World Journal of Microbiology & Biotechnology 13, 103-117. Ribosomal RNA genes of Endomyces fibuliger: isolation, sequencing and the use of the 26S rRNA ...
World Journal of Microbiology & Biotechnology 13, 103-117
Ribosomal RNA genes of Endomyces fibuliger: isolation, sequencing and the use of the 26S rRNA gene in integrative transformation of Saccharomyces cerevisiae for efficient expression of the x-amylase gene of Endomyces fibuliger C.W. Yip, C.W. Liew and B.H. Nga* Endomyces fibuliger is a dimorphic yeast which is homothallic and exists predominantly in the diploid phase with a brief haploid phase. A repeat unit of the ribosomal RNA genes, or rDNA, from E. fibuliger 8014 met has been isolated, cloned and sequenced. In this report, the sequences of the 17S, 5.8S and 26S rRNA genes are presented. Homology between the sequenced rRNA genes and those of closely-related yeast strains, particularly Saccharamyes cerevisiae and Candida albicans, was observed. As a step towards the eventual development of a transformation system for the yeast E. fibuliger, an integrative plasmid containing the 5.8S and a part of the 26S rRNA gene, a selectable marker conferring resistance to the G418 antibiotic and a reporter gene, the a-amylase (ALP1) gene of E. fibuliger, was constructed. This plasmid was linearized at a unique restriction site within the 26S rRNA gene, and transformed into S. cerevisiae INVSC2 MATa his3 ura3 using the lithium acetate method to test the functionality of the vector system. Transformation into S. cerevisiae INVSC2 MATa his3 ura3 was by virtue of the extensive homology between the sequenced 26S rRNA gene of E. fibutiger 8014 met and that of S. cerevisiae, so that homologous pairing and integration into the recipient chromosome was possible. The G418-resistant S. cerevisiae transformants produced halos on starch medium due to hydrolysis by a-amylase, and they were further analysed by Southern hybridization with the ALP1 gene and the gene encoding the aminoglycoside 3'phosphotransferase I enzyme which confers resistance to the G418 antibiotic. A band of 13.7kb which corresponded to the linearized size of the transforming plasmid DNA was obtained on the autoradiogram, suggesting that tandem copies of the plasmid DNA are present in the chromosome. Finally, an assay of the aamylase enzyme secreted extracellularly was performed on the transformants. Key words: 0e-amylase, Endomyces fibuhger, G418 resistance, integrative transformation, ribosomal RNA genes, Saccharomyces cerevisiae.
The eukaryotic ribosome contains four RNA species designated 5S, 5.8S, 17S and 26S ribosomal RNAs. The genes encoding rRNA, also known as rDNA, are organized in tandem head-to-tail reiterations at between 100 and 140
The authors are with the Department of Microbiology, National University of Singapore, Lower Kent Ridge Road, Singapore 119260; fax: 65 7766872. * Corresponding author,
copies per yeast genome (Schweizer et al. 1969; Petes 1979; Mandal 1984). In most eukaryotes, the genes for 17S, 5.8S and 26S rRNAs are cotranscribed in that order as a single transcription unit, together with external and internal spacers (ETS and ITS). Transcribed units are separated by nontranscribed spacers (NTS). The transcription unit is between 8 and 10 kb in size depending on the spacer lengths (Mandal I984). The 5S rRNA gene is found on a separate
9 1997 Rapid Science Publishers
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C. W. Yip, C. W. Liew and B.H. Nga
transcription unit and is often transcribed in the opposite orientation using its own promoter. The 5S rRNA gene is found in the NTS within the rDNA repeat unit in Saccharomyces cerevisiae (Long & Dawid 1980) but in Schizosaccharomyces pombe, it is dispersed in the genome (Mao et al. 1982). Integrative vectors which contain the ribosomal RNA (rRNA) gene sequences are designed to integrate via homologous recombination at the rDNA locus of the yeast genome, and to give high-copy-number integration and increased expression of a homologous or heterologous gene present in the same plasmid. Such vectors have been used for Saccharomyces cerevisiae (Lopes et al. 1989) and Kluyveromyces lactis (Bergkamp et al. 1992). The integrative transformation system of Y. lipolytica has been described by Davidow et al. (1985). Replicative plasraids which carried the Y. lipolytica autonomously replicating sequence ARSJ8 have also been described for this yeast (Fournier et al. 199I, 1993). In a recent publication, a multiple-copy integrative transformation system using an rDNA plasmid in Y. lipolytica was reported by Le Da]l et al. (1994). This report deals with the isolation, cloning and sequencing of the entire rDNA repeat unit of the yeast Endomyces fibuliger and the use of the 26S rRNA gene in integrative transformation of Saccharomtyces cerevisiae based on homologous pairing at the rDNA locus of the yeast genome. Endomyces fibuliger was first isolated from chalky bread by Lindner in 1907. In Asia, strains of E. fibuliger have been used in the production of Chinese rice wine and tape. It is a dimorphic yeast which is homothallic and has a predominant diploid phase and a brief haploid phase (Kreger-van Rij 1984). E. fibuliger produces hat-shaped ascospores within spheroidal asci which are usually borne on lateral branches of the main hyphae. It is a protein secretor and wild type strains produce and secrete glucoamylase (EC 3.2.1.3) and ~-amylase (EC 3.2.1.1). It is a slow-fermenting yeast with the ability to convert sugars such as glucose and maltose to alcohol and carbon dioxide. The construction of a vector which can facilitate transformation into S. cerevisiae by high-copy-number integration is described. This vector consists of a part of the 26S rRNA gene sequence from E. fibuliger, the kan' gene of Tng03 (Jimenez & Davies 1980; Oka et al. 1981; Webster & Dickson 1983) encoding aminoglycoside 3'-phosphotransferase I (EC 2.7.1.95) which confers resistance to the G418 antibiotic, and the co-amylase (ALPI) gene from E. fibuliger.
Materials and Methods Strains of Yeast E. fibuliger 8014 met is a natural isolate from tape ragi, as described by Nga et al. (1994). S. cerevisiae INVSC2 MATa his3 ura3 was obtained from Invitrogen Corporation.
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Bacterial Strain and Plasmid DNAs Escherichia coli DH50r F- ~80d lacZ AM15 A(lacZYA-argF) U169 deoR recAl endAl hsdR17 (rK-mK+) supE44 )L- thi-1 gyrA96 relA1 was from Bethesda Research Laboratories. Plasmid pINA24 (van Heerikhuizen et al. 1985) containing the 7.7 kb HindIII fragment of Yarrowia lipolytica rDNA was from Dr. Philippe Foumier, Institut National Agronomique, Thiverval-Grignon, France. Plasmid pYN12-K13 constructed in this study contained the rDNA repeat unit of E. fibuliger cloned into the KpnI site of the pUC18 vector (Yanisch-Perron et al. 1985). All subclones containing parts of rDNA constructed either for sequencing or as vectors for transformation were derived from this parent plasmid. Plasmid pYN1826ALP1 contained the ALP1 gene of E. fibuliger strain HUT7212 from pSf0r from Professor Ichiro Yamashita, Centre for Gene Science, Hiroshima University, Japan (Yamashita el al. 1985a) cloned into the HindIlI - SalI sites of pYN1826 which contained the 5.8S and 26S rDNA sequences of E. fibuIiger 8014 met cloned into the EcoRI site of pUCI8 vector. P]asmid p371 containing the aminoglycoside 3'-phosphotransferase I structural gene (APH(3')I) of Tng03 between the S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (GAP) gene promoter and transcriptional terminator was a gift from Professor Satoshi Harashima, Department of Biotechnology, Faculty of Engineering, Osaka University, Japan. The template for amplification of the APH(3')-I structural gene was from p18-APH which contained the 1.2 kb SalI- EcoRl fragment of p371 cloned into pUC18. The DNA fragments carrying the E. fibuliger HUT7212 glucoamylase (GLUI) promoter and transcriptional terminator used as templates for PCR were in p18GLU1 which carried the entire GLU1 gene on a 2.54 kb Pstl fragment from pSfGlul, pSfGlul was from Professor Ichiro Yamashita (Yamashita et al. 1985b). Plasmid p18GPT was constructed with the APH(3')-I gene between the GLUI promoter and transcriptional terminator inserted into the SalI - Kpnl sites of the pUC18 vector. Plasmid pYN1826ALP1-GPT was constructed by ligating the insert from p18GPT into the SaII- KpnI sites of pYN1826ALP1. Plasmids were propagated in E. coli DH50r grown in Luria Bertani medium containing (g/l): tryptone, 10; yeast extract, 5; NaC], I0. LB plates contained 1.5% (w/v) agar. Ampicilfin was used at 100 rag/1. Media The recipes of YEA and YEPD were described by Nga et aI. (1994). Modified YEAS contained (w/v): yeast extract, 0.5%; glucose, 0.5%; starch, 1.5%; agar, 1.5%. Medium was supplemented with 50 to 80/xg G418 (geneticin)/ml as necessary. Modified YNBGS medium contained (w/v): yeast nitrogen base without amino acids and ammonium sulfate, 0.15%; sodium glutamate, 0.09%; glucose, 0.8%; starch 0.5%. YNBGS medium was suppIemented with 20 fig histidine/ml and 20/~g uracil/ml. Transformation of E. coli and Plasmid DNA Preparations Preparation of competent E. coli DH50r cells and transformation were according to Cohen et al. (1972). After amplification of plasmid in bacterial culture containing I00 mg ampicillin/l, extraction and purification of plasmid DNA were carried out by alkaline ]ysis according to Bimboim & Doly (1979). Isolation and Cloning of the rDNA Repeat Unit of E. fibuliger Chromosomal DNA of E. fibuliger 8014 met was digested with restriction enzymes and separated by electrophoresis. Southern hybridization of the chromosomal DNA digests (Sambrook et al. 1989) used a 7.7 kb HindIII fragment of pINA24 (van Heerikhuizen et al. 1985) containing the rDNA of Y. lipolytica as probe. Identification of an enzyme that cuts the repeat unit only once
Ribosomal R N A genes of E. Fibuliger Table 1. Materials and conditions used for PCR. Oligonucleotide
Template
Tm*
uncut p18GLU1 or 0.4 kb
64~
TaT
Sail GLU1 upstream
fragment
5' CTCGTCGACCTGCAGTCAACATGCGCATTC 3'
H i n d l l l - X b a l fragment
Xbal
5' CTCTCTAGAAGCAAATAGAAGTTTCAGGAAGG 3'
59~
of p18GLU1
64~
uncut p18GLU1 or 1.54 kb H i n d l l l fragment of p18GLU1
52~ 60~
uncut p18-APH or 1.2 kb
66~
BamHI GLU1
transcriptional terminator fragment
5' CTCGGATCCGTGCTTTGAAAAGTAAGTG 3' Kpnl
5' CTCGGTACCCTGCAGCAACACCGATTTTG 3'
49~
Xba I
APH(3')-I structural gene
5' CTCTCTAGAATGAGCCATATTCAACGGGAAAC 3'
56~
Sail - EcoRI fragment of
BamHI
5' CTCGGATCCTTAGAAAAACTCATCGAGCATC 3'
p18-APH
60~
* Melting temperature, Tm = 2(A + T) + 4(G + C) (without CTC and restriction site) 1 Annealing temperature, Ta = 3 to 5~ below the lower Tm of a pair of oligonucleotides.
was by observation of a signal in one band only, of between 8 and I2 kb, throughout the chromosomal DNA smear. This corresponded to the size of one repeat unit. The entire repeat unit was cloned by digesting the chromosomal DNA with that same enzyme, excising the DNA at the region where the signal appeared, purifying it using the GeneClean kit (Bio I01 Inc.) and ligating it to appropriately digested pUCt8. Selection of clones containing rDNA of E. fibuliger was by probing the insert fragments of about 30 clones with the same Y. lipolytica rDNA probe. All DNA manipulations such as digestions, alkaline phosphatase treatment and ligations were according to Sambrook et al. (1989). DNA Sequencing and Analysis The entire rDNA repeat unit was sequenced in both strands by the dideoxy-chain termination method (Sanger et al. 1977). The strategy involved the construction of subcIones using suitable restriction sites with inserts of 300 to 400 bp each and use of both the 23-mer M13/pUC forward and reverse primers (Bethesda Research Laboratories), and the generation of unidirectional, overlapping subclones with exonuclease III (Henikoff 1984) from the Erase-a-Base kit (Promega). Radioactive labelling was done using [aJsS]dATP (Amersham) with the Bst pre-mixed 7-deaza-dGTP Sequencing Kit (Bio-Rad) which used Bst DNA polymerase at 65~ The labelled reaction mixture was sequenced on the Macrophor unit from LKB Bromma. Sequence homology searches were done using the basic local alignment search tool (BLAST) programme (Altschul et al. 1990) from the EMBL or GenBank nucleotide sequence database release 73.1. Percentage homology based on maximum alignment allowing gaps was determined using the SeqAid II version 3.8I program (developed by D.D. Rhoads and D.J. Roufa) with a k-tuple value of 4. The GenBank accession number for the 5.8S and I7S rDNA sequences is U10409, and that for the 26S rDNA sequence is U09238. Polymerase Chain Reaction (PCR) Primers were synthesized by Oligos Etc., Inc. (USA). Sequences of primers and their annealing temperatures are shown in Table 1. The components of each reaction (I00/21) were: 10 to I00 ng template DNA; 50 pmol each primer; 100/2M each dNTP; 1.5 mM
MgC12; l X T a q polymerase buffer and 2.5 U of Taq DNA polymerase. Mineral oil (75/21) was used as overlay. Reactions were performed with an initial denaturation step at 95~ for 4 min, followed by 30 cycles of denaturation (95~ for I min; annealing (Ta, Table 1) for 1.5 min; and extension (72~ for 2 min, followed by a final extension at 72~ for 10 min. Whole Cell Lithium Acetate Transformation of S. cerevisiae INVSC2 A 100 ml yeast culture grown in YEPD to early exponential phase (16h) containing approximately 1 x 107 cells/ml was harvested and washed twice in sterile water. The cell pellet was then washed twice in 1 x TE/LiOAc, pH 7.5 (0.01 M Tris/HC[, 0.001 M EDTA, O.1 M lithium acetate. The cells were finally resuspended in 0.5 to 0.75 ml of I x TE/LiOAc at a density of 1.5 to 2 x 109 cells/ml. To 50/21 of cells, 3 to 5 #g of linearized plasmid DNA and 50/2g of salmon sperm carrier DNA were added. Li-PEG containing 1 x TE, 1 x LiOAc and 40% PEG (300/21) was then added. The reaction tubes were incubated at 30~ for 30 rain with slight agitation, followed by heat shock at 42~ for 15 rain. YEPD was immediately added to the mixture to dilute the toxic effect of PEG, after which the cells were centrifuged and washed in fresh YEPD before resuspending in I ml of YEPD for recovery at 30~ for 100 min. Following recovery, the cells were centrifuged and washed twice in I ml 1 x TE, pH 7.5 (0.0I M Tris/HC1, 0.001 M EDTA). The cells were then resuspended in I ml of 1 x TE, pH 7.5 and were spread on 5 YEAS plates supplemented with 50 to 80/2g G418/ml. The plates were incubated at 30~ for 3 to 4 days. Isolation of Total Yeast Chromosomal DNA The method used was that of Professor Satoshi Harashima (personal communication), Department of Biotechnology, Faculty of Engineering, Osaka University, Japan, with slight modifications. Yeast cells grown to late exponential phase in 50 rnl YEPD were harvested and the cell pellet was resuspended in 2.5 ml buffer containing 0.2 M Trizma base and 1 M sorbitol with 3 mg/ml dithiothreitol, and incubated at 30~ for 30 rain with shaking. After centrifugation, the pellet was washed in CPES buffer (1 x CPE, I M sorbitol). CPE (2 x ) contains 8 mM sodium citrate, 120 mM Na2HPO,.2H20, pH 6 made up to 92 ml with sterile water before
WorldJournalof Microbiology& Biotechnology,Vo] I3, 1997
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C. W. Yip, C. W. Liew and B.H. Nga Table 2. Differences In the restriction sites of selected rDNA clones. Location
Restriction site
Present in
Absent in
ETS
Xbal
pYN12-K13 pYN12-K16
pYN12-K37 pYN12-K38
ITS2
Hpal
pYN12-K37 pYN12-K38
pYN12-K13 pYN12-K16
pYN12-K13 pYN12-K37 pYN12-K38
pYN12-K16
NTS
EcoRI
addition of 8 ml of 0.5 M EDTA, pH 7.5. The cells were protoplasted in 2.5 ml CPES with 8.75 mg lysing enzyme/ml at 30~ for 5 to 15 min with shaking. The resulting protoplasts were then pelleted at low speed and washed in CPES before resuspending in 1.25 ml buffer containing 0.4 M NaC1, 0.2 M EDTA and 0.1 M Trizma base and 1.25 ml I0% SDS. Proteinase K (2.5/21 of I0 mg/ ml) was added and the mixture was incubated at 50~ for 0.5 to 2 h with shaking until the lysate became clear. The lysate was then extracted with phenol chloroform once, and chromosomal DNA was precipitated by the addition of 2.5 volumes of absolute ethanol. Chromosomal DNA was spooled out, washed with 70% ethanol and air-dried before being dissolved in 100 to 300/21 of sterile water.
Restriction Digestion, Southern Transfer, Labelling of DNA Probes, Hybridization and Detection of Signals DNA manipulations and Southern transfer procedure were performed according to standard methods (Sambrook et al. 1989). Digested DNA fragments were separated by electrophoresis in 0.8% agarose in I x Tris/acetate buffer. Purification of DNA probes from agarose gels was by using the GeneClean kit (Bio 101 Inc.). Labelling of hybridization probes, hybridization and detection of signals were carried out according to the Enhanced Chemiluminescence (ECL) direct nucleic acid labelling and detection system (Amersham). The s-amylase gene probe was the 1.8 kb XhoI - StuI fragment of the pIasmid pSfcr of Yamashita el al. (1985a). The APH (3')-I gene probe was the 0.8 kb XbaI BamHI fragment Of plasmid plSG (described in Table 3).
Extracellular ~-Amylase Assay The method used was that according to Steverson et al. (1984). Approximately 2 x 10~ yeast cells from an overnight preculture were inoculated into 100 mI YNBGS supplemented with the appropriate amino acids. Aliquots (1 ml) from every sample were removed every 6 to 8 h and centrifuged to remove the cells from the culture supematant. Supematant (0.5 ml) was added to 0.5 ml of starch (1% w/v) in 0.05 M acetate buffer, pH 5.0. This was mixed welt and 0.5 ml of the mixture was removed immediately and added to 0.5 ml of DNS reagent (1 g of 3,5-dinitrosalicylic acid, 20 ml of 2 n NaOH and 30 g of potassium sodium tartrate tetrahydrate per 100 ml) to stop the reaction. This was taken as time zero. The remaining 0.5 ml of the mixture was incubated at 30~ for 5 rain for ~-amylase action on starch, after which the reaction was stopped by adding 0.5 ml of DNS reagent. The reactions were boiled for 5 min to allow the orange colour to develop, cooled and diluted with 4 ml of distilled water. The differences in absorbance values between zero and 5 min were measured at 530 nm. Cell counts of samples were also determined.
10~
WorldJournalof Micrabiology& Biotechnology,Vol 13, I997
The values obtained were the average values of triplicate readings from two independent experiments. Maltose standard solutions ranging from 0 to 1 mg/ml were used.
Results and Discussion Isolation, Cloning and Organization of the rDNA Repeat Unit of E. fibuliger Figure 1 shows the autoradiogram of the hybridization of u lipolytica rDNA to the chromosomal DNA of E. fibuliger 8014 met digested with a selection of restriction enzymes. These enzymes were chosen partly on the basis of uniquecutting enzymes in the rDNA of related yeast species such as Y. lipolytica and S. cerevisiae. It was observed that KpnI cuts once only in the rDNA repeating unit of E. fibuliger. Hence, KpnI-digesfed chromosomal DNA of E. fibuliger 8014 met was run on an agarose gel, excised at the 9.3 kb region and the purified fragments were ligated at the corresponding site of pUC18. Upon hybridization of the insert fragments of the recombinant plasmids obtained to the same Y. lipolytica rDNA probe, around 50% of the insert fragments carried rDNA of E. fibuliger. These plasmids were named pYN12-K. The size of the repeat unit was larger than those of Y. lipolytica (7.7kb/8.7kb, van Heerikhuizen et al. 1985) and Kluyveromyces lactis (8.6 kb, Verbeet et al. 1984), comparable to that of S. cerevisiae (9.1 kb, Bell et al. 1977; Bayev et al. 1981), but was smaller than those of Schizosaccharomyces pombe (10.4 kb, Barnitz et al. 1982), Hansenula wingei (11.1 kb, Verbeet et al. 1984) and Candida albicans (12.5 kb, Srikantha et al. I994). The differences in size could be explained by the differences in spacer lengths. The cloned rDNA fragment has been mapped with a few common restriction enzymes (Figure 2). It was observed that restriction site heterogeneity exists in the rDNA of E. fibuliger. When different rDNA inserts of the recombinant plasmids were digested with the same enzyme, the number of sites in the rDNA differed. This was also reported for S. cerevisiae which was found to have two forms of rDNA: those with six or seven EcoRI sites (Petes et aI. 1978). In E. fibuliger, there are at least three restriction enzymes that have a different number of sites in the rDNA repeat unit, as shown in Table 2. Interestingly, the differences all lie in the non-conserved spacers. Through probing separately with the 17S and 26S rRNA genes of Y. lipolytica (van Heerikhuizen et a]. 1985), and comparisons of sequence homology with other yeasts, the organization of the E. fibuliger rDNA repeat unit (from 5' to 3') was found to be as follows: ETS 17S - ITS1 - 5.8S - ITS2 - 26S - NTS1 - 5S - NTS2. Thus, the physical organization of the rRNA genes of E. fibuliger is similar to that of S. cerevisiae, in that the 5S rRNA gene is found within the repeat unit in the NTS and is transcribed from its own promoter and in the opposite orientation as
Ribosomal RNA genes of E. Fibuliger
Figure 1. (a) Chromosomal DNA of E. fibuliger 8014 met digested with A, Apal; B, Bglll; E, EcoRI; H, Hindlll; Hp, Hpal; K, Kpnl; P, Pstl; S, Sail. (b) Southern hybridization of the digested chromosomal DNA with the 7.7 kb Hindlll fragment containing the Y. lipolytica rDNA probe. Kpnl-digested DNA produced a single band of 9.3 kb. Hindlll-digested DNA also gave a single band of 8.8 kb, but there may be another smaller band which was not detected. Both Pstl and Sail do not cut at the rDNA. Other bands obtained were: 4.6, 2.6 and 2.1 kb for Apal; 7.4, 1.0 and 0.9 kb for Bglll; 5.6, 4.5, 3.7 and 1.1 kb for EcoRI; 9.3, 7.3 and 2.0 for Hpal. The band sizes for EcoRI and Hpal-generated fragments are discussed in Results and Discussion.
K [X]H I
X
NTS2 "l,' ETS
X
A
S
I
E
B [p] I 4'm L"J .
17S
B A
X B
P
E
I
[5.8S1 ITS1 ITS2
S
[El I
A HK 1
NTS1
U NTS2
26S
5S 9
~-I
1 kb
I
Figure 2. Physical map and organization of the cloned rDNA repeating unit from E. fibuliger. Horizontal arrows indicate the deduced direclion of transcription. The 17S, 5.8S and 26S rRNA genes are transcribed as 37S pre-rRNA before processing occurs, with its start site mapped at about 700 bp upstream of the 5' end of 17S rRNA, as in the case of Saccharomyces cerevisiae (Bayev et al. t980), Saccharomyces carlsbergensis (KIootwijk et al. 1984), and Saccharomyces rosei (Verbeet et al. 1983). The proposed region for the initiation of the 37S pre-rRNA of E. fibuliger, based on comparison with other related yeasts, is indicated by an asterisk *. Restriction enzymes used were A, Apal; B, Bglll; E, EcoRI; H, Hindlll; K, Kpnl; P, Hpal; S, Sacl; X, Xbal. The restriction sites in brackets [] are non-conserved. The 3.7 kb EcoRI fragment inserted into plasmid pYN1826 is marked by 9
compared with the 3 7S pre-rRNA which gives rise to mature 17S, 5.8S and 26S rRNA. In other yeasts such as u lipolytica and S. pombe, the 5S rRNA genes are dispersed within the genome (Tabata 1981; Mao et al. 1982; van Heerikhuizen et al. 1985).
Sequencing and Sequence Analysis The entire rDNA repeat unit of E. fibuliger has been sequenced in both strands. In this report, the sequences of the 17S, 5.8S and 26S rRNA genes are presented (Figures 3 and 4).
World Journs of Microbiology & Biotechnology, Vd I3, I997
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C. W. Yip, C. W. Liew and B.H. Nga E. C. C. S.
fib. alb. tro. cer.
UAUCUGGUUGAUCCUGCCAGUA m"3 C A U A U G C t ~ G U C U C A A A G A U U A A G C CAUGC-A U G U C U A A G U A U A A G C A A -t~JAUACA UAUCUGGUUGAUCCUGCCAGUA~,UCAUAUGCI~GUCU~GAUUAAGCCAUGC~dG UC U A A G U A U ~ ~ A b A ~ UAUCUGGUUGAUCCUGC CAGUAGUCAUAUGCUUGUCUCAAAGAUUAAGCCAU~UGU~AU~~AUA~ UAUCUGGUUGAUCCUGCCAGUAGUCAUAUGCUUGUCUC3tAAGAUUAAGC C A U G C A U ~ G U A U ~ ~ A U A ~
79 d0 80 80
E. f i b . C. a l b . C. t r o .
GUGAAACUGCGAAUGGCUCAUUAAAUCAGUUAUCGUUUAUUUGAUAGUACCIFu~ACUACUU~-AUAACCGUGGUAAUUC GUGAAACUGCGAAUGGCUCAUUAAAUCAGUUAUCGUUUAUUUGAUAGUACCUU -ACUACUU~ -AU~C C G U ~ C GUGAAACUGCGAAUGGCUCALK/AAAUCAGUUAUCGUUUAUUUGAUAGUACCUU-ACUACUUGG-AUAACCGUGGUAAUUC
158 158 158
S. c e r .
GUGAAACUGCGAAUGGCUCAUUAAAUCAGUUAUCGUUUAUt"JGAUAGUUCCUUUACUACAUGGUAUAAC
160
E. C. C. S.
fib. alb. tro. cer.
UAGAGCUAAUACAUGC -U A A A A A U C C C G A C U C g J U U G G A A G G G A U G U A U U U A U U A G A U A A A A A A C C A A U G C A C U U C G G U G C UAGAGCUAAUACAUGCUUAAAAUCCCGACUGUUUGGAAGGGAUGUAUiK/AUUAGAUAAAAAAUCAAU~ -~ C ~ -~ UAGAGCUAAUACAUGCUUAAAA -U C C C G A C U G U U U G G A A G G G A U G U A U U U A U U A G A U ~ U C A A U G U -CUUC GGA -C UAGAGCUAAUACAUGCUUAAAA -U C U C G A C C C U i ~ G G A A G A G A U G U A U t ~ A U U A G A U ~ U ~ U G U -~ C ~ C
237 235 235 237
E. C. C. S.
fib. alb. tro. cer.
UCUUUGGUGAUUCAUAAUAACUUCUC G A A U C G C A A G G C -U U UCtrt~GAUGAUUCAUAAUAACtKK~CGAAUCGCAUGGCCt~G UCtK~GAUGAUUCAUAAUAACiKA/UCGAAUC GCAUGGCCUUG UCTu-u~JG A U G A ~ C A U A A U A A C U t r u r O C G A A U C G C A U G G C C U U G
316 314 314 316
CGUGGU~C
C A U G C U G G C G A U G G U U ~ ~ C ~ A U ~ -U G C U G G C G A U G G U U C A U U ~ ~ C ~ A U ~ -UGCUGGC GAUGGUUCAUUCAAAUUU~C~AU~ -U G C U ~ ~ U ~ ~ ~ C~AU~
E. f i b .
CUiK/CGAUGGUAGGAUAGUGGCCUACCAUGGLru~CAACGGGUAACGGGGAAUAAGGC4/UCGAUUCCGGAGA~G
C. a l b . C. t r o . S. c e r .
CUUUCGAUGGUAGGAUAGUGGCCUACCAU~GGGUAACGGGGAAUAAGGGUUC
GAUUCCGGAGAGGGAGC~G
394
CUIF~CGAUGGUAGGAUAGUGGCCUACCAUGGUUUCAACGGGUAACGGGGAAUAAGGGI~C CUI~CGAUGGUAGGAUAGUGGCCUACCAUGGUUUCAACGGGUAACGGGGAAUAAGGCg3UC
GAUUCC G G A G A ~ G GAUUC C G G A G A ~ G
394 396
E. f i b . C. a l b . C. t r o . S.cer.
AGAAACGGCUACCACAUCCAA GAAGGC-AGCAGGCGCGC-AAAUUACCCAAUCCUGACACAGGGAGGUAGUGACAAUAUAU AGAAACGGCUACCACAUCCAAGGAAGGCAGCAGGCGC GCAAAUUAC C CAAUCCC GACACGGGGAGGUAGUGACAAUAAAU AGAAACGGCUACCACAUCCAAGGAAGGCAGCAGGCGCGCAAAUUACCCAAUC C CGACAC GGGGAGGUAGUGACAAUAAAU AGAAACGGCUACCACAUCCAAGGAAGGCAGCAGGCGCGCAAAUUACCCAAUCCUAAUUCAGGGAGGUA~U~U
476 474 474 476
E. f i b . C. a l b . C. f r o .
AACGAUGCAGGGC CCUUAC GGGUCt~GUAAUUGGAAUGAGUACAAUUUAAAUACCUUAAC G A ~ ~ ~ AACGAUACAGGGCCCUtK~GGGUCUUGUAAUUGGAAUGAGUACAAUGUAAAUACCUUAAC ~ ~ ~ AACGAUACAGGGCCCtru"JCGGGUCUUGUAA~GGAAUGAGUACAAUGUAAAUACCUUAACGAGGAACAAUUGGAGGGCAA
556 554 554
S. c e r .
AACGAUACAGGGCCCAUUCGGGUCUUGUAAUUGGAAUGAGUACAAUGUAAAUACCIK/AACGAGGAACAA~A~
556
E. C. C. S.
GUCUGGUGCCAGCAGCCGCGGUAAUUC CAGCUCCAAGAGCGUAUAUUAAAGUUGUUGCAGUU~CGUAG~G~ GUCUGGUGC CAGCAGCC GCGGUAAUUC CAGCUC~GUAUAUUAAAGUUGUUGCAGUUAAAAA~C GUCUGGUGC CAGCAGCC GCGGUAAUUCCAGCUCCAAAAGCGUAUAUUAAAGUUGUUGCAGUU~C GUCUGGUGCCAGCAGCCGCGGUAAUUCCAGCUCCAAUAGCGUAUAUUAAAGUUGUU~G~~CGUAG~
GUAG~G~ G U A G ~
636 634 634 636
E. f i b . C.al5. C. t r o . S.cer.
CUt~GGGCUUGGCUGGCC G G U C C G -U -U U U t ~ A A C G A G U A C U G G t r L r u ~ - C A G C C G A G C ~ C ~ C ~ ~ G CCUUGGGCUUGGCUGGCCGGUCC -AUCUUUUUGAUGCGUACUGGAC - -CCAGCCGAGCCUUUCC ....... CCUUGGGCUUGGUUGGCCGGUCC -A U C I Y t ~ C U G A U G C G U A C U G G ~ J C - -CCAACCGAGCCtrur~C C . . . . . . . CtFt~GGGC CCGGUUGGCCGGUCCGAU - Utru-OUCGU GUACUGGAUUUCCAAC GGGGCCIK3UCC .......
U G U A UUCUGGGUA UUCUGGCUA UUCUGGCUA
713 704 704 706
E. f i b . C.alb. C. t r o .
G- -UU~GUCUCAGCUGCAUAU -GAUCCAGGACUAUUACUUUGAAAAAAUUAGAGUGUUCAAAGCAGGCGUUUAGC GCCAU- - -U ............ UAUGGC GAACCAGGACUUUUACUt~GAAAAAAUUAGAGUGUU AAAGCAGGCCUUU GCCUU- -UU ............... GGCGAACCAGGACtKA]UACUUUGAAAAAAUUAGAGUGUUCAAAGCAGGCCIYL~
S.cer.
ACCUUGAGUCCUUGUG
E. C. C. S.
UCGAAUAUAUUAGCAUGGAAUAAUGAAAUAGGAC G -U A U G G U U C U A U U t ~ G U U G G U U U C U A G G A C C A U C G U A A U G A U U ~ UCGAAUAUAUUAGCAUGGAAUA~/JAGAAUAGGACGtr~AUGGUUCUAtK/I~GUUGGUUUCUAGGAC ~ U C ~ ~ UCGAAUAUAt~/AGCAUGGAAUAAUAGAAUAGGACGUUAUGGUUCUAt~u-GUGUUGGUL~CUAGGACCAUCGUAAUGAUUAA UC GAAUAUAUUAGCAUGGAAUAAUAGAAU~GACGUU -U G G U U C U A U U U U G U U G G U U U C U A G G A C C A U C GUAAUGA~
869 848 84 6 863
E. f i b . C. a l b .
UAGGGACGGU•GGGGGCAUCAGUAUUCAAUUGUCAGAGGUGAAAUUCUUGGAUUUAUUGAAGACUAACUACUGCGAAAGC UAGGGACGGUCGGGGGUAUCAGUAUUCAGUUGUCAGAGGUGAAAUUCUUGGAUUUACUGAAGACU~A~~
949 928
C. t r o , S. c e r ,
UAGGGACGGUCGGGGGUAUCAGUAUUCAGUUGU~GAGGU~UUCL~GGAUUUACUGAAUACU~A~~ UAGGGACGGUCGGGGGCAUCGGUAUUCAAUUGUC -G A G G U G A A A U U C U U G G A U U U A U U G A A G A C U A A C U A C U G C G A A A G C
926 942
fib. alb. tro. cer.
fib. alb. tro. cer.
396
- G C U C U -U G G C G A A C C A G G A ~ A C U U U ~ U U A G A G U G U U C A A A G C A G G C
-GC -GC
GUAUUGC
790 768 766 784
Figure 3. Sequence alignment of the 17S rRNA of E. fibuliger, derived from the DNA, with the 17S rRNA sequences from other yeast strains, Candida albicans (C.alb.) (Barns et al. 1991), Candida tropicalis (C.tro.) (Hendriks et al. 1991), and Saccharomyces cerevisiae (S.cer) (Rubtsov et al. 1980). The start of the 17S rRNA for all the yeast strains is at nucleotide 1. The 17S rRNA for each yeast strain ends at the nucleotide number indicated on the right, i.e. 1807 for E. fibuliger, 1787 for C. albicans, 1785 for C. tropicalis and 1789 for S. cerevisiae.
108
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Ribosomal RNA genes of E. Fibuliger E fib. C.alb. C,tro. S cer.
AUL~GCCAAGGA~GULK~CAUUAAUCAAGAA~GAAAGUUAGGGGAU~GAAGAUGAUCAGAUACCGU~GUAGUCUUAA~CA ALrOUACCAAGGACGUUt~CAUUAAUCAAGAACGAAAGUUAGGGGAUC GAAGAU GAU CAGAUAC C GU C GUAGUCUUAAC CA AUUUAC CAAGGAC GULrOUCAUUAAUCAAGAAC GAAAGUUA~UC GAAGAUGAUCAGAUACC GUC GUAGUCUUAAC CA GUUUGCCAAGGACGULrOUCGUUAAUCAAGAACGAAAGUUGAGGGAUC ..... UGAU ..... ACCGUCGUAGUCUUAACCA
E.fib 9 UAAACUAUGCCGACUAGGGAUC GGGUGUUGUU~ACUGACUCACUCGGCACCUUACGAGAAAUCAAAGLK23LrOGGGU C.alb 9 UAAACUAUGCC GACUAGGGAUCGGUUGUUGUUCUI/OUAUUGACGCAAUCGGCACCUUACGAGAAAUCAAAGUCUI/O~U C.tro 9 UAAACUAUGCC GACUAGGGAUCGGUUGUUGUUCULrOUAUUGAC GCAAUC GGCACCUUACGA AAAUCAAAGUCUUUGGGU S . c e r 9 U A A A C U A U G C C G A C U A G - -A U C G G G U G G U G U L K F L K Y O U A A U G A C C C A C U C G G U A C C U U A C G A G A A A U C A A A G U C U U U G G G U
E.fib. C.alb. C.tro. S.cer.
UCU~GUAUGGUCGCAA
- GGCUGAAACUUAAAGGAAUUGACGGAAGGGCAC
1109 1088 1086 1090
CACCAGGAGUGGAGCCUGCGGCL~
1188
UCU~GUAUGGUC GCAA - ~GAAACUUAAAGGAAUUGACGGAAGGGCACCACCAGGAGUGGAGC CUGCGGCUU UCUGGGGGGAGUAUGGUCGCAA -GGCUGAAACUUAAAGGAAUUGAC G G A A ~ C CACCAGGAGUGGAGCCUGC ~ UCU~AGUAUGGUCGCAAA~GAAACUUAAA~UUGACGGAA~CCACUAGGAGUGGAGCCUGC~
1029 1008 1006 1012
1167 1165 -
1169
E.fib. C.alb. C.tro. S.oer.
AAULrOGACUCAACAC GGGGAAACUCAC CAGGUCCAGACACAAUAAGGAUUGACAGAUUGAGAGCUCLrOUCUUGALKA/UGU AAUUUGACUCAACAC~CUCACCAGGUC CAGACACAAUAAGGAUUGACAGAUUGAGAGCUCUUUCUU~GU AAUUUGACUCAACA•GGGGAAACUCA•CAGGUCCAGACACAAUAAGGAUUGACAGAUUGAGAGCUCUUUCUUGAUUUUGU AAUUUGACUCAACAC ~ C U C A C C A G G U C C A G A C A C A A U A A G G A U U G A C A G A ~ ~ ~ G A ~ G U
1268 1247 1245 1249
E. C. C. S.
GGGUGGUGGUGCA•GGCCGUU-CUUAGUUGGUGGAGUGAUUUGUCUGCUUAAUUGCGA•AACGAACGAGA•CUUAACCUA GGGUGGUGGUGCAUGGCCGUU-C[/GAGUUGGUGGAGUGAUUUGUCUGCUUAAUUGCGAUAA•GAA•GAGACCUUAACCUA GGGUGGUGGUGCAUGGCCGUU-CUUAGUUGGUGGAGUGAUUUGUCUGCUUAAUUGCGAUAACGAACGAGACCUUAACCUA GGGUGGUGGUGCAUGGCCGUUUCUCAGUUGGUGGAGUGAUUUGUCUGCUUAAUUGCGAUAACGAACGAGACCUUAACCUA
1347 1326 1324 1329
E. f i b . C. a l b .
CUAAA•AG•ACUGUUAGCGUUUGCUGAUAUAGUU-ACUUCUUAGAGGGACUAUCGGUUUCAAGCCGAUGGAAGUUUGAGG CUAAAUAGUGCUGCUAGCAUUUGCUGGUAUAGU-CACUUCUUAGAGGGACUAUCGACUUCAAGUCGAUGGAAGUUUGAGG
C. t r o . S. c e r .
CUAAAUAGUGCUGCUAGCAUUUGCUGGUAUAG•-CACUUCUUAGAGGGACUAUCGAUUUCAAGU•GAUGGAAGUUUGAGG CUAAAUAGUGGUGCUAGCAUUUGCUGGU-UA-UCCACUUCUUAGAGGGACUAUCGGUUUCAAGCCGAUGGAAGUUUGAGG
1426 1405 1403
E.fib. C.alb. C.tro. S.oer.
CAAUAACAGGUCUGUGAUGCCCUUAGA - C GUUCUGGGCCGCAC GCGCGCUACACUGACGGAGCCAAC GAGUAUAU - CCUU CAAUAACAGGUCUGUGAUGC C CUUAGA - CGUUCUGGGCCGCACGC GC GCUACACUGACGGAGC CAGCGAGUAUAAGCCUU CAAUAACAGGUCUGUGAUGCCCUUAGACGUUCUGGGC C GCAC GCGCGCUACACUGACGGAGCCAGCGAGUAUAAACCUU CAAUAACAGGUCUGUGAUGC CCUUAGAACGUUCUGGGCCGCACGC GCGCUACACUGACGGAGC CAGCGAGU CUAA - CCUU
1504 1484 1482 1486
E.fib. C.alb. C.tro. S.cer.
UGC C GAGAGGUAUGGGUAAUCUUGUGAAACUC GGC C GAGAGGUCUGGGAAAUCUUGUGAAACUC GGCCGAGAGGCCUGGGAAAUCUUGUGAAACUCC GGC C GAGAGGUCUUGGUAAUCUUGUGAAACUC
C GUC GUGCUGGGGAUAGAGCAUUGCAAUUAUUGCUCUUCAAC GAGGAA CGUCGUGCU GGGGAUAGAGCAUUGUAAD-OGLrOGCU~C~ GUC GUGCUGGGGAUAGAGCAUUGUAAUUGUU GCUCUUCAAC GAGGAA CGUC GUGCUGGGGAUAGAGCAUUGUAAUUAUUGCUCUUCAACGAGGAA
1584 1564 1562 1566
E.fib. C.alb. C.tro. S.cer.
UUCCUAGUAAGCGUAAGUCAUCAGCUUGCGUUGAUUACGU•CCUGCCCUUUGUACACAC•GC•CG•CGCUAGUACCGAUU UUC CUAGUAAGC GCAAGUCAUCAGCUUGC GUU GAUUAC GUCCCUGCCCUUUGUACACACCGCCCGUC GCUACUAC C GAUU UUCCUAGUAAGCGCAAGUCAUCAGCUUGCGUUGAUUACGUC CCUGCCCUUUGUACACACCGCCCGUCGCUACUACCGAUU UUCCUAGUAAGCGCAAGUCAUCAGCUUGC GUUGAUUAC GUC CCUGCCCUUUGUACACACCGCCC GUCGCUAGUACCGAUU
1664 1644 1642 1646
E.fib. C.alb. C.tro. S.cer.
GAAUGGCUUAGUGAGGC CUCAGGAUUGGUUU - - -AAAGGAGGAGGCAAC GAAUGGCUUAGUGAGGCCUC C GGAUUGGUUUAGGAAAGGG - - -~ C C U GAAUGGCUUAGUGAGGCUUCCGGAUUGGLrOUAGGAAAGGG - - -GGCAAC GAAUGGCUUAGUGAGGCCUCAGGAUCUGCUUAGAGAAGGG - - -GGCAAC
1740 1720 1718 1722
E.fib.
UUGGUCAUDUAGAGGAACUAAAAGUC GUAACAAGGUUUCC GUAGGUGAACCUGCGGAAGGAUCAUUA UUGGUCAUUUAGAGGAAGUAAAAGUCGUAACAAGGUUUC CGUAGGU GAACCUGC GAAGGAUCAUUA
fib. alb. tro. cer.
C.alb. C.tro. S.cer.
- UCCACCUUGGAACUGAGAAUCUGGUCAAAC C -AUUCUGGAACCGAGAAGCUGGUCAAAC -UCCAUUCUGGAAC C GAGAAGCUAGUCA/kAC - UCCAUCU CAGAGCGGAGAAULrOGGACAAAC
UUGGUCAUUUAGAGGAAGUAAAAGUCGUAACAAGGUUUCC GUAGGCGAACCUGCGGAAGGAUCAUUA UUGGUCAUUUGGAGGAACUAAAAGUCGUAACAAGGUUUCCGUAGGUGAACCUGCGGAAGGAUCAUUA
The 17S rRNA gene is 1807 bp long; longer than some of the I7S rRNA gene sequences to which it was compared, such as those of C. albicans (1787 bp, Barns et al. 1991), C. tropicalis (I785 bp, Hendriks et al. 1991) and 5. cerevisiae
1407
1807 1787 1785 1789
(I789 bp, Rubtsov et al. 1980). It showed extensive homology to the 17S rRNA genes of the three yeast strains mentioned above, with the highest overall percentage homology of 93.9% to C. albicans, 93.2% to C. tropicalis; and
World ]ournal of Microbiology & Biotechnology, Vol 13, I997
109
C. W. Yip, C. W. Liew and B.H. Nga A T G T T A T T T G T T T T T A G A C C T GCC~CT T A A C T G C G C G G T T T A A T A A A C T C T
T A T A C A C A G T G T T T--T G T T ~ G C G A A T m T G G T T T A G T T T G T T G G T T T T ~ T
I00
Bgl I I TCC.~AAGGAT GAAGATTGATTGCTAAATCTTATT~TTTTAAACT~T *-->5.8S ACAAACTAAAAGTTTAAAACTTTCAGCAACGGATCTCTT
CTCTTTTT~TGTATTTTTTTAATTACAACTAGT
C~TTTT
GGTTCTCC~.~TCGATGAAGAACGCAG~GAATT~T~T~TGT
~TT
200
~GATTTTC
3C0
t GT GAAT CATCGAATCTTTGAACC, CATATTGCC.CTCTATAGTATT CTATAGAGCAT GCCTGTTTGAC~CGTCATTTCTCTCTTAAAC~TT~TTT~TAT
400
T G A A G G T T G T GTTAGCTT..C T G C T A A C T C C T T T G A A A T G A C T T
~ A C ~
500
GCT ~ C
600
GGC~ATTGATTGAGTTTTCCATATATTTGCTTAAGGAT~TATTA~TT
Stu I + + + - - > 26S CTTAT TAAATACC CTTTT GC GAAGGACTTACT CGT GTAT CAAGGC CT TATAACTTT GT CATTAATTTTGAC CT CAAAT ~ T ~ T A C C C
TT ~ T A T
CAAT ~
~
A
A
A
C
C
A
A
~
T
T GCCTTAGTAACGGC GAGT GAAGCGGC~JkAAGCT CAAATT T C~J~AGCTAGCACCTT C G
GT GTTCGCGTTGTAATTTGAAGATAGTTTCCTTGAGTAGT
CCTTTATCTATGTTCCTT
GATACTACT CT TT GT GGGAT T CTAT CGAAGAGT CGAGTT GTT T ~
A C C GATA~3CGAAF-AAGTAF-AGT G A T G G A A A F _ ~ T ~
C
T
T
~
CT ~
T
GGAACAGGACGTCATAGAGGGTGAGAACCCCGTAT
CTAAGT GGGT GGTAAAT T CCATCTAAAGCTAAATATT
GAAAAAGTACGT GAAAT T GCT G
ACTTGGTGTTTAATGATTATCAGTTCTTCTTGGACTGTGCACTCGTTTTTCACCGGGCCAATATCA~T
Bgl II CTTGC.GGAGATCTACTT C~'TGGGACTGAGGACTGCGCTTC~T
TCCTT C~TGTTATAGT
GGAAACT CT GGT GGAAG~
T~ C T
T
~
A
A
ATTGGCGTAATGACCT
~
T GAGGT CAG
1300
~
CGTAT CAGT T TTAT GAGGTAAAGCGAAT GATTAGAGG T GT T GGGGACGCAAGGT
AAG T C ~ T
GAACCGAAC$ T GGAGTTAAAGT GCCGGAATATACGCT
C C G C T ~ T
CAT CAG~CACCA~TGTTAGT
1400
C C~kAC~T ~ A G T ~
~TT
1500
C CT TAGC CTAT T C
T T C A A T G T G T A A E . A A G T C C T T G T T A C T T A A T T G A A C G T GC~ACAT T T G A A T G A A G A G C T T T T A G T G G G C C A T T T T T G G T ~ C T
TGC~T
1200
Ap~ I ~CCTTTAAGGGT~
G
~
~
C~CC
1600
1700
Xba I T CATCTAGACAGCC~AC~T~T
GT GTAACAACT CACC GGCCGAAT GAACTAGCCCT GAAAAT GGAT GGC GCT CAAGCGTAT TACT TATA~
I000
II00
TAAGCCGCCCGTCT
C T c-T T G G G A C C C G A A A G A T G G T G A A C T A T G C ~ G ~ T ~ T
C GTAGCGG T T CT GAC GT GCAAAT C GAT C G T C GAATTT GGGTATAGGGGCGAAAGACTAAT
CCT GCCG.~AGT TT CCCT ~
A
800
900
GGC~G
TTTAGCGGTAGAGTACCCCTTGAAATGT~
MIu I TGAAACACC.GACCAAC.GAGT CTAACGTCTAT GCAAGT GTTTGGGTGTAAAACCCGTACC, CGTAATGAAAGTGAACGT~TT
A C A A T C G ~ C C C~AT C C T G A A ~ T T T T C A G A T G G A T T T G A G T A A G ~ T A C ,
GATTTT G
700
1800
GT TA
1900
Bgl II GGGTTGATATGATGCCCTAACGAGT~GGCGTGGAGGTT
GTA~TAF-~---AAATATTC A A A T ~
TGTGACGAAEK~CT T T GGT GT GAACCTGGGTAGAA~CTCTAGT
TT GAAGACT GAAGT ~
T
G
2000
T CCATAT CAACAC~Y~GT T GGATAT GGGTTAGT CGAT CCTAAGAGAT GGG
2100
~CCGTTT~CTGATTTTT~CACCATC~TCTGGTTAAAATTCCAGAACTTGGATATGGATTCTT
T GAAT GT GAAGAC GT CGGTAT ~
CT GG~C.CGCAGTAC~
G C A ~ T
CT C ~
AAGGATC~TTCT~CT
CCT ~
T
~
T
CACGGTAACGTAAC
TAT CTTTT CTT CTTAAF-AC.CTTAACACCCTGGAATT GGT T TAT C CGGAGATAGGGT
.~TT. ~G C T G C G T C C G G T G C G C T T A T G A C G C . C C C T T G A A A A T T C A C A G G A A G G A A T A G T T T T C A T ~ T C G T A ~ Hp~ I TTAACAGC CT CTAGTT GATAGAATAAT GT AGATAAGGGAAGT C GGCAAAATAGAT C CG TAACT T CGGGAT~AT
T C GT CAGACGCAGTGGAACCCGTT
~CTGGCTAT
~ T ~T
G ~ T
CGTGGCCGGACTTCTTTGGGAT
2200
CT TAT GG
2300
~T~CC
2400
T~ T
~
CTTA CT GT T GAC
2500
2600
Figure 4. Nucleotide sequence of the DNA fragment encoding the 5.8S, 26S rRNA and spacer regions. The 5.8S rRNA starts at position 217 and ends at position 374. The borders of the 5.8S rRNA gene are marked by asterisks *. The 26S rRNA starts at either position 565, 566 or 567 and ends at position 3927. The borders of the 26S rRNA gene are marked by +. The regions underlined once indicate base-pairing between the 5' end of 5.8S rRNA (positions 220 to 229) and 26S rRNA (positions 973 to 982). The regions underlined twice indicate base-pairing between the 3' end of 5.8S rRNA (positions 355 to 371) and the 5' end of 26S rRNA (positions 568 to 584). The base-pairings are based on comparison with those of C. albicans (Srikantha et al. 1994). The variable region within the 5.8S rRNA gene (positions 334 to 351) is overlined. The hexanucleotides ATTTGT (underlined by ~) are found twice at the 3' terminus of the 26S rRNA gene. The GenBank accession number for the 5.8S rDNA sequence, which was submitted together with the 17S rDNA sequence and intervening spacer regions, is U10409 and that for the 26S rDNA sequence is U09238.
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Ribosomal RNA genes of E. C-,CK~GTAC,GTAC,C,CTTCGTC,C C G T C C ~ T T
GC.ATTTAACGATCAACTT A C a A A C T G G T A C C s G A ~ T
~ ~
G T ~ T T ~ T ~ T T
G
Fibuliger
2700
CGATGGT CAGAAAGT GAT GTT GACGCAATGTGATTTCTGCCCAGTGCT CTGAAT GTCAAAGT GAAGAAATTCAACCAAC, CGCGGGTAAACGGCGGGAGTA
2800
ACTATGACT CTCTTAAGGTAGCCAAATGCCTCGT
2900
CATCTAATTAGTGACGCGCATGAAT GGATTAACGA~TTCC~CTGTCC~ATCTACTAT
~
G
AAACCACAGCGAAC~GGAACGGCTTCGCAGA~TCAGC~C~AAGA~TGTTGAC~CTTGACT~TAGTTTGACATTGTGAA~CATAGAGGGTGTA
3000
GAATAAGTGGC~AGCTT~C~GCGC~GGTGAAATA~CACTACCT~TATCGTTTCTTTACTTATTCAATTAAGCGAAC~CTGGTCTATC~TTTT~A~GT
3100
TTAAGCGGATTCATTC~GTGATTCGGGTTGAAGACATTGTCAGGTGGGGAGTTTGGCT~GC~C2%CATCTGTTAAACGATAA~GCAGATGTC~
3200
GGGACTCAT~CAGAAAT~TTCAGTAGAATAAAAGGGTAAA~T~CTTGATTTTGATTTTCAGTGTGAATACAAACCATGAAAGTGT~AT
3300
CGATCCTTTAGT CCCT CGGAATTTGAGGCTAGAGGTGCCAGAAAAGTTACCACAGGGATAACTGGCTT
GT G G C A G T C A A G C G T T C A T A G C G A C A T T
Eco RI T T T G A T T C T T C G A T G T C G G C T CT T C C T A T C A T A C C G A A C , C A G A A T T C G G T A A G C G T T G G A T T G T T C A C C C A C T A A T ~ C G T G A G C T
GCTT
3400
G G G T TTAE,A C
3500
•GT•GTGAGACAGGTTAGTTTTAC•CTACTGATGAATGTTAT•GCAATAGTAATTGAA•TTAGTACGAGAGGAAC•GTTCATTCAGATAATTGGTTTTTG
3600
CAGCTGTCTGATcAC~C~CATTGCTGCGAAGCTACCATCTGTTGGATTATGGCTGAA~GCCTCTAAGT~T~CATGCTA~T~TTTATTC~
3700
Sac I TCC•CcCATTTTAGTTGGTACGAATAAGGCAcATTAGTGTCGCTGAACCATATTTACTGGTATGGAGCTCTTGCGGAAAGGCTTGGGTTCTTGCTAGTTTT
3800
CTTAATTTCACTATGAGCGAGGATAAATC~TTTC-CATACGACTTAACTGTACAA~GGGGTATTGTAAGCAGTAGAGTAGCCTTGTTGTTA~GATCT~G
3900
+ AGATTAAc•CCTTTGTTGTCcGATTTGTTATCTCTAAGTATTTGATAGATGGTTCTATTATTTATTTAGAGAATTTGTTTTATATCATAAAGACTTTTGCC
4000
TATAAAcAT~T~TcTTTTAATTTTTTATGATATTcTAGTCATGTGACAAcTT~TATATATATACAATCAGTGTTA~GATATTTcTTC~CTGAG
4100
ATATC
EC Ef Ca Ct SC
4105
AAGUCGUAACAAGGUAACCCUAGGCGAACCUGCGGUUGGAUCACCUCCUUA AAGUC GUAACAAGGUUUC C GUAGGUGAAC CUG C G GAAG GAU CA UUA AAGUCGUAACAAGGUUUCCGUAGGUGAACCUGCGGAAGGAUCA UUA AAGUC GUAACAAG GUUU C C G UAG G C GAACCUGC GGAAGGAUCA UUA AAGUC GUAACAAGGUUUCC GUAGGUGAACCUGC GGAAGGAUCA UUA
Figure 5. The homologous sequences at the 3' ends of the 17S rRNA of Escherichia coli (Ec) (Brosius et al. 1978), Endomyces fibuliger (Ef) (this study), Candida albicans (Ca) (Barns et al. 1991), Candida tropicalis (Ct) (Hendriks et al. 1991), and Saccharomyces cerevisiae (Sc) (Rubtsov et al. 1980). This figure was adapted from Rubtsov et al. (1980). The five conserved nucleotides (in bold) in E. coli 16S rRNA that bind to the ShineDalgarno sequence (Shine & Dalgarno 1974) on the mRNA to initiate translation are not found in these yeast strains.
91.5% to 5. cerevisiae. However, a non-homologous region was observed around bases 697 to 738 of the sequenced 17S rRNA gene. The alignment of the 17S rRNA gene sequences of selected yeast strains is shown in Figure 3. An interesting point to note is that the last 21nucleotides at the 3' terminus of the 17S rRNA genes in the yeast strains compared were identical, suggesting some involvement of this region in essential interactions, perhaps similar to the Shine-Dalgamo model of the ribosome-binding site on the mRNA, which binds to the 3' end of the 16S rRNA
gene in prokaryotes through a consensus sequence for efficient initiation of translation (Shine & Dalgarno I974; Bollon 1982). This consensus sequence, identified at the 3' end of the 16S rRNA gene in Escherichia coli, carries five very conserved nucleotides; CCUCC. However, the sequence associated with this model has not yet been identified in eukaryotic I7S rRNA, and is similarly absent in the 5. cerevisiae 17S rRNA (Rubtsov et al. I980) and in the sequenced E. fibuliger 17S rRNA gene, although a high degree of homology with the adjacent region of the 16S prokaryotic rRNA from E. coli has been observed (Figure 5). The nucleotide sequence containing ITS1, 5.8S rDNA and ITS2 is 564 bp long. The 5.8S rDNA is 158 bp long, similar to those of 5. cerevisiae (Rubin 1973) and 5. carlsbergensis (Veldman et al. I981b), but is I bp longer than that of C. albicans (Srikantha el aI. 1994). It is 91.8% homologous to that of S. cerevisiae , and 90.5% to that of C. albicans. Figure 6 shows a comparison of the 5.8S rRNA genes of various yeast strains. The sequences were shown to be very conserved, with the exception of a region from positions 334 to 351 which was more variable, near the 3' end of the 5.8S rRNA gene. It is interesting that although this region is variable among the yeasts compared, they are all capable
World ]ournal of Microbiology & Biotechnology, Vol 13, 1997
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C. W. Yip, C. W. Liew and B.H. Nga E. fib. C. alb. S. carl. S. cer. S. p o m b e
AAACIYt~CAGCAACGGAUCUCUUGGUUCUCGCAUCGAUGAAGAAC G C A G C G A A ~ U ~ U G U ~ ~ U AAACUUUCAa CAACGGAUCUCUUGGUUCUCGCAUCGAUGAAGAACGCAGCGAAaUGCGAUAcGUAAUaUGAAUUGCAGAU AAACUUUCAa CAACGGAUCUCI~GCgu~CUCGCAUCGAUGAAGAACGCAGCGAAaUGCGAUAcGUAAUGUGAAUUGCAGAa AAACtru-UCAa C A A C G G A U C U C U U G G U U C U C G C A U C G A U G A A G A A C G C A G C G A A a U G C G A U A c GUAAUGUGAAUUGCAGAa A A Ctru-~CAC.CAACGGAUCUCUUGGc U C U C G C A U C G A U G A A G A A C G C A G C G A A a U G C G A U A c G U A A U G U G A A U U G C A G A a
E. fib. C. alb. S. carl. S. cer. S. p o m b e
tK~JC G U G A A U C A U C GAAUCtrt~GAAC GCAUAtr~GC G C U C U A U A G U A L ~ C U A U A G A G C A U G C C U G U L ~ G A G C ~ aUUC GUGAAUCAUCGAAUCUt~GAACGCAc.AUUGCGC cCUcUgGUAUUC o -gg agGCAUGCCUG~u~j~GUC~ U U c C G U G A A U C A U C G A A U C t K ~ C A A C G C A c A U U G C G C cC c u U g G U A U U C c A g g G g G C A U G C C U G U U U G A G C G U C A U U U U U c C ~ J G A A U C A U C G A A U C i K ~ G A A C G C A c A U U G C G C cC c u U g G U A t r U C c . A g g G g G C A U G C C U G U U U G A G C G U C A U U U U U c C G U G A A U C A U C G A A U C U U U G A A C G C A c A U U G C G C c u U u g g G U u c U a c c aAa g G C A U G C C U G U U U G A G u G U C A t W 3 -
80 80 80 80 80
158 157 158 158 157
Figure $. A comparison of the 5.8S rRNA of E. fibuliger, derived from the DNA sequence, and Candida albicans (C.alb.) (Srikantha et al. 1994), Saccharomyces carlsbergensis (S.carl.) (Veldman et al. 1981b), Saccharomyces cerevisiae (S.cer.) (Rubin 1973) and Schizosaccharomyces pombe (S. pombe) (Schaack et al. 1982). Nucleotides of the yeast strains which are different from those of E. fibuliger at similar positions are in lower case. The variable region within the 5.8S rRNA (positions 118 to 135) is overlined. The 5.8S rRNA of E. fibuliger is 158 bp in length, similar to those of S. cerevisiae and S. carlsbergensis, but 1 bp longer than those of C. albicans and S. pombe (157 bp, marked by -).
A
U
U
A U
U
U
G--C
118
G--C
A--U
G--C
U--A
U--A
A--U
U.
U--A
C--G
C--G
C--G
U--A
135
118
G
C--G
C--G
C--G
G--C
G--C
(A)
(B)
A
U
U
U U
C U
G
G--C U.
135
U
G--C
A
G--C G
G--C
C--G
118
U
U--A
U--A
U--A
C--G
U--A
C--G
135
118
C--G
135
C--G G--C
(C)
(D)
Figure 7. Stem-loop structure formation in the variable region (positions 118 to 135, numbered according Figure 6) near the 3' end of 5.8S rRNA found in several yeast strains (A) E. fibuliger (predicted); (B) S. cerevisiae (Rubin 1973; Yeh & Lee 1991); (C) C. albicans (Srikantha et al. 1994); (D) S. pombe (Schaack et al. 1982). Base-pairing is indicated b y - , non-canonical U-G interaction is indicated by e.
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World Journal of Microbiology & Biotechnology, Vol I3, 1997
of forming palindromes, as shown in Figure 7. The predicted secondary structure of the 5.8S rRNA of E. fibuliger is similar to that of S. cerevisiae, as reported by Yeh & Lee (1991). The nucleotide differences in both the 5.8S RNA genes lie in the non-pairing regions and in the variable region which forms a stem-loop structure near the 3' terminus. It is also noteworthy that the 5' end of the 5.8S rRNA (positions 220 to 229) could base pair with a region (positions 973 to 982) in the 26S rRNA, whilst the 3' end of the 5.8S rRNA (positions 355 to 371) could base pair with the 5' end of the 26S rRNA (positions 568 to 584). Another region in the 5.8S rRNA (positions 241 to 249) was also capable of base-pairing with nucleotides 892 to 903 in the 26S rRNA. These features are crucial for the formation of secondary structures in the rRNA. Besides this, other hairpin loops within the 26S rRNA have been identified (data not shown). The 26S rRNA gene of E. fibuliger is between 3361 and 3363 bp in length. The actual number of nucleotides in the 26S rRNA of E. fibuliger is uncertain because this is based on comparison with other closely related yeasts. In 5. carlsbergensis, Veldman et al. (1981a) reported the start of 26S rRNA at TTGA (position 567 in the 26S rRNA of E. fibuliger) whilst in C. albicans, the start of the 26S rRNA is at TTTGA (position 566) (Mercure et al. 1993). Bayev et al. (1981) determined the origin of the 26S rRNA of 5. cerevisiae to be one nucleotide upstream from that of C. albicans (position 565). The 3' end maps at position 3927, with the hexanucleotide ATTTGT identified as the 3' terminal sequence (Bayev et al. 1981). The hexanucleotide ATTTGT was found twice at the 3' end of the 26S rRNA gene of E. fibuliger, whereas in S. cerevisiae, the same hexanudeotide was present three times. However, the role of these repeats remain unclear (Bayev et al. 1981). Overall percentage homology with the 26S rRNA gene of 5. cerevisiae, 3392 bp long (Georgiev et al. 1981), was 88.6%. However, there were stretches where homology
Ribosomal RNA genes of E. Fibuliger Table 3. Sequential cloning strategy of the transforming plasmid pYN1826ALP1-GPT. Vector DNA
Insert DNA fragment*
i. 816 bp Xbal - B a m H I APH(3')I structural gene fragment
Xbal + B a m H I -
ii. 321 bp Sail - Xbal GLU1 upstream fragment
Sail + Xbal -
iii. 657 bp BamHI - Kpnl GLU1 transcriptional terminator fragment
B a m H I + Kpnl -
iv, 1818 bp insert from Sa/I + Kpnl - digested p18GPT
Sail + Kpnl -
(11.9 kb)
digested pUC18
digested p18G digested p18GP
digested pYN1826ALP1
Resulting plasmid P18G (3.5 kb)
p18GP (3.8 kb) p18GPT (4.5 kb) pYN1826ALP1-GPT (13.7 kb)
* PCR product, except (iv).
Ap ori
pYN1826ALP1-GPT ALP1 B A Xb
GLU1 T GLU1P APH(3')-J,,~
)
E
"-----_.___
S
/
/"
Xh
B
Xh
Figure 8. Plasmid map of pYN1826ALP1-GPT. The restriction
sites are as follows: A, Apal; B, B a m H I ; E, EcoRI; H, Hindlll; Hp, Hpal; K, Kpnl; S, Sail; Xb, Xbal; and Xh, Xhol.
between the 26S rRNA genes was greater: 91% for a region (positions 1279 to 2554) and 97% for another smaller region (positions 2741 to 3074) when compared with the corresponding regions of S. cerevisiae 26S rDNA. Overall percentage homology with the 26S rRNA gene of C. albicans, 3363 bp long (Mercure et al. 1993), was 87.5%. From these results, on the basis of the rDNA sequences, we conclude that E. fibuliger may have a relatively closer evolutionary relationship to S. cerevisiae and C. albicans, than Y. lipolytica. As far as we are aware, this detailed description of the organization of the rRNA genes of E. fibuliger constitutes the first report of its kind for this yeast. Integrative Vectors for Multicopy Integration into the rDNA Locus of the Genomic D N A of Yeast The use of rDNA in an integrative plasmid for gene cloning would provide a system for multiple copy integra-
tion into reiterated rDNA loci in the genome of the recipient. The plasmid pYN1826ALP1 which contained the ALPI gene of E. fibuliger was constructed by ligating the 5.5 kb HindIII - Sali ALPI-containing fragment of pSf~I (Yamashita et al. I985a) into the corresponding site of pYN1826 (Yip, C.W., unpublished work), pYN1826 contained the 5.8S and a part of the 26S rRNA gene sequences on a 3.7 kb EcoRI fragment from pYN12-K13 cloned into the EcoRI site of pUCI8. A selectable marker, the karl gene of Tng03 encoding the enzyme aminoglycoside 3'-phosphotransferase I, which confers resistance to G418 (geneticin), was used. The APH(3')-I gene was on plasmid p371 and primers were designed according to the available sequence of the APH(3')-I gene to amplify the 816 bp structural gene, one starting from the ATG start codon and the other from the TAA stop codon (Oka et al. 1981). Using suitable restriction sites tagged onto the primer at its 5' end, the entire GLUI upstream fragment of 321bp (Itoh et al. 1987) which encompassed the promoter was placed just before the ATG start codon of the APH(3')-I structural gene in the same orientation. Likewise, the amplified GLUI fragment of 657 bp containing the transcriptional terminator was joined to the 3' end of the APH(3')-I structural gene, just after the TAA stop codon. The three contiguous parts of the GLU1 upstream fragment, the APH(3')-I structural gene and the GLUI transcriptional terminator in pUCI8 vector was named p18GPT. The sequential cloning steps are shown in Table 3. The replacement with the E. fibuliger promoter and transcriptional terminator was to ensure recognition by the RNA polymerase II of E. fibuliger following transformation of this yeast. The insert from plSGPT was cloned into pYN1826ALP1 at its Sail - KpnI sites, generating pYN1826ALPI-GPT. The map of this plasmid is shown in Figure 8. Transformation of S. cerevisiae INVSC2 M A T a his3 ura3 Because of the ease of transformation into S. cerevisiae with the availability of an established protocol, this vector system was first transformed into this yeast to test its functionality. Moreover, the 26S rRNA gene of E. fibuliger was found to be most homologous to that of S. cerevisiae
World]ournalof Microbiology& Biotechnology,Vol 13, 1997
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C. W. Yip, C. W. Liew and B.H. Nga
recipient was completely inhibited at 50 fig G418/ml. The transformants also showed halos due to starch hydrolysis by the a-amylase enzyme secreted, whereas the recipient, when grown on YEA with 1.5% starch only, did not. This is shown in Figure 9.
Figure 9. S. cerevisiae INVSC2 transformants grown on (a) YEA with 1.5% starch; and (b) YEA with 1.5% starch and 80,ug G418/ ml with the recipient INVSC2 as control. The halos were due to starch hydrolysis by the a-amylase enzyme secreted by the transformants. The recipient did not produce any halo on starch medium and its growth was inhibited by G418. as compared with other yeast strains, raising the likelihood that homologous recombination may occur between the 26S rRNA genes of the two yeasts. In addition the E. fibuliger ALPI and GLUI promoters have been shown to function in S. cerevisiae (Yamashita el al. 1985a, b), but there is no information for the reverse situation. To make the ends more recombinogenic, the 13.7 kb plasmid pYN1826ALP1-GPT was linearized at a unique HpaI site within the 26S rRNA gene prior to transformation into S. cerevisiae INVSC2. At the region of the HpaI site, where homologous recombination and subsequent integration into the S. cerevisiae rDNA locus took place, the percentage homology between the 26S rRNA genes of the two yeasts is 91%. Moreover, about 20 nucleotides directly flanking the HpaI site of the E. fibuliger rDNA were identical to that of S. cerevisiae, but this HpaI site was absent in S. cerevisiae rDNA due to a difference in one nudeotide. Approximately 50 colonies were obtained on each YEA plate containing 1.5% starch and 50/1g G418/ml after 3 days of incubation at 30~ thus giving a transformation efficiency of 50//2g transforming DNA. While the transformants grew well in the presence of G418, growth of the
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WorldJournalof Microbiology& Biotechnology,Vol 13, 1997
Southern Analysis of S. cerevisiae INVSC2 G418-resistant Transformants The chromosomal DNA of three transformants, GPT1, GPT2 and GPT3, was prepared, digested with ApaI (which cut at a unique site in pYN1826ALP1-GPT), and separated by electrophoresis through an agarose gel. Following Southern transfer of the DNA fragments to a nylon membrane, hybridization of the blot was carried out using the 1.8 kb XhoI - StuI fragment of the ALPI gene, and the 0.8 kb XbaI - BamHI fragment spanning the complete APH(3')-I structural gene as DNA probes in separate experiments. In all cases, a band of 13.7 kb corresponding to the linearized size of plasmid pYN1826ALP1-GPT was obtained for transformants GPTI and GPT2, suggesting that tandem copies of the plasmid had integrated into the chromosome at the homologous rDNA locus (Figure 10). In the case of transformant GPT3, two bands were obtained, a slightly more intense one of 13.7kb corresponding to the linearized plasmid present in two or more copies, and a faint band of about 15 kb. The latter band most likely resulted from a single, circular plasmid which had integrated into a region of homology in the rDNA other than that around the HpaI site. There is also a possibility that a single, non-homologous integration could have occurred, as observed by Oakley et al. (1987) in studies of the pyrG gene of Aspergillus nidulans. However, this could only have occurred if the transforming plasmid was in the circular form, because a linear plasmid would possess the recombinogenic ends that would target it to the homologous site (Orr-Weaver et al. 1981). The circular form could arise from in vivo ligation of the linear plasmid once inside the cell, or a small percentage of non-digested plasmid DNA prior to transformation. The intensity of the signal for GPTI was stronger than those for GPT2 and GPT3 with both probes used. This may be caused by a few copies of the plasmid, perhaps in tandem, in the transformant GPT1. G418 Resistance Levels of Transformants Two transformants, GPTI and GPT2, were analysed for their G418 resistance levels. About 5000 cells of each transformant were spread on YEAS with 50, 100, 150, 200, 250 and 300/2g G418/ml. At low G418 concentrations (50 to 200/2g/ml), the number of single, G418-resistant cells that grew was about the same for both transformants. However, at higher G418 concentrations, the extent of G418 inhibition on growth was clearer. At 250/~g G418/ ml, only one single colony of GPT2 grew, whereas about 20 single colonies of GPT1 were obtained after 4 days of
Ribosomal R N A genes of E. Fibuliger excellent correlation between minimum inhibitory G418 concentration and copy number in the transformants of Pichia pastoris. Extracellular a-Amylase Assay of S. cerevisiae INVSC2 Transformants Two transformants, GPT1 and GPT2, were assayed for aamylase production with the recipient, INVSC2, as control. The strains were grown in 100 ml YNBGS supplemented with the appropriate amino acids. At specific intervals, over a period of 100 h, about I ml of culture fluid was withdrawn and the cells were centrifuged. The supernatants were used in the determination of secreted a-amylase. The production of a-amylase started at between 20 and 35 h, and there was an increasing trend of secretion until it reached the maximum level at 71 to 77 h. This period corresponded to the late exponential or stationary phase of cell growth in the batch culture (Figure 11). The increasing level of secretion was most probably caused by the depletion of glucose in the medium, thus requiring the breakdown of starch to simple assimilative sugars. At the period of maximum secretion, transformant GPT1 produced approximately 2.5-fold higher a-amylase levels than transformant GPT2. This could reflect the difference in copy number in the two transformants, consistent with the earlier observations of the signal intensities in the Southern hybridization analysis as well as the G418 resistance levels. a-Amylase secretion was expressed in terms of mg maltose formed/107 cells. This enzyme catalyses the conversion of starch to short chain oligomers, especially reducing sugars like maltose and maltotriose. This occurs very early in the incubation. Only one maltose standard was required since both these reducing sugars contain one reducing group. Measurement of a-amylase secreted was based on the cells/ml and not on total extracellular protein, which could not be measured due to proteins present in the culture medium. Figure 10. (a) Apal-digested chromosomal DNA of the recipient INVSC2 (lane 2); transformants GPT1 (lane 3); GPT2 (lane 4); and GPT3 (lane 5). The sizes of the 1 kb DNA ladder fragments (lane 1) are indicated on the left. Southern hybridizations of the chromosomal DNA with the (b) APH(3')-I and (c) ALP1 gene probes in separate experiments.
incubation at 30~ At 300/lg G418/ml the growth of GPT2 was totally inhibited, and only one colony of GPT1 grew. Hence, GPT1 showed a higher level of resistance to G418, as compared with GPT2. Due to the strong correlation between the signal intensities and the resistance levels of the transformants, GPT1 and GPT2, it is tempting to suggest that a different copy number of plasmid DNA had integrated at the genomic rDNA locus. This was also demonstrated by Scorer et al. (1994) who obtained an
Conclusion We have developed an integrative vector based on the rDNA of E. fibuliger for S. cerevisiae. Such a vector has a great potential as a shuttle vector among different strains of yeast and possibly even fungi due to the homologous nature of the rRNA genes. In view of the fact that a plasmid such as pYN1826ALPI-GPT is ideally suited for transformation in E. fibuliger, preliminary experiments have been carried out in which E. fibuliger A6 met lysI argl has been transformed with it. In these studies, one putativ6 stable G418-resistant transformant has been examined. Work is now being carried out on the analysis of this transformant in greater depth and to improve the frequency" of transformation for this yeast.
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C. W. Yip, C. W. Liew and B.H. Nga s 8 I
6
•
6
%
0
10
20
30
40
50
60
70
80
90
100
Tim~ (11)
Figure 11. c~-Amylase secretion of S. cerevisiae INVSC2 transformants and the recipient in terms of mg maltose per 10 7 cells with respect to time (h) of growth. The values obtained were the average values of triplicate readings from two independent experiments. 9 - - GPT1; 9 - - GPT2; 9 - - INVSC2.
Acknowledgement The work described in this report is part of a project supported by grant RP930356 from the National University of Singapore.
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(Received in revised form 11 March 1996; accepted I9 April 1996)
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