Cloning of the DNA Repair Gene, UVSF, by Transformation … · 2002. 7. 8. · CORVO et al. 1953; KAFER 1977; SCOTT and KAFER 1982). Strains of uusF (KAFER and MAYOR 1986) and pyrG

Copyright 0 1990 by the Genetics Society of America

Cloning of the DNA Repair Gene, UVSF, by Transformation of Aspergillus nidulans

Kalpesh Oza and Etta Kafer

Department of Biology, McGill University, Montreal, Canada H3A 1Bl Manuscript received August 1, 1989

Accepted for publication February 27, 1990

ABSTRACT As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double

mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector. Rare pyr+ uvs+ cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive. Among MMS- resistant transformants, Southerns revealed two types which showed single bands of different sizes when BglII-digested genomic DNA was probed with the vector. Both types produced uvsF- recombinants without vector sequences in homozygous crosses, but only those with the larger band also produced haploid uvs+ progeny. Using BglII-digested genomic DNA to transform Escherichia coli, plasmids of the corresponding two sizes could be rescued. Their inserts had a short internal region in common, giving evidence of rearrangement(s). In secondary transformation of uvsF mutants, only the plasmids with the larger insert showed complementation and these were used to screen Aspergillus libraries. Three types of genomic and two overlapping cDNA clones were identified. The cDNAs hybridized not only to each other, but also to the common region of the rescued plasmids. Therefore, cDNA subclones were used to map the putative uvsF sequences to a short segment in one genomic clone. In Northerns, the complementing large plasmid hybridized to three mRNAs, while the cDNA subclone identified one of these as the probable uvsF message.

D NA repair-defective mutants, sensitive to UV or ionizing radiation, were first isolated in Esche-

richia coli (HOWARD-FLANDERS 1968). Such mutants were assigned to several repair pathways on the basis of extensive genetic and biochemical studies (CLARK and VOLKERT 1978; FRIEDBERG 1985). More recently, gene cloning has provided material for molecular studies of these genes, and of their protein products, interactions and functions (AMUNDSON et al. 1986; CUNNINGHAM and WEISS 1985; SANCAR and RUPP 1983). In yeast, radiation-sensitive ( rad ) mutants have also been isolated in large numbers and genetic analysis has identified epistatic and functional groups of genes which correspond fairly well to those found in E. coli (HAYNES 1975). Many RAD genes have by now been cloned using transformation of mutant strains of yeast with complementing wild type genomic sequences (PEROZZI and PRAKASH 1986; FLEER et al. 1987; FRIEDBERG 1988). Potential function of their gene products often can be identified on the basis of amino acid homologies, as deduced from DNA sequences, to well-characterized proteins from E. coli and its phages or even mammals (e.g., ALANI, SUBBIAH and KLECKNER 1989; CHEN and BERNSTEIN 1988; SUNG et al. 1987).

In Aspergillus and Neurospora, assignment of uus mutants to repair pathways has been difficult, partly because nonepistatic pairs often are inviable (KAFER

Genetics 125: 341-349 (June, 1990)

and MAYOR 1986; INOUE et al. 1981; KAFER 1983), and partly because correspondence to phenotypes of E. coli is much less common than in yeast. Similarly, cloning of genes has lagged behind especially in As- pergillus nidulans, because of the comparatively late development of an efficient transformation system (BALLANCE, BUXTON and TURNER 1983; TILBURN et al. 1983). First, cloned Aspergillus genes were re- covered by their ability to complement homologous genes in E. coli (KINGHORN and HAWKINS 1982) or in yeast (BERSE et al. 1983). More recently, this has been achieved by complementation of mutants using cotransformation of Aspergillus with wild-type library DNA in plasmid vectors that contain a selectable marker, e.g., the Neurospora pyr-4 gene which com- plements pyrG mutants of Aspergillus (BALLANCE and TURNER 1985) and was used in the present work. In Aspergillus, as in Neurospora, complementation of mutations in transformants occurs by stable integration of DNA segments, since no replicating plasmid vectors are available. The pyrG- pyr-4+ selective system has the advantage that incorporation of cloned fragments occurs mainly by homologous recombination and is not influenced by the selective gene which is heterologous.

The first gene of our choice, for molecular analysis of DNA repair in A. nidulans, was uvsF which most likely is an excision repair gene (KAFER and MAYOR,

342 K. Oza and E. Kafer

uvsF ,1 fpaB PYG

I I 19 I I “t“ gal0 ........ ......

1986). Mutant uvsF201 strains show normal growth and fertility and can easily be tested and selected against on media containing MMS. In addition, no revertants have ever been obtained in extensive at- tempts to isolate suppressors. Since many of the repair genes which have been cloned and sequenced from pro- or eukaryotes are involved in excision (LLOYD and HANAWALT 198 1 ; WEISS and FRIEDBERG 1985; VAN DUIN et al. 1986), sequence homologies or inter- species complementation tests may well be able to identify its function at the molecular level. (A prelim- inary report of this work has been published as an abstract; KAFER and OZA 1988.)

MATERIALS AND METHODS

Aspergillus strains and genetic techniques: Standard A. nidulans media and genetic techniques were used (PONTE- CORVO et al. 1953; KAFER 1977; SCOTT and KAFER 1982).

Strains of uusF (KAFER and MAYOR 1986) and pyrG (KA- FER and MAY 1988) were intercrossed to obtain uusF pyrG double mutants. The genotypes of the two recipient strains used for transformation were the following:

M3 10 1 : uusF2Ol pyrG89 riboA1 yA2; wA3; pyroA4 M3115: uusF2Ol pyrG89 riboA1 yA2; wA2; choAl; chaA1.

(Other strains, including various transformants, also have M3100 numbers but are indicated by their last two digits and are characterized in the text.)

The genetic map of the left arm of chromosome I , and the location of uusF, pyrG and linked marker genes within this region, are shown in Figure 1.

Transformation of Aspergillus: Conidia of the recipient strains were germinated in medium supplemented with 10 mM uridine and treated with “Novozyme 234” to obtain protoplasts for transformation, using the method developed by OSMANI, MAY and MORRIS (1987). Uracil-independent transformants were selected on the following regeneration medium: 1 M Sucrose, 0.5% yeast extract, 20 mM glucose, 5 mM MgS04, supplemented with trace elements and vita- min solution. For selection of transformants complemented

FIGURE 1.-Meiotic map of chromosome I (left arm) and possible insertion by homologous recombination of complementing sequences for uvsF during transformation. (=) = genomic DNA of Aspergillus nidulans; ( * * - O * * * ) = centromere region; (-) = uvsF sequences with mutant site; ( ) = corresponding wild type site; (-) = pBR322 in vector; (- - - -) = pyr-4* from Neurospora crassa, complementing the pyrimidine requirement of pyrG- in A. nidulans recipients. B = possible position of a BglII restriction site in the uvsF gene (as found in Tf34). Aspergillus gene sym- bols: uvsF = UV - sensitivity;fpaB = resistance to p-fluorophenylalanine; galD = inability to grow on galactose as carbon source; suAadE = suppressor of adE20.

for uusF, overlays with low concentrations of MMS (0.01- 0.015%) in “complete” medium lacking pyrimidines were added after 16 hours of incubation. This level of MMS inhibited growth of uusF colonies initially, but after several days at 37 O uvs-pyr+ transformants started to grow, because MMS is unstable at that temperature. All conidiating stable pyr+ transformants obtained in this way were tested for UUSF and checked for nutritional markers, by replication to MMS media lacking uracil and to appropriately supplemented minimal media.

Libraries, vectors and E. coli strains: The wild type genomic library of A . nidulans, used for primary transformation of uusF strains, was constructed in the vector pGM3 by OSMANI, MAY and MORRIS (1987). The host for this library was the E. coli strain JM83 (VIEIRA and MESSING 1982). This same strain, as well as DH5a (HANAHAN 1983), were used as hosts for all plasmids and for plasmid rescue experiments. E. coli LE392 (BLATTNER et al. 1977) was the host for the clones from the Aspergillus genomic library in XCharon4A (ZIMMERMAN et al. 1980; ORR and TIMBERLAKE 1982). The A. nidulans genomic libraries in pGM3 and XCharon4A were kind gifts of G. MAY. E. coli C600 (HUYNH, YOUNG and DAVIS 1985) was used as the host for the cDNA library, which was constructed in Xgt 10 and made available to us by S. OSMANI. The pBR322-based vector pGM3 (OS- MANI, MAY and MORRIS 1987) and the pUC18-derived vector pRG3, which both contain pyr-4+ of Neurospora (OSMANI et al. 1988), were kindly provided by G . MAY and R. WARING.

Molecular techniques: The methods used for plasmid rescue from the uvs+ transformants, for preparation of Aspergillus DNA and poly(A+)-enriched RNA and for Northern and Southern analyses, were those described by OSMANI, MAY and MORRIS (1987). Nitrocellulose membranes for Northerns and “nytran” membranes for South- erns were washed at high stringency as described by MAY et al. (1985). For preparation of plasmid DNA, the LiCl method was used, following the protocol of VOLLMER and YANOFSKY (1986). To obtain phage DNA, phages were isolated from glycerol gradients and DNA from all sources was purified by CsCl gradient centrifugation (MANIATIS, FRITSCH and SAMBROOK 1982). For small samples, overnight high-speed runs were carried out in a table top ultracentri- fuge (WEEKS, BEERMAN and GRIFFITH 1986). Phosphatase

Cloning the Aspergillus uvsF Gene 343

37

Probes : Vector pGM3 Insert of pEK6.1 Subclone pOK5.2

34 40 35 36 +R343637 H I I I I I I I I I * I l l

34 36 37

., ..- ~.

* 2.3 * 2.0

.1 ! I i

0.00 0.01 0.02 0.03

MMS Concentration (%) FIGURE 2.-Survi\.al of transformants compared to wild type (+)

and uvsF controls, in platings of conidia onto media containing MMS (methyl-methanesulfonate). For the transformants of the Tf S typt- (Tf35-37) with wild type-like "MS resistance, averages ( ~ s E ) are shown; T f L(34) = incompletely complemented type (data from two experiments).

treatment of vectors, gel electrophoresis, and screening of the cDNA and genomic libraries were as described in MAN- IATIS, FRITSCH and SAMBROOK (1 982). For subcloning, DNA fragments were purified by gel electrophoresis and ligated in low melting agarose (STRUHL 1983).

Conditions were as specified by the supplier for restriction digests, for nick translation of DNA with [a-"PIdATP or [a-"PIdCTP using the BRL kit, and for separation of dNTPs from the nick-translated probes using BRL "NACS PREPAK" columns. Competent E. coli DH5a cells were purchased from GIBCO Canada Inc. (GIBCO/BRL, Bur- lington, Ontario) and transformed according to the protocol supplied.

RESULTS

Primary transformation: Two uvsF pyrG double mutant strains were transformed with an Aspergillus wild-type genomic library, constructed in the vector pGM3 which contains the Neurospora pyr-4+ gene (MAY et al. 1985). We tested >3000 pyr+ transformants that were selected on medium lacking pyrimidines for complementation of the highly MMS-sensitive uvsF mutant (and for nutritional markers). Four formed resistant colonies similar to wild type on MMS medium, but one of these transformants (Tf34) grew somewhat more slowly. While the other three strains were indistinguishable from wild type in survival curves (Tf 35-37; Figure 2), Tf34 showed lower survival on MMS media than wild type, but its resistance to MMS (and to UV) was considerably increased compared to uvsF. (A few additional transformants showed very slightly increased growth on MMS.) All of the pyr+ colonies checked had retained the residual markers of the recipient strains (riboA, and pyroA or

a) b) C> FIGURE 3,"Southern anaysis 01' BgIII-digested genomic DNA

from transformants, their progeny, and controls, hybridized to three different probes. (a) Primary transformants T f L(34). T f S(35-37) and a very slightly complemented type. Tf(40). probed with vector DNA (pCM3); (b) wild-type control (+ = standard biA I strain), uvs+ Pur- recombinant (R76, FI of Tf34). and primary transformants, probed with putative uvsF sequences ( i e . , the Pstl insert o f pEKG. 1, derived from the complementing plasmid: Figure 5); (c) primary transformants probed with putative uvsF plus vector sequences (Le., EcoRI subclone pOK5.2 from pEK5/6; Figures 4 and 5).

choA). This confirmed that none of them were con- taminants, but also indicated that not a single case of transformation for a nutritional marker was obtained.

Southern analysis of transformants: Genomic DNA from the four MMS-resistant transformants and some of their progeny from crosses was digested with BgZII and probed with vector DNA (pCM3, which has no BglII sites). T w o types could be distinguished which all showed a single band, but of different sizes (Figure 3a). A larger band (about 13.8 kb) was found in the incompletely complemented transformant Tf34 and in some of its progeny, which were therefore called "Tf L" types. A shorter band (approxmately 9.7 kb) was observed in the three wild type-like cases which were called "Tf S" types (Tf35-37; Figure 3a; the few transformants which showed very slight complementation, e.g., Tf40, were characterized by multiple plasmid bands; these were generally sterile and have not been analysed further).

The same bands of different sizes which hybridized to the vector sequences in transformants were also seen in subsequent tests, when putative uvsF sequences were used as probes for BgZII-digested genomic DNA (Figure 3, band c). In addition, such probes identified two smaller bands for the genomic uvsF copy. The latter were identical in both types of transformants as well as in wild type controls. In contrast, results obtained with StuI digests and various types of probes, showed a single band of similar large size for wild type


TABLE 1

Meiotic mapping of plasmid insertion sites in crosses between transformants and markers of chromosome Z

Percent crossovers Sample size

transformant ( u u s F Eal')" (uus+ galD-)

crosses tested No. of Total Parental

T f Lb (Tf 34, its 26 f 3 5 605 FI and secondary Tf)

T f Sb (Tf 35) 51 f 3 3 358 T f Sb (Tf 37) 46 f 4 3 258

Parental relevant genotypes: Tf: uusF/+pyr-4+/pyrG gal'; haploids: uusF galD pyrG (or py@, very close to gaZD; Figure 1).

a Estimate represents an upper limit, since recombination between plasmid and genomic copy also produces uusF gal+ types (Table 2).

T f L = transformants with large band in Southern of BglII- digested DNA; T f S = transformants with smaller band.

as well as for both types of transformants (1 6- 18 kb; results not shown).

Mitotic mapping of the incorporated plasmids: For the four complementing transformants, the chromosome into which the pyr-4+ uvs+ plasmids had integrated was identified by haploidization of heterozygous diploids. In all cases, haploid segregants showed complete linkage of uvs+ and pyr+ to markers of chromosome Z (65-95 haploids tested from each of 5 heterozygous diploids). These results are compatible with incorporation by homologous recombination (Figure 1). They also confirmed the observation that the MMS resistant transformants, once purified from single conidia, were stable in mitosis.

Meiotic mapping and meiotic stability of transformants: While in most cases normal recombination values between markers on linkage group I was observed in over 20 hetero- and homozygous crosses for all MMS-resistant transformants, results from such crosses consistently differed in other respects for the two types identified in Southern analyses. In the case of Tf L types, meiotic linkage of the uvs+::pyr+ sequences to galD was as expected for incorporation by homologous recombination in the uvsF region (26%; Table 1 and Figure 1; unfortunately, the closer markers fpaB, and also trpB, could not be used for mapping because of interaction with PyrG). On the other hand, in all crosses of the Tf S types, recombination between uvs+::pyr+ and galD appeared to be significantly larger. Since these latter transformants also produced a high frequency of uvs-pyr- (gal+) intrachromosomal recombinants (Table 2), which cannot be distinguished from uvs- gal+ crossovers between homologs, the site of insertion of the complementing sequences in the Tf S types remains uncertain. Furthermore, only Tf L types but not any of the Tf S, produced haploid UVSF' pyrG- recombinants in which uvsF+ sequences presumably were replacing those of the mutant (Table 2). These, as well as the more frequent

uvs- pyr- intrachromosomal recombinants, had lost the vector sequences and were indistinguishable from wild type in Southerns, whatever the probe (shown for one case, R76; Figure 3b). When backcrossed to wild type, the uvs+ pyr- types from Tf L strains never produced uvsF- progeny in crosses.

Marker rescue experiments: For plasmid rescue, genomic DNA from the four complemented transformants was digested with BgZII, since in pGM3 neither the pBR322-based plasmid sequences, nor pyr- 4 of Neurospora, contain BglII sites (NEWBURY, GLAZEBROOK and RADFORD 1986). When ligated and used to select ampr transformants of E. coli, 5-1 0 colonies were obtained in each case. Plasmids isolated from the resulting E. coli colonies were of the expected sizes (13.8 kb from Tf L, and 9.7 kb from Tf S types; Figure 4). They were checked by restriction analysis and, as expected, contained the 2.2-kb EcoRI fragment of pyr-4 and a single BglII site in the middle of the A. nidulans insert. Plasmids rescued from the three primary Tf S type transformants had indistinguishable restriction patterns in digests with many enzymes (pEK8 from T F S35 in Figure 4). On the other hand, they differed considerably from those found in plasmids pEK5 and 6, isolated from Tf L34 in two independent rescue experiments and indistinguishable from each other (pEK5/6 in Figures 4 and 5). Detailed comparison of the restriction maps revealed a short (<0.6 kb) common region adjacent to the BglII site which had, however, reverse orientation relative to the vector (Figure 5, top part). Evidently the different inserts do not represent a simple overlap, and their restriction maps suggest that some rearrangement may have occurred in at least one of them.

Secondary transformation and subcloning from complementing plasmids: Unexpectedly, only the plasmids with the larger insert (pEK5/6 from Tf L) were able to complement uvsF strains in secondary transformation. Such secondary transformants produced the incompletely complemented phenotypes typical for Tf L(34) (Figure 2). They also showed genetic behavior similar to that observed for the various Tf L types, and when tested in Southern analyses the larger BglII bands were found. In contrast, the rescued plasmids with the smaller insert never produced uvs+ transformants (>400 pyr+ colonies tested).

T o identify the smallest complementing sequence from the large insert of the plasmid pEK5/6, three restriction fragments were isolated and subcloned (Figure 5). Of the two EcoRI fragments, one contains the pBR322 sequences and this was simply religated (Figure 5, pOK5.1; ability to complement uvsF of Aspergillus was tested by cotransformation with a Pyr- 4+-containing vector). The other, larger, EcoRI insert segment, and the PstI fragment which spans the BglII and EcoRI sites, were subcloned in the pRG3 vector


TABLE 2

Meiotic stability of transformants ~~~~ ~~~~ ~

Recombinants involving plasmid and genomic sequences Frequencies for different types of transformants

Tf L Tf S Phenotypes

uvs PYR x Total tested x Total tested

Haploid strains 4 Heterozygous crosses (Tf X haploid) ~

uvs+ Pur+ - -0

- +b

uvs+ pyrG - - +

uvsF pyrG - + uvsF pyr+ - -

- + - + -

Homozygous transformation crosses (Tf X Tf) Selfed, or one type - -

+ homozygous

- + -

Intercrosses of the - - two Tf types - +

+ -

14 3

14 2 0

7 f 4 1 3 f 5 19 f 7

0

0 14

95 20 f 4 425 8 f 2

172 25 f 2 314 0

230 0 151 0

484 30 f 3 53 1 0

101 33 f 2 553

0.5 f 0.3 0

619 34 f 2

4 f 2 2 f l

* Elimination of vector sequences confirmed in Southern analyses. * Elimination of vector and pyr-4+ sequences plus crossing over between homologues in crosses to haploid pyrG', or rare "movement" of

plasmid which eliminates complementation of uvsF (one case mapped to chromosome V). Elimination of vector and pyr-4+ sequences and replacement of mutant wild-type uusF sequences.

Anpergillua

H

P E SO p y r - L pEK 0

Neuronporn

/,BR 322 ( 2.2 kb )

Vector ( 4.4 kb 1 E FIGURE 4.-Restriction maps of original plasmids rescued from

the primary Aspergillus uvs' transformants usingBglI1 digests. Both types contain the pBR322 and Neurospora pyr-4 sequences of the library vector. pEK5/6 = complementing plasmid from transformant Tf L34 with larger A. nidulans insert; pEK8 = noncomple- menting plasmid from Tf S35 with shorter insert; double line indicates "common" sequence of the plasmids (BglII-PstI segment, <0.6 kb). Restriction sites (also of Figure 5): B, EamHI; Bgl, BglII; C, ClaI; D, DraI; E, EcoRI; H, HindIII; K, KPnI; P, PstI; Pv, PvuII; S, SalI; Sp, SphI; St, SstII; Stu, StuI; X, XhoI; Xb, XbaI.

(pOK5.2 and pEK6.1; Figure 5). When used for transformation, neither of the EcoRI subclones (pOK5.1 and 5.2) complemented uvsF. Therefore, regions crucial for complementation must be located on both sides of the EcoRI site in the complementing plasmid. Unexpectedly, even though the third subclone (pEK6.1) spanned the EcoRI region and, in addition,

. E>$... P P P

1 kb

FIGURE 5.-Restriction maps of inserts, and of subclones from the complementing rescued plasmids. Top line: insert of pEK8 from Tf35 (3.1 kb; inverted compared to Figure 4); second line: insert of complementing plasmid pEK5/6 from Tf L(34), (7.2 kb). Remaining lines: three subcloned fragments of pEK5/6, namely both EcoRI segments (in pOK5.1 and 5.2), and the PstI fragment (>2.3 kb in pEK6.1) which contains the "common" region (enzyme abbreviations as above, Figure 4).

contained the short common (overlap) sequence, it also failed to complement (250-890 pyr+ transformants tested on MMS media for each subclone).

Screening of cDNA and genomic libraries: Since only the original plasmid from Tf L(34) complemented the uvsF mutation, this plasmid (pEK5/6 with the large insert) was used as the probe for primary


Restriction : E P E ‘/H “ - - “-

N r c

.. - ”

kb

2.2 - 0.8 , 0.7 ’

kb

4 23

4 9.4 4 6.1

* 4.2

- 2.3

a) b) FIGURE 6.--Southern blots probed with Xgt 10.93 to identify

hotnologous Sequences between the cDNAs m d specific restriction fr;lgments in rrscuetl plasmids and their putative uvsF subclones. a) < ~ r o s s - l l y l ~ r i d i ~ ; t t i ~ ~ ~ ~ between cDNAs 10.93 and 10.66, and hybridization t o the complenlenting plasmid pEK6/5, especially its Psfl segment (P), sulxloned in pF.KG. 1 ; b) differential hybridization of rDS.4 X10.93 to the two LmRI (E) segnwnts of pEK5/G, and the c-orrt*sponding subclones (pOK.5.2 and 5 . 1 : first seven lanes); last three h w s : hybridization of cDN,4 to the RgIII-Hind111 (BgI-H) fr.;lgment of the noncomplen~er~t in~ plasmid pEKR (different amounts of D S A lo;tded in duplicate lanes for double and triple digests).

screening of two available Aspergillus libraries. T w o clones were obtained from a cDNA library in the XgtlO vector (of OSMANI, MAY and MORRIS 1987). These two clones, Xgt10.93 and 10.66, contained overlapping inserts of similar size (-1200 bp) which produced slightly different cross-hybridizing EcoRI fragments (the larger ones visible in Figure sa).

Screening of the wild type genomic library (ZIM- MERMANN et al. 1980) with the complementing plasmid identified five clones. Since three gave indistinguishable restriction patterns in single and double digests by EcoRI and BglII, these five genomic clones represent three different types.

Identification of uvsF sequences in cDNAs: To check whether the cloned cDNAs are likely to represent uvsF sequences, the cDNA clone Xgt10.93 was used as a probe for Southern analysis, using various digests of the rescued plasmids and their subclones (Figure 6). I t is evident that this cDNA hybridized to the “common” region of both types of rescued plasmids: strongly to the PstI band and subclone (pEK6.1) of the larger plasmid (pEK5/6; Figure 64 , and to a smaller extent to the BglII-Hind111 fragment of the smaller plasmid, which contains the overlap (shown for pEK8, last two lanes of Figure 6b). In addition, the cDNA hybridized to both of the EcoRI subclones, pOK5.1 and 5.2; ie., to regions on both sides of the RcoRI cut in pEK5/6 which abolishes complementation. However, hybridization is much stronger to

FIGURE 7,”Variousdigests of the XCharon4A. 137 clone, probed with the subcloned cDNA fragment. pOKl0-93L ( i t - . , the larger EcoRI fragment of the cDNA insert from Xgt10.93, subcloned in pRG3). T h e same results were seen after probing with pEK6.1 (i .e.. the Psfl segment spanning the Bg111 and KcoRI sites of the complementing plasmid). Restriction sites (including Figure 8): B, BamHI; D, Dral; E, KcoKI; H, HindIll: K, K p n l ; P, Psfl; S, Sall ; St, SslI; S f U , slul; x. XbaI.

pOK5.2 which contains the common region (Figure 6b).

Mapping of uvsF sequences in the genomic clone X4A.137: To locate the putative genomic sequences of uvsF in one of the XCharon4A clones, the largest EcoRI fragment of the cDNA inserts was subcloned in the vector pRG3. The resulting plasmid, pOK10.93L, when used as a probe against genomic clones, gave the same result as the PstI subclone from the original complementing plasmid (pEK6.1; Figure 5). Both probes hybridized to only one of the three types of genomic clones (X4A.137). When the non- complementing rescued plasmid with the shorter insert (pEK8) was used as a probe, X4A. 137 also hybridized strongly, but in addition one of the other two types of X4A clones, identified by screening with pEK5/6, hybridized to a smaller extent (results not shown). Therefore, the most likely clone to contain genomic sequences of uvsF is X4A. 137.

To map the uvsF sequences within the large insert of X4A.137 by Southern analysis, a large number of single, double and triple digests were probed with the cDNA subclone (Figure 7). For most enzymes these probes hybridized to more than one band (including BglII, and RcoRI; the latter in lane 12, Figure 7).


genomic 4A-137

E stu A B H St K ] E E S t X X S t X - X S t E I E I I I I I I I I f I I I I

yum

A, ! A, “ “. .

1 kb

FIGURE 8.-Restriction map of the A. nidulans insert in XCharon4A. I37 clone (total size, 14.5 kb). Indicated between ar- r o w : rcgions o f maximum (c-). 4 kb. and minimum (- - -) possible. extent o f hybridization to the cDNA subclone ( i t - . , the 1argc.r EroRI fragment of Xgt 10.93). XL and XR = left and right arm of XCharon4A vector. Restriction sites as in Figure 7 (for the restriction map of the vector arms. see MANIATIS, FRITSCH and SAMBROOK 1982).

However, single short bands were obtained in two cases; namely in the double digest of StuI and SalI, and in the single digest with PstI (bands of 7.5 and 2.2 kb, lanes 9 and 10, respectively; Figure 7; however, in the case of PstI, hybridization of both of the double bands visible in the gel, is not ruled out). In addition, after digestion with DraI, the probe hybridized mainly to a single band (of 4.0 kb, lane 1 in Figure 7), but after longer exposure a smaller band became visible as well. From the combined results of these digests and their blots, it was possible to deduce that the uvsF gene sequence was located within a region of about 4.0 kb at one end of the large insert of X4A. 137 (Figure 8). The cDNA probe hybridized to fragments on both sides of the three restriction sites indicated in that region. (BglII and DraI sites could not be mapped with certainty, because of the many small fragments in the X vector arms.)

Northern analysis: In Northern analysis of poly(A+)-enriched wild-type RNA with pEK5/6 as the probe, three bands were obtained (1.4 kb, 1 .O kb and 0.8 kb), but by far the brightest band was that of approximately 1.0 kb (Figure 9a). When after strip- ping this R N A blot was reprobed with either pEK6.1, or the cDNA subclone pOK10.93L (Figure 9, b and c), only the 1.0 kb band was seen which, therefore, may represent the uvsF mRNA.

DISCUSSION

The results presented here demonstrate some of the similarities and differences between Aspergillus transformation systems and those of yeast and Neu- rospora, when genes are cloned by complementation of mutations using genomic libraries. As in yeast, stable single-copy incorporation of complementing DNA plus linked vector sequences was found in the same chromosome in which uvsF is located. Incorpo- ration presumably occurred by homologous recombination, as shown clearly for one case. This type of transformant regularly produced, by intrachromosomal meiotic recombination, progeny of two types

R N A markers

Probes

kb PEK6 pEK6.1 hgt10.93

9.5 b 7 .5 * 4.4 - 2.4 - 1.4 -

.24 -

- 1 kb

a) b) c ) FIGURE 9.”Northern analysis 01. poly(A+)-enriched RNA from

uusF+ strain (different amounts loaded in duplicate lanes): (a) probed with the complementing plasmid pEK5/6; (b) with its Pstl subclone, pEK6.I; or, (c) with the cDNA subclone (pOK10.93L). RNA markers provide for approximate estimates of mRNA size (HAUGE 1988).

which all had lost the vector sequences and the dupli- cation for uvsF: they were either uvs- and indistinguishable from uvsF201, or uvs+ but pyrG- and identical to wild type in Southern analyses and crosses. The observed frequencies of 8- 15% are very similar to those obtained for “marker loss” by MAY et al. (1985) in the case of the &tubulin gene, but considerably higher than is known to occur in yeast. From this transformant, complementing plasmids could be rescued, and the crucial sequences could be mapped to both sides of the EcoRI site in the insert, since neither of the two subcloned EcoRI segments alone complemented. These results clearly indicate the pres- ence of UVSF+ sequences in the rescued library fragment.

However, several unexpected features indicate that the situation in Aspergillus is more complex than in yeast. Not only can incorporation occur at non-ran- dom heterologous sites (DIALLINAS and SCAZZOCCHIO 1989) but, in addition, rearrangements are not un- common (e.g., UPSHALL 1986). Similar findings are seen here for primary transformants and the plasmids rescued from these. For example, in contrast to three other wild type-like transformants, complementation was incomplete in the primary transformant from which similarly complementing plasmids could be rescued. This suggests that minor alterations occurred or sequences are missing, which cause the reduced uvsF enzyme activity. Rearrangements must have occurred, since the two types of rescued sequences ob-


tained from the MMS-resistant transformants are not colinear, even though they crosshybridize and have a short sequence in common. It seems likely, that the larger rescued plasmid contains the rearranged sequence, because only a single original transformant of this type had been isolated and three identical rearrangements seem very unlikely (unless they were derived from a single case that occurred during library construction and amplification in E. coli) . In addition, complementation in the other three cases was perfect, suggesting perfectly normal sequences, even though rescued plasmids (from genomic BglII digests) did not complement and only contained a small part of the putative uvsF sequence. An explanation for these findings might be, that in all three cases plasmids had inserted unlinked to UVSF, but nonrandomly, into the same region of chromosome I (since they are indistinguishable in all types of Southern analyses). Such incorporation of a functional uvsF gene, far and prob- ably distal from the genomic uvsF locus, might explain not only the inability of rescued plasmids to complement, but also the absence of intrachromosomal uvs+ pyrG- recombinant from these transformants. It would then also suggest, that the large bands in South- erns, probed with vector or insert sequences, are of similar size by chance when StuI digests are analysed from wild type and from these transformants which do not contain a StuI site in their rescued plasmids. Additional evidence would be needed to exclude or confirm any of these possibilities. In either case, it is evident that normal uvsF sequences might be difficult to obtain from the rescued plasmids. The usual procedure, which is to use such rescued complementing sequences as probes for the screening of genomic (BALLANCE and TURNER 1986) and/or cDNA libraries (OSMANI et al. 1988), was therefore also adopted here. A complementing fragment from the identified genomic clone (XCharon 4A. 137) should provide the normal uvsF sequence and comparison of restriction maps in this and the obtained cDNAs may provide some answers to the above questions.

Aspergillus also seems to differ from Neurospora, where an unusual mechanism for inactivation of duplicate gene sequences has recently been discovered (SELKER and GARRETT 1988; CAMBARERI et al. 1989). Apparently, during the mitotic divisions which pre- cede meiosis in the ascogeneous hyphae, one or both copies of duplicate segments undergo high frequencies of G-C to A-T mutation (as well as methylation). In our analysis of the many crosses between transformants and normal-sequence strains, the corresponding types would presumably become uvs-, while remaining pyr' (since the Neurosporapyr-l+ sequences which are heterologous would not be inactivated by mutation). Such types were either very rare or absent in crosses in which they could be identified, and similar

inactivation mechanisms, if they exist in Aspergillus, would have to be considerably less active.

Part of this work was carried out by E.K. during extended stays in the Department of Pharmacology, Rutgers Medical School, New Jersey. We are very grateful to RON MORRIS for his generous hospitality, and to several members of his group for providing us with material which made this project possible. Thanks are due especially to GREG MAY, and to STEVE and AYSHA OSMANI, for their help, encouragement and provision of know-how and infor- mation. We thank DOT LUK for her excellent technical assistance in the genetic analysis of many crosses and the preparation of figures, and SANDRA BLASKOVIC for the careful analysis of one of the transformants. Some of the results presented here were obtained by K.O., in partial fulfillment of the requirements for an M.Sc. degree. This work was supported by the Natural Science and Engineering Research Council of Canada.

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Documents

Cloning of the DNA Repair Gene, UVSF, by Transformation … · 2002. 7. 8. · CORVO et al. 1953; KAFER 1977; SCOTT and KAFER 1982). Strains of uusF (KAFER and MAYOR 1986) and pyrG