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Nucleotide Sequence of 9.2 kb Left of CRY1 on Yeast Chromosome I11 from Strain AB972: Evidence for a Ty Insertion and Functional Analysis of Open Reading Frame YCR28 M.L. AGOSTONI CARBONE*, L. PANZERI*, M. MUZI FALCONI*, C. CARCANO*, P. PLEVANI* AND G. LUCCHINI*+

*Diparthento di Genetica e di Biologia dei Microrganismi, via Celoria 26,20133 Milano, Italy TIsrituto di Genetica, via Mancini 5,07100 Sassari,Italy Received 14 February 1992; accepted 2 March 1992

We report the 9210 bp sequence from a segment of yeast chromosome I11 cloned from strain AB972 in hPM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed functional analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on sensitivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium. KEY WORDS-Chromosome

INTRODUCTION

111; Ty insertion; gene disruption.

MATERIALS AND METHODS

In the context of the EEC Biotechnology Action Strains and media Program for sequencing chromosome I11 from Saccharomyces cerevisiae, we sequenced 9.2 kb of a Saccharomyces cerevisiae strains S288C (MATa), chromosome I11 fragment from the S288C derivative TD28 (MATa, ura 3-52, inol, canl), F762 (MATa, strain AB972 (Olson et al., 1986) camed by the trplA1, ura 3-52)and F763 (MATa, trplAl, ura 3-52) recombinant phage clone hPM3270. This region over- were kindly provided by G.R. Fink (Whitehead laps contiguous BamHI segments cloned into Ylp5 Institute-MIT, Cambridge). Strain DFB20 is the (Newlon et al., 1986) from another S288C derivative diploid resulting from the cross F762 by F763. strain, XJ24-24A (Strathem et al., 1979), which were Escherichia coli strains used were JM109 (recA 1, also sequenced during this project (Oliver et al., 1992), endAl, thi, hsdR17, supE44, gyrA96, relAl, A(1acproAB) [F’tra36, proAB+, laclq, lacZAM151) and allowing comparison between the two strains.

0749-503X/92/090805-08$09.00 01992 by John Wiley & Sons Ltd

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M.L. AGOSTONI CARBONE ETAL.

DB6507 (hsdR, hsdM, recA, leu, pro, thi, thr, endA, supE44, pyrF 74::Td). E. coli media were according to Sambrook et al., (1989). Yeast media: YPD and YPE are 1% yeast extract, 2% peptone, supplemented respectively with 2% glucose and 2% ethanol. Synthetic minimal medium 40 has been described (Magni et al., 1977); when requested, bases or amino acids were added to the final concentration of 25 pg/ml. For allantoate medium, a 0.17% solution of DIFCO yeast nitrogen base whitout ammonium sulfate was adjusted to pH 6 with sodium citrate and supplemented with 2% glucose and 0-1% allantoate. Sensitivity to 3-aminotriazole (3-AT) was tested in synthetic medium 40 containing 30 mM-3-AT and excess (500 pg/ml) leucine. Phages and plasmids DNA from recombinant phage hPM3270 was received from S.G. Oliver (UMIST, Manchester). The phage is a hGM3 derivative containing a DNA region of about 21 kb (Figure 1) from chromosome I11 of strain AB972 (Olson et al., 1986). As shown in Figure 1, the 5.7 kb, 1.0 kb and 3-7 kb EcoRI fragments were cloned into the EcoRI site of

sm.l

AL yt ...................

I

I

-224a

,

Bmnl I 111

Y

1

plasmid pGEM3-Zf(-) (Promega), to give the corresponding plasmids pYGA1, pYGE2, pYGCl and pYGC2. Plasmids pLA535 and pLA536 were obtained from pYGE2 by ligating respectively a SmaI-DraI digest and a Smal-NcoI digest after filling in the NcoI ends with Klenow enzyme. The 2.8 kb and the 5.4 kb SmaI-XhoI fragments were cloned into the SmaI-XhoI sites of plasmid pGEM7-Zf(+) to give plasmids pLA5 15 and pLA5 14 respectively. Plasmid pLA534 derives from a pLA514 deletion plasmid, which was digested with XhoI+HindIII and ligated after filling in the ends with Klenow enzyme. Sequencing strategy and methods As shown in Figure 1, sets of unidirectional deletions about 150 bp apart in the 3.7 kb EcoRI fragment and in the 5.4 kb SmaI-XhoI fragment were generated according to the procedure of Henikoff (1984), by exonuclease I11 treatment of plasmids pYGCl and pYGC2 digested with BamHI+SphI and of plasmid pLA5 14 digested either with ApaI+XhoI or with BamHI+SacI. SP6 and T7 promoter primers were used to determine on both strands the sequence of the 9.2 kb segment depicted in Figure 1, using as templates the

sm* I .......X ..R..........

1

pYGA1 pYGE2

A

pLA515

-

2 Kb

A

9210

pLA536 +A535

pLA514

A

pLA534

Figure 1. hPM3270 yeast DNA restriction map and sequencing strategy. The top line represents the main features of the 21 kb yeast DNA fragment inserted between the left and the right (hR) arms of hGM3 (dotted line), with vertical short bars indicating the positions of the EcoRI sites and open boxes representing the duplicated region discussed in the text. The second line from the top gives an enlarged view of the segment used for subcloning and sequencing in our laboratory and positions 1 to 9210 delimit the region of contiguous sequence reported in Figure 2. Position -2248 for the second EcoRI site from the left is given with respect to position 1 and is approximate from restriction analysis data. The fragments used for subcloning (see Materials and Methods) are indicated under the restriction map, flanked by triangles indicating the position of the SP6 (open triangle) and T7 (solid triangle) promoter sequences in the derivative plasmids, whose names are indicated on one side of each fragment. Arrows in the bottom part of the figure indicate the extent and directions of the sequenced fragments. A wavy line preceding the arrow indicates the use of purpose-synthesized primers.

(a)

NUCLEOTIDE SEQUENCE OF 9.2 KB LEFT OF CRY1 ON YEAST CHROMOSOME 111FROM STRAIN AB972

pYGC1, pYGC2 and pLA514 deletion plasmids, as well as plasmids pYGE2, pLA535, pLA536, pLA534 and pLA5 15. Appropriate synthetic oligonucleotides were used to sequence the region encompassing the two XhoI sites in plasmid pYGAl and to fill in some gaps in the sequence of pYGCl and pLA514 inserts. The dideoxynucleotide chain-termination method (Sanger et al., 1977) was used for sequencing reactions (T7 sequencing kit, Pharmacia). Sequence analysis software Sequence analysis and comparison of nucleotide and amino acid sequences were performed at MIPS (Martinsried) using the MIPS package.

807

Other techniques Southern and Northern blot analysis, plasmid DNA preparation, and DNA restriction analysis were performed as described by Sambrook et al. (1989). RESULTS AND DISCUSSION Sequence analysis The major features of the hPM3270 yeast DNA insert are described in Figure 1, as well as the strategy we used to determine the 9210 bp of contiguous sequence in the left half of it, which is reported in Figure 2. Computer-assisted analysis of this sequence, its comparison with the sequence of corresponding

Figure 2. Nucleotide sequence of contiguous 9210 bp in the left half of hPM3270 yeast DNA. Only the sequence of one strand is given and nucleotides 1 and 9210 are referred to the positions given in Figure 1. Putative ORFs are underlined, with arrows indicating the direction of translation and dots pointing out the stop codons. 6 sequences are boxed. Intron sequence in YCR29 is indicated by a bold line, with a box including the STACTAAC3' splicing signal and arrows pointing out the intron splicing sites. A dashed line is used to indicate the sequence that is found repeated also in the right half of the hPM3270 insert (boxes in Figure 1, top).

808

M.L. AGOSTONI CARBONE ETAL.

Figure 2. (cont'd)

fragments derived from strain XJ24-24A (Oliver et al., 1992) and with the sequence of the right half of the hPM3270 insert (Rodriguez et al., 1991) allowed the following major observations. (1) Nucleotides (nt) 1-515 in our sequence match almost perfectly with nt 5408-59 18 in the sequence of the yeast transposable element Ty912 (Clare and Farabaugh, 1985), including the carboxy-terminal end of the Ty open reading frame (ORF; nt 1-152) flanked by a 6 element (Figure 3). The presence of part of a Ty ORF in the left portion of the hPM3270 insert was confirmed by determining the sequence of the EcoRISphI segment starting about 2200 nt upstream to nt 1 in Figure 1. As shown in Figure 4, this sequence also shows very high homology to the sequence of a similar restriction fragment found in Ty912. These data indicate that a Ty element is located in this position in strain AB972, while a solo 6 is found in the same position in strain XJ24-24A (Oliver et al., 1992). Since an additional &like sequence is found in our fragment, adjacent to the first one (nt 544-859, boxed in Figure 2), we suggest that the AB972 pattern is due

to insertion of a complete Tyl element nearby a preexisting 6 sequence. The region of insertion is distal to the right-arm transposition hot spot previously identified on chromosome I11 (Oliver et al., 1992; Warmington et al., 1987). (2) The hPM3270 insert carries a 2-2kb duplication, as indicated in the top part of Figure 1. In fact, the region between nt 6973 and nt 9210 in our sequence is found repeated in the right part of the phage insert, between nt 5186 and nt 7418 of the sequence determined by Rodriguez et al. (1991). Subsequent analysis of new phage stocks indicates that the h clone sequenced probably contains a rearrangement (C.S. Newlon, personal communication). However, this region does form part of a much larger duplication in the chromosome I11 of strain AB972 (Wicksteed et al., in preparation). Restriction analysis (Newlon et al., 1992) show clearly that this region is unique in the chromosome I11 of strain XJ24-24A. It is likely that the large rearrangement which has occurred in strain AB972 is due to the interaction of flanking Ty elements.

NUCLEOTIDE SEQUENCE OF 9.2 KB LEFI OF CRY1 ON YEAST CHROMOSOME 111FROM STRAIN AB972 5 4 0 9 TTTTCCUCAAAGGCMTCAGACTT-TGAAGTATWTAATAATT

5 458

IIIIII I I I 1 IIIIIIIIIII IIIIIIIIIIIIIIIII I l l I I 2 TTTTGGTACT1AU;CUTOTAAGAGATGAAGTATWTCATS 5 4 5 9 TATACGTATACTACATCC-CATTCCTGATGTGATGICA

51 5508

IIIIII Ill IIIII IIIII IIIII IIIII II II IIIII

I

5 7 T G C A C C T A T G C T A T A T C C A A A C ~ T A T T G C A G A C G T A A T G A C ~101 5 5 0 9 AAACCTCTTCCGAT-TTTAAACTATTAACTAACAAAT-TTU

IlIIlIllIlIIlIIIlIl

5558

I l l IIIIIIIIIII IIIIIIIIII’III

1 0 2 A A A C C T C T T C C G A T A A A A A C T T ~ C T A T T A A C W A C 3 A A T G C A T T ~1 5 1 5 5 5 9 TTAGATCTATTACATTATGGGTGGTATGTTGGAATWTCAACTATCA

5608

IIIIIIlIIIIIIIIIIIIIIiIlIIIIIlIlIlIl IIIII IIIIII

-..

1 5 2 TTAGATCTATTAUTTATGGGTOGTATGTTGGAATAAAAATCCACTATCG

201

5 6 0 9 TCTACTAACTAGTATTTACATTACTAGTATATTATCATATACGGTGTT A 5 6 5 7

IIII

IIIII II Ill Ill1

I IIIIIIIIIIIIIIIIIIIII I

2 0 2 TCTATCAACTAATAGTTATATTATCAATATATTATCATATACGGTGTTAA 2 5 1 5 6 5 8 GAAGATGACGCA&&TGATGAGAAATACTCATCTA&ATTAGTGGAAGCTGA

I I IIIIII

II I IIIIIII

IIIIII I

5707

IIII IIIIIIIII

2 5 2 GATGATGAUTAAGTTATWTGTCATCGATGTTAGAGGAAGCTGA 3 0 1 5 7 0 8 AACGCAAGCATTGATAATGTAATAGGATWTGAATATAAACATATAbAa

5757

IIIIllIIIlIIlIlIIIIIlIIIlIIIIIIIIIIlIIIIIlIIIIIIII 3 0 2 AACGCXAGGATTGATAATGTAATAGGATWTGAATATIUUCATATAbAa 5 7 5 8 TG

I

351

ATGA T A A T A A T A T T T A T A G L 4 T T G T G T A W T T C C C T T 5 8 0 4 Ill1 IIIIII I Ill I I IIIIIII I IIIIIII I I

352 CGGAATCU;CMTMTCCTAATATTAGTATCTWTATAGATTCUTT 4 0 1 5 8 0 5 TTATGGATTCCTAAATCCTTC~CCLGUCTTCTTCTAGTATATTCTGTATACC 5854

/ I

IIIOIIII IIIII OIIIIIIIIIIIIIIIIIIIIIIIIIIII

4 0 2 TT-TTCCTATATCCTCGUGAGAACTTCTACTATATTCTGTATACC

As indicated in Figure 2, YCR29 contains an intron splicing signal, boxed in the figure (5’TACTAAC3’, Langfordetal., 1984) betweennt4117 and4111,295nt from the first ATG codon. 5‘ and 3’ to this signal, respectively 243 nt and 323 nt from the start codon (positions 4169 and 4083, pointed out by arrows in Figure 2), we find the consensus sequences S’GTATGT3’ and 5’PyAG3’, which usually delimit intron sequences in S. cerevisiae (Woolford, 1989). Splicing of this 83 nt intron would generate a mature transcript containing a 135 codon ORF, while, in the absence of splicing, the corresponding ORF would be only 99 codons, with a stop signal TAA just inside of the S’TACTAAC3’ sequence. YCR29 has been recently cloned independently (Van Dyck and Foury, personal communication) as the RIM1 gene, a suppressor of a mitochondria1 temperature-sensitive defect of pifl null mutants (Foury and Van Dyck, 1985) and splicing of the hypothetical 83 nt intron has been confirmed by Van Dyck (personal communication), who is performing further analysis on this gene.

451

5 8 5 5 TAATATTATAGCCTTTATCTGGAATCCCAACAATTATCTCAACAT

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I

809

5904

I

452 TAATATTATICCCTTTATWWTGGAATCCCAAWTTATCTAATTAC 5 0 1 5 9 0 5 ‘TACCCATTTCTCA 5 9 1 8

Ill IIIIIIIII 5 0 2 CCACACATTTCTCA 5 1 5

rEcolll~ ’ 3 9 2 9 GUTTWCTGATTGCAGCTCT-TAAAATWT-CCAATA

3978

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Figure 3. Sequence comparison between h3270 yeast DNA and Ty912. Upper row, Ty912 nucleotides 5409-5918 (Clare and Farabaugh, 1985); lower row, hPM3270 nucleotides 2-515. The carboxy-terminal sequence of Ty912 ORF is underlined, with arrows indicating the direction of translation and dots indicating the stop codon. A dashed line points out the sequence of the 6 element.

1 GAATTWCTUTTGCAGCTGT-T-TWTCAAACCAATA 3 9 7 9 CGGACAKCTTACGATACGATGNXC%ATCACCTATMT-TATTM

50 4028

IIIIIOIIIII lIlIIlIIIIIIIlIIIIIIIIIIII:IIlIIIIIII 5 1 C C G A U C C T T A A G & T A C G A T G T C A C C T A T M T A A & G k T A T T A A 100 4 0 2 9 -TATATCGXGCC%TACCA-TCAATCAACTGT

4078

IIIIII IOIIIIIIIIIII IIIIIIIIIIIIIIIIIIII I I I I I I 1 01 - T A T A T C m T A C C A W T W C C A A C T A T

150

4019 T W U L G I I T G A A A A C T T U C T G A C G A A T A T T A T G A ~ ~ T 4 A1 2 8

Ill1 IIIII IIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIII 1 5 1 T W V I U T W T A C T T G G G A C A C T G A C A A A T A T T A T G A ~ T A 200 4 1 2 9 GACCCTAAAAGWTAATITCAATGTTTATCTTCAA-CGTGI

4178

IIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIII IIIIIIIII 2 0 1 GACCCTAAAAGWTAATAAATTCAATGTTTATCTTWWCGTGA 250

(3) Search for putative ORFs in the 9210 bp sequence revealed that, due to the above duplication, the 668 codon ORF starting at position 6802 and terminating at position 4798, and the divergent ORF of at least 646 codons starting at position 7273 represent in fact only parts of ORFs found in the corresponding region in strain XJ24-24A, namely YCR30 and YCR32 respectively (Oliver ef al., 1992). For this reason we did not analyze them further. The two additional ORFs underlined in Figure 2, which are found in our sequence, both have identical counterparts in the corresponding region of strain XJ24-24A: YCR28, 512 codons, starting with an ATG at position 3395 and terminating at position 1860, and YCR29, with the same orientation, between positions 441 1 and 3923.

4 1 1 9 COGTACTCATIUUCGTAGATTTGTTGCUGAGGCGATATTCAGCATCCTG 4 2 2 8

IIIIIIIIIIIII

IIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIII

2 5 1 CGCTACTCAT-TAGATTTGTTKAAGAGGTGATATTCAGCATCCTG

300

.

rlphl4 2 2 9 AUCTTACGACTUGGCITGCUTCCAATACCGTACATCACTATGCATTA 4 2 1 8

IllIIIIIlIlIIIIIIIIIIIlIlIIIIIIIllIII’III/IIIIIIII 3 0 1 ACACTTACGACTUGGCITGCUTCCAATACCGTACATCACTATGCATTG

350

4 2 7 9 ATGAUTCCCTGTWTTOUTTAGACAATMCTACTATATTACACAATT 4 3 2 8

IIIIIIIIIII IIIIII IIIIlIlIIIIIIlIIIIllIIllIIIIIIl 3 5 1 ATGICATCCCTATUCTTTCATTmCAATAACTACTATATTACAWTT 4 0 0 4 3 2 9 AGACATATCTTCGGCLTA 4 3 4 6

IIIIIIIIIIIIIIIIII 4 0 1 AGACATATCTTCGGCATA 418

Figure 4. hPM3270 contains part of a Ty ORF. Nucelotides 1 4 1 8 in the lower row represent the DNA sequence of the EcoRI-SphI region found on the left end side of the hPM3270 insert (see Figure I), compared to the sequence of a similar fragment found in Ty912 (upper row; Clare and Farabaugh, 1985).

M.L. AGOSTONI CARBONE ETAL.

810 Functional analysis of YCR28 As shown in Figure 5, YCR28 encodes a putative 5 12 amino acid protein, which shows a weak homology to the yeast allantoate permease, encoded by the DAL.5 gene (Ray et al., 1988). No other significant homologies with known protein sequences were found. Southern blot analysis of total yeast DNA from strains DFB20, TD28 and S288C digested with several restriction enzymes and probed with the PstI-EcoRI fragment between positions 1166 and 43 16 indicates that this region is unique in the yeast haploid genome (data not shown). Northern blot analysis (Figure 6) on yeast poly(A)+ RNA probed with a 638 bp Sun fragment internal to YCR28 reveals two mRNA species, a more abundant 2.5 kb transcript and a less represented 1.9 kb mRNA. This result indicates that the YCR28 region is transcribed; a more detailed analysis will be necessary to verify the direction of transcription and possible different roles for the two mRNA species. In addition, the 3150 bp PstI-EcoRI probe used for Southern analysis identifies a very abundant 0.68 kb mRNA, that could be related to the YCR29 ORE These data are in agreement with analogous findings from Yoshikawa and Isono (1990). To verify whether YCR28 had a functional significance in yeast metabolism, we performed one-step 1 MMKESRS~

.~~~~

I TG

Figure 6. Northern blot analysis. Poly (A)' RNA from strain S288C (10 Fg) was electrophoresed, transferred to nitrocellulose filter and hybridized with the SalI (lane A) and the PstI-EcoRI (lane B ) probes described in the text.

~~

gene disruption experiments (Rothstein, 1983), using the two different deletion-substitution strategies described in Figure 7A. * ,**. Disruption I, leading to the replacement of the 1 3 DLTVSNTVFH 1c;Y IVGHVFNNI.HLLCVP 1 2 1 QYSh'VTSAFYFCYLFHNLGPV3F I FVRT 1256 bp SphI-SalI region with a 1100 bp Hind111 frag*.* .* + f.. ment carrying the URA3 gene, was performed on the 1 3 0 A I R F F Q A L F E S C T F S G T H F V L G j k " ( K E D E L ~ I R S A I F ~ S C ' V G S * I F - i ~ F H Q T i I F T 1 l L N1 8 9 i a i VLRVLLGCAESWTPCFTIlTAQYWKTEEQFTRVSlWFGMN~LCSILlNAlAY~VYlH 2 3~ 9Q diploid strain DFB20 and two URA+ transformants, **. .+ .*. * * 1 9 0 C R N ~ L A G W R W L F I ~ C F ~ l T L P ~ A ~ Y ~ F l ~ F P ~ L P ~ ~ ~ A J ~ ~ F ~ M T R Y2l4F9N ~ Q Eshown L H Y A R R by Southern analysis to be heterozygous for 2 4 0 D S Y A I K G W R T L F V I ~ V I T l F l G l L l F L W I P U U P ~ ~ R F L S K R E K L M- WQ R I P S N W 2 9 6 * ... ..*. ..+.. . * * . . . * .* the disruption, were sporulated and subjected to tetrad 2 5 0 RLPARDESTRLDWSTIPRVLKRWHWMFSLW-VLG analysis. As summarized in Figure 7B, the disruption 2 9 7 GF G N H E I K K Y Q I I E A L K D V R I W L Y F L F W S S N I P N * * * . * did not affect either spore viability or growth on rich 308 A Q R N N Y P S G I F A V G l V S T L C S A V Y M ~ K l P ~ H A U H W H V ~ V ~ I S L Y H Y I V A V L l R A ~ P1l .6NbP K medium with glucose (YPD) at any temperature. 3 5 6 MGLPTGAVELVGCPLFG I LAVY 4ANKY I PFWKY KLSWA I FAA'ILAITAS