sequencing analysis of a 4·1 kb subtelomeric region from yeast chromosome iv identifieshxt15, a new...

YEAST VOL. 12: 1005-1011 (1996)

oo O OO 0 0 0 IV '0 0 Yeast Sequencing Reports 0 O O O O

Sequencing Analysis of a 4.1 kb Subtelomeric Region from Yeast Chromosome IV Identifies HXTl.5, a New Member of the Hexose Transporter Family M. BARGUEST$. D. SALOMf, A. GOMEZI, N. PARICIOt. M. PEREZ-ALONSOtS AND J . E. PEREZ-ORTIN*fV

i. Departuniento de GeriPticu rind 1' Departartimto tic Bioqtiimica. Uniwrsitut Ui. Vali.nci;li' Dr. kloliner 50. E-46100 durjussot. Spurn $Laboratorio Boehrirrger M~inrrheit~i-l~rii~~er.c.itctt tie Vuleiicia, Prirnr~lo Reig 102. E-46010. I~iileircin. Spain r(hstiruro rle Agroqtrirsiico J! Tmdogic i de Alirm~tos. C. S.I. C.. Po/igono Lri Coriiu sln. Paternti. Sprrirr

Received 70 January 1996: accepted 5 March 1996

The DNA sequence of a 4.1 kb region of Sric~c~liur~oni~re,~ cerevisiae chroinosome IV was determined. This region contains a single open reading frame which codes for a member of the hexose transporter family. This new gene has been named HXTIS according to yeast gene data bases. The sequence has been entered in the EMBL data library under Accession Number X92891.

KEYWORDS - genome sequencing; Succlirir.om,vces c'ererisirre; yeast; chromosome IV; H X T I S ; hesose transporter

INTRODUCTION

In the framework of the European yeast genome sequencing project, we have determined the sequence of a 4144 bp fragment of chromosome IV carried by the lambda clone 2417. The sequence shows only one open reading frame (ORF) which is highly homologous to the yeast hexose transporter family. This family includes to date, in Saccha-roi?ijws cerevisiae. 16 members named H X T plus the galactose transporter GAL2 (Bisson et al., 1993; Reifenberger et al.. 1995). We have named our gene HXT1.5 according to the chronological order of publication for those genes.

*Corresponding author. M.B. and D.S. contributed equally to this paper

MATERIALS AND METHODS

Bacterial stlviitu The EscJzerichici coli strain DH5a was used for

all subcloning and sequencing work. Lambda clone 2417 was provided by C. Jacq acting as chromosome IV coordinator. This clone was generously given by L. Riles and M. Olson. Routine culture and DNA inanipulations followed standard protocols (Sambrook rf al., 1989).

Seq ueii citig s t m t egx The yeast insert was divided into four restriction

fragments which were subcloned into pUC vectors. Subfragments were sequenced from universal primers and this information was used to design new primers following a 'primer walking' strategy.

CCC 0749~503></96/101005~07 ( 1996 by John Wiley & Sons Ltd

1006 M . BARGUES ET AL.

Lefi telornere Centrornere, - H B P P E I I I I I

0 5 kh .- y------H

Figure 1. Partial restriction map deduced from the 4144 bp sequence showing EcoRl (E), Hind111 (H), BumHI (B) and PstI (P) sites. The arrow indicates the location and direction of the ORF named HXTIS.

Restriction sites and both ends of the insert were directly sequenced from the original clone using primers designed from the insert or from the vector respectively. Double-stranded templates were pre- pared either by the Wizard 373 DNA purification system (Promega Co.) or by alkaline lysis mini- preps (Sambrook et al., 1989). Sequence analysis was performed by three different methods: auto- mated sequencing (ABI 373A) using fluorescent dideoxy terminators, dATP 35S manual sequenc-

ing and the ‘GATC’ system in combination with the DIG-Taq DNA sequencing kit (Boehringer Mannheim).

Sequence analysis DNA and protein sequences were analysed

using programs from the GCG Software Package (University of Wisconsin).

RESULTS AND DISCUSSION Chromosome IV is the largest yeast chromosome (excluding the rDNA repeat of chromosome XII). It has been divided into four parts according to the yeast genome sequencing world wide agreement (Mordant et al., 1995). The European Yeast Genome Sequencing Consortium has been in charge of the 600 kb proximal to left telomere. Some cosmid libraries were made from this part of the chromosome and a set of ordered cosmid clones were distributed between the various par- ticipant laboratories. Due to contamination with

A

P ETIGLSLEEI QLL AKKKSWKEVL KFPKSFN

B 1-

’ Phobic

EPhilic 0 -

L .I J J I 1 1 1 ~ 1 1 1 1 ~ 1 1 1 1

0 200 400 Figure 2. (A) Deduced amino acid sequence of HxtlSp. The predicted transmem- brane segments deduced from the hydropathy index and from the comparison with the other known hexose transporters according to Maftahi et ul. (1995) are shown shadowed. (B) Hydrophobicity plot of HxtlSp. The hydropathy index (Kyte and Doolittle, 1982) was calculated for a window of 20 residues at each position in the protein sequence, and this value was plotted against residue number. Values above the abscissa indicate hydrophobic regions, values below the abscissa indicate hydrophilic regions.

NEW MEMBER OF HEXOSE TRANSPORTER FAMILY

Table 1.

1007

List of sequences used for dendrogram construction.

Protein name

Amino Accession acid S. cerevisiae

Gene number residues chromosome Reference ~

Hexose transporter 1 H X T l M82963 569 VIII Lewis and Bisson (1991) Hexose transporter 2 HXT2 248613 541 XI11 Kruckeberg and Bisson (1990) Hexose transporter 3 HXT3 LO7080 567 IV KO et al. (1993) Hexose transporter 4 HXT4 M81960 576 VIII Prior et al. (1993) Hexose transporter 5 HXTS XI7961 592 VIII Reifenberger et al. (unpublished) Hexose transporter 6 HXT6 X31691 570 IV Reifenberger et al. (1995) Hexose transporter 7 HXT7 231692 570 IV Reifenberger et al. (1995) Hexose transporter 8 HXT8 234098 569 X Vandenvol et al. (1 994) Hexose transporter 9 HXT9 234098 567 X Vandenvol et ul. (1994) Hexose transporter 10 HXTlO 246255 546 VI Barrel et al. (unpublished) Hexose transporter 11 H X T l l X82621 567 XV Wesolowski-Louvel (unpublished) Hexose transporter 12 HXTIZ 247047 567 IX Barrel et al. (unpublished) Hexose transporter 13 HXT13 U18795 564 V Dietrich et al. (unpublished) Hexose transporter 14 HXT14 246259 540/565* XIV Maftahi et al. (1995) Hexose transporter 15 HXTIS X92891 567 IV This paper Putative hexose transporter YJR158 W 249658 567 X Zagulski et al. (unpublished) Galactose transporter GAL2 M68541 574 XI1 Szkutnicka et al. (1989) Maltose permease MAL6I X17391 614 VIII Cheng and Michels (1989) Sugar transporter S T L l LO7492 536 IV Zhao et al. (1994)

K. lactis low-affinity

High-affinity

Human glucose transporter

K. lactis hexose transporter 2 KHT2 247080 566 - Weirich et al. (unpublished)

glucose tansporter RAG1 X53752 567 - Goffrini et al. (1990)

glucose transporter SNF3 503246 884 IV Celenza et al. (1988)

type 4 GLUT4 M20741 509 Fukumoto et al. (1989) ~

*Amino acid length depends on whether or not a putative intron is used.

chromosome VIII DNA (Jacq et al., 1995) the cosmid assigned to us was found to correspond to a piece of chromosome VtII (Bargues et al., 1995), previously sequenced by the group of M. Johnston (Johnston et al., 1994). The gap between the adja- cent cosmids was not contained in any cosmid (Jacq et al., 1995) and, for that reason, it was necessary to use a lambda clone from the Riles and Olson library.

The 4.1 kb EcoRl-Hind111 was difficult to subclone into pUC vectors and when finally subcloned it conferred slow-growing phenotype to the bacterial strain. This prompted us to divide it into minor fragments in order to facilitate sequencing work. One of these sub-fragments, BamHI-PstI (see Figure l), was impossible to subclone so it seems to contain DNA sequences toxic for E. coli. The sequence of that fragment was obtained work- ing directly on the original unfragmented clone.

The restriction map of the 4144 bp fragment is shown in Figure 1.

The analysis of this sequence revealed an overall G+C content of 38.4%. The region contains a single putative ORF (see Figures 1 and 2). The deduced protein has 567 amino acids and a predicted molecular weight of 62 928. It is highly similar to a group of yeast proteins known as ‘hexose transporters’ or ‘hexose permeases’ (Bisson et al., 1993). The percentage of identity ranges from 99.6% with YJR158w to about 26% with either Stllp or Snf3p. The Saccharomyces Genome Database Project at Stanford (CA, U.S.A.) has assigned the name HXT15 to our sequence because it is more similar to the Hxtp family than to Snf3p. As for the other HXT proteins, Hxtl5p has 12 putative membrane-spanning domains (Figure 2). Its codon bias index is low (0.26). This circum- stance is similar to that observed for other

1008

Hxtl5

Yjr158w

Hxtl3

1 Hxt06

Hxt07

I Ma161 Figure 3 . Dendrogram of the available Hxt proteins and some other sugar transporters. Pairwise comparisons were made using the PILEUP program and the results of the comparisons ranked to determine the relative protein sequence similarities. The dendrogram was constructed using the DISTANCE and GROWTREE programs. The sequences used are listed in Table I . Alignments were made over the whole protein sequences except for SnDp in which only the first 570 amino acids were used. Note that this plot is not an evolutionary tree.

members of this family (HxtSp, Hxt8p-Hxtl4p), suggesting that they are low abundance proteins.

We have further compared this new gene with other known Saccharomyces hexose transporters (see Table 1). In this analysis we have included for comparison a yeast disaccharide transporter (Mal6lp) and hexose transporters (Kht2p, Raglp) from another yeast (Kluyveromyces lactis) and from humans (Glut4). The comparison showed significant similarities among all these proteins. The relationships among them were determined by means of the PILEUP and GROWTREE programs (GCG package). As can be seen in Figure 3, Hxtl5p is grouped with YJR158w (a new ORF from chromosome X) and Hxtl3p. Some other subgroups are also visible but with different

M. BARGUES ET AL.

degrees of homology. For instance Hxt6p and Hxt7p (Reifenberger et al., 1995) or Hxt9p, Hxtl l p and Hxtl2p are very similar but other groups show lower similarity. It is interesting to note that Gal2p clusters to Hxt6p, Hxt7p, Hxt4p and Kht2p so it is more related to some Hxt proteins than others, as previously noticed by Reifenberger et al. (1995). Because of that, and also because the function of Hxt8p-Hxtl5p is not known, the possibility that some of the more recently described Hxt proteins were not glucose permeases but mere transporters for other monosaccharides cannot be ruled out. From our comparison and from previously pub- lished data (Bisson et al., 1993) it can be concluded that there is an evolutionary related group of hexose permeases which extends over different substrates (galactose, glucose and, perhaps other monosaccharides) and different yeast species. This group is distantly related to other hexose transporters, both from yeast (Snf3p, Stllp) and other eukaryotes (human glucose transporters) and all of them are separated from disaccharide transporters, as Mal6lp.

Comparison of nucleotide sequences suggests that some of the H X T genes were originated by gene duplication and (sometimes) recombination between different chromosomes. There are three kinds of data which support this hypothesis. First, some of the H X T genes are clustered in certain small regions of one chromosome: HXT3, HXT6 and HXT7 are closely placed in tandem in chromosome IV (Reifenberger el al., 1995); H X T l , HXT4 and HXTS in chromosome VlII (Johnston et al., 1994), and HXT8 and HXT9 in chromosome X (Vandenbol et al., 1994) are similarly tandemly placed. Second, some of the H X T genes have a subtelomeric location which is a postulated mech- anism of evolution for other yeast gene families, such as SUC (Carlson et al., 1985), M A L (Charron et al., 1989) and R T M (Ness and Aigle, 1995) genes. Third, we have found that some of the more closely related H X T genes extend their homology through both 5' and 3' flanks. For instance, HXT9, H X T l l and HXT12 are almost identical at their 5' flank, although they are in different chromosomes; HXT6 and HXT7 are highly homologous not only through their coding sequences but also in 100 bp in each flank (data not shown). Finally H X T l 3 (chromosome V, subtelomeric) and HXTI.5 (chromosome IV, subtelomeric) are also similar in both flanks (see Figure 4).

It seems that sugar transporter genes have been originated from a common ancestor previous to

NEW MEMBER OF HEXOSE TRANSPORTER FAMILY 1009

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

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HXTlS HXT13

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HXT15 HXT13

HXTlS HXT13

HXTlS HXT13

HXT15 HXT13

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AACTCAAGTATGATAACTCCAGTGTT--GGCCTGTAATCACCAACTTTCAAA-CGTATAA CGTTCAAGGGTGTGAGGTCGTATCTTCCAGCAAGTAATTGC-AACTAAAAAGTCATATAA

+ t t * l t t t * * * * * *t * * * * * * * + * * t t * * * t t

AGAAAGTAAGTAATTCATTGTTTGCTTCAGGTTTTAGTCT-TATTACTATCCATAGAAGTA AAGAGGCAGATGAAACAGAGTT-GTTTGAGGTAAAATTTTCTATTCCTACTT-TGCACGTA

* t * * *t* t t * * * * * t * * * * + + * * * * +

AAAC-TATTGATATTATTGACAGAAAAGGAGGAGGA?+AGG--AAACTAAAGG~@&C A G G C A T A A C A A T U A A ? G A A G A A A A A A G A A A C A A A A G T T A A A C ~ TC

* * * +t * * * * * * *I* * * * * * * * * * * t * t * * f *

AAGCGAACAGTCCTCACCAGAAATTAATGCAGATAATCTAAACAGTAGTGCAGCTGACGT TAGTGCGCAATCCTCTATTGATAGCGATGGTAGATGTTCGAGAT---------GCTGATAT t * * * * * * * * t t * * * t * t t * * * * * * * *

TCATGTACAGCCACCCGGAGAGAAAGAATGGTCAGACGGGTTTTATGAWGAAGTCAT TCATGTCGCACCACCCGTGGAAAAAGAGTGGTCAGATGGATTTGATGACAACGAAGTCAT * * * * * * * * * * * * * * * t t * t * * * * * * * * * t t * * * * * * * * * * * * * * * * * *

TAATGGAAATA-CGCCAGACGCACCGAAGAGAGGCTTTTTAGGTTACCTTATTATCTACT AAACGGGGATAACGTTGAGC-CACCXVLZiAGAGGGCTCATAGGTTATCTTGTCATTTACT

* * * * * * * * * t t t * * * * * * * * + t * t * * t * * * * t * * ***t

TACTATGCTATCCTGTATCCTTTGGC~TTTTTTACCT~TT~TAGTGGTATTACTG TACTGTGTTATCCAATATCCTTTGGGGGTTTTCCTGCCTGGTTGGGATAGTGGTATCACAG * t t * *+ * f t * t * * * * t t t t * * * t t * * * t * * * * t * * * * * * * * t * t t * ,* *

CAGGCTTCATCAATATGGATAACTTTAAAATGARTTTTGAATTTTGGTTCTTACAAGCACAGTACTG CAGGTTTCATTAACATGGACAACTTTAAAATGAACTTCGGTTCTTACAAGCATAGCACTG * * * * * * * * * * * ***tt * * * * * * * * * * * * * * *t * * * * * * * * * * * * * * * * * * * *

GTGAGTATTATTTGAGCAACGTGCGTATGGGTCTTCTCGTGGCCATGTTCAGTGTAGGAT GTGAATATTATTTGAGCAACGTGCGTAT~TCTTCTTGTGGCTATGTTCAGTATTGGAT ***t * * * * * t * * t * * + t t + * * * * * * * * * * * * * * f * * * * * * * * * * * * * * * * + * * * * * *

G T T C C A T T G G C G G T G T T G C T T T T G C G A G A C T T G C T G A T A G G T A G A A GTGCCATAGGTTGGCCTTATTTTTGCCCGTCTTGCTGATACTTTAGtTAG~GGCTGGCAA ,* * * * * * * * * * * * * * * * * * *****t**********t****tf*t* * * * *

TTGTAATCGTGtTTTTGGTATATATGGTTTGGTGCAATTATTCAGATCAGTTCGAATCACA TTGTGATCGTGGTGTTGGTATATATGGTTGGTGCAATTATTCAGATCAGTTCAAATCACA t t t f * * * * * * * * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * * * * * * *

AATGGTACCAATACTTTGTCGGTAAGATCATCTACGGTCTTGGTGCTGGTGGCTGTTCGG AATGGTACCAGTACTTTGTCGGTRAGATCATCATCTACGGTCTTGGTGCTGGT~TGTTCGG *... '*...* * * .+* t ~ . . . . * . * t t * * . r * . . . . . * . * t * t r * t . ~ * * * * . t . . * . I I

TGTTGTGTCCAATGCTTTTATCTGAAATAGCCCCC9CAGCCCCCACAGATTTGAGAGGTGGACTT~TCT TGTTGTGTCCAATGCTTTTGTCTGAAATAGCCCCCACAGATTTGAGAGG~~ACT~TCT t.*.*.*..r*..*t..*t * l t . * r * * " t * t t . t . * l . f * * ~ . ~ ~ . . ~ ~ ~ " ~ . ~ t.."

CATTGTACCAACTTAACATGACCTTCGGTATTTTCTT~TTATTGTAGCGTTTAT~ CATTGTACCAACTGAACATGACGTTCGGTATTTTCTT~TTATTGTA~GTTTATGGTA ...*... ..*..* .t...*t* f...*r.*.rt~.t"~""..*...*."~"~.~*.* . CAAGGAAGTATAGTAATACTGCG~TGGAGGATTCCTGTGtGACTATGCTTTCTGTGGG CGAGAAAATACGATAAZACTGCACAATGGAGAGTCCCCCTTGGG€TTTGCTTTTTATGGG . t t * t r t + r * r r t t . .It"*.** f .* *. t. . r * t t . . t f * .

CTCTAATTATCATCGTTGGTTATTAGTTCCAGAGTCCCCAAGATATCTGATTGAAT CTTTGATTATCATCATTGGTATGTTATTGGTTCCAGAGTCCCCAAGATATCTGATATCTGATTGAAT t. ,....*,.* * * * . . , . t t f * * tttr.ltr.t**.tC.t.*.**~,*~tr*..

GTGAGAGACATGAAGAGGCCTGTGTCTCCATCGCCAAGATCAACAAGGTTTCAZCAGAGG GTGAGAGACACGAAGAGGCCCGTGCTTCCATTGCCAAAATCAACAAGGTTTCACCAGAGG *..*..*.** ,. ."*.... .*. ..t*r ..t*t rtt....~**..t~.*.***..

ATCCATGGGTACTCARACAGGCTGATGWTCAACGCCGGTGTCCTTGCCCAAAGAGAAC ATCCATGGGTACTCAWCAGGCTGATGAMTCAACGCCGGTGTCCTTGCCCAAAGGGAAC *~,~.,**.... "tt.*t**,.****~~**~*,~~**".**.***.*~*.~.~,, t r t *

TAGGGGAAGCCTCATGGAAAGAACTTTTCTCCGTCAAAACAAAAGTCCTTCAACGTTTGA TAGGAGAAGCTTCATGGAAAGAACTTTTCTCTGTAAAAKTAAAGTCCTTCAACGTTTGA *.** .*,.* ***.*,..*... tt**.*t* I. ..*** **"l.l*ttt*..*.***t

TCACAGGTATTCTTGTGCCTTTTTGCAACTTACTGGTGA?AACTACTTCTTCTTCT T C A C A G G T A T T C T T G T G C A C T T T T T G C A A C T T A C T G G T T C T T C T T C T .r**.'.,.*~t..**rt..Ir.l.tl...".**.*.."*~.*"..~.**~~..~..*"~*..

Figure 4 ( ' i i i t ~ i n t i ~ i l oit tic \ I pity1

1010 M. BARGUES ET AL.

HXT15 HXT13

HXT15 HXT13

HXTl5 HXT13

HXTl5 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

HXT15 HXT13

Figure 4

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ACGGAACTACCATTTTCAAATCAGTTGGGCTTACTGATGGGTTTGAGACTTCGATCGTCC ACGGAACTACCATTTTTAAATCAGTCGGTCTTACTGATGGGTTTGAGACGTCGATCGTCC * * * * * * * * * * * * * * * * ******** * * . . . . . . . . . . . . . . . . . . . . * * * * * * * * * *

TAGGTACAGTGAATTTCTTCTCCACTATTATTGCTGTTATGGTCGTAGACAAAATAGGCC T A G G T A C A G T G A A C T T C T T C T C C A C T A T T A T T A T C * * * * * * * * * * * * * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * * * *

GTCGTAAATGTCTGTTATTCGGAGCGGCTTCAATGATGGCTTGTATGGTAATCATATTTGCAA GTCGTAAATCTCTGTTATTTGGTGCAGCTGGGATGAT~TTGTATGGTCATATTTGCAA rrr*t*.***..rrrr.tr t t * * ..* ."*t**t."t**tt***"*.*".,**.~

GTATCGGGGTAAAATGTCTTTACCCTCATGGCCAGGATGGTCCATCCTCGAAAGGTGCAG G T A T C G G G G T G A A A T G T C T T T A C C C T C A T G G C C A G G A C G G G ***.**..** .* t * . . * * . * t * * r * . . * . . . * I . I * i.tr* * * **.**. *.*.... GTAATGCCATGATTGTGTTCGCTTCTATATATTCTGCTTTGCAACGACATGGGCCC GTAATGCCATGATTGTGTTCCTTGTTTCTATATATTCTGCTTTGCAACGACATG~TC t . * t t t * l t * t . ~ t * * * , * * ~ * *. tt****ttt.....**t.t***.,*",~",,~

C T G T T G C T T R r A T T G T G G T T ~ C C G A G T C A T T C C C T T C G A T G T CTGTTGCTTATATTGTGGTTGCCGAGTCGTTCCCTTCGAAGGTCAAGTCTAGAGC~TGT ..lr~.tr..t.t.rt*rtr*Ir*r*r* *.*..*..****.**.. r * r t .*(I ".*. CAATTTCGACTGCATTCAACTGGTTATGGCAATTCTTCTTGATTGGTTTTTTCACACCATTCA CGATTTCAACTGCATGCAA;TGGTTAT~AATTTTT~AT~GGTTTTTTCA~ACCAT~CA . " * " * * ***..*. * * * * . t t r * . t * * r r r * * * * * t t *rtr*"..**t*r"*t"r.

TTACTGGGTCTATCCACTTCTATTATGGTTATGTGTTCGTAGGTTGTTTffiTTGCTATGT TTACTGGGTCTATCCACTTCTATTATGGTTATGTGTTCGTAGGTTGTTTGGTTGCTATGT tt.***tt.....r.*.t.*~*.*.~.**rlt,****.**"."****..,*~

T T T T G T A ~ G T T T T C T T C T T T T T A C C A G A A A C A A T T G G T C T TTTTGTACGT'PTTCTTCTTTTTACCAGAAACGATTGGTCTATCTTTGGAGGAAATCCAAT t. t . . . * . t + . . t * t t * t * " . * r . r * - t t l t *rt****l*..*t*..t*****...~ (I

TACTATATGAAGAAGGTATACATGGAAATCTGCATCTTGGGTACCACCCTCAAGGA TACTATACGAAGAAGGTATAAAACCATGGAAATCTGCATCTTGGGTCCCACCTTCTAGGA * r * r r r * **~**"*.tt*"t**l**l."***.****.*~~*"*"* * * * * * * * f " . *

GAGGAXTTCTTCCAGGGAAACTGAGGCTAAGAAGAXAAGCTGGAAAGAAGTTTTGAAGT GAGGTATTTCTTCCGAAGAAAGTAAGACCGAGAAGAAGGATTGGAAGAAATTTTTGAAGT ..," ,..=*** ..*. . t. * t t * * * * * t * * r f. ..*.*..*. TCCCAAAGAGTTTTAA-CAATATGCGCGCTTGATAAACATGCTAAGAGATAAATTC TCTCAAAGAATTCTGA~~&&TCATAGAGTGCATATGTTTGTCTTGATAGGCAATTCCA- - - - - * * * * , * * * I * * * , * * * * * * * t * * *. t " * . TTGIVVL9ARAATAG;GCCTACTGTARAACGAAGTGAAGTGACACGTAATTTCAAATTTAATCAAC TTGTATAT---TATAGTTTAC-ATAA-------TAATGTGTAATTTTAA-TTTG-TCAC- f f . I f *. " .** t * * * I ...**XI t l * * * t r l i

AAAAAACTACGAGTTTCTTTGCAAACTCCCTTTTTCGGACTACTAGTACTATATPAWlTA --GAATTTATGAAGATTATCG-GA~T-----ATGAAGATCAACA-TA-TGTACA;\ACTG

* * * * * * * 1 . * * * * * * t * * * * **. *

A C G A G T T A T T T A G T G ~ G A A A G C G T T T G G T T A A G G C A A T T C T G T T T C GCATGTG--TCATAAGTAAAAAGCATTCA-TTAAGACPAWlTTGTGTCTAATTCTTAGTC

* f t . - t r r r * * * * r * r r * t t * * * * . .. * t * * f .

TTTAAGGCAAAGCAGAGTATAAA----AAGTAGGATGCTTATGGAGTGAAAACGGAGTGG ATTTATTCAATTTTTTTTTTTTTTTTTACGTAAA~GGG-GTGGAGTGAAA-TGATATGG

* * * t t * * * * **. t **.*****.* t I * .

TGATGAAGATGGGGAAGTCAACATAGGTCCACTTGAGTGAGTGCTTATGGAAGATCCTTM GGATGGYGATGAGAAACTCTAAATGGATCCATTTGACGATCGATTAAAGATCATGAAAGA

.*.* * * t * * f . * I I r * f t t * * * * * *. I * . + * + * * t * %

TGCATTTCCAGGAACGTACTGTACCATTCCTCTAAATAAAGTG-TATACAATAATAT TGCGATTGAAGGAACATAACACGTTACTATTTGTCTAAATWCAATATAC~TAC * * * * * ( I * * * - * * . * * * * * * * ."" .** * * . * * * * * * . * * * * "tt.

AGAA--ATATAGATTCTTCCCAA-ATATTCAGTTGTTACTATAAAGGGAATATT--TGAG GACATTATATATACTCTACCCAAGATATTTAGTCATTACTATCGAGGGCGCGTAGATCAG * ***** * * * ***** * * * * * * * * * * * * * * * **** * * *

Figure 4. Continued from previous page

. Nucleotide sequence comparison for HXTl3 and HXTIS genes including coding regions and flanks. Translation start and stop codons are shadowed. Asterisks below the sequences denote identical nucleotides. Sequences are numbered taking the translation start for HXTIS as + 1.

NEW MEMBER OF HEXOSE TRANSPORTER FAMILY

eukaryote diversification. After that the hexose permease ancestor might have evolved to different transporters more or less specialized and, finally, in the S. cerevisiae species there would have been a ‘explosive’ expansion of this family, probably due to the high dependence of this species on the fermentable sugar carbon sources. More work on the function of each H X T gene and on the pres- ence of this family in natural strains is needed to further understand this topic.

101 1

ACKNOWLEDGEMENTS

We are grateful to Dr Claude Jacq for providing the lambda clone. We would like to thank MIPS and SERB10 (Universitat de Val6ncia) for help with sequence analysis. This work was supported by the Commission of the European Communities (BIOTECH programme) and by BI094-1277-CE (to M.P.-A.) and B1094-1271-CE (to J.E.P.-0.) grants from the Comision Interministerial de Ciencia y Tecnologia (Spain).

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sequencing analysis of a 4·1 kb subtelomeric region from yeast chromosome iv identifieshxt15, a new...

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