4 Yeast Molecular Techniques

In this brief overview, we will concentrate on approaches that have been useful in yeast recombinant DNA technology rather than consider the plethora of genetic and biochemical techniques that have made yeast biology so successful in the past decades. Some standard compilations of general procedures employed in studying structural, genetic or biochemical aspects of yeast cells [Broach et al., 1991; Guthrie & Fink, 1991; Mortimer et al., 1992; Johnston, 1994] have already been mentioned in the Introduction (chapter 1).

4.1 Isolation of Particular Cell Types and Components

Figure 4-1: Isolation of specific components from yeast cells. Figure 4-1 summarizes the approaches which are still in use to produce yeast cells synchronized in terms of cell cycle phase and isolation of yeast spheroblasts, for the isolation of intact nuclei, or respiratory competent mitochondria, and cellular components. A specialized device used to isolate synchronized cells by continuous flow centrifugation is the so-called elutriator. The preferred technique, however, to synchronize yeast cells is blocking of the cell cycle by mating factor.

4.2 Yeast Vectors

Genetic engineering, i.e. transformation of yeast cells with recombinant DNA, became feasible for the first time in 1978 [Beggs, 1978; Hinnen et al., 1978]. Since then, recombinant DNA technology in yeast has established itself, and a multitude of different vector constructs are available. Generally, these plasmid vectors (shuttle vectors) contain genetic material derived from the E.coli vector

pBR322 (or its derivatives) and a genetic element (origin of replication) which enable them to be propagated in E.coli cells prior to transformation into yeast cells and a selectable marker (mainly the lactamase gene, amp) for the bacterial host (Figure 4-2).

Figure 4-2: Yeast shuttle vectors. Additionally, the shuttle vectors enharbor a selectable marker (Figure 4-3) to be used in the yeast system. Conventionally, markers are genes encoding enzymes for the synthesis of a particular amino acid or nucleotide, so that cells carrying the corresponding genomic deletion (or mutation) are complemented for auxotrophy or autotrophy. Further, these vectors contain a sequence of (combined) restriction sites (multiple cloning site, MCS) which will allow to clone foreign DNA into this locus. Convenient markers developed for the screening of large collections of mutant cells are the lacZ gene or the kanamycin-resistance gene (Kan) gene. The chloramphenicol-resistance gene (cat) [Mannhaupt et al., 1988] or the luciferase gene can be integrated into vectors in combination with promoter sequences from yeast to monitor expression levels.

Figure 4-3: Markers in yeast recombinant DNA technology. Principally, four types of shuttle vectors can be distinguished (Figure 4-2) by the absence or presence of additional genetic elements: - Integrative plasmids (YIp) which by homologous recombination are integrated into the host genome at the locus of the marker, when this is opened by restriction and linearized DNA is used for transformation. This (normally) results in the presence of one copy of the foreign DNA inserted at this particular site. - Episomal plasmids (YEp) which carry part of the 2 plasmid DNA sequence necessary for autonomous replication. Multiple copies of the transformed plasmid are propagated in the yeast cell and maintained as episomes. - Autonomously replicating plasmids (YRp) which carry a yeast origin of replication (ARS sequence) that allows the transformed plasmids to be propagated several hundred-fold. - Cen plasmids (YCp). In addition to an ARS sequence these vectors carry a centromeric sequence (derived from one of the nuclear chromosomes) which normally guarantees stable mitotic segregation and reduces the copy number of self-replicated plasmid to just one. To date, transformation of yeast cells may be achieved by three principal approaches:

- Permeabilization of cells by treatment with Li-acetate [Ito et al., 1983] - Electroporation - Bombardement of cells by DNA-coated tungsten micro projectiles.

4.3 Yeast Expression Vectors 4.3.1 Regulated Promoters

Yeast expression vectors will employ promoter and terminator sequences in addition to the gene of interest. It is advantageous to use yeast-derived (homologous) rather than heterologous sequences, because the former are more efficient, and heterologous elements will sometimes not work in yeast. Figure 4-4 lists some of the promoter modules that are in use. Constitutive promoters are derived from genes of the glycolytic pathway, because these lead to high-level transcriptional expression.

Figure 4-4: Promoter elements in yeast expression vectors. On the other hand, regulated promoters can be controlled by controlling the availability of certain nutrients. This allows to augment yeast cell mass prior to heterologous gene expression, so that the cell population can be optimized before the regulated promoters are turned on.

4.3.2 Secretion of Heterologous Proteins from Yeast

Protein secretion in yeast is a complex process and there is no generally accepted signal sequence which directs secretion. Although several foreign proteins can be secreted under the direction of their

own signals, homologous signal sequences are much more successful and can result in highly expressed heterologous proteins recoverable from the extracellular medium. Frequently used signal sequences in S. cerevisiae include those derived from invertase (SUC2), acid phosphatase (PHO5) or -factor pheromone (MF1; Figure 4-5). It is of value that the specificity of the signal processing enzymes for the -factor precursors allows for the production of heterologous proteins with authentic N-termini.

Figure 4-5: Processing and secretion of -factor in yeast.

4.3.3 Post-translational Processing and Modification of Heterologous Proteins in Yeast

Another important molecular aspect of recombinant proteins expressed in yeast are the features of post-translational processing and modification processes specific to yeast, particularly with attention to therapeutic agents produced in yeast. N-and O-linked glycosylation patterns in yeast may prove to be different from those in the native host. For example, yeast adds mannose units to threonine or serine residues, while higher eukaryotes prefer sialic acid O-linked side chains. Such differences may affect the folding, stability, activity and immunogenicity of proteins produced in yeast. By contrast, N-linked glycosylation in yeast largely resembles that of higher eukaryotes. Attention has also to be paid to possible differences in phosphorylation, acetylation, methylation, myristylation and isoprenylation of proteins in yeast towards other organisms. Once synthesized and modified, heterologous proteins produced in yeast may undergo intracellular proteolytic degradation before they can be purified. In S. cerevisiae, proteolysis may be unspecific and associated with the vacuole, or specific and coupled to the ubiquitin-proteasome system.

4.3.4 GFP Fusion Proteins

A relatively recent development of labelling proteins involves the green fluoresent protein (GFP) as a reporter molecule for intracellular localization and in vivo gene expression studies [e.g., Niedenthal et al., 1996]. Fusion proteins with the conventional GFP moiety (some 200 amino acids in length) can be visualized by fluoresecence microscopy at 395 nm (blue light). Interestingly, two variants of GFP, having particular amino acid replacements, are now available which will emit fluorescent light of lower (red) or higher (blue) wavelengths. In most cases, the globular extension in the modified protein will not influence its intracellular localization nor its function as compared to the native protein, independent of whether the GFP moiety has been fused to the N-terminus or to the C-terminus. However, this has to be checked for each protein of interest individually. Variants of the native GFP are available, the genes of which have been modified such that they are adapted to codon usage in plants, and these have proven to be advantageous in expression also in the yeast system (Figure 4-6).

Figure 4-6: GFP vector constructs.

4.4 Yeast Cosmid Vectors

Cosmid vectors have proven to be very convenient for cloning and sequencing of large segments of yeast chromosomal DNA. To construct a library with as complete coverage as possible with as few clones as possible, the cloned DNA fragments should be randomly distributed on the DNA. Under

these conditions, the number of clones (N) in a library representing each genomic segment with a given probability (P) is N = ln (1-P)/ln (1-f) where f is the insert length expressed as fraction of the genome size [Clarke & Carbon, 1976]. For example, with the size of 12,800 kb for the yeast genome and assuming an average insert length of 35 kb, a cosmid library containing 4600 random clones would represent the yeast genome at P=99.99%, i.e. about twelve times the genome equivalent. The actual number of cosmid clones obtained by the usual procedures is very high (>200,000/g DNA).

Figure 4-7: Yeast cosmid vectors. One of the first yeast cosmid vectors, pHC79, was developed in 1980 [Hohn and Collins]. In connection with the yeast genome sequencing programme, two major types of cosmids have been employed (Figure 4-7). (i) pYc3030 generated from pCH79 by adding the yeast 2m plasmid origin of replication and the yeast HIS3 marker is a shuttle vector that most conveniently allows DNA to be shuttled between E. coli and yeast cells [Stucka and Feldmann, 1994]. It contains a BamH1 cloning sites which is suitable for

accommodating yeast DNA fragments of ca. 30-45 kb in size obtained by partial digestion of high molecular weight DNA with Sau3A. For cloning, the vector arms comprising the -phage cos-sites have to be prepared separately and are ligated to a mixture of partial Sau3A fragments that have been size-fractionated by centrifugation of the digestion mixture in NaCl gradients. Replica plating which is one of the common procedures used for the storage and screening of cosmid libraries has been successfully applied to yeast cosmid libraries. Colonies can be easily purified, and cosmid DNA can be prepared by one of the 'mini-prep' procedures. We found that yeast cosmid can be stored at -20C for several years without damage. Cosmids have not only been used successfully for chromosomal walking, but also in complementation analyses; cosmids are maintained in yeast cells in only one or a few copies. (ii) pWE15 (and pWE16) are cosmid vectors that have been designed for genomic walking and rapid restriction mapping [Dujon et al., 1993]. They contain bacteriophage T3 and T7 promoters, respectively, flanking a unique BamH1 cloning site. By using the cosmid DNA containing a genomic insert as a template for either T3 or T7 polymerase, directional 'walking' probes can be synthesized and used to screen genomic cosmid libraries (or sublibraries) These vectors contain additional genes (SV2-neo or SV2-dhfr, respectively) which allow the expression, amplification and rescue of cosmids in mammalian cells. NotI restriction sites have been placed near the BamH1 site which allow the insert to be removed as a single large fragment.

4.5 Yeast Artificial Chromosomes (YACs)

The construction of YACs follow a similar strategy as that of the ARS/CEN plasmids [Burke et al., 1987]. In addition to the usual components, they are endowed with telomere sequences flanking a yeast marker gene (HIS3 in pYAC4; Figure 4-8); restriction sites flanking the telomere sequences can later be used to linearize the plasmid DNA for yeast transformation. The insertion site for large foreign DNA segments is located within a second 'marker' gene, the SUP4 gene encoding a suppressor tRNA, which allows selection of transformed cells that possess the appropriate genetic background. As the linearized plasmids behave like endogenous chromosomes, they are maintained and replicated in the same manner as resident yeast chromosomes. The only caveat in the use of YACs, which has been noticed particularly in conjunction with the Human Genome Project, is that YACs might undergo recombination in yeast.

Figure 4-8: Yest artificial chromosomes.

4.6 The Yeast Two-Hybrid System

The yeast Two-hybrid system has been developed as a potent tool to identify cDNAs, carried on one plasmid, which code for proteins that interact with a target protein specified by a DNA sequence carried on another plasmid [Fields and Song, 1989]. The two-hybrid assay is based on the fact that the yeast Gal4p transcriptional activator is composed of two physically separable, functionally independent activation and binding domains (Gal4-AD and Gal4-BD, respectively). The cloning vectors, which are endowed with different markers, are used to create fusions of the GAL4 domains with genes for proteins that potentially interact (Figure 4-9). After introduction into a yeast strain that carries an appropriate reporter gene (HIS3 or lacZ) with a GAL4 UAS element in its promoter, if the two domains interact, the DNA-BD will be tethered to the AD, and will reconstitute the Gal4 transcriptional activator, which results in the activation of the reporter gene. Selection can be made by screening for His+ or lacZ+ positives, and the GAL4-AD/library fusion plasmid can efficiently be retrieved from such colonies. The method has been improved since its invention, particularly to minimize the appearance of false positives, which however still seems to be a problem not completely overcome.

Figure 4-9: Principle of the yeast two hybrid system.

4.7 The Yeast One-Hybrid (Matchmaker) System

The yeast one-hybrid system provides the basic tool for conducting a one-hybrid assay - an in vitro genetic assay used for isolating novel genes encoding proteins that bind to a target, cis-regulatory element or any other short, DNA binding sequence. The one-hybrid assay offers maximal sensitivity because detection of the DNA-protein interactions occur while proteins are in their native configurations in vivo. In addition, the gene encoding the DNA binding protein of interest is immediately available after a library screening (Example in Figure 4-10). To conduct a one-hybrid assay, a sequence consisting of tandem copies of a known DNA element is inserted upstream of the HIS3 and lacZ reporter gene promoters (present on separate vectors). Subsequently, the reporters are integrated site-specifically into the yeast genome to create the new yeast reporter strains. After construction of the reporter strains, the cDNA candidates encoding the protein of interest (sometimes from a complete yeast genomic plasmid library) are expressed as fusion proteins with a target-independent GAL4 activator domain. A GAL4-AD library can be screened for a cDNA encoding a DNA-binding protein of interest. After transforming the modified yeast reporter strain with an AD fusion library that contains candidate cDNA clones and plating, if an AD/library hybrid protein interacts with the target element, the HIS3 reporter is expressed, allowing colony growth on minimal medium lacking histidine. If a HIS3/LacZ reporter strain is used, a -galactosidase assay can be performed to verify the DNA-protein interaction and help eliminate false positives. The -gal assay can be conducted as an 'overlay' test. Figure 4-10 presents a specific example, in which the factor Rpn4 interacting with a particular UAS element (PACE) has been cloned [Mannhaupt et al. 1999].

Figure 4-10: Application of the Matchmaker system.

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