variety of dicotyledonous plants – such as tomato, tobacco, potato, peas, beans etc. (Figure 11.1). A wound on the stem of the plant allows the bacteria to invade and cause a cancerous proliferation of the stem tissue. A plasmid carried within the bacterium is responsible for its ability to cause crown gall disease (Zaenen et al., 1974; Van Larebeke et al., 1974). This tumour inducing (Ti) plasmid is large ( 200 kbp) and carries a number of genes that are required for the infection process (Figure 11.2) (Suzuki et al., 2000). After infection, part of the Ti plasmid, called the T-DNA, becomes integrated into the plant genome at an apparently random position through non-homologous recombination. T-DNA, approximately 23 kbp in size, contains not only the genes responsible for the cancerous properties of the transformed cells (e.g. those controlling the production of the plant hormones auxin and cytokinin to stimulate cell division and growth) but also those responsible for the synthesis of opines, which are amino acid derivatives (Figure 11.2). Different Ti plasmids direct the synthesis of different opines. The opines produced by the infected plant, e.g. nopaline, which is formed through the reductive condensation of arginine and α-keto-glutarate, can be used by Agrobacterium cells as their sole source of carbon and energy. Other soil bacteria are unable to metabolize opines, and thus these molecules serve to promote the growth of more Agrobacterium cells, and may provide a selective advantage to Agrobacterium over competition from other microorganisms present in the soil. Within the Ti plasmid, the

Figure 11.1. Crown galls formed by the invasion of Agrobacterium tumefaciens into wounds on the stem of a potato plant. These images were kindly provided by Professor Paul Hooykaas (Leiden University, The Netherlands)

T-DNA is flaked by two 25 bp imperfect direct repeats, known as the left and right border sequences (Figure 11.3). These sequences are necessary for the integration of T-DNA into the plant genome (Yadav et al., 1982), and any DNA sequence located between them will be integrated into the plant genome. Once integrated into the plant, the T-DNA is stably maintained and is passed on to daughter cells.

In addition to the T-DNA, the Ti plasmid also carries the genes needed for opine utilization, a series of approximately 35 genes required for virulence (vir) and an Agrobacterium origin of replication (ori) (Figure 11.3). Since the T-DNA fragment becomes integrated into the plant genome, the Ti plasmid offers an excellent mechanism for the introduction of novel or manipulated genes into the plants. First, the T-DNA needs to be ‘disarmed’ so that it is unable to promote the formation of cancerous growths that would be disruptive to gene cloning experiments. To do this, the genes encoding the proteins for the production of auxin and cytokinin are simply removed from the T-DNA fragment (Zambryski et al., 1983). New DNA can then be inserted between the left and right border repeats and will be integrated without concurrent tumour

formation. We have seen previously, however, that large DNA molecules are difficult to manipulate in vitro (Chapter 3) and the size of the Ti plasmid precludes simple cloning procedures. Two basic strategies have been devised for inserting new sequences into the T-DNA region.

•Co-integration vectors. The insertion of new DNA into a Ti plasmid results from the recombination of a small vector plasmid, for example an E. coli vector, and a Ti plasmid harboured in Agrobacterium. The recombination takes place through a homologous region present in both of the plasmids (Matzke and Chilton, 1981).

•Binary vectors. The T-DNA does not need to be physically associated with the virulence genes in order to become integrated into the plant genome (Bevan, 1984). Therefore the disarmed T-DNA can be cloned into a small plasmid and manipulated appropriately. The plasmid can then be transformed into Agrobacterium that possesses a Ti plasmid that still contains all of the virulence genes, but from which the T-DNA has been removed. The virulence proteins provide all the function required to integrate the T-DNA into the plant genome.

The transformation of plants using modified Agrobacterium Ti plasmids, like the transformation of E. coli (Chapter 2), requires selection to identify the transformed cells from their untransformed counterparts. Disarmed T-DNA still contains the genes responsible for opine production, and screening for transformants on the basis of this new biochemical activity is possible, but technically difficult. Most vectors used today contain a selectable marker gene between the left and right border repeats for transformant identification. This is commonly the kanamycin-resistance gene (see Chapter 3), but may also be a gene required for resistance to other antibiotics or herbicides. For example, the csr1-1 gene from Arabidopsis, encoding a form of the enzyme acetolactate synthase, renders plants resistant to sulphonylurea herbicides such as chlorsulphuron (Haughn et al., 1988). As we will discuss later, however, the introduction of resistance genes into plants has caused widespread concern about their dissemination into the environment.

Plant transformation using Agrobacterium is most commonly performed using leaf tissue (Horsch et al., 1985). Small discs are taken from the leaf, using a paper punch, and incubated with the recombinant Agrobacterium. Bacterial invasion occurs at the ‘wounded’ edges of the disk. The infected leaf disc is transferred to a medium containing high levels of cytokinins to induce shoot formation prior to incubation with a mixture of the antibiotics kanamycin and ampicillin – to both select for the transformed plant cells and kill the bacteria.

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Cells surviving this treatment are then used to regenerate plants from tissue culture using plant hormones.

Agrobacterium has proved to be an incredibly useful tool for the insertion of genes into plants (Hooykaas, 1989), but its scope is limited to plants that can be infected by the bacteria. Monocotyledonous plants, such as wheat, barley, rice and maize, are attractive targets for genetic manipulation, but are generally resistant to Agrobacterium infection. The reason for this is not entirely understood. It is possible that the induction of cell division, and consequent synthesis of DNA, that occurs upon wounding in dicotyledonous plants aids the integration process. Wounding of monocotyledonous plants tends to result in lignification. Agrobacterium mediated transformation of rice can occur at high frequency if acteosyringone, a phenolic compound produced by wounded dicotyledonous plant tissues, is included in the transformation reactions (Hiei et al., 1994). A variety of other monocotyledonous plants have also been transformed successfully using Agrobacterium either in the presence of chemicals, or using plant embryos, in which cell division may occur sufficiently rapidly to allow integration of T-DNA (Cheng et al., 1997). Another difficulty with Agrobacterium mediated transformation is the requirement to regenerate intact plants from cell cultures. The ease with which this can be achieved is species dependent and can form a major stumbling block to the generation of transgenic plants.

If the foreign DNA that has been inserted into the plant genome is to be expressed, it must be under the control of a plant promoter. The promoters from the Agrobacterium opine synthesis genes have been widely used to generate high levels of transgene expression that is inducible by wounding (An, Costa and Ha, 1990). In dicotyledonous plants, the promoter from the cauliflower mosaic virus (CaMV) 35S RNA gene is capable of driving high levels of transgene expression (Rathus, Bower and Birch, 1993), but the promoter is not active in monocotyledonous plants. Additionally, a number of promoters have been used to direct the expression of genes in particular tissues. For example, the promoters from several seed storage proteins can be used to target gene expression in the seeds only (Schubert et al., 1994). Other factors, in addition to promoter strength, also play a role in dictating the levels to which transgenes can be expressed. The expression of the transgene will generally occur to higher levels if 5 untranslated sequences and an intron are included within the transgene, and a polyadenylation site is located at its 3 -end. However, the expression of transgenes has been found to be quite variable. Much of this appears to be a result of the location into which the transgene has integrated. The non-homologous recombination that is required to insert a foreign gene into the plant genome means that the foreign gene is inserted randomly.

At some locations in the genome, the expression of a particular transgene may be extremely high, while at other locations the same transgene may be barely expressed at all. Positional effects such as this are particularly noticeable if the foreign DNA is inserted near telomeres. Additionally, the random nature of the integration event often results in multiple copies of the transgene being inserted into the plant genome – either in the form of tandem repeats at a single locus, or scattered throughout the genome of the plant. Rather than increasing the levels at which the foreign gene is expressed, this can lead to silencing of the transgene so that it is not expressed at all (Plasterk and Ketting, 2000). Transgene silencing is a complex process. In an excellent demonstration of the process, Garrick et al. (1998) constructed a transgene that contained multiple repeated copies of a gene. Each of the genes also contained a loxP site (see Figure 3.7). The multiple repeats were expressed very poorly, but if the Cre recombinase was also expressed in the cell (to recombine the loxP sites and eliminate all but one of the gene sequences) then the transgene was highly expressed. In addition, the reduction in gene copy number was also associated with a decrease in chromatin compaction and a decrease in DNA methylation at the transgene locus. These appear to be critical factors for repressing gene activity. Attempts have been made to develop a system for targeted transgene insertion into plants through the introduction of elements of a homologous recombination system, e.g. the Cre/lox system (Kumar and Fladung, 2001). To date, however, these have not yielded consistent results.

11.1.2Direct Nuclear Transformation

The relatively limited spectrum of Agrobacterium infection led to the search for alternative methods for introducing DNA into plant cells. These generally fall into two categories.

(a)Protoplast transformation. A protoplast is a cell that has had its cell wall removed. In plants, this can be achieved by treating tissue with pectinase and cellulase enzymes to, respectively, break up cell aggregates and digest away the cell wall. Protoplasts will synthesize a new cell wall within 10 days, after which they are capable of undergoing cell division and, under appropriate conditions, the regeneration of new plants. In the protoplastic state, cells can be persuaded to take up DNA through either chemical (Negrutiu et al., 1987), electroporetic (Shillito et al., 1985) or liposomal (Hansen and Wright, 1999) means. The DNA inserted into cells in this way is not capable of independent replication. However, it may become randomly integrated into one of the plant chromosomes through a process