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ION x XND CELL t~IOL~Y 5~ Ann ~e~, In~, Da~I~ Pp. 35~371 Review Article Schizosaccharotnyces pombe: A Model for Molecular Studies of Eukaryotic Genes YUQI ZHAOt and HOWARD B. LIEBERMAN ABSTRACT Several features of the fission yeast Schizosaecharomyces pombe make it exceptionally well suited for the study of eukaryotie genes. It is a relatively simple eukaryote that can be readily grown and manipulated in the laboratory, using a variety of highly developed and sophisticated methodologies. Schizosaecharomyces pombe cells share many molecular, genetic, and biochemical features with cells from multicelluiar organisms, making it a particularly useful model to study the structure, function, and regulation of genes from more complex species. For examples, this yeast divides by binary fission, has many genes that contain introns, is capable of using mammalian gene promoters and polyadenylation signals, and has been used to clone mammalian genes by functional eomplementation of mutants. We present a summary of the biology of S. pombe, useful features that make it amenable to laboratory studies, and molecular techniques available to manipulate the genome of this organism as well as other eukaryotic genes within the fission yeast cellular environment. INTRODUCTION NItJMEROIIS F-'~XTURE.q OF THE FISSION YEAST Schizosac- charomyces pombe make it an attractive model system for molecular genetic studi~ of eukaryotic genes. This small, relatively simple eukaryote possesses many of the same fundamental cellular propetlies of larger, multicellular organisms. However, it can be manipulated in the laboratory readily and has a relatively short doubling time, making it amenable to many types of experiments. Furthermore, S. pombe has a relatively small genome size, approximately 1.5 x 107 bp in the haploid state, which is about three times larger than that ofEscherichia coli. Allolh~r useful featare of S. pombe is that it usually exists as a haploid organism. This characteristic facilitates the creation and identification of mutants. In addition, ceils c~n be mated to constrt~:t with desired genetic backgrotmds and to study genes in haploid a> ~,ell as diploid ceils. Haploids, diploids, and tetrads can be purillcd and used for subsequent genetic analysis. The budding yeast S, wcharomyces cerevisiae has been studied extensively. Thcretbre, what makes S. pombe, at the least, another attractive nmdel system for studying eukary- otie genes? Even though S. cerevisiae andS. pombe are both ascomycetes, they are not very closely related evolution- arily. Protein sequence comparisons have indicated that S. pombe is nearly as distant from S. cerevisiae as it is from mammals (Russell, tqSg) At the gene level, mammalian introns can be spliced properly in fission yeast (Kaufer et al.. 1985L and inlrons within S. pombe genes are also appropri- ately spliced when a component of the yeast spliceosome, 02 snRNA, is replaced by a human equivalent (Shuster and Gathrie, 1990L In addition, mammalian promoter and poly(A) ~ign~s ,ae l'mtctiortal in S. pombe b~t not in S. cere~i~tae (Russell, 1950 ). The con~er~ation of gene ber~.een S, pombe ar~,~ mammals wa~ clearly demonstrated Center for Rad~ological Research. Col!ege of Ph~ sicia~ and S~rgeor, x. Columbia Uni* or,., ,. N ~ ~ Yo:~.. NY 10032. ~P~eser~ add*e~s: l)¢par~men~ of ecd:amcs. N o.-'r.hweslem Uni~,ers.D Med~¢a! Cen:er. 23~ ,, ~i.-i' .lr:-i', Plaza. CTeica~,. IL f'X)514. 359

~ T I~ L'q %T'£F~' O E.FECTI ON O~ Zi~\ C F'RO?,$ I I IV - I s~ L~ clinical ~sol~:~ of ~'-I, i~cl~ding s~n~ n~cleoside in,biter 3' ~d~T.3'-di~eoxy~hymi~i~e (AZ~ or to the ca~p~unds wcr~ also foun~ to be s}narg~s~ic wi~ A~. 2'.3'- at (1995] ~e. in pre~). ~ md eo~rkers have ~m~nd~ ~t by "at~ng" ~h~ two zi~ fi~g¢~ of ~V nucle~apsid protein (NCpT). E~idene* will he presented hhat ~n~O~ of ~e ~-HIV disul~d¢ ~n~ni~ e~t ~e from ~V N~7 in ~i~o. ~ m~nimmd by ~c zinc-sg~tfie tluor~nI ~r~ TSQ. S~c~ly si~ di~a~de ~ides ~at ~e ~ot ~6-~1 do not ej~ ~. ~r do ~og$ of ~ ~dv~ com~aa~ "xi~ ~c disulfide ~pla~d wi~ a me~yl~e sat~de. ~* ~ of NCp7 zi~ ~mdt$ w~ found to ~ ~n-tat~abte .~M bicx~a~ffl. mm of ejecdon ~om ~= C-m~ fi~c finger 7-fold f~ ~ ~at ~m • e N-~. ~e ~v~M ¢om~ were fou~ to inh[bh ~ fine- ~nd~t b~ing of NCp7 to ~ ~A. ~ smfli~ by gel z~t ~, ~ ~elamd well with ~ z~e e~eedon da~. ~afi-~ disulfide ~n~d~ ~peeific~y e~t NC~7 zi~ ~d ~o2ish ~e pro~in% abitiw to bind V RMA in rico. providing eviden~ for a possible ~tke~ovizA meeh~ism of aet~n of ~e~ commune. ~ngene~ of under adv~¢~ preeli~i~l ewluatioa ~ a ~*tcntial chemo~e~py for g~uked i~u~efieiency GRO~ArFH PROPERTI~q OF HSIVnot : HIV CONTAINING THE NEF GENE FROM PAT/"IC-X3EIqlC MOLECULAR CLONE SFVmnc239, Yoont, Mi Ran Kangt, Hye Young Leet, Harry W. Kestler, IIIz, Sung S. R.he~, and Sunyo',mg KimL tInstitute for Molecular Biology and Genetics, Se~.tl National University, Korea; 2The Cleveland Clirdc Foundation, US.A., ~San'tsuix$ Biomedical Re*a=rch Institute, Korea To elucidate the function of he/, we constructed infectious chimeric don~ between HIV-I and SIV o~ macaques by deleting a part of the mf sequence from the HIV-I genome arid in~Iting the eatlre he[ gene from the pathogenic molecular clone SlVmac239" resu,tting in H$1Vne[. HSIV di~ers from SHIV previouMy reported by the fact that our virt~ contains the HIM-I baeJtboa~ Two full-length proviral HIV-1 donma, HXB2 and NL432, were used to construct HSIV by deleting 250 bp of the nef gene startkng from its start ¢odon and installing a tmique Noti site. This deletion left the HIV-I lmlypurine tract and the ease U3 and R regio~ intact but removed the ne, f gene, Th~ con~truct~ were generated for each proviral done: (1) An~, which has a the n~ reglorq (2) ~-NgF, which ¢ontalns SIV net in a • er~e orientation; (3) HSIV-FEN, whir.h harbors SlY n~ in an antisense orientatlon. Together with the original provlra,l clones, the~e cor~tructs were transft<:ted tnto the human embr,/or2e kidney cell line 293T. Ala c~nsWacts produced I0-30 ng/rrd p2~ a.r~tige~ three days s~ggesting that the hybrid c~nstnac~ could normally express and pro<e~ gene products. Fartherm0re, cel~-free vh.-ust~ obtruded ~rom these transfect~ons pro<l~c~vdy EXPI.IEJ'.',IO~'I OF HIV-I ~ GEI'~E INDUCES ClIAPIGE23. I, ' FISSIO;~ YE~.~F ~¢hlrol|cchuro~¥¢~ 12~_~_~SIMILA.R TO INDUCED IN IIUMAN CKLLS, Yaq| Z~o'-~o Ji~ ~o'. O'G~t z. Jun I ~s~', ~,~ g~ Yogtvt-t 'HIM ~m f~ ~ildrca and ~ aS a m~:l S~(~ [o ~dy CCt~ ¢ff~ Of HIV-1 ~ e~pve~ion, ~c ~ ~.~ac '~ cl~ into ~¢~ deriva~v~ of~c inducible ceil culture. 5 <:

A~ST?-.:~CT~ OF THE -~:-~ CONFE27.NC: Sessio~ 8. Slide Virology: Reverse Transcription and Regulation Monday, 9:45 a.m. P.ETI2-.OVI:UL':~, AND 072OXTU.NI:C INEEC"TIOXg CharacleriT~ti+w~ ~f HIV.I Matdx Function: Analysis 0f %qral P.e+ertaat+. ERIC O. F~D" AND M&LCOLM A. NIAID. NIH. h=lh:~a biD Ihos~ that: i) :,~.<ked virus assembly, ii) rcditcctcd compar/mcnH. Hi) :nlcrfercd with virus entry, or iv) blocked Env i.,.U,l:uration into virions without affecting for two posit+~m 12 mutations which blocked Ear lncor~oration i~t< :'~ric, a5, ~nd a Gln->Lys s~bstituti~n I~S[~C 27 (i~ d,c L~A hlgbly-ha~ic do~tln) rcvet~¢d the undcrslandi~ MA structur~functlon rclalions~ips and dclincating tim role , MA in [he HW-I life cyct¢ will 21 C)'cle C2 .*.r~c+t in Fk,~I~o

DN,~, £~D CELL P~. 35~-371 Review Article Schizosaccharomyces pombe: A Model for Molecular Studies of Eukaryotic Genes YUQI ZHAO~ and HOWARD B. LIEBERMAN ABSTRACT Several features of the fission yeast Schizosaccharomyces pombe make it exceptionally well suited for the stud.v ofeukaryotic genes. It is a relatively simple eukaryote that can be readily grown and manipulated in the laboratory, using a variety of highly developed and sophisticated methodologies. Schizosaccharomyces pombe cells share many molecular, genetic, and biochemical features with cells from multicellular organisms, making it a particularly useful model to study the structure, function, and regulatio~ of genes from more complex species. For examples, this yeast divides by binary fission, has many genes that contain introns, is capable of using mammafian gene promoters and polyadenylation signals, and has been ~ to clone mammalian genes by functional complementation og mutants. We present a summary ofthe biology of S. pombe, useful features that make it amenable to laboratory studies, and nmlecuim" techniques available to manipulate the genome of this organism as well as other eukaryotic genes within the fission yeast cellular environment. INTRODUCTION NUMEROUS FI~ATURES OF THE FISSION YEAST Schizosac- charomyces pombe make it an attractive model system for molecular genetic studies of eukaryofic genes. This small, relatively simple eukaryote pos~sses many of the same fundamental cellular properties of larger, multicellular otganisrns. However, it can be manipulated in the laboratory readily and has a r~latively short doubling time, making it amenable to many types of experiments. Furthermore, S. pombe has a relatively small genome size, approximately !.5 >: 107 bp in the haploid state, which is about three times larger than that of Escherichia call Another useful future of S. pombe is that it usmally exL,'ts as a haploid organism. This ch~,'acteri~tie facilitates the creation and identification of mutants. In addition, celIs can be mated to cotmruct strains wilh desired genetic backgrounds and to study genes in haploid as ~©I1 as diploid cells. Haploid% diploids, and tetrads can be purified and used for subsequent genetic analysis. The budding yeast Saccharorayces cerevisiae has bex)n studied extensively. Therefore, what makes S. pombe, at the least, another attraetive model system for studying eukaty- otic genes? Even though S. cerevisiae and S. pombe ate both ase, omycetes, they are not very. closely related evolution- arily. Protein sequence comparisons have indicated that S. pombe is nearly as distain from S. cerevisiae as it is from mammals (Russell, 1989). At the gene level, mammalian inttons can be spliced properly in fission yeast (Kaufer et al., 1985), and introns within S. pombe genes axe al~o appropri- ately spliced when a component of the yeast spliceosome, U2 snRNA, is replaced hy a human equivalent (Shaster and Gtlthrie, 1990). In ~ddifion, mammalian promoter and poly(A) signals are fm~cti~nal in S. pombe but nr>t in S. cerevisiae lRussell, 1989). The conservation of gene activity between S. pvmbe and rn:~rnmals was clearly demonstrated Ce~a~r for R~ological Research. College c,t Physician~ 'Pre~erl ~dd~zs~: Dep~"tme~ ,~f Pediatrics, 59~4~16

c~'c2 Cell c2,,-Ie control llaman CDC2tls Lee ~d Nar~z 1P5S71 cd,:25 Cell c~cle control Haman CDC25Hu2 Nagata ¢t al. (1991) ~'¢el Cell c:~c]e ¢~ntrol Human WEEIHu Igamshi et aL { I§91 ) rad9 Rafliore~sis ~tan~ Mous~ homo!og~ Zhao a al. (!~2) urn4 Uracil biosyn~esis M~e ho~log O~i etal. (1~) by the functional compIementation of a number ofS. pombe mutants by cognate genes from mammalian cells ~Tablc 1 ). Schizomccharomyces pombe can therefore provide a novel host system to investigate the structure, function, and expres- sion of cukaryotic genes, especially those of mammalian origin. Many molecular genetic techniques are now available to pursue these goals. General reviews concerning aspects of S. pombe cell and molecular biology, as well as classical genetic approaches to study this organism, have been publishe, d previously (Miteh- ison, 1970; Gutz etal., 1974; Egel etal., 1980; Nasim etal., 1989; Moreno etal., 1991). The goal of this minireview is to provide an outline of the molecular teelmiques currently available for manipulating S. pombe in the laboratory and to draw attention to the usefulness and experimental tractability of this relatively simple organism for studying eukaryotic genes of both yeast and mammalian origin. BIOLOGY OF S. POMBE Schizosaccharomyces pombe is an ascomycete that di- vides by binary fission. Hence, it is referred to as a "fission ],,east." Each cell is normally 3-4 I~m in diameter and 7-15 p,m in length. Its specific length corresponds well with its growth phase in the cell cycle (Mitehison, 1970; Alfu et al., 1993), which is similar to that of other eukaryotes, "and includes the Gt, S, G2, and M (mitosis) phases (see Fig. 1 ). However, S. pombe contains an extended G2 pha~ that carl make up as much as 75% of the cycle (Mitchison, 1970). In addition, the S. porabe nuclear envelope remains intact throughout mitosis. Therefore, all transactions involving the chromosomes during this phase occur intranuelearly. Fttr- thermore, there is a delay between mitosis and tl~ complete separation ofdaughter cells, resulting in the conclusion of Gt before cellular division is finished. Thus, ceils initially exidng mitosis contain the 2C content of DNA (Alfa etal., 1993). Schizosaccharomyces pombe grows optimally at approxi- matel~' 3&C in the laboratory and has a doubling time of 2-4 hr, depending upon the genetic background of the cell and the growth medium used for culturing. On agar medium, S. pombe forms cream to tan, bubrous colonies fBarnett etaL, 1990). The life ~,cle orS. pombe ~ haplontic, Le., ascxuaI ceils are normally haploid IFig. IL Tbere are prirmmly t~o Ol:-,posite mating t}pes, designated as h ' lheterolhalhc plusl or h- (hetert-~hallic minus). The most commonly u~d strains contain the h+s and h-s versions, respectively, of the mating types. Mating can occur between heterothalli¢ h* and h- cells or as a self-cross of homothallie strains (e.g., h%, which possess both mating type characteristics (Gutz etal., 1974). Mating can be induced by the stre~ of nutritional depletion (Egel and Egel-Mitani, 1974). Both h÷ and h- ceils secrete a diffusible mating pheromone that attracts cells of the opposite mating" type (Egei and EgeI-Mitani, 1974; Leupold, 1987). Fusion of the two cells results in the formation of diploid zygotes. Spomlation is followed imme- diately by meiosis to produce four haploid round or oval ascospores that are 1-2 Ixm in diameter and enclosed within an aseus. The ascus wall will disintegrate, and aseospores will germinate "and eventually divide to foml haploid clones, when the cells are refed appropriate nutrients (Leupold, Ascospores FIG. 1. Zygote Meiosis ~"~~S po r u lati o n The Schizosacd',arom~ces pembe cell cycle.

MODEL FOR ~IOLECULAR STL~IES 2Ual ~a::Iff ct ~., 1994). P-th=t~r '~iI1 c~m~rcially (~ alpha fa:tor is fcrS. cere:isi:eL allowing g~'a'~ ~st in G~ ~ ~e pa~uit of m~ny cu~ently im~siMe to T~e gentle linage ~oups were identified in S. ~mb¢ by ~lysis of recombination t?~uencies ~tween {Kehli ct aL, 1977; Munz ct at., 1989). Coffes~ndingly, ~ c~omosomes have ~so ~n vis~ized ~ micros- copy (Robinow, 1977). ~ c~omosomes we~ sub~- quenfly found to ~ 5.7, 4.6, ~d 3.5 Mb (~gab~s) long, ~fively, ~ ~e~i~ by pulse-field gel elec~ophore- sis (PFGE; Smi~ et al., 1988). A ~ysicM ~p of the S. ~echmmo~ h~ also ~en ~lis~d ~d it consists of ~ Not I ~s~cfi~ e~ si~s p~nt wit~ ge~ (F~ el al., 1989). ~is m~es it ~ible to ~sign ge~ of inte~st to s~ific res~fi~ e~yme-genem~ DNA ~nts by using Sou~em blott~g ~ related techn~ues (S~lhem, 1975). More ~ 5~ S. pombe genes Mve ~n idenfifi~ ~ f~, ~d ~e map ~ti~ of at l~t 2~ have ~n ~te~in~. A list of ~own gen~ and, if ~te~in~, ~ir c~omo~ map ~siti~s can ~ found in Ko~i (1987), Mu~ et al. (1989), ~d Lennon ~d ~hrach (1~2). STRAIN SOURCES Schizosaccharomyces pombe can b~ found naturally in a variety of habitats including arak marshes, molasses, cane sugar, grapes, apples, palm wine, and Bantubac, r (Barnett et al., 1990). However, most of the currently studied strains of S. pombe have been derived from th~ same haploid wild-type parents, 972h-, 975h÷, and 968h~ (Moreno et al., 1991). These three strains arc isogenic and were obtained originally by Leupold (1950) from a culture of S. pombe Lindner str. liquefaciens (OsterwaldeO. Numerous mutant derivatives have been created and successfully maintained. Collections of S. pombe mutant swains have been amassed by and are available from a variety of organizations, including ATCC (Am~iean Ty~ Cultu~ Collection, 12301 Paridawn Drive, Rockville, MD 20852-1776, USA), CBS (Cent~dbure~u ,,'oor ScMmmclcultums, P. O. Box 273, 3740 AG BAARN, Th~ Neth~lands), and th~ National Collection of Yeast Cultures (AFRC Institute of Food Research, Colncy Lane, Norwich NR4 7UA, UK). CONSTRUCTION OF MUTANT $. POMBE STRAINS The generation of a mutant S. pombe strain is normally the first step toward the initial isolation of a specific gene and the under~tanding of its biological function. A number of muta- genie agems can be used to alter me genome ofS. porabe, and some of these, as well as a procedme to enrich for mutant c¢lis in a predormnmatly wild-type population, are described below. F~be, incl~/l~g h~d;,.,>.~la:~ir2, niuc~s a:iJ. e~yl rc~:h- an~,ulfe~t~ ~ EMS L ~:~ ?,'-m~lhyl-?,"ni:ro-,~uni~os~g~m:i- din~ (N~G). EMS is thz most comm:nly u~ed ~gent. mmagens often used IMorcno ct a[., 19~1}. It ~ferentiMly i~duces GC-t~AT transitions ~Sega, 1984) and is also ca~ab~ of generating point mutations in yeast ~t~hon~al DNA (Smolinska, 1987). In Mditi~n, the num~r of muta- tions indued ~r su~.iving cell is high, m~ing efficient at inducing genomic Mtemfiom (Mo~ et 1~1). ~ot~ols t~r EMS mutagenesis ~ ~ found in Hayles etaL { 19~} and Moreno et at. (1~1). NTG is also a ve~" efficient mutagen ~M~cno et af., 1~1) ~d is used often to c~ate mutations. ~e ~¢hanism of mutation induction by ~G is not clear, M~oa# it h~ ~en s~cu- luted that this command reacts wi~ DNA to in~u~ d~g DNA replication IAdelberg et aL, 1~5). ~ols d~fibing the um of NTG have ~en descried by Uemum ~ Y~a#~ (1984) and Moreno et al. (1~1). Me~ for t~ u~ of nilus acid :~s a mutagen ~e pre~nted by Gu~ (1961) ~d Guglielminetti et al. (1967). ~e use of hydrox- ylmine is de~fi~d by Guglielminetti et al. (1967). Physical agents as mumgens Ultraviolet (UV) light and ionizing radiation le.g., X-rays, ~/-rays. etc.) are useful for introducing mutations. It is known that UV predominantly generates pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts in yeast DNA (Bimboim and Nasim, 1974). These lesions can lead to mutations if not removed (Fabre, 1972; Genmer and Wemer, 1975). A recent study by Zhao et al. (1994), using a gene located on a plasmid as a target, suggested that thymi- dine dimers, in particular, play an essential role in generating mutations in S. porabe. Ionizing radiation can induce several lesions, including DNA base damage, but single- and dou- ble-strand DNA breaks are primarily formed and can also lead to mutation if not properly repaired (Bomham and Mitchel, 1991). However, relative to EMS, radiation, in getmtal, is not an efficient mutagenizing agent and is not used as frequently as chemicals to alter the S. pombe genome. Enriched selection qf mutants Becaus~ induced or especially spontaneous mutatio~s aris¢ with low frequency, attempts were made to develop varies methods for the "enriched selection" of fission yeast mutants. Methods simJk, r in principle to the pe~ieillin tech- nique for selecting auxotrophic mutants in bacteria, as des~ibed in the early 1960s (Gorini and Kaufman, 1960), have been applied to S. pombe. This strategy involves the of agents that eliminate or 6gnificantiy inhibit the growth of nonmutated, actively dw~cting cell% while spanng mutated, nondi~iding cells. 2-D~,ox.~gluconase 12-DG) is tree such chemical agent and ~t i~ a~ed as a cell division inhibitor. 2-DG inhibits phosphog i acorn atase and ~ha~ blocks cell wall bio~,nthesi~. Ac:c, cl5 db. ,ding 5. p,~mbe celis, Lrcated wiLh

ZILXO ,~ND LIEt3ERM~N ~rctreated vvifia 2-DG ~ill cnh~¢e ~: s~l=ctl~n { Fere~.ez el.. 1975; McAtE=)anJ KiI~ey, 1976; SiliCa ~J Far- er.ezy, 197S1. Thi~ method h~; be2n u:ed ~<ucce~. fulI:, for and also au~o:rop~s tMe~met, 1985bL Ino~tol, a vi~in that is ~sentiM for S. Fombe cell ~owth, is mno~er agent us~ for the enriched selection mut=t ~pulatio~ (Megnet, I ~l. On minimal ag~ devoid of i~sitol, growing pmtetrophic cells will die d~ to the lack of ~is vitamin. Nongrowing mutant cells, however, are e~entially un~u~d dung Nis prc~s ~d ¢~ ~ recov- e~d by simply adding inositol ~10 days later. N~edures c~ ~ us~ to enrich for mut~ts ~sing s~nta- neously or t~se i~uc~ either by chemical or physicM agents (Megnet, 19~). MANIPULATION OF S. POMBE DNA DNA extraction In general, protocols used for preparing S. cerevisiae DNA can be readily adapted for S. porabe. Methods for purifying S. pombe genomic DNA include large-scale (Durkacz et al., 1985) and mini-scale (Hoffman and Win- ston, 1987; Rose et al., 1990) preparation procedures. It is difficult to isolate large amounts of plasmid DNA directly from S. pombe because of the low copy numbers present within cells. Plasmids harbored in S. pombe are usually recovered by transforming purified total DNA into E. coli and selecting bacterial cells that have received plasmid (Hoffman and Winston, 1987: Rose etal., 1990; Moreno et al., 1991). In this way, S. pombe plasmids can be readily amplified and purified using procedures well established tbr E. coli (Ausubel et al., 1994). However, transformation of total DNA into E. coli often results in a low efficiency of plasmid recovery, perhaps due to unidentified inhibitory factor(s) in the DNA preparation (Rose, 1987). To circum- vent this problem, modified versions of the Hoffman and Winston (1987) protocol were developed by Robzyk and Kassir (1992) for small-scale preparations and by Zlmo and Lu (1995) for larger yields. Using these procedures, plasmid DNA with presumably le~ inhibitors can be obtained and used to achieve higher transformation effieiencies. How- ever, a greater problem is the tendency of $. pombe to multimerize and rearrange plasmids. This problem is more difficult to solve, but the bacterial swain used for transforma- tion can influence the efficiency of plasmid recovery (Alia et aL, 1993). In addition, the recovery of plasmid DNA from S. pombe by transformation into E. coli is significantly im- proved by using electroporation rather than other methods of bacterial transformation. Methods for DNA tran~Jbrmarion Variotm methcxt~ are a~ailable for the introduction of DNA it.to S. porabe. These procedures inclnde the ttse of lithium acetate (Ito et aL. 1983; OkazaM et aL, 19901 or lithium chloride (Broker, 19~i7), lhe sphereplast methtxi (Beth arm ~er.tice, If~)2). ~ e~ly ~r~a~0n cx~Vmzn~a Fro- deced effiN~zi~s of approxima~ely 5 '.: 10 ~sIk,~ka'gg of DNA. Later, ~ elfi~e~cy of 7. ~fo~anka~g of DNA w~ obtained ~i~g a of ~e sph~repl~t m=lk~5 e6ginally dzscfi~d by Beach a~d Nurse 119811. ~is new pr~ed~ inelud~ the use of the cationic ~some-fo~ing r~gent Li~fecan/GIBCO/B~ Cat# 18292011). ~is chemicN fo~ cemplexes with ~d cell membr~es, which ~sumably enh~e the up~e of DNA (Allshi~, 1990). A simil~ t~nsfo~ation effi- ciency w~s ~so achieved by ~ing ~ improvod lithium acetic meth~ (Oka~ et eL, 1~). Conventionally, freshly p~ed comNtent cells ~e ~de I~r tmsfo~a- tion. However, it was ~cently found that comNtent S. ~e cells, ~ed using t~ spheroplast meth~, can stered fm~n forat l~t 3 monks without signifie~t loss of tr~st~ation e~aciency (~aoetal., 1~3). S. pom~cells ~ comNtent by a ~r~ed~e using li~ium acetate can al~ ~ c~oprese~ed IBr~er, 1~31. ElectroCution is, however, ~e quickest ~d ~siest meth~ for high-efficiency t~sfo~afion and may well be the pmt~ol of choice in ~st la~mtoNes utilizing S. pombe. FISSION YEAST VECTORS General features Typical S. porabe cloning vectors are capable of "'shut- tling" between yeast and bacteria. They consist of an auton- omous replication sequence (ars), a yeast selectable genetic marker and unique restriction sites for cloning. In addition, a bacterial origin of replication (ori) and a selection ~ker for bacteria (i.e., usually encoding ampieillin or tetracycline resistance) are Mso part of the vector. A stabilizing element (stb), which is sometimes added to the vector, enhances the stability of the plasmid within transformants (Moreno et al.. 1991). Depending upon the specific purpose, the cloning plasmids can be classified as "general cloning vectors," vectors for constructing gene banks, or expr~sion vectors (Russell, 1989). However, this classification is somewhat arbiwary since, for example, vectors used for subeloning and for constructing gene banks are essentially interehangeal~le. Markers for selection in 3"east It is important to be able to select S. pombe cells that have received vector DNA. For this Ixtrpose, most vectors contain a genetic marker that would confer a growth advantage uixm transfomaanls, relative to cells that have not received plus- mid. Probably the most widely used selectable rrmrker systems involve genes that complement S. porabe mutations which prevent growth on medium devoid of either leueine (i.e., leul-32, etc.), be~tuie of the lack of 3-isoprop]/lmalate dehydrogenase, or uracil (i.e., ura4.204, ura4-Dr, ura4- D18, ttc.), as a result of the inactivity of orotidine-5'- phosphate decarboxylase (tbr review, see Russell, 1989). Both the S. pombe teal and the S. certvisiae LEU2 genes complement the deficiency in leul- fission yeast cells. The S. pombe ura4 and S. cere~.tsiae URAA genes can comple- ment the S. pombe uro¢- growth detect. Ho~,'e',cr, the 5".

~:e ~ft~n req~r~fl t~ ~c~v~ f~I! cempl~m~n~cn ~d gc~d In ~d~i~n to ~ ~puI~ ~Iec~on s~em~, m~y ethers ha~e ~e~n d~vis~d. Complementafion of S. ural (S~<a~chi ~d Yam~o{o, 19~2). hi~ (Burke ~d Gould, 19~}, and h~ (A~lin~o etal., 1~3) cell~ wi~ • e co~nding fission ~east wtld-~ genes, ~ ~'eil ~ ~c6-7~M mu~t cots ~5~ ~e cl~ suppressor ~NA gone sup3-5 (~, 1987), have ~en d~cfi~d. Vectom that m~e use of ~g-r~is~ce m~kem have Mso ~en u~ for selecfion of S. ppmbe w~fo~ants. ~is cl~s of m~ke~ incl~e the ~' gone of ~ns~son Tn~3 for seleefi~ of ~sist~ce to ~e =tibiotie ~18 (S~M et M., 19~), ~d~e BSD gone of~pergill~ terreus for ~lecfion of ~sist~ce to the fungici~ bl~ticidin S (Kimum etal., 1~). However, w~ch ever selectable mmker syaem is u~d, ~re must ~ a low genomie mve~ion fr~y to ~ able to det~t ~e tmsfo~ation events. Cells containing deleti~s ofge~c ve~ions of exogenous genes, if coffes~ng e~omo~l genes exist, ~ ~st suited for ~em stud,s, ~ ~ey have ~e low~t reve~ion freque~y a~ mud not to undergo gen~ conversion even~ if.e, ~n-reciproeM exch~ge of DNAL Vectors for library construction and subeloning When choosing a plasmid to construct a gone bank or to subelone, a vector that is stable in S. porabe and can carry a relatively large DNA fi'agment should be selected. The vector systems developed by Wright and co-workers (1986), a prototype being pWHS, are particularly well suited for cloning. Insertions of DNA fragments into the kcI represser gone within pWH5 will knock out suppression of the tetra- eyeline resistance gone (under control of the k PR promoter) ~so present on the plasmid. Consequently, only bacterial colonies transformed by pla.smids containing an insert will be tetracycline resistant. However, the presence of a S. core. visiae 2p, element in this vector appears to lead to frequent rearrangements. Recently, a new vector (peNS) with similar features, except for the presence of a S. porabe arm in place of the S. cerevisiae 2p, and a suppressed transposon-derived neomycin resistance gone instead of the regulmed tetracy- cline-resistance gone, has been described tbr use with S. pombe (Weilguny et al., 1991 ). Another series of plasmids (Barbetetal., 1992), derived from pUC 19, and containing S. pombe arm and the E. coli lacZ gone for blue-white screen- ing to detect the presence of inserts, has also been developed. In addition, Cottarel and co-wafl~ers (1993) have developed a set of multicopy plasmids (pSPI, pSP2) that 'also contain S. porabe arsl. Plasmids ealmbte of supporting the maintenance and rep- lication of several hundred kilobase pairs of DNA, such the yeast artifi~al chromosome (YAC) vectoes for S. cere- visiae (Burke et t~l., I987L have, in addition, been devel- oped for S. pombe tHahnenberger etal., 1989; Allshire, i990). Ordered YAC~ PI~ and cosmid clones, bearing large fragments of the S. pombe scheme, have been generated by two independent genome studies, conducted at the Imperial C:mcer R~,earch Fund in London (Hohetsel etal., 1993; N~' York togcth:r ~e~e m; vM~ble inter~t. Expression vectors and other specialized ptasraids Kxi~ression vectors have also been develo~d to mediate transcription of cDNAs of interest by inclusion of promoter sequences in plasmid~. For example, the S. pombe adh (alcohol dehydroge, ase~ sere prom,~ter has been used. In addition, unlike for the yeast S. cerevisiae, promoters from many different organisms are also capable of driving tran- scription in S. p~mhe. The SV40 early promoter is active in S. pombe and has been used in several fission yeast vectors, ineludingpSM2andpDB248(KauferetaL, 1985;Moreno et aL, 1991). This promoter mediates transcription constitu- tively at a relatively low level, and is about 500-fold less active than the adh gone promoter (Forsburg, 1993). Even the tomato nia gone promoter functions in S. pombe and a vector making use of this regulator), region has been con- structed (Truong etal.. 1992). Interestingly, the human immunodefieiency v~ru.~ long terminal repeat promoter (Toyama et al., 1992), tile hunmn chorionic gonadotropin alpha gone promoter, and human cytomegalovims promoters (Toyama and Okayama, 1990) are extremely active in S. pombe. Several of these promoters could potentially b~ well suited for expressing human genes in fission yeast, especially when high levels of transcription may be important to achieve cross-species biological activity. Inducible promoters have also been described for S. pombe vector systems. For example, the promoter for the fructose bisphosphatase gone is capable of driving transcrip- tion of S. pombe genes in a controllable fashion (Hoffman and Winston, 19891. cDNAs fused to this promoter can be induced by glucose repression over a 100-fold range. This allows tight control of gone expression levels by the manip- ulation of growth conditions. However, the drawback of this system is the very slow growth of S. pombe on glycerol. A tetrucyeline-inducible promoter, which is a derivative of the cauliflower mosaic virus 35S gone, was also used for gone expression studies in S. pombe (Faryar and Gatz, 1992). Likewise, a glucocorticoid-respoosive promoter from the rat glueoeoaieoid receptor gone has been used for an inducible promoter system in S. pombe (Heard etaL, 1990). However, probably the most popular vectors currently used for regu- lat~l expression of cDNAs in S. pombe are t~ pREP/pRIP series of plasmids (B~.~i e2 ~I., 1993; Maundrell, 1993). They contain the thiamine-repressible nmtl gone promoter from S. pombe. The pREP plasmid and derivatives replicate extra- chromo~omally, whereas the pRIP plaxmid lacks S. pombe arm and therefore tend~ to integrate into the chromosome. Systematic modification of the nmtl promoter region in pREP has resulted in ~he generation era series of derivatives that together provide :~ aide range of regulated expression levels for a DNA sequence of interest. Although repression in this s~,stem is xtct c,,~plcIe, m~mduced baseline levels of expression are ~e~ k,,~

ger.c~e of S. ~-~e at a s~ecitic si:~. Keer.~y ~d Bceke (I~4) b~va ~vcIwed vact~ f,~r ~-~e~ i~Ik, n via k~m~.,~a~ recombination m:o lcal ~=d ura4 gone regien~. MecMnisms that ~iaze ~get~ integration e~en~, ~d • eir useNIne~. ~e ad&essN ~elow in ~spla:e~nt. N¢¢ ~1 veeto~ for use in & p~e se~'e to faei~ the cloning ~d ~pre~i~n of genes. Some have mo~ s~ciN- iz~ functions. For exmp~, a shuttle v~t~ to d~ect mamge~sis in S. po~ h~ ~n de~d a aL, 19~1. ~is v~tar, ~RI, ~plieat~ ~ E. cofi cells ~ ¢~es ~n E. coli ~NA su~p~ssor ge~e ~ the mutagen t~get. By using ~g o~ or induced mu~ons of ~ supF ge~ gene~d in S. pombe ¢~ ~ deteet~ ~r in~uc~en ~t~ a ~mbi~ bac~a~pl~mid K~IFKY241 re~er system (~ aal., 1~2), by sel~g fornNi~ie~id~si~tbactefiN calls ~or by ~ning for wNte coloNes on 5-bmm~ cMom-3-indolyl-~-D-gaNctopy~ ~-~iog~act~yr~side (I~) ~ ag~ (i.e., the I~Z- phenot~). MOLECULAR CLONING OF S. POMBE GENES Several strategies used routinely for the isolation of S. pombe genes are described below. Metho~ employed to construct S. pombe genomic DNA libraries (Beach et al., 1982; Wright et al., 1986) or eDNA libraries (Fikes et al., 1990; Olesen et al., 1991) have been described previously. In addition, cloning by nucleic acid hybridization and poly- merase chain reaction (PCR) are also possible, but are not unique to S. pombe and will not be addressed in this section. Cloning by functional complementation Molecular cloning by functional complementation is the mo~ lmpular method for isolating S. pombe genes. In brief, a genomic or eDNA library is introduced into $. pombe cells containing a mutation in a gone whose wiki-type is to be isolated. Traasformaals receiving plasmid are lectad by viflue of a gea~ on the library vector that provides a growth advantage. Cells receiving the geae of interest, and therefore having a rescued murat phenotype, c~n eilber be sel~-~d simullaneonsly with selection for receipt of pl~mid or tramformant populations can be screened at a later date for the presence of the gene of interest and, consequently, a complanented mutant txhenotype. Many features of S. pombe make it amenable to the functional complementation approach for gene cloning. The genome size of ~his organism is relatively small so that a manageable number of transformants can be generated and soreemxl to identify a gene of interest successfully. Because the $. pombe haploid genome is 1.5 × I ~ kbp long, approx- irrmtely 1,500 plasm~ds containing 1O-kbp inserts v, ould theoreticalb represent one librat)' of DNA. However, for screening purposes, at least three to tour timeg that number ~hould be tesled to ensure a high probability of obtaining ~St o:ic sro~~ in ~= ak~nce of 5elec~e gre~N (Rus~II, 19891. ~s ~Ween loss of pI~m!d ~d loss of~ phenog;ge ~c-:iated wi~ a clon~ gent, ruling out t~ ~;~ibility of isNating and ~g gentle reverts. Wi~ lib~ m~e in rela- tively l~ge ~ectom, such as pDB248, them have ~en ~ol~ed incidents of re~ge~n~ o~ed in pl~mi~ rec~ve~ ~ E. coil ~eyer et aL, 1986; H.B.L. =d Y.Z., ~publis~fl da~l. However, ~c u~ of libmes ~ in relatively s~l venom, s~h ~ pUR ( B~t el al., I ~2), in c~binatian wi~ r~oveD" in E. coil s~in J~26 {a recBC- s~), f~ ex~ple, e= vi~ly climate ~e ~tentiM ~gemen~ ~t ~ ~c~ (A~ a al., ! 985; ~N a al., 1~3). ~hegchia coil HB 101, a re~ - recBC* s~in, has M~ ~n u~d succe~lly for pl~mid recove~ (Ok~i al., IN). ~e m in,t, active DNA fmg~nt is i~lated, sub~nt stu~es, m~ng u~ of ~ ~nspl~ement techniques (~ ~tion on Gone ~nspl~e~nt, ~low), whe~by the No~ gone mplae~ i~ homolog~s counter- p~ in ~e germ, c~ confi~ ~at t~ isolated gone is the wiM-~ vemion of ~ altered Mlele. Alternatively, cr~s-sNcies complemenmfion strategies c~ld ~ K~d to cloue S. ~e genes. Schizoxacc~romy- cespo~e urn1 ~ ~o3 were cloud by funcfio~ comple- menmtion of co~e~dMg g. coil mumn~ (Ya~om e~ al., 1981; N~sM ~ Ym~to, 19~). Saceharo~ees cerevisiae h~ p~ ge~. Ge~ enc~ing alc~ol dehydrogen~ (Rus- sell ~ HMI, 1983), ~om-#~ate isomer~ (Rus~ll, 1~5), ~ ~e C&42p ~ase (Miller and Johnson, i~) from & ~mbe have, for ex~ple, Men cloud by comple- ~n~tion of eo~es~n~ng S. cer~,isiae mutm~. The tenfiM ~blem with ~ing ~is m~ is that ~c~pfion of S. p~be ge~s in $. cerevisi~ is usu~ly ~M ~d incident (Ru~ll, 1983, 1985). A morn ~fi~s pr~lem ¢o~e~ ~e p~v~e of in~ons wi~ S. ~e genes that Lack ~liee con~us ~ne~s ~ogni~ble by S. eer~isi~e (Rus~ll, 1989). E~Nent ~d ~eu~e s#ic~g wouM ~ ~ to nerve g~ ~p¢~sion ~d ~emfore ~ co~l~nmfion. To c~mvent lores, S. p~ c~A lib--, driven by a S. eerev~iae pm~r, have ~y ~n ~et~ ~ ¢~ m i~lme ~ fi~ion ye~ ge~s by reNuing mu~t pMno~s in b~ing y~t ~es el., 1~1). ANALYSES OF A CLONED GENE BY MUTAGENESIS AND TRANSPLACEMENT Once a gene is isolated on a plasmid, aside from the deterraiaation of its DNA sequence/Maxim and Gilbert, 1977; Sa~ger et al.. I977"), functional domains and biologi- cal actMty can be ch,~'a(:lenzed asing a variety of mutagcn- esis technklues, either alone or in concert with germ trans- placement.

S. FI2MBE. A .'~IODEL FOR ~IOLECUL.~R S'TEDIES :qi.~ =~z~ f~n~on. If a cDNA f~m containing a geze of interest is cloned, at l~at one of ~verM tech~ques mast pr~i~ lc~fioa of ~e gene. Fmgment~ of DNA c~ ~ su~lo~ed ~ ~d in vivo for acfivib, after MLng ~sfm~ imo ~e appropriate murat ~lls, t~by I~ali~ng g¢~ ~on to a subfmg~nt of DNA isolmed. As ~ ~mly in~ed into ~, ge~mic DNA ~ ~ co~l~ %5~ 1~ ~ retenfi~ of ~on, a~n ~fiMng ~gions ¢mc~ for acfi~ty =d m~t l~ely ~siding wi~M ~e ~ of int~st (~e~ et M., 19~; Snyder a al., 1986; Huis~ a al., 1987). Mmy t~h~qu~ m avMlable to ~tcr ge~s in vitro, ~ a st~ tow~ ~ ~mb~s~nt of s~cmwd~fi~ ~lafion- ships by co,lining s~cific alterations ~fion of ~fivib,. S~veml of ~ ~,#es co~nly u~ to alter a relmi~ly smMl, def~ regi~ of a ge~ fi~d in Ausu~l et al. quences left intact ~r sy~emafi¢ ~g~fion wi~ exonu- cl~s, ~ ~n~ ~ming ~ of a ~ of in~r~t, ~ ~ ~ed for ~tivity back~unds. P~mially, genes ~uM also ~ mumgeMz~ in vivo, whil~ ~iding in ~ ~fiMd f~ using synt~tic oligonucleoti~s, ~ sho~ as 20 bp, to ~ffo~ site-d~t~ m~e~sis, buffing y~t S. cerevisMe. ~se stall DNA ~nts haw ~n ~nsfo~ into S. cereviMae, ~d inse~on muttons in g~o~c regions h~dog~s to ~ tmnsfo~- ins DNA ~v¢ su~u~tly ~¢n i~nfified (M~h~ et al., l~g). AI~u# ~is has not yet ~n ~ffo~ in fimion y~, i~ ~¢~ im~ to do ~ s~ in S. po~e. Gene transplacement Another powerful aHxoach to analyze g¢ne function in yeast involves the creation of dismp6on mmants tagged with a marker that can be used for ~leclion (Roth~in, 1983; Stmhl, 1983). "l'his rr~thod consists oft~ physical intetra!m. tion of a clomxi germ in vitro and the replacememt of the inherent cellular g,ne with tim modifmd version (i.e., trans- pl~men0. A ramkxn piece of DNA er ideally a selectable rrmrker such as S. porabe leul or ura4 (Grimm et al., 1988), or the corr~pom~ng S. cerevixiae functionalcognat~ LEU2 or URA3 (Beach and Nur~, 1981; Los~on and Lacro~te, 1983), Call l~ il~rted within a gone. However, if ore ofth~ aforementiomxl markers is used, the S. pombe genes are preferr~ be.cau.~ the lmdding yeast germs are expressed ~.aldy in f'~fion yeast and would, therefore, not provide a strong s~lection for an insert present as a single copy (see section, Matker~ foe selection in yeaxt). Iftl~ goal is to create a true ~null" mutation, part or all of the open re~ing frame of the gone of interest can be ~leted Mfore the interscuing piece of DNA is in_~z~al. However, r¢gatdles~ r.c,t s~I~c~b'~c on i~ c~wa. f~nhz~c, rc. hh~ ~-ker ~-~uld tag • ~ gen~ of ~tere~t so ti~at it could t~ ~te¢~d e~ily progeny of genetic cro~se~, a usefal fea~re for subsequent studies ~volving strain con~waction. DNA con~ng an autonomous replica~on ~quence (ars) t~ to replicate ind¢~ndently of ~c S. c~mosomes. When DNA d~void of ars is ~ into g. pombe, the DNA cannot rcpli~te unle~ it inte~tes into • e genoa, htegmtion can ~xear mdomly, md m sites tMt ~ no signifi~nt homulogy to t~ exoge~us, ~o~ing DNA. Howev~, ~e DNA ~mn also ~ ~et~, via ho~l- ogous ~combimtion, to a genomic re#on tMt h~ in commn. ~ latter integration e~ents, ~ well ~ the p~ie~ ge~c s~ctures generate, ~ su~ed by Ro~mM (1~1). S~veml events ~¢ ob~'ed ~ter ~for- m~on ~ a ~cul~ pl~mid, devoid ofar, md c~tMning ~ a gone of interest (altered in vigo if ap~p~te) =d a ~l~tabM m~ homologous to gen~ l~amd in a chromo- rome. A s~# reciprocal exchmge ~tween ~ ~l~mble m~er or the gone of interest, and a c~es~nding site in germ, will r~ult in the intention of~e fl~mid adj~ent to ~ genomic site having s~es in commn. ~- times, multiple tandem integrations of ~ plmmid ~ ~m~fively, tMre will ~ a replacement of ~e ~i~table m~ in ~ g~no~ by only the m~er ~ the plasmid, probably ~ia gone conversion or a double crossover. If t~ pl~d is inifiMly line~zed with a r~ct~n enzyme at a sire ~ ~ clon~ yeast DNA a~acent to t~ gone of ~temst, ~fom ~sfo~ng ~lls, the same even~ c~ ~cur, but the relative ~encies will differ. A signific~tly l~ger pro~r- fion of inm~t~ns will ~cur at the site of~e ge~ ofint~st in t~ genome, ~d the overall efficiency of ~o~ation will inc~ d~aticMly. ~e ~sition of t~ cut in t~ pl~mid ~d ~ length of s~nces homlogom to t~ g~me will aff~t ~e frequency of ~c inte~ti~ events, M~ugh ~owl~ge of these p~ete~ will not Mways ~t a g~ ~iction of tM ~sfomtion out~. S~m bl~ ~flyses (Sout~m, 1975) cm ~ u~ to ~ce~ ~ namm of the integration H~s intention events ~ ~c~ at a hi~ q~y ~ $. ~. Tmnsfo~ c~ ~ ~ for ~is ~ of inm~on or. alt~ati~ely, a s~ial v~tor-host s~ ~v~ by G~len et al. (1~3) ~ ~ ~ to ~l~t ~y for such events. Howev~, regals of w~r~ ~eefion system is u~d, ~ ~pl~nt by homI~s ~ombin~iun c~ ~ ~comgefl if li~ ~r ~ e~l~ DNA is u~d ~ ~ ~le-s~ DNA e~, ~nding to tM ~t~on si~ of im~t, ~ ~ ~ ~e exogenous DNA u~ for ~f~ti~ ~R~te~, 1~1). In ~e ye~t S. cer~i~i~, ~ v~t ~r- i~ of in~n e~en~ ~cur at sit~ ~1~o~ to ~e ~co~g pi~e of DNA ~Ro~stein, I~1). For S. ~l~om ~ombina6on e~en~ c~ M less f~m even w~n ~ ~ e~ of tr~sfo~mg DNA ~ ~mologous to c~~ DNA regions. G~ ~d Kohli 119~ • at in~on of the ura4 gem into a co~s~ing ~e mu~t ~cu~ed ~ia homologom ~imfion, ~sL 67% of ~ time. Gral:¢~ e~ ul. (I~3) ~zed ~0~22