YEAST

VOL. 8: 903-922

(1992)

Structural and Putative Regulatory Sequences of Kluyverornyces Ribosomal Protein Genes G. KARIN BERGKAMP-STEFFENS, RUURDTJE HOEKSTRA AND RUDI J. PLANTA Laboratorium voor Biochemie en Moleculaire Biologie, Vrije Universiteit, de Boelelaan 1083.1081 HV Amsterdam, The Netherlands Received 10 March 1992; accepted 29 June 1992

The transcription of the majority of the ribosomal protein (rp) genes of Saccharomyces cerevisiae is activated by cis-acting elements, designated RPG boxes, which specificallybind the multifunctional protein RAPl in vitro. To investigate to what extent this global system of transcription regulation has been conserved, we have isolated a number of rp genes of the related yeast species Kluyveromyces lactis and Kluyveromyces marxianus, whose counterparts in Saccharomyces are controlled by RAPl . The coding regions of these genes showed a sequence similarity of about 90% when compared to their Saccharomyces counterparts. In contrast, little or no sequence similarity was found between the upstream regions and the intervening sequences of Kluyveromyces and Saccharomyces homologs. However, the occurrence and the position of the introns is conserved. The sequence data also show that the physical linkage that exists in S. cerevisiae between the rp genes encoding RP59 (CRYI), S24 and L46 is conserved in Kluyveromyces. Northern analysis demonstrated that each of the isolated Kluyveromyces genes is transcriptionally active. By sequence comparison we identified a number of conserved sequences in the upstream region of each of the Kluyveromyces rp genes, which we designated the X, Z and RPG, boxes. The last one is highly similar, though not identical, to the S . cerevisiae RPG box. Functional analysis of the intergenic region between the genes encoding Kluyverornyces ribosomal proteins S24 and L46 showed that the RPG, box ( + Z box) functions as a transcriptional activator, while the X box acts as a transcriptional repressor. Band-shift assays confirmed the existence of a RAP1-like protein in Kluyveromyces that binds to the RPG, box but not to the S. cerevisiae RPG box. In contrast, S. cerevisiae RAPl did recognize the RPG, box. KEY WORDS -Yeast;

Saccharomyces; Kluyveromyces; ribosomal protein gene; RAP].

INTRODUCTION The synthesis of Saccharomyces ribosomal proteins (rp) is coordinately regulated at the transcriptional level (reviewed by Planta and RauC, 1988; Rauk and Planta, 1991). Sequence comparison of the upstream regions of the rp genes (Leer et al., 1985b) identified a 14 nucleotides long element, the RPG box, shared by the majority of these genes. In front of most of these genes duplicate copies of this element are present, in either orientation, at a distance of 25CL500 bp upstream from the ATG codon. Mutational analysis of a number of rp genes has shown these RPG boxes to be crucial cis-acting elements in the coordinate control of rp gene expression (Woudt et al., 1986, 1987b). Band-shift assays and footprinting analyses demonstrated that the RPG box acts as a binding site for an abundant Address correspondence to: Prof. Dr. R. J. Planta, Lab. voor Biochemie en Mol. Biol., Vrije Universiteit, de Boelelaan 1083, 108 I HV Amsterdam, The Netherlands. 0749%503X/92/011903-20 $15.00 0 1992 by John Wiley & Sons Ltd

protein, RAPl (also designated T U F or G R F l ; Shore and Nasmyth, 1987; Vignais et al., 1987; Kimmerly et al., 1988). The same protein is also involved in transcription activation of a variety of other yeast genes, including those involved in glycolysis, cellular differentiation, nutrient transport and the cell division cycle (Mager and Planta, 1991). Furthermore, RAPl binds to telomeres (Buchman et al., 1988; Berman et al., 1986) and plays a role in anchoring DNA to the nuclear scaffold (Hofmann et al., 1989). So far, the precise mechanism by which RAPl activates transcription is not well understood. In particular, it is not known whether the coordinate changes in rp gene transcription in S. cerevisiae in response to changing physiological conditions are mediated directly through RAPl, e.g. by modification, or through additional factors interacting with RAPl. In order to determine to what extent the regulatory machinery controlling rp gene transcription in S. cerevisiae has been

904

G. K. BERGKAMP-STEFFENS, R. HOEKSTRA AND R. J. PLANTA

conserved in other yeast species, we decided to investigate the rp genes of the related yeasts Kluyveromyceslactis and Kluyveromycesmarxianus. Here we report the isolation and sequence analysis of the Kluyveromyces homologs of the S. cerevisiae L25, L46, S10, S24 and RP59 genes, all of which have been shown to harbor RPG boxes. By sequence comparison of the upstream regions of the different Kluyveromycesrp genes, we identified three types of putative cis-acting elements, including one that has a sequence highly similar to the S. cerevisiae RPG box. We have analysed the possible functional role of these elements by deletion mapping in vivo, as well as in vitro band-shift experiments. The results show that the RPG, box acts as a binding site for a Kluyveromycesprotein that is probably functionally homologous to S. cerevisiae RAPl, though considerably smaller in size. One of the remaining two Kluyveromyces-specific elements, the X box, appears to act as a transcriptional repressor. The function of the third element, the Z box, remains to be established. MATERIALS AND METHODS Strains, plasmids and media Escherichia coli strains JMlOl and JM109 (Yanisch-Perron et al., 1985) were used for cloning and subcloning of the Kluyveromycesrp genes. The yeast strains used were K. lactis SDl 1 (a lac4 trpl derivative of CBS2360; Das and Hollenberg, 1982), K. lactis wild-type strain CBS2359 and K. marxianus wild-type strain CBS6556. S. cerevisiae strainHR2. (Maarseetal., 1988)and Saccharomyces carlsbergensis strain S74 (Bollen et al., 1981) were used in control experiments. Recently it has been shown that both the latter two strains belong to the species cerevisiae (Klootwijk and Planta, 1990). Functional analysis of the Kluyveromyces rp genes was carried out in K. lactis strain MSKllO (MATa, uraA, trpl::URA3; Stark and Millner, 1989). Cloning and sequencing of the Kluyveromyces rp genes was carried out with the aid of plasmids pUC18, pUC19 and M13 (Yanisch-Perron et al., 1985; Genbank Association #VB026 Fecbase: p u c 19Cl). For transformation of K. lactis we constructed the plasmid LEpTRP (K. lactis Episomal plasmid containing the T R p l gene from S. cerevisGe). To this end we cloned the NarI-Nar-I fragment from pKARS2 (Das and Hollenberg, 1982) into the NarI

site of YIplac204 (Gietz and Sugino, 1988), a plasmid containing the TRPl gene from S. cerevisiae. This NarI-NarI fragment bears the KARS2 sequence, required for autonomous replication in K. lactis. E. colicells were grown on YT medium consisting of 0.8% (w/v) Bactotrpyton, 0.5% (w/v) yeast extract (Difco Laboratories) and 0.5% (w/v) NaCl. For selection, ampicillin was added to a final concentration of 100 pg/ml. Untransformed yeast cells were grown on YPD medium, consisting of 1YO(w/v) yeast extract, 2% (w/v) Bactopeptone (Difco Laboratories) and 2% (w/v) glucose. Transformed yeast cells were grown on minimal medium, consisting of 2% (w/v) glucose and 0.67% (w/v) Yeast Nitrogen Base w/o amino acids. Transformation

E. coli JMlOl was transformed by the CaCl, method, described by Maniatis et al. (1982). E. coli JM 109 was transformed, using RbCl according to the method described by Lopes et al. (1984) or the one described by Maniatis et al. (1989). K. lactis was transformed by the LiAc method, described by Ito et al. (1983). Probes Radioactive labeled DNA fragments from S. cerevisiae were used as probes for the isolation of the KluyveromycesS10 and L25 genes. S10: clone pBMCYll3 digested with HindIII (Leer et al., 1982) L25: clone pBMCY138 digested with KpnI (Leer et al., 1983) Oligonucleotide probes for the isolation of the Kluyveromyces S24-1 and L46 genes were derived from the open reading frames of the respective S. cerevisiae counterparts. They were synthesized on an Applied Biosystems 381A DNA synthesizer. S24- 1: 5' -CTTGATAATGACCTTGGAGGATGGTCTGATTAAAACT- 3' L46: 5' -GGTTCTCAATCTGATCCATTGTGGCAATGGTCTG- 3' Double-stranded oligonucleotide probes used in the band-shift experiments were: S. cerevisiae RPG box: 5'ACACCCATACATTTGCATG- 3' 3' -ACGTTGTGGGTATGTAAAC - 5' K . marxianus RPG, box:

REGULATORY SEQUENCES OF KLUYVEROMYCES RIBOSOMAL PROTEIN GENES

5’- CACAAACCCACACACCACGTA - 3‘ 3‘- GTGTTTGGGTGTGTGGTGCAT - 5‘ DNA manipulation

Restriction endonucleases, T4 DNA ligase, Klenow enzyme and T4 polynucleotide kinase were obtained from Gibco BRL, Bioexellence,Pharmacia or Biolabs. Calf intestine alkaline phosphatase was obtained from Boehringer Mannheim. All enzymes were used in accordance with the supplier’s recommendations. DNA sequencing was performed as described by Sanger et al. (1977), using the pUC Sequencing Kit (Boehringer Mannheim) or the Sequenase Kit (Sequenases version 2-0, U.S.B., Cleveland, U S .A.). DNA fragments were labeled by the Random Primer method (Promega). Oligonucleotides were labeled with the aid of T4 polynucleotide kinase. Radiolabeled nucleotides were purchased from Amersham. DNA preparation

Plasmids were purified according to Birnboim and Doly (1979). Total DNA from Kluyveromyces was isolated as described by Pedersen (1983) or by the method of Struhl et al. (1979). DNA fragments were isolated by fractionation of restriction digests on a 1 or 2% agarose gel in TAE (0.04 M Tris-HAc, 0.02 M NaAc, 0.01 M EDTA; pH 7.8). The desired bands were cut out of the gel and DNA was extracted by using either the freezesqueeze method (Tautz and Renz, 1982) for fragments smaller than 1 kb, or the Geneclean Kit@(Bio 101, California, U S A . ) for larger fragments.

905

in 2Ox SSC (0.3~-sodiumcitrate, 3111 NaCl; pH 7.0), according to the method described by Southern (1975). Hybridization was performed in 6 x SSC, 5 x Denhardt’s solution (100 x Denhardt’s: 0.1% (w/v) bovine serum albumins, 0.1 % (w/v) Ficoll and 0.1YO (w/v) polyvinyl pyrrolidon) and 100 pg/ml herring sperm DNA at 65°C. Hybridization with oligonucleotides was performed at 48°C. Northern blotting onto nylon filters was performed in 20 x SSC. Filters were baked for 2 h at 80°C. Hybridization was carried out under the same conditions as described above, accept that 5 x SSPE (20 x SSPE: 0.9 M NaCI, 50 mM Na,HPO, and 5 mM EDTA; pH 7.7), instead of 6 x SSC, was used. Colony hybridization was performed as described by Maniatis et al. (1982). Band-shifr assays

Cell extracts were prepared according to the procedure of Olesen et al. (1987). The protein content of the extracts was determined by measuring the OD260nm and OD280nm as well as by the method of Bradford (1976). The incubation mixture typically contained per 20 pl: 5-10 fmol of labeled DNA probe, 10 pg of total cell protein, 1 pg poly [dI/dC], and 500 ng competitor DNA (pUC) in 50mM-Tris-HC1 (pH 8.0), 40 mM (NH,),SO,, 2.5 mM MgCl,, 0.25 mM EDTA and 2.5% (v/v) glycerol. After 25 min incubation at room temperature, the mixture was loaded on a 4% vertical polyacrylamide gel in TBE buffer ( 9 0 m Tris-HCI, ~ pH 8-0, 9 0 m ~boric acid, 2 . 5 m ~EDTA) at 4°C. The sample was electrophoresed at 400 V for 2-4 h. RESULTS AND DISCUSSION Isolation and cloning of Kluyveromyces rp genes

Various restriction digests of both K . lactis and K . marxianus total DNA were analysed by Southern Twenty ml of yeast culture, grown to an OD660nm blotting with probes derived from the coding region of 0.5, were broken with glass beads and total RNA of the S. cerevisiae L25, L46, S10 and S24 genes (see was isolated essentially as described by Zitomer et Materials and Methods). al. (1979). Samples containing 10 pg of total RNA In all cases, we observed strong hybridization were denatured in a solution containing 1 M glyoxal signals (not shown), indicating a high degree and 50% (v/v) dimethylsulfoxide for 2 h at 50°C and of homology between the Kluyveromyces and fractionated on 1.5% agarose gels. Saccharomyces genes. Surprisingly, we found only the gene for S24 to be present in two copies in Kluyveromyces. This is in striking contrast to the Hybridization situation in S. cerevisiae, where most of the rp genes After fractionation on agarose gels, DNA was are duplicated, the one encoding L25 being one of blotted onto nylon filters (Hybond-N, Amersham) the few exceptions (Woudt et al., 1986). The S24 RNA preparation

906

G . K. BERGKAMP-STEFFENS, R. HOEKSTRA A N D R. J. PLANTA

A. S. cerevisiae

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Figure 1. Physical maps of the isolated rp genes of K . lactis and K . marxianus and their counterpartsof S. cerevisiae. Closed bar: coding region; open bar: intervening sequence. B =BgflI, E = EcoRI, H = HindIII, K = KpnI, S =Sun, Sp= SphI.

gene described in this paper is transcribed at a much higher rate than the second copy (G. K. BergkampSteffens, unpublished results) and will be called S24-1. From the genomic hybridization data it was possible to construct physical maps for the various genes (Figure 1) and hence to choose fragments for cloning these genes, including sufficiently long stretches of their 5'- and 3'4anking regions. One important conclusion to be drawn from the mapping data is that the linkage of the S24-1 and L46 genes observed in S. cerevisiae is conserved in K. marxianus. In order to obtain the desired genes we isolated the respective restriction fragments by preparative gel electrophoresis and cloned them in pUC18 or pUC19 (S10: HindIII-HindIII, k2.7 kb; L25 K . lactis: HindIII-HindIII, f3.8 kb; L25 K . marxianus: HindIII-HindIII, f2-6 kb; RP59-S241-L46: EcoRI-EcoRI, f4-2 kb). Several positive clones for each gene were selected by colony hybridization with the same probes used for the Southern analysis. These clones were further characterized

by restriction analysis in order to devise suitable sequence strategies. Primary structure of the ribosomal proteins from Kluyverom yces The nucleotide sequences and deduced amino acid sequences of the genes isolated from Kluyveromyces, including about 700 nucleotides upstream and 350 nucleotides downstream from the coding region, are presented in Figure 2. Comparison of the deduced amino acid sequences with those of the Saccharomyces proteins are presented in Figure 3. The presented sequence data reveal several interesting features. Firstly, sequence analysis not only confirmed the linkage of the S24-1 and L46 rp genes in a head-to-head arrangement, but it also revealed that the RP59 gene is located closely downstream from the S24-1 gene in the opposite orientation. Inspection of the sequences in and around the S24-1 and L46 genes, published by Leer et al., (1985a), and the RP59 sequence, published by Larkin et al. (1987), showed a similar linkage between the three genes to exist in S . cerevisiae.

REGULATORY SEQUENCES OF KLUYVEROMYCES RIBOSOMAL PROTEIN GENES

However, the S. cerevisiae RP59 gene linked to the S24-1 gene must be RP59-2 (CRY2) since its 3‘flanking region differs from that of the RP59- 1 gene sequenced by Larkin et al. (1987). Analysis of the 3’-flanking region from the recently isolated RP59 gene of K. lactis (Larson and Rossi, 1991) revealed the same genomic organization of the RP59 and S24 genes also in this strain. Secondly, the coding regions of the Kluyveromyces genes closely resemble those of their Saccharomyces counterparts, showing about 90% sequence similarity at the nucleotide and 85-95% at the amino acid level. This resemblance is exceptionally high when compared to, for instance, the K . lactis LAC9 gene and the functionally homologous GAL4 gene from S. cerevisiae (Wray et al., 1987). Furthermore, the lengths of the coding regions of the Kluyveromyces genes are the same as in Saccharomyces (Raue et al., 1991; Table 1). This is in contrast to the recently isolated LEU2 gene from K. marxianus (Bergkamp et al., 1991), which lacks five codons with respect to the corresponding gene in S. cerevisiae. Finally, both the occurrence and the position of the intron has been conserved in each of the Kluyveromyces rp genes. The K . marxianus S24-1 gene lacks an intron, as it does its S. cerevisiae counterpart, and only in the case of the K . marxianus RP59 gene is there a slight shift in intron position (one codon). However, in the latter case it should be kept in mind that the comparison is with the RP59-1 gene of S. cerevisiae, while the K. marxianus RP59 gene, according to its chromosomal location, corresponds to the S. cerevisiae RP59-2 gene. The intron sequences do not show any conservation in either length or sequence, except for the 5‘- and the 3’-splice sites and TACTAAC branchpoint sequences (Table 1). The position of the TACTAAC box is not conserved between the two species (see Table 1) but in all cases falls within the limits observed in S. cerevisiae (Cellini et al., 1986). The conservation of the splicing signals is in agreement with the finding of Deshler et al. (1989) that the actin gene of K. lactis is properly spliced in S. cerevisiae. The 5‘- and 3’-flanking regions of the Kluyveromyces rp genes show no significant sequence similarity with the corresponding regions of their respective counterparts from Saccharomyces, with one important exception. In the upstream region of each of the six genes, we detected one or more copies of a sequence with a high degree of similarity to the S. cerevisiae RPG box (Figure 2). These elements are discussed below.

907

Transcription of the Kluyveromyces rp genes

Because, except for S24, all the newly isolated Kluyveromyces rp genes are present in single copy, it was possible to determine their transcriptional activity by Northern analysis. Total RNA, isolated from S. cerevisiae, K. lactis and K. marxianus cells, was hybridized with probes derived from the various rp genes of K. marxianus. The results (Figure 4) show that each of the genes is indeed transcribed. For S24-1 we have to discriminate between transcripts from rp gene S24-1 and S24-2. Experiments with the S24-2 gene on a multicopy plasmid transformed to K . lactis, followed by Northern analysis with a S24-2-specific probe, indicate that this gene is transcribed at a very low level (G.K. Bergkamp-Steffens et al., unpublished results). Thus, the signal visible in Figure 4 should indeed represent S24-1 transcripts. The lengths of the transcripts from the individual rp genes varied between the different yeast species. For example, the transcripts from the gene encoding ribosomal protein S 10 were significantly longer in Kluyveromyces than in S. cerevisiae (Figure 4). This analysis showed also that the S10 transcript of K . marxianus is longer than its counterpart of K . lactis. Putative upstream regulatory sequences

Sequence comparison of the upstream regions of S. cerevisiae rp genes has been successfully used to identify potential regulatory elements (Leer et al., 1985).The same approach was, therefore, applied to the upstream regions of the six Kluyveromyces rp genes described above and the K, lactis RP59 gene isolated by Larkson and Rossi (1991). As a result we detected three types of a conserved sequence element, present in one or more copies and either orientation, upstream from the coding region of each of the genes. These elements are indicated in Figure 2 and a schematic representation of their position and orientation is given in Figure 5. The three elements have been designated the X, Z and RPG, boxes, respectively. The first two do not have an equivalent in Saccharomyces. The RPG, box, however, is very similar, though not identical, to the RPG box shared by the majority of the rp genes in S. cerevisiae. The X box consists of eight nucleotides, 5’GGGACTGT-3’, and in most cases occurs in the coding strand. The S24-1-L46 intergenic region, however, contains only a single X box located in the non-coding strand of the S24-1 gene.

---------

-

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-

B.

560

640

MTATTCCTTTCGACTACGCAGTTTC~TGCGACCTTGTATCTGTATTGTTTCAT-GTAATATTGAACGTTTT

160

80

1

77

-

-

+

i

..

TTAAACAACTACTATTTCGATTAGATCCTTAAATTGGAATTACT~ATCATGATACGTC-~TACGATGCGTAGTT

ATACGGTTCATCGGTGTTTATGAGTTAAGTTTTTTG~CATGTT~GGCCCACTCGACGCTTTCCCTGTACCGTTC

AGAGCATAAAATACACCTTACATTTAATACACTTAAAGTCAATTCTCTAGTTTT~CAGAAG~TCATGCCGTT~TCGA

ACTAGTTATCCTTCAATCAACATTT~TCCAGTTTTACTAAC~TTTCCTATTATTATTTTTTCCACAG CT AAG

GCT ACT GCT GCT AAG AAG GCT GTT GTT AAG GGT ACC RAT GGT AAG AAG GCT TTG M G GTC

AGA ACC TCT GCT TCC TTC AGA TTG CCA AAG ACC TTG AAG TTG GCT AGA TCT CCA AAG

TAC arg thr ser ala ser phe arg l e u pro lys thr leu lys l e u ala arg ser pro lys tyr

GCT ACC AAG GCT GTC CCA CAC TAC AAC AGA TTG GAC TCT TAC AAA GTC ATC GAA CAA CCA ala thr lys ala val pro his tyr asn arg leu asp aer tyr lys val i l e glu gln pro

ATC ACT TCT GAA ACT GCC ATG AAG AAG GTT GAA GAT GGT AAC ACT TTG GTT TTC AAG GTC ile thr ser glu thr ala met lys lys Val glu asp gly asn thr leu Val phe lys val

TCT CTA AAG GCT AAC AAG TAC CAA ATC AAG AAG GCT GTC AAG GAA TTA TAC GAA GTT GAT sex leu lys ala asn lys tyr gln lle lys lys ala val lys glu l e u tyr glu Val asp

GTT TTG TCT GTC AAC ACC TTG GTT AGA CCA AAC GGT ACC M G AAG GCT TAC GTC AGA TTG Val leu ser Val asn thr l e u val arg pro a s n gly thr lys lya ala tyr Val arg l e u

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i i

1057 CAACTTCCGTTGATCTTGCCGTTTATCCCCCACCATTT~TCTTTCAAAGATTCCCAAT~TGAATGTTATTTACTGTA

977 ATAAATCGTGTATTTCAATTTCATTTTAGTTTCAATAAGATGTTTAGCCGAGGGGATGTCAGAGTTAAGAAGGGGT

t

+

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910 GAT GCC TTG GAT nTc GCC AAC AGA ATT GGT TAC ATC TAA TCATTTTTAATGTATACATRAAAGCTC asp ala leu asp ile ala asn arg ile gly tyr ile stop

asn the leu val arg pro asn gly thr lys lys ala tyr val arg l e u thr ala asp tyr

TAC CAA ATC RAG AAG GCT GTC AAG GAA TTA TAC GAA GTT GAT GTC TTG AAC GTT as" lys tyr gln i l e lys lys ala Val lya glu leu tyr glu Val asp Val leu asn Val

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730 ACT GCC ATG AAG AAG GTT GAA GAC GGT AAC ACC TTG GTC TTC CAA GTC TCC CTA AAG GCC thr ala met lys lys Val glu asp gly asn thr leu Val phe gln Val ser leu lys ala

670 GTC CCA CAC TAC AAC AGA TTG GAC TCT TAC AAG GTT ATC GAA CAA CCA ATC ACT TCT GAA Val pro his tyr asn arg leu asp ser tyr lys Val i l e glu gln pro ile thr ser glu

ser phe arg l e u pro lys thr l e u lys leu ala arg ser pro lya tyr ala thr lys ala

TCC TTC AGA TTG CCA AAG ACC TTG AAG TTG GCT AGA TCC CCA AAG TAC GCT ACC AAG GCT

AAT GGT AAG AAG Gcp TTG AAG GTC AGA ACC TCT GCT lys lys ala val val lys gly thr a m gly lys lya ala leu lye val arg thr a e r ala

AAG AAG GCT GTT GTT AAG GGT ACC

CT AAG GCT ACT GCT GC

.../hr lys ala thr ala a1

476 GAACCATCTTTTACTAACATTTCCCCATCAAATGGGTTATTCTTTTTTTACAG

396 ATCACAATTCTAAAAGCGAGTTTT~GGTGTACTCATAATCATAATCTTTCAA~CTATCAGTTCCAATGTGAT

316 TATCCAGAA~CATAACCGAATAGACGTTTGAATGAAATTG~TAGTTATTAATAACTT~GAATAGGATTTGG~

t

+

t

ACATTCCACGCACAGGATATAAGAATTTAGA~GCAACCCATTTATT~GAGTGAATGAAATCCTTGACCTTAC

236 TTTTATAACGTCGAGCCGTATGAC~GAAGACTTAGAGTTGGATGATCTATTTGAACGTGGTATATTTC~TCT

156

76 G A A C A T T T A G A C C T C A A C T T ~ T ~ C C G T C A A G A C T T A G A A T ~ C ~ C A T G G T A G T T A A A A G C A T T A A

1 ATG GCT CCA TCT A G T A T G T T T A G A ~ C T T G A T G C T G A T A G T A T T ~ ~ C G A G A T T G A A m t ala pro ser t/. ..

80 AACAATATTTCAGCGTTTGGTAGCATAATAGAGTTCTTATACGTTATTCGACAAGGACGAACAC~C~GACA-

160 TCCCTGTACTGTACACTGGATGGACCCATCA~TATT~GAT~TATTGCTCTAAGTTAAAGTATTATTG~GTTTT

240 CTCCTAACGAAGTCCTGCCTAGCGGTCTGCCTACCACCTGCCTA~CT~TTGTATCCTC~TAATCCAAAGACT

+ 610

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Figure 2 (A and B)

TATACATATCAACTGAATITCGTGTATTTCAATTAAATCATTTTAGTTTCAAAT~~GAAT~TTT~TTAA

thr ala asp phe asp ala l e u asp lle ala asn arg ile gly tyr ile stop

ACT GCC GAC TTC GAT GCC TTG GAC ATT GCC AAC AGA ATC GGT TAC ATC TAA TCATTTTTAATG

ala thr ala ala lys lys ala Val val lya gly thr asn gly lys lys ala l e u lys Val

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T t A A C A T C A A A T G A A A C G A T G A A C A C A A A C A C G C T t C A A

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. . ./hr lys

TACGCTAATGAGAAACCAACTACGATCTGAATAATTGTACATGATATTACCTATGTGTGAACCTGTCATGAAAGCGGA

+ 157

GCGGGCTGAACAAGTGAGAATGAAGATATTACCTTGAGTGTAATTACACACCTGTGGGA~CGAATGGTAGAATAAG

met ala pro ser t/.

ATG GCT CCA TCT A GTATGTTACTGAGATGCGCAACTGAAAGCTACATGCTGATAATGGT~ATAACGACTTACC

AAAGAATATTTCAATATCATTCGGTATTAGAGTACACTTGATTA~TT~GAGGCC~CACAACCAAGACACAAAA

CCTGAGCCTGGAGCCATATCTGTACCTG~T~~GTGCGATTGTGCTACCTGGCCAGGGATACCCTGTTCGTGCTG

- 240

-

320

-

ACTCTCCCCTCTACTCGCACTGACATTCT~GGT~GTA~A~CTGCTGTCCATCCAGCCTATCATCCATTCCTTC

GGCTGCCAGGTTCTCACCGTCTCCTTCTGTC~~CG~GAGTCTAGTCTTCCCAGCGAGAGGACTC~TTCTGCCC

400

i

------- -

ACGGW LCTGTTGT~TC;r A C GGATTCC~GTTTCTGTAGCTTCCACT~TGATGTTTCTGCAAGACTGT~TC

400 G C C C A G A C C C C A T T C C A G T C C T C C C T G G O C T T A C G C A W L G T C

-

-

TA~TATTTTAGTATTCCATTTTA~~~AAMT~CA)LTT~GGT~T~GGAAACCCGA~GGCGTTCC

t

TAAGCAAGTATTAGAGAGGGACAAMTACATTCACTGATATCTGTTGGTTGATATTAGA CTATCTCTACA)LGTGCCTATTTTCCCAGA~TTMTTGGT~CTCCAGAGATGG~rCTTTTTTTTTTTATTCCAGAT

L25 K.marxianus 700

A A ~ ~ A A A A T T T T C C G G T ~ G C ~ G ~ ~ A T ~ C ~ T T T ~ T T T A G A T T T T T T G T ~ T T G T - 480 CCTTTGTTCATTCAGGTTCGTTTTAAATTTCTGAAAMMTGGTACGGGCGGCCCAGTGGCATATCTC 4

---*

TAAGACAGCAATTCCTATGATTTCACACA)LTTATGACATT~CATGCCTTATAATCTCT~TGCTAATTTCATCG

640

----------

AACAAATAATGCATGTATATAAATTTAACGTAAC~CCA~TTTTCGTTTAAATACAT

560

700

L25 K.loctis

- 480

-

-

A.

-

G T C C T G G T G G G G C T T C A C T G G A A A C C A A T C T C T C T C T A G A

400

320

240

-

-

-

-

AGAAAATACTATTAGGAGAAAGTCTTTACTGAGAACAG~CGTAACCTAGGATATAGTTTGAAGGTT~CAATCA

80

1

79

+

+

+

+

CTTTCATTTTACCTGCTGTTTGCAACATTGTTTTGTCGACTATTTACTAACGAACTTGAAATTCTATTTCATATTTAATG

CAG TTG AAC ATC TCT TAC CCA ATT AAC GGT ACT CAA AAG TGC GTT GAA ATC GAT GAT GAA /leu a m i l e ser tyr pro i l e asn gly thr gln lys cys Val glu ile asp asp glu

CAC CGT GTC CGT GTT TTC TAC GAC AAG AGA ATC GGT CAA GAA GTT GAT GGT GAA GCC GTT his arg Val arg Val phe tyr asp lys arg ile gly gln glu val asp gly glu ala Val

GGT GAT GAA TTC AAG GGT TAC GTC TTC AAG ATT GCC GGT GGT AAC GAC AAG CAA GGT TTC gly asp glu phe lys gly tyr Val phe lys ile ala gly gly asn asp lys gln gly phe

CCA ATG AAG CAA GGT GTC TTG TTG CCA ACC AGA GTC AAG TTG TTG ATG GCC M G GGT ACC pro met lys gln gly Val leu leu pro thr arg val lys leu leu met ala lys gly thr

TCT TGT TAC AGA CCA AGA AGA AAC GGT GAA AGA AAG AGA AAG TCC GTC AGA GGT GCT ATC aer cys tyr arg pro arg arg asn gly glu arg lys arg lys ser Val arg gly ala ile

GTC GGT CCA GAT TTG GCT GTT TTG GCT TTG GTT ATC GTC AAG AAG GGT GAC WLA GAA ATT val gly pro a8p leu ale Val leu ala leu Val ile val lys lys gly asp gln glu ile

GAA GGT GTC ACC AAC GAG ACT GTT CCA AAG AGA TTG GGT CCT AAG AGA GCC AAC AAC ATC glu gly Val thr asn glu thr Val pro lys arg leu gly pro lys arg ala asn asn ile

AGA AAG TTC TTC GGT TTG ncc AAG GAA GAT GAT GTC CGT GAC TAC GTT ATC AGA AGA GAA arg lys phe phe gly leu thr lys glu asp asp Val arg asp tyr Val ile arg arg glu

319

+ 399

+ 459

519

799

979

+

+

+ 859

919

+

+ 679

739

+

+

+

GAA GTT AGA AAG AGA AGA GCC TCC TCT TTG AAG GCT GCT GAA TAA ACGAATTTCAATATCTAGTC glu Val arg lys arg arg ala ser ser leu lys ala ala glu stop

+ 1159

+

GCT GCT GCT GAA TAC GCT CAA TTG TTG GCT AAG AGA TTG GCT GAP. AGA AAG GCT arg glu ala ala ala glu tyr ala gln leu leu ala lys arg leu ala glu arg lys ala

+ 1099

CTTGAGTATATATATACACTTGGIVLACTGT

1301 TTTTGCGGTCTTGTAGCGCTTTTAATATTGTGTATTAATTTTGTACGTTTT~TTGATTAATCAGTATGTA~TATATG

TAGTTGTGATACAACAGTTTATGAAGATATATCCTATACCT~TTAAAGAGCGCTT~T~CTT~AGCGT

1141 TAAGTTTTGAGAATATAATATAGCATTTCAAT~ATCCTAATTTTTAATTATTCGTGAATAGTTTAATTATT~TTGAAT

+1381

+

GTT ACT CCA GTT CCA TCT GAC TCT ACC AGA AAG AAG GGT GGT AGA AGA GGT AGA AGA TTA va1 thr pro val pro ser asp ser thr arg lys lys gly gly arg arg gly arg arg leu

1062 TGA TTTGCTTCATTATTATCTCTCAGTATTTULCAATTGTATAA~TGTGTGTTT~GCTTTACATATCTTAC~ stop

+ 1221

+

+

GCC GTC CAC ATC AAG ATC AGA GCT ACT GGT GGT ACT AGA TCC AAG ACT CCA GGT CCA GGT ala val his ile lys ile arg ala thr gly gly thr arg ser lys thr pro gly pro gly

942 GGT CAA GCT GCT TTG AGA GCT TTG GCT AGA TCC GGT TTG AGA ATC GGC CGT ATC GAA GAT gly gln ala ala leu arg ala leu ala arg ser gly leu arg ile gly arg ile glu asp

882

+ 1002

+

ACT ATC GCC AGA GTT ACT GGT GGT ATG AAG GTC AAG GCT GAC AGA GAT GAA TCT TCT CCA thr ile ala arg Val thr gly gly met lys Val lys ala asp arg asp glu ser sex pro

AGA ATC TTC GCT TCT TTC AAC GAC ACT TTC GTG CAT GTC ACC GAT TTG TCT GGT AGA GAA arg ile phe ala ser phe a m asp thr phe Val his Val thr asp leu ser gly arg glu

822 TAC GCT GCC ATG TTG GCT GCT CAA GAT GTT GCT GCC AAG TGT AAG GAA GTT GGT ATC ACT tyr ala ala met l e u ala ala gln asp val ala ala lye cys lya glu Val gly ile thr

+ 762

+ 102

...

AGGTTTATMTCAAATGAGGATAACAATACTATTGTTGTACTCGTTTGTTTTATTAATAC~CTGTTTTACTAACAATT

Figure 2 continued (C and D).

1224 ATTGIVLATTTTTTTAATCTTTTCTTCATTACTCTTTTGTTAACATCGAACTCCATTATGCTGCAAAAGCTT

AGA GAA

CAA AGA TTG CAA AGA AAG AGA CAA CAA AAG GCT TTG AAG ATC AAG M C GCT W GCT CAA gln arg leu gln arg lys arg gln gln lys ala leu lys ile lys a m ala gln ala gln

+ 1039

GTT ACC AAG GGT GAA AAG ACT TAC ACC AAG GCT CCA AAG ATC CAA AGA TTG GTT ACT CCT Val thr lys gly glu lys thr tyr thr lys ala pro lys ile gln arg leu Val t h r pro

...

+

GAATGATTTTATTGAAATTTTTTGAGTG~TGTACTTTTATWTTATGAATGTTCTCAGTGAGGG~CCAATGAGGT

637 CATTTCTCATATTATTTTAACAG TT GTT CAA GCT AAG GAT AAC TCT CAA GTT TTC GGT GTT GCT /a1 val gln ala lys asp a m ser gln Val phe gly val ala

+ 551

TTGTATGAATAACGTCGTAGAGG~TCATAGAGGG~GCATTGCATTAT~TAGACTACAATAGTGGGTTGTAAAG

239

+

477 T C A A G T A C T A C T A G C T C A G T G ~ G G C A G C G C A T C C T G T A T ~ G A A T A T G C A T T C A T T T C ~ ~ ~ G A C T ~ G A G

TCATTATCACAAACGGGATTTATGGATCGCAAACAATTAACTTGGAGTCAATTGTTTGTTAACT~CTTATTTACTG

+ 391

+

TTATTCTWLACGAGAGCCTCGTTGTATTCTCTTCGATAACTTCCATCTAAAATACGGAACGGTCGACGTTGATAATGAGT

237 AAGTAGGCACTCAATCGTCTGAGGAT~GTG~TGTGTTG~GATGATCTACTTTCCCTCTGATT~TGTTGATGAGC

GGGACAI\CAGATCAGTTGTTCTGAACGACACCTATCTGTCACTCTTTTULGT~TC~GTGATTCCA~~CAGAAG

77 A C U L T G C G G C A T A T G A T A A A T A A G ~ C T C A A G A A T ~ A ~ T G C T ~ G A C A T ~ T T C ~ C ~ T T A G T A A T A A T G

.

1 ATG GCT AAC G G T A C G T G C T G A G G G G T G G T A T C C C G T T T ~ C C C T T T A G T T G ~ G T T W G A A ~ T ~ T T T A A met ala asn vl..

80 A A A A C C T G T T T G G A T A T T A T A G T A T A A T T G T T T G T T G T A T A G T A G A A ~ C C T C A A G ~ C C A G T ~ U L T C A A G A

+ 317

+

---- ----

160 TCGTCCATTGGGTTTTCCAACTTTCCRAAATGTCATCATTCAATTTC~TAATATATAACGAATGCATTCTACTCA~T

+ 151

+

_.

-

GAATTCGCCGCGATGAGATTACTTTCATGAAGTCTTAACTT~ACTGTTTGTATAAGTACA~T~TT~T

~ K.5mrxianus 9

240 T T A T T T C T T T C T G G T T C T G A T C T T C C T G T T T T G G A A A A T A C T T T T T T ~ ~ T ~ ~ C A C C ~ C A T C A A C A A C C A ~ T

+ 159

.

ATG AAG G T A T G T G G A G G G T C A A T A T A T T G A ~ G C A G G C T T G T A A T ~ G A A G A A C C T A T T G A G A A C A T C ~ C A met lya/. .

TCGCCAGTCTGGAAATCATGTCTGAAGCTAAACTTTC~TATCACACATTGTTGAAATCGT~~CATTGTATGT

ATTAAAACTGGATATAGAWCAACATATATCAGATAGGATATTCAGCAATACAAGGTAGTCATAGCA~CTAAGCCATC

160

-

GTCTCTCACCCTCATTCCCTGGTACCACGGTGTA~GAGTTTCTCT~TCC~TGT~TTCGCCGGTCGGCCATGCT

A A C T G T C C T C C A G A G A C C T T A C G A T C C C C A G A A T ~ T G T A ~ C C T U L T T C C T C T T C C T A A ~ T T T ~ T ~ G A+

-----*

AATATCTTGCTGAAGACAGGTGACATTTTTTCTCTTGTA~ACGAGAGGAGCCCGCCAGTTTCCTTCCTTCTAGCTTAG

480

~

320

D.

CCGTACACCATGTATTTTATATTATATTTAAGGATATGTTTTT~TTT~GGA~CCTATTATAGTAATTTAA~GCGAA -

-

-

560

640

-

.-

ACAAATATGAAARTTAGG~TTTTTAGCCTTGCATCAACCGAAC~CGAT~CAGTC

--* GGATGGACAACCAGIVLAGTTGAAACACACTAATCCAAGTATCACGTGATGTTGA~ACTGTTTGTATTTTATTTTCCAT - -----___* 4 .---

s 10 K.manrianus

700

-

c.

720

640

560

480

400

-

-

-

-

-

-

----------

CAATTTTCATATACAGAATAGTAACTAAAWLGG~AATTT~TCTTCATCTACTCAAACCCA~CACCACGTATAAA

-4---*.-------_ - * - --------*

GCACCTGGGCAGCTCGCGTAGAGCAGAGAAACAACATTC~TACTACGAA~GTTGT~AT~TCATTTTT~TATT

GGT AAG CGT

CAA GTT TTG ATT AWL CCA TCC TCT AAG GTC ATC ATC AAG TTT TTG CAA GTT gly lys arg gln Val leu ile arg pro ser ser lys Val ile i l e lya phe leu gln Val

TAC ATC GAT GAC CAC AGA TCT GGT AAG met gln lys his gly tyr ile gly glu phe glu tyr ile asp asp his arg ser gly lys

61

121

181 ATC GTC GTT CAA TTG AAC GGT AWL TTG AAC AAG TGT GGT GTT ATC TCT CCA AWL TTC AAC ile Val Val gln leu asn gly arg leu aan lya cys gly Val ile ser pro arg phe asn

241 GTT AAG ATT TCC GAT GTC GAA AAG TGG ACT GCT AAC TTG TTG CCA GCC AGA CAA TTC GGT Val lys ile ser asp Val glu lys trp thr ala asn leu leu pro ala arg qln phe gly

t

t

+

+

M T A C A T A C T G A T T A A T C A A T C C A A A A C G T A C A A A A T T A A T T G

ACAATAATTAAACTATTCACGAATAATT~TTAGGATAGATTGAAATGCTATATTATATTCTC~CTTATTGGTA

-

-

-

400

480

560

479

+

670 750 830

910

+ + t

609

+

t

549

399

t

319

239

t

t

159

+

+

79

1

80

160

240

+

+

-

-

Figure 2. DNA sequences ofthe isolated r p genes of K . lactis and K . marxianus and amino acid sequences of the derived proteins. Nucleotidesare numbered starting from the +=Xbox; - - - h = Z box. first nucleotide of the initiation codon. Arrows indicate the putative regulatory sequences:---------*=RPGbox;

Figure 2 continued (E and F).

+ 670 AGATATGTAAAGCTTTAAAUCACATTTGTTATACAATTGTGAAATACTWLGAGATAATAATGAA~AAA

+ 590

510 CCAAGTGCCCATTTTAAGC~TCT~AAT~TTAGGTATAGGATATATCTTCATAAACTGTTGTATCACAACTAATTCUL

+ 430

361 GTT TCC GGC AAG ATC TTG GGT TTC GTT TAC TAA ACAGTTTCCULGTGTATATATATACTCAAGCATATA Val *or gly lys lle leu gly phe Val tyr stop

tyr Val ile leu thr thr aer ala gly lle met asp his glu glu ala his arg lys his

GTT ATC TTG ACT ACT TCC GCT GGT ATT ATG GAC CAC GAA GAA GCT CAC AWL AAG CAC

ATG CAA AAG CAC GGT TAC ATT GGT GAA TTT GAA

+ 301 TAC

t

GCC GAA AAG ACC

net thr arg thr ser Val leu ala asp ala leu asn ala ile aan asn ala glu lye thr

M C

1

+

TCC GTT TTA GCT GAT GCT TTG AAC GCT ATC AAC

AGTAAAGAGTGTAACAGTAAATTAGGATTATTAGGTAGCTAAGGTCCTTCGATTCCGTGCCCGAAGAGTATACGCCAAG

80

-

ATG ACC AGA ACC

160 AGCTTGAGCAGAGTTGGCATGCCTAACGATTTTTCAATTTAAGAATTTCATGTAAATGGTACTATTA~ATTGTATAT

t

720

800

834

TAWLGOGAATCGGCTCCCTA~~C~~~~~CTAC~TG~CGC~CACAAGGAATTTCCCACCAGCGAG

-

-

-

-

A C T C C ~ G C G T A C G T C C T G C C T A ~ ~ ~ ~ - ~ ~ ~ G T A G T G T A A ~ T T ~ ~ T C T C T G C C A C G ---------) 640

A C A T A C T M T T T A C A G A A G G T G C A M G C W U C G O W L W L C C T C G

-

-

ATTTGATTCTTATCCTTGGTGTCTTAG~

AGGATATAGCTAAACACTAATCTAACT~GCACCTATATAACCTTGTTCATT~CGTTTACTTGCAACATTAACTA

L46K.monrianus

320 T A C A T A A T A C A ~ C A W L T A A C A C C C A T G A A ~ A ~ C T ~ T G ~ ~ T C C ~ T ~ C A T T ~ C T U G T T G A - 320 240 T T T T ~ C I L I V L M G C C C G T A C G C ~ C ~ ~ ~ ~ ~ G T A G T T ~ ~ ~ ~ T G C C C T T ~ A G G T T T G T G G A

800

-

-

834

-

F.

marxianus cerevisiae

lacti8 marxianus cereviaiae

I.

marxianus cerevisiae

A

t

*

t

* * * V * *

S

* * T F

C V

E I D D E H R V R V F Y D K * * * * * * + 1 * * + I+ *

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

S.

I(.

S.

X.

t

*

t

t

*

t

* *

t

*

S.

K,

S.

I.

marxianus cerevisiae

marxianus cerevisiae

E. L46

S.

* * * * * * * * * *

V S G K I L G F V Y

* * * + r * * * * * * * * * * * * + t * * * * * * * a * * *

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

. . . . . . . . . . . . . . . . . . . . . . . . .

I V V Q L N G R L N K C G V I S P R F N V K I S D V E K W T

* * * * * L t * * * ~ * * * * * * * * * * t t * * * * * * *

S K V I I K F L Q V M Q K H G Y I G E F E Y I D D H R S G K

* * * S " * * * * * ' * * * * * ' * * " * * * * * * * *

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

S K T P G P G G Q A A L R A L A R S G L R I G R I G D V T P . . . . . . . . . . . . . . . . . . . . . . . . . . .

* * r * * * * * * * * R + " ~ * * * * ~ ~ * * * * * , . *

M L A A Q D V A A K C K E V G I T A V H I K I R A T G G T R

* * * r K * r r * + r * * * * * * t t * r * * + * * * * * * * * *

T D L S G R E T I A R V T G G M K V K A D R D E S S P Y A A

Figure 3. Comparison of the extrapolated amino acid sequence of the isolated genes encoding rp genes of K . lucris, K . marxianus and of S.cerevisiae. Differing residues are in bold type.

L L A K R L A E R K A E V R K R R A S S L K A A E * t + * * t S * , * * ' I * * * * * * * * * * * *

* * * *

marxianus cerevisiae

I* * * * * * * * *.

P Q R L Q R K R Q Q X A L K I K N A Q A Q R E A A A E Y A Q * * * * * * * * g * R * * * V R * * * * * * * * * * + * + *

* * * * *

marxianus cerevisiae

s. cerevisiae

I[.

s.

K.

D D V R D Y V I R R E V T K G E K T Y T K A P K I Q R L V T

.

marxianus

.

X.

.

S.

.

I E G V T N E T V P K R L G P K R A N N I R K F F G L T K E L t t L * D T * * * * * * * * * * * * * * * * * L t * s t *

.

marxianus cerevisiae

.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

E R K R K S V R G A X V G P D L A V L A L V I V K K G D Q E

.

marxianus cerevisiae

.

F P M K Q G V L L P T R V K L L M A K G T S C Y R P R R N G I(. * * * * * * * * * * * * I * * * L T * N " ~ * * D * S.

.

marxianus cerevisiae

s. cereviriae

cerevisiae

1. marxianus

S.

I. marrianus

S.

K.

marxianus cerevisiae

S24-1

D.

S.

marxianus cerevisiae

marxianus cerevisiae

marxianus cerevisiae

marxianus M A N V V Q A K D N S Q V F G V A R I F A S F N D T F V H V * t * * * * * t * 1 * " * * * * y * cerevisiae(Rp5g-l) " s * * * t * R

I.

S.

K.

I. S.

S.

K.

c. RP59

K.

R I G Q E V D G E A V G D E F K G Y V F K I A G G N D K Q G

t

M K L N I S Y P IN G T Q K

* * + t r r t * * y * * * * * * * * * * * *

. . . . . . . . . . . . . . . . . . . . . .

K A Y V R L T A D I D A L D I A N R I G Y I

marxianus CRERViEi4.

s.

CRXRVi8i.R

marrianus

K.

S.

marxianus cerevisiae

marxianus cerevisia.

K.

s.

I[.

B. S 1 0

S.

X.

K.

IaCtis I . marxianus cerevisiae S.

S.

K.

S.

K.

1. 1aCtis

A. L25

97 84

91 87

393 156

S. cerevisiae K . marxianus

S. cerevisiae K . marxianus

L46

94

92

414

S24- I

89

90

71 1

S . cerevisiae K . marxianus

93 95 98

RP59

91 90 96

at aa level

S. cerevisiae K . marxianus

(S.C.vs K.l.) (S.C.vs K m . ) (K.l. vs K.m.)

at bp level

s10

429

Homologies (in %):

S. cerevisiae K. lactis K . marxianus

Yeast

Length of ORF (in nucl.)

L25

Ribosomal protein gene

383 513

-

307 649

GTATGT; after 2nd GTATGT; after 2nd

GTATGT; in 3rd GTACGT; in 4th

GTATGT; after 2nd GTATGT; after 2nd

GTATGT; in 5th GTATGT; in 5th GTATGT; in 5th

41 5 616 519 352 395

5’-Splice site; codon position

Length of intron (in nucl.)

- 53 - 36

-47

- 26

-31

- 20

- 38

- 28

- 39

TACTAACa position

TAG TAG

-

-

TAG CAG

CAG CAG

TAG CAG CAG

3’-Splice site

TAA TAA

TAA TAA

TGA TGA

TAA TAA

TAA TAA TAA

Stop codon

Table 1. General structure, homologies and intron sequences within the isolated Kluyveromyces rp genes and their counterparts from Saccharomyces

P p

REGULATORY SEQUENCES OF KLUYVEROMYCES RIBOSOMAL PROTEIN GENES

913

Figure 4. Northern analysis of RNA from S. cerevisiae(S), K . lactis (L) and K . marxianus (M) hybridized with gene-specificprobes from the isolated ribosomal protein genes of K . marxianus. The positions of 17s and 26s rRNA are indicated.

The Z box consists of 15 nucleotides with the consensus 5’-GRARATTTNNRRYYY-3’ and is also usually found in the coding strand. In the intergenic region of the genes encoding S24-1 and L46, the Z box is present in two orientations. The position of this element varies from - 353 to - 684. The Z box shows a strong similarity to the binding site of the product of the regulatory gene LAC9 (5’-CGGAAATTTGTGGTCCG-3’; Ruzzi et al., 1987; Leonard0 et al., 1987). The LAC9 protein activates the transcription of the LAC4 gene, involved in lactose and galactose metabolism, and is homologous to the GAL4 protein of S. cerevisiae. No Z box was found in the upstream region of the gene encoding RP59, possibly due to the fact that only about 300 bp of this region are present in the sequenced fragment. We identified RPG, boxes at positions varying from - 603 (S24) to - 232 (L46) of the rp genes of Kluyveromyces, of which the transcription in S. cerevisiae is activated by RAPl (Table 2). The majority of these sequences were found at a distance of about 550 nucleotides to the ATG codon, which is about 200-300 nucleotides further away than in Saccharomyces (Leer et al., 1985b). Scrutiny of the RPG, boxes of the isolated genes of Kluyveromyces and the RAP 1-binding sequences of the same genes of Saccharomyces shows a clear homology. ‘Vignais et al. (1990) showed that in the consensus sequence of the RPG box in Saccharomyces, - R A/C AY CC RY NC AY Y -, the nucleotides 2,3,4,5,6,7, 10 and 12 were important for the in vitro binding with the RAPl protein. The importance of respective nucleotides of the RPG box was supported by their frequency of occurrence

(Nieuwint et al., 1989). The frequency of occurrence of specific nucleotides at positions 2, 3, 4, 5, 6 and 7 of the RPG, box of Kluyveromyces suggests a similar importance for the binding with a protein (Figure 6) that might be the RAPl analogue of Kluyveromyces. In contrast, no strong conservation of nucleotides 10 and 12 could be found in these sequences, indicating that the binding domain of the binding protein of Kluyveromyces differs slightly from that of RAPl of Saccharomyces. The orientation of the RPG, boxes in each of the Kluyveromyces rp genes is identical to that of the RAP 1-binding sequences in the corresponding Saccharomyces gene, except in the case of RP59 (Figure 5). The number of RPG, boxes in the upstream region of each of the Kluyveromyces rp genes is the same as in the upstream region of its counterpart in Saccharomyces, except in the intergenic region of the genes encoding L46 and S24. Two additional RPG,! boxes could be found in this region of K . marxianus. The upstream region of the gene encoding ribosomal protein S10 in K . marxianus also harbours a CPl-binding site (5’-GTCACGTG-3’) at position -601. CP1 is an abundant protein which binds to the highly conserved CDEl element of S. cerevisiae centromeres. CPI -binding sites have also been detected outside centromeres, e.g. in the upstream regions of the GAL2 gene, close to the GAL4-binding site (Bram and Kornberg, 1985) and of the S. cerevisiae L45 rp gene (Kraakman et al., 1991). However, the CPl-binding site does not appear to be involved in transcription activation of these genes (Kraakman et al., 1991).

914

G. K. BERGKAMP-STEFFENS, R. HOEKSTRA A N D R. J. PLANTA

7

I

I

-800

I

I

-600

I

I

I

-200

-400

i 0

s10

b

K . marxianus

D D W

S. cerevisiae

DD-

( s10-1)

D D-

(S10-2)

L25

a, ww a

K.lactis K . marxianus

,a a-

S. cerevisiae

RP59 AAA D a

K . marxianus

k

S . cerevisiae

S24-1

a-

D

K . manrianus

M D D4-D

S . cerevisiae

L46

-Da Q M -D

K . marxianus

W - b x

D

a

a a-

S. cerevisiae

P

-

B

x-box

,

z-box

-

T-stretch

non-sequencedregion

Figure 5. Position and orientation of the conserved sequence elements upstream of the isolated rp genes of K. lactis and K. marxianus and of S. cerevisiae relative to the initiation codon. The upstream region of the gene encoding RP.59 of K. marxianus is limited to 313 nucleotides. The arrows indicate the orientation of the sequence shown in Table 2. Thin lines indicate T-stretches.

Functional analysis To determine whether the conserved sequence elements found in the upstream regions of the Kluyveromyces rp genes are indeed important for transcriptional activation, we determined the transcriptional activity of K. marxianus S24-1 and L46 genes flanked by various parts of the S24-1-L46 intergenic region. The constructs used in these experiments are shown in Figures 7A and 8A, respectively. Each of the constructs was cloned into the multicopy vector LEpTRP. This vector was derived from the plasmid YIplac 204 (Gietz and

Sugino, 1988) by insertion of the KARS2 sequence from K . lactis (Das and Hollenberg, 1982), which ensures autonomous replication in K. lactis cells. The various LEpTRP derivatives were transformed into K. lactis MSKl 10 and the level of S24-1 or L46 transcript was determined in duplicate with labeled DNA fragments (BglII-BglII for S24-1 and HindIII-EcoRI for L46). The hybridization data are shown in Figures 7B and 8B and in graphical form in panel C of each of these figures. The latter data were obtained by laser-scanning densitometry of the Northern blots. The signal intensity was corrected for the copy number of the plasmid, as determined

RtAYCCRYNCAYY (- 250/ - 500)

ACATCCATTGCTA (- 602/ - 233) ACATCCACACAAC ( -41 5 / -420) AAACCCACACACC (- 330/ - 505) ACACCCATGAACC (-285/-550)

AAATCCAAACACC (- 25 1)

TAATCCAAGTATC (-601) CCATCCGTACACC ( - 564)

GCATCCAGAGATT (- 584) GCATCCACAAAAC (- 530) ACATCCACAACAG (- 553) TCACCCAGTGGAA ( - 5 15)

RPG, box

Not found

GAAGATTTGAAATTT (-353/-482) GGAAATTTCAAATCT (-35.5-480)

Not found

Not found

GGGACTGT (-266/- - 569)

GGGACTGT ( - 272)

GGGACTGT (- 586)

GGGACTGT (- 557)

GGAAATTTCAGGTTC ( -464)

GGAAATTTTTAGCCT (- 684)

GGGACTGT (- 534)

X box

GAAGATTTAAAATTT (- 561)

Z box

The position of the first nucleotidein each box is given. Differing nucleotidesof the Kluyveromyces RPG boxes with respect to the RPG consensus sequence in Saccharomyces (Vignaiset al., 1990) are in bold type.

rp genes Saccharomyces

S24- 1/L46 K . marxianus

RP59 K . marxianus

K . marxianus

s10

K. marxianus

L25 K . lactis

rp Gene

Table 2. Homologous sequences upstream from the isolated rp genes of Kluyveromyces

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G. K. BERGKAMP-STEFFENS, R. HOEKSTRA AND R. J. PLANTA

RPG-box (nucl.)l Kluyveromyces

2

3

‘8 G2 A3

4

5

6

7

9 10 11 12 13

‘4 A9 ‘ 5 ‘ 1 T 3 G2 A3 c1 A3 A2 A 2 T2 G3 G1 T3 G1 G2 c1 T1 T1

T8 ‘11 ‘11 A1O ‘4 c3

G1

T2 C1 Saccharmnyces

8

A5 c 6 Al

TS c9

‘8

A5

T 8 A9 c9 Al T9 ‘ 5

G3 A2 c1 T1 G1 G1

c4

T1

G4

c1

c2

T3 A1

Figure 6. Frequence of occurrence of the nucleotides of the putative RPG boxes of the isolated rp genes of Kluyveromyces and of their counterparts of Succhuromyces.

by Southern analysis, as well as the background level from the genomic copy and the different RNA amounts loaded. As shown in Figure 7, introduction of S24-1 genes lacking all of the conserved upstream elements ( - 137/S24) does not increase the amount of S24- 1 transcripts over the background level (panels B and C, lanes 3 and 4). Thus, - 137/S24 genes are transcriptionally inactive. This situation is not improved by extending the 5’-flanking region to include the X box (- 289/S24; lanes 5 and 6). A further extension, however, to include the group of three RPG, and two Z; boxes (- 453/S24, lanes 7 and 8; - 413/S24, lanes9 and 10) leads to clearly detectable transcription activation, amounting to about 30% of the transcriptional activity observed for the control plasmid (control) that encompasses the complete RP59-S24-1-L46 cluster (lanes 1 1 and 12). Figure 8 shows the results of a complementary analysis of the role of the S24-1-L46 intergenic region in transcription of the L46 gene. Deletion of the group of three RPG, and two Z boxes (- 362/ L46) in this case also reduces transcription to a value only slightly above background level, even though the remaining 5’-flanking region still contains one RPG, box and two slightly divergent X boxes (panels B and C, lanes 3 and 4). Including the group of RPG, and Z boxes, but not the upstream X box, in the construct - 546/L46 activates transcription to about 50% of the level shown by the L46 gene in the control construct (control, compare lanes 5 and 6 to 9 and 10). Surprisingly, transcription is reduced to virtually background level again when the 5‘flanking region is extended beyond the X box upstream of the RPG,/Z cluster (- 698/L46). From these data it is clear that sequences important for transcription activation of both the S24- 1 and L46 genes of K. marxianus are to be found within the cluster of three RPG, and two Z boxes present in the region separating the two genes. A

more detailed analysis will be required to establish whether individual elements act in a gene-specific manner or are shared by the two genes, as in the case of S. cerevisiae. Furthermore, additional activating sequences appear to be present further upstream from both the S24-1 and L46 gene. Either gene is transcribed at a considerably higher level when part of the large EcoRI-EcoRI fragment encompassing the complete RP59-S24-1-L46 gene set, as compared to the L46 construct extending to just beyond the RPG,/Z cluster. The picture is complicated further by the striking difference in transcription between the - 698/L46 and -546/L46 genes (Figure 8). Apparently, the region upstream of the RPG,/Z cluster, up to the SphI-site, is able to suppress the activating effect of the cluster on L46 transcription completely. However, this inhibitory effect is in turn negated by the more distally located activating sequences. Finally, the region between the SphI site and the RPG,/Z cluster has a much less severe, if any, negative effect on transcription activation of the S24-1 gene by the cluster. The latter may have a positional or orientational cause. While the element upstream from the RPG,/Z cluster that represses transcription cannot yet be identified unambiguously, it is tempting to hold the conserved X box responsible for this effect. The lack of transcriptional activity of the - 362/L46 gene may indicate that the single RPGK box present in this construct is either non-functional or insufficient to cause detectable levels of transcription. However, it should be noted that this RPG, box is preceded by two slightly divergent X boxes orientated away from the L46 gene. Because we do not yet know precisely what constitutes a functional X box, in terms of sequence and/or position, we cannot exclude the possibility that these two divergent X boxes suppress the activating effect of the single RPG, box. Band-shgt assay It has been well established that the S. cerevisiae RPG box constitutes the binding site for multifunctional protein RAPl. In order to ascertain whether in Kluyveromyces, cells contain a protein binding to the RPG, box, related to S. cerevisiae RAP1, we performed band-shift assays with two different probes. One contained the consensus RPG box from S. cerevisiae, the other one a sequenceidentical to one of the RPG, boxes from the S24-1-L46 intergenic region of K. marxianus (see Materials and Methods). Using extracts from S. cerevisiae cells,

A.

Lanes

-1371S24

394

I

-2891S24

5.6

-4531S24

7,8

-4731S24

9,10

Control

-

E

RP59

L46

S24-1

B.

E

-

11,12

C.

- 26s

- 17s

4. 3

2

- S24

1

0 1 2 3 4 5 6 7 8 9101112

1 2 3 4 5 6 7 8 9 1011'12

Figure 7. (4) Constructs made for analysis of the putative regulatory sequences, in S24 context. The total intergenic region is also represented. The S24 constructs all end at the EcoRI site upstream of RP59. Negative control: LEpTRP. - 137/S24: starting at the SphI site. -289/S24: starting at the NlaIII site upstream of the SphI site. -453/S24: deletion with Exonuclease I11 up to nucleotide -453 with respect to the S24-1 ATG. -473/S24: starting at the ScrFI site (essentially the same as -453/S24 plus 20 additional nucleotides). Positive control: ('control') 4.2 kb EcoRI-EcoRI fragment bearing the RP59, S24-1 and L46 genes. 1= RPG box, -+ = X box, 9 = Z box. (B) Northern analysis of K. lactis transformants. Lanes 1 and 2: LEpTRP; lanes 3 and 4: - 137/S24;lanes 5 and 6: - 289/S24;lanes 7 and 8: -453/S24; lanes 9 and 1 0 -473/S24; lanes 1 1 and 12: positive control. (C) Graphic representation of mRNA levels of the transformants, corrected for the amount of RNA loaded, the copy number and mRNA background of the genomic copy.

918

G . K. BERGKAMP-STEFFENS, R. HOEKSTRA AND R. J. PLANTA

A.

Lanes

-36uL46

394

-546/L46

5,6

L 7,8

Control

-

E

RP59

S24-1

L46

B

9,lO

+1

B.

C.

- 26s

lo

1

8-

- 17s

64-

REGULATORY SEQUENCES OF KLUYVEROMYCES RIBOSOMAL PROTEIN GENES

A

919

B

c1+ c2-

1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 Figure 9. Band-shift assay using oligonucleotides encompassing the RAP1-bindingsite of S. cerevisiae and K. marxianusas probes. (A) The oligonucleotide encompassingthe RAPl site of K. marxianuswas incubated with S. cerevisiae cell protein (lanes 1 4 : 0,0.5,2.5and 10 pg) and K. marxianuscell protein (lanes 5-9: 2.5,5, 10,20 and 30 pg). (B) The oligonucleotide encompassing the RAPl site of S. cerevisiae was incubated with S. cerevisiae cell protein (lanes 10-13: 0,0.5,2.5 and 10 pg) and K . marxianuscellprotein (lanes 1416: 2.5,5,10pg). F indicatesthe position of the free probes, C1 and C2 indicate the position of the complexes after incubating the probes with protein from S. cerevisiae and K. marxianus respectively

we observed complex formation with either probe (Figure 9, lanes 2 4 and 11-13). Moreover, the amount of complex formed at a given concentration of the cell extract is virtually the same for both types of probe. Consequently, we conclude that the binding affinities of the s. cerevisiae RAPl protein for the homologous RPG and the heterologous RPG, box are closely similar. Competition experiments demonstrated that both complexes could be competed out by adding non-labeled double-stranded oligonucleotides representing the other box, indicating that the binding protein is the same for either box (results not shown). Using extracts from K. marxianus cells, we observed complex formation only with the RPG, (Figure 9, lanes 5-9) and not the RPG probe (lanes 1416). Thus, K . marxianus cells contain a protein that recognizes the RPG, box but has no, or at best a very low, affinity for the Saccharomyces RPG box. Furthermore, the complex of the RPG, probe with the K. marxianus protein migrates faster than that with S. cerevisiae RAPl factor, indicating that the Kluyveromyces protein is smaller than S. cerevisiae RAPl .

Comparing the sequences of the S. cerevisiae RPG box and the consensus of the Kluyveromyces RPG, box (see Figure 6), it seems likely that the difference in binding affinity can be ascribed to the presence of the extra 5’-flanking bp in the RPG, oligonucleotide. Taken together, the data indicate that the Kluyveromyces strains contain a protein that is functionally homologous but structurally different from the RAPl protein in Saccharomyces. The resemblance between presence, orientation and number of RPG boxes in the upstream regions of rp genes of Kluyveromyces and Saccharomyces and their capacity to bind a protein from both strains makes it likely that the regulation of the transcription of the ribosomal proteins of Kluyveromyces is controlled in a similar fashion as in Saccharomyces. This conclusion is also supported by the finding that a Kluyveromyces rp gene can be expressed in Saccharomyces and vice versa (Larson and Rossi, 1991; Rentenaar and Hoekstra, unpublished results). Expression on a single-copy CEN plasmid of the essential L25 gene from K . marxianus and the L25 gene from S. cerevisiae in a conditional

920

G. K. BERGKAMP-STEFFENS, R. HOEKSTRA A N D R. I. PLANTA

expressible L25 mutant from S. cerevisiae (Rutgers e t al., 1991) showed that there is no difference in growth rate between the different transformants (Rentenaar and Hoekstra, unpublished results). On the other hand, the greater distance of the RPG, boxes to the ATG codon of Kluyveromyces, the loss of strict conservation of nucleotides 10 and 12 of the putative RAPl-binding boxes regarding the consensus sequence of the RAPl-binding sequence of Saccharomyces and the presence of two additional conserved sequences, also show that the regulation of the transcription of the rp genes in Kluyveromyces displays some differences as compared with the situation in Saccharomyces. ACKNOWLEDGEMENTS The authors are indebted to Trudy Wassenaar and Arne Poortinga for isolating and sequencing the L25 gene of K . Iactis and Jan Boesten for synthesis of the oligonucleotides. They also thank Dr W. H. Mager for stimulating discussions and gratefully acknowledge Dr H. A. Rauk for critically reading the manuscript. This work was supported in part by grants from the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Research (NWO). It was also financially supported by Gist Brocades, Delft, and Unilever Research Laboratories, Vlaardingen, The Netherlands. REFERENCES Bergkamp, R. J. M., Geerse, R. H., Verbakel, J. M. A., Musters, W. and Planta, R. J. (1991). Cloning and disruption of the LEU2 gene of Kluyveromyces marxianus CBS6556. Yeast 7,963-970. Berman, J., Tachinaba, C. Y. and Tye, B. K. (1986). Identification of telomere-binding activity from yeast. Proc. Natl. Acad. Sci. USA 83,3713-3717. Bicknell, J. N. and Douglas, H. C. (1970). Nucleic acid homologies among species of Saccharomyces. J. Bact. 101,505-512. Birnboim, H. C. and Doly, J. C. (1979). A rapid alkaline extraction procedure for recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1525. Bollen, G. H. P. M., Cohen, L. H., Mager, W. H., Klaassen, A. W. and Planta, R. J. (1981). Isolation of cloned rp-genes from the yeast Saccharomyces carlsbergenesis. Gene 14,279-287. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dyeing. Anal. Biochem. 72,248-254.

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92 1

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