Vol. 12, No. 9

MOLECULAR AND CELLULAR BIOLOGY, Sept. 1992, p. 3843-3856

0270-7306/92/093843-14$02.00/0 Copyright © 1992, American Society for Microbiology

PTAJ, an Essential Gene of Saccharomyces cerevisiae Affecting Pre-tRNA Processing J. PATRICK O'CONNORt AND CRAIG L. PEEBLES* Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received 19 December 1991/Returned for modification 11 February 1992/Accepted 17 June 1992 We have identified an essential Saccharomyces cerevisiae gene, PTA], that affects pre-tRNA processing. PTA] was initially defined by a UV-induced mutation, ptal-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. ptal-) does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from ptal-) strains had normal pre-tRNA splicing endonuclease activity. ptal-) was suppressed by the ochre suppressor tRNA gene SUPII, indicating that the ptal-I mutation creates a termination codon within a protein reading frame. The PTAI gene was isolated from a genomic library by complementation of the ptal-) growth defect. Episome-borne PTA) directs recombination to the ptal-) locus. PTAI has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTAI gene product have been identified within the EMBL or GenBank data base. Disnrption of PTA) near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA) gene product are discussed.

endonuclease subunits (51). Three mutant alleles of SEN2 have been identified. The sen2-1 allele causes temperaturesensitive (Ts) pre-tRNA splicing endonuclease activity but not pre-tRNA accumulation (62), while the sen2-3 allele exhibits aberrant pre-tRNA splicing endonuclease activity that causes the accumulation of a splicing intermediate composed of the 5' exon and intron (2/3 molecule [25]). Two other genes control activities required for end maturation. RPR1 encodes the RNA component of the nuclear RNase P (35), while CCA1 encodes tRNA nucleotidyltransferase (1). The 10 remaining genes (RNA1, LOS), SEN1, SPLI, STPI, and TPD1 to TPD5) are represented by mutant alleles that exhibit various effects on pre-tRNA processing. Mutant alleles of RNAJ and LOS1 lead to accumulation of endtrimmed but unspliced pre-tRNAs from all 10 tRNA gene families with intervening sequences (27, 28). A mutant allele of SENI displays reduced pre-tRNA splicing endonuclease activity and accumulation of end-trimmed but unspliced pre-tRNAs (62). Null alleles of STPJ lead to accumulation of end-trimmed but unspliced pre-tRNAs for a subset of the gene families (61). The TPD1 to TPD5 genes are defined by the mutations tpdl-l, tpd2-1, tpd3-1, tpd4-1, and tpd5-1; these mutations were identified by a modified loss-of-suppression assay (59). A mutant allele of SPLI has a dominant restoration-of-splicing phenotype, activating splicing of the mutant suppressor tRNA sup3-e A39 (34, 46). These 18 genes can be grouped into two classes; those with known biochemical defects (CCAI, MM1, MOD5, RLG1, RPR1, SEN2, TRMI, and TRM2) and those with unknown modes of action (LOS), RNAI, SEN1, SPLI, STPJ, and TPDI to TPD5). This simple distinction suggests that the pre-tRNA processing pathway may be substantially more complex than any current model emphasizes (12, 44). Direct screening of mutagenized strains has recovered multiple mutant alleles for just two of these genes, LOS) and SEN2 (25, 28, 62). This implies that additional genes affecting pre-tRNA processing remain to be identified. We know that pre-tRNA splicing is essential for cell growth, since every known member of 10 different S. cerevisiae tRNA gene families contains intervening sequences

The pre-tRNA processing pathway consists of many steps catalyzed by a large number of enzymes (15). A fairly complete picture of pre-tRNA processing reactions in Saccharomyces cerevisiae and other eukaryotes has emerged from experiments with crude extracts and purified activities (9, 21, 35, 38, 48), microinjection of Xenopus oocytes with cloned tRNA genes (14, 39, 41), and analysis of processing intermediates from S. cerevisiae cells (33, 44). It is now clear that primary transcripts from tRNA genes have both 5' leader and 3' trailer sequences, lack -CCAOH tails and nucleoside modifications, and contain introns if transcribed from genes with intervening sequences. Processing of these transcripts requires several nucleolytic reactions and many nucleoside modifications. Some of the processing reactions occur in a dependent order; for example, 5' end trimming by RNase P normally occurs before 3' end trimming in eukaryotes (35, 44); likewise, -CCAoH tail addition must occur after 3' end trimming (16). The steps of splicing are cleavage at the 3' and 5' splice sites, respectively (44, 48), exon ligation (21), and finally removal of the 2' phosphate (38). However, some processing reactions do not occur in a dependent order, since splicing can take place either before or after end trimming (44). Genetic approaches are an important complement to direct biochemical dissection of complex cellular functions. To date, 18 S. cerevisiae genes that affect pre-tRNA processing have been identified by a variety of assay procedures. Four genes (TRM1, TRM2, MU), and MOD5) encode nucleoside modification activities (26). The order of tRNA nucleoside modifications does not appear to be obligatory. However, some nucleoside modifications important for the function of certain tRNAs are added only to unspliced pre-tRNAs (30, 58). Two genes encode enzymatic activities previously implicated in pre-tRNA splicing. RLGI is the tRNA ligase gene (49), and SEN2 encodes one of the pre-tRNA splicing *

Corresponding author.

t Present address: Howard Hughes Medical Institute, University

of Pennsylvania Medical School, Philadelphia, PA 19104.

3843

3844

MOL. CELL. BIOL.

O'CONNOR AND PEEBLES

TABLE 1. S. cerevisiae haploid strains A364A BJ3341 BJ3342 BJ4065 BLAY5-2c BUB-24c CLP1-la DK365-28B EElb LA4-22 LABJ3-S17c LABJ3-S17d POC1-13a POC8-23c POC8-23d POC8-32c POC8-32d POC8-36a

POC9-15d POC9-9c POC18-ld SS2A-3C tsxl7-6b tsx24-5b IIId-2c a

Source

Genotype

Strain

YGSCa E. W. Jones E. W. Jones E. W. Jones This study This study This study D. Kaback A. K. Hopper This study This study This study This study This study This study This study This study This study This study This study This study J. Abelson M. Culbertson M. Culbertson A. K. Hopper

MA4Ta adel ade2 gall his7 lys2 tyrl ural MATa gal2 leu2-Al lys2-801 mal mel trpl-AIOI ura3-52 CUPI SUC2 AL4Ta gal2 leu2-Al lys2-801 mal mel trpl-AJOl ura3-52 CUP1 SUC2 AL4Ta his3-A200 M4Ta leu2-Al lys2-801 trpl-AlOJ ura3-52 PTA1::LEU2 (YCpPTAI-HindIIIB) M4Ta FUN40::URA3 leu2-Al ura3-52 MATa ade2-1 his3-A200 trpl-AlJO ptal-l AL4Ta ura3 cdc24 MATa ade mal-l tyrl ura3-52 UV-mutagenized isolate from A364A ALATa leu2-Al lys2 trpl-AlOJ tyrl ura3-52 ATcTa ade2-1 his7 leu2-AJ lys2 ptal-l trpl-A101 tyrl MATa ade2-1 leu2-Al lys2ptal-l trpl-AIOI M4Ta ade2-1 leu2-Al lys2 trpl-AJOJ MATa ade2-1 leu2-Al lys2ptal-l trpl-AJOJ ura3-52 MATa leu2-Al lys2-801 ptal-l t7pl-AlOl ura3-52 MATa ade2-1 leu2-Al lys2 trpl-AJOJ ura3-52 MATa ade2-1 leu2-Al lys2-801 ptal-l trpl-A101 ura3-52 AMTa gal2 leu2-Al mal mel trpl-AlOJ ura3-52 CUPJ SUC2 MATa gal2 leu2-Al lys2-801 mal mel ura3-52 CUPJ SUC2 ptal-J sen2-1 MA Ta ade2-101 his3-A2000 lys2-801 sen2-3 ura3-52 GAL SUC2 MATa leu2-3,112 pep4-2 sen2-1 ura3-52 AL4Ta leu2-3,112 pep4-3 senl-l ura3-52 MATa ade2-1 canl-100 losi-A VSUP-4o ura3-1

YGSC, Yeast Genetic Stock Center, Berkeley, Calif.

(44). Increased steady-state levels of intron-containing pretRNAs, referred to here as pre-tRNA accumulation, is one phenotype characteristic of mutations affecting pre-tRNA splicing and pre-tRNA processing in general. Accordingly, we chose to look for pre-tRNA accumulation in strains harboring conditional growth mutations, believing that these two phenotypes would identify additional pre-tRNA processing mutants with defects in essential genes. Here we describe a new mutation leading to pre-tRNA accumulation, ptal-1. We have analyzed pre-tRNA processing in ptal-1 cells, isolated and sequenced the PTAJ structural gene, and shown that PTAI is an essential gene.

MATERIALS AND METHODS S. cerevisiae strains, media, cultivation, and genetic methods. S. cerevisiae strains are listed in Tables 1 and 2. YPD contained 2% peptone, 2% glucose, and 1% yeast extract. YPDS was YPD supplemented with 1 M sorbitol. Synthetic complete (SC) medium was prepared according to the recipe of E. W. Jones, Carnegie-Mellon University (63). SCS was SC supplemented with 1 M sorbitol; SCS-Leu and SCS-Ura are SCS without leucine or uracil, respectively. 5-Fluoro-orotic acid (5-FOA) medium was prepared as described elsewhere (7). Solid media contained 2% agar. Cul-

TABLE 2. S. cerevisiae diploid strains Strain

BLAY5 BUB CLP6

CLP11b LABJ3

POCi POC4 POC5 POC6 POC7 POC8

POC1O POCll POC14 POC16 POC17 POC31 POC34 POC40

Relevant genotype

Cross

POC17::p2.3-BL14(YCpPTAI-HindIIIB) POC17::pCB2-U17 CLPl-la x BUB-24c POC8-36a x BLAY5-2c LA4-22 x BJ3341 LABJ3-S17d x BJ3342 LABJ3-S17d x tsx24-5b LABJ3-S17d x tsxl7-6b LABJ3-S17d x LA4-22 LABJ3-S17d x SS2A-3C POCl-13a x BJ3341 LABJ3-S17c x BJ3342 LABJ3-S17c x LA4-22 LABJ3-Sl7c x tsx24-5b LABJ3-S17c x SS2A-3C POC9-15d x POC9-9c POC8-32c x POC8-23d (YCpPTA1) POC8-32c x POC8-23d::YIpPTA1(SnaBI) BJ4065 x POC8-23d::YIpPTA1(SnaBI)

+/PTAI::LEU2 (YCpPTAI-HindIIIB) +IFUN40::URA3 FUN40::URA3/ptal-I

ptal-1/PTAJ::LEU2 ptal-lI+ ptal-1/+ ptal-l/senl-l ptal-l/sen2-1

ptal-l/ptal-l

ptal-l/sen2-3 ptal-l/+ +/+

+Iptal-l

+Isenl-l +/sen2-3

leu2-AJ/leu2-Al ura3-52/ura3-52 ptal-l/ptal-l (YCpPTA1) ptal-1/ptal-1::PTA1

+/ptal-1:YPTAl

S. CEREVISIE PTAI GENE

VOL. 12, 1992

3845

TABLE 3. Plasmids Plasmid

YIp5 YCp5O YEp24 pSEYC58 pUN50 pUN60 YEpRNAI YCpLOSI pYEP11 pYEP99 pC6 YCpPTAI YCpPTAl-HindIIIB

YCpPTAI-HindIII-SaII YCpPTAI-HindIII-ClaI YCpPTAI-BamHI-HindIII

YCpPTAI-HindIII-BamHI YIp5-XhoIA YIpPTAI

YEp13-G pUN-PTAI pUN-BamHI pCB2 pCB2-U17 pBLUE-URA3 p2.3 p2.3-BL14

S. cerevisiae gene(s) and origin of replication

URA3 URA3 CEN4 ARS1 URA3 2pm URA3 CEN4ARSI URA3 CEN4 ARS1 URA3 CEN4ARSI SUPJJ RNAI in YEp24 LOSI in YCp5O RLGI in pSEYC58 RLG1 in YEp24 STPI in YEp24 PTAI in YCp5O 5.3-kbp HindIII fragment of YCpPTAI in YCp5O 2.5-kbp HindIII-SalI fragment of YCpPTAI in YCp5O 3.8-kbp HindIII-ClaI fragment of YCpPTAI in YCp5O 4.2-kbp BamHI-HindIII fragment of YCpPTAI in YCp5O 1.1-kbp HindIII-BamHI fragment of YCpPTAI in YCp5O 9.5-kbp XhoI fragment of YCpPTAI in YIp5 5.3-kbp HindIll fragment of YCpPTAI in YIp5 4.8-kbp EcoRI fragment spanning FUN40 in YEp13 4.0-kbp SacI-SalI fragment of YEp13-G in pUN50 1.7-kbp fragment of YEp13-G in pUN50 4.0-kbp Sacl-Sall fragment of YEp13-G in pUC119 1.2-kbp KprnI-BamHI fragment of pBLUE-URA3 in pCB2 1.2-kbp HindIII fragment containing URA3 in pBluescribe 2.5-kbp HindIII-EcoRI fragment of YCpPTAI in pUC119 3.1-kbp BgAII fragment of YEpl3 containing LEU2 in p2.3

tivation, plating, and tetrad analysis were performed as described previously (44, 56). Diploid strains were identified by selection for prototrophs, microdissection of zygotes, or screening for sporulation ability. Sporulation was induced with PSP medium (1% potassium acetate, 0.8% nutrient broth, 1% yeast extract, 2% agar). Transformation, nucleic acid isolation, and manipulation. S. cerevisiae cultures were transformed with plasmid DNA for complementation analysis and with linear DNA for gene disruption or targeted integration (54), using the lithium acetate method (56) followed by selection on SCS-Ura or SCS-Leu. Plasmid DNA was isolated from transformed S. cerevisiae strains as described previously (56). Escherichia coli DH5a and JM101 were transformed by the protocol of Hanahan (23). Plasmid DNA was isolated from transformed E. coli strains by a modified alkaline lysis procedure (4) and further purified by CL-4B gel filtration chromatography. Plasmids are listed in Table 3. Restriction endonucleases were obtained from various manufacturers and used as recommended by New England Biolabs (Beverly, Mass.). Other DNA manipulations were done as described elsewhere (4). S. cerevisiae total RNA was isolated as described previously (44). Northern (RNA) blots were made by separating 10 p,g of total RNA per lane on partially denaturing 7% polyacrylamide gels followed by electrophoretic transfer to GeneScreen as described previously (44). Northern blots were prehybridized, hybridized, and stripped as described previously (44). Northern blots were hybridized with an oligonucleotide complementary to an S. cerevisiae pretRNA intron (for pILE, pSERcga, pSERgcu, and pTRP probe sequences; see reference 44), stripped, and then hybridized with an oligonucleotide complementary to the 214- and 179-base US RNA (snR7) species (probe 5E7; 5'-CCT CCG CCA TTG ATC TGT A-3'; a gift from D. Brow

Source (reference)

J. Woolford J. Woolford J. Woolford E. Phizicky J. Woolford (20) J. Woolford (20) A. Hopper (2) A. Hopper (29) E. Phizicky (49) E. Phizicky (49) A. Hopper (61) This study This study This study This study This study This study This study This study J. Pringle (19) This study This study This study This study J. Pringle This study This study

and C. Guthrie [45]). Pre-tRNA, tRNA, and oligonucleotide probe nomenclature is as described previously (44). DNA sequencing and data analysis. The chain termination method of sequencing used Sequenase (U.S. Biochemical, Cleveland, Ohio) according to the manufacturer's instructions. Templates were prepared as plasmids or as singlestranded DNA from libraries of DNA fragments or by construction of specific subclones from convenient restriction endonuclease sites (4). Sequencing primers were purchased from commercial sources (M13 universal and reverse primers; New England Biolabs) or prepared by the University of Pittsburgh DNA Synthesis Facility. Sequence data were compiled and checked with the assistance of the program DNA Strider (37). Protein sequence comparisons, pattern searching, structure prediction, and figure preparation relied on the GCG package, version 7.0, as implemented at the Pittsburgh Supercomputing Center (17). Identification of pre-tRNA-accumulating strains. S. cerevisiae A364A was mutagenized with UV light to 2 to 5% survival and plated at 23°C on YPD. Individual Ts isolates were identified by lack of growth at 37°C. Each Ts strain was grown for 12 to 16 h at 23°C in YPD and then shifted to 37°C for 30 to 60 min. Cells were collected from 1-ml cultures by centrifugation, and the supernatant was carefully removed. The cell pellet was placed on ice and suspended in 200 ,l of 1% sodium dodecyl sulfate-1% 2-mercaptoethanol-3.7% formaldehyde-50 mM EDTA. The suspension was incubated at 65°C for 15 min and placed on ice before addition of 50 ,ul of 0.5 M KCl. The mixture was vortexed, held on ice for 10 min, and centrifuged for 5 min at 12,000 x g. Samples containing 100 ,ul of supernatant were applied to GeneScreen, using a Schleicher & Schuell Mini-Fold apparatus. The RNA was covalently cross-linked onto the GeneScreen by exposure to UV light (10) and hybridized with 32P-labeled oligonucleotide probes complementary to pre-tRNA introns

3846

!ew -. . . . . - . . .

O'CONNOR AND PEEBLES

as described previously (44). After autoradiography, pretRNA accumulation was compared with that of wild-type and rnal-1 strains (42, 43). Pre-tRNA splicing endonuclease assays. S. cerevisiae pretRNA splicing endonuclease fractions equivalent to fraction II of Peebles et al. were prepared essentially as described previously (48). Cultures were grown overnight in 2 liters of YPDS at 30°C. Cells were harvested by centrifugation, washed, and broken by a single pass through a French press at 20,000 lb/in2. The 2,000 x g supernatant was centrifuged at 250,000 x g for 2 h at 4°C. The membrane pellet was suspended to a final protein concentration of 10 mg/ml. Pre-tRNA splicing endonuclease assays were done as described previously (48), using 1 pmol of pre-tRNA PheGAA (52) and 20 ,ug of pre-tRNA splicing endonuclease fraction in 10-pl reactions. For pre-tRNA splicing endonuclease thermal inactivation, nuclear extracts were prepared and assayed as described previously (62) except that pre-tRNA PheGAA was used as the substrate. Nucleotide sequence accession number. The PTAI sequence has been submitted to GenBank (6) and is available under accession number M95673.

RESULTS Identification and characterization of a pre-tRNA-accumulating strain. Ts strains from UV-mutagenized A364A cells were assayed for pre-tRNA accumulation. RNA was isolated from each Ts strain and analyzed by filter hybridization with 32P-labeled oligonucleotides complementary to pretRNA introns. From approximately 500 strains, the one designated LA4-22 displayed particularly strong pre-tRNA accumulation (data not shown) and was chosen for further study. As we will demonstrate, Ts growth and pre-tRNA accumulation in LA4-22 are both phenotypes of ptal-1, a single recessive mutation. We next determined whether LA4-22 accumulated just one, some, or all 10 intron-containing pre-tRNAs. If LA4-22 accumulated a single pre-tRNA, the accumulation could be attributed to a tRNA gene mutation. If the ptal-1 mutation present in LA4-22 acts in trans to affect pre-tRNA processing, all or several pre-tRNAs should accumulate. Total RNA was isolated from LA4-22 and A364A cells cultured at various temperatures. No change in the overall pattern of RNA was evident by ethidium bromide staining after separation on polyacrylamide or agarose gels (data not shown). The RNA preparations were subsequently assayed for pretRNA accumulation by Northern analysis (Fig. 1). Substantial accumulation of end-trimmed pre-tRNAs IleUAU and SerGCU was clearly evident in LA4-22 cells and was independent of growth temperature. The amounts of pre-tRNAs IleUAU (Fig. 1A) and SerGCU (Fig. 1B) were consistently much lower in A364A cells grown under the same conditions. Similarly, all eight of the other intron-containing pre-tRNAs accumulated in LA4-22 cells compared with A364A cells (43). The amounts of U5 snRNA (Fig. 1C) and end-extended pre-tRNAs IleUAU and SerGCU were approximately the same between A364A and LA4-22. We also examined the pattern of pre-tRNAs accumulated in an outcrossed strain harboringptal-I (POC8-23d) by metabolic labeling with 32Pi (43). The ptal-1 strain was found to accumulate the same labeled pre-tRNAs as did an mal-1 culture shifted to 37°C (27, 32). We conclude that the PTA1 gene product acts in trans to affect the processing of all intron-containing pre-tRNAs. A single recessive mutation controls both phenotypes of

MOL. CELL. BIOL.

Shtamin 3 Grown at '3 Shifted t 3o

A364A 3t7 33 3 A37

L A4-2 2 7_

_E"V _A_OK _ Ol-_ __ *_qw

.A*,

..

.....

Pre-tRNA Ile-UAU Intron

B

AiIHIML.

Pre-tRNA SER-GCU Intron

-inUwm". ImaI

u . t. i

U5 (SNR7) RNA 5'End

FIG. 1. Accumulation of unspliced pre-tRNAs inptal-1. Northern blot analysis of total RNA from A364A (wild-type) and LA4-22 (ptal-1) cells grown at different temperatures in YPD. Cultures were grown at the indicated temperatures. Some cultures were temperature shifted for 30 min to the indicated temperature prior to harvesting. Lanes: 1 to 6, A364A total RNA; 7 to 11, LA4-22 total RNA. (A) pILE probe; (B) pSERgcu probe; (C) 5E7, an snR7 (U5 snRNA)-specific probe. Arrows in panels A and B indicate endmature pre-tRNAs.

LA4-22. Twelve independent Ts+ revertants were selected from LA4-22 and tested for pre-tRNA accumulation by Northern analysis, using four different pre-tRNA intron probes. The pre-tRNA accumulation phenotype was lost from all 12 Ts+ revertants for every pre-tRNA tested (43). These results indicate that the same mutation controls both pre-tRNA accumulation and Ts growth. All ptal-JIPTAI heterozygotes grew at 37°C on complete media (data not shown); all of those heterozygotes tested by Northern analysis failed to accumulate pre-tRNA (Fig. 2, lanes 1, 6, and 11; however, theptal-1lsen2-3 heterozygote is an exception [see below]). The small amount of pre-tRNA accumulation seen in the diploid strains (lanes 1, 6, and 11) appears to be a general property of diploid strains and is not an indication of ptal-1 semidominance (43). In contrast, a ptal-l/ptal-I homozygous diploid is Ts and accumulates pre-tRNAs (for example, see Fig. 7, lane 3). These observations show that both the RNA processing and the Ts phenotypes of ptal-1 are recessive. The ptal-I mutation from LA4-22 was outcrossed three times in succession to strain BJ3341 or BJ3342 to yield diploids LABJ3, POC1, and POC8 (Tables 1 and 2). Since BJ3341 and BJ3342 are wild type and do not accumulate

S. CEREVISIE PTA1 GENE

VOL. 12, 1992 LANE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Strain

I

1A

IB

IC

1D

2

2A

Growvn at

37

30

30

30

30

37

30

Accumulation Growth at37°C

l _I 1+ _ l+ _I 1+ -_ T _ _ [-1 + 1_ l+ 1+ 1_ + 1_ 1+ l+ + 1_-I+ +

2B

2C

3 2D

30 30

30

3 l 3A

3B

3C

30

30

3847

13D WTIIWT21pta1WlVT 30

-

3

30

-

3

30

+ _

-+ I+

Pre-tRNA TRP-CCA Intron

B

U5 (SNR7) RNA 5'End FIG. 2. Tetrad analysis of the ptal-1 pre-tRNA accumulation phenotype. Total RNA from spore clones of one tetrad from each of three successive outcrosses of LA4-22, their parentalptal-1 heterozygotes, the two wild-type strains used for the outcrosses, LA4-22, and A364A (the strain from which LA4-22 was derived) were assayed for pre-tRNA accumulation by Northern analysis. Each lane of a partially denaturing 7% polyacrylamide gel was loaded with 2 ,ug of total RNA. The gel was run for 1.5 h at 200 V. The blot was hybridized with the pTRP probe (A), stripped, and then hybridized with the 5E7 probe (specific for snR7) (B). Lanes: 1, LABJ3 (+/ptal-l); 2 to 5, the a, b, c, and d spore clones, respectively, of tetrad LABJ3-S17; 6, POC1 (+Iptal-l); 7 to 10, the a, b, c, and d spore clones, respectively, of tetrad POC1-13; 11, POC8 (+/ptal-l); 12 to 15, the a, b, c, and d spore clones, respectively, of tetrad POC8-23; 16, BJ3341 (wild type [WT1]); 17, BJ3342 (wild type [WT2]); 18, LA4-22 (ptal-1); 19, A364A (wild type [WT]; LA4-22 parental strain). The Ts growth and pre-tRNA accumulation phenotypes of each strain are indicated. The temperature at which each strain was cultured prior to harvesting and RNA extraction is also indicated.

pre-tRNAs (43), any pre-tRNA accumulation displayed by the meiotic products from these diploids can be attributed to the mutation(s) originally present in LA4-22. Several asci from these diploids and from a cross to a sen2-1 strain (POCS) were dissected on YPDS. The spore clones were tested for Ts and other genetic markers. In 34 of the 36 tetrads examined, Ts segregated in a 2:2 pattern to yield a total of 70 Ts- and 74 Ts' spore clones. In the two exceptional tetrads, three spores were Ts' and one was Ts-. We suspect that the two extra Ts+ spore clones arose from either gene conversion or second-site suppression of ptal-1 (see below). All other genetic markers segregated normally. Spore clones from three tetrads of the sen2-1 cross (POC5), three tetrads of the first and second outcrosses, and nine tetrads of the third outcross of ptal-1 were assayed for pre-tRNA accumulation (Fig. 2). In these 18 tetrads, pretRNA accumulation segregated 2:2 and always cosegregated with Ts growth, demonstrating that ptal-1 is a single, recessive, nuclear mutation controlling both phenotypes. In the course of this genetic analysis, we found that ptal-1 strains grow better and can grow at 37°C on media supplemented with 1 M sorbitol or other osmotic supplements. However, the pre-tRNA accumulation phenotype was not

suppressed by these conditions (43). The ptal-1 growth phenotype may best be described as temperature sensitive, osmotic remedial (Tsor [55]). ptal-) defines a gene affecting pre-tRNA processing. Complementation analysis was performed to determine whether ptal-1 represented an allele of any known pre-tRNA processing mutation. An outcrossedptal-1 strain (LABJ3-S17d) and a wild-type sibling (LABJ3-S17c) were mated to individual strains harboring pre-tRNA processing mutations (mal-1, losl-AV, senl-1, sen2-1, and sen2-3). Each diploid was assayed for growth at 37°C on YPD and for pre-tRNA accumulation by Northern analysis. In each instance, the heterozygotes grew at 37°C. The heterozygotes also failed to accumulate pre-tRNA (representative data are shown in Fig. 3 and summarized in Table 4; see also reference 43). Since sen2-3 is a bona fide pre-tRNA splicing endonuclease mutant, we were concerned that the accumulation of 2/3 molecules in the ptal-l/sen2-3 strain (Fig. 3B, lane 7) might indicate some functional interaction of Ptalp with Sen2p (the protein products of the PTAI and SEN2 genes, respectively). However, the +Isen2-3 heterozygote also accumulated an equivalent amount of pre-tRNA SerCGA 2/3 molecules (Fig. 3B, lane 9), demonstrating that the sen2-3 allele is

3848

t,.':M,Itl+}lt;'7s-l)'l''^$ixjZl-'''w'-']\;r V "

r ~ ~ ~~~~ stsr>->

Str.ailn

7

.........

:

SEtt-CCG.A lilti-oni

.!

::

:.

.

.:

':

7

;r >'S'Ss' Ss-

v§

':.

6

.*_lX.je.a Sm;_.. ;* :..;G:J

MOL. CELL. BIOL.

O'CONNOR AND PEEBLES

:.

',

:: ::'

U5 (SNR7) RNA 5END U5 (SN171 SHNA 5iEnd FIG. 3. Complementation of the ptal-1 pre-tRNA accumulation phenotype. Northern analysis was performed on total RNA extracted from tsx24-5b (senl-l) and aptal-1 heterozygote made with tsx24-5b (POC4) (A) and on total RNA extracted from SS2A-3C (sen2-3) and a ptal-1 heterozygote made with SS2A-3C (POC7) (B). The blots were hybridized with the pSERcga probe (top panels), stripped, and then hybridized with the 5E7 probe to ensure that intact and equivalent amounts of RNA were present in each lane (bottom panels). (A) Lanes: 1 to 5, tsx24-5b; 6, blank; 7, POC4 (senl-llptal-1); 8, LABJ3-S17d (ptal-1); 9, POC14 (+Isenl-1); 10, LABJ3-S17c (wild type [WT]). (B) Lanes: 1 to 6, SS2A-3C; 7, POC7 (sen2-3Iptal-1); 8, LABJ3-S17d (ptal-1); 9, POC16 (+/sen2-3); 10, LABJ3-S17c (wild type [WT]). Cultures were grown at the indicated temperatures. Some cultures were temperature shifted for 30 min to the indicated temperature prior to harvesting.

semidominant and that there is no evidence for a functional interaction between Ptalp and Sen2p. Several different intron-specific pre-tRNA hybridization probes were used, and all gave similar results (data not shown). Since pretRNA accumulation can result from a deficiency of pretRNA splicing endonuclease, we showed that the senl-l and sen2-1 mutations assort independently of ptal-1, establishing that ptal-1 represents a gene distinct from either SENI or SEN2 (43, 49b). Complementation was also performed with episomal copies of genes involved in pre-tRNA processing. POC8-32c (relevant genotype, ptal-1 ura3-52) was transformed with either an RNA1, LOS1, RLG1, or STPJ plasmid (2, 29, 49, 61). In each case, the transformed strains remained Tsor and accumulated pre-tRNAs (Fig. 4). Complementation analysis of tpdl to tpdS strains with ptal-1 was reported by van Zyl et al. (59), andptal-1 is not an allele of any one of the TPDJ to TPDS genes. SPLI and RPRI have been mapped to chromosomes III and V, respectively (34, 35), while PTAJ maps to chromosome I (see below). Therefore, PTA1, SPL1, and RPRI are distinct genes. Comparison of restriction maps and sequences (see Fig. 8 for PTA1) shows that PTAJ is different from CCA1, the gene for tRNA nucleotidyltransferase (1). These results (summarized in Table 4) clearly indicate that ptal-I defines PTA1, a previously undescribed gene affecting pre-tRNA processing. Pre-tRNA splicing endonuclease activity is normal in ptal-l extracts. Pre-tRNA splicing endonuclease has been purified as a complex of three distinct protein subunits (51). The SEN2 gene encodes one subunit of the pre-tRNA splicing endonuclease (25), but the genes for the remaining two subunits have yet to be identified. We wanted to test whether PTAI encoded one of the pre-tRNA splicing endonuclease subunits, since ptal-I results in accumulation of unspliced

pre-tRNA and is not an allele of SEN2. We prepared extracts from ptal-1, senl-1, sen2-1, and wild-type strains and assayed for pre-tRNA splicing endonuclease activity. The levels of activity from ptal-1 and wild-type strains were identical (Fig. 5A). In contrast, an extract prepared from a senl-l strain had reduced pre-tRNA splicing endonuclease activity, as expected (62). We also examined the thermal inactivation kinetics of pre-tRNA splicing endonuclease activity prepared fromptal-1 strains and found that the rate of inactivation was indistinguishable from the wild-type rate (Fig. SB), whereas extracts prepared from sen2-1 strains were more heat sensitive, consistent with the described phenotype of this mutation (62). We conclude that theptal-1 mutation does not directly affect pre-tRNA splicing endonuclease activity as assayed in vitro and that PTAJ probably does not encode any of the pre-tRNA splicing endonuclease subunits. Nature of the ptal-l lesion. Genetic analysis of the ptal-1 mutation was hampered by a high reversion frequency. Approximately 2% of progeny derived from a ptal-1 single colony were revertants after the strain was grown in YPD at 30°C for 10 generations. The frequent reversion of ptal-1 suggested that second-site mutations suppress ptal-1. Most ptal-1 strains also carry ade2-1, derived from the parental A364A strain. The ade2-1 allele is an ochre mutation, and ade2-1 colonies appear red. Almost all of the Ts' revertant colonies were white and Ade+ (data not shown). Simultaneous suppression of ade2-1 indicates that most second-site suppressors of ptal-1 are ochre suppressor tRNAs. To show that the ptal-1 lesion is an ochre mutation, POC8-23d (relevant genotype, ade2-1 ptal-1 ura3-52) was transformed with either pUN50 or pUN60, two low-copy-number plasmids (20). These plasmids are identical except that pUN60 carries SUPJ1, a tyrosine-inserting ochre suppressor tRNA

3849

S. CEREVISIAE PTA1 GENE

VOL. 12, 1992 TABLE 4. ptal-1 complementation analysis

compleEpisomal mentation

Classic genetic complementation Gene

ptal-I diploid phenotype

Mutant phenotype

Chromo-

(trans-

formed into a

ptal-l

some

Mutation

RNAI LOSI RLGI SENI SEN2

STPI TPD1 TPD2 TPD3 TPD4 TPD5 CCAlf RPR1 SPL1 PTAI

XIII XI XII

IV

V III I

Splicing Growth endonudefects defects clease

Pre-tRNA accumulation

Growth defects

Pre-tRNA accumulation

Recombines

strain)

Is gene essential?

defects

defects

Pre-tRNA accumulation

Tsor Tsor

Yes Yes

Yes No

Tsor

Yes

Yes

Growth

W1l rnal-1 losl-A

None Ts None

WT WT WT

No Yes Yes

None None None

No No No

senl-l sen2-1 sen2-3 stpl::URA3 tpdl_ld tpd2-1

Ts

Reduced

Yes

None

No

Yes

Yes

None

Ts Abnormal

No Yes Yesc

None

No Slight

Yes

?

Yes

None

tpd3-1 tpd4-1 tpd5-1 ccal-I

Ts Ts Ts Ts

Wr WT WT WT WT

Yes

None None None None

ptal-1

Tsor

Noneb None Ts Ts

None

Tsor

Yes

No

No

? ? ? ? ? ? Yes ? Yes

Abnormalg WT

Yes

Tsor

Yes

None

WT, wild type. sen2-3 causes a cold-sensitive spore germination defect. The STPI disruption causes the accumulation of some but not all pre-tRNA families. d ptal-1 complementation analysis with all tpd strains was performed by van Zyl et al. (59). Pre-tRNA splicing endonuclease activity of the tpd strains was assayed by Pippert (49a). f The PTAI and CCAI genes have different sequences, and their loci have different restriction endonuclease maps. g The ccal-I mutation causes the accumulation of tRNAs without the -CCAOH tail and aberrant mRNA synthesis at the nonpermissive temperature. a

b

c

gene. Both plasmids complement ura3-52 (Fig. 6C). The ade2-1 mutation was suppressed by pUN60 but not by pUN50 (data not shown). Only pUN60 permitted POC8-23d to grow at 37°C on either YPD or SC-Ura (Fig. 6). Identical results were obtained with a secondptal-1 strain, POC8-32c (data not shown). POC8-23d strains that had lost pUN60 were selected by resistance to 5-FOA; the resulting plasmidloss strain was capable of growth only at 30°C on YPDS (Fig. 6). This result shows that ptal-J had not reverted in the pUN60-transformed strain. SUPJJ also suppressed pretRNA accumulation in ptal-I (Fig. 4, lanes 1 and 2). Accumulation of pre-tRNA TrpCCA occurred in POC8-32c transformed with pUN50 but not with pUN60. Identical results were obtained for pre-tRNA LysUUU and with transformed POC8-23d strains (data not shown). These results indicate that the ptal-1 lesion is a point mutation that disrupts the translational reading frame of PTAJ with an ochre stop codon. Isolation and mapping of the PTA] gene. The PTA1 gene was isolated by complementation of the ptal-1 mutation. We used an available library prepared by partial Sau3AI digestion of S. cerevisiae genomic DNA ligated into the BamHI site of YCp50 (53). This vector is maintained at one or two copies per S. cerevisiae cell with URA3 as a selectable marker. Approximately 64,000 transformants of POC8-23d were selected on SCS-Ura at 30°C and screened. Two characteristics were used to identify those transformants that contained the PTAJ gene: growth at 37°C at low osmolarity and pink colony color. Only two transformants met both criteria. Plasmid DNA was isolated from both positive strains and transformed into E. coli DH5a. Plasmid DNA prepared from single-colony isolates of the two transformed E. coli strains complemented ptal-1 at high fre-

quency. Restriction endonuclease analysis demonstrated that the two independently isolated plasmids were identical; this plasmid has been designated YCpPTAJ (see Fig. 8). Growth of a ptal-1 strain transformed with YCpPTAI is illustrated in Fig. 6. YCpPTAJ also complements the pretRNA accumulation phenotype ofptal-1 (Fig. 7). Total RNA was prepared from a wild-type diploid, a ptal-1 heterozygote, aptal-I homozygote, and aptal-1 homozygote transformed with YCpPTAJ. Pre-tRNA accumulation in the transformedptal-1 homozygote was dramatically reduced to near wild-type levels. Three different pre-tRNAs were examined, and all gave similar results. The same result was obtained with haploid ptal-1 strains transformed with YCpPTAI (data not shown). Several subclones of YCpPTAI and an independent clone, YEp13-G (18), were assayed for PTA1 function (Fig. 8B). The smallestptal-l-complementing region was found to be a HindIII-SalI fragment of YCpPTAI (YCpPTA1-HindIII-

SalI; Fig. 8). To show that DNA sequences present on YCpPTAI map to the ptal-1 locus, plasmid YIpPTAI was constructed by subcloning the ptal-l-complementing HindIII fragment of YCpPTAl into YIp5 (the same HindIII fragment is present in YCpPTAI-HindIIIB; Fig. 8). This integration vector contains URA3 as a selectable marker. YIpPTAJ was cut with SnaBI and transformed into POC8-23d, an integrative transformant was selected on SCS-Ura, and the transformant was mated to POC8-32c (relevant genotype, ptal-1 ura3-52) and to BJ4065 (relevant genotype, PTA1 URA3) to produce diploids POC34 and POC40, respectively. We tested 20 of 22 tetrads from POC34 that produced four viable spore clones. The outside genetic markers segregated in a normal 2:2 pattern. Ts' and Ura+ cosegregated 2:2 in 17 tetrads (Table

3850

O'CONNOR AND PEEBLES

LANE Plastmidt1 Gelie

1

2

IPtN pL.NI

Al

Al

3 YC

4 YEE,24

UP17

5

6

MOL. CELL. BIOL.

7

8

9

RA

0, I

I; W

A.

10 11

p'pC6. rpR.":A1 i.f 11 pYL E 1'9 |Y 9 C6

STP1

I

I

90. g.

IN'T

80

K..

O 70 S X 60-

1

50-

T40I

_A

a. 3020-

P're-tRNA TRP-CCA Intron

100

10

20

30 40 minutes at 300C

50

60

B.

1.000

1,qf VO,

a

FIG. 4. Episomal complementation of the ptal-1 pre-tRNA accumulation phenotype. POC8-32c (ptal-1 ura3-52) was transformed with several different plasmids. Total RNA was extracted from the transformed strains and assayed for pre-tRNA accumulation by Northern analysis. The strains were grown at 30°C in SCS-Ura medium. The blot was hybridized with a 32P-labeled pTRP probe (A), stripped, and hybridized with a 32P-labeled 5E7 probe (specific for snR7), (B). Lanes: 1, pUN50 (vector) 2, pUN60 (SUPJl); 3, YCp5O (vector); 4, YEp24 (vector); 5, pSEYC58 (vector); 6, YEpRNA1; 7, YCpLOSJ; 8, pYEP11 (RLG1 [also called LIG1]); 9, pYEP99 (RLG1); 10, pC6 (STP1); 11, untransformed POC8-32d (wild type) grown in SCS medium at 30°C.

5); three exceptional tetrads contained no Ura+ colonies but had two (one tetrad) or one (two tetrads) Ts' clones that probably represent URA3 plasmid excision events that left behind a PTAI allele (Table 5). These results show that the sequence derived from YCpPTAl and the URA3 marker are linked in the YIpPTAJ transformant. We analyzed 24 tetrads from POC40. The outside genetic markers segregated in a normal 2:2 pattern. None of the 96 spore clones were Ts or Tsor for growth, establishing that the integrated sequences

derived from YCpPTAl are tightly linked toptal-1 (Table 5). Hybridization analysis of genomic DNA extracted from the parental strains and from the colonies of several tetrads demonstrated that a single copy of YIpPTAl was integrated at the intended locus and segregated as expected (data not shown). These results indicate that sequences derived from YCpPTAl direct integration to the ptal-I locus. Thus, YCpPTAJ contains the bona fide PTAJ gene. The PTAI gene was mapped to chromosome I by hybridization analysis of S. cerevisiae chromosomal DNAs resolved by pulsed-field gel electrophoresis and probed with PTAJ DNA (data not shown). A similar analysis using genomic DNAs from S. cerevisiae strains carrying fragments of chromosome I refined the location of PTAJ to the left arm (CDC24 side) of chromosome I. Comparison of the YCpPTAI and CDC24 restriction maps suggested that YCpPTAJ overlapped CDC24 (Fig. 8) (19, 40). To confirm this assignment, strain DK365-28b (relevant genotype, ura3 cdc24) was transformed with YCpPTAI. Several Ura+ transformants were tested, and all grew at 37°C, indicating that

15 0.1002

-

v~~~~~~~

.a0.010n

rni. 0

1o

20

30

40

minutes at 370C FIG. 5. Splicing endonuclease activity in ptal-I extracts. Extracts were prepared from wild-type and mutant strains of S. cerevisiae and assayed for pre-tRNA splicing endonuclease activity. 32P-labeled pre-tRNA PheGAA was used as the substrate. All cultures were grown in YPDS at 30°C for extract preparation. (A) 0, Wild type (average of two independent A364A extracts and one POC8-23c extract); 0, ptal-I (average of two independent POC823d extracts and one POC18-ld extract); A, senl-l (results from one tsx24-5b extract); A,ptal-l/senl-l (equal amounts of aptal-1 and a senl-l extract were mixed and then assayed); V, mock (wild-type extract was heat inactivated at 65°C for 10 min and then assayed). The extracts were assayed at 30°C. (B) Crude nuclear extracts were prepared from wild-type (POC8-23c; 0),ptal-l (POC8-23d; 0), and sen2-1 (tsx17-6b; Y) strains. Each extract was preincubated at 37°C prior to assay at 25°C for 15 min. Data are normalized for eacb extract to 100% activity at time zero of preincubation and presented as the natural logarithm of the fraction of product relative to the total of remaining substrate plus product.

YCpPTAJ complemented the Ts growth defect of cdc24. This finding demonstrates close linkage between PTA1 and CDC24. A transcript map is available for this region of chromosome 1 (18, 19) (Fig. 8). Three transcripts overlap the HindIII fragment of YCpPTAJ-HindIIIB; these transcripts have been designated FUN9 (1.4 kb), FUN39 (3.1 kb), and FUN40 (0.6 kb). (The acronym FUN stands for function unknown.) While the direction of transcription was not established empirically for these RNAs (18, 19), our partial sequence analysis suggests that FUN9 is transcribed toward the centromere and that FUN39 and FUN40 are transcribed away from the centromere. The smallest fragment comple-

RLASMID

pUN3SO

pUNSO

|

p

'CpS

UUN6

SCpI

I|

L~Lost { O(1A IL c}tor tfU II lolie -cct,1r I|

GE:NE

3851

S. CEREVISLAE PTA1 GENE

VOL. 12, 1992

AI

LANE

SC-iflF FOA |

|

A

2

3

4

I+/+ I+/- 1-/ I-/-I

GENOTYPE YPDS(300C)

B

1

YCpPTAl

I

I

I

I

+

I

z...

YPD (37CC)

SCS-ura (30CC)

D

SC-ura (37CC)

FIG. 6. Episomal complementation of the ptal-1 growth defect. POC8-23d (ade2-1 ptal-l ura3-52) was transformed with pUN50, pUN60 (SUP11), YCp5O, and YCpPTAI. The transformed strains were grown for 2 days on different media at 30 or 37°C and then photographed. The pUN60- and YCpPTAJ-transformed strains were induced to lose the plasmid DNA by 5-FOA selection. The plasmid loss strains were then assayed for growth as described above. Growth conditions: (A) YPDS, 30°C; (B) YPD, 37°C; (C) SCS - Ura, 30°C; (D) SC - Ura, 37°C.

menting ptal-1 is the 2.5-kbp HindIII-SalI fragment (YCpPTAI-HindIII-SalI; Fig. 8), which contains a large C-terminal portion of the putative FUN39 open reading frame, lacking promoter and translation initiation sites, and a large N-terminal segment of the putative FUN40 open reading frame with its promoter intact. Since FUN9 does not overlap this fragment, FUN9 is not PTA1. Gene disruptions identify FUN39 as PTAI. We constructed gene disruptions to assign PTAJ unambiguously to one of the transcribed regions within theptal-l-complementing region. A FUN40::URA3 disruption was constructed by replacing the Kjpn1-BglH fragment of FUN40 in plasmid pCB2 with a 1.2-kbp fragment containing URA3 to make plasmid pCB2U17 (Table 3 and Fig. 8). Diploid POC17 was transformed with BamHI-digested pCB2-U17 to obtain the FUN40 disruption in a heterozygous condition. Asci were dissected from three Ura+ transformants (strains BUB, PUA, and PUB). Four viable spores were obtained for most asci with Ura+ segregating 2:2 in the tetrads (data not shown). Hybridization analysis was performed on genomic DNAs extracted from each diploid and from each clone of several tetrads. The pattern of hybridizing fragments confirmed that the intended replacement had been recovered and that the restriction fragments diagnostic for the gene disruption always cosegregated with Ura+ (data not shown). Thus, FUN40 is not an essential gene for growth on complete medium. One Ura+ haploid strain, BUB-24c (relevant genotype, FUN40::URA3), was mated to CLP1-la (relevant genotype, ura3-52 ptal-1) to determine whether FUN40::

Pre-tRNA TRP-CCA Intron FIG. 7. Complementation by YCpPTAI of the ptal-1 pre-tRNA accumulation phenotype. Northern analysis was performed on total RNA extracted from POC10 (+/+; lane 1), POC11 (+/ptal-1; lane 2), POC6 (ptal-lIptal-l; lane 3), and POC31 (ptal-l/ptal-1) transformed with YCpPTAI (lane 4). Five micrograms of RNA was applied to each lane. The blot was hybridized with the pTRP probe and autoradiographed. The blot was subsequently stripped and then hybridized with the 5E7 probe to ensure that intact and equivalent amounts of RNA were present in each lane (data not shown).

URA3 was allelic to ptal-1. The resulting Ura+ diploid (CLP6) grew as well as the wild type at all temperatures, indicating that the ptal-1 Tsor defect had been complemented. Therefore, PTAI is not FUN40. FUN39 was disrupted by inserting a 3.1-kbp BglII DNA fragment containing LEU2 into the BamHI site of FUN39 to make plasmid p2.3-BL14 (Table 3 and Fig. 8). p2.3-BL14 was digested with BglII plus SnaBI and transformed into diploid POC17 to obtain the FUN39::LEU2 disruption in a heterozygous condition. Asci were dissected from two Leu' transformants of POC17 (strains BILA and BLB) on either YPD or YPDS. Most asci produced just two viable colonies (Table 6). All but one of the survivors were Leu-, while the other genetic markers segregated about 50%+ and 50%-. The single, exceptional Leu+ spore may have resulted from ectopic recombination of the LEU2 allele integrated at the PTA1 locus with the leu2 locus (36) or by nondisjunction leading to chromosome I disomy (2, 3). Microscopic examination of the nongrowing spore clones revealed that germination had occurred but that growth and cell division failed after a few cycles. The microcolonies contained between 5 and 89 cells (36 colonies were examined). All of the cells found in these microcolonies were extremely large, arrested either as unbudded cells or as large cells with one or two equally large buds. No further cell division was detected after 18 to 24 h at 30°C, although the cells continued to increase in size. These observations show that FUN39 is not required for germination but is essential for normal growth.

TABLE 5. Tetrad analysis: YIpPTAI integration Diploid

POC34 POC40 a

b

c

Asci with given no. of viable spores

dissected 44 36

4

3

2

1

0

22 24

14 5

5 4

2 3

1 0

No. of tetrads tested

20 24

Tetrads with given no. of: Ts' spores/tetrad Ura+ spores/tetrad 4

3

2

1

0

4

3

2

1

0

0 24

0 0

18 0

2 0

0 0

0 2

0 19

17a 3

0 0

3b OC

All Ura+ spores are also Ts'. Two tetrads had no Ura+ spores and one Ts' spore. Two outside markers segregated 2+:2- in all tetrads. One tetrad had three Leu- spores and one Leu+ spore. Five outside markers segregated 2+:2- in all tetrads.

3852

O'CONNOR AND PEEBLES

A.

MOL. CELL. BIOL.

Left Arm Chromosome I E Sc BH HKG BX

,M-.r -I

)I

S EXb CE

s irI:

FUN42 FtN41 FUJN40

PTA1

HEPEEP

1)Ar

FUN9

(9J9)

CDC24

G

HPE

H

II

E

xssc

CYC3

WI-

-*CEN Transcripts

Complementaton S3

B.

Plasmid

S3

J'

1hOp

HS3

ptal-1 +

YCpPrAl

+

H

YCpPTAl-HindIIIB H S3

+

+1

YCpPTAl-HindIII-ClaI

+

ND

YCpPTAl-HindfI-SalI

+

ND

YCpPTAl-HindHII-BamHI

-

ND

YCpPTAl-BamHI-HindIII

-

ND

pUNPTAl

+

C

~~~~~~~~~~~~~~~~~~~~~~I

'a

HS3

PTAI.::LEU2

B

H

B

Sc

S

I E

_

E _

_.

x

Sc B H H

B

B

ND

YIp5-XhoLA

ND

S

I 11 ) H S3

YEpl3-G x

SE

pCB2-U17

+

ND

p2.3-BL14

-

ND

FIG. 8. Restriction map of YCpPTAI and subclones. (A) The restriction map of the PTAI locus is diagrammed to scale. Restriction sites: B, BamHI; C, CiaI; E, EcoRI; G, BglII; H, HindIII; Hp, HpaI; K, KpnI; P, PvuII; S, SalI; Sc, SacI; S3, Sau3AI; X, XhoI; Xb, XbaI. (B) The original PTAI clone, YCpPTA1, is a partially digested Sau3AI fragment inserted into the BamHI site of YCp5O. Subclones of YCpPTAI and other clones from this locus are diagrammed. The complementation ability of each plasmid for both ptal-1 and PTAI::LEU2 strains is indicated. ND, not determined.

To recover haploid strains carrying FUN39::LEU2, plasmids with FUN39 sequences were introduced into diploids BLA and BLB, and more asci were dissected. Only plasmids containing the complete FUN39 gene, such as YCpPTA1, rescued the lethal defect of FUN39::LEU2 and allowed complete tetrads to be recovered, with Leu+ segregating 2:2 (Table 6). In the Leu+ colonies, a complementing plasmid was always retained, although plasmids were frequently lost from the Leu- colonies. This segregation pattern is expected for a centromeric plasmid complementing a disruption of an essential gene. Furthermore, the Leu+ colonies failed to produce 5-FOA-resistant colonies, while the Leu- colonies did so readily. The Leu+ phenotype was also linked to the expected, altered pattern of genomic DNA restriction endonuclease fragments (data not shown). To determine whether FUN39::LEU2 is allelic to ptal-1, the haploid BLAY5-2c [relevant genotype, ura3 leu2 FUN39::LEU2 (YCpPTA1)J was mated to POC8-36a (relevant genotype, leu2 ura3 ptal-1). The resulting diploid CLP11 readily lost YCpPTAI to give the Ura- diploid CLP11b, which was Tsor for growth. Transformation of CLP11b (relevant genotype, FUN39::LEU2Iptal-1) with YCpPTA1, pUNPTAI, or pUN60 restored growth at high temperature (data not shown). These results confirm that the observed Tsor growth of CLP11b results from expression of the ochre-suppressible ptal-J mutation and confirms that FUN39::LEU2 (hereafter referred to as PTA1::LEU2) is allelic to ptal-1.

Sequence analysis of PTA]. We have determined the sequence of 2,760 bp spanning PTAI (Fig. 9). This segment includes the entire region mapped for the PTAJ transcript (18). There is a single large open reading frame that could encode a protein of 785 amino acids, assuming that translation initiates at the first available methionine codon. The

TABLE 6. Tetrad analysis: PTA1::LEU2 disruption Diploid

No. of No. of 4-spore asci complete dissected tetradsa

Tetrads with given no. of viable spores/tetrad 4

BLA(YPDS, 30°C) BLB(YPDS, 30°C) BLB(YPD, 23°C)

BLA(pUN5O) BLA(pUNPTAI) BLA(YCp5O) BLA(YCpPTAI) BLA(YCpPTAI-HmndIIIB) BLB(YCp5O) BLB(YCpPTAI)

23 22 12 6 6 30 18 22 12 12

18 19 10 4 6 30 18 22 12 11

3

2

1 0

0 0

18

0 0 1 0 0 0 0 0 0 0

0 0 1b 16C 2 0 0 9 0 4 0 0 0 0 0 6 0 0 0 29 1 4 7 7 0 7 9 5 1 0 0 12 0 2 7 2 0

a All four spores successfully germinated as judged by microscopic visual

inspection. b c

All three spore clones were Leu- in this tetrad. A single Leu+ spore was recovered in this group.

S. CEREVISIAE PTA1 GENE

VOL. 12, 1992 -240

------------------------------------------------------------

TGTGTTATATCACTGTCCTACTCCGCATGTATACGAGACATAAAACTAGCTTTCCTTTAT

-181

-180

AATGGCGTGGTTTCCACTCCATCTTGATGGAAAATCGCCGTTCTGATGTTCAAGTCGTAT

-121

-120

ATAGTGACTTGATCTAAAAGTGAAAAGTTGCCATATTTCGATACCTTTTAACCGTTAGTA

-60 1 1 61 21

121 41

381

CCTGTGTCCGCACCTGCAACGGGCTCTTCAACCGAAAACATGCTTGATCAACTGAAGATA P V S A P A T G S S T E N M L D Q L K I

1200 400

-61

1201 401

TTGCAAAAATACACCCTCAACAAGGCTTCACACCAGGGCAATACTTTTTTCAACAACTCA

1260 420

CTTGACTGAACAAATGGTTAAATCGTGGCAAGAAGCATATAACTACCACTAAGCAGCGCA

-1

1261 421

60 20

CCCAAACCAATCAGCAACACCTACTCATCTGTGTACTCATTGATGAACAGTTCGAACTCC P K P I S N T Y S S V Y S L M N S S N S

ATGTCATCTGCAGAGATGGAACAATTGTTACAGGCCAAGACACTGGCCATGCACAACAAT

1320 440

1321

AACCAGGATGTGACCCAGCTACCCAATGACATACTTATCAAGCTGTCCACAGAGGCCATC

1380 460

M

S

S

A

E

M

EQ

L

L

Q

A

K

T

L

A

M

H

N

N

P

T

E

M

L

P

K

V

L

E

T

T

A

S

M

Y

H

N

G

N

CTCAGCAAGCTGAAGTTGCCTTTGGCCAAGTTTTTTACACAGTTAGTTCTAGACGTGGTG L

S

K

L

K

L

P

L

A

K

F

F

T

Q

L

V

L

D

V

V

120 40

180 60

TCGATGGACTCTCCAATTGCGAATACTGAGAGACCGTTTATTGCTGCTCAATATCTGCCA S M D S P I A N T E R P F I A A Q Y L P

240 80

241

81

CTACTTCTTGCTATGGCGCAATCCACCGCGGACGTACTAGTGTACAAGAATATCGTGCTT L L L A M A QS T A D V L V Y K N I V L

300 100

301 101

ATTATGTGCGCTTCATACCCGCTGGTGTTGGATCTGGTTGCTAAGACATCAAACCAGGAA

360 120

361 121

ATGTTTGATCAGTTGTGTATGCTGAAGAAGTTCGTGCTCTCGCACTGGAGAACTGCATAT M

421 141

CCTTTGCGTGCCACCGTTGACGATGAAACGGATGTCGAACAATGGCTGGCGCAGATTGAC P L R A T V D D E T D V E Q W L A Q I D

480 160

481 161

CAAAATATCGGCGTGAAATTAGCGACCATCAAGTTCATATCTGAGGTCGTGCTGTCGCAA QN I G V K L A T I K F I S E V V L SQ

540 180

541 181

ACTAAATCACCCAGCGGCAACGAGATTAATTCATCTACCATCCCGGATAACCACCCTGTG

600 200

601 201

TTGAACAAACCGGCTTTGGAGAGCGAGGCTAAGAGGCTTCTTGATATGTTGCTAAACTAC

661 221

CTAATTGAGGAACAGTACATGGTCTCGTCCGTTTTCATTGGTATCATCAATTCTTTATCC

721 241

TTCGTCATCAAAAGAAGGCCGCAGACAACAATAAGAATTCTTTCCGGGCTGTTGCGTTTC

I

T L L F

M F

K N

I V

C D

S K

E I

A

Q

P P E

K

S L

S A

Q R

Y

C

G L Y R

P M

N E

M P

L

L

E

S V

Q

V

K

I E

S T

L K

N A

S T

D F

S K V I

L V

S R F R

V L

T

L I

I

A

S

I

L

G L

K

H

P

D I

S

T W

D

M

I

G

S R

N

L N L

N

T

H L

S L

Q A

P

N

L R

E

Y

V

Y

S F

AACGTCGACGCCAAGTTTCCCCTAGAGGGCAAGTCTGACTTGAACTACAAACTATCCAAG N

V

D

A

K

F

P

L

E

G

K

S

D

L

N

Y

K

L

S

K rT I

420 140

660 220 720 240

260 840 280 900 300

R

901 301

ACAAAATCCCTCTCATCCGGATCAGGGTCATCGATCTACTCCAAGCTGACCAAGATTTCT T K S L S S G S G S S I Y S K L T K I S

960 320

961 321

CAAACTTTACACGTTATTGGCGAAGAGACCAAGAGCAAGGGAATTTTGAACTTCGACCCT T L H V I G E E T K S K G I L N F D P Q

1020 340

1021 341

TCCAAGGGCAATAGCAAGAAAACGTTGTCCAGGCAGGACAAACTAAAATACATCTCACTA S K G N S K K T L S R Q D K L K Y I S L

1080 360

1081 361

TGGAAAAGGCAATTATCCGCGTTATTGTCTACTCTAGGGGTGTCCACAAAGACCCCCACG W K R Q L S A L L S T L G V S T K T P T

1140 380

V

E

R

A

Y

K

N

F

V

Q

F

G

L

K

N

Q

I

I

1381 461 1441

481

L

N

Q

Q

K

D

Y

V

T

T

L

Q

N

L

K

P

A

N

S

D

H

I

Q

L

G

I

N

K

T

L

F

S

F

T

N

E

N

A

S

I

TTGCAAATGGACAGCACGAAACTGATCACCGGATTGTCTATCGTTGCTTCGAGGTACACG L

Q

M

D

S

T

K

L

I

T

G

L

S

I

V

A

S

R

Y

T

GATTTAATGAATACGTACATCAATTCTGTACCGTCCTCGTCATCATCAAAGAGGAAATCC D

L

M

N

T

Y

I

N

S

V

P

S

S

S

S

S

K

R

K

S

1440 480

1500 500

1501 501

GACGATGATGACGACGGCAACGACAATGAAGAAGTTGGAAACGATGGCCCAACGGCTAAT

1561 521

AGCAAGAAAATCAAAATGGAAACAGAACCACTAGCGGAGGAACCAGAGGAGCCCGAAGAC

1621 541

GATGACCGAATGCAGAAGATGCTTCAAGAAGAGGAAAGCGCCCAAGAAATCTCAGGAGAT

1681 561

GCCAACAAATCAACTTCTGCCATTAAGGAGATCGCACCCCCCTTTGAACCTGACTCATTG

1741

581

ACGCAGGATGAAAAACTAAAGTACCTCTCAAAGCTGACCAAGAAACTGTTTGAATTATCC T Q D E K L K Y L S K L T K K *L F E L S

1800 600

1801 601

GGTCGCCAGGATACTACCCGGGCCAAATCTTCGTCTTCCTCCTCCATATTACTGGACGAT

1860 620

1861 621

D

S D

A

G

D K D

N

D K

R K

R Q

D I M

S

D

D K Q T

T

G M K

S

T

N

E M A

R

D T L I

A

N

E Q

K

K

E P E E

S

E L

E I

S

V A E A

S

G E S P

S

N

E A P

S

D P Q

F

S

G E E E

I

P E I

P

L

T P S D

L

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G S

D

N D D L

D

GACGACTCCTCGTCATGGTTACACGTCTTAATCAGATTGGTTACGAGAGGAATCGAAGCA D

D

S

S

S

W

L

H

V

L

I

R

L

V

T

R

G

I

E

A

1921 641

CAAGAGGCCAGTGACCTGATTCGTGAAGAACTGCTTGGCTTCTTCATCCAGGATTTCGAG

1981 661

CAACGTGTCAGTCTGATCATTGAATGGCTCAATGAAGAATGGTTCTTCCAAACCTCGCTG

2041 681

CATCAAGATCCCTCTAACTACAAAAAATGGTCCTTAAGAGTTCTCGAGTCTCTGGGTCCA

2101 701

F

Q Q

E R

A V

S S

D L

L I

I I

R E

E W

E L

L N

L E

G E

F W

F F

I F

Q Q

D T

780

841 281

F

1141

441

CCAACGGAGATGCTGCCCAAGGTGCTCGAAACTACGGCATCCATGTACCACAACGGTAAT

181 61

781 261

3853

2161 721 2221 741

2281

H

Q D

P

S

N

Y

K

K

W

S

L

R

V

L

E

S

L

F

S G

E L P

TTCCTTGAAAACAAACACAGACGATTCTTCATCAGACTTATGAGCGAACTGCCCAGTCTT L

E

N

K

H

R

R

F

F

I

R

L

M

S

E

L

P

S

L

1560 520 1620 540 1680 560 1740 580

1920 640

1980 660 2040 680 2100 700 2160 720

CAAAGCGATCATCTTGAGGCACTGAAGCCTATCTGCCTGGATCCGGCAAGAAGTTCCCTT S D H L E A L K P I C L D P A R S S L

2220 740

GGTTTCCAAACGCTAAAGTTTCTCATTATGTTTAGACCCCCAGTGCAGGACACTGTTCGC

2280 760

Q

G

F

Q

T

L

K

F

L

I

M

F

R

P

P

V

Q

D

T

V

R

761

GACCTGCTGCATCAGCTAAAGCAAGAAGATGAAGGCTTACACAAGCAGTGCGATTCACTG D L L H Q L K Q E D E G L H K Q C D S L

2340 780

2341 781

CTTGACAGGCTAAAATGATCACAACCATTACATACATCATATATACGCATGTGTAGGGTC

2400

2401

TTCCTTCAACTTGCAAATCAATGTTTTATCTATCATTCTGTGCTATAGGGACCTCGCAAT

2460

2461

TTTGACACTTCCGTAAGGAGTCATTCAGCGGCGGAGTCACTTTCGCTAACTCTTTCTTCT

2520

L

D

R

L

K

FIG. 9. DNA sequence of the PTAI gene. The sequence of the entire PTAI gene is shown along with flanking regions corresponding approximately to the ClaI-SphI fragment. The DNA sequence is numbered from 1 at the first ATG of the open reading frame; negative numbers indicate sequences upstream of the first ATG. The predicted protein sequence is shown below the DNA sequence in the single-letter code, with an asterisk at the first termination codon.

calculated molecular weight is 88,417 with an isoelectric point of 6.17. The insertion of LEU2 at BamHI removes or replaces the C-terminal 51 amino acids (P735 to K785). No significant sequence similarities to known protein sequences in the GenBank (6) or EMBL (8) data base were identified by FASTA or TFASTA (47) sequence comparison analysis, using the entire PTAI protein-coding region as the query sequence. For example, the best score from a FASTA search represented 16 identities spanning a segment of 58 amino acids and included just two tripeptides, two dipeptides, and six single amino acid matches. Shuffled Ptalp sequences frequently produced better scores, and no segment of Ptalp was repeatedly found to match several other proteins. No motifs were identified by the pattern-matching program PROFILESCAN (22). Only short or ambiguous patterns were identified by the program MOTIFS, using the PROSITE data base (5). As with the FASTA searches, shuffled Ptalp sequences produced equal or higher numbers of matches. Self-comparisons by dot matrix methods revealed no internal repeated amino acid sequences. The simplest interpretation of these results is that PTAJ is not closely related to any previously sequenced gene.

DISCUSSION We have identified a recessive nuclear mutation called ptal-I that causes accumulation of end-trimmed, introncontaining pre-tRNAs under all growth conditions (Fig. 1) and exhibits temperature-sensitive but osmotic-remedial growth. The same set of 10 pre-tRNA families is accumulated in several other pre-tRNA processing-defective mutants, including rnal-1, losl-l, and senl-l mutants (27, 28, 32, 43, 62). The ptal-I mutation also causes a slight accumulation of 2/3 molecules in cells grown at low osmolarity (Fig. 1) as well as accumulation of both 5'-2/3 (a 2/3 molecule with the 5' leader) and 2/3 molecules in cells grown at high osmolarity (43). We did not detect accumulation of 35S pre-rRNA or actin pre-mRNA inptal-1 strains on Northern blots (data not shown), indicating that the ptal-I allele specifically affects pre-tRNA processing. ptal-I segregates as a simple Mendelian character (Fig. 2). Complementation tests, gene mapping, and sequence comparisons have established that PTAI is different from any previously described gene that affects pre-tRNA processing or splicing. The ptal-1 lesion is apparently an ochre stop codon, as indicated by suppression with SUP11 (Fig. 4 and 6). The recessive

3854

MOL. CELL. BIOL.

O'CONNOR AND PEEBLES

character of ptal-1 indicates that the absence or truncation of Ptalp inptal-I strains results in the observed phenotypes. The ptal-l-derived ochre fragment is probably not deleterious per se. ptal-1 apparently affects the cleavage step of pre-tRNA splicing, the known role of pre-tRNA splicing endonuclease. However, extracts prepared fromptal-1 cells contain normal amounts of pre-tRNA splicing endonuclease activity with normal heat stability (Fig. 5). This observation was somewhat unexpected, since pre-tRNA accumulation in ptal-I cells is so drastic. These results suggest that Ptalp may be responsible for an unknown step in pre-tRNA processing or may control the activity of a known pre-tRNA processing activity. The PTA] gene was isolated on plasmid YCpPTA1, which was identified by complementation of ptal-1. YCpPTAI restores wild-type growth and dramatically reduces pretRNA accumulation when transformed into ptal-I strains (Fig. 7). Sequences present in YCpPTAI direct recombination to the ptal-1 locus, which is tightly linked to CDC24 on chromosome I (Table 5, Fig. 8, and data not shown). The complete sequence of PTA] has been determined, and inspection of this sequence reveals a single large open reading frame that extends 785 amino acids from the first methionine (Fig. 9). The predicted molecular weight of Ptalp is 88,417 with an isoelectric point of 6.17. The amino acid composition does not appear to be remarkable, and there are no stretches of unusual or monotonous amino acid composition. There are no protein sequences in the data base that are significantly like PTA]. Moreover, we were unable to identify any familiar motifs that might be anticipated for a protein with an essential role in the pre-tRNA processing pathway. In particular, there are no evident membranespanning, single-stranded RNA-binding, zinc finger, or nucleotide-binding motifs. The predicted size of Ptalp is too large for any of the three proposed pre-tRNA splicing endonuclease subunits (51). Further investigation of the physiological role of PTAI is in progress. The smallest subclone that complements ptal-1 has 2.3 kbp of the YCpPTAl insert that includes a large N-terminal portion of FUN40 and a C-terminal part of FUN39 sufficient for 524 amino acids (18) (YCpPTAI-HindIII-SalI; Table 3 and Fig. 8). Since the FUN39 information was separated from its promoter, we initially thought that FUN39 would not be expressed from the 2.5-kbp fragment and that FUN39 could not be PTA]. After disruption of FUN40 established that PTA] is not FUN40, we tested the possibility that the C-terminal half of FUN39 complementsptal-1. FUN39 was disrupted with LEU2 to make the PTAI::LEU2 allele (Fig. 8). We found that PTA1::LEU2 is a recessive lethal mutation that fails to complement ptal-]. Curiously, the 4.0-kbp fragment on pUNPTA] that complements ptal-] fails to complement PTA1::LEU2 (Fig. 8). At least three different restriction fragments cloned in three different plasmids serve to complement ptal-] by providing similar-size C-terminal gene fragments (Fig. 8). This situation is reminiscent of the complementation pattern seen for sen]-]; in that case, a large C-terminal gene fragment is expressed from a promoter that is internal to the gene and normally is not used. Nonetheless, the protein translated from the abbreviated transcript complements both point mutations and disruptions in SEN] (13). We cannot fully explain the observed complementation pattern for the PTA] locus, but we suggest that this may be an example of intragenic complementation. It is possible that an episome-encoded C-terminal fragment of Ptalp is made that associates with the N-terminal ochre fragment of Ptalp synthesized from the genomicptal-] allele

to form an active complex. Alternatively, the C-terminal

Ptalp fragment and the ochre fragment of Ptalp could be separate functional domains that can independently carry out different functions. Neither of these explanations are completely satisfactory, since the disruption allele PTA]:: LEU2 should make an N-terminal segment of 734 amino acids, and this allele fails to complement eitherptal-] or the C-terminal segment of PTA] present on pUNPTAl. The precise mechanism of complementation of ptal-] by the large C-terminal gene fragment found on pUNPTAI remains obscure for now. Although the original ptal-] isolate, LA4-22, grows on YPD at 23 or 30°C, outcrossed strains that harborptal-] are difficult to grow on media without 1 M sorbitol (43). Growth is very slow, and revertants arise frequently when ptal-] strains are grown at low osmolarity (data not shown). All ptal-] strains are now routinely cultured at high osmolarity by using 1 M sorbitol, which alleviates the severe growth defect due to ptal-] and also selects against newly arising nonsense suppressor mutations (57). However, growth on 1 M sorbitol does not prevent pre-tRNA accumulation in ptal-] strains (43). The observation thatptal-] is suppressed by the ochre suppressor tRNA gene SUP]] suggested that ptal-] might represent the null phenotype. However, this is not the case, since PTA]::LEU2 spores fail to grow even with 1 M sorbitol in the medium (Table 5). Theptal-] ochre fragment apparently performs some essential function. Alternatively, occasional translational read-through of the ochre stop codon might generate a low level of full-length gene product sufficient for the essential activity of Ptalp. Identification of pta]-1 demonstrates that screening of mutagenized Ts collections for the accumulation of precursor RNAs by filter hybridization analysis remains a feasible approach for identifying other essential genes affecting preRNA processing. One version of this approach was originally described in 1986 (42), and similar methods have been successfully used to identify S. cerevisiae pre-mRNA and pre-tRNA processing mutants (1, 25, 60). Since only 2 (LOS] and SEN2) of the 18 known genes affecting pre-tRNA processing are represented by multiple alleles that were identified after direct screening, there are probably more S. cerevisiae genes affecting pre-tRNA processing that remain to be found (26). Recovery and analysis of mutant alleles of these other genes will help define the pre-tRNA processing

pathway more completely.

There are about 100 transcribed regions on chromosome I of S. cerevisiae. Of these genes, only 15 were identified by classical genetic methods. This disparity has been termed the gene-number paradox (31, 50). Recently, transcribed regions of chromosome I were mapped and systematically disrupted by targeted recombination to determine which transcripts represent essential functions (18, 19). In one interval, six transcribed regions (FUN]], FUN12, FUN]9, FUN20, FUN21, and FUN22) were disrupted; three (FUN12, FUN]9, and FUN20) were required for growth on YPD. In a complementary approach, an extensive mutagenesis of chromosome I was conducted (24). Of 21 independent Ts mutations analyzed, 17 mapped to just three previously identified complementation groups. Of the remaining four Ts mutations, two mapped to a known gene involved in cysteine metabolism, another was too leaky to map, and the last mapped to FUN24, an essential gene of unknown function. PTA] represents only the eighth gene identified as a Ts allele on chromosome I. These studies. of chromosome I genes demonstrate that there are many essential genes in S. cerevisiae that may be difficult to identify from conventional

S. CEREVISIAE PTAI GENE

VOL. 12, 1992

Ts strain collections. Our experience with the Tsor phenotype ofptal-1 suggests that additional essential genes will be found in Tsor or osmotic-remedial collections. There are several possible roles that the PTAJ gene product may have in the cell. (i) Ptalp normally functions in the turnover of excess pre-tRNA. Accumulation occurs in ptal-I because transcription routinely produces more pretRNA than required, but the excess is destined for turnover. (ii) Ptalp functions as a negative regulator of tRNA gene transcription that responds to some unknown signal. For instance, Ptalp could sense the level of charged or uncharged cytoplasmic tRNA relative to translational need and inhibit tRNA gene transcription whenever an excess of tRNA develops. Lack of Ptalp function leads to uncontrolled tRNA gene transcription and pre-tRNA accumulation. (iii) Ptalp functions to retain pre-tRNA in the nucleus. Lack of Ptalp allows inappropriate nuclear export of unspliced pre-tRNA, and accumulation results from separating unspliced pre-tRNAs from splicing activities that are retained in the nucleus (11, 39). (iv) Ptalp functions as a positive effector of pre-tRNA splicing activity. Pre-tRNA accumulation in ptal-I strains might then reflect a compensation mechanism of increasing pre-tRNA substrate concentration so that the down-regulated pre-tRNA splicing activities can still supply adequate amounts of mature tRNA. Additional studies are needed to distinguish among these models for Ptalp function. ACKNOWLEDGMENTS We thank Shirley Wang, Anita Hopper, Eric Phizicky, Calvin Ho, John Abelson, Mark Winey, Mike Culbertson, Emil van Zyl, and James Broach for providing yeast strains and plasmids and for communication of unpublished results. We are grateful to John Woolford, Elizabeth Jones, and members of their laboratories for advice, yeast strains, and plasmid DNAs used throughout these studies. We express our appreciation to Graham Hatfull for material assistance and advice on sequencing PTAI. We thank Leslie Lyons, Bonnie Diehl, David Kaback, and John Pringle for providing yeast chromosomal DNAs as hybridization filters, providing yeast strains and cloned DNAs pertaining to chromosome I, and communication of unpublished results. We specifically thank David Kaback for identifying the map location of PTAI by comparing restriction maps. We wish to emphasize our appreciation of the open communication and sharing of materials among the yeast pre-tRNA processing laboratories. We are grateful to Robert Duda for assistance in sequence comparison analysis and to the Pittsburgh Supercomputing Center for use of their facilities. We also thank Lisa Altomari, James Brayer, Holly Anderson, Todd Pippert, Viktor Stolc, Chadd Nesbitt, Mincheng Zhang, and Xiaodong Zhu for technical assistance. This work was supported in part by grant GM37166 from NIH and by grant NP-729 from the American Cancer Society.

REFERENCES 1. Aebi, M., G. Kirchner, J.-Y. Chen, U. Vjayraghavan, A. Jacobson, N. C. Martin, and J. Abelson. 1990. Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 265:16216-16220. 2. Atkinson, N. S., R. W. Dunst, and A. K. Hopper. 1985. Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype. Mol. Cell. Biol. 5:907-915. 3. Atkinson, N. S., and A. K. Hopper. 1987. Chromosome specificity of polysomy promotion by disruptions of the Saccharomyces cerevisiae RNAI gene. Genetics 116:371-375. 4. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1987. Current

3855

protocols in molecular biology. John Wiley & Sons, New York. 5. Bairoch, A. PROSITE: a dictionary of protein sites and patterns, 7th release. University of Geneva, Geneva, Switzerland. 6. Bilofsky, H. S., and C. Burks. 1988. The GenBanks genetic sequence data bank. Nucleic Acids Res. 16:1861-1864. 7. Boeke, J. D., J. Trueheart, G. Natsonlis, and G. R. Fink. 1987. 5-Fluoro-orotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154:164-175. 8. Cameron, G. N. 1988. The EMBL data library. Nucleic Acids Res. 16:1865-1867. 9. Chen, J.-Y., G. Kirchner, M. Aebi, and N. C. Martin. 1990. Purification and properties of yeast ATP (CTP):tRNA nucleotidyltransferase from wild type and overproducing cells. J. Biol. Chem. 265:16221-16224. 10. Church, G. M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995. 11. Clark, M. W., and J. Abelson. 1987. The subnuclear localization of tRNA ligase in yeast. J. Cell Biol. 105:1515-1526. 12. Culbertson, M. R., and M. Winey. 1989. Split tRNA genes and their products: a paradigm for the study of cell function and evolution. Yeast 5:405-427. 13. DeMarini, D. J., M. Winey, D. Ursic, F. Webb, and M. R. Culbertson. 1992. SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae. Mol. Cell. Biol. 12:2154-2164. 14. DeRobertis, E. M., and M. V. Olson. 1979. Transcription and processing of cloned yeast tyrosine tRNA genes microinjected into frog oocytes. Nature (London) 278:137-143. 15. Deutscher, M. P. 1984. Processing of tRNA in prokaryotes and eukaryotes. Crit. Rev. Biochem. 17:45-71. 16. Deutscher, M. P. 1990. Ribonucleases, tRNA nucleotidyltransferase, and the 3' processing of tRNA. Prog. Nucleic Acids Res. 39:209-240. 17. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 18. Diehl, B. E. 1990. The gene-number paradox. Ph.D. thesis. University of Michigan, Ann Arbor. 19. Diehl, B. E., and J. R. Pringle. 1991. Molecular analysis of Saccharomyces cerevisiae chromosome I: identification of additional transcribed regions and demonstration that some encode essential functions. Genetics 127:287-298. 20. Eliedge, S. J., and R. W. Davis. 1988. A family of versatile centomeric vectors designed for use in the sector-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70:303-312. 21. Greer, C. L., C. L. Peebles, P. Gegenheimer, and J. Abelson. 1983. Mechanism of action of a yeast RNA ligase in tRNA splicing. Cell 32:537-546. 22. Gribskov, M., M. Homyak, J. Edenfield, and D. Eisenberg. 1988. Proffle scanning for three dimensional structural patterns in protein sequences. CABIOS 4:61-66. 23. Hanahan, D. 1983. Studies on transformation of Eschenichia coli with plasmids. J. Mol. Biol. 166:557-580. 24. Harris, S. D., and J. R. Pringle. 1991. Genetic analysis of Saccharomyces cerevisiae chromosome I: on the role of mutagen specificity in delimiting the set of genes identifiable using temperature-sensitive-lethal mutations. Genetics 127:279-285. 25. Ho, C. K., R. Rauhut, U. Vijayraghavan, and J. Abelson. 1990. Accumulation of pre-tRNA splicing '2/3' intermediates in a Saccharomyces cerevisiae mutant. EMBO J. 9:1245-1252. 26. Hopper, A. K. 1990. Genetic methods for the study of transacting genes involved in processing of precursors to yeast cytoplasmic tRNAs. Methods Enzymol. 181:400-421. 27. Hopper, A. K., F. Banks, and V. Evangelidis. 1978. A yeast mutant which accumulates precursor tRNAs. Cell 14:211-219. 28. Hopper, A. K., L. D. Schultz, and R. A. Shapiro. 1980. Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs. Cell 19:741-751. 29. Hurt, D. J., S. S. Wang, Y.-H. Lin, and A. K. Hopper. 1987. Cloning and characterization of LOSI, a Saccharomyces cerevisiae gene that affects tRNA splicing. Mol. Cell. Biol. 7:12081216.

3856

O'CONNOR AND PEEBLES

30. Johnson, P. F., and J. Abelson. 1983. The yeast tRNA` gene intron is essential for correct modification of its tRNA product. Nature (London) 303:681-687. 31. Kaback, D. B., P. W. Oeller, H. Y. Steensma, J. Hirschman, D. Ruezinsky, K. G. Coleman, and J. R. Pringle. 1984. Temperature-sensitive lethal mutations on yeast chromosome I appear to define only a small number of genes. Genetics 108:67-90. 32. Knapp, G., J. S. Beckmann, P. F. Johnson, S. A. Fuhrman, and J. Abelson. 1978. Transcription and processing of intervening sequences in yeast tRNA genes. Cell 14:221-236. 33. Knapp, G., R. C. Ogden, C. L. Peebles, and J. Abelson. 1979. Splicing of yeast tRNA precursors: structure of the reaction intermediates. Cell 18:37-45. 34. Kolman, C., and D. Sill. Personal communication. 35. Lee, J.-Y., C. E. Rohlman, L. A. Molony, and D. R. Engelke. 1991. Characterization of RPRI, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P. Mol. Cell. Biol. 11:721-730. 36. Lichten, M., R. H. Borts, and J. E. Haber. 1987. Meiotic gene conversion and crossing over between dispersed homologous sequences occurs frequently in Saccharomyces cerevisiae. Genetics 115:233-246. 37. Marck, C. 1988. DNA Strider: a C program for the fast analysis of DNA and protein sequences on the Apple MacIntosh family of computers. Nucleic Acids Res. 16:1829-1836. 38. McCraith, S. M., and E. M. Phizicky. 1991. An enzyme from Saccharomyces cerevisiae uses NAD+ to transfer the splice junction 2'-phosphate from ligated tRNA to an acceptor molecule. J. Biol. Chem. 266:11986-11992. 39. Melton, D. A., E. M. DeRobertis, and R. Cortese. 1980. Order and intracellular location of the events involved in the maturation of a spliced tRNA. Nature (London) 284:143-148. 40. Miyamoto, S., Y. Ohya, Y. Oshumi, and Y. Anraku. 1987. Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae. Gene 54:125-132. 41. Nishikura, K., and E. M. DeRobertis. 1981. RNA processing in microinjected Xenopus oocytes: sequential addition of base modifications in a spliced transfer RNA. J. Mol. Biol. 145:405420. 42. O'Connor, J. P. 1986. Analysis of tRNA biosynthesis in the yeast Saccharomyces cerevisiae using oligodeoxynucleotides as hybridization probes. M.S. thesis. University of Pittsburgh, Pittsburgh, Pa. 43. O'Connor, J. P. 1990. Pre-tRNA processing in the yeast, Saccharomnyces cerevisiae. Ph.D. thesis. University of Pittsburgh, Pittsburgh, Pa. 44. O'Connor, J. P., and C. L. Peebles. 1991. In vivo pre-tRNA processing in Saccharomyces cerevisiae. Mol. Cell. Biol. 11: 425-439. 45. Patterson, B., and C. Guthrie. 1987. An essential yeast snRNA with a U5-like domain is required for splicing in vivo. Cell 49:613-624. 46. Pearson, D., I. Willis, H. Hottinger, J. Bell, A. Kumar, U.

MOL. CELL. BIOL.

Leupold, and D. SoIl. 1985. Mutations preventing expression of sup3 tRNAser nonsense suppressors of Schizosaccharomyces pombe. Mol. Cell. Biol. 5:808-815. 47. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence analysis. Proc. Natl. Acad. Sci. USA 85:

2444-2448. 48. Peebles, C. L., P. Gegenheimer, and J. Abelson. 1983. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell 32:525-536. 49. Phizicky, E. M., R. C. Schwartz, and J. Abelson. 1986. Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene. J. Biol. Chem. 261:2978-2986. 49a.Pippert, T. Unpublished data. 49b.Pippert, T., and J. P. O'Connor. Unpublished data. 50. Pringle, J. R. 1987. The gene number paradox and the complexity of the cell-division cycle and other cellular processes, p. 299-306. In C. Waymouth (ed.), Molecular mechanisms in the regulation of cell behavior. Alan R. Liss, New York. 51. Rauhut, R., P. R. Green, and J. Abelson. 1990. Yeast tRNAsplicing endonuclease is a heterotrimeric enzyme. J. Biol. Chem. 265:18180-18184. 52. Reyes, V. M., and J. Abelson. 1987. A synthetic substrate for tRNA splicing. Anal. Biochem. 166:90-106. 53. Rose, M. D. 1987. Isolation of genes by complementation in yeast. Methods Enzymol. 152:481-504. 54. Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. 101:202-211. 55. Russell, R. R. B. 1972. Temperature-sensitive osmotic remedial mutants of Escherichia coli. J. Bacteriol. 112:661-665. 56. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 57. Singh, A. 1977. Nonsense suppressors of yeast cause osmoticsensitive growth. Proc. Natl. Acad. Sci. USA 74:305-309. 58. Strobel, M. C., and J. Abelson. 1986. Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo. Mol. Cell. Biol. 6:2663-2673. 59. van Zyl, W. H., N. Wills, and J. R. Broach. 1989. A general screen for mutants of Saccharomyces cerevisiae deficient in tRNA biosynthesis. Genetics 123:55-68. 60. Viayraghavan, U., M. Company, and J. Abelson. 1989. Isolation and characterization of pre-mRNA splicing mutants of Saccha-

romyces cerevisiae. Genes Dev. 3:1206-1216. 61. Wang, S. S., and A. K. Hopper. 1988. Isolation of a yeast gene involved in species-specific pre-tRNA processing. Mol. Cell. Biol. 8:5140-5149. 62. Winey, M., and M. R. Culbertson. 1988. Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae. Genetics 118:609-617. 63. Zubenko, G. S., F. J. Park, and E. W. Jones. 1982. Genetic properties of mutations at the PEP4 locus in Saccharomyces cerevisiae. Genetics 102:679-690.