158 CHAPTER 9 Schiestl and Petes (1991) developed a method for forcing illegitimate recombination by transforming yeast with BamH1-generated fragments in the pres- tence of the BamE1 enzyme. Not only did this increase the frequency of transformants but the transformants which were obtained had the exogenous DNA integ- rated into genomic Baml{1 sites. This technique, which is sometimes referred to as restriction-enzyme- mediated integration (REMI), has been extended to other fungi, such as Cochliobolus (Lu et al. 1994), Ustilago (Bolker et al. 1995) and Aspergillus (Sanchez, etal, 1998). Plasmid vectors for use in fungi Ifthe heterologous DNA introduced into fung! 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). All of them have features in common. First, they all contain unique target sites for anumber of restriction endonucleases. Secondly, they can all replicate in E. coli, often at high copy number. This is important, because for many exper- iments it is necessary to amplify the vector DNA in E, coli before transformation of the ultimate yeast recipient, Finally, they all employ markers that can be selected readily in yeast and which will often complement the corresponding mutations in E. coli as well. The four most widely used markers are His3 Leu2, Trp] and Ura3. Mutationsin the cognate chro- mosomal markers are recessive, and non-reverting, mutants are available, Two yeast selectable mark- ers, Ura3 and Lys2, have the advantage of offering. both positive and negative selection, Positive selec- tions for complementation of auxotrophy. Negative selection isfor ability to grow on medium containing, @ compound that inhibits the growth of cells ex- pressing the wild-type function. In the case of Ura3, it is 5-fluoro-orotic acid (Boeke et al. 1984) and for Lys2 it is c-aminoadipate (Chatoo et al. 1979). ‘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 itis 4 reni—4 Cla 185901 rey —! ea a wot pipr2011 Soh Sorat Fig. 9.2. Schematic representation ofa typical yeast episomal plasmid (YBp 24). The plasmid can replicate both in P. colt {duet the presence ofthe pBR322 origin ofreplication) and Ins. cerevisiae (due tothe presence ofthe yeast 2 4m origin of replication}, The ampicilin and tetracycline determinants, are derived from pBR322 and the URA3 gene from yeast. large and contains sites within the coding sequence for many of the commonly used restriction sites. Yeast episomal plasmids YEps were first constructed by Beggs (1978) by recombining an E. coli cloning vector with the nat- urally occurring yeast 2 um 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 repres- entative YEpisshown in Fig, 9.2, Yeast replicating plasmids YRps were initially constructed by Strubl etal, (1979) ‘They isolated chromosomal fragments of DNA which, carry sequences that enable E. coli vectors to replic- ate 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.Cloning in S. cerevisiae and other fungi 159 Although plasmids containing an ars transform yeast very efficiently, the resulting transformants areexceedingly unstable. For unknown reasons, YRps tend to remain associated with the mother cell and are not efficiently distributed to the daughter cell (Note: S. cerevisiae does not undergo binary fission but buds off daughter cells instead.) Occasional stable transformants are found and these appear to bbe cases in which the entire YRp has integrated into a homologous region on a chromosome ina manner identical to that of YIps (Stinchcomb et al. 1979, Nasmyth & Reed 1980), 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 ew2, ede10 and pgk loci on chromosome Ill of yeast. ‘As expected for plasmids carrying an ars, most ofthe recombinants were unstable in yeast. However, one of them was maintained stably through mitosis and meiosis. The stability segment was confined toa 1.6 kb region lying between the leu2 and cde10 loci and its presence on plasmids carrying either of two ars tested resulted in those plasmids behaving like minichromosomes (Clarke & Carbon 1980, Hsiao & Carbon 1981). Genetic markerson the minichromo- somes acted as linked markers segregating in the first meiotic division as centromere-linked genes and, ‘were unlinked to genes on other chromosomes. Structurally, plasmid-borne centromere sequences have the same distinctive chromatin structure that ‘occurs in the centromere region of yeast chromo- somes (Bloom & Carbon 1982). Functionally YCps; exhibit three characteristics of chromosomes in yeast cells, First, they are mitotically stable in the absence of selective pressure. Secondly, they segreg- ate during meiosis in a Mendelian manner. Finally, they are found at low copy number in the host cel. Yeast art ficial chromosomes Allthree autonomous plasmid vectors described above are maintained in yeast as circular DNA molecules — even the ¥Cp vectors, which possess yeast cen- tromeres. 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 linea have unique structures that are called telomeres. ‘Telomere structure has evolved as a device to pre- serve the integrity of the ends of DNA molecules, which often cannot be finished by the conventional mechanisms of DNA replication (for detailed dis- cussion see Watson 1972). Szostak and Blackburn (1982) developed the first vector which could be maintained asa linear molecule, thereby mimicking a chromosome, by cloning yeast telomeres into a YRp. Such vectors are known as yeast artificial chro- ‘mosomes (YACS). One advantage of YACs is that, unlike the other plasmid vectors, their stability Increases as the size of the insert increases. Thus, there is no prac- tical 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. ‘eukaryotic chromosomes, 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). Bach delta element contains a promoter as well as sequences recognized, by the transposing enzyme, New copies of the trans- poson arise by a replicative process, in which the Ty transcript is converted to a progeny DNA molecule by a Ty-encoded reverse transcriptase. The comple- mentary DNA can transpose to many sites in the host DNA. The Ty element has been modified in vitro by replacing its delta promoter sequence with pro- moters derived from the phosphoglycerate kinase or galactose-utilization genes (Garfinkel etal. 1985, Mellor etal. 1985), When such constraints are intro- duced into yeast on high-copy-number vectors, the ‘Ty element is overexpressed. This results in the formation of large numbers of virus-like particles160 Target DNA, Feoat cloning ste CHAPTER 9 “TRP ARS CEN @__Sa_a iT) Una ‘TRP ARS CEN ta «a_i aa —_—_—____® Primary wanscipt ‘one fF 2 Structure ofa typical Ty element. ORF 1 and ORF 2 represent the two open reading frames. The delta soquences are indicated by LTR (long terminal repeats) (VLPs), which accumulate in the cytoplasm (Fig. 9.5). ‘The particles, which have a diameter of 60-80 nm, have reverse-transcriptase activity. The major struc~ tural components of VLPs are proteins produced by proteolysis of the primary translation product of Fig.9.3 Construction ofa yeast artificial chromosome containing large pleces ofcloned DNA. Key regions ofthe BYAC vector are asfollows: TEL, yeast felomeres: ARS I, autonomously replicating sequence; CEN 4, centromere from chromosome 4; URA3 and TRP', yeast marker genes; urate | ampicillin-resistance determinant of pBR322: or, origin ofreplication of BR322. ORF 1. Adams et al. (1987) ha proteins can be produced in cells by inserting part of ‘a gene from human immunodeficiency virus (HIV) into ORF 1, Such fusion proteins formed hybrid HIV.Ty-VEPs. 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). Providing the inserted gene lacks transcription- termination signals, transcription of the 3° delta sequence will occur, which is a prerequisite for transposition. Such constructs act as amplification cassettes, for, once introduced into yeast, transpost- tion of the new gene occurs to multiple sites in the genome (Boeke et al. 1988, Jacobs etal. 1988). shown that fusion