Gene, 101 (1991) 97-104 0

1991 Elsevier

GENE

Science

Publishers

97

B.V. 0378-l 119/91/$03.50

03993

Cloning, sequence and chromosomal NCYC396 (Recombinant

DNA;

a-galactosidase;

Hilkka Turakainena, ‘I Department

location of a MEL gene from Sacckzromyces

melibiase;

mRNA;

codon usage; homology;

carlsbevgensis

brewing lager yeast)

Matti Korhola b and Sirpa Aho b

Helsinki (Finland) Tel. (358-O)191 I and b Research Laboratories of the Finnish State (Alko Ltd.), SF-00101 Helsinki (Finland)

of Genetics, University of Helsinki, SF-00100

Alcohol Company

Received by J.K.C. Knowles: Revised: 16 November 1990 Accepted: 5 December 1990

10 August

1990

SUMMARY

Yeast strains producing cc-galactosidase (aGal) are able to use melibiose as a carbon source during growth or fermentation. We cloned a MEL gene from Saccharomyces carlsbergensis NCYC396 through hybridization to the MEL1 gene cloned earlier from Saccharomyces cerevisiae var. uvarum. The aGal encoded by the newly cloned gene was galactose-inducible as is the crGa1 encoded by MELI. A probable GALCprotein recognition sequence was found in the upstream region of the NCYC396 MEL gene, The gene was transcribed to a 1.5-kb mRNA which, according to the nucleotide sequence, encodes a protein of 47 1 amino acids (aa) with an M, of 52 006. The first 18 aa fulfilled the criteria for the signal sequence, but lacked positively charged aa residues, except the initiating methionine. The enzyme activity was found exclusively in the cellular fraction of the cultures. The deduced aa sequence was compared to the aa sequences of other clGal enzymes. It showed 83 y0 identity with the S. cerevisiae enzyme, but only 35 y0 with the plant enzyme, 30% with the human enzyme and 17% with the Escherichia coli enzyme. With pulsed-field electrophoresis, the MEL gene was located on chromosome X of S. carlsbergensis, whereas the S. cerevisiae var. uvarum MEL1 gene is located on chromosome II.

INTRODUCTION

Only some strains of the genus Saccharomyces are able to produce aGa1 and thus completely utilize raflinose as a carbon source (Yarrow, 1984). Normal baker’s yeast is unable to produce this enzyme. Introduction of aMEL gene Correspondence to: Dr. H. Turakainen Laboratories

of the Finnish

Box 350, SF-00101 Tel.(358-0)13311; Abbreviations:

Helsinki

at her present

State Alcohol

aa,

ing aGal;

amino

frame; medium.

S.,

bromide;

(Finland)

acid(s);

aGal,

kb, kilobase

nt, nucleotide(s);

electrophoresis;

Research

(Alko Ltd.), P.O.

Fax(358-0)1332781. a-galactosidase;

5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside; EtdBr, ethidium

address:

Company

oligo,

OFAGE,

bp,

YPD,

aXGal, pair(s);

or 1000 bp; MEL, gene encodorthogonal

oligodeoxyribonucleotide;

Saccharomyces;

base

yeast

extract,

field alternation ORF,

open

peptone,

gel

reading dextrose

coding for crGa1 into a commercial baker’s yeast strain should result in a more complete use of substrate sugars during propagation and thus in a decreased effluent load in the baker’s_yeast-producing factory. S. carlsbergensis strains have traditionally been used to make beer; introduction of the MEL gene from a brewing lager yeast into baker’s yeast would not add anything to the human diet that had not been there before. The MEL1 gene from an S. cerevisiae strain MPY1041 (Post-Beittenmiller et al., 1984) and from a strain of S. cerevisiae var. uvarum ATCC9080 (Ruohola et al., 1986) and the MEL genes from E. coli (Liljestrom and Liljestrom, 1985), plant (Cyamopsis tetragonoloba; Overbeeke et al., 1989), and human (Bishop et al., 1986) have been cloned and sequenced. We have cloned and sequenced a MEL gene from S. carlsbergensis NCYC396, derived from the authentic

98 S. ca~~sbergensjsbottom-fermenting strain No. I described by E.C. Hansen in 1908 (Pedersen, 1986). In a preliminary report (Turakainen et al., 1988), we called the new gene MEL2 because it was the second yeast MEL gene sequenced. However, while this work was proceeding, we

I

2

E

I

E

I

1

B

IO kb

PPB

\ . .

I Bg

I Ho

XH

I

J

‘.

‘.

AAA II HB

I

Ha

&.A 1 Hc

I Ha

H

3.6 kb

3

ma1 location different from any other known MEL gene. Until allelism tests have been performed we call the new

Fig. 2. Restriction

map ofthe IO-kb EcoRI fragment

gene. The restriction

gene NCYC396 MEL according to its strain number in the National Collection of Yeast Cultures (Norwich, U.K.).

from the pBR322

E

II

\ . .

A

II BgHo

X

III

‘--. AA

and called MEL3 and MEL4 (Naumov, 1989). Here, we show that we have cloned another Saccharomyces MEL gene with divergent sequence homology and a chromoso-

I

6

Ii

HB

I I I I I

learned that the MEL gene from an S. cerevisiae strain was also called h4EL2 and another S. cerevisiae strain contained multiple MEL genes two of which were genetically isolated

RESULTS

I 4

I

0

containing

sites were derived from the digestions

clone containing

in section a or from its subclones.

the IO-kb EcoRI fragment Detailed

described

sites were con-

in Fig. 3. A, AIuI; B, BarnHI;

firmed from the nt sequence

shown

BglII; E, EcoRI;

Ha, HaeIII;

H, HindIII;

restriction

the MEL

of the DNA

Bg,

Hc, HiptcII; P, PstI; X, XbaI.

AND DISCUSSION

obtained. Its restriction map is shown in Fig. 2. No sites were observed for AvaI, BclI, BglI, PvuII or SalI. Hybridization of a Southern blot, containing fragments generated by several restriction enzymes, with the MEL1 probe showed that the MEL gene was located mainly in the 2.3-kb EcoRI-BamHI fragment. Also the adjacent 1.3-kb HindIIIBarnHI fragment showed weak hybridization. The lo-kb EcoRI fragment but not the 2.3-kb EcoRIBamHI fragment, when inserted into the E. co&yeast shuttle vector and transformed into yeast, conferred the Mel+ phenotype upon the S. cerevisiae Mel- strain (data not shown). Nutrient agar plates containing the chromogenic substrate clXGa1 (Sigma) were used to differentiate Mel+ from Mel- colonies (Tubb and Liljestrdm, 1986).

(a) Isolation of the MEL gene Genomic DNA was isolated from S. carlsbergensis NCYC396 and digested with EcoRI, Southern blotted, and hybridized using S. cerevisiue var. uva~~ MELI gene as a probe (Fig. 1). A weak signal was detected as a IO-kb band. EcoRI-generated DNA fragments about 10 kb in size were extracted from agarose gel and ligated to the EcoRI-cut pBR322. An E. cd’ transformant containing the pBR322 with a IO-kb insert hybridizing to the MEL1 probe was

1 2 *y-1Okb

(b} Nucleotide sequence of the MEL gene We determined the complete nt sequence of the 2.3-kb EcoRI-BamHI fragment and the 1.3-kb HindIII-BamHI fragment (Fig. 2). An oligo primer was used to confirm the sequence around the Barn HI site. The sequence is presented in Fig. 3. The nt sequence revealed a single ORF of 1413 bp, sufficient to encode a polypeptide of 471 aa, with a deduced M, of 52006. The direction of this ORF was consistent with the direction of the MEL transcript which was determined by using RNA-RNA hybridization and probes generated in vitro from the strands of the MEL gene as template for RNA polymerases. Only one of these clones generated a transcript which hybridized to yeast RNA in a dot-blot hybridization experiment (data not shown).

5 kb-

Fig. 1. Southern

hybridization

0.4 pg) and NCYC396

analysis

of ATCC9080

DNA (lane 2,1.0 pg). Genomic

with EcoRI endonuclease,

electrophoresed

DNA

(lane 1,

DNA was digested

on a 0.5% agarose

gel, trans-

ferred to a nitroceIlulose filter (Southern, 1975), and hybridized with MELI RNA probe (a 2%kb genomic BarnHI-Sal1 fragment in pGEM3 plasmid; Ruohola et al., 1986). The probe was prepared by the in vitro transcription system and labelled with [a-32P]UTP (PB10163, Amersham) (Melton

et al., 1984).

Fig. 3. The nt sequence of the MEL gene and the deduced aa sequence (GenBank accession number M58484). The nt numbering begins from the A in the start codon, ATG. The asterisk indicates the stop codon. The TATA-like sequence preceding the coding region and the consensus sequences for transcription termination are underlined. The putative sequence for GALB-mediated regulation is shown in brackets, DNA fragments were subcloned into pIBI76 plasmid (International [a-?S]thio-dATP

Biotechnologies,

(SJ304, Amersham).

to the first nt in each row.

Inc.) and the nt sequence

The oligo primers

were prepared

was obtained

by the chain-termination

using a DNA synthesizer

(Applied

method of Sanger et al. (1977) with

Biosystems

model 381A). Numbers

refer

AGACTGA~TTAAAGTGTTCA~CCCTATGTTCACTAACAACTAACTAkATCTATATA TTGGTCATCTTAGAGATTACAGTACCTAGAGTACTCGAEAAATATTCATGAC ACCTGAGACAACTAACCAGATCATATCGATATATCAGAAGATTTTCTCACGCCATGCCCG TTTTGATTGAGCACCTTATTTATACTTCCTGTTCACTAAAAAATACCTT~ACTCTCACA AATGAATTATTTEAAATATTGAACCACATCGC~TCAATACAATATC~AGTACACATC TCTTCACAAGGTTTCTCTAGCACGTATAATACGGCTTGGT AATTTTTCTCAAAAATGCTTTTTTCATAGTCTTTTAGTTGCGGTTTAGATCTCGGTATTG CCAAGTACTTCAACAGATCCACCACTGCCCTACTCTTTACCGATGGTGAAGG~CCTCAA TACTGAAACATTGGTTCTGCTGAGAGAATAAAATAAAAT~~ATTGATTAGTATTGGGAAAAT ACTACTCCARGACTATTACATCTGTATGCCTGTTATCGCATAGATCAAAGAGTGGAGTAA TTTGAAAAGTACAGAAAGCGGAAATTGAATTTTTCAAATCATTGAGTTATTTTTTTAGAA CTAGTTAAGAGTGCTGATGACATTGTAACAACATTTTTTCCCAC~ACCTTCCGATTCA TCATATTGCGGGGAATATTATGATATAGTAAGGCAAACX;GGACACGTTTAGATAGGTACC AGATTCTGTTTTAAACGACGCTACTAATCTCTCTTTTATTAGATGAATTACCATCAAAGGAT CGTGGGGTATCACTCAACATACACCGTCTGCTATTCATTTCATTCAATGCACCCTAGCTC CCTACTCCCAACAATGTTAAACCCCTACGAACCCTGACAAGGCGGATCGGAGACGAATAA TTCTGAAACATACTCCAGAGCTCCGAACTGACCCTGACCCTTTTTGAACTCAACACAAAAATTAAC CGCAGTTAAAGCTGATAATGATTATGTGTTTGTTAGGATATCAAAGATCTTGTGCTTAGA CATTCTCGTTAAGGTTTTAGTTGGGACCCCTTAGACCCCTCCCGA~TACGACAGTCCAT TACT~GGCCTAGTGTTGTCC~GACGGGGCAAAATTkATGCCT TCTCTATACTACATGATGATATTATGAACCATAGGATGA~GGTAGCTGATG~GGTT ATTAAAATGATAAGATAGGATGAAGCTAGCTGATGAAG(;TTATAAAAATGGAGATAAGAT TAATGATAAAATTCAAGCGGTATACAGTTGTTGTTTGTTCCTAGTGA~AAA~GTATCA 1 ATGTTTCTTTTATATCTTTTTACCTCTTTCGCCGCTGTGAGCGGTGTTCTCGGTTCGTCA MetPheLeuLeuTyrLeuPheThrSerPheAlaAlaValSerGlyValLeuGlySerSe~ 61 CCGAGTTACAATGGCCTTGGCCTCACCCCTCAAATGGGTTGGGACAATTGGAACACATTT ProSerTyrAsnGlyLeuGlyLeuThrProGlnMetGlyTrpAspAsnTrpAsnThrPhe 121 GCCTGCGACGTCAGTGAGCAATTGCTTTTGGATACCGCTGACCGGATTTCTGAAATAGGA AlaCysAspValSerGluGlnLeuLeuLeuAspThrAlaAspArgIleSerG~uIleGly 181 CTAAAGGATTTGGGTTACACTTATGTTATATTGGATGACTGCTGGTCTAGTGGCAGAACT LeuLysAspLeuGlyTyrThrTyrValIleLeuAspAspCysTrpSerS~rGlyArgThr 241 GCAAATGGTACACTCGTTGCAGACAAGGAAkftGTTTCCCATCATGTGGCT AlaAsnGlyThrLeuValAfaAspLysGluLysPheProAsnGlyMetSerH~sValAla 301 GACCACCTGCATAACAACAATTTTCTCTTTGGCATGTACTCATCTGCTGGTGAGTACACT AspHisLeuHisAsnAsnAsnPheLeuPheGlyMetTyrSsrSerAlaGlyGluTyrThr 361 TGTGCAGGATACCCTGGATCTTTAGGACACGAGGAAGAAGATGCGGAGTTTTTTGCTAGC CysAlaGlyTyrProGlySerLeuGlyHisGluGluGluAspAlaGluFhePheAlaSer 421 AATGGTGTTGATTACTTAAAGTACGACRRCTGTTACARCAhAGGTCAGTTTGGTGCACCA AsnGlyValAspTyrLeuLysTyrAspAsnCysTyrAsnLysGlyGlnPheGlyAlaPro 481 GAAACTTCCTACAAACGTTACAAGGCTATGTCTGATGCTTTGAACAAAACAGGCAGACCT GluThrSerTyrLysArgTyrLysAlaMetSerAspAlaLeuAsnLysThrGlyArgPro 541 ATATTTTATTCTTTGTGTAACTGGGGTC~GATTTGACGCATTATTGGGGGTCTGATATA IlePheTyrSerLeuCysAsnTrpGlyGlnAspLeuThrHisTyrTrpGlySerAspIle

-1390 GAATTCGAGA

TAGGTTTTTTCTTCAGATTAGTTTAAAGGGTACA~TTTATACGTTATGCAAAGCGTTT TGAAATTTTTACCAGCAAGTGACGAGACGACAGTATTTATATCTTTACAGACGCAATCAC GGGTAGCGCCGCATGGTTAAGAGTGCCCGAATTTTATTAAATCAGTCTTAGTATAATGATG GGTCTAAGTTATAATAGACTATGTTGCAAGTGCCAAAATAAATTA ATTTCCCTGCGGTTTTTAAACCATAAAGTATCGTATCGTTTG~TTGTTAGCA~TCTGGCCTAC GTGAGAGTTATCAAACACTATTGGTAGTGTCCGCAAAAAATGATG CTCCCCTAACATCATGGTTGCTTGACGTGAAAGTATGATATGATA~G~GATTCATTGTAAGC GCTAGAATAACTCAATAGCTCTTCCTTGTTATTTCAGCTTTGGGTTTTTTTTTT~GTTG TGCAACAGACCGATCATATTCAATTTTCATTTATTCATATTTAGCTCGAA~AATAAAAT TAATTTCAGGTGATATTAGTAGTTCAACTGCAATGAAGTCA TTTAGTATATATATATATATATATCTATCAATTAGAATAC ACACATGCCCTGGTATGTCCTTGGTTTCTGCTAGTTCCAACTC GATGGCGGATTTTTCGTTATGGAATCGCCTTATTTGTTTCTAGCGGTATTTGCCTT~AA GCT

601 GCAAACTCCTGGAGAATGAGTGGGGATATTTATCCTCAGTTTACTCGTCCTGACAGTAGG AlaAsnSerTrpArgMetSerG~yAspIleTyrProGlnPheThrArgProAspSerArg 661 TGTCCTTGCGATGGTGACCAGTTTGATTGCGCATATGCATATGCTGGTTTTCATTGTTCTATCATG CysProCysAspGlyAspGlnPheAspCysAlaTyrAlaGlyPheHisCysSerIleMet 721 AATATTCTARACAAAGCAGCTCCAATGGGGCAAAATGCGGCGAT AsnIleLeuAsnLysAlaAlaProMetGlyGlnAs~laGlyIleGlyGlyTrpAsnAsp 781 CTGGATAACTTAGAAGTCGGTGTCGGAAACTTGACAGATGATGAAGAGAAAGCGCATTTC LeuAspAsnLeuGluValGlyValGlyAsnLeuThrAspAspGluGluLysAlaHisPhe 841 TCTATGTGGGCAATGGTTAAGTCTCCACTAGTCATCGGCGCTGATGTGAATCACTT~~ SerMetTrpAlaMetValLysSerProLeuVa~I~eGlyAlaAspValAsnHisLeuLys 901 GCGTCTTCATATTCAATTTACAGTCAAGCTTCCGTAATAGCAATCAACCAGGATCCCAAG AlaSerSerTyrSerIleTyrSerGlnAlaSerValIleAlaIleAsnGlnAspProLys 961 GGAGTACCAGCAACAAGAGTTTGGAGACATCAAGTGCCACAAACTGACAAGTATGGTCAA GlyValProAlaThrArgValTrpArgHisGlnValProGlnThrAspLysTyrGlyGln 1021 GGTGAAATTCAATTTTGGAGCGGTCCACTTGATAACGGGTCTCTTA GlyGluIleGlnPheTrpSerGZyProLeuAspAsnGlyAspGlnVa~IleAlaLeuLeu 1081 AATGGGGGAATCAAACCAAGACCAATGAATACGAACTTGGAAGAAATTTTCTTTGACAGC AsnGlyGlyIleLysProArgProMetAsnThrAsnLeuGluGlu~lePhePheAspSer 1141 TACTTAGGTTTCGAGCAATTGTCCTCAAACTGGGATATTTATGACTTATGGGCTAACAGA TyrLeuGlyPheGluGlnLeuSerSerAsnTrpAspIleTyrAspLeuTrpAlaAsnArg 1201 GTTGACAATGCAACATCGGCTAACATTCTGAATAATAACGCTACTATT ValAspAsnAlaThrSerAlaAsnIleLeuAsnAsnAsnSerValG~yAsnAlaThrIle 1261 TACAATGCTACCGCACTATCATACAAGGATGGGATGGCAAAGAATGATACCAGATTATTC TyrAsnAlaThrAlaLeuSerTyrLysAspGlyMetAlaLysAsnAspThrArgLeuPhe 1321 GGTACCAAAATCGGCAGTATTTCTCCAGATGGCTTGCTTAACAC~CTGTTCCAGCACAC GlyThrLysIleGlySerIleSerProAspGlyLeuLeuAsnThrThrValProAlaHis 1381 GGAATTGCTTTCTATAGGTTAAGACGGTCTACTTAATAAGAAGATTTCCATCAACCGGG GlyIleAlaPheTyrArgLeuArgArgSerThr *

100

123

5-10% have extra AUG codon upstream from the known translation start site (Kozak, 1986). The promoter region of the NCYC396 MEL gene lacked the C + T-rich block and the CAAG sequence, found at the tsp in a number of yeast genes encoding abundant mRNAs (Dobson et al., 1982). The entire 5’-untranslated sequence of most S. cerevisiae mRNAs is very rich in A residues (Hamilton et al., 1987). In the MEL1 gene, the noncoding 5’ region preceding the AUG start codon is composed of 20 nt, 9 of them A residues (Liljestrem, 1985). In the NCYC396 MEL gene, A comprised 11 out of 20 nt upstream from the AUG start codon (-1 to -20). These values agree with the average occurrence of 47% A residues within 5’-untrans-

Ukb

lated regions (Cigan and Donahue, 1987). The expression of the MEL1 gene is controlled

Fig. 4. Northern

hybridization

analysis

(lane 3) conditions. same medium,

galactose

4 h. The culture

to one portion.

noninduced isolated

The cells were grown

to McAlister

cells was denatured

on 1% agarose/2%

cellulose

filter and hybridized

RNA

probe

Hind111 fragment

and Finkelstein

with formamide

phoresed MEL

and the cells were grown for

into two portions,

sample was taken before galactose

according

(2.3-kb

in YPD medium with 3 y0

glucose. The culture was diluted in the

(2%) was added,

was divided

in

(lane 2) and glucose-repressed

Cells were grown overnight

glycerol and 2 % ethanol replacing

added

of total RNA from NCYC396

(lane l), galactose-induced

noninduced

and glucose for another addition.

(9%) was 1.5 h. The

Total RNA was

(1980). RNA from 10’

and formaldehyde,

and electro-

formaldehyde gel, transferred to nitrowith a 32P-labeled anti-sense NCYC396

EcoRI-BumHI

fragment

+ 1.3-kb BarnHI-

in pIBI76). The arrows show the migration

ofribosomal

RNA-subunits.

(c) Northern hybridization analysis The size of the mRNA in vivo was estimated from the Northern blot hybridized with the NCYC396 MEL RNA probe. The mRNA was estimated to be 1.5 kb (Fig. 4), which is in good agreement with the size of the presumed protein (52 kDa). mRNA encoded by MEL1 gene has been found to be slightly larger (Post-Beittenmiller et al., 1984; Ruohola et al., 1986). Because both genes code for a protein of 471 aa, mRNA from the NCYC396 gene must contain shorter untranslated regions. Human clGa1 mRNA has been estimated to be 1.45 kb (Bishop et al., 1986). (d) The 5’4anking region The 5’-TATAAA sequence, the proposed transcription initiation signal, was located between nt -80 and -75, which is a typical distance from the start codon. The NCYC396 MEL gene had two out-of-frame ATG-triplets between this TATAAAA sequence and the suggested translation start codon. These two ATG triplets were both in the same frame and were followed by two stop codons TAG and TGA just before the third ATG. Of eukaryotic mRNAs

by GAL4

protein and thus its 5’ region contains a sequence required for the binding of this protein (Liljestrdm, 1985). The consensus GAL4-protein recognition sequence proposed by Giniger et al. (1985) is a 17-bp dyad symmetric sequence 5’-CGGA(G/C)GAC(A/T)GTC(G/C)TCCG. A related sequence, 5’-CGGCCTAGTGTTGTCCG, was found in the 5’-flanking region of the NCYC396 MEL gene (nt -239 to -223). In yeast genes, the -3 position flanking the AUG start codon strongly favours A and avoids pyrimidines (75 y0 A, 13 % G, 8 % C and 3 % U ; Cigan and Donahue, 1987). In NCYC396 MEL mRNA, the -3 position was occupied by U, as is the case in another galactose-inducible gene, GAL10 (Johnston and Davis, 1984). An A+ U transversion at -3 may decrease the efficiency of translation initiation (Zitomer et al., 1984; Werner et al., 1987). In animal cells, R- 3 --f Y transversion has been shown to decrease the mRNA translation efliciency about 20-fold (Kozak, 1986). Incidentally, the aGa1 activity in NCYC396 yeast strain was remarkably lower than the activity in the

TABLE

I

Production strains

of aGa1 in the absence

NCYC396

and presence

of galactose

by the yeast

and ATCC9080 aGa1 activity” Medium -gal

NCYC396

0

ATCC9080

0.02

Cells + gal

-gal

0

+ gal

0.0004

26.8

1.26

2.29

82.3

il The yeast cells were grown for 8 h on YPD medium,

which contained

3% glycerol and 2% ethanol instead of glucose in the absence (-gal) presence ( + gal) of 2% galactose. The enzyme activity was measured the medium and in cell samples as described

by Kew and Douglas

Enzyme activity is expressed in nmol substrate pyranoside) hydrolyzed/min/lO’ cells.

or in

(1976).

(p-nitrophenyl-a-galacto-

101 E.&i lMM-A-KITFI-AGSTIFVKSSPSYNGLGLTPQMGWDNWNTFAC S.url. 1 V___________--------____ s.cf?r.1 AE----Q--P---NS--H-Gplant 1 LD---AR--T---LH-ER-M-NLHumn 1

ILG

D _ -

-FHREA-K--HIALMDVSEQLLLDTA DRISEI __________ ____DL IN-NVVRE--AMVST I--K-FMEM-ELMV--

iDCQEEPDSC' * DPTR-EESHI-VRKLMD - A-ASGKI-CHTQQ--ALEDADFVV-E.coli42 GYTYVILDDCW SSGRTANGT LVADKE KFPNGMSHVA S.car1.42 GLKDL ___-M --K-I-__----__DSD_F ____EQ _---e-C_S.cer.42 --Q-IN-----AELN-DSE-N M-PNAA A--S-IKAL--AAPlant 39 --E-LCI----MAPQ-DSE-R -Q--PQ K--H-IRQl.Humn 49 -W--A

E.&i 86 S.car1.83 s.ccr.83 Plant 81 Hm 91

FQICCYEPCTVTD-EVCKRH-L-Q-I DHLHNNNFLFGMYSSAG -----_S_--______-YV-SKGLKL-V--D-NYV-SKGLKL-I-ADV*

-DTL-PG-IMRALRTIPH EYTC AGYPGSLGHEEEDAEFFA ___~ ________R-m-_-Q__NQ--SKRM--------Q--KT--_F_--F-yyDI_mQTm_ NK--

E.coll130 S.Carl.l22 s.cer.122 Plmt 121 Htmm 1W

LWQICEDMTEV-PDATMLNYVNPMAMNTW--yARYPHIKQVGLCH SNGVDYLKYDNCYNKGQFGAPETSYKRYKAM N-R---------__--___T--I--H----IS"K--PP- w_---------E-LDw--_L__FmG-_ CDSL-NLADG--H-

E.cdi 175 S.carl.1~ S,cer,l& Plant 163 Hmm 176

- VQGTAEE-ARDLNI-P-TLRY-CA-INHMA-YLELE-KTA--St SLCNWGQDLTHYWGSDIA NSWRMSCDIYPQFTRPDSRCPCDG ----------F----G--_~-----VTAE-----_~-----M-E--WEDPQI-AKS-G ----TT--EDNWNPYM-PFQKP YTERYCNHW-NFA-I - -E-

E.wli 2.X s.car1.m s.cer.m PLanL 193 !!.xm 21"

VNLYPELLA--E--QAPKPN-HG-TRCQNIVRYEMFKKL-YFVTE DQFDCAY AG FHCSI MNILNKAAP~GQ~AGIGGWNDLD -Ey--K__ ~~___ ___~--_~~----~-~~----~_Sy--p-----p_ MT-ADSND-W3 c r.1 !'" '! ? S!!K SI !'s I I," 1.: TSF

E.&i 265 s.car1.245 S.m.245 Plant 217 Hmm 246

ssHFAEY-PWFI-PGREDLIERY-v--DEYPKRc-EQ-ANWHK NLEVGVGNLTDDEEK AHFSNWAMVKSPL VIGADVNHL ____~__________ ____~______~~ I---N--NM----N-GM-TE-YR S---I--LA-A-I.V-C-IRAM M-VT-NFG-SWNQQV TQMAL--IMAA-FMSN-LR-J *

E.di 311) s.car1.285 s.cer.285 Plant 257 Hurm 2%

ELEE-KK--R-D-KPSREYASTIMNAIWTGEPSVI--NVRNDGLI SYSIYSQASVIAINQDPKGVPATRVWRHQVPQTDKYGQGEIQFb~S ----______~-____SN-I______-yy_SD---_--~~THELI-N-E----V---KL-QGKK-KSAKALLQDKD--------LKQGY-LR-G-NFEVW-

SDALNKTGRPIFY _____________ GK--LSS-----F -L---R---S-V*

"

i

NDL-

DNPQ-CC-EV-C-VDAN--Q-TKVG-LPSHLAALMQTNI E.&i 3% GPL DNGDQV IALL NGGIKPRPMNT NLEEIFFDSYLG s.car1.m S.cer.3x) --_ -----V___ _mmSVS___mT__----e-N-- -W - RSSS-ATVPlant 297 ---S--KVAASWS-IGfkmn 3x) - - AWAVAM -XRQ EIG-SY-AVAt c E.di 397 s.car1.369 s.cer.369 Plant 329 Hmm 362

* * * MMDPHTAAVL-IDE-SANILNNNSVGNATIYNATALSYKD --GR-KTATGIL----EQ----GE-SAEIDSHACKM-VL-PR-37C YEWTSRLR-HI-P-GTVLLQ-ENTM * *

TLLTEA-LTENRD--YH-A LSSNWDIYDLWANRVDNAT -T-T-----___----_S_A-QGTTV-AR---EHSTQSLV CNPACF-TQ-LPVKRKLGF *

*

KAS ___ DDT SPQ

V-A RPL-

NVFEQ SKK --KGVA

--

VD-

*

E.&i 437 LI-AHGDW-P-WLHR451 s.car1.413 GMAKNDTRLFGTKIGSISPDGLLNTTVPAHGIAFYRLRRST471 s.cer.413 _LS-____---Q----~--~~I-___----__--------~-~~~~ Flant

Q-SLK-

HImma

L-414 *

Fig. 5. Protein

sequence

alignment

of aGal

*

from yeasts

tetrugonoloba; Overbeeke

et al., 1989) human

(Bishop

shown

single-letter

are aligned

by their standard

N-glycosylation

sites in yeast sequences

codes,

(S. cereviriae:

Sumner-Smith

et al., 1986) and E. coli (Liljestrom to maximize

(above the sequences:

identity

et al., 1985; S. corlsbergensb:

this study),

and Liljestrbm,

show the identical

with the NCYC396

1987). Dashes

MEL sequence.

S. carlsbergensis; below: S. cerevisiae).

The asterisks

plant denote

(Cyamopsis aa. The aa, the putative

102 strain ATCC9080

(Table I), which has A _ 3 in the MEL1

mRNA (Liljestrdm, 1985). The highly expressed yeast genes have A - 3 in all 18 cases (reviewed by Hamilton et al., 1987). This biased nt usage is more pronounced in highly expressed genes than in genes in general. Yeast mRNAs

codons were used (not shown). The codon bias index (Bennetzen and Hall, 1982) of the mRNA was 0.23 which is the same as the index of MEL1 (Liljestrbm, 1985). The aa sequence contained nine putative N-glycosylation sites, six of which were conserved compared to MEL1 (Fig. 5).

show a preference for U at positions + 4 (39%) and + 6 (47%) (Cigan and Donahue, 1987). The NCYC396 MEL mRNA also had U at these positions.

To characterize the aGa1 protein as deduced from its gene, the aa sequence was analyzed for hydropathic profile.

(e) The characteristics

similar.

of MEL signal sequences

The signal peptides of yeast secretory proteins contain 17-20 aa, as reported also for ctGa1 (MEL1 product; Sumner-Smith et al., 1985; Ruohola et al., 1986). Although the signal sequence of the MEL1 (ATCC9080) protein and the presumed signal sequence of the NCYC396 MEL protein had only 7 identical aa out of 18 aa, they both fulfill the requirements of a signal sequence: they are very hydrophobic and the last residue is a small neutral aa, Gly, which allows the cleavage by signal peptidase (von Heijne, 1983). At least one positively charged residue is present in yeast signal sequences (von Heijne, 1985). In the deduced signal sequence of the MEL1 protein a Lys residue is present at aa -5. The NCYC396 MEL protein lacked any charged residues at the signal peptide region except the initiator Met which is assumed to provide one positive charge in eukaryotes (von Heijne, 1985). On the other hand, no clGa1 activity was secreted into the culture medium by the strain NCYC396 in conditions where the strain ATCC9080 secreted 25 y0 ofthe enzyme activity into the culture medium (Table I). This supports the importance of the charged residue for the functional signal sequence of yeast secretory proteins. (f) Comparison of MEL genes and their protein products The differences in the restriction maps and in the intensities of the hybridization of MEL1 probe to the two MEL genes suggested that the genes are not identical. The rule of thumb that 10% dissimilarity in the nt sequence reduces the hybridization intensity by 50% (Maniatis et al., 1982) suggested 70-80% identity between the two yeast MEL genes. The comparison of nt sequences revealed 77.1% identity in the protein coding region. Based on the alignment shown in Fig. 5, the yeast MEL proteins exhibit 83.0% sequence identity of mature proteins (as defined by Beckman’s Microgenie program). The degree of similarity reached 87.9% when accepted aa replacements were included (Be-Leu-Val, Ser-Thr, Phe-Tyr, Arg-Lys, Asp-Glu). The homology between the NCYC396 MEL protein and ctGal from other species was lower (17.3% between yeast and E. coli; 29.6% between yeast and human; 35.3% between yeast and plant). A summary of the codons used in the coding region of NCYC396 MEL showed that 59 out of the 61 possible

Comparison of the profiles of NCYC396 MEL protein and MEL1 protein indicated that the two proteins are very Only minor

differences

were observed,

the most

remarkable changes being a hydrophobic peak in NCYC396 MEL protein lacking in MEL1 protein (aa 142 in Fig. 5) and a hydrophobic peak in MEL1 protein lacking in NCYC396 MEL protein (aa 185-190 in Fig. 5). The putative N-glycosylation sites were found in the hydrophilic regions, except Asn404 and Asn436, suggesting that they are located on the surface of the protein and available for addition of sugar side chains. (g) The 3’-flanking region The tripartite consensus sequence 5’-TAG...TAGT. ..TTT has been found in many yeast genes and has been proposed to be important for transcription termination and polyadenylation (Zaret and Sherman, 1982). In the 3’-flanking region of the NCYC396 MEL gene one set

Fig. 6. Pulsed-field NCYC396

gel electrophoresis

of chromosomes

(lanes 1 and 3) and ATCC9080

show the EtdBr-stained

gel, lane 3 shows the corresponding

gram of lane 1 hybridized

with MEL probe

shows the autoradiogram

of lane 2 hybridized

reman

numerals

from the strains

(lanes 2 and 4). Lanes 1 and 2

on the left correspond

autoradio-

from NCYC396 with MELl

to the positions

and lane 4 probe.

The

of the chromo-

somes in the standard yeast strain YNN295 (BioRad). Yeast chromosomes were prepared as described by Carle and Olson (1985) and separated by OFAGE (Carle and Olson, 1984) for 17 h at 280 V and 110 mA at 16°C with a switching interval of 50 s. Gels were stained with EtdBr. DNA was denatured and transferred onto a nitrocellulose filter after a 30-min depurination

step in 0.25 N HCl. Arrowheads,

origin.

103 could be identified

between

nt 21 and 58 after the trans-

lation stop codon TAA. The NCYC396 MEL gene contained only 20 nt between the stop codon and the tripartite sequence, whereas MELI has 110 nt (Liljestrom, 1985). Poly(A) addition usually takes place lo-40 bp downstream from the TAGT-sequence of yeast mRNAs (Zaret and Sherman, 1982). This places the proposed end of the NCYC396 MEL mRNA between nt 1470 and 1500 from the start codon

(Fig. 3).

T.F.: Sequence

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initiator

and structural

regions

features

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M.J., Tuite, M.F., Roberts,

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R.E., Conroy,

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Acids Res. 10 (1982) 2625-2637.

E., Vanum,

S.M. and Ptashne,

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C.K. and deBoer,

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