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Clarke.Pdf (13.44Mb) STADLER SYMP. Vol. 13 (1981) University of Missouri, Columbia 9 THE STRUCTURE AND FUNCTION OF YEAST CENTROMERES (Saccharomyces cerevisiae, yeast transformation, centromere cloning, functional minichromosomes, recombinant DNA, nucleo­ tide sequencing) LOUISE CLARKE, MOLLY FITZGERALD-HAYES, JEAN-MARIE BUHLER*, and JOHN CARBON Department of Biological Sciences, University of California, Santa Barbara, California 93106; *Service de Biochemie, C.E.N Saclay, 91190 Gif-sur~Yvette, France SUMMARY Segments of Saccharomyces cerevisiae DNA have been iso­ lated that contain centromeric sequences (CEN3 and-CENll) from chromosomes III and XI. When present on a plasmid vector capable of replica~ion in yea~t (pLC544 or Yrp?), centromeric DNA e~ables _that p~asm"d t~ funct"on as an ordinary monosome (or extra d"some "f dupl"cated) "nan aneuploid yeast cell. Genetic markers on a "mini~hrom~so"!e" are stably maintained through mitosis and segregate "n me"os"s I and II as centromere-linked genes. Subcloning experiments have indicated that the functional centromeric sequences, CEN3 and CENll, are each confined to a unique segment of DNA less than one kilobase pair in length. The complete nucleotide sequence of the 627-base-pair CEN3 segment is presented, along with a preliminary structural com­ parison of this region with that contained on a 900-base-pair CENll fragment. These cloned centromere DNAs represent ex­ cellent probes for studying the molecular mechanism of chro­ mosome segregation in mitosis and meiosis. INTRODUCTION The centromere is one of the most important and least un­ derstood structural elements of the eukaryotic chromosome. The presence of a centromere ensures the stable maintenance and segregation of a chromosome through the complex processes of mitosis and meiosis, presumably by providing an attachment point for spindle fibers or membrane sites involved in chromo­ some movement. The yeast, Saccharomyces cerevisiae, is an ideal organ­ ism for the study of centromere structure and function. Ex­ tensive genetic mapping analysis in yeast has identified several genes that are very closely linked to centromeres. 10 CLARKE, FITZGERALD-HAYES, BUHLER & CARBON It is the typical meiotic segregation patterns exhibited by these markers that have established genetically the presence of functional centromeres on yeast chromosomes. Centromeric region DNA can be isolated by cloning these centromere-linked genes and obtaining flanking DNA segments by overlap hybridi­ zation screening (chromosome "walking"; CHINAULT & CARBON 1979). In addition, yeast chromosomes do not contain highly repetitive DNA sequences in the centromere regions, making possible the isolation of relatively long contiguous DNA seg­ ments by overlap hybridization screening of yeast genomic libraries. Autonomously replicating plasmid vectors are available that carry both bacterial and yeast replicators along with genetic markers for selection in either Escheri­ chia coli or yeast (STINCHCOMB et al. 1979; KINGSMAN et al. 1979). After insertion into a "shuttle vector," a segment of DNA can be assayed for centromere function in vivo by trans­ formation of yeast cells. Finally, yeast is particularly tolerant of additional chromosomes (aneuploidy), and will readily maintain synthetic minichromosomes containing a functional centromere plus a suitable replicator and genetic marker for selection. In this paper we shall describe the isolation and genetic and biochemical characterization of functional centromeric DNA (GEN) from yeast chromosomes III and XI. Both centromeres are contained on unique DNA segments that are less than one kilo­ base pair in length, and either segment, when present on a plasmid carrying a yeast replicator, enables that plasmid to function as a chromosome both mitotically and meiotically. Minichromosomes containing a GEN sequence are stable in mitosis and segregate as ordinary yeast chromosomes in the first and second meiotic divisions. Thus, they are excellent model systems for the study of centromere function. ISOLATION OF CENTROMERIC DNA (CEN3) FROM CHROMOSOME III Because the size and properties of yeast centromeric DNA were unknown, our initial approach to the isolation of a cen­ tromere involved the molecular cloning of genes that map on opposing sides of a centromere, followed by the subsequent isolation of all the DNA between the genes. Therefore, DNA segments from around the centromere-linked leu2, cdclO, and pgk loci on yeast chromosome III were obtained on hybrid plasmids by a number of techniques, including complementation of auxotropic mutations in E. coli (RATZKIN & CARBON 1977), overlap hybridization (CHINAULT & CARBON 1979), complementa­ tion of mutations in yeast through transformation (HINNEN et al. 1978; CLARKE & CARBON 1980a), and immunological screening (CLARKE et al. 1979; HITZEMAN et al. 1980). In this way, a contiguous region of about 25 kilobase pairs of DNA,extending from the LEU2 gene on the left of the centromere through the GDGlO gene on the right, was obtained on a set of over-1-a-pJc>ing plasmids (Fig. 1) (CHINAULT & CARBON 1979; CLARKE & CARBON 1980a; CLARKE & CARBON 1980b). Among these is the plasmid, pYe(GDGlO)l, which contains the yeast GDGlO gene included on an 8-kilobase-pair segment of yeast DNA. Within the limits of a standard Southern blot hybridization (SOUTHERN 1975), this YEAST CENTROMERES 11 DNA segment was shown to be unique in the yeast genome (CLARKE & CARBON 1980a) .. Plasmid pYe (CDClO) 1 was isolated by comple­ mentation of trpl and temperature-sensitive edelO mutations in yeast from a DNA recombinant library that contained random fragments of yeast DNA sealed with poly(dA•dT) connectors into the E. eoZi-yeast shuttle vector, pLC544 (CLARKE & CARBON 1980a). This vector consists of the yeast TRPl gene and the replicator, arsl, cloned into plasmid pBR313, and it transforms both trpC E. eoZi and trpl yeast auxotrophs to Trp+ with high efficiency (KINGSMAN et al. 1979). /eu2 centrvmere PKk 1~----8cM------1-2cM-I LEU2 ___ _ CEN;---- CDC/()_____ ----------------PGK 21 kbD--------l----l--- ->17kbp---------l +--3-.4-----f-3-.6-+-l.4+--2.-s-+--3-_3----,~1.o-lf-1.2+---,3-_4-t-:-2_.,..6 --t,...0_9:'l--:-1.6-++IH-:-,-,-_3 t-----:-s.-=-o --ff-········ l1 ? 1 +----t---t--t-·· pYe(L£U2)10 in pYe57E2 +--+---+---,,....;.-,.... pYe 40C3 6 If ~ I 6.--K-·+· pYe 4682 .. ~--· pYe (CDC IO ) I --H'tr+-+------- pYe98F4T ------- pYe65H3T pYelOICJT ~ py e3 5 ---.1:::_::_::_::t,+-+-- pYe(CENJ) 11 ---<I pYe(C£NJ)21.41 Fig. 1. Genetic and physical maps of the centromere region of S. eerevisiae chromosome III. The restriction map gives the location of EeoRI ( -+-- ) , HindIII (-X-) , and BamHI (-t,- ) sites in the DNA. Numbers denote kilobase pairs (kbp). The order of inserts in various overlapping plasmids is indicated below the restriction map. Small dots at the ends of inserts pertain to those sheared segments of yeast DNA that are joined to their respective plasmid vectors by poly (dA•dT) connec­ tors. The isolation and characterization of plasmids pYe(LEU2) 10 (RATZKIN & CARBON 1977), pYe40C3 and pYe46B2 (CHINAULT & CARBON 1979), pYe(CDClO)l, pYe98F4T, pYe65H3T, and pYel01C3T (CLARKE & CARBON 1980a & b), and pYe57E2 (HITZEMAN et al. 1980) have been described. Plasmids pYe(CDClO)l, pYe98F4T, pYe65H3T, and pYel01C3T all contain the TRPl arsl vector, pLC544 (KINGSMAN et al. 1979). The vector portion of the remaining plasmids is ColEl. Unlike other arsl--bearing plasmids, which can replicate autonomously in yeast, but are mitotically very unstable, plasmid pYe(CDClO)l transforms appropriate yeast strains efficiently and is relative stably maintained through mitotic cell divisions in all the transformants. After 15 to 20 generations of nonselective growth, approximately 90% of cells that originally harbored pYe(CDClO)l still carry the plasmid (Table I), whereas the shuttle vectors Yrp7 (STINCHCOMB et al. 1979) or Yrp7' (TSCHUMPER & CARBON 1980), other arsl-con­ taining plasmids such as pYe98F4T, pYe65H3T, or pYel01C3T (Fig. 1), and plasmids carrying two ars elements are almost 12 CLARKE, FITZGERALD-HAYES, BUHLER & CARBON completely segregated under the same conditions (Table l; CLARKE & CARBON 1980b). Table 1. Relative mitotic stabilities of plasmids in yeast Percentage of Trp+ or Leu+ cells Plasmid after 15-20 generations of non-selective growth pYe(CDClO)l 97% pYe(CEN3)41 91% pYe(CEN3)30 55-89% pYe(MET14)2 60-85% pYe(CEN11)12 73-87% pYe(CEN11)5,10 64-72% pYe(arsl-ars2)1 5-8% YRp7(arsl) 1% YRp7' (arsl) 1% pYe98F4T 1-5% pYe65H3T 1-2% pYel01C3T 1-2% Yeast strains XSB52-23Ca(ede10 gal leu2-3 leu2-112 trpl), Jl7a(adel his2 met14 trpl ura3) or RH218a(CUP1 gal2 mal SUC trpl) were transformed with the above plasmid DNAs and in each case several individual transformants were isolated and grown nonselectively overnight on YPD (rich) medium. Each culture was subsequently streaked for single colonies on YPD agar, allowed to grow for 2 days, and replica-plated onto minimal agar medium with or without tryptophan or leucine, depending on the marker carried by the plasmid. Beween 40 and 100 individual clones of each original transformant were scored for plasmid loss after nonselective growth. Preparations of media and transformation of yeast were carried out as de­ scribed by HSIAO and CARBON (1979). Because stability in mitosis is a predicted property of a replicating unit carrying a functional centromere, the unique mitotic stability of pYe(CDClO)l led us to examine the behav­ ior of this plasmid through meiosis and sporulation. Yeast strain XSB52-23Ca/pYe(CDC10)l(ede10 leu2 trpl/CDC10/TRP1) was crossed with X2928-30-1Aa(trpl leul adel met14), diploids were sporulated,and the resulting asci were dissected for genetic analysis. Data from 16 tetrads (cross 1, Table 2) indicate that the plasmid (marked by the wild type TRPl allele) in at least 60% of the asci segregates in the first meiotic division as a chromosome,and is thus found in the two sister spores, the products of the second meiotic division. Only parental ditype and nonparental ditype asci were obtained in this cross using as reference centromere markers leul, leu2, met14, and adel (complete data not shown).
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