gene order in a 10 275 bp fragment of yarrowia lipolytica, including adjacent ylura5 and ylsec65...

Yeast Sequencing Report

Gene order in a 10 275 bp fragment of Yarrowialipolytica, including adjacent YlURA5 and YlSEC65genes conserved in four yeast species

Manuel Sanchez and Angel Domınguez*Departamento de Microbiologıa y Genetica, Instituto de Microbiologıa Bioquımica/CSIC, Universidad de Salamanca, 37071 Salamanca, Spain

*Correspondence to:A. Dominguez, Departamento deMicrobiologıa y Genetica, Institutode Microbiologıa Bioquımica/CSIC,Universidad de Salamanca,37071 Salamanca, Spain.E-mail: [email protected]

Received: 23 December 2000

Accepted: 10 February 2001

Abstract

We have determined the sequence of a 10275 bp DNA segment of Yarrowia lipolyticalocated on chromosome VI. The sequence contains six complete open reading frames

(ORFs) longer than 100 amino acids and two more partial ORFs at both ends. Two of the

ORFs encode for the well-characterized genes YlURA5 (orotate phosphoribosyl-

transferase) and YlSEC65 (encoding a subunit of the signal recognition particle). These

two genes show an identical organization—located on opposite strands and in opposite

orientations—in four yeast species: Saccharomyces cerevisiae, Kluyveromyces lactis,

Candida albicans and Y. lipolytica. One ORF and the two partial ORFs code for putative

proteins showing significant homology with proteins from other organisms. YlVI-108w

(partial) and YlVI-103w show 39% and 54% identity, respectively, with YDR430c and

YHR088w from S. cerevisiae. YlVI-102c (partial) shows significant homology with a

matrix protein, lustrin A from Haliotis rufescens, and with the PGRS subfamily (Gly-rich

proteins) of Mycobacterium tuberculosis. The three remaining ORFs show weak or non-

significant homology with previously sequenced genes. The nucleotide sequence has been

submitted to the EMBL database under Accession No. AI006754. Copyright # 2001 John

Wiley & Sons, Ltd.

Keywords: Yarrowia lipolytica; Saccharomyces cerevisiae; Kluyveromyces lactis;

Candida albicans; gene order; genome organization

Introduction

Completion of the Saccharomyces cerevisiae genomesequence (Goffeau et al., 1996), together with theextensive work carried out on the systematicsequencing of the genomes of another two yeasts(Schizosaccharomyces pombe and Candida albicans),has opened the possibility of analysing to whatextent gene order is conserved in yeast genomes.

Another two yeasts, Yarrowia lipolytica andKluyveromyces lactis, which are quite divergentfrom the evolutionary point of view (Barns et al.,1991) but amenable to classic and molecular geneticstudies, are currently under extensive research byseveral European groups within the framework ofthe Biotech Programme (Cell Factory Area, Grienglet al., 1999). The total haploid genome size is21–22 Mb for Y. lipolytica (Casaregola et al., 1997)

and 12 Mb for K. lactis (Wesolowski-Louwel andFukuhara, 1995). The electrophoretic patterns ofchromosomal DNA suggest that both yeast speciescontain six DNA bands (numbered I–VI, fromsmallest to largest) and about 150 genes have beencloned and located physically in each yeast (Weso-lowski-Louvel et al., 1996; Casaregola et al., 1997;Domınguez, A., unpublished).

Several authors have reported conservation ofgene order in ascomycete fungi, e.g. S. cerevisiaeand Saccharomyces douglasii (Adjiri et al., 1994);S. cerevisiae and K. lactis (Wesolowski-Louvel andFukuhara, 1995; Bai et al., 1999); S. cerevisiaeand Ashbya gossypii (Attman-Johl and Philippsen,1996) and S. cerevisiae and C. albicans (Hartunget al., 1998). Extensive analyses aimed at showingthe order of genes along chromosomes amongS. cerevisiae and other yeast species by comparing

YeastYeast 2001; 18: 807–813.DOI: 10.1002/yea.735

Copyright # 2001 John Wiley & Sons, Ltd.

data from DNA sequences contained in the data-bases have been performed by Keogh et al. (1998).The global degree of synteny for the conservation,or non-conservation, of neighbouring gene couplesbetween S. cerevisiae and 13 hemiascomycetousyeasts species has also recently been described(Llorente et al., 2000). Both studies have shownthat the extent of gene order conservation decreaseswith increasing evolutionary distance. However, thecomparative analysis of genomic DNA has beenevaluated on the basis of the sequences of smallDNA fragments (500 bp at both ends). To furtherextend the data on gene order, sequence compar-ison, genome compactness and analysis of theterminator–promoter environment among yeastspecies, we have chosen a different approach: thesequencing of larger fragments of DNA (8–16 kb)from several yeast species. This study reports thesequence of a 10.2 kb Y. lipolytica region containingin its central part two genes previously character-ized by us: YlURA5 and YlSEC65 (Sanchez et al.,1995, 1997). The two genes are adjacent, with aninverted orientation. This distribution is conservedin four yeast species (S. cerevisiae, K. lactis, C.albicans and Y. lipolytica).

Materials and methods

Plasmids and strains

The YlURA5 and YlSEC65 genes were isolated asplasmid pMP47 (Sanchez et al., 1995). This plasmidcontains a 10.2 kb insert from a DNA libraryconstructed from Y. lipolytica W29 strain partiallydigested with Sau3A and cloned in the BamHI siteof the pINA62 vector (Xuan et al., 1988). TheEscherichia coli strain used as host for transfor-mation and amplification of plasmids was DH5asupE44 DlacU169(ø80 lacZ DM15)hsdR17 recA1endA1 gyrA96 thi-1 relA1 (Hanahan, 1983). E. colitransformants were selected on LB media supple-mented with 100 mg/l ampicillin.

Manipulation of nucleic acids

Routine DNA manipulations, Southern blotting,restriction enzyme digestions, agarose gel electro-phoresis and E. coli transformation were performedaccording to standard techniques (Sambrook et al.,1989). Plasmid preparations were carried out usingWizard miniprep columns (Promega).

Sequencing strategy

The sequence was determined using universal andreverse primers with the ABI377 automatic sequen-cer (Applied Biosystems Inc.) using the Taq DyeDeoxy Terminator Cycle Sequencing Kit as sup-plied by the manufacturer. Junctions were sequen-ced with walking primers using the entire plasmid.The quality of the final sequence was ensured byvisual inspection of the sequencing profiles at eachposition on each DNA strand. The sequence wasconsidered final only when an unambiguous readingof each nucleotide on each strand was achieved.

Software used

Walking primers were designed using the DNAsiscomputer program (Pharmacia Biotech). Assemblyof the sequences was accomplished with theSeqMan program of the DNASTAR programmepackage (DNASTAR Ltd.). ORFs were predictedusing DNA Strider software (Marck, 1988). Foreach ORF the first ATG was assumed to be theinitiation codon. ORFs were named with the prefixYl (Yarrowia lipolytica), the chromosome number(VI) and the MIPS working nomenclature forS. cerevisiae. Searches for homologies were doneusing the BLAST (Altschul et al., 1997) or FASTA(Pearson and Lipman, 1988) programmes. Multiple-sequence alignments were obtained using theCLUSTAL programme (Thompson et al., 1994) orPILEUP (GCG package).

Results and discussion

Plasmid pMP47, containing an insert of 10275 bpfrom chromosome VI of Y. lipolytica, was sequen-ced. A search for coding regions revealed six clearORFs (two of them partial) longer than 100 aminoacids and two more overlapping fragments(Figure 1). The major characteristics of the ORFsare listed in Table 1. The most upstream ATGcodon was arbitrarily considered as the initiationcodon. The ORFs, considering YlVI-104c, occupy69.5% of the complete sequence, a value slightlylower than the 72% described for S. cerevisiae(Dujon, 1996) but higher than those obtained forSz. pombe (54–59%; Sanchez et al., 1999; Xianget al., 2000). The sequence region has an overallG+C content of 48.5%, in good agreement withpreviously reported data (49.6–51.7%; Nakase and

808 M. Sanchez and A. Domınguez

Copyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: 807–813.

Komagata, 1971; Kurtzman and Fell, 1998; Kucket al., 1980). The coding region alone (including theYlVI-104c) has a slightly higher G+C content(51.7%).

ORFs and ORF products

The YlVI-108w ORF is truncated at its 5k end andis located at one end of the genomic fragmentcloned into pM47 (Figure 1). Therefore, thededuced amino acid product is only 780 resi-dues long. However, this portion is sufficient todetect 39.4% identity with the YDR430cp of

S. cerevisiae (aminoacyl-transfer RNA synthetase

class-I signature).The deduced protein product of YlVI-107c is 382

residues long and shows no significant homology

with known proteins or ESTs. ORFs YlVl-104c

and YlVI-104w represent partially antiparallel over-

lapped ORFs. Both have lower G+C contents than

average (46.98% and 47.55%, respectively). Neither

the predicted products nor homologies were found

in databases, raising the question of their biological

significance. YlVI-103w encodes a putative protein

of 333 amino acids in length. FastA analysis

(EMBL database) revealed 63.44% identity with

Figure 1. DNA Strider plot showing the ORFs in the six possible frames. ATG codons are represented by half-height verticalbars and stop codons by full-height bars. In the lower part, arrows indicate positions and directions of the ORFs on the twostrands. WSc and CSc are Watson and Crick strands in S. cerevisiae

Table 1. Characteristics and homologies of ORFs and deduced amino acid sequences of the 10.2 kb fragment

ORF name Coordinates

Strand

orientation*

Length

(aa)

Molecular

mass (kDa) Homologies

FastA scores

Initn Init1 optn

YlVI-108W ?–2343 W – – Similar to S. cerevisiae YDR430C 1541 520 1941

YlVI-107C 2910–4058 C 382 43107 No similarity found – – –YlVI-106W 4549–5208 W 219 23667 S. cerevisiae URA5 782 656 807

YlVI-105C 5471–6403 C 310 35464 S. cerevisiae SEC65 368 253 372

YlVI-104C 6803–7186 C 127 14521 No similarity found – – –

YlVI-104W 6640–7011 W 123 13556 No similarity found – – –YlVI-103W 7755–8756 W 333 39329 Similar to S. cerevisiae YHR088W 1232 946 1263

YlVI-102C 9519–? C – – Similar to Lustrin A 471 471 555

*W=Watson strand; C=Crick strand.

The URA5–SEC65 region of ascomycetous yeasts 809


S. cerevisiae YHR088wp, a protein of unknown

function (Figure 2). The Y. lipolytica protein

appeared to be 38 amino acids longer than the

S. cerevisiae protein. Some of the Y. lipolytica

proteins are longer than their S. cerevisiae coun-

terparts [i.e. those encoded by YlSEC14 (Lopez

et al., 1994) and YlSEC65 (Sanchez et al., 1997)]. In

YlVI-103w, two methionines are located at the

N-terminal region (Figure 2, bold and underlined),

the second at amino acid 40. If transcription starts

at this point, a protein of 239 amino acids with a

size more similar to that of S. cerevisiae will be

obtained.YlVI-102c is also truncated at its 5k end

(Figure 1). The deduced amino acid product is 257

residues long. The protein shows a moderate degree

of homology with lustrin A (a matrix protein of

1482 amino acids from shell and pearl nacre of

Haliotis rufescens; Shen et al., 1997) and with

Rv3507 (a protein of 1381 amino acids), a mem-

ber of the glycine-rich PGRS subfamily of the

Mycobacterium tuberculosis PE protein family (Cole

et al., 1998). However, in both cases the similarity

is restricted to a GS-rich region located in the

C-terminal region and hence the N-terminalsequence would be necessary if more accurateconclusions are to be drawn.

Genomic URA5–SEC65 regions of Y. lipolytica,S. cerevisiae, K. lactis and C. albicans

Analysis of the DNA fragments harbouring theURA5–SEC65 genes revealed that both ORFs arearranged in the same order and orientation in thefour ascomycetes (Figure 3). The sizes of the genesare similar. The level of protein identity between thepairs varies (81.9–56.6% for URA5, 59.7–23.3% forSEC65). In Y. lipolytica and C. albicans, both genesare located on the largest chromosome (VI and R,respectively). In S. cerevisiae, they are locatedon chromosome XIII, while their chromosomallocation is unknown in K. lactis. Our results offerone of the best examples of the conservation ofsynteny and gene orientation between differentand yeast species. The lack of homology of threeY. lipolytica genes (out of eight) with the genesequences described in the databases are in goodagreement with the only 1187 genes identified after

Figure 2. Predicted amino acid sequences of the proteins YHR088w of S. cerevisiae and YlVI-103w of Y. lipolytica. Gaps havebeen introduced to give the best alignment. Identical residues (asterisks) and conservative amino acid substitutions (dots) areindicated



analysing 4940 Y. lipolytica random sequence tags

(Casaregola et al. 2000).In all cases the highest identities were obtained

between the two most closely related yeasts

(S. cerevisiae and K. lactis), the data being con-

sistent with their phylogenetic position (Figure 4;

Barns et al., 1991, Bolotin-Fukuhara et al., 2000,

Llorente et al., 2000). The intergenic regions do not

display many significant sequence similarities.

Moreover, the sizes of the intergenic regions of

Y. lipolytica are similar to those of the other three

yeasts. These observations raise an interesting

question about genome organization in this yeast.The size of the Y. lipolytica genome has been

estimated to be 21–22 Mb (Casaregola et al., 1997).

Taking into account a rDNA cluster size of 3 Mb

(slightly overestimated, Casaregola et al., 1997), we

obtain a size of 18 Mb.Assuming that the coding region occupies 63%

of the total (average 60–69.5%; this work;

Domınguez et al., in preparation); thus, 11.3 Mb

remain, which is 2.3 Mb or 3.7 Mb larger than

those of S. cerevisiae (9.0 Mb, 12.6r72%; Dujon,

1996) or Sz. pombe (7.8 Mb, 13.6r55%; Sanchez

et al., 1999; Xiang et al., 2000). Only a few Y.

lipolytica genes have introns (Barth and Gaillardin,

Figure 3. Map of the Y. lipolytica loci of URA5–SEC65 and its comparison with. S. cerevisiae, C. albicans and K. lactis. The ORFsand their orientation are represented by arrows. Thin lines represent intergenic regions

Figure 4. Phylogenetic tree of URA5 (A) and SEC65 (B)proteins from Y. lipolytica, S. cerevisiae, C. albicans and K. lactis,using the MegAlign document



1996) and to date no chromosomal duplicationshave been described. Establishing a theoreticalstandard size of 500 amino acids per gene, theextra 2.3 Mb implies that Y. lipolytica has1500–2000 genes more than S. cerevisiae. Whetherthis reflects a higher metabolic capacity of Y.lipolytica or is a consequence of its divergentphylogenetic position remains to be elucidated.

We have also analysed gene order conservationin the flanking regions of the URA5–SEC65 genesbut the extent of linkage conservation falls off. InY. lipolytica the two nearest putative ORFs donot show similarity to any known sequence. Thenext two genes, YlVI-130w and YlVI-108w, showgood consensus with the S. cerevisiae genesYHRO88w and YDR430c (Table 1), located onchromosomes VIII and IV, and not on chromosomeXIII (where the URA5–SEC65 gene pair is located).In C. albicans and K. lactis, no ORFs have beendescribed in the 5k region upstream from the URA5gene (1600 and 617 bp, respectively). In C. albicansthe CaCDC4 gene is located 425 bp in the flankingregion of the CaSEC65 gene. The S. cerevisiaeCDC4 gene is located on chromosome IV. Finally,in Sz. pombe the URA5 and SEC65 homologues arelocated on different chromosomes (II and III,respectively).

Several analyses have been carried out on thedegree of synteny and gene order conservationbetween related yeast species (Keogh et al., 1998;Ozier-Kalogeropoulos et al., 1998; Sychrova et al.,2000; Llorente et al., 2000), but all of them haverelied on the comparison of small DNA fragments.Our results suggest that in order to understandgenome evolution at the chromosomal level, moreclosely related organisms, or at least an entirechromosome from some of them, must be fullysequenced.

Acknowledgement

We wish to thank N. Skinner for revising the English version

of this manuscript. This work was partially supported by a

grant from the EU (BIO4-CT96-0003).

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gene order in a 10 275 bp fragment of yarrowia lipolytica, including adjacent ylura5 and ylsec65...

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