Cloning, mapping and molecular analysis of the pyrG (orotidine-5'-phosphate decarboxylase) gene of Aspergillus nidulans

Gene, 61 (1987) 385-399 Elsevier

385

GEN 02249

Cloning, mapping and molecular analysis of the pyrG (orotidine-5’-phosphate of Aspergillus ~id#l~n~

decarboxylase)

gene

(Recombinant DNA; nucleotide and amino acid sequences; introns; S 1 nuclease mapping; complementation; transformation; pyrimidine pathway; fungal genetics; transcriptional start point)

Berl R. Oakley *, Janet E. Rinehart a, Brenda L. Mitchell”, C. Elizabeth Oakley *, Cynthia Carmona b, Gregory L. Gray b and Gregory S. May c9* a Department ofMolecular Genetics, The Ohio State University, Columbus, OH 43210 (U.S.A.) Tel. (614)292-3472; b Genencor Inc., South San Francisco, CA 94080 (U.S.A.) Tel. (415)588-3475,and’ Department ofPharmacology, UMDNJ- Robert Wood Johnson Medical School, ~cataway, NJ 08854 (U.S.A.j Tel. ~2Oij463~166 Received 11 May 1987 Accepted 17 September 1987

SUMMARY

We have modified the transformation procedures of Ballance et al. [ Biochem. Biophys. Res. Commun. 112 (1983) 284-2891 to give increased rates of transformation in Aspecgi~lw nidulans. With the modified procedures we have been able to complement pyrG89, a mutation in the orotidine-5’ -phosphate decarboxylase gene of A. n~u~ns, by ~~sfo~ation with a library of wild-type (wt) sequences in pBR329. We have recovered, by marker rescue from one such ~~sfo~~t, a plasmid (pJRl5) that carries an A. ~j~Za~ sequence that complements pyrG89 efficiently. In three experiments, this plasmid gave an average of 1985 stable ~~sfo~~ts/~g of transforming DNA. We have analyzed ten of these genetically and by Southern hybridization. In five transformants a single copy of the transforming plasmid had integrated at the pyrG locus, in one transformant severdl copies of pJRl5 had integrated at this locus, in one transformant several copies of the plasmid had integrated into other sites, and in three transformants, the wt allele had apparently replaced the mutant allele with no integration of pBR329 sequences. Sequence and Sl nuclease protection analysis revealed that pJR15 contains a gene that predicts an amino acid sequence with regions of strong homology to the orotidine-5’-phosphate decarboxylases of Neurospora crassa and Saceharomyces cerevisiae. We conclude that this gene is the wt pyrG allele. Finally, we have compared the 5’- and 3’-noncoding sequences and intron splice sequences to other genes of A. n~uZa~ and have mapped the pyrG locus to a region between the fpaB and galD loci on linkage group I.

Correspondence to: Dr. B.R. Oakley, Department of Molecular Genetics, The Ohio State University, 484 West Twelfth Ave., Columbus, OH 43210 (U.S.A.) Tel. (614)292-3472.

* Present address: Department of Cell Biology, Baylor College of Medicine, 1200 Moursund Ave., Houston, TX 77030 (U.S.A.) Tel. (713)799-4756. Abbreviations: bp, base pair(s); Cm, chloramphe~coi; S-FOA, 5-fluoro erotic acid; kb, 1000 bp; OMPdecase, orotidine-5’-

phosphate decarboxylase; ORF, open reading frame; PEG, polyethylene glycol; Pyr + , pyrimidme prototrophic; Pyr -, pyrimidine auxotrophic; SDS, sodium dodecyl sulfate; Sl mix, see MATERIALS AND METHODS, section e; T6, TlO see RESULTS, section b; TBE buffer, 89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA; Tc, tetracycline; TE buffer, 10 mM Tris pH 8.0, 1 mM EDTA; wt, wild type; YAG, 5 mg&l of yeast extract, 20 mgjml of dextrose, 15 mg/mI of agar; YG, 5 mg/ml of yeast extract, 20 mg/ml of dextrose.

0378-l 119/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

386

mation studies and extends the rather limited knowledge of gene structure in A. nidufans. Cloning genes by complementation of mutations has become one of the most important techniques of modern molecular genetics, for organisms in which it is possible, because it permits the isolation of virtually any gene for which a selectable mutation has been identified. For cloning by complementation to be feasible, two requirements must be met: (i) the tr~sfo~ation efficiency must be sufIiciently high that one can complement mutations with manageable amounts of DNA and numbers oftransfo~able protoplasts and (ii) one must be able to recover the transforming gene from transformants. Successful attempts to clone genes by complementation in the lilamentous fungus A. nidulans have relied on a cosmid vector (Yelton et al., 1985) which carries large fragments ofA. niduluns DNA or plasmids that give unusually high frequencies of transformation (Johnstone et al., 1985; Ballance and Turner, 1985). We have attempted to improve tr~sfo~ation frequencies with conventional vectors and have developed procedures that give tr~sfo~ation frequencies of more than 800 stable transformants/pg with a vector, pDJB1 (Ballance and Turner, 1985) that carries the pyr4 gene of Neurospora crassa. This gene encodes OMPdecase and complements the A. niduluns mutation, pyrG89 (Ballance et al., 1983). We have used these procedures to complement pyrG89 with a library of A. nidulans sequences constructed in plasmid pBR329 (Covarrubias and Bolivar, 1982), and we have recovered, by marker rescue, a plasmid carrying an insert of A. nylons origin that complements pyrG89 efficiently and integrates preferentially at the pyrG locus. We have sequenced the insert and found that a portion of it specifies an amino acid sequence that has homology to the OMPdecases of S. cerevzkiae and N. crassa. We conclude that this sequence includes the wt pyrG allele. By S 1 nuclease protection analysis we have determined that the gene is translated from multiple initiation sites and have identified an intervening sequence within the gene. We have also mapped the pyrG locus to a region between the fpaB and galD loci on linkage group I. Because of the high frequencies obtained, our transformation procedures are likely to be of value in complementing other mutations. Our analyses expand our knowledge of thepyrG locus which has been important in transfor-

MATERIALS AND METHODS

(a) Strains and media A. njdu~o~sstrain G191 (pabaA 1, pyrG89; fivA 1, uaY9) was obtained from Dr. G. Turner (University of Bristol) via Dr. CF. Roberts (University of Leicester). FGSC4 (Glasgow wt), FGSC154 (adE20, biA1; wA2, cnxE16; sC12; methG1; nicA2; IucAl; choA1; chuA l), FGSC475 (jjuB37, gulD5, suAladE20, riboA1, anA 1, pabaA 1, yA2, adE20, biA 1; sD85, JwA2) and FGSC515 (jjaB37, galD5, suA ladE20, riboA l,yA2, adE2O;pyroA4; facA303; chaA 1) were obtained from the Fungal Genetics

Stock Center, University of Kansas Medical Center, Kansas City, KS, via Dr. N-R. Morris (University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School). GCR2-13 (tubCZ 13, pyrG89,pabuA 1; benA 22) was constructed in the laboratory of Dr. N.R. Morris. E. coli K-12 strain DHl (gyrA96, recA 1, relAl?, end4 1, t&-l, hsdR 17, supE44, ,I-) (Harmhan, 1983) was obtained from the E. coli Genetic Stock Center (Yale University School of Medicine). DB6656 (trparn, &am, r;, rnz , pyrF: : Mu) was obtained from Drs. M. Rose and D. Botstein (Massachusetts Institute of Technology). Plasmid pDJB1 (Ballance and Turner, 1985) was obtained from Dr. G. Turner via Dr. C.F. Roberts. YG (5 mg/m of yeast extract, 20 mg/ml of dextrose) and YAG (YG with 15 mg/ml of agar) were used as complete media. These media do not contain sufftcient uracil or uridine to support growth of strains carrying pyrG89. Strains carrying this mutation were grown on YG or YAG supplemented with 10mM uridine. Since ascospores carrying pyrG89 do not germinate well on YAG + 10 mM uridine, the following medium was used for the germination of ascospores: 20 mgjml of malt extract, 1.0 mg/ml of peptone, 20 mg/ml of dextrose, 1.0 mg/ml of uracil, 15 mg/ml of agar, 10 mM uridine. Nutritional supplements were added to this medium as required to allow the germination of ascospores that carried nutritional markers from parental strains. Additional media were as previously described (Oakley et al., 1985).

387

(b) Purification of DNA Plasmid DNA for transformation of A. nidulans was purified by an alkaline lysis procedure, a lysozyme/EDTA lysis procedure (Maniatis et al., 1982) or by the method of Summe~on et al. (1983) followed by pu~cation through CsCl density gradients. Maximum transformation rates were obtained with plasmids purified through two C&l gradients. A. niduluns DNA for library construction was prepared as follows. FGSC4, grown overnight in liquid culture, was harvested by filtration, washed with cold distilled water, and the hyphal mat was pressed to remove excess water and weighed. Hyphal mat (5 g) was then frozen in liquid nitrogen and pulverized while frozen. The powder was resuspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4,lO mM EDTA, 0.25 M sucrose, 10mM KCI), SDS was added from a 10% solution to give a final concentration of 1% and RNase A was added to a final concentration of 200 pg/ml. The suspension was heated rapidly to 60’ C and maintained at that temperature for 20 min. KC1 was added to give a concentration of 0.5 M and the suspension was placed on ice for 15 min. The suspension was then centrifuged at 5000 x g for 5 min at 4 oC. The supernatant was extracted once with phenol, twice with ether, and eth~ol-precipitated. After the precipitate was pelleted by centrifugation, it was resuspended in TE buffer (10 mM Tris pH 8.0,l mM EDTA) and subsequently the DNA was further purified by two CsCl density gradients. For Southern hybridizations A. nidulans DNA was prepared by a rapid miniprep procedure previously described (Oakley et al., 1987). DNA from bacterial DHl was purified by the method of Schleif and Wensink (1981). N. crassa DNA was a gift from Dr. G. Marzluf (The Ohio State University). (c) Transformation of Aspergillus niduluns Freshly harvested conidia (conidiospores) were incubated in liquid complete medium at a concentration of 5 x 106/ml until they swelled and a few began to form germ tubes. They were then harvested by centrifugation (5 min, 1800 x g), washed in YG, resuspended in YG to a concentration of 1.2 x 107/m1and mixed with an equal volume of 2 x protoplasting solution prepared as follows. The pH

of a 1.1 M KCl, 0.1 M citric acid solution was adjusted to 5.8 with 1.1 M KOH. Driselase (Sigma) was added to a concentration of 20 mg/ml, and after 15 min on ice, the starch carrier was removed by cent~ugation (5 min, 1800 x g). Novozym 234 (Novo Industri) was added to 4 mg/ml, bovine serum albumin (Sigma No. A-7096) to 20 mg,‘ml and /I-glucuronidase (Sigma No. G-0672) to 20 ~tyml. The solution was then filter-sterilized through a low-protein binding filter (Millipore SLGVO25LS or GVWPO2500) and left on ice until use. Alter incubation with gentle agitation for approx. 5 h at 30°C the resultant protoplasts were harvested by centrifugation (10 min, 1800 x g), resuspended in 0.6 M KC1 and washed three times by centrifugation (3 min, 2400 x g in microcent~uge tubes) and resuspended in 0.6 M KCL They were then washed in 0.6 M KCI, 0.05 M CaCl, and resuspended in the same solution such that the protoplasts from 5 x IO7 conidia were resuspended in 100 ~1. DNA in less than 15 ~1 of TE buffer was mixed gently into each 100~~1 sample and 50 ~1 of PEG solution [ 25% PEG, approx. N, 3350 (Sigma), 0.6 M KCl, 0.05 M CaCl,, 10 mM Tris pH 7.5, filter-sterilized immediately before use] was mixed into the sample and the sample was incubated on ice for 25 min, One ml of PEG solution (at room temp~at~e) was then mixed into the sample and the sample incubated at room temperature for 30 min before plating directly onto selective medium (YAG + 0.6 M KCI, or minimal medium with appropriate supplements and 0.6 M KCl). (d) Library construction DNA from FGSC4 was purified on two CsCl density gradients and partially digested with Sau3AI. Fragments 4-6 kb in length were purified from a low melting point agarose gel using an NACS prepack column (Bethesda Research Labs) and ligated into the BamHI site of 2 x CsCl-gradientpurified pBR329. Bacterial strain DHI was transformed with the ligation mixture following the procedure of Hanahan (1983) and more than 100000 Cm-resistant colonies were obtained. Approximately 100 colonies were analyzed for Tc sensitivity and minipreps of Tc-sensitive colonies verified that they carried plasmids with inserts. The results of these experiments indicated that approx. 34000 colonies

388

carried plasmids with 4-6-kb inserts. Calculations based on the equation derived by Clark and Carbon (1976) indicated that the ~~sfo~~ts with inserts should include more than 99.9% of the sequences in FGSC4. To reduce the fraction of colonies without inserts, all the colonies were harvested and plated onto media that select for Tc-sensitive colonies (Maloy and Nunn, 1981). Several hundred thousand colonies were obtained and minipreps revealed that approx. 90% had inserts. Approximately 90% of the inserts were 4-6 kb in length with the remaining 10% 8-12 kb in length, probably representing double inserts. (e) SI m&ease protection analysis Single-stranded Sl probes were generated using subclones in M13mp18 and M13mp19. For the 5’ end analysis the 364-bp SalI, Sac1 fragment extending from 138 bp into the coding region to 228 bp upstream from the initiator methionine (see Fig. 3) was subcloned into M 13mp 19. To map the 3 ’ end of the intron, a 142-bp SacI-SalI fragment spanning the intron was subcloned into Ml3mp18. Probes were synthesized and hyb~dizations were performed as described by Nasmyth (1983). Hybridizations were terminated by the addition of 275 pl of ice-cold Sl mix, which consisted of 30 mM sodium acetate, pH 4.4, 0.25 M NaCl, 5 mM ZnSO,, 5% glycerol and 1000 units/ml Sl nuclease. Sl reactions were incubated for 1 h at 37°C. The reactions were terminated by the addition of 30 ~1 of 0.5 M Tris * HCl, pH 8.0, 0.25 M EDTA, 10~1 tRNA and 0.7 ml ethanol. The precipitate was sedimented, washed in 70 % ethanol and dried. The final product was dissolved in 95% formamide, 10 mM EDTA and separated on an 8% sequencing gel. A set of dideoxynucleotide sequencing reactions of a template of known sequence was run in adjacent lanes to size the products accurately. (f) Other methods Restriction digests, nick translations, plasmid minipreps and ligations were carried out as described by Maniatis et al. (1982). Southern hyb~d~ations were as described previously (Oakley et al., 1987). Sequencing was via the dideoxy method (Sanger et al., 1977) in Ml3 vectors (Yanisch-Perron et al., 1985).

RESULTS

(a) Improvement of t~sfoFmation frequencies For experiments in improving transformation frequencies we used A. nidulans strain G191 which carries pyrG89, a mutation in the OMPdecase gene (Palmer and Cove, 1975). Our transforming plasmid was pDJB1 (Ballance and Turner, 1985) which carries the OMPdecase (pyr#) gene of N. crassa (Buxton and Radford, 1983). This gene functions in A. niduluns, complementing pyrG89 (Ballance et al., 1983). Observations of protoplasts throughout the tr~sfo~ation procedure of Ballance et al. (1983) revealed considerable lysis upon the addition of 2.5 ml of 25 % PEG solution to 250 ~1 of protoplast solution. Since PEG 6000 is a very large molecule, the molarity of a 25 % solution is quite low (0.042 M) and althou~ the PEG solution specified by Ballance et al. (1983) contains 50 mM CaCl, the final molar&y of the solution is only approx. 0.1 M. Lysis is, thus, probably due to osmotic shock. Ballance and Turner (1985) have recently modified their procedure by, among other things, reducing the amount of PEG solution added to the protoplasts. This should reduce the osmotic shock and transformation frequencies of 50-100 transformants/pg are now obtained with a plasmid (pFB6) that carries pyr4 (Ballance and Turner, 1985). We modified the procedure of Ballance et al. (1983) by adding KC1 to the PEG solution to a final concentration of 0.6 M and obtained much less lysis and an immediate lOO-fold increase in transformation frequencies. We have subsequently made a number of additional modi~cations (detailed in MATERIALS AND METHODS, sectionc) such as preparing protoplasts from swollen conidia instead of hyphae and eliminating top agar, plating directly on medium containing 0.6 M KCl. We now routinely obtain more than 1000 tr~sfo~~ts/~g, when transforming protoplasts prepared from 5 x IO’ swollen conidia with 5 pg of pDJB1, and have obtained figures as high as 24OO/pg. Approximately 40% of these transformants retain their ability to grow on selective media when subcultured.

389

(b) Complementation of pyrG89 with a wild-type 4

5

6

23-

2 L32 on

Fig. 1. Hybridizations of A. nidtdum strain G191 transformed with a wt A. niduhns library and of untransformed strains. The scale at the left shows the position of fragments of a Hind111 digest of bacteriophage the sizes of which are given in kb. Lanes l-3 were probed with [32P]pBR329. Lanes I and 3 contain DNA from a library transformant, TlO. The DNA in lane 1 was digested to completion with EcoRI while the DNA in lane 3 was not digested. Lane 2 contained a similar amount of DNA from FGSC4, au untransformed wt strain which was also digested with EcoRI. Careful ex~inat~on of radioauto~aphs exposed for various times revealed at least 16 bands in lane 1. Lanes 4-6 are EcoRI digests of FGSC4, N. massa, and DHI, an E. coii strain, respectively. Each was probed with [“2P]pJRi5. FGSC4 DNA contained a single band which hybridized to pJRl5 while there was no hybridization to N. craw or E. cob DNA. Electrophoresis was for 16 h at 25 V in 1% agarose with TBE buffer (89 mM T&-borate, 89mM boric acid, 2mM EDTA). Gels were strained in 1 ng/ml ethidium bromide, photographed before denaturing, drying, hybridizing and washing as previously described (Oakley et al., 1987). The specific activity of the probe was 2 x lo7 cpm and autoradiography was with Kodak XAR-5 x-ray film for 24 h.

library

A Sau3AI partial digest library of wt sequences was constructed in pBR329 and protoplasts from 10’ conidia of G191 were ~~sfo~~ with 250 pg of library DNA. Ten stable pyrimidine prototrophic (Pyr + ) colonies were obtained, two of which (designated T6 and TlO) were chosen for further study. DNA from each of them and from the wt strain FGSC4 was digested with EcoRI and analyzed by Southern hybridization, probing with pBR329. Results for TlO and FGSC4 are shown in Fig. 1. Plasmid pBR329 did not hybridize to wt DNA but hybridized to 16 or more bands in TlO. Similar res&s (not shown) were obtained with T6. This result demonstrate that TlO is indeed a ~~sfo~~t and not merely a revertant. The multiplicity of bands suggested that several plasmids from the library in addition to the plasmid carrying the wt pyrG allele had integrated into the chromosomal DNA of the transformant. (c) Recovery by marker rescue of sequences that complement pyrG As reported (Tilbum et al., 1983; Yelton et al., 1984; May et al., 1985; Upshall, 1986), ~~sforrn~g plasmids generally integrate by homologous recombination in A. nidulans. The wt pyrG allele should, thus, be closely linked in the transformant genome to the antibiotic resistance markers on pBR329. By cutting transformant DNA with an appropriate restriction endonuclease and subsequently ligating, one should produce circular molecules which are capable of transforming E. caii cells to antibiotic resistance and which carry the wt allele of the transforming gene (Hicks et al., 1979; Yelton et al., 1984; Bahance and Turner, 1985). For recovering the wt pyrG allele from our transformants there were two complications. The first was that several plasmids, only one of which carried pyrG, appeared to have integrated into the chromosomal DNA of the transformants, and thus only a minority of recovered plasmids would carry pyrG. The second was that since we did not know the restriction sites in pyrG nor the position of pyrG89 in pyrG, we did not have sufficient info~ation to allow us to choose an enzyme that would allow the recovery of the wt pyrG allele.

390

We reasoned, however, that if we partially digested transformaut DNA, ligated and recovered sufficient numbers of transforming bacteria, some of the plasmids should carry the wt pyrG allele. Such plasmids could be identified by their ability to transform Gl91 to p~midine prototrophy. We partially digested DNA of tr~sfo~~ts T6 and TlO separately with Suu3AI and DNA of transformant TIO with EcoRI, sized the digests on agarose gels, ligated and transformed the bacterial strain DH 1. We obtained 56 transformants from the Suu3AIdigested material and 333 from the EcoRI-digested material. We next prepared a plasmid pool from a culture inoculated with all the transformants from the Suu3AI digest and a separate pool from all the transfounts from the EcoRI digest. G191 was transformed with each of the two pools. The Sau3AI pool and a no-DNA control gave no Pyr + transformants, but the EcoRI pool gave a substantial number of Pyr’ transformants. This result demonstrated that the EcoRI pool contained one or more plasmids that complemented pyrG89. To identify a single such plasmid, we subdivided the EcoRI bacterial transformants into ten groups of 33 and prepared pools of plasmids from each group. Two pools transformed G191 to Pyr’ . One of these pools was subdi~ded and the process was repeated until a plasmid that complemented pyrG89 was isolated. We have designated this plasmid pJR15. Subsequent transformation experiments with pJR15 have yielded very high transformation frequencies. Three separate experiments in which protoplasts from 5 x lo7 germinating conidia were transformed with 4.2 pg of pJRl5 yielded 3870,7674 and 3346 transformants/~g. As noted with other genes (Ballance et al., 1983; Tilburn et al., 1983), the colonies tr~sfo~ed with pJRl5 varied considerably in size and growth rate on selective medium. Approximately 15% of the colonies conidiated after three days of incubation and approx. 40% of all colonies retained their Pyr + phenotype upon subcultering on selective medium. Thus the average transformation frequency for these experiments was 1985 stable transformants/~g.

TABLE I Frequency of pyrG_ segregants among progeny of crosses between Aspergibt.s nidulans strain FGSCl54 and pyrG + transformants of strain G191” Tr~sfo~ants

B D E F N 0 S

V W

b Type of integration’

1

2 3

I 1 1 3 3 1 4

Frequency of pyrG ascospores d (pyrG_/total tested) 21100 21/100 o/100 o/100 o/100 4/100 o/100 o/100 3/103 491103

a Crosses were made as described by Morris et al. (1982). b Transformants B-W correspond to lanes 2-1 I in Fig. 2. c Types of integration: 1, single integration, apparently into genomic pyrG sequence; 2, multiple integrations, apparently none into genomic pyrG sequence; 3, gene replacement transformation; 4, multiple integrations, apparently at least one of which is into the genomic pyrG sequence. d Ascospores from hybrid cleistothecia were allowed to germinate on non-selective medium to form colonies. Conidia from each colony were tested for growth on complete medium and medium lacking uridine. Colonies the conidia of which were able to form colonies on complete medium, but not on medium lacking uridine, were scored as pyrG_ . Since each colony is derived from a single ascospore, the fraction of pyrG- colonies reveals the fraction of pyrG- acospores.

(d) Plasmid pJR15 contains an insert of Aspergilfus nidulans origin that directs integration at the pyrG locus

These data suggest strongly that pJRl5 carries the wt pyrG allele, but there are two other possible explanations. One is the trivial explanation that we have accidentally cloned the E. coli or Neurospora OMPdecase gene and that it functions efficiently in A. nidulans. A second is that we have cloned another A. nidulans gene that suppresses pyrG89. We were able to rule out the first possibility by probing EcoRI digests of E. co&, AJ. crassu and untransformed A. nidulans DNA with pJR15. A single baud in the A. niduI~ns digest showed homology while the N. crama and E. co&digests showed no homologous

Fig. 2. Hybridizations ofA. nidz&ns strain G191 transformed with plasmid pJRl5. Ail lanes were probed with [32P]pJRtS and the scale at the left is fragment size in kb as determined from a Hind111 digest of bacteriophage. Lanes 1-12 contain DNA digested with &I. Lane 1 contains 1.0 ng of pJR15 DNA and lanes 2-l 1 contain DNA from various transformants. The DNA miniprep procedure used to prepare these samples of DNA causes absorbance readings which do not accurately reflect the amount of DNA present. We consequently do not know the exact amount of DNA in these lanes. Observation of the gel stained with ethidium bromide, however, suggests that all of the lanes have about the same amount of DNA (approx. 5 pg) except for lane 10 which contains about half as much. Lane 12 contains a simifar amount of ~transfo~ed FCSC4 DNA. The tr~sfo~ants are arranged in the same order as in Table I. Thus lane 2 contains DNA from transformant B, lane 3 contains DNA from transformant D, and so on. Lane 13 contains undigested pBR329 DNA. Lanes 2,5,6,7, and 12 have two bands of homology, one slightly smaller than the fragment in the untransformed strain and one slightly larger than pJR15. The size differences are barely apparent in this gel but were more obvious on gels run longer to spread out the low mobility, large fragment size, region of the gel. Lanes 4, 8 and 9 each contain one band of homology of the same size as the band in the untransformed strain. Lane 3 shows one homologous band, the mobility of which is the same as the band in the un~ansformed strain and, visible in shorter exposures, four additional bands, one of which has the same mobility as pJRl5. Shorter exposures of lane 1I reveal three bands, two with mobilities similar to the bands in lanes 2,5,6,7, and 10 and one with a mobility similar to pJR15. Electrophoresis and hybridization were as described in Fig. 1.

bands (Fig. 1). Plasmid pJRl5 thus carries sequences from A. nidulans and not N. crassa or E. coli. In A. nidzduns the majority of transformation events involve the insertion of the plasmid-borne sequences into homologous chromosomal sequences (Tifbum et al., 1983; Yelton et al., 1984; May et al., 1985). If pJRI5 carries, as we expect, the wt pyrG allele, it should integrate preferentially at the pyrG locus. We can determine if pJR15 integrates at pyrG

by crossing transformants to a wt strain. If the transforming gene integrates at a site unlinked to pyrG, approx. 25% of the progeny of the cross should be pyrimidine-auxotrophic (Pyr - ). If the transforming gene integrates at the pyrG locus, Pyr - segregants should be rare. Results of crosses of ten pJRl5 transformants of G191 to awt strain are shown in Table I. In eight of the ten, the transforming sequences are linked to pyrG and in the remaining two they are not.

1 101

201 301 401 501 601 701 801 901 1001 TIDXGAA-EATG

BmCC4Amm

1101 ~UmmmC 1

Met

1199TcTTcG~TGccFiccTcccCTACGcAATTcGcGcAAcC,aAC~TccCAACccT~AcA~AAA~~TcC 2 Ser Ser Lys Ser His Ieu Pro Tyr Ala Ile Arg Ala Thr Asn His Fro Asn pro I_EU'IhrSer Lys Zeu phe Ser

27 Ile A&t Glu Glu Lys Lys lhr AsnVal Thr Val Ser Ala Asp Val 'Ihrlkr Ser fia Glu leu Leu Asp Leu Ala 1349G4cc 52 Asp A

mc4DmCmmc444TA~

GTGAGTACAG GCSX4KXccCTATATCccAGIT rg I..eu Gly Pro Tyr Ile Ala Val

Sal1 1435~~AcC~CA~GACATccI;CACCGATcTGAcCccGTcGACCcTTTccTcGcM:cAATcCcTcGcGAccI~ 61 Leu Lys Th.rHis Ile Asp Ile I&u lhr Pro Ser Thr Pro Ser 'IhrJ.EUSer Ser hu Gln Ser Leu Ala 'IhrLys 1510CACAACTM:CrCATC~GAGGACCCC~TM:ATCGACAn:GGCCACC~CAAAAGCIU;TAC~~GGT~ 86 His Asn I%e Leu Ile Pne Glu Asp Arg Lys Pne Ile Asp Ile Gly Am Tnr Val Gln Lys Gln Tyr His Gly Gly 1585 GCTCICUXATCTCC ~TGG~cACATcATc~CTGcGGc~cTGccGGGcGAcI.GGGATcGM:GAGGcCcTC 111Ala IEU Arg Ile Sex Glu Trp Ala His Ile Ile Asn Cys Ala Ile Leu Pro Gly Glu Gly Ile Val Glu Ala Leu 1660GCACAS:ACAACX:AAGTCT~~Cm~GACGCG~TT~CCSlOGTCTC~AZT~GCCAATGACGAGT I.36Ala Gin 'k 'IhrLys Ser Pro Asp Fhe Lys Asp Ala Asn Gln Arg Gly Leu Leu Ile Leu Ala Glu Met 'IlwSer 1735AAGGGAn;-rCrrGcG~~GAG~cAGGcAcGcTcGGTT~TAGGGG~AAL;~TAAGoGG~~ATG 161 Lys Gly Ser Glu Ala Thr Gly Glu Ser Gin Ala Arg Ser Val Glu Tyr Ala Arg Lys 'Qr Lys Gly Phe Val Met 1810GGA~Gn;~ACAAGGGOGTIY:AGTGAGGTGCT(:CCCGPACAGAAAGAGGAGACX:~GATmGTCGTCm 186 Gly F&o Val Ser 'I&rArg Ala Leu Ser GluVal Ieu Pro Glu Gln Lys Glu Glu Ser Glu Asp Re Val Val Phe 1885AcGAcTGcGGTGAAT~Tcx:c$T~GGGGAT~cI‘GGGGcAG~~TcAGAcAccTcGGT(JGGcGGTTGGG 211 l'hr'IhrGly Val Asn Leu Ser Asp Lys Gly Asp Lys Leu Gly Gln Gln Q-f Gln 'JhrPro Gly Ser Ala Val Gly 196OcGAcx=rGcGGAC~ATcATTGcGGGTAGGGGcATc~TApb:GcGGAcGfir~~C~GcGGTTcPG~~C 236 Arg Gly Ala Asp Phe Ile Ile Ala*GlyArg Gly Ile Tyr Lys Ala Asp Asp Pro Val Glu AlaVa

Gln Arg Tyr

BTf_xxAAmTrGAmm 2035cGGGAGGAAGGcTGGAbAGcTTACWAAAAGAGIT~c?TTGA 261 Arg Glu Glu Gly Trp Lys Ala Tyr Glu Lys Arg Vd Gly Leu OP-

Fig. 3. Nucleotide and predicted amino acid sequences of the insert in pJR15 containing the wt pyrG gene. Transcription start points are under the asterisks. The restriction sites used for S 1 nuclease protection analysis are shown and the putative 3’ splice signal sequence for the intron is underlined. The tr~sc~ptional start points and the 3’ intron boundary were determined by Sl protection analysis (Fig. 4). The 5’ intron boundary was not determined directly but all other possible sites predict an unreasonably short (13 bp or Iess) intron and/or disrupt the ORF. We are, thus, confident that the 5’ intron boundary is as shown. The numbering ofthe nucleotide sequence starts with the EcoRI site at one end of the fragment and ends with the Sat&AI site at the other end. The predicted amino acids of the protein encoded by the wt pyrG gene arenumbered separately.

393

5’

lntron

al

a

MACGT

;

RNA -+

RNA

:

-+

;

ACGT

M 217

217

Fig. 4. Determination of 5’ transcriptional start points and the intron 3’ splice site by Sl nuclease protection assays. Lanes M contain end-labeled Hinff fragments of pUC19 used as size markers with sizes indicated at the right and left of the figure. Lanes A, C, G and T are d~deo~nucleotide se~uenciug products of a template for accurate sizing. Lanes marked ‘probe’ contain ill-len~h undigested probes and represent 1% of the amount of probe used in the hybridizations. Lanes marked ‘-’ are S 1 products of probehybridized to yeast tRNA and those marked ‘ + ’ are products protected by hybridization to total RNA from A. nidulans. Electrophoresis was at 1800 V for 2 h 30 min in an 8% polyacrylamide gel containing 8.0 M urea with TBE buffer. Autoradiography was with Kodak XAR-5 film for 24 days.

394

These results were clarified by Southern hybridization studies of each transformant. DNA of each transformant was digested with PstI and probed with labelled pJR15 (Fig. 2). Since PstI cuts pJR15 once, in the ampicillin-resistance gene, hybridizations of pJR15 to PstI digests of transformants in which a single copy of pJRl5 has integrated by homologous recombination into the chromosomal pyrG gene should show two bands of homology equal in combined M, to the combined M,s of pJRl5 and the PstI fragment carrying pyrG in untransformed strains. Non-homologous integration events produce three bands of homology, two due to the integrated sequences and a third due to the resident pyrG allele. Gene replacement transformation events have also been reported in A. niduluns (Miller et al., 1985; Upshall, 1986). In such transformants the resident gene is replaced by the transforming gene and a single band of homology indentical to that found in wt strains should be found.

Five of the transformants (Fig. 2, lanes 2, 5, 6, 7, 10) probed with pJR15 revealed two bands of homology indicating a single integration into the ho-

mologous resident gene. Thesebandsalsoshowed homologyto pBR329 (results not shown). Three transformantsrevealed a singleband homologous to pJRl5(Fig.2, lanes 4,8,9)and no homologyto pBR329(results notshown). Such a pattern could be produced by gene replacement (or, more accurately, mutation replacement) transformations or by reversions. Since the reversion frequency for pyrG89 has proven to be less than one in lo9 in our hands (not shown), it is extremely likely that these are gene-replacement transformants. One transformant (Fig. 2, lane 3) gave five bands of homology (more easily visible in short exposures than in Fig. 2) including an intense band with an M, similar to pJR15 (approx. 6.2 kb) and one band the size of the genomic fragment. This result is consistent with integration at two sites other than the resident gene and

pYrG urd pYr4

MSSKS HLPYA IRATN HPNPL TSKLF SIAEE KKTNV TVSAD ADR-G PYIAV LKTHI M-SKA TYKE,--RAAT HPSPV AAKLF NIMHE KQTNL CASLD REA-L PKICL LKTHV MSTSQ ----- ----T H--PL TSYLF RLMEV RQSNL CLSAD ADKVG PSIW LKTHY

pYrG ura3 +

DILTD LTP-S TLS-- -SLQS LATKH NFLIF EDRKF IDIGN TVQKQ YHGGA Lm DILTD FSMEG TVK-- -PLKA LARKH GFLLF EDRKF ADIGN TVKLQ YSAGV YRIAE DLITG WDYHP HTGTG AWLAA LSAKY NFLIF EDRKF VDIGS TVQKQ YTAGT ARIVE

pyrG WAHII NCAIL PGEGI VEALA QTT-K SPDFK DANQR GLLIL AEMTS KGSLA TGESQ urd WADIT NAHGV VGPGI VSGLK QAA-E EU--- TKEPR GLLML AELSC KGSLA TGEYT @ WAHIT NADIH AGEAM VSAMA QAAQK WERIP Y#LDR GLLIL AQMSS KGCLM DGKYT pyrG ARSVE YARKY KGFVM GFVST RALSE VLPEQ KEESE DFWF TTGVN LSDKG DKLGQ ura3 KGTVD IAKSD KDFV? GFIAQ RDMGG RDEGY DWLIM ----- TPGVG LDDKG DALGQ pyr4 WECVK AARKN KGFVM GYVAQ QNLNG ITKEA LAIHT PGCKL PPPGE EAPQG DGLQQ pyrG QYQTP GSAVG --RGA DFIIA GRGIY KADDP VEAVQ RYREE ----- GWKAY EKRVG ura3 QYRTV DDWS --TGS DIII- ----Y KADDA KVEGE RYRKA GWEAA GWEAY LRRCG 3 QYNTP DNLYA NIKGT DIAIV GRGII TAADP PAEAE RYRRK ----- AWKAY QDRRE pyrG L* urd QQN* pYr4 RLA* Fig. 5. Comparison of predicted amino acid sequences of orotidine-5’-phosphate decarboxylase genes ofA. niduluns (pyrG), 5’. cerevisiae (uru3) and N. crassa (pyr4). Regions of most obvious homology are overlined. The ura3 sequence was taken from Rose et al. (1984), the pyr4 sequence from Newbury et al. (1986) and the pyrG sequence is as shown in Fig. 2. The single-letter designations for amino acids are used. The protein encoded bypyr4 contains 99 additional aa at the position designated ‘8’. A single ammo acid in the ura3 sequence is undetermined and is designated ?‘. Stop codons are designated by asterisks. To achieve optimal alignment small gaps had to be introduced into the sequences and they are designated by dashes.

395

the intensity of the 6.2-kb band suggests that a tandem repeat of pJRl5 is present in the transformant. The final lane (Fig. 2, lane 11) shows three bands of homology (more readily visible in short exposures than in Fig. 2), two as predicted if integration occurred into the resident gene and a third, more intense, band of the same size as pJR15. This result suggests that integration at the pyrG locus has occurred and that there is a multiple tandem repeat of pJRl5 at this site. A comparison of Fig. 2 and Table I reveals that the transformants in which a single copy of pJRl5 has integrated into homologous resident sequences or in which mutation replacement tr~sfo~ation has occurred produce few or no Pyr- progeny when crossed to a pyrG + strain. Thus the A. niduluns sequences on pJRl5 direct integration at a site closely linked to pyrG. (e) !Sequence and transcript analysis of the insert in pJRl5 To verify that the insert in pJRl5 includes the pyrG gene we have sequenced it. Examination of the sequence (Fig. 3) suggested that it contained an ORF that predicts an amino acid sequence homologous to the OMPdecase genes of S. cerevisiae and N. crassa and that the ORF was interrupted by an intervening sequence. S 1 nuclease protection analyses of the ORF (Fig. 4) verified that an intron was present and the boundaries of the intron and the initiation sites for mRNA synthesis are shown in Fig. 3. Fig. 5 shows the predicted amino acid sequence of the ORF as well as the sequence for the S. cerevisiae and N, crassa OMPdecases. (f) The insert in pJR15 does not complement an Escherichia coli pyrF mutation Dmochowska et al. (1980) have reported the complementation of an E. eoli OMPdecase (pyrF) mutation with an A. niduluns library. The cloned DNA fragments appear, however, to be markedly rearranged and no evidence has been obtained that they contain the pyrG gene (Berse et al., 1983). To test whether the sequences on pJRl5 can complement a pyrF mutation, we transformed the E. coli strain DB6656, which carries a mutation in pyrf’, with pJR15. Five transformants carrying pJR15

were tested for uracil prototrophy. Each of the five transformants retained its uracil auxotrophy and, consequently, the sequences on pJRl5 do not complement the pyrF lesion. (g) Mapping of the pyrG locus Since pyrG89 has become a very valuable marker for ~~sfo~ation, it is useful to know the map

TABLE II Meiotic recombinant frequencies ofAspergi1ia.tnidulans mutation pyrG89 with markers on linkage group I” Markers b

pyrG89, fpa337

Number of recombinants’/ total progeny tested

Recombination frequencyd

2421982 771470 266/716

(%)

fiaB37,

galD5

127/470

24.6 16.4 37.2 27.0

fpaB31,

riboAl

3&t/716

42.5

galD5,

r&A 1

1451457

31.7

pyrG89, galD5 pyrG89, riboA 1

a These data were obtained from two crosses, CGRZ 13 x FGSC475 and GCR2-13 x FGSC515. Crosses were performed as described by Morris et al. (1982) and ascospores were analyzed as described in footnote d to Table I. Ascospores from hybrid cleistothecia were allowed to germinate and form colonies on complete medium. Conidia from the colonies were used to test for the markers. ’ Data from the two crosses were combined to determine the recombination frequencies between each pair of markers. ’ Recombinants are those progeny in which a crossover has occurred between the two markers. For example, GCR2-13 carries pyrG89 and is wt for the fpaB locus while FGSC475 carries fpaB37 but is wt at the pyrG locus. Thus, in a cross between these two strains, progeny that carry both pyrG89 and fpaB37 or are wt at both loci are recombinants. d Recombination frequency is the number of recombinant progeny/number of total progeny tested. These data indicate that the pyrG locus maps between the fpaB and gau) loci at a map distance of 24.6 units from fpaB37 and 16.4 units from g&X. These data were verified by further analyzing progeny of the GCR2-13 (j$aB+, pyrG89, gaZD+) x FGSC515 cfpaB37, pyrG + , gaZD5) cross for single and double crossovers. The results were as follows (genotypes of the classes of progeny are given in parentheses): total progeny tested: 470; parentals: 307 lfpa337, pyrG + , galD5; fpaB + , pyrG89, galD + ); single crossover: 127 (&I#‘, pyrG+, galD5; J&B+, pyrG89, golIf5; fpoB37, pyrG89, gaID + ; &B37, pyrG+, galD + ); double crossover: 36(&1B+,pyrG+,guZD+ ; fpaB37,pyrG89,guZD5). These data verify that the pyrG locus lies between the fpaB and galD loci.

396

position of pyrG. Palmer and Cove (1975) mapped pyrC to linkage group I but were unable to establish

linkage to any other markers. We have found (Table II) that pyrG89 maps approximately 24.6 map units to the right of fpaB37 and 16.4 map units to the left of galD5.

DISCUSSION

We have cloned an A. nid~a~ sequence that efficiently complements pyrG 89, that directs integration into a chromosomal sequence tightly linked to the pyrG locus and that predicts an amino acid sequence homologous to the OMPdecases of S. cerevisiae and N. crassa. These facts argue strongly that this sequence contains the wt pyrG allele. Moreover, the fact that pJR15 can complement pyrG89 even when it integrates at another site in the genome argues that pJRl5 carries the entire functional pyrG structural gene. The ~~sfo~ation procedure we have developed gives consistently high transformation frequencies and should be of value for transforming A. nidulans and, in particular, for complementing mutations with libraries of wt sequences. It could be argued that the high transformation rates we obtain are, for unknown reasons, unique to the pyrG locus. We obtain significantly higher transformation rates, however, with pDJB1 than the highest reported values (Ballance and Turner, 1985) and we have successfully complemented each of four additional mutations attempted including three nutritional markers and one conditionally lethal mutation (Oakley et al., 1987 and unpublished). While the approach we have taken to clone pyrG may seem laborious, the entire procedure can be carried out easily in less than six weeks. The recovery of the gene of interest would be facilitated greatly if there were but a single integration event in the transformant. Single tr~sfo~ation events should be favored if the ratio of protoplasts to DNA were increased and the amount of library DNA we used in these experiments was probably much greater than optimal. In this regard, Dr. S. Osmani and others in the laboratory of Dr. N.R. Morris have used transformation procedures similar to ours to complement several mutations with a library, obtaining single in-

tegrations by using low DNA/protoplast ratios (personal communication). Analysis of the sequence of pyrG and S 1 nuclease protection analysis reveals several similarities among pyrG and the small number of published A. nidulans sequences. As with the argB (Upshall et al., 1986), trpC (Mullaney et al., 1985) and tpiA (McKnight et al., 1986) genes but unlike the ADH3 (McKnight et al., 1985) and 3-phosphoglycerate kinase (Clements and Roberts, 1986) genes, pyrG does not contain a consensus TATA sequence upstream from the start sites, though p~midine-huh regions are present. In the trpC (Mullaney et al., 1985), argB (Upshall et al., 1986) and @iA (McKnight et al., 1986) genes, most transcription start points are within a consensus sequence (CAAG). This is not the case in pyrG or ADH3 (McKnight et al., 1985). One of the start points in pyrG is immediately 5’ to a sequence (AGTTT) which is found at the 5’ end of the tpiA cDNA. Two similar sequences (AGTCT and AGTTC) are associated with inscriptions start points in the avB gene (Upshall et al., 1986). Although we have not determined the polyadenylation sites for pyrG, no consensus polyadenylation site was found in the 3 ’ -noncoding region of our clone. While the argB (Upshall et al., 1986), trpC (Mullaney et al., 1985) and tpi4 (McKnight et al., 1986) genes contain a consensus polyadenylation sequence, ADH3 does not (MeKnight et al., 1985). Most A. nidulans introns sequenced to date contain consensus higher-eukaryote splice sequences. The pyrG intron contains the typical 3’ splice sequence, CAG (Lerner et al., 1980) and the higher-eukaryote putative 3’ splice signal sequence, CTGAT (Keller and Noon, 1984) (Fig. 3). The putative 5’ splice sequence, GTACAT, however, differs from the typical higher-eukaryote splice sequence, GTAAGT (Lemer et al., 1980) in two of five positions and from the typical N. crussa sequence, GTA$GT (Orbach et al., 1986) in one position. The 5’ splice sequences in the introns of the ADH3 gene (GTATTT and GTGTGT) are also somewhat atypical (MeKnight et al., 1985). Interestingly, the introns of the pyrG and ADH3 genes all contain a common sequence (GCTGAT) which includes the putative 3’ signal sequence. A portion of this sequence (CGCTGA) is repeated two bases upstream from the putative 5’ splice site of the pyrG intron (Fig. 3) and one base upstream from the 5’

391

splice site of one of the two ADH3 introns (M&night et al., 1985). A similar sequence (GTTGA) is present three bases upstream from the 5’ splice site of the second ADH3 intron (McKnight et al., 1985). The presence in each case of these sequences which contain a portion of the 3’ signal sequence immediately upstream from atypical 5’ splice sites is striking and suggests the possibility that these sequences may be involved in 5’ splice site recognition in these introns. Our results also confirm and extend the rather limited results available on the types of integration that occur in transformation in A. nidufuns. Transformation occurred primarily at chromosomal sequences homologus to sequences carried on the plasmid. The most common type of transformation was a single integration event at the homologous site. Other events occurred, however, including multiple integrations at the homologous site, non-homologous integrations and mutation replacement transformations in which bacterial sequences from the plasmid were lost during transformation. These results are similar to those obtained by Upshall (1986) with the argB locus although Upshall examined more transformants and found some types of integrants not found in our smaller sample. The similarity of results obtained with the two loci suggests that these patterns may be general. As expected, when mutation-replacement transformants were crossed to a pyrG + strain no pyrG progeny were obtained. Crosses of some transformants in which a single copy of pJR15 had integrated into the genome by homologous recombination at the pyrG locus, to a pyrG + strain produced some pyrG - segregants. If integration was, indeed, at the pyrG locus, the mutant and wt alleles would be less than one map unit apart and pyrG - progeny would occur at a frequency of 0.25% or less. The fact that substantially greater numbers of pyrG - segregants were found among progeny of some crosses would seem to indicate that pJR15 had integrated near but not at the pyrG locus. The Southern hybridization analysis data (Fig. 2), however, appear to rule out this possibility and a much more likely explanation is that the integrated sequences are meiotically unstable as has been demonstrated for other loci (Tilbum et al., 1983; Yelton et al., 1984; Upshall, 1986). In this regard, it is notable that pyrG progeny occurred among segregants of a cross

between transformant W, in which several copies of pJR15 had integrated at the pyrG locus, at a very high frequency. .The pyrG gene should be of value for several reasons. The OMPdecase genes of several organisms have been cloned (Bach et al., 1979; Buxton and Radford, 1983; Donovan and Kushner, 1983; Gillum et al., 1984; Floyd and Jones, 1985) and comparisons of the sequences of these genes should be of value in determining functional domains of the proteins they encode. An extensive analysis of these sequences is underway (Dr. Alan Radford, University of Leeds, personal communication) but the results of our limited comparison (Fig. 4) reveal several highly conserved regions. In some cases pyrG may be a more useful marker than pyr4 for transforming A. niduluns because it gives higher transformation frequencies and because the transformants appear more quickly after transformation. OMPdecase genes have proven extremely useful for transformation systems in Sacchuromyces because it is possible not only to select for OMPdecase function but, with 5-FOA, to select strains in which OMPdecase function is lost (Boeke et al., 1984). The 5-FOA selection also works in A. niduluns (C.E.O. and B.R.O., unpublished) and this has permitted the selective eviction of a plasmid carrying the N. crussu pyr4 gene (P.W. Dunne and B.R.O., unpublished), which should facilitate in vitro mutagenesis experiments as it does in Succhuromyces (reviewed by Botstein and Shortle, 1985). It may also be possible to use the cloned pyrG gene to create identical pyrG mutations rapidly in many strains. Following the suggestion of Boeke et al. (1984), one should be able to create pyrG - mutants rapidly by transforming PyrG + strains with a cloned mutant pyrG allele (pyrG89 or a mutant allele created in vitro). Miller et al. (1985) have demonstrated that direct and indirect gene replacements occur in A. niduluns and our results suggest that in approx. 30% of transformants the resident allele will be replaced by the transforming allele. It should thus be possible to select, on 5-FOA, for transformants in which the wt allele has been replaced by the mutant allele.

398 ACKNOWLEDGEMENTS

This work was supported by grants GM31837 to Dr. B.R. Oakley and GM29228 to Dr. N.R. Morris from the N.I.H. Dr. G.S. May was supported by a postdoctoral fellowship from the N.I.H. and from the Anna Fuller Fund.

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