The sequence of a 15 769 bp segment of Pichia anomala identifies the SEC61 and FBP1 genes and five new open reading frames

Yeast Sequencing Report

The sequence of a 15 769 bp segment of Pichiaanomala identifies the SEC61 and FBP1 genes and fivenew open reading frames

Teresa Ruız1, Manuel Sanchez2, Jose M. De la Rosa1, Luis Rodrıguez1* and Angel Domınguez2

1 Departamento de Microbiologıa y Biologıa Celular, Facultad de Farmacia, Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain2 Departamento de Microbiologıa y Genetica, Instituto de Microbiologıa Bioquımica, CSIC-Universidad de Salamanca, 37071 Salamanca, Spain

*Correspondence to:L. Rodrıguez, Departamento deMicrobiologıa y Biologıa Celular,Facultad de Farmacia,Universidad de La Laguna,38071 La Laguna, Tenerife,Spain.E-mail: [email protected]

Received: 16 March 2001

Accepted: 3 May 2001

Abstract

We have determined the sequence of a 15 769 bp DNA segment of Pichia anomala. The

sequence contains seven complete open reading frames (ORFs) longer than 100 amino

acids and a putative tRNA gene. Two of the ORFs code for the well-characterized genes

SEC61 (which codes for the core subunit of the ER translocation complex) and FBP1(encoding fructose-1,6-bisphosphatase). A gene coding for a protein similar to S. cerevisiaeYDL054c was found between the two genes. These three genes show a different

organization (intermingled triples) in three yeast species: Saccharomyces cerevisiae,Candida albicans and P. anomala. Two out of the four remaining ORFs show weak

homology with different proteins from other species and the other two show non-significant

similarity with previously sequenced genes. The nucleotide sequence has been submitted to

the EMBL database under Accession No. AJ306295. Copyright # 2001 John Wiley &

Sons, Ltd.

Keywords: Pichia anomala; Saccharomyces cerevisiae; Candida albicans; gene order;

genome organization; intermingled triples

Introduction

Comparative genomics is a new field of research thatprovides insight into the understanding of molecularevolution. To date, only one yeast, Saccharomycescerevisiae, has been completely sequenced (Goffeauet al., 1996). Two others are close to completion,Schizosaccharomyces pombe (http://www.sanger.ac.uk/Projects/S_pombe/) and Candida albicans (http://www-sequence.stanford.edu/group/candida/). The sequenceof 45 000 000 nucleotides belonging to 13 selectedyeast species of the Class Hemiascomycetes hasbeen described (Souciet et al., 2000; Artiguenaveet al., 2000; Tekaia et al., 2000). An extensiveanalysis of the global degree of synteny betweenS. cerevisiae and each of the other 13 yeast speciesto determine the conservation, or non-conservation,of neighbouring gene couples has been published(Llorente et al., 2000). Gene order evolution hasalso been studied by comparing S. cerevisiae wholegenome sequence data to the available C. albicans

data obtained from whole genome shotgun andcosmid sequencing (Seoighe et al., 2000).

On the other hand, several authors have alsoobtained (limited) comparative genomic inform-ation about related yeast species: S. cerevisiae andKluyveromyces lactis (Wesolowski-Louvel andFukuhara, 1995; Ozier-Kalogeropoulus et al., 1998);S. cerevisiae and Ashbya gossypii (Altman-Johland Philippsen, 1996; Prillinger et al., 1997) andS. cerevisiae and C. albicans (Hartung et al., 1998).Comparative analysis of genomic DNA in theabove reports was carried out on the basis ofthe sequences of DNA fragments up to 1 kb. Thesequences were obtained by single-pass sequencingat both ends of random genomic DNA libraries,prepared by generating fragments in the size range4–5 kb (Souciet et al., 2000). To further extend thedata on gene order, sequence comparison, genomecompactness and analysis of the terminator–promoter environment among yeasts, we havechosen a different approach: the sequencing of

YeastYeast 2001; 18: 1187–1195.DOI: 10.1002 / yea.766

Copyright # 2001 John Wiley & Sons, Ltd.

larger fragments of DNA (8–16 kb) containing pairsof adjacent genes with respect to the map loca-tion of their S. cerevisiae homologues in fouryeast species. One is K. lactis, very close, fromthe evolutionary point of view, to S. cerevisiae(Wesolowski-Louvel and Fukuhara, 1995; Bolotin-Fukuhara et al., 2000); another, quite distant inevolutionary terms, is Yarrowia lipolytica (Barnset al., 1991; Casaregola et al., 2000). Finally, thereare two putative pathogens: Paracoccidiodes brasi-liensis (Restrepo, 1993) and Pichia anomala (alsodesignated as Hansenula anomala and, in its asexualstate, Candida pelliculosa; Kurtzman, 1984). P.anomala plays an important role in the occurrenceof the off-flavour associated with the spoilage ofconfectionery (Lanciotti et al., 1998) and has beenreported to be one of the organisms that can beused efficiently for the treatment of wastewatercontaining high concentrations of organic com-pounds (Moriya et al., 1990). As killer yeast,P. anomala has potential as an antimycotic bio-control agent, since it shows inhibitory and killingactivities against a range of fungal pathogens(Mathews et al., 1998). Currently, however, itsmain interest derives from its role as an emergentfungal pathogen, since cases of fungaemia byP. anomala have been reported in neonates, inpatients suffering of leukemia, and in immuno-compromised AIDS patients (Salesa et al., 1991;Thuler et al., 1997; Wong et al., 2000).

This study reports the sequence of a 15.5 kbP. anomala region containing two genes previouslycharacterized by us: PaSEC61 and PaFBP1 (T. Ruızet al., manuscript in preparation). Both genes andthe homologue of the S. cerevisiae YDL054c offer apeculiar case of intermingled triples and geneinversion and support the notion that, for theunderstanding of genome organization, our experi-mental approach of sequencing large fragments ofDNA of different yeast species is correct.

Materials and methods

Vectors and strains

Lambda phage TR-D was selected from a Pichiaanomala genomic library constructed in thelEMBL3 vector (Garcıa-Lugo et al., 2000). Itcontained about 15.5 kb of DNA from the P.anomala strain CECT1112 (CBS1982, NCYC435)obtained by partial digestion of chromosomal DNA

with Sau3A and cloned into the BamHI site ofthe vector. The Escherichia coli strain used as hostfor infection and phage isolation was Y1090(Stratagene). E. coli strain DF4116 (Fraenkel,1967), obtained from the E. coli Genetic StockCenter (Yale University, USA), was employed fortesting the expression of PaFBP1 gene. Subcloningof this gene was carried out in the pRS316 shuttlevector (Sikorski and Hieter, 1989).

Manipulation of nucleic acids

Routine DNA manipulations, cosmid preparation,subcloning, Southern blotting, restriction enzymedigestions, agarose gel electrophoresis, ligationof DNA fragments and E. coli transformationwere performed according to standard techniques(Sambrook et al., 1989).

Sequencing strategy

The sequence was determined using universal andreverse primers with the ABI377 automatic sequen-cer (Applied Biosystems Inc.), employing the TaqDye Deoxy Terminator Cycle Sequencing Kit assupplied by the manufacturer. Junctions weresequenced using walking primers using the entirephage. The kit uses dITP as a standard substitutefor dGTP, which effectively eliminates compres-sions formed during polyacrylamide gel electropho-resis. In total, 150 sequences (70 direct and 80reverse reads) were performed. Altogether, raw datafrom 63 044 bases were aligned to assemble the finalcontig, the average reading number per base pairbeing 4 and each base being sequenced on bothstrands and at least twice (upper and lower strandtogether). The quality of the final sequence wasensured by visual inspection of the sequencingprofiles at each position on each DNA strand. Thesequence was considered final only when anunambiguous reading of each nucleotide on eachstrand was achieved.

Computer-assisted sequence analysis

Assembly of the sequences was performed with theSeqMan program of the DNASTAR programpackage (DNASTAR Ltd.). ORFs were predictedusing the DNA Strider software (Marck, 1988). Foreach ORF, the first ATG was assumed to be theinitiation codon. ORFs were named with the prefixPa (Pichia anomala), and the MIPS workingnomenclature for S. cerevisiae. Detection of tRNA

1188 T. Ruız et al.

Copyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: 1187–1195.

genes was carried out using the tRNAscan-SE program (Lowe and Eddy, 1997). The databasescan for similar sequences was accomplished usingthe BLAST (Altschul et al., 1997) and FASTA(Pearson and Lipman, 1988) programs (para-meters: BLOSUM62 matrix for BLAST; andKtup=2 for FASTA). Multiple-sequence align-ments were obtained using the CLUSTALW pro-gram (Thompson et al., 1994) or PILEUP (GCGpackage). Protein patterns (motifs) were identifiedby the ProfileScan and ScanProsite programs ofthe ExPASy WWW server (Appel et al., 1994) inthe PROSITE database of protein sites and pat-terns (Bairoch et al., 1997). Transmembrane do-mains were identified using the TMpred software(http://www.ch.embnet.org/software/TMPRED_form.html). Data for C. albicans were obtained from ourprevious results (de la Rosa et al., 2000) and from theC. albicans sequencing project database (http://www-sequence.stanford.edu/group/candida/).

Results and discussion

Sequence analysis

Lambda phage TR-D was isolated in a screen-ing for the Pichia anomala SEC61 gene (T. Ruızet al., manuscript in preparation). The insert of

P. anomala genomic DNA included in this phagewas completely sequenced and the sequence ana-lysed as described in Materials and methods. The15769 bp sequence has a GC content of 36.18%, ingood agreement with previously reported data(Ouchi et al., 1970; Nakase and Komagata 1971)and contains seven ORFs (Figure 1) and a putativetRNA gene, giving a density of one gene per 2 kb.The ORFs occupy 51% of the complete sequence, avalue clearly lower than the 72% described forS. cerevisiae (Dujon, 1996) but similar to thatobtained for Sz. pombe (54–59%) (Sanchez et al.,1999; Xiang et al., 2000). The coding region alonehas an average GC content of 35.93% (36.85%highest and 34.14% lowest values).

Sequences with known or putative functionsassigned

PaFBP1 codes for fructose-1,6-bisphosphatase(Fbp1p; see Table 1). A 2.2 kb NruI–NruI fragmentfrom the 5k-end of the analysed sequence, includingthe complete PaFBP1 ORF, was subcloned into thepRS316 plasmid and introduced by transforma-tion into E. coli DF4116. Mutations lacY1 and fbp-5 carried by this strain render it unable to uselactose and gluconeogenic carbon sources forgrowth, due to the lack of active lactose permease

Figure 1. DNA strider plot showing the open reading frames (ORFs) in the six possible frames. ATG codons arerepresented by half-height vertical bars and stop codons by full-height bars. In the lower part, arrows indicate positions anddirections of the ORFs on the two strands. WSc and CSc are Watson and Crick strands in S. cerevisiae

The SEC61–FBP1 region of ascomycetous yeasts 1189


and fructose-1,6-bisphosphatase, respectively. Selec-ted E. coli transformants were able to growefficiently in MacConkey medium, indicating thatthe cloned fragment was able to complement thefructose-1,6-bisphosphatase deficiency, allowing the

cells to use peptones as gluconeogenic carbon source(results not shown). Amino acid sequence compar-ison of FBP1 gene products revealed an identity of70% between P. anomala and C. albicans and 67%between P. anomala and S. cerevisiae (Figure 2).

Table 1. Characteristics and homologies of ORFs and deduced amino acid sequences of the 15.5 kb fragment

ORF name Coordinates

Strand

orientation*

Length

(amino acids)

Molecular

mass (Da) Closest homologues

FASTA scores

Initn Init1 Optn

PaFBP1 1378–2400 C 340 36 763 S.cerevisiae FBP1 1049 573 1495Pa054C 2716–4185 C 489 54 195 S.cerevisiae YDL054c 631 296 696

PaSEC61 4406–5976 intron

(4416–4546)

C 479 52 890 C. albicans SEC61 2457 2457 2459

Pa380c 6473–8440 C 655 75 701 D. melanogaster CG5613 375 184 566

PatK(UUU)L 8745–8856 intron

(8782–8820)

C — — S. cerevisiae tK(UUU)L

Chromosome XII

— — —

Pa381c 9072–9674 C 200 22 566 No similarity found — — —Pa382c 10 775–12 361 C 528 59 313 No similarity found — — —

Pa383w 14 456–12 597 W 619 69 194 S. coelicolor Q9KY46 524 323 672

*W, Watson strand; C, Crick strand.

Figure 2. Multiple alignment of fructose-1,6-bisphosphatase (FbPase) amino acid sequences deduced from P. anomala,C. albicans and S. cerevisiae FBP1 genes. The consensus sequence of the catalytic motif found in FbPases is boxed. Gaps havebeen introduced to give the best alignment. Identical residues (asterisks) and conservative amino acid substitutions (dots) areindicated



PaYDL054c encodes a putative membrane pro-tein of 489 amino acids with 40% and 34% identityto the C. albicans and S. cerevisiae YDL054c geneproducts, respectively (Figure 3). A similar numberand topology of putative transmembrane domainswere identified in these products using the TMpredsoftware.PaSEC61 codes for Sec61p, a protein that in

other organisms has been shown to play a crucial

role in the insertion of secretory and membranepolypeptides into the ER and that is essentialfor cell growth (Rapoport et al., 1999; Stirling,1999). This ORF includes an intron of 131 bp afterthe first 10 nucleotides containing all the canon-ical sequences for processing (T. Ruız et al., inpreparation).

A putative tRNA gene, 111 nucleotides long,including an intron of 39 bp, is placed between

Figure 3. Multiple alignment of predicted amino acid sequences of putative proteins PaYDL054c, YDL054c (S. cerevisiae) andCaYDL054c (C. albicans). Gaps have been introduced to give the best alignment. Identical residues (asterisks) andconservative amino acid substitutions (dots) are indicated



ORFs Pa380c and Pa381c (see Table 1). Onceprocessed, the mature tRNA from this gene isidentical (except in two nucleotides) to the maturetRNA-Lys obtained by transcription and intron-processing of the S. cerevisiae tK(UUU)L gene,located between SEC61 and FBP1 genes inchromosome XII. As in other yeast tRNA genes,

there is no sequence conservation between theintron of the putative PatRNA-Lys gene and theone of its homologue in S. cerevisiae and, also asin other cases, the intron interrupts the anti-codon loop immediately 3k to the anticodon (Abelsonet al., 1998). No homologue for this tRNA gene isfound in the C. albicans sequence database.

Figure 4. Map of the P. anomala loci of the FBP1–PaYDL054c–SEC61 genomic region and its comparison with S. cerevisiae (A)and C. albicans (B and C). Gene orientation is indicated by the arrow



ORFs with weak similarities or no assignedfunctions

The Pa-380c ORF may encode a protein exhibitingweak similarity (27.7% identity over 486 aminoacids) with the CG5613 protein of Drosophilamelanogaster (Swall Q9VX51; Adams et al., 2000)and 31.4% identity over 382 amino acids with Arab-idopsis thaliana endo-b-N-acetylglucosaminidase(Kaneko et al., 1998).

Pa381c and Pa382c ORFs code for hypotheticalproteins that show no significant homology withknown proteins or ESTs.

Pa383w ORF could encode a protein exhibit-ing weak similarity with putative membrane trans-porters from Streptomyces coelicolor (Redenbachet al., 1996) and Sz. pombe (Dimitrov and Sazer,1998). In spite of its relatively low amino acidsequence identity (23%), the protein encoded byPa383w resembles fnx1, a fission yeast multidrugresistance protein, in the number (14) and thetopology of their transmembrane domains predictedby using the TMpred software (Dimitrov and Sazer,1998).

Genomic SEC61-FBP1 regions of P. anomala, S.cerevisiae and C. albicans

The gene organization of the FBP1–SEC61 regionin P. anomala, S. cerevisiae and C. albicans is shownin Figure 4. From the analysis of gene orienta-tion between P. anomala and S. cerevisiae, it ispossible to predict the existence of an intermingledtriple, in which the two-homologous-gene pair,FBP1–SEC61, is interspersed by one interveninggene whose homologue lies on S. cerevisiae chromo-some IV (Figures 3, 4A). However, comparison ofP. anomala and C. albicans suggests the exis-tence of a gene inversion and translocation forthe SEC61 gene in C. albicans (Figure 4B) or agene inversion without disruption of synteny(Figure 4C). Whether the putative ORF predictedfrom the C. albicans sequence (SPTREMBL,Q9P8Q5), located between SEC61 and FBP1genes, encodes for a protein of 108 amino acidsremains to be elucidated, since no homologue forthis putative polypeptide has been described in anyother organism.

The conservation of small clusters of geneswithout absolute conservation of order or orienta-tion lends support to the proposal that small DNAinversions have contributed significantly to the

evolution of ascomycete genomes (Seoighe et al.,2000). Upon comparison of genome sequence databetween S. cerevisiae and C. albicans, it has beenfound that small clusters of C. albicans genes whoseS. cerevisiae homologues are also clustered aregenerally shorter than 10 genes and often areinterspersed with genes from other S. cerevisiaechromosomes (Seoighe et al., 2000). This is thesituation that we have found for the gene organiza-tion of the FBP1–SEC61 chromosomal region inS. cerevisiae and C. albicans. However, since themechanism by which small inversions occur is stillunknown, to date we have no rational explanationfor the opposite orientation of the three genesobserved upon comparing P. anomala and C.albicans with S. cerevisiae. Reciprocal transloca-tions between segments tend to conserve geneorientation and inversions of fragment orientationdo not change gene order. Also at this moment weare not able to predict the role of the tRNA-Lysgene, if any, in the organization of this genomicregion.

Taken altogether, our results validate the need tosequence larger fragments of DNA or, probablybetter, at least an entire chromosome of severalyeast species in order to understand the mechanismsof molecular evolution in eukaryotes.

Acknowledgements

This work was partially supported by Grant PI1999/054

from Direccion General de Universidades e Investigacion

(Gobierno de Canarias). J.M. de la Rosa was the recipient of

a FPU predoctoral fellowship from the Ministerio de

Educacion y Cultura, Spain. We wish to thank N. Skinner

for revising the English version of this manuscript.

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Documents

The sequence of a 15 769 bp segment of Pichia anomala identifies the SEC61 and FBP1 genes and five new open reading frames