Folia Microbiol.37 (3), 176-180 (1992)
Cloning of Candida boidinii DNA Fragments Promoting Autonomous Replication of Plasmids in Saccharomyces cerevisiae I. JANATOVA and O. NAVRATIL* Institute of Microbiology, CzechoslovakAcademy of Sciences, Videfiskd1083, 142 20 Prague4, Czechoslovakia ReceivedJanuary 7, 1992
ABSTRACT. Fragmentsof Candida boidinii chromosomalDNA were inserted into the integrativevector Ylp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated $6/4 and $6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridizationdata confirmed that the plasmids carry sequences from the C. boidinii chromosome.Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequencythan cloningvectors based on the replicationorigin of the 2#m plasmid. Mitoticstabilityof the constructedplasmidsis similar to that of the 24~-basedvector pNF2 in S. cerevisiae. The yeast strain Candida boidinii 11Bh belongs to the group of yeasts capable of utilizing methanol as the sole source of carbon and energy (Anthony 1982). Transformation systems were developed in two methylotrophic yeast species - Pichia pastoris and Hansenula polymorpha (Cregg et al. 1985; Gleeson et al. 1986; Roggenkamp et al. 1986; Tikhomirova et al. 1986) but no gene manipulations in methylotrophic Candida strains have been reported so far. One of the strategies for constructing new vectors for gene manipulations in yeasts is random cloning of DNA fragments into selectable plasmids which lack the origin of DNA replication functional in yeasts (Struhl et al. 1979; Stinchcomb et al. 1980). In this way DNA sequences acting as ARS-elements in S. cerevisiae have been found in nuclear and mitochondrial DNA of various eukaryotic microorganisms including several yeast species (Hsu et al. 1979; Kawamura et al. 1983; Tikhomirova et al. 1983; Teixeira et al. 1986; Sibson et al. 1988). In the present work we used chromosomal DNA of the methylotrophic yeast strain Candida boidinii l l B h (from which no ARS elements have as yet been cloned) as the source of sequences supporting autonomous replication of plasmids in S. cerevisiae cells.
MATERIALS
AND
METHODS
Strains and plasmids. Eschenchia coli HB101 (Maniatis et al. 1982) was used for propagation of plasmids. A wild-type strain of Candida boidinff l l B h was provided by Dr. O. Volfowi (Volfov~i and Pil~it 1974). Saccharomyces cerevisiae $2072A (a, arg4 trp l leu I gal2) and Saccharomyces cerevisiae PJF1 (a, ura3 trpl) were gifts from L.P. Tikhomirova (Institute of Biochemistry and Physiology of Microorganisms, Pushchino, Russia); S. cerevisiae AH215 (a, his3 leu2) was kindly supplied by Z. Palkowt (Institute of Biotechnology, Charles University, Prague, Czechoslovakia). The plasmids used in this work were YIp5-kan r (Rao and Reddy 1986) - a yeast integrative vector (7.2 kb) bearing S. cerevisiae URA3 gene and the gene coding for aminoglycoside phosphotransferase (APT - Km r and G418 r) from the bacterial transposon Tn903; pNF2 (Naumovski and Friedberg 1983) - a vector (10.3 kb) carrying the origin (oH) of replication of the 2/zm plasmid, URA3 gene of S. cerevisiae and APT gene from Tn903; pJDBV5 (Z. Palkov~i) - a derivative (8.0 kb) of the plasmid pJDB207 (Beggs 1981) carrying oH of the 2#m plasmid, LEU2 and URA3 genes from S. cerevisiae for selection in yeasts. Media, enzymes and chemicals. LB broth (Maniatis et al. 1982) was used for cultivation of E. coli cells; kanamycin or ampicillin was added to a final concentration of 25 m g / L when necessary. The yeast growth medium was YEPD ( 1 % yeast extract, 2 % bactopeptone, 2 % glucose). Restriction endonucleases and T4 DNA ligase were from Boehringer, Mannheim (Germany), the antibiotic G418-sulfate (Geneticin) was purchased from Sigma (USA). D N A isolations. Plasmid DNAs were extracted from E. coli by alkaline lysis (Ish-Horowicz and
*Presentaddress: Central Research Institute for Plant Production,Ruzyn6507, 161 06 Prague 6, Czechoslovakia.
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Burke 1981) and further purified by CsCI-ethidium bromide gradient centrifugation. Chromosomal DNA of C. boidinff was isolated according to Cryer et al. (1975). Total yeast DNA for retransformation ofE. coli was prepared according to Hoffman and Winston (1987). Transformation techniques. A standard calcium chloride procedure was used for E. coli transformation (Dagert and Ehrlich 1979). S. cerevisiae protoplasts were transformed according to Kurtz et al. (1986) and regenerated in a mixture of 30 % polyethylene glycol 4000 in YEPD (Svoboda and Piedra 1983). After 18-h incubation at 30 ~ the suspension was washed with glucitol (1 mol/L) and plated onto YEPD agar containing G418 (300 rag/L). Transformants appeared after 4 - 6 d at 30 *C. D N A - D N A hybridization. For hybridization analysis electrophoretically separated DNA fragments were transferred to nitrocellulose membranes according to Southern (1975) and hybridized with nick-translated probes (Rigby et al. 1977) by a standard procedure (Maniatis et al. 1982). Measurement ofplasmid stability. To assess plasmid stability cultures were grown under nonselective conditions to stationary phase, plated onto a nonselective medium and then replica.plated onto a selective medium. The copy number of the plasmids was determined using the method Cashmore et al, (1986).
RESULTS A N D DISCUSSION Chromosomal DNA of C. boidinff was partially digested with restriction endonuclease Sau3A and DNA fragments were ligated with BamHI-cleaved yeast integrative vector YIp5-kan r. E. coli HB101 cells were transformed with the ligated DNA and ampicillin-resistant clones were selected. Plasmid DNA isolated from pooled transformants and purified by CsCI-EtBr gradient centrifugation was used to transform S. cerevisiae protoplasts. From ten randomly picked G418r transformants plasmid DNA was recovered by transformation of E. coli with their total DNA. One of the examined S. cerevisiae clones yielding the highest number of E. coli kanamycin-resistant colonies was chosen for further analysis of its plasmid content. We assumed that this clone contains a plasmid carrying a chromosomal fragment with a potent ARS which assures a high level of plasmid DNA replication. Eco RX
Eco RI
_L_ , B/Sa
I
[i
_.L__f,~fl/$a
~.V
lOakh
|
II /
LH
l!~m
11.6kb
~H e
AvaI
Ava.,.
Ava.~
AvaZ Sa! I
Fig. 1. Physical maps of the plasmids $6/4 and $6/5. Chromosomal DNA inserts from C. boidinii are indicated by the thick line flanked in both plasmids by the hybrid restriction sites BamHI/Sau3A. Restriction enzymes sites: B, BamHI; E, EcoRI; H, HindIII; $, Sail; Sa, Sau3A; V, PvuII.
Screening ofE. coli Km r clones obtained after transformation with total DNA from this G418 r clone revealed the presence of two plasmids of different size - $6/4 (10.4 kb) and $6/5 (11.6 kb). According to restriction analysis the plasmids did not differ in the YIp5-kan r vector moiety but in the size of the insert (3.2 and 4.4 kb, respectively). The restriction patterns of these inserts did not share any similarities (Fig. 1). Hybridization experiments proved that the inserted sequences came from C. boidinii chromosomal DNA (data not shown). To elucidate whether there is any homology between the inserts in the plasmids $6/4 and $6/5, cross-hybridization was performed (Fig. 2). Weak signals in lanes 2 and 5 in Fig. 2 are the result of hybridization of short vector sequences flanking the inserts in YIp5-kan r present in the labelled probe as well as on the hybridizing fragments. It seems that the inserts in the plasmids $6/4 and $6/5 do not share larger homology. More probably, the presence of two different plasmids in one yeast cell could be due to cotransformation of one S. cerevisiae protoplast
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with two independent plasmids rather than a structural modification of one plasmid. Cotransformation is rather frequent in yeast cells and it was used for construction of new vectors as well (Hicks et al. 1979; Jimenez and Davies 1980; Sakai et al. 1984).
Fig. 2. Southern hybridization analysis. LeO: agarose gels; r/g/u: autoradiographs of Southern hybridization with the 32p-labelled EcoRI fragment of the plasmid $6/5 (A) and the EcoRISail fragment of the plasmid $6/4 (B) as probes. Lanes 1 and 5, plasmid $6/5 DNA digested with EcoRI; lanes 2 and 4, plasmid $6/4 DNA cleaved with EcoRI and Sail; lane 3, 2 DNA cleaved with HindIII.
We transformed several S. cerevisiae strains with our constructs and with the 24xm-based vectors pNF2 and pJDBV5 to compare the frequency of transformation (Table I). Usually 5/-~g of plasmid DNA was used for transformation. The transformation efficiency when using the plasmids $6/4 and $6/5 was at least four times higher than when the 24zm-based vectors were used. It made no difference whether S. cerevisiae transformants were selected for their resistance to the antibiotic G418 or for complementation of an auxotrophic mutation. The 2#m-based vectors are of similar size and carry the same selection markers as new constructs. The number of transformants we obtained with plasmid pNF2 is similar to those published earlier for S. cerevisiae transformation using G418 resistance as the selection marker (Lang-Hinrichs et aL 1989; Hadfield et al. 1990). Measurement of mitotic stability of plasmids $6/4, $6/5 and pNF2 showed a comparable stability of plasmid $6/5 with that of pNF2 which carries the entire region of the 2~m plasmid responsible for plasmid partitioning. The plasmid $6/4 was found to be more stable in S. cerevisiae cells than was pNF2 (Table II). This cannot be due to plasmid integration into the genome of S. cerevisiae because we were able to isolate E. coli clones after transformation with total DNA of yeast cells carrying plasmids $6/4 and $6/5. Moreover, the presence of plasmids $6/4 and $6/5 in an autonomous state was proved by D N A - DNA hybridization of intact total DNAs of the transformants harboring these plasmids.
CLONING OF C. boidinii DNA FRAGMENTS
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Table I. Transformation frequencies of plasmids in various S. cerevisiae strains
Strain
Plasmid
$2072Aa
YIp5-kan r
0
s6/4
26
$6/5
23
YIp5-kanr
0 16 14 4
$6/4 AH215 a
Number of transformants per/~g of DNAc
$6/5 pNF2
179
Table II. Mitotic stability and copy number of plasmids in S. cerevisiae AH 215 cells
Generationa
Copy
Plasmid
pNF2 $6/4 $6/5
10
20
30
number
91 97 94
82 92 93
35 90 28
3 3
al'he percentage of G418 r clones after a given number of doublings in nonselective media.
The copy number determination revealed that the plasmids $6/4 and $6/5 are mainPJF1 b tained in S. cerevisiae cells in a low copy numpJDBV5 I00 ber, similar to plasmid YRpl7 bearing ARS1 from S. cerevisiae chromosomal DNA aSeleetion of G418 r transformants. (Roggenkamp et al. 1986) (Table II), while bSelection of ura + prototrophic transformants. plasmid pNF2 is a derivative of a high-copyc/vie.an values from 3 experiments. number vector Yep24 (20-40 copies per cell) (Cashmore et al. 1986). Colonies of pNF2harboring clones were approximately 3 times larger than those with the $6/4 and $6/5 plasmids. The smaller size of the colonies of plasmid $6/4- and S6/5-harboring cells can be due to the lower copy number of these plasmids in S. cerevisiae cells which results in a lower gene dose and a lower level of aminoglycoside phosphotransferase in cells (thus in a lower level of resistance). In this study we isolated two fragments from C. boidinff chromosomal DNA functioning as ARSs in S. cerevisiae. From the experiments we carried out so far it is evident that the isolated fragments do not support autonomous replication of plasmids in Candida boidinii. It is a frequent event that heterologous ARSs functioning in S. cerevisiae cells do not have the same function in cells of the original host (Hsu et al. 1983; Cregg et al. 1985; Williamson 1985). $6/4 $6/5
460 582
We thank Dr. M. P~tek and Dr. J. Hubfi~ek for critical reading the manuscript.
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