,B-Galactosidase in Escherichia coli - Journal of Bacteriology
Nucleotide Sequence of α-Galactosidase MEL Gene from Zygosaccharomyces mrakii
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News & Notes Nucleotide Sequence of a-Galactosidase MEL Gene from Zygosaccharomyces mrakii Yuji Oda, 1 Tomoko Fujisawa 2 1 Upland Agriculture Research Center, Hokkaido National Agricultural Experiment Station, Memuro, Kasai, Hokkaido, 082-0071, Japan 2 Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan Received: 10 February 2000 / Accepted: 29 March 2000 Abstract. The region encompassing the a-galactosidase MELr gene was amplified from Zygosaccha- romyces mrakii IFO 1835 T by inverse-PCR and then sequenced. The nucleotide sequence of this region revealed a single open reading frame of 1410 bp encoding a 470 amino acid protein with a molecular weight of 51,909. The similarity of the deduced mature protein to other yeast a-galactosidases was 63.3% to Zygosaccharomyces cidri, 71.5% to Torulaspora delbrueckii, and 70.7–73.9% to Saccharomyces species. The nucleotide and amino acid sequences are deposited in the DDBJ/EMBL/GenBank database under Accession Number AB030209. Yeasts were originally classified on the basis of their morphological and physiological characteristics, but these classifications are now supported by molecular techniques such as nuclear DNA-DNA hybridization [12]. These methods have not only contributed to accu- rate delimitation of species, but also have uncovered new species. The genera Zygosaccharomyces and Torula- spora are related to each other; some of these species have been included in the genus Saccharomyces [4]. James et al. [3] have analyzed the sequence of 18S rRNA and two internal transcribed spacers, ITS1 and ITS2, and proposed Z. microellipsoides and Z. mrakii should be combined with T. delbrueckii, T. pretoriensis, and T. globosa. Using ITS data, they found a close relationship between Z. mrakii and T. delbrueckii. Yeast MEL genes encoding a-galactosidase have been studied in detail by Naumov et al. [5] and provided significant information on the delineation of Saccharo- myces species. In the genus Zygosaccharomyces, the MEL gene of Z. cidri has been cloned, sequenced, and used as a probe to detect the corresponding genes in the other Zygosaccharomyces strains [11]. Although the Torulaspora species usually do not ferment melibiose, we have amplified the MELt gene from the genomic DNA of T. delbrueckii IFO 1255, an exceptional melibi- ose-fermenting strain, and determined its sequence [8]. These structural homologies showed that T. delbrueckii is phylogenetically more related to Saccharomyces sensu stricto species than to Z. cidri. This paper reports isola- tion and characterization of the MELr gene of Z. mrakii. The cells of Zygosaccharomyces mrakii IFO 1835 T were grown and used for the preparation of genomic DNA as described previously [8]. Sequences of primers and those target positions corresponding to MELt gene of T. delbrueckii [8] are as follows: TDM-16F (59-CAGAT- GGGTTGGAACAACTGGAACA-39, 91–115), TDM- 17R (59-TTGCAGACTTAACAATAGCCCACAT-39, 868 – 844), ZRM-21F (59-ACCCAGGTGGTTGGAAT- GATCTAGA-39, 761–779), ZRM-22R (59-AAAG- CAATTGCTCCGTGACGTGCAC-39, 156 –132). The PCR reaction was first done with primers TDM-17F and TDM-18R, and the sequence of the amplified product was determined. Primers ZRM-21F and ZRM-22R were successively synthesized to obtain the entire region span- ning the MELr gene. The nucleotide sequence was de- termined for 2400 bp (Accession No. AB030209) of a 6-kb fragment amplified by inverse-PCR from genomic DNA that had been previously digested to completion with HindIII and then self-ligated. The sequence con- tained a single ORF of 1410 bp sufficient to encode a Correspondence to: Y. Oda; e-mail: [email protected] CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 220 –222 DOI: 10.1007/s002840010123 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2000
Transcript of Nucleotide Sequence of α-Galactosidase MEL Gene from Zygosaccharomyces mrakii
News & Notes
Nucleotide Sequence ofa-GalactosidaseMEL Gene fromZygosaccharomyces mrakii
Yuji Oda,1 Tomoko Fujisawa2
1Upland Agriculture Research Center, Hokkaido National Agricultural Experiment Station, Memuro, Kasai, Hokkaido, 082-0071, Japan2Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan
Received: 10 February 2000 / Accepted: 29 March 2000
Abstract. The region encompassing thea-galactosidaseMELr gene was amplified fromZygosaccha-romyces mrakiiIFO 1835T by inverse-PCR and then sequenced. The nucleotide sequence of this regionrevealed a single open reading frame of 1410 bp encoding a 470 amino acid protein with a molecularweight of 51,909. The similarity of the deduced mature protein to other yeasta-galactosidases was 63.3%to Zygosaccharomyces cidri, 71.5% toTorulaspora delbrueckii, and 70.7–73.9% toSaccharomycesspecies. The nucleotide and amino acid sequences are deposited in the DDBJ/EMBL/GenBank databaseunder Accession Number AB030209.
Yeasts were originally classified on the basis of theirmorphological and physiological characteristics, butthese classifications are now supported by moleculartechniques such as nuclear DNA-DNA hybridization[12]. These methods have not only contributed to accu-rate delimitation of species, but also have uncovered newspecies. The generaZygosaccharomycesand Torula-spora are related to each other; some of these specieshave been included in the genusSaccharomyces[4].James et al. [3] have analyzed the sequence of 18S rRNAand two internal transcribed spacers, ITS1 and ITS2, andproposedZ. microellipsoidesand Z. mrakii should becombined withT. delbrueckii, T. pretoriensis, and T.globosa. Using ITS data, they found a close relationshipbetweenZ. mrakii andT. delbrueckii.
Yeast MEL genes encodinga-galactosidase havebeen studied in detail by Naumov et al. [5] and providedsignificant information on the delineation ofSaccharo-mycesspecies. In the genusZygosaccharomyces, theMEL gene ofZ. cidri has been cloned, sequenced, andused as a probe to detect the corresponding genes in theother Zygosaccharomycesstrains [11]. Although theTorulasporaspecies usually do not ferment melibiose,we have amplified theMELt gene from the genomic
DNA of T. delbrueckiiIFO 1255, an exceptional melibi-ose-fermenting strain, and determined its sequence [8].These structural homologies showed thatT. delbrueckiiis phylogenetically more related toSaccharomycessensustricto species than toZ. cidri. This paper reports isola-tion and characterization of theMELr gene ofZ. mrakii.
The cells ofZygosaccharomyces mrakiiIFO 1835T
were grown and used for the preparation of genomicDNA as described previously [8]. Sequences of primersand those target positions corresponding toMELt gene ofT. delbrueckii[8] are as follows: TDM-16F (59-CAGAT-GGGTTGGAACAACTGGAACA-39, 91–115), TDM-17R (59-TTGCAGACTTAACAATAGCCCACAT-39,868–844), ZRM-21F (59-ACCCAGGTGGTTGGAAT-GATCTAGA-39, 761–779), ZRM-22R (59-AAAG-CAATTGCTCCGTGACGTGCAC-39, 156–132). ThePCR reaction was first done with primers TDM-17F andTDM-18R, and the sequence of the amplified productwas determined. Primers ZRM-21F and ZRM-22R weresuccessively synthesized to obtain the entire region span-ning theMELr gene. The nucleotide sequence was de-termined for 2400 bp (Accession No. AB030209) of a6-kb fragment amplified by inverse-PCR from genomicDNA that had been previously digested to completionwith HindIII and then self-ligated. The sequence con-tained a single ORF of 1410 bp sufficient to encode aCorrespondence to:Y. Oda;e-mail: [email protected]
CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 220–222DOI: 10.1007/s002840010123 Current
MicrobiologyAn International Journal© Springer-Verlag New York Inc. 2000
polypeptide of 470 amino acids with a deduced molec-ular weight of 51,909.
In the 59-non-coding region ofMELr gene, the pro-posed transcription initiation signal, 59-TATAAA-3 9,was located between283 and277. TheSaccharomycesMEL genes are induced by galactose through the actionof the positive regulatory protein GAL4, which recog-nizes the sequence 59-CGGA(G/C)GAC(A/T)GTC(G/
C)TCCG-39 [2]. In Z. mrakii, a related sequence, 59-AGGACTACTGTAGTCCG-39, was found upstream tothe 2198 to 2182 region. In addition,Z. mrakii se-quence, 59-GAGTGTGGG-39 (2317 to 2308), resem-bled the binding sequence ofS. cerevisiaeMIG1 protein,59-(A/T)(A/T)(A/T)T(G/C)(T/C)GGG-39, which medi-ates catabolite repression [7]. The presence of theserecognition sequences, located a typical distance from
Fig. 1. Dendrogram constructed by the neighbor-joining method from amino acid sequences of yeasta-galactosidases. Bar5 10% differencebetween sequence. Retrieved sequences used in the present study areZ. cidri MELz(L24957);T. delbrueckii MELt(AB027130);S. cerevisiae MEL1(X03102), MEL2 (Z37508), MEL5 (Z37511), andMEL6 (Z37510); S. paradoxus MELp(X95505); S. pastorianus MELx(M58484), andSaccharomycessp.MELj (X95506).
Y. Oda, T. Fujisawa:MELr Gene ofZygosaccharomyces mrakii 221
the start codon, is found for theMEL genes ofZ. cidriandSaccharomycesspecies.
In the deduced amino acid sequence of theMELrgene product (Accession No. AB0302069), a signal pep-tide composed of 18 residues was observed at the aminoterminus. There were eight putativeN-glycosylation sites(N-X-S/T), and five sites coincided with those ofZ. cidri,T. delbrueckii, andS. cerevisiae[6, 8, 11].
The amino acid sequence was aligned by using theCLUSTAL W program [10]. A distance matrix and neigh-bor-joining phylogenetic tree were obtained with the PRO-TDIST, NEIGHBOR, and DRAWGRAM programs, in thePhylogeny Inference Package (PHYLIP), version 3.5c [1].Similarities of amino acid sequence of the presumed matureprotein were 63.3% with MELz, 71.5% with MELt, 71.4%with MEL1, 72.1% with MEL2, 72.9% with MEL5, 73.2%with MEL6, 70.8% with MELp, 70.7% with MELx, and73.9% with MELj. The tree topology was similar to thosebased on ITS sequences [3, 9] except for extended branchesin the two groups of closely related species,Z. mrakiiandT.delbrueckiiandSaccharomycessensu stricto species (Fig.1). This difference may be related to the fact that sequencesof MEL genes are more variable than those ofMRS, NAM2,or ARG4genes [6]. The depicted tree shows that divergencebetweenMELr and MELt is larger than that among theMEL gene family ofSaccharomycessensu stricto species.
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