Copyright 0 1994 by the Genetics Society of America

The SPTlO and SPT21 Genes of Saccharomyces cerevisiae

Georges Natsoulis,*” Fred Winstont and Jef D. Boeke*

*Department of Molecular Biology and Genetics, Johns Hopkins School ofMedicine, Baltimore, Maryland 21205 and tDepartment of Genetics, Haruard University Medical School, Boston, Massachusetts 021 15

Manuscript received June 18, 1993 Accepted for publication September 30, 1993

ABSTRACT Mutations in the SPTIO and SPT21 genes were originally isolated as suppressors of Ty and LTR

(6) insertion mutations in Saccharomyces cereuisiae, and the genes were shown to be required for normal transcription at a number of loci in yeast. Now we have cloned, sequenced, mapped and mutagenized SPTlO and SPT21. Since the sptlO mutation used to clone SPTlO resulted in very poor transformation efficiency, a novel method making use of the karl-1 mutation was used. Neither SPT gene is essential for growth, and constructed null alleles cause phenotypes similar to those caused by spontaneous mutations in the genes. sptlO null alleles are strong suppressor mutations and cause extremely slow growth. Certain sptlO spontaneous alleles are good suppressors but have a normal growth rate, suggesting that the SPTlO protein may have two distinct functions. An amino acid sequence motif that is similar to the Zn-finger motif was found in SPT 10. Mutation of the second Cys residue in this motif resulted in loss of complementation of the suppression phenotype but a normal growth rate. Thus, this motif may reside in a part of the SPTlO protein that is important for transcriptional regulation but not for normal growth. Both the SPTlO and SPTPl proteins are relatively tolerant of large deletions; in both cases deletions of the C-terminus resulted in at least partially functional proteins; also, a large internal deletion in SPT2I was phenotypically wild type.

T he T y elements of Saccharomyces cerevisiae are a group of retrotransposons that can inactivate

the expression of genes. They can do this either by inserting into structural genes, or into the upstream regions that regulate gene expression. The SPT genes of yeast constitute a group of genes defined by their ability to mutate in such a way that expression of a gene inactivated by a T y element (or solo LTR ele- ment) insertion is restored. Although the SPT genes are not a natural assemblage in that they all participate in one specific biochemical pathway, they all do affect some aspect of transcription (BOEKE and SANDMEYER 1991). Typically, transcription of a number of genes or types of genes is known to be affected. One SPT gene, S P T l 5 , encodes a known transcription factor, the TATA box binding factor (EISENMANN, DOLLARD and WINSTON 1989; HAHN et al. 1989). The S P T l l and S P T l 2 genes encode histones (CLARK-ADAMS et al. 1988), and many SPT gene products are thought to encode proteins that are a part of chromatin or somehow regulate it (WINSTON and CARLSON 1992). The subset of regulated genes and gene families is different for different classes of SPT genes; SPT3 regulates T y l as well as the mating factor genes, whereas SPTlO affects transcription at T y l elements, at P H 0 5 , STE6 and ADH2.

sptlO and spt21 mutations suppress a distinct class of T y and LTR insertion mutations (NATSOULIS et al.

California 9450 1.

Genetics 136: 93-105 (January, 1994)

’ Present address: Avigen, Inc., 1201 Harbor Bay Parkway, Alameda,

199 1). One example of this phenomenon is the ura3- 153 mutation. This mutation is a Tyl insertion in the 5’ region of the URA3 structural gene that results in a Ura- phenotype (NATSOULIS et al. 1989). Although this insertion results in an in-frame TYA-URA3 fusion ORF that encodes a potentially functional URA3 gene product, the fusion protein is not expressed because it is under the control of the 3’ LTR, which is tran- scriptionally silent in wild-type strains. sptlU and spt22 mutations suppress this mutation by activating tran- scription from the 3’ LTR, resulting in a Ura+ phe- notype (NATSOULIS et al. 199 1).

In addition to expression mediated by 3’ LTRs, inappropriate transcription at a variety of transcrip- tionally regulated genes that are not known to be in the neighborhood of a T y element or LTR sequence has also been observed in the sptlO and spt21 mutants (Table 1). In these mutants, the typical phenotype at an affected gene is an increase in the repressed level of transcript, as well as a decrease in the derepressed level of the same transcript. Hence, the SPTlO and SPT21 proteins appear to have both a positive and a negative effect on the expression of these transcrip- tionally regulated genes. The sptlO mutations gener- ally confer a more severe phenotype than do the spt21 mutations, and most sptlO mutations also cause a severe growth defect (NATSOULIS et al. 1991).

In this work, we describe further molecular and genetic analysis of SPTlO and SPT21. These SPT genes were cloned, sequenced and mapped in an effort

94 G. Natsoulis, F. Winston, and J. D. Boeke

TABLE 1

Promoters whose activity are affected by sptlO and spt2l mutations

Promoter Type of effect($ References

Tyl 3’ LTR Increase in promoter activity NATSOULIS et al. 199 1 Tyl 5’ LTR Promoter becomes SPT3-independent NATSOULIS et al. 199 1 ADH2 Increase in promoter activity (sptlo) DENIS 1984 STE6 Derepressed in MATa cells NATSOULIS et al. 199 1 pH05 Basal transcription increased (spt21) NATWULIS et al. 1991 HTA 1 Production of HTAl transcript in presence SHERWOOD AND OSLEY 199 1

HTB2 Transcription greatly reduced DOLLARD et al. 1994 HHFl Transcription slightly reduced DOLLARD et al. 1994 HHF2 Transcription greatly reduced DOLLARD et al. 1994

of hydroxyurea (Hir phenotype)

to learn more about the unusual properties of the spt mutants and, eventually, to divine the function of these genes. As a method to clone SPTlO, we describe the use of crosses in which a karl-1 mutation is used to allow transfer of recombinant centromeric plasmids between one nucleus and another (LIVINGSTON 1977; DUTCHER 1981) (which we refer to as “plasmiduc- tion”). This maneuver was necessitated by the poor growth properties and poor transformation efficiency of sptlO mutants. This method may be generally useful for the cloning of yeast genes by complementation when the mutation to be complemented results in poor growth, high reversion frequency, and/or low transformation efficiency.

The SPTlO and SPT21 gene sequences indicate that both genes encode relatively large, hydrophilic pro- teins. The SPTlO protein contains a Zn-finger-like motif that when mutated, results in an Spt- pheno- type, suppression of the ura3-153 mutation. There is also a weak homology to an ATP binding site consen- sus in the SPTlO protein. SPT2 1 contains numerous SPXX motifs. Thus, these proteins are candidates for nucleic acid binding proteins or components of chro- matin, although their exact cellular function remains unknown.

MATERIALS AND METHODS

Strains, plasmids and media: The yeast strains used in this work are described in Table 2. The plasmids are de- scribed in Table 3. The media were prepared as described (ROSE, WINSTON and HIETER 1990) except that supplements to minimal plates were as follows: canavanine (Can) 20 pg/ ml; cycloheximide (Cyh), 2 pg/ml; 5-fluoroorotic acid (Foa), 1 mg/ml.

Scoring SPTlO and SPT22 function: Complementation of sptl0 and spt21 mutations can be scored in a number of ways; however, for the experiments in this paper, comple- mentation (Spt+) was scored as loss of suppression of ura3- 153 (i.e., as restoration of the Ura- phenotype). For certain experiments involving sptl0 mutants, additional phenotypes were scored, namely the colony size on YPD (Spt-, small; Spt+, large) and acid phosphatase expression on high Pi medium (Spt-, expression; Spt+, no expression) (NATSOULIS et al. 1991).

Plasmiduction cloning method: A CEN LEU2 library made in the vector YCp50-LEU2, containing inserts of 10-

12 kb (F. SPENCER, unpublished data) was first introduced into strain GN56, a MATa karl-1 leu2Al ura3-52 SPTlO+ strain, by transformation (ITO et al. 1983); 1 104 colonies were patched onto the surface of SC-Leu agar plates at 12 patches per plate. These patches were mated by replica- plating to lawns of strain GN51 (MATa cyh2 can1 leu2 KARl+ ade2 ura3-153 sptl0-2) for 14 hr at 30” on YPD plates. The mating plates were then replica-plated to SD+Ade+Ura+Can+Cyh plates in order to select Leu+ Can‘ Cyh‘ plasmiductant papillae. True plasmiductant papillae are red because they carry the ade2-101 mutation as an unselected recessive (red color) marker, whereas diploids that leak through this selection (by gene conversion of the recessive drug resistance markers) would be white. Under these conditions, each patch produced an average of 10 Leu+ Can’ Cyh‘ papillae; 90% of such papillae were red and 10% were white. The papillae were then replica-plated to SD+Ade+Cyh plates to identify plasmiductants that had become Ura-. Red papillae were identified as true “plasmi- ductants” with the nuclear genotype of GN51 and bearing an unstable Leu+ plasmid. White colonies are diploids in which both drug resistance markers have become homozy- gous; they are Ade+Lys+Ura- and sporulation-proficient. These diploids are Ura- (as is the plasmiductant of interest) because their genotype is ura3-153 sptlOlura3-52 SPT 10. Because they are Ura-, these diploids do not grow after the second replica-plating and thus do not contribute any back- ground. Thus, for the cloning of SPTlO, the patch derived from the transformant of interest lacked papillae that grew after the second replica plating, although red papillae were present after the first replica-plating.

Construction of TRPl insertions in SPT10: Plasmid pGN990 was partially digested with SauJA, and full-length linear fragments were excised from an agarose gel. These were ligated to a BglIl fragment containing TRPl from plasmid pPH330 (the kind gift of P. HIETER) and trans- formed into Escherichia coli strain MH5. Trp+ colonies were picked and the position of the inserts was roughly mapped using an XbaI digest. Note that some of these plasmids may contain deletions of SPTlO or flanking DNA in addition to the insertion of TRPl. SPTlO subclones, insertion mutants, deletion mutants

and site-directed mutants: A variety of subclones, including pGN 1048 and 1 10 1, were made in plasmid pRS3 15 (SIKOR- SKI and HIETER 1989) and tested for complementation. Subclone pFW217 is a SalI-Hind111 fragment containing SPTlO cloned in pRS316. Deletion derivatives pGN1256- 1265.1 were constructed from pGNllO1 (Figure 1). TnlO- LUK derivatives were made in plasmid pFW2 17 according to the method of HUISMAN et al. (1987). These insertions were roughly mapped and helped to define the position of the SPTlO region (data not shown). A point mutation was

SPTlO and SPT21 Genes 95

TABLE 2

Strains

Strain Genotype

GN43 MATa adel-I00 his4-519 leuZ-3,112, lys2-66 ura3-52 GN46 MATa adel-I00 his4-519 leu2-3,112 lys2-612 ura3-52 GN50 MATa ade.2-101 canl leu2AI sptl0-2 trplA1 ura3-153 GN5 1 MATa ade2-101 canl cyh2 leu2A1 sptl0-2 trplA1 ura3-153 GN56 MATa karl-1 leu2A1 lys2 ura3-52 GNlOO MATa lys2-503 sptl0-I t rp lAl ura?-153 GNlOl

GNX41-25B GNX41-25D GNX68-2C GNX68-3A

MATa ade2-I01 can1 leuZAl sptl0-2 trplAl ura3-153 MATa lys2-503 trpl AI ura3-153 MATa ade2-101 lys2-503 trplAl spt2I-2 ura3-153 MATa his3A200 lys2-503 t rp lAl ura?-I5? MATa ade2-IO1 leuZAI spt21-2 trplAl ura3-153 MATa ade2-101 leu2AI spt21-2 trplAl ura?-I53

GNX192-7A MATa his3 leu2AI lys2 s ~ t l 0 L : T ~ l trplAl ura3-52

TABLE 3

Plasmids

Plasmid name Description Reference ~~~ ~

pFW2 17 pGN961

pGN984 pGN990

pGN967.1-969.10

pGN997.1-997.20 pGN1035-1037.10 pGN 1069 pGNl101

pGN 1262 pGN1263

pGN 1268 pGN 1269 pGN1622 pRG275 pRG295 pRS22

pGNl146-1147

pGN1264.4-1266.5

pRS303-305 pRS3 15-3 16 YCF3-4 YIPS

Subclone of SPTlO Initial clone of SPT21 TRPl insertions in pGN961 XhoI subclone of SPT21 in pRS22 Initial clone of SPTlO TRPl insertions in pGN990 Subclones of SPT2 1 derived from pGN984 SPT21 BamHI fragment in YIp5; internal SpeIA HindIII-SpeI subclone of SPTlO in pRS315 Subclones of SPT21 in pRS3 14 SPT21 A::lJRA3 plasmid SPTI0A::TRPI plasmid SPTlO subclones derived from pGNl101 SPT2 I A::HIS3 plasmid SPTl0A::LEU.Z plasmid SPTlO Cys-Ser mutant made in pRS304 TRKl clone TRKl subclone CEN LEU2 vector Integrating vectors Centromeric vectors Fragmentation vectors Integrating vector

MATERIALS AND METHODS

Figure 2

Figure 2 Figure 1 MATERIALS AND METHODS Figure 2 Figure 2 Figure 1 Figure 2 Figure 2B Figure 1B Figure 1 Figure 2B Figure 1B Figure 1

MATERIALS AND METHODS

0 3

4 5

References: 1, GABER, STYLES and FINK 1988; 2, R. SIKORSKI, unpublished data; 3, SIKORSKI and HIETER 1989; 4, GERRING, CONNELLY and HIETER 1990; 5, BOTSTEIN et al. 1979.

introduced into the putative metal-binding domain, con- verting Cys codon 387 to Ser (C387S) by oligo-directed mutagenesis (KUNKEL 1985) using oligonucleotide JB99 (5' TGCCAAAAATEAAGATGAGGTAC 3'; mutant bases underlined). SPT21 subclones and mutants The vectors for the

SPT2l subclones and mutants in Figure 2 were as follows: pGN984, 1035, 1037.1, 1037.10 are in pRS22; pGN1069 is in YIp5; and pCNl146 and 1147 are in pRS314 (Table

Restriction mapping: The partial digestion method of SMITH and BIRNSTIEL (1976) was modified to carry out rapid restriction maps of the rather large inserts in the SPT gene plasmids. For the mapping of the SPTlO (and SPT21) regions, pGN990 (and pGN961) were digested to comple- tion with POuI (which has only a single site in each of these plasmids; Figures 1 and 2). Then 1 r g of the plasmids was digested with 1 unit, 0.2 unit and 0.04 units of the restriction

3).

enzyme to be mapped and incubated at the appropriate temperature and buffer for 20 min. These partial digests were then electrophoretically separated on 0.6% agarose gels, transferred to nitrocellulose and hybridized to a probe made from the PvuI-EcoRI fragment of the vector (which is immediately adjacent to the insert) by the random hexamer method (FEINBERG and V~CELSTEIN 1983). This probe served as an indirect end-label for the insert.

Chromosome fragmentation: Chromosomes were frag- mented using subclones of SPTlO and SPT21 inserted in plasmids YCF3 and YCF4 according to the methods of GERRING, CONNELLY and HIETER (1 990). Each of two BglII- Hind111 fragments from the SPTlO region (of 1.5 and 4.0 kb; shaded in Figure 1) were inserted into the centric YCF3 and acentric YCF4 vectors between the BglII and Hind111 sites in these vectors. This resulted in four constructs which were linearized with BgEII; as expected, only two of these gave rise to stable pink transformants in diploid strain

96 G. Natsoulis, F. Winston, and J. D. Boeke

A.

spel 1

€COR1 X&/ EcuRl P s d Hirdlll 1807 2041 2140 2451

- .. c 1 I / I \ Colony size Ura Aci

lo00 ATG AT0 290338 € C O R 1

m TL2210 \ Phosph-

pGN1101 +++ +++ +++

pGN1264.4 +++ +++ +++ I €*I

I Xbal pGN1264.5 +++ + I pGN1266.5 ~

psn v pGN1265.1 ++ +

Cvs + Ser

B. pGN1263 spt1M::URfU

pGN1269 sptlad::HIS3

FIGURE l.-SPTlO restriction map, subclones, mutants and disruption alleles. (A) The heavy black bar indicates the yeast sequences in pGN990, the original clone of SPTlO. In the inset, the uppermost bar represents the sequenced region, with the ORF indicated by an open box. The remaining bars indicate the DNA carried by certain subclones and insertion mutations generated by filling in restriction sites (diamonds). For plasmids pGN1101, pGN1264-1266 and pGN1622, three phenotypes were examined: colony size on YPD (assayed in GNX192-7A), complementation of spt10-2 as assayed by reversal of suppression of ura3-153 (assayed in GN50), and acid phosphatase activity [assayed in GNX192-7A (NATSOULIS et al. 1991)]. Note that pCN1622 was assayed in integrated form whereas the other inserts were checked in the form of CEN plasmids. Open boxes, SPTlO ORF; dark shaded boxes, fragments used in chromosome fragmentation analysis; heavy linea, SPTlO locus DNA in various subclones; S curves, boundaries with vector sequences; light shaded box, region of ORF not required for full SPTlO function; diamonds, restriction site frameshift mutants. (B) Null alleles of SPTIO. SPTlO disruption alleles were constructed by the y transformation method (KUPIEC and SIMCHEN 1984; SIKORSKI and HIETER 1989). The indicated recombinant plasmids were digested with PstI and transformed into yeast. pGN1263 is in the pRS304 vector, a parallel construction, pGN1269, is in pRS305 and differs only in that it contains LEU2 as the selectable-marker.

YPH49, one in YCF3 and one in YCF4, with the inserts in opposite orientations relative to their normal positions in the chromosome. The pink transformants were picked and analyzed by pulsed field gel electrophoresis and Southern blotting as described (GERRING, CONNELLY and HIETER 1990). Stable pink transformants were only produced with the 4.0 kb fragment in the YCF4 vector, indicating that SPTlO transcription is toward the telomere.

A similar strategy was applied to SPT21 except that BamHI-EcoRI fragments of 1.3 and 1.1 kb were used (Figure 2) and cleavage prior to transformation was with EcoRI.

RESULTS AND DISCUSSION

Plasmiduction, a novel method for cloning SPTlO: Mutations in SPTlO suppress T y l insertions such as u r d - 1 5 3 , resulting in a Ura+ phenotype. The SPTlO gene was cloned by complementation of the sptl0-2 allele in a urd-153 strain by identification of colonies that became Ura- as the result of a recombi- nant plasmid. Because the sp t l0-2 allele causes slow growth and very poor transformation efficiency, a novel conjugation-based method, plasmiduction, was used to introduce a library of CEN LEU2 plasmids into the strain.

Previous work has shown that in matings in which at least one of the parents is a kar l -1 strain, hetero- karyons are produced rather than stable diploids. These heterokaryons are unstable, giving rise to cy- toductants, which are progeny cells containing one or

the other parental nucleus in a mixed cytoplasm (CONDE and FINK 1976). Furthermore, exceptional cytoductants can be identified which acquire either (1 ) a plasmid (LIVINGSTON 1977) or (2) one or more chromosomes from the opposite nucleus during such a karl-1 cross (DUTCHER 1981; JI et al. 1993). We refer to the former process (acquisition of a plasmid from the opposite nucleus during a karl-1 cross) as plasmiduction. This method, which circumvents the requirement to directly transform the sptl0-2 strain, was used to clone SPTlO and is described in detail in MATERIALS AND METHODS. The plasmid library of interest is first introduced into a readily transformable karl-1 strain. The karl-1 transformants are then mated (as patches of cells on the surface of agar plates) to lawns of the strain bearing the mutation to be complemented, in this case sp t l0-2 . Plasmiductants are then selected by selecting for the nucleus contain- ing the mutation to be complemented and for the marker used in the plasmid library (in this case, LEU2). Recessive drug resistance mutations, such as cyh2 and can l , are introduced into the recipient strain prior to the plasmiduction; selection for these drug resistances reduces the appearance of undesired diploid colonies. This technique is generally useful for complementa- tion cloning in cases where the mutation to be com- plemented results in poor transformation efficiency. A single plasmid containing the SPTlO gene was re-

97

pGNIW7.1

7' +

pGN1147 I + pGNIW5

pGNI 089 Ai; pGN1037.10

Ned

BamHl I

pGNI 148 I

B. pGN1262 spt2lA::URAJ pGNl268 spt2lA::HlS3

FIGURE 2.-SPT22 restriction map, subclones, mutants and disruption alleles. Symbols are as in Figure 1. (A) Ability to complement an spt21 mutant is indicated to the right of each subclone/mutant. These subclones were tested for ability to complement the spt22-2 uru3-253 strains GNX68-2C or GNX68-3A. The insert in pGN1069 was tested differently: GN43 and GN46, which contain spt2O and spt21- suppressible LYS2 alleles, were transformed with pGN 1069 to Ura+ to give a partial duplication; then FoaR derivatives were selected and all of these were Lys-, indicating an Spt+ phenotype. Five of eight Lys+ FoaR strains tested were shown to contain the deletion by genomic DNA blot analysis (data not shown), confirming that this deletion, in the native SPT21 chromosomal locus, has an Spt' phenotype. (B) SPT22 disruption plasmids. SPT21 disruption alleles were constructed by the y transformation method (KUPIEC and SIMCHEN 1984; SIKORSKI and HIETER 1989). The indicated recombinant plasmids were digested with XhoI and transformed into yeast. The URA3 disruption plasmid, pCNl262, is in the YIp5 vector; digestion for linearization is with XhoI. The HIS3 disruption plasmid, pGNl268, differs only in that the EcoRI/SalI fragment of pGN 1262 containing URA3 was replaced by EcoRIIXhoI cleaved pRS303. pGN 1268 can thus also be linearized for targeting to SPT22 with XhoI.

covered by screening 1 104 transformants in this man- ner. Thus, this represents an efficient method for complementation cloning of yeast genes.

The complementing plasmid, pGN990, was shown to encode a complementing DNA fragment by plas- mid segregation tests (the Leu+ and Ura- phenotypes cosegregated in the plasmiductants) and by retrans- formation of other sptlO strains; the Spt- phenotypes of strains bearing sptlO and ura3-153 markers as well as sptl0 lys2-612 (lys2-612 is another sptlO- and spt21- suppressible allele) was complemented by plasmid pGN990 in these strains. The insert in pGN990 was shown to derive from the sptl0 locus as follows. A family of plasmids, pGN997-1 to pGN997-20, derived from pGN990 was constructed which contain inser- tions of the TRPl gene at random SauSAI sites within the insert fragment (see MATERIALS AND METHODS for details). These plasmids were transformed (intact) into strain GN51 in order to determine whether or not they complemented the sptl0-2 mutation. In addition, they were separately transformed following digestion with EcoRI (which releases the TRPl-marked insert fragment from the vector sequences) and selection for the Trp+ phenotype, into diploid strain GNlOl,

whose relevant genotype is ura3-153 +/ura3-153 sptl0-2. The Trp+ Leu- diploid transformants with these plasmids were selected and further character- ized as to their meiotic behavior segregation patterns and growth phenotypes (Table 4). The data indicate tight linkage between the inserted TRPl marker and the sptlO locus.

Plasmid pGN990 was restriction mapped by a mod- ification of the partial digestion method (SMITH and BIRNSTIEL 1976) (Figure 1). Subclones and insertion and deletion derivatives of pGN990 were generated in order to identify the SPTlO gene within the insert (Figure 1). A number of TnlO-LUK insertions in SPTlO were obtained and roughly mapped. Together, these data indicated that the essential complementing region of SPTlO lay within a SpeI-Hind111 fragment of 2.5 kb. However, we subsequently found that cer- tain insertions and deletions within this fragment did not completely eliminate SPTlO function (see section below on mutagenesis).

Cloning and subcloning of SPT21: The SPT21 gene was cloned by complementation of the spt21-2 allele. This mutation also suppresses ura3-153, result- ing in a Ura+ phenotype. Strain GNX68-2C was di-

98 G. Natsoulis, F. Winston, and J. D. Boeke

TABLE 4

Mapping of DNA inserted in pGN990 to the sp t l0 locus

Plasmid Spt phenotyp in GN5 1 phenotype

Diploid Sph Segregation of markers' N d

pGN990 + N.A.' N.T.' pGN997.3 + + 2Trp+Spt-: 2Trp-Spt' 33 pGN997.5 - + 2Trp+Spt-: 2Trp-Spt+ 39 pGN997.5 - - 2Trp+Spt-: 2Trp-Spt- 33 pGN997.6 + + 2Trp+Spt+: 2Trp-Spt- 51 pGN997.15 + + N.T. pGN997.20 - + N.T. pGN997.20 - - 2Trp+Spt-: 2Trp-Spt- 64

a GN51 (relevant genotype: ura3-153 spt1O-2 leu2) was transformed to Leu+ with the indicated plasmid and the Ura phenotype was scored; Ura+ indicates an Spt- phenotype.

GNlOl (relevant genotype: MATa ura3-153 sptIO-2 leu2/MATa ura3-153 S P T leu2) was transformed to Trp+Leu- with the indicated plasmid following complete digestion with EcoRI. Spt phenotype was scored as in footnote a. Note that for the constructs that fail to complement the sptIO-2 mutation in GN5 1, both Spt+ and Spt- diploid transformants were obtained. This is expected for integration of the TRPZ-marked DNA into the spt1O-2 or SPT+ homolog, respectively.

One or more diploids for each construct were sporulated and dissected. Spore viability was low in these crosses but at least 50% of the spores came from complete tetrads. There were no exceptions to the segregation patterns noted.

N . total number of mores analvsed in tetrad analvsis of diploids. N.'A., not applicable.N.T., noitested.

rectly transformed with the CEN LEU2 library using spheroplast transformation. Approximately 2000 transformants were obtained and screened as follows. Top agar containing the transformant colonies was homogenized through a sterile syringe (30 ml) and the resulting suspension of cells was diluted and the dilutions were plated on SC/5-Foa plates. The stability of the Leu+ and Ura- phenotypes of several 5-FoaR transformants was determined after overnight growth on YPD plates. Approximately 5% of the cells became Ura' following growth on YPD, but none of these colonies were Leu-, as expected for simultaneous loss of the LEU2 and SPT21 genes. This result is consistent with these FoaR transformants containing several dif- ferent LEU2 CEN plasmids from the library, as often happens during spheroplast transformation. Presum- ably only one of these plasmids would carry SPT21 and confer the Ura- phenotype. A total DNA prepa- ration was made from such a Ura- Leu+ colony and transformed into E. coli. Plasmids recovered in this manner were purified and retransformed into GNX68-2C. Only one such plasmid of three tested, pGN96 1, was able to complement the spt21-2 muta- tion. The insert in a derivative of pGN96 1 was shown to map genetically to the sfit21 locus (Table 5).

Plasmid pGN961 was restriction mapped as de- scribed in MATERIALS AND METHODS; subclones and insertion and deletion derivatives were generated in order to identify the SPT21 gene within the insert (Figure 2). A number of subclones from pGN961 were tested (not shown) and a subclone containing a 3.0 kb XhoI fragment was found to complement spt21-2. Further derivatives of this plasmid were then tested for complementation. A 2.5-kb XhoI-BglII fragment was the smallest subclone able to complement spt21- 2, although this subclone does not contain an intact open reading frame.

Nucleotide and predicted amino acid sequences of SPTlO and SPT21: The nucleotide sequences of both SPTlO and SPT21 were determined in their entirety on both strands. SPTlO encodes a predicted protein of 640 amino acids, and the M, of the predicted protein is 72876 Da (Figure 3). An antibody directed against a synthetic peptide from the C-terminal pep- tide sequence recognizes a ca. 80-kDa protein from S P P strains that is absent in an sptlOA strain, in reasonable agreement with the sequence data (C. DENIS, unpublished data). Although no transcript could be detected in wild-type strains by total RNA blotting, strains bearing a high copy number plasmid containing SPTlO produced a transcript of the appro- priate size (data not shown), suggesting that these transcripts are relatively rare.

Searches of GenBank and other databases using FASTA and TFASTA failed to reveal significant lev- els of amino acid sequence identity with known pro- teins or ORFs. However, two amino acid sequence motifs of possible relevance were found: (1) a CX2CX21HX5H motif that is similar to metal binding domains such as the putative metal-binding domain of SV40 T-antigen and (2) a motif similar to an NTP- binding domain found in certain helicases (GORBA- LENYA et al. 1988; HODGMAN 1988; COMPANY, ARE- NAS and ABELSON 1991) (Figure 4). The Zn-finger- like region from SV40 has been shown to be important for hexamerization of the protein as well as for making appropriate contacts with the SV40 replication origin; mutants in the site are completely defective for initi- ation of SV40 DNA replication in vivo and in vitro (LOEBER, PARSONS and TEGTMEYER 1989; LOEBER et al. 1991). These SV40 mutants still show specific DNA binding, but they cause an aberrant DNAse I footprinting pattern relative to wild-type T antigen;

SPTlO and SPT21 Genes

TABLE 5

Mapping of DNA inserted in pCN961 to the spt21 locus

99

Plasmid Spt phenotype in GNX68-2Ca

Diploid Spi phenotype Segregation of markers N

pGN96 1 + N.A. N.T. pCN967.1 + + (transft 1) 2 Trp+Spt-: 2 Trp-Spt+ 23

+ (transf t 2) 2 Trp+Spt+: 2Trp-Spt- 54 pGN969.10 - - 2 Trp+Spt-: 2 Trp-Spt- 20

+ 2 Trp+Spt-: 2 Trp-Spt’ 40

a GNX68-2C (relevant genotype: ura3-153 spt21-2 leu2) was transformed to Leu+ with the indicated plasmid and the Ura phenotype was ’ GNX41-25B X GNX41-25D (relevant genotype: MATa ura3-153 spt21-2 lysZ/(MATa ura3-153 S P r lys2) was transformed to Trp+ Leu- sc red; Ura+ indicates an Spt- phenotype.

with the indicated plasmid following complete digestion with EcoRI. Spt phenotype was scored as in footnote a.

the mutant proteins are also defective in forming the stable hexamers characteristic of native T antigen. The relevance of the putative NTP motif is uncertain because it is not a perfect match to the consensus and additional amino acid sequences usually found in these proteins are not obviously present in the SPTlO se- quence.

SPT21 encodes a predicted protein of 758 amino acids, and the M, of the predicted protein is 84,645 Da (Figure 5). Although no transcript could be de- tected in wild-type strains, strains bearing a high copy number plasmid containing SPT21 produced an ap- propriately sized transcript, again suggesting a rela- tively low level of expression (data not shown). Searches of GenBank and other databases using BLAST, FASTA and TFASTA failed to reveal sig- nificant levels of amino acid sequence identity with known proteins or ORFs. The central third of the predicted SPT2 1 protein is similar in sequence to the central globular domain of histone H1 from a variety of organisms; however, the most highly conserved residues among histones H1 were not conserved in SPT2 1. As is also true of histones HI, the SPT2 1 protein sequence contains numerous (eight) occur- rences of the SPXX sequence, which has been impli- cated as a candidate DNA binding motif (SUZUKI 1989; SUZUKI 1990a; SUZUKI 1990b). In addition, both the N-terminus and the C-terminus of the pre- dicted SPT21 protein contain highly acidic stretches (N-terminus: 13/18 Glu and Asp; C-terminus: 9/11 and 11/26 Glu and Asp). The protein also contains 19% serine and threonine residues, and 9.5% aspara- gine residues, and has very high hydrophilicity.

Mutagenesis of SPTlO: SPTlO was mutagenized by filling in restriction sites to create frameshift muta- tions, by insertion of TnlO-LUK, and by oligonucle- otide-directed mutagenesis. Four frameshift muta- tions were made within the SPTlO ORF; these were tested for SPTlO function with three assays: colony size, reversal of suppression of ura3-153 by sptl0-2, and level of acid phosphatase activity (spt lo alleles result in elevated acid phosphatase activity (NATSOU- LIS et al. 1991)) (Figure 1). The first frameshift mu- tation, at a PstI site, resides between the first and

second ATGs of the ORF and almost completely abolishes SPTlO function. This strongly suggests that the first AUG is in fact the initiation codon for SPTlO translation; however, the small amount of residual SPTIO activity suggests that some initiation may occur at the second AUG codon. A frameshift downstream of the second ATG abolishes SPTlO function com- pletely, by all assays. A frameshift mutation at an EcoRI site about three-fourths of the way into the SPTlO ORF results in partial SPTlO function as judged by large colony size, but the ura3-153 suppres- sion phenotype is not complemented. Finally, a frame- shift mutation that results in loss of the C-terminal 24 amino acids is phenotypically wild type in all three assays of SPTlO function. Thus, the C-terminus of SPTl 0 protein is dispensable.

The putative metal binding region of SPTlO was mutagenized by oligonucleotide-directed mutagenesis as described in MATERIALS AND METHODS, generating mutant allele sptlO-C387S (Figure 4). This changes the second Cys residue in the putative metal-binding region to Ser, a substitution that inactivates all Zn- finger proteins (BLUMBERG et al. 1987) (and J. BERG, unpublished data). A sptlO-C387S plasmid and the parental SPTlO+ plasmid were transformed into strain GNlOO (relevant genotype sptl0-1 ura3-153). The sptlO-C387S mutant failed to complement ura3- 153 suppression, but it did complement the slow growth defect of this mutant (Figure 1). This result suggests that the domain of the SPTlO gene product containing the putative metal binding domain is re- quired for transcriptional control and that a different part of the protein is responsible for normal growth rate; a phenotype similar to that seen with the EcoRI site frameshift mutant. Alternatively, both of these mutations might result in the synthesis of unstable, low abundance proteins with an “intermediate” phe- notype. Distinguishing between these possibilities de- finitively will require immunoblotting experiments or other methods to directly assess protein levels.

Mutagenesis of SPTZI: We made a small number of deletion mutations and frameshifts in SPT21 in the course of mapping it. The phenotypes of these mu- tants, in combination with the sequence data, indicate

100 G. Natsoulis, F. Winston, and J. D. Boeke

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CtCGGGATGAGTAAAMGTGTACCGATCMGMCMCTCTAGACTTCCGCCAAAGTGATTATCMCAMMTCGTMTMTTAGCTTCMTGCTAAATCA M L N Q

GCACACMGTTCAGTACCG~TGAC~CATCTGCAGATGGCACACC~TAGCAGTTCAGMGTGAGMATGffiGCAGCGGTCCCAGATCMCTTTTG H T S S V P D D E H L Q M A H Q N S S S E V R N E A A V P D Q L L

ACTCCTTTACMCCGTATA~ATTTTATTGAAAGATGGAGAAACMTAGCTACGATGTATCCTATACCTGCGTATCCTGATTTATTGCCGCTAGGGCTCT T P L Q P Y T l L L K D G E T I A T M Y P l P A Y P D L L P L G L L

T A M T T T C C T T C T f f i A T G M T T T M C A T G G M G T G G A T G T C T G G T T N F L L D E F N M E V E K G D S F P Y Y E T L S L E E F K N V W F

TCATMT~TGGTCACGTTTGTATTATGGTGTTAGGCGAMTACCAGMCTGGATTATAGTATGGATACTGMGCGGACACAMTGATAATTTTGGMCA H N D G H V C I H V L G E I P E L D Y S M D T E A D T N D N E G T

GAAATAGAMCCACGMGCACACCACCCCM~ATMGAAACG~GMCG~CGCMTTTAAATCTMGTATGCAGTGGGAGMGCMTGTCTA~CATAT E I E T T K H T T P Y K K H K E R R N L N L S M Q W E K Q C L G I F

TTGACCTC~CCAGCATACCCTGGACGGTCAGCGCATGTGGTCACAGGMCATTTCTGGTCMTGCTGGTATTCGTGG~GGTATTGGGAAAACACT D L K P A Y P G R S A H V V T G T F L V N A G l R G K G I G K T L

GGATTGMCTTTAGGCGGATTGGAAAGCTGCCTGAAGCCGGGATTCTCMGGGCTTTG~TGTCCCAGTAGATTCTTTCATGTATGGTAAAGAGTTCACAC G L N F R R l G K L P E A G I L K G E D V P V D S F M Y G K E F T H

ATATTAC~GTATAGATTTGTTACGAGATCCTC~GAGTATCG~TTGGGAAATATGAACGATTAAAGCATTTTCTGGAAACAGGCMGTATCC I T K S I D L L R D P Q K S l E l G K Y E R L K H F L E T G K Y P

TTTACATTGTGbTffiAAACCGGCTAGATTMGAGTACTTTCC~CTCATTCGGTATTGAATGGCMGTTGATGAC~GG~GAAATCATT L H C D R N E K A R L R U L S K T H S V L N G K L M T K G K E I I

TATGACACGGATCMCAAATCAGATTGCATTAGAGATACATTTMTGGMCATTTAGGCATCAACM~TGACCTCC~TTGGTGAMAATACCATT Y D T D Q Q l Q l A L E l H L H E H L G I N K V T S K l G E ~ Y H W

~ G A G G M T C M ~ G T A C T G T T T C ~ A G G T M T C T C T C G G T G C C ~ T G C A A G A T G A G G T A C A M G A C G G G A C A G G G G T M T M T T G A A C A A A A G A G R G I ~ S T V S E V I S R C Q K C K M H Y K D G T G V l l E Q K R

~GCAGTTMGCAGGCGCATITGCTACCGACGCAGCATATAG~CTATCMCAATCCMGMMAGC~AAGCATGATAATGCGTTACTTGGGACAGGCA A Y K Q I H M L P T Q H I E T l N N P R K S K K H D N A L L G Q A

ATAAATTKCCTC~TATTATTTCCAGTACGCTGMTGATG~GAGGGTGMCCCAC~CCGCCAGACACTAATATCGTAC~CCCACATTCCAGAACG I N F P Q N l I S S T L N D V E G E P T ~ P D T N l U Q P T E Q N A

CCACGMTAGTCCCGCMCMCAGCTGMGCCAACG~CGMT~GATCCGAGTTTCTTTCCTCMTACAG~CACGCCACTATTGGACGACGAACA

T N S P A T T A E A N E A N K R S E F L S S l Q S T P L L D D E Q

GTCMTGMTTCCTTCMTffiATTCGTGGAG~GMMCTCAffiAAAAAGACGAAAATACTTGGATGTTGCCTCCMCGGMTTGTACCCCATTTMCG S M N S F N R F V E E E N S R K R R K Y L D U A S N G l U P H L T

MTMCGMTCGCffiGATCACGCGMCCCCGTCMCCGTGATGMCGAGATATGMCCATTCAGTTCCCGATTTAGATAG~TGACCATACTATAATGA N N E S Q D H A N P V N R D E H D M N H S V P D L D R N D H T I M N

ACGATGCTATGTTGAGCCTCGAAGATAACGTCATGGCCGCTCTAGAAAT~TCC~GGAACAACMCAGMGATAMTCATCGA~TGMGATGTGAC D A M L S L E D N V M A A L E M V Q K E Q Q Q K I N H R G E D V T

TGGCCMCMATCGATTTGMCMCffiTGMGGTMT~GMTTCAGTCACTMGATTGTTMCMCG~TCTMTACATTTACAGffiCACMTTCTMT G Q Q I D L N N S E G N E N S V T K I V N N E S N T F T E H N S N

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FIGURE 3.-SPTIO DNA and pre- dicted protein sequence. The DNA sequence has been submitted to GenBank (accession number L24435).

2401 TGCTTGCATGACGMAGGTTCTGTTACAGTTTTACGTTGAAACCTAGG 2418

SPTlO and SPT2I Genes 101

Zn-binding-like domain

SPTlO C X2 C X21 H X5 H

SV40 TAg C X2 C X7 H X3 H (X2 H)

TFIIIA C X2-5 C X12 H X2-3 H

Steroid receptors C X2 C x13-15 C X2 C

-9 proteins C X2 C X4 H X4 C

NTP binding site homology

SPTlO G - K G I G K T

Cons. G X P G T G K T S

FIGURE 4.-Sequence motifs in SPTlO protein. A putative metal binding domain is indicated and compared to other putative and known Zn-binding proteins. A putative NTP-binding domain char- acteristic of helicases (GORBALENYA et al. 1988; COMPANY, ARENAS and ABEISON 199 1) is also indicated.

that the C-terminal region of SPT2l protein and a large region in the middle of the protein are dispen- sable for function. Specifically, sequences distal to the BglII site (see pGN 1 147; Figure 2), and the sequences between the SpeI sites (which give rise to an in-frame deletion in pGN1069; Figure 2) are dispensible for function.

Physical and genetic mapping of SPTZO: Hybrid- ization probes for SPTlO and SPT21 were generated and hybridized to yeast chromosome-sized DNA frag- ments separated by CHEF (Figure 6). sptlO mapped to chromosome X and spt21 to chromosome XZZZ. Chromosome fragmentation analysis (GERRING, CON- NELLY and HIETER 1990) indicated that SPTlO is located approximately 2 10 kb from one of the chro- mosome X telomeres. Furthermore, the insert of pGNllO1, was found to cross-hybridize with plasmid pRG275 (but not pRG295), from the TRKl region of chromosome X , providing a precise location on X . Restriction mapping of plasmids pGN990, pRG275 and pRG295 (GABER, STYLES and FINK 1988), frag- mentation analysis (Figure 6) and genetic analysis (Table 6) placed SPTlO centromere-proximal to TRKl and the tightly linked gene PBS2 (BOGUSLAWSKI and POLAZZI 1987). Tetrad analysis confirmed that sptlO mutations are weakly linked to inol, and extremely tightly linked to trkl (Table 6; Figure 7).

Physical and genetic mapping of SPT2Z: Follow- ing identification of SPT21 on chromosome XIZZ, we mapped the gene further using a combination of physical and genetic methods. Fragmentation analysis of chromosome XZZI using probes from SPT21 and GAL80 indicated that SPT21 was located on the right arm of XZZZ approximately 3 10 kb from the telomere,

placing it in the vicinity of ilv2 and cin4. We oriented SPT21 relative to chromosome XZIZ as described above and determined that SPT21 is transcribed toward the right telomere. Tetrad analysis of spt21 mutations indicated no linkage to lys7 (not shown), linkage to ilv2 and tighter linkage to cin4 and ctfl3 (Table 7; Figure 8).

Null alleles of SPTZO and SPT21: The DNA se- quences facilitated the construction of null alleles of SPTlO and SPT21. SPTlO was disrupted (separately) with TRPl and LEU2 and SPT21 was disrupted with URA3 and HZS3. The structures of these alleles are indicated in Figures 1B and 2B. These null alleles are readily introduced into wild-type haploid strains by y- transformation (SIKORSKI and HIETER 1989) and se- lection of the appropriate auxotrophies. The pheno- types of the spt21 null alleles have been extensively tested (NATSOULIS et al. 199 1) and are similar to those of the spontaneous alleles obtained for spt21. Simi- larly, null alleles of sptl0 are similar to the “severe” class of sptlO alleles obtained spontaneously; namely, they show a severe growth defect as well as the suppression pattern and acid phosphatase overexpres- sion characteristics observed in most sptlO mutants.

Conclusions: We have described a novel comple- mentation cloning method, plasmiduction, that we used to clone SPTlO. In this method, a gene library is introduced into a Karl-1 CAN1 CYH2 strain bearing an appropriate auxotrophic marker(s) (e.g., ura3) and that is otherwise wild type. Colonies or small patches of this yeast library are then mated to the donor patches by replica-plating to lawns of the recipient ( i e . , the mutant strain of interest). This recipient strain must be of the opposite mating type, and carry can1 and cyh2 mutations (SIKORSKI and BOEKE 1990). Plasmiductants are then selected from the mating mixtures by replica-plating to SC-ura+can+cyh plates. Each population of plasmiductant papillae that arises contains a single species of recombinant plasmid in the mutant genetic background of interest. These populations of papillae can then be tested for the phenotype of interest, typically by replica-plating.

This generally useful method can be applied to the cloning of yeast genes in a variety of special situations in which standard complementation cloning strategies are unsuccessful or especially difficult. One such situ- ation arises when the mutation of interest results in a low transformation frequency, as was the case with our sPtlO mutation. Alternatively, the mutation might be present in a poorly transformable genetic back- ground, and that background must be maintained intact in order to score the mutant phenotype ( i e . , ability to score the mutant trait effectively depends on multiple genes in the background). A third situa- tion in which plasmiduction cloning is useful is when mutant alleles with a high reversion rate are the basis for complementation cloning. In this situation, con-

102 G. Natsoulis, F. Winston, and J. D. Boeke

1 TTCTGIULCTTGA~CMGTTCTCGMCCAGGAGAGCTGMTACCCGAGTATTTAGMCTATGTATAGATATAGATTTTTTTTTTG~AGC~GTCATC 100

101 T C A G T C R T C G C C G G T A G T T G C G C T T T C T T C T C M C A A I \ I C T G T T A 200

201 G W C T T C M T C G C I A A A G T ~ C A T T T G ~ T G C R T R T C C G ~ G A C C ~ C C A ~ M M ~ T A T A T A A T T C A T T C A A T A G A T A C A A C T G C T C C A T G A 300

301 M G G ~ C C T T C A T T T A C G G T C G C G T T T T T T T T C G T G A T A T ~ A A A G G T A A M M A A C G C G T C G C G T T A G A ~ A A A G G T G T G T G T G I U L G A G ~ A G A C 400

401 C C A T T G M T G G A G A G T T C G G T T C T T ~ M T T G C C G C T G A A C 500

501 C T A G T C M C A C A C A T M C T G U G T C M T T T G A C C A G G T G C A A C T G T C T G 600 n s ~

601 MCTCTCTC~TGACATTGM~~CCT~TACACATTGGATAATGGATCCIULTGGTAGTTA~TTAGCCCGCTC~GGGCTCC~IULAC~GGTAAGAGTT~ 100 L S Q M T L K I L Y T L D N G S N G S Y L A R S R A P K Q U R V A

701 T M T A T T C C T R G T C C A T T C C C G A C A G A T T C G M T G A G C M A C A T A T A C T T A A A T 800 N I P S P F P T D S N E Q T E L R I G A I H L K T I L H E I Y L N

001 TCACCTGMGTGTTRGACCATGACACTTTA~GATGGATATGATTATAACTTGTATTATCGTGAC~TATGTGAAGTGG~TGAGCCATTAGTIULGCCTTG 900 S P E U L D H D T L K D G Y D Y N L Y Y R D I C E U D E P L V S L G

901 GTCTTCTTTCCGGGCTTCGATTTCATAAGAATAGCCCCTACCAGTATACAGAAA~CIULTATT~TGAAGAAGA~TGAAGAG~GAGATGAGG~ 1000 L L S G L R K K F H K N S P Y Q Y T E N N I G E E E S E E R Q E U

1001 MCTGIULGAAGAGTRTGIULGATGMTCTTTTATAGTTACTGGA~GGGTATGCTCGAATGTTTCCGCTTTACTAC~AAGATCATATAGCAACATCTCGAAT 1100 T E E E Y E D E S F I U T G R U C S N V S A L L R R S Y S N I S N

1101 ~GGGMGGGTCGTMACAACCAAATTCCGGAAGMACTTTGGAGGTTAMCTMGAT~CACAAMGTT~TIULCCAATTTAAGGACGTCTGGTAACA 1200 K K G R U V N N Q I P E E T L E U K L R F T K U l T N L R T S G N N

1201 A T A C T R C ~ T T C T C G G I T A T C G T G T C I T C A A A T G C C G T C 1300 T T N S R I S C L Q M P S S L P S A T L P F T P K S Q S L F K T N FIGURE 5 . S P T 2 1 DNA and pre-

dicted protein sequence. The DNA

1301 C F A A A T ~ T T C M L L M T t i ~ A A t i L A C C A C L A T A A C A A T A M T A A T A C T A A T A ~ t i L t i A C T G T

Q I K N S R N A R T T I T I N N T N S G T U

.". ."."....."""".""....""" ~ "..."..".. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ " . ~ _ ~ ~ GGGAAGAAGACAGACGAATCCTATGCCTGCTCCA 1400 sequence has been submitted to

L24436). G R R Q T N P M P A P GenBank (accession number

1401 AAAGCTGTCAGMCTCAGTCTTTACCCATCT~AATCTTAMCCAAATATAGCGAATACTGGTTTCCCIULG~ATTCAATTGCGCACAIULATCTACTTAG 1500 K A U R T Q S L P I W N L K P N I A N T G F P R N S l A H K I Y L A

1501 C f f i A T A G ~ C A G M G C C M T C M C ~ C A A C C A G C A T C A 1600 D R K T E A N Q Q N H Q H Q N I A Y E I N T L Q N D N T I Q R T K

1601 GATCGATGATTCGGTMGCMGAGGTTCG~TTTT~TGCTCMC~AGAIULGTCTACGAAAAMGTGTCACCTGGTATAGCAACGATAGCAAMAMCCA 1700 I D D S V S ~ R F D F M L N K R K S T K ~ U S P G I A T I A K K P

1101 G C T T C M T ~ C A T ~ T C C ~ G C M C C G C C G A A G A C T A G T G G T G A I U L M A A A G C ~ A T G A T A A G C ~ C G A T T G T C A A A G T T A A G A A T T C G A A T T C C A 1800 A S I N I N P K Q P P K T S G E K K A N D K Q T I U K V K N S N S K

1801 A M A T T C U ; C T M G l C T A C A C A A G C A G G A T G T A G G C G I \ T C G G A 1900 N S A K S T Q A G C R R S S V l E H L N D H D D S I L S D l L S E

1901 ACCAGGCATTGMCtGCAGIULATTGCAGCIULMACAAMGGGGCGTA~GATATCTTTAACTAGTGMAATG~T~IULGAAAATATTCC~CCCCMAGC~T~ 2000 P t I E G Q K L Q Q K Q K G R K I S L T S E N D K E N l P P Q S I

2001 ACTAGT~GAGMCMGCTTGAAGGTGACTTGGATTTTAACGCTGAGTTCCCCATGAGTGACTT~TCGGATGTAGTA~TTAAAG~TG~GATGGGATGGT 2100 T S K E N K L E G Q L D F N A E F P M S D F S ~ U U F K D E U G W F

2101 TTTCCIULTTTCRATTGCMTTTTTTTG~ICACCAACTTCTGCMGTGCATCACAACTCAATCAGCIUL~TTTGMGCCTTCTATAACACTCIULCGATCC 2200 S N F N C N F F E S P T S A S R S Q L N Q Q N L K P S l T L N D P

2201 AMCMCTGTMCACCATT~CTCTC~~TGAAGATGTCAGTGAATTGGAMCAGCGCAAAATAATAMATATCTTTGCCTAGCGATGTTGACAAAACC 2300 N T C N T I A L E N E D U S E L E T A Q N N K I S L P S D U Q K T

2301 T C C C C M T A G R C T C G T T G T C T A ~ A C C ~ ~ ~ ~ ~ ~ G A ~ C ~ ~ A C ~ C A ~ ~ C ~ ~ G C ~ C G A C M C A I U L C ~ ~ G C A G C G ~ A ~ A T C C ~ ~ T A A A G ~ ~ G G A ~ C G ~ C A C ~ M 2400

S P I D S L S l P L I E L T H S S S T T N M Q R I S l K E G S T L N

2401 ATATMCAGATAGTM~MTGCCACCCCATGTGATAATGATATMMGATAGAAAGGCATCTGTMTAGATTCGGACAATACAAAACCTCAGGCAGGGCT 2500 I T D S N N I T P C D N D I K D R K A S V I D S D N T K P Q ~ G L

SPTIO and SPT2I Genes 103

3101 CTTGMTATAGGATCC 3116

B

Chr X -

SPTlO

-780 Kb

-70 Kb

+210 Kb

FIGURE 5,"Continued

SPT21 GAL80

& .o a c Chromosome size DNA fragments

rn .- FIGURE 6.-Chromosomal frag- > 950Kb +b C Z c r .- 5 '= mentation mapping of SPT genes.

9 were separated according to the CHEF method of CHU et al. (1986) and probed with the indicated radio- active DNA. Acentric and Centric refer to diploid yeast strains in which fragmentation was performed using subclones made in YCF4 and YCFS. respectively. Approximate molecular weights were determined from the position of known chromosome e 640 Kb + bands. (A) Fragmentation of chro- mosome X by SPTlO. (B) Fragmen- tation of chromosome X111 by SPT21.

0

Chr X// / - 4 e

a e 310Kb +

TABLE 6

Tetrad analysis, SPTlO

Markers PD NPD T cM

inol sptlO 15 0 5 12 sbtlO trkl 47 0 0 <1

ventional complementation cloning is often rendered difficult to impossible by the large number of rever- tant transformants that must be screened through in order to identify true complementing plasmids. When positive donor patches bearing the plasmid of interest are identified by the plasmiduction method, they will give multiple drug-resistant papillae bearing the plas- mid marker as well as the phenotype of interest. Since the plasmiductant papillae result from independent mating events, they cannot all be the result of rever-

sion. The revertants will arise at some constant fre- quency within the mutant lawn. Thus, in contrast to donor patches bearing the plasmid of interest, rever- tants will show up at a constant frequency in all of the mating patches that contain irrelevant plasmids.

Once a CEN or 2-pm plasmid library has been introduced into the karl-1 strain by transformation, it can be saved and used for multiple complementation cloning experiments. If saved in the form of an array of transformants of the karl-1 strain in microtiter plates, it could readily be rescreened to clone addi- tional genes of interest without transformation.

The SPTlO and SPT2I genes encode large, hydro- philic proteins whose transcripts are of relatively low abundance. Both genes can sustain sizeable deletions and still retain significant function. The proteins con- tain putative amino acid sequence motifs that resem- ble those found in a variety of DNA-binding proteins;

104 G. Natsoulis, F. Winston, and J. D. Boeke

" A / . w

/ . . - / .

/ . . .

. I \

0 0

\

\ 0

0 1.6 CM (N=93) u 0

\ 0 \

0 4 . 8 CM (N=26) 0

0

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0

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\ roquenced regions 1 pRG275

DRG295 ffiN990

FIGURE 7.-Physical and ge- netic mapping of SPTlO. Heavy lines indicate the genetic map of chromosome X . First inset shows relevant genetic mapping data. Te- trad data are from the following sources: cdc6-ino1, inol-ura2 and ura2-trkl (CABER, STYLES and FINK 1988); inol-sptl0 and sptl0-trkl, Table 6. Second inset indicates the physical map of the SPTlO PES2 TRKl region, with the direction of transcription of these genes indi- cated by arrows. The sequence data (double ended arrows) that support this map come from this study, from published work (BOGUS- LAWSKI and POLAZZI 1987; CABER, STYLES and FINK 1988) and from unpublished DNA sequences that span the PBSP-TRKl interval (kindly provided by G. Bocus- LAWSKI).

TABLE 7

Tetrad analysis, SIT21

Markers PD NPD T cM

ilv2 spt21 12 2 33 47 cin4 spt2l 82 0 48 18.5

ccfl3 cin4 42 2 56 34" cy3 spt21 24 6 70 53"

These tetrad data taken from DOHENY et al. (1 993) and unpub- lished data of K. DOHENY.

L I 1 I I - X/ / /

$.$ 8 3 % b J $ - 20cM

FIGURE 8.-Genetic mapping of SPT21. Tetrad data are from Table 7.

however, these proteins have effects at many loci scattered throughout the yeast genome, suggesting that these SPT proteins are not GAL4-like transcrip- tion factors that bind to a very restricted target se- quence.

SPTlO and SPT21 probably are part of the same transcriptional regulatory pathway, because mutations in the genes lead to similar phenotypes. Because spt l0 null alleles are more severe that spt21 null alleles in terms of growth rates, SPTlO may more directly affect target gene expression. These genes do not appear to encode redundant functions, because sptl0 spt21 dou- ble mutants have the same phenotype as that of the sptlO mutant alone; i .e. , the mutations are not syner- gistic (NATSOULIS et al. 1991). Furthermore, overex- pression of SPTlO (via 2-pm plasmid constructions) in spt2l strains and SPT21 in sptlO strains has no impact on the mutant phenotypes (data not shown).

The most drastic transcriptional effects observed thus far for spt l0 and spt21 mutants are on a subset of the histone genes; thus some of the phenotypes observed in sptlO and spt21 mutants may be indirect, resulting from reduction in intracellular histone con- centration and consequently, altered chromatin struc- ture. On the other hand, it is not possible to recapit- ulate the spt l0 or spt21 phenotypes by eliminating the SPT-dependent histone gene copies, so the SPTlO and SPT21 proteins must be acting at other loci as well (DOLLARD et al. 1994). Thus, sptlO and spt21 mutant phenotypes represent a complex interplay be- tween altered chromatin resulting from reduction in histone quantity and quality and the direct effects of the SPT proteins themselves.

We thank P. HIETER and D. KOSHLAND for helpful discussions and CLYDE DENIS for criticisms of the manuscript. We thank GEORGE BOGUSLAWSKI, CLYDE DENIS, KIM DOHENY, RICK CABER, BOB SIKORSKI and MARC VIDAL for providing unpublished data, and for gifts of plasmids. We thank VINCENT BERNARD and KATE DOLLARD for technical assistance. G.N. was supported by DAMON RUNYON/WALTER WINCHELL Postdoctoral research Award DRG- 044 and a Merck Postdoctoral Fellowship. F.W. was supported in part by grant GM32967 from the National Institutes of Health. J.D.B. was supported in part by grants CA16519 and GM36481 from the National Institutes of Health and by an American Cancer Society Faculty Research Award.

LITERATURE CITED

BLUMBERG, H., A. EISEN, A. SLODZIEWSKI, D. BADER and E. T. YOUNG, 1987 Two zinc fingers of a yeast regulatory protein shown by genetic evidence to be essential for its function. Nature 328: 443-445.

BOEKE, J. D., and S . B. SANDMEYER, 1991 Yeast transposable elements, pp. 193-261, in The molecular and cellular biology of the yeast Saccharomyces, edited by J. Broach, E. Jones and J.

SPTlO a n d SPT21 Genes 105

Pringle. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

BOGUSLAWSKI, G., and J. 0. POLAZZI, 1987 Complete nucleotide sequence of a gene conferring polymyxin B resistance on yeast: similarity of the predicted polypeptide to protein kinases. Proc. Natl. Acad. Sci. USA 84: 5848-5852.

BOTSTEIN, D., S. C. FALCO, S. E. STEWART, M. BRENAN, S. SCHERER, D. T. STINCHCOMB, K. SmuHLand R. W. DAVIS, 1979 Sterile host yeasts (SHY): a eukaryotic system of biological contain- ment for recombinant DNA experiments. Gene 8 17-24.

CHU, G., D. VOLLRATH and R. DAVIS, 1986 Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234 1582-1585.

CLARK-ADAMS, C., D. NORRIS, M. A. OSLEY, J. S. FASSLER and F. WINSTON, 1988 Changes in histone gene dosage alter tran- scription in yeast. Genes Dev. 2: 150- 159.

COMPANY, M., J. ARENAS and J. ABELSON, 1991 Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature 3 4 9 487-493.

CONDE, J., and G. R. FINK, 1976 A mutant ofS. cerevisiae defective for nuclear fusion. Proc. Natl. Sci. Acad. USA 73: 3651-3655.

DENIS, C. L., 1984 Identification of new genes involved in the regulation of yeast alcohol dehydrogenase 11. Genetics 108:

DOHENY, K. F., P. K. SORGER, A. A. HYMAN, S. TUCENDREICH, F. SPENCER and P. HIETER, 1993 CTFl3 is an essential compo- nent of the Saccharomyces cerevisiae kinetochore. Cell 73: 761- 774.

DOLLARD, K., S. L. RICUPERO, G. N. NATSOULIS, J. D. BOEKE and F. WINSTON, 1994 SPTlO and SPT21 are required for tran- scription of particular histone genes in Saccharomyces cerevisiae. manuscript in preparation.

DUTCHER, S. K., 1981 Internuclear transfer of genetic informa- tion in hrI/KARl heterokaryons in S. cereuisiae. Mol. Cell Biol.

EISENMANN, D. M., C. DOLLARD and F. WINSTON, 1989 SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcriptional initiation in vivo. Cell 58:

FEINBERG, A. P., and B. VOCELSTEIN, 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Bioch. 137: 266-267.

CABER, R. F., C. A. STYLES and G . R. FINK, 1988 TRKl encodes a plasma membrane protein required for high-affinity potas- sium transport in Saccharomyces cereuisiae. Mol. Cell Biol. 8:

GERRING, S. L., C. CONNELLY and P. HIETER, 1990 Positional mapping of genes by chromosome blotting and chromosome fragmentation. Methods Enzymol. 194: 302-318.

GORBALENYA, A. E., E. V. KOONIN, A. P. DONCHENKO and V. M. BLINOV, 1988 A conserved NTP-motif in putative helicases. Nature 333: 22.

HAHN, S., S. BURATOWSKI, P. SHARP and L. GUARENTE, 1989 Isolation of the gene encoding the yeast TATA binding factor TFIID: a gene identical to the SPTIS suppressor of T y element insertions. Cell 58: 1 173-1 18 1.

HODGMAN, T. C., 1988 A new superfamily of replicative proteins. Nature 333: 22-23.

833-844.

1: 246-253.

1183-1191.

2848-2859.

HUISMAN, O., W. RAYMOND, K. u . FROEHLICH, P. ERRADA, N. KLECKNER, D. BOTSTEIN and A. HOYT, 1987 A TnlO-lacZ- kan"URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes. Genetics 116

ITO, H., Y. FUKUDA, K. MURATA and A. KIMURA, 1983 Transformation of intact yeast cells with alkali cations. J. Bac- teriol. 153: 163-168.

JI, H., D. P. MOORE, M. A. BLOMBERG, L. B. BRAITERMAN, D. F. VOYTAS, G . NATSOULIS and J. D. BOEKE, 1993 Hotspots for urnelected Tyl transposition events on yeast chromosome I l l are near tRNA genes and LTR sequences. Cell 73: 1007-1018.

KUNKEL, T. A., 1985 Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82:

KUPIEC, M., and G. SIMCHEN, 1984 Cloning and integrative dele- tion of the RAD6 gene of Saccharomyces cerevisiae. Curr. Genet. 8: 559-566.

LIVINGSTON, D. M., 1977 Inheritance of the 2pm DNA plasmid from Saccharomyces. Genetics 86: 73-84.

LOEBER, G., R. PARSONsand P. TEGTMEYER, 1989 The zinc finger region of simian virus 40 large T antigen. J. Virol. 63: 94-100.

LOEBER, G., J. E. STENGER, S. RAY, R. E. PARSONS, M. E. ANDERSON and P. TECTMEYER, 1991 The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting. J. Virol. 65: 3 167-3174.

NATSOULIS, G., C. DOLLARD, F. WINSTON and J. D. BOEKE, 199 1 The products of the SPTlO and SPT21 genes of Saccha- romyces cerevisiae increase the amplitude of transcriptional reg- ulation at a large number of unlinked loci. New Biol. 3: 1249- 1259.

NATSOULIS, G., W. THOMAS, M. C. ROCHMANN, F. WINSTON and J. D. BOEKE, 1989 T y 1 transposition in Saccharomyces cereuis- iae is nonrandom. Genetics 123: 269-279.

ROSE, M. D., F. WINSTON and P. HIETER, 1990 Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Labo- ratory, Cold Spring Harbor, N.Y.

SHERWOOD, P. W., and M. A. OSLEY, 1991 Histone regulatory (hir) mutations suppress d insertion alleles in Saccharomyces cerevisiae. Genetics 128 729-738.

SIKORSKI, R. S., and J. D. BOEKE, 1990 In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Meth. Enzymol. 194: 302-3 18.

SIKORSKI, R. S., and P. HIETER, 1989 A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cereuisiae. Genetics 122: 19-27.

SMITH, H. O., and M. L. BIRNSTIEL, 1976 A simple method for restriction site mapping. Nucleic Acids Res. 3: 2387-2395.

SUZUKI, M., 1989 SPXX, a frequent sequence motif in gene regulatory proteins. J. Mol. Biol. 207: 61-84.

SUZUKI, M., 1990a The heptad repeat in the largest subunit of RNA polymerase I1 binds by intercalating into DNA. Nature

SUZUKI, M . , 1990b SPKK, a new nucleic acid-binding unit of protein found in histone. EMBO J. 8: 797-804.

WINSTON, F., and M. CARLSON, 1992 Yeast SNF/SWI transcrip- tional activators and the SPT/SIN connection. Trends Genet. 8: 387-391.

191-199.

488-492.

344 562-565.

Communicating editor: M. CARLSON