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HAPTER 9 Cloning in Saccharomyces cerevisiae and other fungi Outline ●Introduction, 156 ● Introducing DNA into fungi, 156 ● The fate of DNA introduced into fungi, 156 ● Plasmid vectors for use in fungi, 158 ● Retrovirus-like vectors, 159 ● Expression of cloned genes, 163 ● Overexpression of proteins in fungi, 165 ● Specialist vectors, 166 ● Yeast surface display, 168 ● Identifying genes encoding particular cellular activities, 171 ● Determining functions associated with particular genes, 171 1. Introduction The analysis of eukaryotic DNA sequences has been facilitated by the ease with which DNA from eukary-otes can be cloned in prokaryotes, using the vectors described in previous chapters. Such cloned sequences can be obtained easily in large amounts and can be altered in vivo by bacterial genetic techniques and in vitro by specific enzyme modifications. Ideally these eukaryotic genes should be reintroduced into the organism from which they were obtained. In this chapter we shall discuss the potential for cloning these genes in Saccharomyces cerevisiae and other fungi and in later chapters we shall consider methods for cloning in animal and plant cells. 2. Introducing DNA into fungi Like Escherichia coli, fungi are not naturally transformable and artificial means have to be used for introducing foreign DNA. One method involves the use of spheroplasts (i.e. wall-less cells) and was first developed for S. cerevisiae (Hinnen et al. 1978). In this method, the cell wall is removed enzymically and the resulting spheroplasts are fused with ethylene glycol in the presence of DNA and CaCl2. The spheroplasts are then allowed to generate new cell walls in a stabilizing medium containing 3% agar. This latter step makes subsequent retrieval of cells inconvenient. Electroporation provides a simpler and more convenient alternative to the use of spheroplasts. Cells transformed by electroporation can be selected on the surface of solid media, thus facilitating subsequent manipulation. Both the spheroplast technique and electroporation have been applied to a wide range of yeasts and filamentous fungi. 3. The fate of DNA introduced into fungi In the original experiments on transformation of S. cerevisiae. Hinnen et al. (1978) transformed a leucine auxotroph with the plasmid pYeLeu 10. This plasmid is a hybrid composed of the enterobacterial plasmid ColEl and a segment of yeast DNA containing the LEIJ2* gene and is unable to replicate in yeast. Analysis of the transformants showed that in some of them there had been reciprocal recombination between the incoming LEU2+ and the recipient Leu2~ alleles. The results described above can be confirmed by restriction-endonuclease analysis, since pYeLeu 10 contains no cleavage sites for Hindlll. When DNA from the Leu2~ parent was digested with endonucle-ase Hindlll and electrophoresed in agarose, multiple DNA fragments were observed but only one of these hybridized with DNA from pYeLeu 10. During transformation, the integration of exogenous DNA can occur by recombination with a homologous or an unrelated sequence. In most cases, non-homologous integration is more common than homologous recombination (Fincham 1989), but this is not so in S. cerevisiae (Schiestl & Petes 1991). Schiestl and Petes (1991) developed a method for forcing illegitimate recombination by transforming yeast with BamHl -generated fragments in the pre-ence of the BamHl enzyme. Not only did this increase the frequency of transformants but the transformants which were obtained had the exogenous DNA integrated into genomic BamHl sites. This technique, which is sometimes referred to as restriction-enzymemediated integration (REMI), has been extended to other fungi, such as Cochliobolus , UstUago and Aspergillus . 4. Plasmid vectors for use in fungi If the heterologous DNA introduced into fungi is to be maintained in an extrachromosomal state then plasmid vectors are required which are capable of replicating in the fungal host. Four types of plasmid vector have been developed: yeast episomal plasmids (YEps), yeast replicating plasmids (YRps), yeast centromere plasmids (YCps) and yeast artificial chromosomes (YACs). These inhibitors permit the ready selection of those rare cells which have undergone a recombination or loss event to remove the plasmid DNA sequences. The Lys2 gene is not utilized frequently because it is large and contains sites within the coding sequence for many of the commonly used restriction sites. 5. Yeast episomal plasmids YEps were first constructed by Beggs (1978) by recombining an E. coli cloning vector with the naturally occurring yeast 2 |xm plasmid. This plasmid is 6.3 kb in size, has a copy number of 50-100 per haploid cell and has no known function. A representative YEp is shown in Fig. 9.2. 6. Yeast replicating plasmids YRps were initially constructed by Struhl et al. (19 79). They isolated chromosomal fragments of DNA which carry sequences that enable B. coli vectors to replicate in yeast cells. Such sequences are known as ars (autonomously replicating sequence). An ars is quite different from a centromere: the former acts as an origin of replication (Palzkill & Newlon 1988, Huang & Kowalski 1993), whereas the latter is involved in chromosome segregation. 7. Yeast centromere plasmids Using a YRp vector, Clarke and Carbon (1980) isolated a number of hybrid plasmids containing DNA segments from around the centromere-linked leu2, cdclO and pgk loci on chromosome III of yeast. As expected for plasmids carrying an ars, most of the recombinants were unstable in yeast. However, one of them was maintained stably through mitosis and meiosis. The stability segment was confined to a 1.6 kb region lying between the leu2 and cdc10 loci and its presence on plasmids carrying either of two ars tested resulted in those plasmids behaving like minichromosomes . 8. Yeast artificial chromosomes All three autonomous plasmid vectors described above are maintained in yeast as circular DNA molecules even the YCp vectors, which possess yeast centromeres. Thus, none of these vectors resembles the normal yeast chromosomes which have a linear structure. The ends of all yeast chromosomes, like those of all other linear eukaryotic chromosomes, have unique structures that are called telomeres. Telomere structure has evolved as a device to preserve the integrity of the ends of DNA molecules, which often cannot be finished by the conventional mechanisms of DNA replication . Thus, there is no practical limitation to the size of a YAC and they are essential tools in any genome-sequencing project. The method for cloning large DNA sequences in YACs developed by Burke et al. (1987) is shown in Fig. 9.3. 9. Retrovirus-like vectors The genome of S. cerevisiae contains 30-40 copies of a 5.9 kb mobile genetic element called Ty (for review see Fulton et al. 1987). This transposable element shares many structural and functional features with retroviruses (see p. 193) and the copia element of Drosophila. Ty consists of a central region containing two long open reading frames (ORFs) flanked by two identical terminal 334 bp repeats called delta (Fig. 9.4). The Ty element has been modified in vitro by replacing its delta promoter sequence with promoters derived from the phosphoglycerate kinase or galactose-utilization genes (Garfinkel et al. 1985, Mellor et al. 1985). When such constraints are introduced into yeast on highcopy-number vectors, the Ty element is overexpressed. This results in the formation of large numbers of virus-like particles (VLPs), which accumulate in the cytoplasm (Fig. 9.5). The Ty element can also be subjugated as a vector for transposing genes to new sites in the genome. The gene to be transposed is placed between the 3' end of ORF 2 and the 3' delta sequence (Fig. 9.6). 10. Choice of vector for cloning There are three reasons for cloning genes in yeast. The first of these relates to the potential use of yeast as a cloning host for the overproduction of proteins of commercial value. Yeast offers a number of advantages, such as the ability to glycosylate proteins during secretion and the absence of pyrogenic toxins. Commercial production demands overproduction and the lactors aliecting expression ol genes in yeast are discussed in a later section (see p. 165). Yeast is also used in the production of food and beverages. For many biologists the primary purpose of cloning is to understand what particular genes do in vivo. Thus most of the applications of yeast vectors have been in the surrogate genetics of yeast. One advantage of cloned genes is that they can be analysed easily, particularly with the advent of DNA-sequencing methods. The availability of different kinds of vectors with different properties (see Table 9.1) enables yeast geneticists to perform manipulations in yeast like those long available to E. coli geneticists with their sex factors and transducing phages. Thus cloned genes can be used in conventional genetic analysis by means of recombination using YIp vectors or linearized YRp vectors 11. Plasmid construction by homologous recombination in yeast During the process of analysing a particular cloned gene it is often necessary to change the plasmid's selective marker. Alternatively, it may be desired to move the cloned gene to a different plasmid, e.g. from a YCp to a YEp. Again, genetic analysis may require many different alleles of a cloned gene to be introduced to a particular plasmid for subsequent functional studies. Suppose we wish to move the HIS 3 gene from pBR328, which cannot replicate in yeast, to YEp420 (see Fig. 9.7). Plasmid pRB328 is cut with PvuI and PvuII and the HIS3 fragment selected. The HIS3 fragment is mixed with YEp420 which has been linearized with EcoRI and the mixture transformed into yeast. 12. Expression of cloned genes When gene manipulation in fungi first became possible, there were many unsuccessful attempts to express heterologous genes from bacteria or higher eukaryotes. This suggested that fungal promoters have a unique structure, a feature first shown for S. cerevisiae (Guarente 1987). Four structural elements can be recognized in the average yeast promote (Fig. 9.8). First, several consensus sequences are found at the transcription-initiation site. Two of these sequences, TC(G/A)A and PuPuPyPuPu, account for more than half of the known yeast initiation sites . These sequences are not found at transcription-initiation sites in higher eukaryotes, which implies a mechanistic difference in their transcription machinery compared with yeast. The second motif in the yeast promoter is the TATA box (Dobson et al. 1982). This is an AT-rich region with the canonical sequence TATAT/AAT/A, located 60-120 nucleotides before the initiation site. Functionally, it can be considered equivalent to the Pribnow box of E. coli promoters . The third and fourth structural elements are upstream activating sequences (UASs) and upstream repressing sequences (URSs). These are found in genes whose transcription is regulated. 13. Qverexpressiort of proteins in fungi The first overexpression systems developed were for S. cerevisiae and used promoters from genes encoding abundant glycolytic enzymes, e.g. alcohol dehydrogenase (ADH1), PGK or glyceraldehyde-3-phosphate dehydrogenase (GAP). These are strong promoters and mRNA transcribed from them can accumulate up to 5% of total. They were at first thought to be constitutive but later were shown to be induced by glucose (Tuite et al. 1982). Now there is a large variety of native and engineered promoters available (Table 9.2), differing in strength, regulation and induction ratio. These have been reviewed in detail by Romanos et al. (1992). The ideal promoter is one that is tightly regulated so that the growth phase can be separated from the induction phase. This minimizes the selection of nonexpressing cells and can permit the expression of proteins normally toxic to the cell. The ideal promoter will also have a high induction ratio. One promoter which has these characteristics and which is now the most widely used is that from the GAL1 gene. Galactose regulation in yeast is now extremely well studied and has become a model system for eukaryotic transcriptional regulation (see Box 9.1). Following addition of galactose GAL1 mRNA is rapidly induced over 1000-fold and can reach 1% of total mRNA. However, the promoter is strongly repressed by glucose and so in glucosegrown cul-tures this induction only occurs following depletion of glucose. To facilitate galactose induction in the presence of glucose, mutants have been isolated which are insensitive to glucose repression (Matsumoto et al. 1983, Horland et al. 1989). In recent years, methylotrophic yeasts, such as Pichia pastoris, have proved extremely popular as hosts for the overexpression of heterologous proteins. There are a number of reasons for this. First, the alcohol oxidase (AOX1) promoter is one of the strongest and most regulatable promoters known. Second, it is possible to stably integrate expression plasmids at specific sites in the genome in either single or multiple copies. Third, the strains can be cultivated to very high density. To date, over 300 foreign proteins have been produced in P. pastoris (Cereghino & Cregg 1999,2000). Promoters for use in other yeasts are shown in Table 9.2. 14. Specialist vectors Many different specialist yeast vectors have been developed which incorporate the useful features found in the corresponding E. coli vectors (see p. 70), e.g. an fl origin to permit sequencing of inserts production of the cloned gene product as a purification fusion, etc. Some representative examples are shown in Fig. 9.9. Two aspects of these vectors warrant further discussion: secretion and surface display. Fig. 9.9 Two specialized vectors for use in Saccharomyces (YES vectors) and Pichia (pPICZ). V5. Express, 6XHis and c-myc encode epitopes which can be readily detected and purified by affinity chromatography. The YES vectors offer a choice of 2 ųm origin for high copy or CEN6/ARSH4 origin for low copy in yeast, a choice of UR A 3 or TRP1 genes for auxotrophic selection on minimal medium, blasticidin resistance for dominant selection in any strain and an f 1 origin to facilitate DNA sequencing. In the pPICZ vectors, the zeocin-resistance gene is driven by the EM-7 promoter for selection in E. coli and the TEF1 promoter for selection in Pichia. More details of these features can be found in Chapter 5. In yeast, proteins destined for the cell surface or for export from the cell are synthesized on and translocated into the endoplasmic reticulum. From there they are transported to the Golgi body for processing and packaging into secretory vesicles. Fusion of the secretory vesicles with the plasma membrane then occurs constitutively or in response to an external signal (reviewed by Rothman & Orci 1992). Of the proteins naturally synthesized and secreted by yeast, only a few end up in the growth medium, e.g. the mating pheromone a factor and the killer toxin. The remainder, such as invertase and acid phosphatase, cross the plasma membrane but remain within the periplasmic space or become associated with the cell wall. Polypeptides destined for secretion have a hydrophobic amino-terminal extension, which is responsible for translocation to the endoplasmic reticulum (Blobel & Dobberstein 1975). The extension is usually composed of about 20 amino acids and is cleaved from the mature protein within the endoplasmic reticulum. 15. Yeast surface display S. cerevisiae can be used to elucidate and dissect the function of a protein in a manner similar to phage-display systems. Either can be used to detect protein ligand interactions and to select mutant proteins with altered binding capacity (Shusta et al. 1999). However, phage-display systems often cannot display secreted eukaryotic proteins in their native functional conformation, whereas yeast surface display can. In practice, the gene fusion is somewhat more complicated. Usually the cloned gene product is sandwiched between two simple epitopes to permit quantitation of the number of fusion proteins per cell and to determine the accessibility of different domains of the fusion protein (Fig. 9.10). 16. Detecting protein-protein interactions Chien et al. (1991) have made use of the properties of the GAL4 protein to develop a method for detecting protein-protein interactions. The GAL4 protein has separate domains for the binding to UAS DNA and for transcriptional activation. Plasmids were constructed which encode two hybrid proteins. The second consisted of residues 768-881 of the GAM protein, representing the activation domain, fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the test protein (bait) and a protein encoded by one of the library plasmids (prey) led to transcriptional activation of a reporter gene (Fig. 9.11). A major operational problem with the twohybrid system has been the high frequency of false positives, i.e. cells in which the reporter gene is active even though the bait and prey do not interact in nature and/or in yeast. There are two general solutions to this problem. In the first of these (James elal. 1996), a host strain is used that contains three reporter genes under the control of different GAL4-inducible promoters. 17. Identifying genes encoding particular cellular activities As a result of a major genome-sequencing project, the sequences of genes encoding every biochemical activity of S. cerevisiae are known. The next task is to connect specific biochemical activities with particular genes. Martzen et al. (1999) have developed a rapid and sensitive method for doing this which is applicable to almost any activity. To correlate genes with activity, the different trans-formants were dispensed into 64 96-well microtitre plates (64 x 96 = 6144). Initially, all the clones from each plate were pooled and the 64 pools analysed for a specific biochemical activity. Analysis is facilitated since the GSTORF fusions are readily purified from other cellular activities by affinity chromatography using immobilized glutathione. Once a plate was identified as having the desired activity, pools of clones were made from each of the eight rows and 12 columns of wells and these 20 pools reassayed. If only one clone expresses the desired activity, then one row and one column should be identified (Fig. 9.13) and the point of intersection identifies the wanted clone. Sequencing of the ORF then allows identification of the gene corresponding to the activity. 18. Determining functions associated with particular genes Determining the function of a particular gene product usually involves determining the expression profile of the gene, the subcellular location of the protein, and the phenotype of a null strain lacking the protein. Conditional alleles of the gene are often created as an additional tool. These procedures usually require multiple independent manipulations. To use this transposon system, yeast genes are cloned in an E. coli strain that overexpresses the Tn3 transposase. The transposon, carried on a derivative of the sex factor F, is introduced by mating and transposition ensues. Yeast DNA containing the transposon is excised from the E. coll vector and transformed back into yeast, with selection made for URA3 activity. An alternative approach to the large-scale mutational analysis of the yeast genome is to individually delete each gene using a polymerase chain reaction (PCR)-based methodology (Winzeler et al. 1999). For each gene, a deletion cassette is constructed (Fig. 9.15) that contains a kanamycin resistance gene, two 'molecular barcodes' and yeast sequences homologous to the upstream and downstream flanking sequences. Fig. 9.15 'Knockout' strain collection, (a) The bar-coded deletion cassette is generated in two rounds of PCR. It contains a kanamycin-resistance gene for selection in yeast and two molecular bar-codes 20 bp long that are unique to each deletion. The molecular bar-codes are flanked by common sequence elements that allow for the PCR amplification of all the bar-codes in a population of yeast strains. Atthe end of the cassette are45 bp sequences that are homologous to sequences flanking the gene of interest, (b) The deletion cassette is transformed into diploid yeast strains. The 45 bp of yeast sequences allows one copy of the gene of interest to be replaced with the deletion cassette via homologous recombination. The resulting heterozygous strains (c) can then be sporulated and dissected to generate a collection of haploid mutant strains, if viable (d). Homozygous mutant strains (e) are made by the selective matings of haploid segregants. The molecular bar-codes are particularly important since they are unique to each deletion strain within a pool of many different strains (Shoemaker et al. 1996). The bar-code can be amplified by PCR, using common primer sequences that flank the bar-code. A pool of yeast mutant strains can thus be analysed in a single experiment by amplifying the bar-codes and probing a DNA microarray (p. 116) containing sequences complementary to the bar-codes. Variations on this technique have been used in other organisms to identify key functions associated with particular activities. See you next time! Thank you