MOLECULAR AND CELLULAR BIOLOGY, Jan. 1987, p. 209-217
`0270-7306/87 /010209-09$02.00/0
`Copyright © 1987, American Society for Microbiology
`
`Vol. 7, No. 1
`
`Development of Autonomously Replicating Plasmids
`for Candida albicans
`M. B. KURTZ,* M. W. CORTELYOU, S. M. MILLER, M. LAI, AND D.R. KIRSCH
`Department of Cellular Biology, Squibb Institute for Medical Research, Princeton, New Jersey 08543-4000
`
`Received 23 July 1986/Accepted 14 October 1986
`
`A pool of Candida albicans Rsal fragments cloned onto a vector containing pBR322 sequences and the
`Candida ADE2 gene was used to transform a Candida ade2 mutant to adenine protrophy. A potential
`autonomously replicating sequence (ARS) in Candida DNA was identified by two criteria: (i) instability of the
`selectable marker in the absence of selection and (ii) the presence of free plasmid in total DNA preparations.
`Plasmids carrying the ARS transformed C. albicans at a high frequency (200 to 1,000 ADE+ transformants per
`fl,g of DNA), and Southern hybridization analysis of these transformants indicated that multiple copies of the
`plasmid sequences were present and that, although they were present in high-molecular-weight molecules,
`these sequences had not undergone rearrangement. Orthogonal field alternation gel electrophoresis indicated
`that the high-molecular-weight transforming sequences were not associated with any chromosome. The
`simplest interpretation to account for these data is that the transforming sequences are present as oligomers
`consisting of head-to-tail tandem repeats. The transformed strains occasionally yield stable segregants in which
`the transforming sequences are integrated into the chromosome as repeats. The Candida sequence responsible
`for the ARS phenotype was limited to a single 0.35-kilobase Rsal fragment which is present in one copy per
`haploid genome.
`
`Candida albicans is the major fungal pathogen of man
`(34). With increased use of immunosuppressive therapy and
`the increased incidence of immunosuppressive disease, C.
`albicans infections are a rapidly growing cause of noso(cid:173)
`comial infections, morbidity, and mortality (20). Several
`laboratories have pursued genetic studies in C. albicans, in
`part as a means to understand the nature of pathogenic
`determinants in C. albicans and to develop improved thera(cid:173)
`pies for candidal infections. Genetic analysis in C. albicans
`is frustrated by the fact that the organism has a diploid
`genome and no known sexual cycle (40). New techniques are
`continuously being developed to facilitate genetic studies in
`C. albicans. Parasexual analysis after fusion of protoplasts
`has been simplified by the observation that diploids can be
`selected from tetraploid fusion products by heat shock (18)
`or by recessive fluorocytosine resistance (46, 47). Recombi(cid:173)
`nant DNA technology can also be applied to C. albicans by
`using an integrative transformation system that we have
`described previously (25).
`In several fungal species, autonomously replicating se(cid:173)
`quences (ARSs) have been identified subsequent to _the
`development of protocols for integrative transformation (10,
`11, 14, 24, 38, 43, 44, 45). Vectors carrying such sequences
`transform at higher frequencies anq facilitate cloning of
`nuclear genes by mutant complementation. Sequences on
`autonomously replicating vectors are generally present at
`high copy numbers and provide the potential to study the
`effects of gene dosage on gene expression.
`This report describes the isolation of such a sequence and
`its behavior in C. albicans transformants. Similar to ARS
`elements in Saccharomyces cerevisiae, the Candida ARS
`element produces an increased transformation rate and
`unstable transformants which show a high copy number of
`transforming DNA sequences. Unlike chromosomal ARSs
`
`* Corresponding author.
`
`209
`
`from S. cerevisiae, the cloned C. albicans ARS promotes the
`formation of head-to-tail tandem repeats which are either
`present as unstable free plasmids or integrated stably into
`the chromosome. We also show that a C. albicans gene can
`be overexpressed by cloning onto these high-copy-number
`vectors.
`
`MATERIALS AND METHODS
`Materials. J3-Glucuronidase (type H-2), o-sorbitol, poly(cid:173)
`ethylene glycol 4000, trimethoprim, methotrexate, sulfanil(cid:173)
`amide, and dihydrofolic acid were obtained from Sigma
`Chemical Co. (St. Louis, Mo.). [a-32P]deoxynucleotide tri(cid:173)
`phosphates (7,600 Ci/nmol) were from New England Nuclear
`Corp. (Boston, Mass.), BioTrace RP was from Gelman
`Sciences, Inc. (Ann Arbor, Mich.), Elutip-d was from
`Schleicher & Schuell, Inc. (Keene, N .H.), and restriction
`enzymes and DNA-modifying enzymes were from Bethesda
`Research Laboratories, Inc. (Gaithersburg, Md.) or New
`England BioLabs, Inc. (Beverly, Mass.).
`Strains. Escherichia coli RRl (5), a recA + strain, was used
`for routine amplification of plasmids. When a recA strain
`was desired E. coli HB101 was used (6). To detect the
`function of the C. albicans URA3 gene, E. coli DB6656
`(pyrF::µ trp lacZ hsdR) was transformed to antibiotic resist(cid:173)
`ance and tested on defined medium lacking uracil.
`The ade2-J-containing S. cerevisiae strain, W343-4A,
`kindly provided by R. Rothstein, has the following markers:
`mata ade2-l ura3-JJ leu2-3 leu2-112 lys2-l his3-JJ his3-15
`canl-100. Strain A-3 (mata leu2-3 leu2-112 his3-JJ his3-15
`was used as the recipient for some YEp13 constructions.
`Strain S288C (mata SUC2 mal mel ga/2 CUPJ) was used as
`the S. cerevisiae wild type for chromosome separations.
`A spontaneous MET+ derivative (strain SGY-129) of C.
`albicans hOG300 (35) has the markers ade2 pro. SGY-129
`was used as the recipient for most of the transformation
`experiments described below. The other independently iso(cid:173)
`lated ade2 C. albicans strains employed in the ADE2
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1062, Page 1 of 9
`
`

`

`210
`
`KURTZ ET AL.
`
`MOL. CELL. BIOL.
`
`subcloning were A81-Pu (ade2) (26) and hOG480 (ino ade2)
`kindly provided by R. Poulter. Strain SC5314 is a clinical
`isolate and is the source for wild-type DNA for library
`construction. Strain SGY-243, a homozygous ura3 deletion
`mutant that lacks a 4-kilobase (kb) fragment encompassing
`the entire -1.2-kb URA3 coding region and flanking DNA,
`has been described elsewhere (23a).
`Plasmid and library construction. Standard recombinant
`DNA techniques were used to subclone the 5.2-kb ClaI
`(:andida fragment containing the ADE2 gene from pMC2
`(25). pSM7, the plasmid used for library construction, con(cid:173)
`tains the left-hand EcoRV insert fragment of pl\fC2 (which
`includes 190 base pairs of pBR322 sequence from EcoRV to
`the BamHI-Sau3A junction) inserted into the EcoRV site of
`pBR322. Small C. albicans DNA fragments of -1.5kb were
`prepared by digesting strain SC5314 total genomic DNA to
`completion with RsaI which produces blunt ends. The
`fragments were ligated to Pvull-cleaved pSM7 DNA, and
`the reaction mix was used to transform E. coli RRl cells to
`ampicillin resistance. A total of 24,000 individual transfomi(cid:173)
`ants were recovered. Plasmid from 16 colonies chosen at
`random was isolated by the rapid boiling procedure of
`Holmes and Quigley (19). The restriction nuclease pattern of
`the plasmids indicated that at least 40% had inserts. DNA
`was isolated from a pool of these 24,000 E. coli transform(cid:173)
`ants. Subcloning p56 and its derivatives was accomplished
`by standard recombinant DNA techniques. A description of
`the plasmids is provided in the Results section. The integra(cid:173)
`tive URA3-containing plasmids, pET5 and pUR3, have been
`described previously (6, 23a). pET5 contains a 15-kb insert
`of the C. albicar,,s Ura3 gene region in YEp13. pUR3 has a
`6.1-kb PstI fragment containing the URA3 region in pBR322.
`Isolation and subcloning the C. albicans dihydrofolate
`reductase (DHFR) gene is described in the Results section.
`The gene was isolated from a library of Sau3A fragments in
`YEp13 (13).
`Media. A rich medium, YEPD (2% Bacto-Peptone [Difeo
`Laboratories, Detroit, Mich.], 1% yeast extract, 2% glu(cid:173)
`cose), was used to grow C. albicans and S. cerevisiae.
`Synthetic media were prepared by addition of the appropri(cid:173)
`ate amino acids or nucleic acid bases to 0. 7% yeast nitrogen
`base (without amino acids) and 2% glucose (41). Media were
`solidified with 1.5% agar. Transformation media contained 1
`M sorbitol. To test for DHFR overexpression in C. albicans,
`we used the selective medium of Miyajima et al. (32). This
`medium contains 5 mg of sulfanilamide per ml and 10 µ.g of
`methotrexate per ml to inhibit the dihydrofolate pathway. To
`test for DHFR expression in E. coli, we grew cells in Luria
`broth or minimal medium containing the appropriate antibi(cid:173)
`otic for plasmid maintenance (31).
`DNA isolation and hybridization. Plasmid DNAs in E.coli
`were amplified and isolated by standard procedures (28).
`Plasmid DNA from S. cerevisiae was isolated by the small(cid:173)
`scale rapid method of Sherman et al. (41). Total genomic
`DNA from S. cerevisiae or C. albicans was prepared by
`standard methods (41). CsCl gradient purification was some(cid:173)
`times omitted. Digestion of genomic or plasmid DNA with
`restriction enzymes was carried out as described by Maniatis
`et al. (28). DNA fragments were separated by electrophore(cid:173)
`sis on 0.5 to 0. 7% agarose slab gels in 89 mM Tris borate
`buffer, pH 8.0. For orthogonal field alternating gel electro(cid:173)
`phoresis, the conditions described by Carle and Olson (9)
`were reproduced with a 1.5% agarose gel, a 300-V field, and
`a 20-s switch interval. Gel-fractionated DNA restriction
`fragments were transferred to BioTrace RP by the procedure
`recommended by the manufacturer. Hybridizations with
`
`DNA labeled by nick translation were performed by stan(cid:173)
`dard procedures (28).
`Transformation. Calcium chloride-treated E. coli cells
`were transformed by the method described by Maniatis et al.
`(28). S. cerevisiae spheroplasts were transformed by the
`method of Beggs (4). C. albicans spheroplasts were trans(cid:173)
`formed by the Saccharomyces protocol described by Kurtz
`et al. (25). Candida transformants were selected on the
`surface of selective medium with 1 M sorbitol as osmotic
`support.
`DHFR assay. The spectrophotometric DHFR assay of
`Baccanari et al. (1) was used to determine enzyme activity of
`crude extracts of E. coli cells. One enzyme unit is defined as
`the amount of enzyme required to reduce 1 µ.mol of
`dihydrofolate per min and is expressed per milligram of total
`protein. Yeast cell extracts were prepared and assayed by a
`variation of the above method described by Nath and Baptist
`(33) except that cells were broken by vortexing with glass
`beads. In all cases, values reported are the average of two
`independent experiments.
`Plasmid copy number determination. Total DNA from C.
`albicans transformants was digested with HindIII, and
`Southern blots of the serially diluted, cut DNA were hybrid(cid:173)
`ized with 32P-labeled pSM7. The two genomic HindIII
`fragments of the ADE2 gene served as a single-copy gene
`standard. The intensity of the hybridization signal from
`vector sequences was quantified by visual inspection of the
`dilution series as compared with the single-copy control.
`These estimations were based on the assumptions that all the
`DNA fragments transferred with equal efficiency and that
`hybridization was proportional to the homology of the probe
`to the DNA fragment. No correction was made for the fact
`that a large fraction of cells do not have plasmid even under
`selective pressure. In two determinations, the appropriate
`region· qf the gel was identified on the autoradiogram, cut
`out, and counted. A blank region of the gel of equal size was
`used for background determinations. The counts were cor(cid:173)
`rected to account for the different homology length· of the
`probe and compared with the single-copy ADE2 gene. Es(cid:173)
`sentially the same results were obtained by visual inspection
`and by counting the fragments.
`·
`
`RESULTS
`Isolation of putative ARS sequence from C. albicans. We
`had previously used a 5.2-kb C. albicans fragment carrying
`the ADE2 gene in pBR322 or YEp13 as an integrative vector.
`Subcloning experiments indicated that a 2.3-kb region of this
`fragment was sufficient to produce ade2 complementation in
`both Candida mutants tested (see Materials and Methods
`and Fig. 1). This region along with 190 base pairs of pBR322
`sequence was subcloned as an EcoRV fragment into the
`EcoRV site of pBR322 to produce plasmid pSM7. This
`vector was then used to construct a library of Candida DNA
`RsaI fragments as described in Materials and Methods.
`Based on the characteristics of chromosomally derived
`ARS plasmids in S. cerevisiae, two criteria were chosen to
`screen potential ARS plasmids in C. albicans: (i) instability
`of the selectable marker (ADE+) in the absence of selection
`and (ii) the presence of free plasmid in total yeast DNA
`preparations. C. albicans SGY-129 (ade2 pro) was trans(cid:173)
`formed with the RsaI library, and 12 unstable ADE+ trans(cid:173)
`formants were selected from 260 adenine prototrophs repre(cid:173)
`senting 70 to 90 kb of insert. Total DNA from these 12
`unstable transformants was used to transform E. coli to
`ampicillin resistance to test for free plasmid.
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1062, Page 2 of 9
`
`

`

`VoL. 7, 1987
`
`C. ALB/CANS AUTONOMOUSLY REPLICATING PLASMIDS
`
`211
`
`Plasmid
`
`Vector
`
`pMC2
`
`pMC3
`pMK3H
`
`pMK5
`
`pMK7
`
`pMK6
`pSM7
`
`pBR322
`
`pUC12
`
`YEp13
`
`pBR322
`
`pBR322
`
`pBR322
`
`pBR322
`
`pMKB
`
`pET1
`
`Insert DNA
`
`H BR
`I I I
`
`E::)(Bg
`
`s
`I
`
`X
`I
`X
`
`H
`
`B
`
`s
`s
`s
`r
`s
`
`R
`I
`B
`
`K X,Bg HR EC
`II
`11
`I
`I
`X
`
`H
`
`C
`
`K
`
`Bg
`
`------
`
`E
`
`'g
`
`y
`
`Complementation
`+
`
`+
`+
`+
`
`pBR322
`pMK4
`FIG. 1. Subcloning of C. albicans insert DNA and localization of the ade2-complementing region. Details of plasmid construction are
`given in Materials and Methods. Selected restriction sites are shown: S, Sau3A; X, Xbal; H, Hindill; R, EcoRl Bg, Bgfll; K, Kpnl; and C,
`Clal. Complementation was determined by the appearance of ADE+ transformants of C. albicans SGY-129, A81-Pu, and hOG480. Cloning
`vehicles pETl (13), pBR322 (5), YEp13 (7), and pUC12 (30) have been described previously.
`
`The plasmids from each of the E. coli transformants were
`isolated and used to retransform C. albicans. Only one of
`these plasmids, p56, transformed at a high frequency (200 to
`1,000 ADE+ transformants per µg of plasmid DNA) com(cid:173)
`pared with the parent plasmid, pSM7 (0.5 to 5/µg). The
`remaining plasmids transformed at the same frequency as
`the parent plasmid. Plasmid p56 also conferred the unstable
`adenine prototrophy expected of an autonomously replicat(cid:173)
`ing vector. After eight generations in nonselective medium,
`only 1 to 2% of the progeny were still ADE+. We define the
`sequence(s) producing these properties as Candida ARS 1
`(CARS!).
`Characterization of CARSl. The unstable adenine
`prototrophs produced by p56 transformation are small pink
`colonies on selective medium, while stable integrated pSM7
`transformants produce white, vigorously growing colonies.
`The pink color of these prototrophic colonies is probably due
`to the unstable nature of the chromosomal ARS. In S.
`cerevisiae, the C. albicans ADE2 gene on an S. cerevisiae
`ARSl plasmid resulted in pink colonies, while the same gene
`on the more stable 2µm plasmid gave white colonies. Trans(cid:173)
`formation of C. albicans with p56 also produces a small
`number (5/µg) of stable, white transformants, probably the
`result of plasmid integration into the Candida chromosome.
`The pink colonies are unstable on selective medium as well
`as on adenine-containing medium, producing white fast(cid:173)
`growing papillae after extended incubation. These white
`prototrophs are presumably due to integration of the auton(cid:173)
`omously replicating plasmid into the Candida genome as
`they are stable.
`To confirm the presence of free plasmid in p56 transform(cid:173)
`ants, total uncut DNAs from the original Candida isolate
`(SGY-159), three pink retransformants (R-1, R-2, R-3), and
`one white colony (W-1) were isolated and analyzed by
`hybridization in gel blots. Unexpectedly, the results of this
`experiment showed that hybridization with pBR322 (or p56
`[data not shown]) occurred at the position of chromosomal
`DNA in all cases (Fig. 2A). However, the high intensity of
`the hybridization bands compared with that of the control
`strains with pSM7 integrated into the chromosome suggested
`that multiple copies of the plasmid sequences were present
`(compare lanes 8 and 9 with lanes 2 to 5). These integrated
`pSM7 transformants were chosen on the basis of having the
`strongest hybridization to vector sequences in colony hy(cid:173)
`bridization experiments. Southern blot analysis indicated the
`presence of multiple (at least two) integrated copies of the
`
`vector. Three Pvull bands (10, 8, and 4.8 kb) are expected
`when DNA from a transformant carrying a single integrated
`copy of pSM7 is probed with vector sequences. These two
`transformants, however, contained four or five hybridizing
`bands respectively (Fig. 2C, lanes 8 and 9,), indicating the
`presence of additional integrated plasmid copies. This there(cid:173)
`fore demonstrates a bona fide increase in copy number
`produced by p56 relative to what can be achieved via simple
`integrative transformation. The untransformed recipient,
`SGY-129, does not hybridize to the pBR322 probe.
`The unexpected hybridization of vector sequences as a
`high-molecular-weight band suggested that most, if not all,
`of the p56 sequence was not present as a simple, monomeric
`plasmid. To determine whether the p56 sequence had been
`rearranged in the transformants, total DNA from several
`transformants was digested with HindIII, and Southern blots
`were probed with 32P-labeled pSM7. All the pink transform(cid:173)
`ants (Fig. 2B, lanes 2, 3, and 5) showed the pattern expected
`for unrearranged p56 DNA (1.3- and 7.2-kb bands), as did
`the white transformant (lane 4). As expected, all the trans(cid:173)
`formants have the 3.4- and 4.0-kb HindIII fragments pro(cid:173)
`duced by hybridization to the chromosomal ADE2 se(cid:173)
`quences. The intensity of the 1.3- and 7 .2-kb plasmid bands
`indicates that they are present in greater copy number than
`the genomic ADE2 gene. In addition to the plasmid and
`genomic copy bands, some of the transformants (Fig. 2B,
`lanes 5, 6, 11, and 13) display other fragments which
`hybridize to pSM7. One of these fragments (2.45 kb) was
`also seen in strain SGY-416, a pSM7 transformant (lane 9),
`and may indicate the existence of another potential site for
`plasmid integration.
`To test whether multiple single copies of the plasmid are
`integrated at various sites in the genome, we cut total DNA
`from transformants with Pvull. Plasmid p56 has no internal
`Pvull sites as a result of the RsaI cloning. Thus, for
`integrated plasmids, the fragment size generated by cleavage
`of transformant DNA with Pvull would depend on the
`chromosomal location of the plasmid and the nearest Pvull
`sites. Multiple single copies would produce many different(cid:173)
`sized bands hybridizing to the pSM7 probe in addition to the
`known chromosomal ade2 locus. Integrated tandem repeats
`would appear as a single very large fragment since there
`would be no Pvull sites within the entire repeated unit.
`Oligomeric free plasmid would also hybridize as a single
`large fragment. The Southern hybridization results in Fig. 2C
`indicate that the plasmid sequences are present as a large
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1062, Page 3 of 9
`
`

`

`212
`
`KURTZ ET AL.
`
`MOL CELL. BIOL
`
`p56
`transformants
`.:!' !£?
`!£?
`,5?.> R?
`pMK16
`~ ,:._v""'""'
`k
`"' G G J J transformants ~
`,.... <v
`>:-
`,,£:)
`<'/ Q: Q:: ~ Q:: 0 0
`0 0 ...-------. Q
`
`A
`
`B
`
`C
`
`-
`
`-
`
`-
`
`23.6
`9.5
`6.7
`4.3
`
`2.3
`2.0
`
`2 3 4 5 6 7 8 9 10 11 12 13 14
`
`-
`
`2
`
`3 4 5 6 7 8 9 10 11 12 13 14
`
`7.2
`4.0
`
`3.4
`
`1.3
`
`23.6
`9.5
`6.7
`
`4.3
`
`2.3
`
`9 10 11 12 13 14
`7 8
`2 3 4 5 6
`FIG. 2. Autoradiograms of Southern blot hybridizations of
`genomic DNAs from p56 and pMK16 transformants of SGY-129.
`Numbers on the right indicate size in kilobases. (A) Equivalent
`amounts of uncut genomic DNA were run in each lane on a 0.7% gel,
`and blots were probed with 32P-labeled pBR322. Lanes: 1, uncut
`p56; 2 to 5, p56 transformants; 6, original p56-containing isolate,
`SGY-159; 7, untransformed SGY-129; 8 and 9, pSM7 transformants
`SGY-415 and SGY-416; 10 to 13, pMK16 transformants; 14, uncut
`pMK16.(B) HindIII-cut genomic DNAs were run on a 0.7% gel, and
`blots were probed with 32P-labeled pSM7. Equivalent amounts were
`used in each lane except for lane 7, in which seven times as much
`DNA was used to visualize clearly the position of the genomic ADE2
`fragments . The slightly greater mobility of the ADE2-hybridizing
`bands compared with that of the other lanes is probably due to
`relative overloading. Lanes are as in panel A. Hybridization to pure
`plasmid is extremely faint in lanes 1 and 14 and was visualized by
`longer exposures. (C) Equivalent amounts of Pvull-cut genomic
`DNAs were run in each lane on a 0.5% gel, and blots were probed
`with 32P-labeled pSM7. Lanes are as in panel A.
`
`unresolved smear above 30 kb in size, consistent with
`tandem multimers. The single-copy Pvull fragment of the
`ADE2 gene (8 kb) is clearly resolved in this gel, and
`Pvull-treated p56 DNA runs as covalently closed circles
`(compare lanes 1 in Fig. 2A and C). Last, to rule out
`
`concatenated circles as a mechanism to produce these re(cid:173)
`sults, DNA from p56 transformants was digested with
`topoisomerase II and run on gels. No change in hybridiza(cid:173)
`tion pattern was observed relative to untreated DNA (data
`not shown).
`Additional evidence favoring the presence of large
`multimeric plasmids comes from E. coli transformation
`experiments. In general, Candida DNA preparations trans(cid:173)
`formed E. coli cells poorly, and this was at least in part due
`to an inhibitor of transformation in the preparations. Trans(cid:173)
`formation was especially poor with recA cells (HBlOl) when
`compared with otherwise largely isogenic recA + cells (RRl).
`The lower transformation rate observed with recA cells
`could potentially be due to the inability to convert large
`oligomeric plasmids to monomers which are inherited more
`efficently. In one rare HBlOl transformant, a large
`oligomeric plasmid was isolated after transformation with
`total Candida DNA. When partially digested with ClaI, the
`oligomeric plasmid produced fragments of ~26, 17, and 8.5
`kb indicating a tandem repeat of three or more (data not
`shown). Large plasmids were transiently seen in recA + cells,
`but were rapidly converted to the monomer, presumbably by
`homologous recombination. Similar difficulties in recovering
`multimeric plasmids from Schizosaccharomyces pombe cells
`in E. coli have been reported (38).
`Direct proof for free plasmids in the p56 transformant was
`obtained by separating the CARS plasmid from the Candida
`chromosomes on pulsed orthogonal field gels. Conditions
`were chosen (Fig. 3A) so that all the large chromosomes of
`C. albicans remained near the well. Under these conditions
`some of the smaller S. cerevisiae chromosomes were re(cid:173)
`solved (compare lanes 1 to 3 with lane 4). When blots of
`these gels were probed with 32P-labeled pBR322 for plasmid
`sequences, strain SGY-415, a pSM7 integrative transform(cid:173)
`ant, hybridized at the position of the Candida chromosomes.
`In contrast, DNA from SGY-417, a p56 transformant, hy(cid:173)
`bridized intensely both at the well and at a position well
`below the Candida chromosomes. Since free plasmids run
`anomalously in these gels, the size of the plasmid could not
`be determined. We consistently observed significant hybrid-
`
`B
`
`!P
`-"
`!;f
`
`:fl
`;r-
`!;f
`
`..
`
`:::
`,,.
`-"
`!;f
`
`j
`(J
`
`-t
`
`- well
`
`- Candida
`chromosomes
`
`- free plasmid
`
`2 3 4
`1
`1 2 3 4
`FIG. 3. Orthogonal field alternation gel electrophoresis gels of C.
`albicans transformed with integrative and CARS vectors. (A)
`Ethidum bromide-stained gel. Lanes: 1, SGY-129, untransformed
`control; 2, SGY-415, an integrative pSM7 transformant; 3,
`SGY -417, a pink p56 transformant; 4, S288C, a wild-type S . cerevi(cid:173)
`siae strain included to show chromosome separation. (B) Autora(cid:173)
`diogram of gel in panel A blotted and probed with 32P-labeled
`pBR322.
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1062, Page 4 of 9
`
`

`

`VOL. 7, 1987
`
`C. ALB/CANS AUTONOMOUSLY REPLICATING PLASMIDS
`
`213
`
`A
`
`Hind Ill
`Clal
`
`Pst I
`
`Amp"
`
`lOd
`
`pSM7
`8.7kb
`
`ind Ill
`
`Eco RI
`
`Rsa I
`
`Barn HI
`
`PYu 11/Rsa I
`
`Eco RI
`
`p56
`8.5kb
`
`L•CARS
`
`I
`
`Rsa I
`Eco RI
`
`R
`
`Pvu 11/Rsa I
`
`Eco RI Rsa I
`
`Eco RI
`
`URA 3
`
`TEP
`
`pMK18
`7.8kb
`
`Eco RI
`
`Xba I
`
`pMK22
`8.0kb
`
`Pvu 11/Rsa I
`
`Rsa I
`
`Rsa 1/Pvu II
`
`B
`
`I
`1 Kb
`FIG. 4. (A) Restriction maps of some plasmids used in this study. Bold lines, pBR322 sequences; boxed lines, C. albicans sequences; L,
`left-hand Rsal fragment; I, internal Rsal fragment; R, right-hand Rsal fragment. Selected restriction sites are shown for each plasmid which
`are referred to in the cloning manipulations discussed in the text. Ori, Origin. (B) Restriction map of C. albicans inserts which contain the
`DHFR gene region.
`
`Dlhydrofolate Reductase Gene Region
`pML22 t-1 --------+l+-1 --+-----+-----+l-+-11 !
`: ..
`ii
`_i=!~
`..
`~
`~
`::ii;
`a:: - M
`8 ~ fl
`~wg
`i
`~
`W:I: Cl)
`:I:,
`pML20 ii-----1l1-f---+-----+i-----+-i +---f+-l -l.+-1--fi
`1--------4.9 Kb
`
`c(
`
`: ,
`
`•
`
`I
`
`£
`
`i3
`
`as
`
`-
`:,
`
`!'-
`••
`:,
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1062, Page 5 of 9
`
`

`

`214
`
`KURTZ ET AL.
`
`MOL. CELL. BIOL.
`
`TABLE 1. Transformation frequencies of various
`CARS-containing derivatives
`
`CARS insert
`
`Transformation frequency•
`
`Plasmid
`
`pMK16
`pMK17
`pMK13
`pMK15
`p56
`pSM7
`pMK22
`pET5
`pUR3
`
`Rsal
`fragment
`
`L
`L
`L
`I
`L, I, R
`None
`L, I, R
`None
`None
`
`No. of
`copies
`
`1
`2
`3
`2
`1 each
`0
`1 each
`0
`0
`
`ADE•
`
`URA•
`
`900
`1,300
`470-1 ,500
`14
`666-1,400
`24
`
`250
`1.1
`50
`
`• Transformation frequency was determined by calculating the number of
`ADE• or URA+ colonies per microgram of DNA. The number of revertants
`was subtracted when appropriate.
`
`ization at the well with the p56 transformant but not with
`other strains, suggesting that either the CARS plasmid does
`not enter the gel efficiently or it is present in two forms, one
`of which is unable to enter the gel.
`A rough estimate of copy number was achieved by a series
`of dilutions of HindIII-cut transformant DNA. Gel blots
`were probed with pSM7, and the relative intensities of
`plasmid and chomosomal bands were compared visually. An
`overall average copy number of 5 to 10 per diploid genome
`was determined as in Materials and Methods. An alternative
`method which counted the radioactivity in a band directly
`gave similar results.
`Subcloning the insert of p56. Restriction mapping of p56
`indicated that the insert region had three Rsal fragments
`(Fig. 4A). The three fragments may be the result of incom(cid:173)
`plete Rsal digestion of Candida DNA used to construct the
`library and therefore represent three contiguous fragments
`or may be a cloning artifact of fragment excess and represent
`three nonadjacent fragments . It was therefore of interest to
`determine whether one or more of the fragments was suffi(cid:173)
`cient for ARS activity. Initially, genomic blots of Rsal-cut
`DNA were probed with a p56 subclone lacking the ADE2
`gene (pMK12) which hybridized with three bands of 0.37,
`0.61, and 3.3 kb (data not shown). The smaller bands
`correspond in size to the two smaller Rsal fragments in p56.
`The largest band is much larger than any of the insert
`fragments, indicating that a rearrangement must have oc(cid:173)
`curred either during cloning or in vivo. Southern gel analysis
`of HindIII-, EcoRV-, and Seal-cut Candida DNA probed
`with p56 or pSM7 showed three bands in addition to the
`fragment derived from the ADE2 region (data not shown).
`The intensity of the bands indicated that the Rsal fragments
`are not reiterated relative to the single-copy ADE2 gene.
`In view of the fact that the Rsal fragments are likely
`derived from nonadjacent DNA sequences, the two com(cid:173)
`plete Rsal fragments (I and L, Fig. 4A) were subcloned and
`used to transform SGY-129 to adenine prototrophy to test
`for ARS activity. The data in Table 1 show that the L
`fragment but not the I fragment confers ARS activity.
`Furthermore, increasing the number of L tandem repeats
`from one to three does not increase transformation fre(cid:173)
`quency within the accuracy of this determination (Table 1).
`The majority of the pMK16 (one L fragment) transformants
`were pink unstable ADE+ colonies identical to p56 trans(cid:173)
`formants. Growth rate in minimal selective media was the
`same for all pink transformants tested (data not shown). The
`third Rsal fragment present in p56 was not subcloned.
`
`HindIII- or Pvull-digested and undigested total DNAs
`from pMK16 transformants were analyzed by hybridization
`of pSM7 to gel blots. As in the p56 transformants, the
`pMK16 plasmid sequences were reiterated and present as
`oligomers (Fig. 2). No free monomer band was seen (Fig.
`2A, lanes 10 to 13). Although the activity of the right-hand
`(R) fragment remains undetermined, we can define the L
`fragment as CARSl, since the L fragment was necessary and
`sufficient to confer all the properties of the p56 plasmid.
`CARSI function with the Candida URA3 gene. The unusual
`properties of CARSl made it desirable to determine the
`general usefulness of the cloned CARS. Construction of
`autonomously replicating vectors with a different selectable
`marker, the URA3 gene of C. albicans, was accomplished by
`first deleting the ADE2-containing EcoRV fragments of p56
`to generate pMK12 (Fig. 4A). A 1.5-kb Pstl-Scal fragment
`containing the URA3 gene was inserted into the Pstl-Scal
`sites of pMK12. The resultant plasmid, pMK22, was used to
`transform C. albicans SGY-243, a ura3 deletion mutant
`which lacks all but a 0.6-kb region of homology to the URA3
`insert (23a), to uracil prototrophy. The frequency of appear(cid:173)
`ance and characteristics of pMK22 transformants were com(cid:173)
`pared with those of integrative plasmids pET5 and pUR3.
`The transformation of the CARS-containing plasmids was
`much higher than that of the integrating plasmids (Table 1).
`The latter, however, produced numerous pinpoint colonies
`most of which did not grow when subcultured to medium
`lacking uracil. Plasmid pMK22 transformants were unstable;
`only 5% were still URA+ after 10 generations on uracil(cid:173)
`containing media, while the pET5 and pUR3 transformants
`were stable. Southern analysis of uncut DNA showed a
`pattern consistent with a multicopy plasmid present as an
`oligomer (Fig. 5).
`Uncut total DNA from three pMK22 transformants hy(cid:173)
`bridized to the Pstl-Scal fragment of pMK22 (the URA3
`region) at the position of chromosomal DNA (Fig. 5, lanes 5
`to 7). Since the probe contains a small piece of DNA
`homologous to the chromosome in this deletion strain, the
`faint band seen in the untransformed control SGY-243 indi-
`
`cO'
`~ I Q
`
`3.6
`2.7
`
`- - •
`7 8 9 10 11
`12
`1 2 3 4 5 6
`FIG. 5. Autoradiograms of Southern blot hybridizations of
`genomic DNAs from URA+ transformants probed with the Scal(cid:173)
`Pstl fragment of the URA3 gene (Fig. 4A). Lanes: 1 to 7, uncut
`DNAs; 8 to 12, Eco RI-digested DNAs. Equivalent amounts of DNA
`were run in each lane except for purified plasmids. Numbers on right
`are in kilobases.
`
`1.3
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1062, Page 6 of 9
`
`

`

`VoL. 7, 1987
`
`C. ALB/CANS AUTONOMOUSLY REPLICATING PLASMIDS
`
`215
`
`TABLE 2. Enzymatic assay of DHFR activity in strains
`transformed with the Candida DHFR gene
`
`Strain
`
`DHFR
`activity
`(mU/mgof
`protein)
`
`Trimethoprim-
`resistant
`DHFR
`activity
`(mU/mgof
`protein)
`
`%
`Trimethoprim
`resistant
`
`Ratio
`pMK18/p56
`
`E.coli
`pML23
`pBR325
`
`C. albicans
`pMK18
`p56
`
`2.22
`1.47
`
`12.84
`1.95
`
`1.17
`0.24
`
`53
`16
`
`6.6
`
`cates the migration of uncut chromosomal DNA (lane 4). As
`expected, the intensity of the pMK22 transformant bands
`was much greater than that o