a gene coding for the uric acid-xanthine permease of aspergillus

Copyright 0 1989 by the Genetics Society of America

A Gene Coding for the Uric Acid-Xanthine Permease of Aspergillus nidulans: Inactivational Cloning, Characterization, and

Sequence of a &Acting Mutation

George Diallinas and Claudio Scazzocchio

Znstitut de Microbaologie, Unite' Associe'e au CNRS 136, Uniuersiti Paris-Sud, Orsay Cedex 91 405, France Manuscript received November 14, 1988

Accepted for publication February 16, 1989

ABSTRACT In Aspergillus nidulans, integration of transforming sequences can proceed through recombination

with homologous sequences or at heterologous sites in the genome. In a strain with a large deletion in the gene coding for acetamidase (amdS), a plasmid carrying this gene integrates into and inactivates uapA, the putative structural gene for uric acid-xanthine permease, with a frequency of 0.3%. The integration event occurs 3' to the open reading frame of amdS. A 10-nucleotide sequence which occurs in this region is also found within the open reading frame of uapA. We have taken advantage of this integration event to clone the permease gene and to characterize a cis-acting mutation, uap- 100, as a duplication of 139 bp located in the upstream region of uapA. Northern and dot blot analyses confirmed earlier results measuring the uptake of uric acid: the transcription of the uapA gene is inducible and the uap-100 mutation results in a bypass of the need for induction while having an 8-fold up-promoter effect under inducing conditions.

I N Aspergillus nidulans, as in other filamentous fungi, integration of transforming sequences can

occur at both homologous and heterologous locations. In particular, heterologous integration events of plasmids carrying the amdS gene, coding for acetamidase, have been previously described (TILBURN et al. 1983; WERNARS et al. 1985). TILBURN (1988) has shown, using amdS deletions, that when the homology between the transforming amdS plasmid and the resident chromosomal DNA was about 1.5 kbp, only 1 out of 19 transformants analyzed contained an integration in the residual chromosomal amdS sequences. If heterologous integration occurs at random, it may be possible to use it as a means of cloning genes by inactivation in every case where a seletion for loss of function is available.

T o investigate the randomness of integration events we devised the following system. We used a strain with a large deletion in the acetamidase gene, amd- S368 (HYNES 1979), and a plasmid containing the entire amdS structural gene plus essential control sequences but shorter than the sequences deleted in amdS368 (HYNES et al. 1988). T o detect integration events at a number of different genes we used a technique described by ALDERSON and SCAZZOCCHIO (1967). In the presence of the xanthine analog 2- thioxanthine, wild-type strains of A. nidulans give yellow rather than green conidia. This effect requires the uptake of the analog and its oxidation to 2-thiouric acid which presumably acts by chelating the copper of laccase (polyphenol oxidase) which is involved in the

Genetics 122: 341-350 (June, 1989)

conversion of yellow to green pigment (SCAZZOCCHIO 1966; ALDERSON and SCAZZOCCHIO 1968). Mutations at any locus necessary for this process will result in resistance to 2-thioxanthine, i.e., in the production of green conidia even in the presence of 2-thioxanthine. Loci at which mutations result in 2-thioxanthine resistance, and the rationale for the mutant phenotype, are shown in Table 1. Very easy growth and/or complementation tests permit the assignment of any given mutation, or integration event, to a given gene (DAR- LINGTON and SCAZZOCCHIO 1967; ALDERSON and SCAZZOCCHIO 1968). In this article we show that a plasmid carrying amdS sequences integrates prefer- entially at the uapA gene encoding the uric acid- xanthine permease. We made use of one such event to obtain genomic clones containing this gene.

The expression of uapA requires specific induction by uric acid or its thio-analogs and the presence of the narrow-domain uaY and the wide-domain areA regulatory gene products. The expression of uapA is im- paired specifically in strains carrying the areAl02 al- lele. We have previously described a cis-dominant and trans-recessive mutation, uap-100, which maps adja- cent to uapA and has a number of effects on the regulation of the permease gene (ARST and SCAZZOC- CHI0 1975; SCAZZOCCHIO and ARST 1978). This mutation results in a 2.5-fold up-promoter effect, a bypass of the need for induction still leaving the mutant dependent on the presence of an active uaY product (SCAZZOCCHIO and ARST 1978; SCAZZOCCHIO, SDRIN and ONG 1982), and a bypass of the areA102 pheno-

342 G. Diallinas and C. Scazzocchio

TABLE 1

2-Thioxanthine-resistant classes of mutants

Nitrogen source and purine analogs

Ammonium Ammonium d-tartrate

Class of Ammonium + 2-thiouric mutant d-tartrate 2-thioxanthine acid Hypoxanthine allopurinol

Sodium Uric acid nitrate

d-tartrate + Hypoxanthine +

hxA, hxB + r S - - + + uaY + r r - - - + cnx ABC,E,F,G,H + r S - - + uapA Wild-type +

- + r r + + * +

S S + - + + + indicates normal growth; - indicates lack of growth; * indicates the leaky growth typical of uupA mutants; r and s indicate resistance

and sensitivity to 2-thioxanthine and 2-thiouric acid, respectively. Allopurinol specifically inhibits the utilization of hypoxanthine as a nitrogen source but is taken up through the uric acid-xanthine uptake system. Mutations in the hxA and h d genes, as well as in the cnx genes, can be easily distinguished from each other by complementation tests. For the rationale of all these tests see DARLINCTON and SCAZZOCCHIO (1967), ALDERSON and SCAZZOCCHIO (1968) and SCAZZOCCHIO and ARST (I 978).

type (ARST and COVE 1973) still leaving the mutant dependent on the presence of an active areA product (ARST and SCAZZOCCHIO 1975). In this article, we show that uap-100 is a 139-bp duplication which increases uapA transcription and that the amdS integration events occur downstream from the uap-ZOO duplication site, within the uapA open reading frame. Also, we present results giving interesting insights on the effect of uap-IO0 on the expression of the uapA gene.

MATERIALS AND METHODS

Strains: The following Aspergillus nidulans strains were used: amdS368 amdIl8 amdA7 niiA4 biA1; amdS368 amdIl8 amdA7 niiA4 uapA24 biAl pantoB1OO;fiAl areA102pyroA4; uap-100 biA1; biA1; yA2 amdS320 amdIl8 amdA7 niiA4 PantoBlOO biAl; and yA2 uapA24 pantoB100.

amdS368 is a 6.0-kbp deletion of the amdS gene coding for acetamidase and amdS320 is a 2.8-kbp deletion of the same gene (HYNES 1979; HYNES et al. 1983). uapA24 is a mutation in the structural gene for the uric acid-xanthine permease (ARST and SCAZZOCCHIO 1975). uap-100 is a cis- acting mutation which has been discussed in earlier sections. areA102 is a mutation in the wide domain regulatory gene areA involved in nitrogen metabolite repression (ARST and COVE 1973). It results in enhanced expression of a number of genes involved in the utilization of nitrogen sources, but abolishes the expression of the gene for uric acid-xanthine permease and diminishes the expression of the gene coding for formamidase (HYNES 19'72; ARST and COVE 1973; ARST and SCAZZOCCHIO 1975). amdA7 and amdIl8 are mutations resulting in elevated levels of amdS expression and have been described in detail by HYNES (1978a,b, 1979) and HYNES et al. (1988). niiA4 is a mutation in the structural gene for nitrite reductase (PATEMAN and COVE 1967). yA2 andfwA1 result in yellow and fawn conidiospores, respectively. biA1, pantoBlOO and pyroA4 indicate auxotrophies for biotin, pantothenic acid and pyridoxine, respectively.

Escherichia coli K 12 strain DH 1 (LOW 1968) was used for routine plasmid preparations. E. coli strains JMlOl and JM109 (YANISCH-PERRON, VIEIRA and MESSING 1985) were used for the propagation of M13 phages. E. coli Q359 (KARN et al. 1980) was used for the propagation of h EMBL4 recombinant phages.

Media and growth conditions: The standard media and growth conditions for A. nidulans were used as described by COVE (1 966). Mycelia from which protoplasts were prepared were grown as described by TILBURN et al . (1 983). Mycelia for DNA preparations were grown in appropriately supplemented minimal media for 18 hours at 25", shaken at 120 rpm. Mycelia for RNA preparations were grown for 21 hr at 120 rpm on appropriately supplemented minimal media, plus 1 % glucose as carbon source and 5 mM urea as nitrogen source (noninducing conditions; NI) or on the same media to which 27 p~ 2-thiouric acid was added after 16 hr and growth allowed to continue for a further 5 hr (inducing conditions; I).

Isolation of nucleic acids: A. nidulans DNA and RNA preparations were as previously described (TILBURN et al. 1983; LOCKINCTON et al . 1985). Plasmid preparations were as described by MANIATIS, FRITSCH and SAMBROOK (1 982). poly(A)+ RNA was isolated as described by WERNER, CHEMLA and HERZBERG (1984).

Transformation: A. nidulans protoplasts were prepared as described by TILBURN et a l . (1 983). In each transformation experiment, protoplasts were aliquoted. pAMD21 was used as the transforming plasmid in three experiments. Experiment 1 involved 12, experiment 2, 15 and experiment 3, 30 aliquots, respectively. DNA (25, 10 and 10 pg in experiments 1, 2, and 3, respectively) and polyethylene glycol were individually added to such aliquots and the procedure was continued as detailed in TILBURN et al. (1983). Each aliquot was used to inoculate a tube containing 4 ml of soft agar which was used to overlay a single Petri dish. Controls without DNA and regeneration controls were performed in an analogous manner. E. coli transformation was performed according to HANAHAN (1 983).

Genetic techniques: Aspergillus genetic techniques (crossing, selfing, etc.) were performed as described by PONTECORVO et al . (1953).

DNA manipulations and sequencing: Restriction digests of Aspergillus or plasmid DNA were performed as described by MANIATIS, FRITSCH and SAMBROOK (1982). Ligations were as described by MURRAY (1986). DNA sequences were determined with the dideoxynucleotide chain termination method (SANGER, NICKLEN and COUUON 1977) using M 13mp 18 and M 13mpl9 single-stranded templates as described by MESSING (1983). Single- and double-stranded DNA of M13 subclones was prepared as described by the Amersham M 1 3 Cloning and Sequencing Handbook (1983).

The uapA Permease of A. nidulans 343

["SIdATP labeled DNA molecules of different lengths were separated on 6% acrylamide/8 M urea gels. All possible Sau3AI and NheI restriction fragments of the wild-type and uap-100 Bglll-PstI region (see Figure 5) were cloned into M 13 vectors and sequenced in both directions.

Transfers and Hybridizations: Probes were labeled by nick-translation as described in the Amersham Bulletin (1987). Southern and Northern transfers, as well as dot blots, were as described by MANIATIS, FRITSCH and SAM- BROOK (1982). Nitrocellulose was baked for 2 hr at 80" under vacuum. Prehybridization and hybridization were performed as described by BOYER (1986). To monitor poly(A)+ RNA concentrations we used an actin-specific probe (plasmid pSF5). Densitometry readings enabled us to compare the levels of uapA expression under non-induced and induced conditions in a wild-type and a uap-100 background.

Plasmids and gene libraries: Plasmid pAMD2I was a gift from M. HYNES. I t carries an intact A. nidulans amdS gene and essential control sequences contained in an EcoRI- SmaI fragment cloned into pUC9 (HYNES et al. 1988). Plasmid pSF5 was a gift from R. MORRIS. I t carries the actin gene from A. nidulans cloned as a Hind111 fragment into pUCl8. Plasmid PILJl6 was a gift from I. JOHNSTONE. I t contains the argB gene from A. nidulans cloned as a Sal1 fragment into pUC8 (JOHNSTONE, HUGHES and CLUTTER- RUCK 1985). The A. nidulans X EMBL4 gene library was a gift from C. M. LAZARUS.

RESULTS

Isolation of transformants resistant to Z-thioxanthine: Reconstruction experiments with regenerating protoplasts made from a strain which is 2-thioxanthine-resistant (uapA24 biA1) showed that it was easy to detect green colonies on the background of wild- type (&AI) yellow conidiating colonies. A strain carrying a complete deletion of the amdS gene [amdS368 amdll8 amdA7 niiA4 biA2 (HYNES 1979)] was transformed with plasmid pAMD2l carrying an intact amdS gene and bearing no sequences homologous to the genomic DNA of the recipient strain. Transform- ants were selected on acetamide as nitrogen source in the presence of 2-thioxanthine (0.1 mg/ml final con- centration). Among approximately 12,000 transformants, 34 were 2-thioxanthine-resistant, i.e., showed green conidia. These results represent the aggregate from three different experiments. In experiment 1, one 2-thioxanthine-resistant colony was isolated among 3400 amdS+ colonies; in experiment 2, the yield was one 2-thioxanthine-resistant colony among 2000 amdS+ colonies; and in experiment 3, we obtained 32 2-thioxanthine-resistant colonies among 6500 amdS+ colonies. In this last experiment, 30 different transformation mixtures were plated onto sep- arate Petri dishes. In a similar experiment, a strain carrying a point mutation in the gene coding for ornithine carbamyl-transferase (argB2 b i A l ) was transformed with 200 pg of plasmid pILJ 16 carrying an intact argB gene (JOHNSTONE, HUGHES and CLUTTER- RUCK 1985). Transformants were selected on ammonium d-tartrate as nitrogen source in the absence of

1 2 3 4 5 6 7 8 9

"6.3kb

FIGURE 1 .-Southern blot analysis of DNA from transformants resistant to 2-thioxanthine and classified as uapA-. DNA (5.0 pg) of six transformants (TI, T2, T3, T4, T19, T33; tracks 2-7) and 2- thioxanthine-sensitive transformants (TA; track 1) from strains 6iA I (wild-type strain; trdck 8) and amdS368amdIlRamdA7niiA46iAI (recipient strain; track g), was digested with EcoRI. After fractionation on an agarose gel (0.8%), DNA was transferred to nitrocellulose filters and hybridized with nick-translated pAMD2l plasmid (see MATERIALS AND METHODS).

arginine and in the presence of 2-thioxanthine. Among the 10,000 transformants recovered, no 2- thioxanthine resistant colonies were found.

The 34 2-thioxanthine-resistant transformants were purified and tested on the media described in Table 1 in parallel with a number of 2-thioxanthine sensitive transformants as controls. All 34 were shown to grow on acetamide and acrylamide as nitrogen sources and to maintain their 2-thioxanthine resistance. It was striking, however, that all belonged phenotypically to the uapA class. No hxA, hxB, cnx or uaY mutants were found.

Analysis of the integration events in transformants showing the uapA phenotype: Six transformants showing the uapA phenotype were analyzed by South- ern blot. The Southern blot includes the two transformants obtained in experiments 1 and 2 and four transformants obtained in experiment 3. Each of these four transformants was isolated from a different Petri dish and thus originated in a different transformation mixture. Figure 1 shows an EcoRI digest of DNA from these transformants. All transformants showed a com- plex but nearly identical pattern when probed with pAMD2 1. This plasmid does not hybridize with the residual acetamidase sequences. These can be detected only when probing with plasmid p3SR2 (TIL- BURN et al. 1983); all transformants retain, as ex- pected, the residual flanking sequences of the amdS

344

a c e t a m i d e

G. Diallinas and C . Scazzocchio

h y p o x a n t h i n e A B

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 I

e

. '. .I -

h y p o x a n t h i n e + a l l o p u r i n o 1

FIGURE 2.-Meiotic analysis of selected transformants. Trans- formants T1 and T I 9 were selfed as described in MATERIALS AND

METHODS and progeny were analyzed on different nitrogen sources (acetamide, acrylamide, hypoxanthine and hypoxanthine/allopurinol). Three distinct classes of progeny were found: progeny with parental phenotype for all nitrogen sources tested ( T I .4), progeny with reduced amdS activity (T1.5, T19.3, T19.4), and progeny which have lost amdS activity (Tl.1, T1.2, T1.3, T19.1, T19.2, T19.5). N o uapA+ progeny, i.e., those sensitive to allopurinol were recorded. T33 is another original 2-thioxanthine-resistant transformant and ts is a 2-thioxanthine-sensitive transformant. wt indicates wild type (biAI) and r is the recipient strain (amd- S368amdl 18amdAAiiA4biAI).

locus (data not shown). pAMD21 detects five main bands. Integration should have given a 6.3-kbp band corresponding to the size of the plasmid, plus two bands of the flanking sequences. In each case, two additional bands could be seen, implying a second integration event. This interpretation was supported by Southern blot analysis of DNA digested with XhoI, a restriction enzyme which does not cut pAMD2l. Two high molecular weight bands were visible (data not shown). It is difficult, however, to understand why a second integration event should always be associated with the integration event which led to uapA inactivation.

Meiotic analysis of selected transformants: A. nidulans is a homothallic organism and we have shown that selfing leads to rearrangement of integrated sequences (TILBURN et a l . 1983; DURRENS et al . 1986). Analysis of selfed cleistothecia of transformants T 1 and T19, both being 2-thioxanthine-resistant, showed three classes of progeny. Class 1 is indistinguishable from the parental transformants. Class 2 (including T1.5 and T19.3) shows reduced growth on acetamide and loss of growth on acrylamide, implying a reduc-

FIGURE 3.--Southern blot analysis of DNA from selected selfed progeny. DNA (5.0 ag) from T1.5 and T19.3 (tracks 3 and 7) (of phenotype amdS+uupA-), T19.2 and TI . I (tracks 4 and 5) (of phenotype amdS-uapA-), T19 (track 1) (an original transformant) and T B (track 6) (a 2-thioxanthine-sensitive transformant) as well as from strains biAl (track 8) and amdS36RumdllRamdA7niiA4biAI (track 2; recipient strain carrying the umdS deletion), was digested with EcoRl. After transfer to nitrocellulose filters, DNA was hybridized (as already described) with nick-translated pAMD2l (A) and pAN501 (B). Molecular weight markers in kbp are shown at both sides.

tion in the number of copies of the amdS gene. Finally, class 3 (including T1.1 and T19.2), like the recipient strain, does not grow on acetamide or acrylamide, showing a complete loss of amdS function. These results, shown in Figure 2, corroborate those described earlier (TILBURN et al. 1983; DURRENS et al . 1986). All progeny maintain the uapA- phenotype. Figure 2 shows the distribution of progeny for two selfed cleistothecia isolated from transformants T 1 and T19.

Southern blot analysis of selected selfed progeny is shown in Figure 3A. EcoRI digests, probed with pAMD21, showed a substantial rearrangement and simplification of integrated copies. It was obvious that among the progeny analysed, all strains showing the amdS+uapA- phenotype retained the 7- and 9-kbp bands, and that progeny showing the amdS-uapA- phenotype have a single band, of 12-1 3 kbp, which is not present in digests from the original transformants. XhoI digests, probed with pAMD2 1, were in agreement with these results. A single high molecular weight band is maintained in the amdS+uapA- progeny but is replaced by a new, higher mobility, band in the amdS-uapA- progeny (results not shown). Prog- eny from both T1 and T19 showed identical band patterns in agreement with their phenotypes. The significance of this result will be apparent later.

The umdS gene maps at the uupA locus: Trans- formants T 1 and T19 and their meiotic progeny T1.5 and T19.3 (amdS+uapA-) were crossed with strain yA2 amdS320 amdIl8 amdA7 niiA4 pantoBlOO biAl (amdS-uapA+). The results are shown in Table 2A. While the presence of amdS-uapA- progeny was ex-


TABLE 2

Mapping the integration of amdS sequences in 2-thioxanthine resistant transformants

A. Cross

yA2 amdS320 amdlld amdA7 niiA4 pantoB100 biAl

X

Phenotype of progeny T1 T1.5 T19 T19.3

amdS-uapA+ 78 120 101 96 amdS+uapA- 45 108 93 34 amdS-uapA- 21 0 19 3 amdS+uapA+ - 0 - 0 - 0 - 0

144 228 213 133

B. Cross

yA2 uapA24 pantoBlOO X

of progeny Phenotype

T1.5 T19.3

uapA- 138 140 uapA+ - 0 0

138 140 -

T 1 and T19 are original amdS+uapA- transformants obtained with an amdS368 amdll8 amdA7 niiA4 biAl strain. T1.5 and T19.3 are selected selfed progeny from the above transformants sharing an amdS+uapA- phenotype.

pected, given the results of the selfing experiment, in no case were amdS+uapA+ recombinants found. This is strong evidence that functional amdS sequences have integrated into, and inactivated, the uapA locus. The number of amdS-uapA- progeny seems lower in the crosses involving the T1.5 and T19.3 progeny than in those involving the original transformants. This might not be significant, as excision events can well be premeiotic and vary from one cleistothecium to another (SELKER et a l . 1988; TILBURN 1988). Al- ternatively, this may be related to the reduced number of copies in the progeny, which will result in an increase in the meiotic (or premeiotic) stability of the inserted sequences.

T 1.5 and T19.3 were also crossed with strain yA2 uapA24 pantoB100. No uapA+ recombinants were recovered among the progeny analyzed (Table 2B), giving additional evidence for amdS integration at the uapA locus.

Cloning of uupA: The evidence indicates that at least one copy of the amdS gene has integrated into the uapA gene, resulting in its inactivation. The EcoRI restriction pattern of progeny T19.3, probed with pAMD21 (Figure 3A), with the 3.6-kbp EcoRI-SmaI fragment comprising the amdS sequences of pAMD2 1, and with pUC9 (results not shown), dem- onstrates that one copy of the amdS gene from pAMD2 1 has recombined heterologously, presumably in the uapA locus, and that this event has taken place via sequences of the amdS gene or flanking. A. nidulans sequences and not via plasmid sequences.

Genomic DNA from T19.3 and T1.5 was used to transform competent E. coli (DH1) cells as described by JOHNSTONE, HUGHES and CLUTTERBUCK (1985). Ten ampicillin-resistant clones were obtained from T19.3 DNA and three from T1.5 DNA. All 13 rescued plasmids were larger than pAMD21 and, when cut with restriction enzymes EcoRI, Sal1 or PstI, gave identical band patterns, different from those of pAMD21 cut with the same enzymes (results not shown). One of the plasmids, named pAN501, was selected for further study. Restriction analysis (not shown) of pAN501 showed that most Aspergillus sequences downstream from the amdS gene, which were present in the original transforming plasmid pAMD21, had been lost. The ClaI site, which is 189 nt before the end of the amdS open reading frame (CORRICK, TWOMEY and HYNES 1987), is conserved. Plasmid pAN5Ol contains a functional amdS gene, because it transforms a strain carrying the amdS368 deletion. The same plasmid does not transform a strain carrying a uapA24 mutation to prototrophy.

pAN501 was used to probe a nitrocellulose membrane identical to the one shown in Figure 3A (Figure 3B). The results strongly suggest that the 10-kbp band which is detected by pAN501 in uapA+ strains repre- sents intact uapA sequences. This band is also present in 2-thioxanthine-sensitive transformants but is replaced by two bands of 7 and 9 kbp in P-thioxanthine- resistant transformants and in progeny of class 2. The integration of pAMD21 (6.3 kbp) into sequences rep- resented by a 10-kbp EcoRI restriction fragment in- troduces a plasmid EcoRI site which splits this fragment into two new fragments of total size 16.3 kbp. This is clearly visible in the simplified progeny T19.3 and T1.5. Progeny T19.2 and T1.l (class 3) seem to have lost plasmid sequences, including the EcoRI site, and thus a single band (1 2- 13 kbp) could be seen that was different from those in original transformants, progeny of class 2 and wild-type strains. However, this meiotic excision event, which took place in T1.l and T19.2 and led to an amdS-uapA- phenotype, is different from the mitotic excision event which yielded plasmid pAN501. From the Southern blot analysis presented in Figures 1 and 3 (band pattern, size and intensity) and the structure of the rescued plasmid pAN50 1, we propose that the integration-excision events which led to the isolation of plasmid pAN501 are those shown in Figure 4.

An A. nidulans X EMBL4 gene library was probed with pAMD21 and pAN501 and clones which hybri- dised with only pAN501 were selected. T w o such clones gave phages LAN501 and LAN502, the first completely overlapping the second. LAN50 1 carries a 15.5-kbp insert, including a 10-kbp EcoRI fragment (which hybridizes with pAN5Ol) equivalent to the one detected earlier in Southern blot analysis and which


Bg Xb

" - I I

Sm I N T E G R A T I O N

Xb E a

amdS c 1

$. E X C I S I O N

1.0 kb

Sm

I S Xb

FIGURE 4.-A possible integration-excision pathway for the inactivation of u p A by the transforming plasmid pAMD2 1 and the subsequent generation of rescued plasmid pAN50 1. The white bar indicates the uaPA region, the black bar indicates amdS sequences, and lines indicate pUC9 sequences. Excision points are shown with arrows (Bg = BglII, Xb = XbaI, S = SalI , E = EcoRI, C = ClaI, Sm = SmaI).

should correspond to intact uapA sequences (results not shown). Figure 5 shows the relationship of the pAN501 and LAN501 physical maps.

LAN501 was used in co-transformation experiments with pAMD2l to transform an amdS-uapA- double mutant (amdS368 amdl18 amdA7 niiA4 uupA24 biAl pantoB100). Among 40 umdS+ transformants selected, 10 were also uapA+, suggesting that LAN501 carries a functional uupA gene.

The 4.2- and 3.5-kbp BglII-Sal1 restriction fragments, contained in the I0-kbp EcoRI fragment of LAN501, were subcloned into the bluescript (KSf) M13 vector, and the new plasmids were named pAN502 and pAN503 respectively. In analogous co- transformation experiments with pAMD21 and an amdS-uupA- double mutant, only pAN503 gave transformants with a uapA+ phenotype, while no such transformants were found with pAN502. The high frequency of co-transformation with pAN503 (30 uapA+ out of 40 amdS+ transformants) suggested that a complete functional uapA gene is included in the 3.6-kbp BglII-Sal1 fragment of this plasmid (Figure 5). This was subsequently confirmed by Northern blot analysis (see below).

Characterization of the uap-100 mutation: We have available four cis-acting mutations affecting the

expression of uapA. The best studied, uup-100, was discussed in the Introduction. Three others, uap-302, uap-310 and uap-349 (D. GORTON and C. SCAZZOC- CHIO, unpublished results), have no phenotype except that they result in a bypass of the areA102 mutation. Southern blot analysis of genomic DNA from all four mutants, probed with pAN503, revealed that only uap-ZOO results in a detectable change in the restriction pattern (Figure 6). This change corresponded to an approximately 200-bp insertion or duplication in- ternal to the 1.8-kbp BgZII-PstI fragment (Figure 5).

Genomic DNA from uap-100 was digested with EcoRI, ligated to a X EMBL4 vector and packaged in vitro. A collection of 4000 clones from the uap-100 library was screened with pAN503 and hybridizing clones were isolated. One of them, LAN5 10, was used to further characterise the uap-ZOO mutation. Restric- tion analysis of a uap-100 subclone including a 3.6- kbp BgZII-Sal1 fragment indicated that this mutation is an approximately 150-nt direct duplication including an NheI site (Figure 5).

The BglII-PstI region, including the uap-100 mutation, was subcloned into M 13 vectors and sequenced in parallel with the same region from a wild-type strain. The results showed that uap-100 is a 139-bp direct duplication in the putative cis-acting region of


uapl00 wt uapl00 wt

3.6 -3.4

21) - 1.8 - 1.2 -

\

\

; y P P m n

1 . 1 I I /

'S I

uapl00 ,' *, / pAN503 , . , . , . U a P a

1 k b

~ T T ~ T T G ~

FIGURE 5.-Physical map of A. nidulans sequences in vectors pAN501, LAN501 and pAN503. pAN501 includes a complete functional amdS gene but does not have uapA transforming activity. The black bar indicates the amdS coding region while the white bar shows rescued sequences from the uapA locus. LAN501, selected from an A. nidulans library using pAN501 as a probe, contains a functional uapA gene. The white bar indicates sequences of the uapA region. pAN503. constructed by cloning the BgllI-Sal1 fragment from LAN501 into bluescript (ks+) M13 vector (striped bar), includes a complete uapA gene and its flanking sequences (white bar). The proposed borders of the uapA transcript are shown. The region in the cis-acting area of uapA duplicated in the uap-100 mutation (black bar) and the proposed sequence (CTTTTTTTGG) involved in the uapA/amdS integration-excision event are also shown (Sm = SmaI, Bg = BgllI, Xb = Xbal, S = SalI, E = EcoRI, C = Clal , B = BamHI, N = h'hel, H = Hincl l ) .

uapA, 11 5 bp upstream from the start of translation (Figure 7). No other change was found in the DNA of this region. The complete uapA sequence will be published separately.

To confirm that this duplication of 139 bp causes the uap-100 phenotype, we transformed an areAI02 strain fwAI areAIO2 pyroA4, which cannot grow on uric acid as sole nitrogen source (ARST and SCAZZOC- CHI0 1975) with 5 fig of M13 single-stranded subclones including the BglII-PstI region, using both wild- type and uap-100 DNA, and selected for growth on uric acid as sole nitrogen source. Only the uap-100 subclone gave transformants able to grow on uric acid. Four such transformants were further tested on different nitrogen sources and shown to have an areA102 uap-100 phenotype (ARST and SCAZZOCCHIO 1975). Southern blot analysis of DNA from these transformants, probed with pAN503, showed that all are the result of sequence replacement events, i e . , all showed the uap-I00 duplication when digested with BglII and PstI (results not shown). This result is in agreement with results obtained from single-stranded transfor- mations and subsequent gene replacement events using other cis-acting regulatory mutations (V. So- PHIANOPOULOU, G . DIALLINAS and C. SCAZZOCCHIO, unpublished data). The technique of sequence replacement by single-stranded transformation follows

Bg-P Bg-S FIGURE 6.-Southern blot analysis of uap-IO0. DNA (5.0 pg)

from strains biAI and uap-100 biAI were doubly digested with Bglll/Pstl or Bglll/Sall, fractionated on an 0.8% agarose gel, transferred, and probed with nick-translated pAN503. Molecular weight markers are indicated in kbp.

the methodology of GoYoN and FAUCERON (1 988) for the fungus Ascobulus immersus.

The uupA transcript in wild-type and uup-100 strains: Northern and dot blot analyses of poly(A)+ RNA from wild-type and uap-100 strains are shown in Figure 8. A uapA-specific transcript of approximately 2.1 kbp is transcribed from BglII to PstI (see Figure 5). This direction of transcription is consistent with the uap-I00 mutation having occurred 5' to the gene. The mRNAs present in the wild-type and uap- 100 strains have identical sizes, within the limits of the Northern blot analyses. The 5' ends lie for both mRNA species within the 1.8-kbp BglII-PstI region. Both transcripts end before the XbaI-Sal1 0.8-kbp region (data not shown).

The relative amounts of the uapA transcript in wild- type and uap-100 strains grown under inducing and non-inducing conditions were estimated by dot-blot densitometry and normalized after hybridization of the same membranes with plasmid pSF5 containing the actin gene (Figure 8C). uapA is 2.5-fold inducible by the gratuitous inducer 2-thiouric acid (27 PM). The uap-100 mutation results in a 5-fold increase of the uapA transcript in mycelia grown under noninducing conditions and in an 8-fold increase in mycelia grown under inducing conditions. Thus, the uap-I00 mutation increases both the basal level of uapA expression, resulting in an apparent constitutivity, and the net inducibility of the gene. These results are also in agreement with those found earlier in uptake studies (ARST and SCAZZOCCHIO 1975; SCAZZOCCHIO and ARST 1978).

DISCUSSION

The results presented here describe the inactivational cloning and initial characterization of the uric acid-xanthine permease gene (uapA) in A. nidulans.


110 120 130 160 150 160 170 180 190 T C C C T T T G T G G C C A T T A T A G C G T T A T G C T G G A G T A C G A T G A A A G A A A A C T A C A G A C G A T G A G T C G C G G A C C A T C A A A G A T A G A A A A C A T T C T G G T C

210 220 230 260 250 260 270 280 290 AGCATAGCTA GCTCAGCTCG AGATCAAGGA CTATCCACCC TGTATGGTAG ACACTAGGTG CTAGCCTCGG CCCGGCAGAC CCTAAGCACC CAAAGC

310 320 330 360 350 360 370 380 390 GGGCCGCTAC ATGGGTAGCC ATGGTGGTCT T T A G A A C A T A GACGAAGGCA AGTTAGACGC A T A T T A T A T C T A T T C C C C G T TTCGAGGAAC AGCATT

610 430 &LO 650 660 670 680 690 AATGGAGACG ACCTGCCGGG GAAGAAGGTC CTCCAGACGA GTTTTGGATG TTTTCTCATC CTCCGCACCC GCTGTCTGAA CGTCGGAGGC CGCCGA

A A C C A A G G C C G T C C A A G C C A C G C T A G C T T G A C A G T T T G C A T T A C T C G A T C G C G T T T C T T T A C C G G T T C A A C T G T G G C T G A C G C C A C G C T A A T C G T C 510 5?0 530 5LO 550 t 560 570 530 590

610 620 630 660 650 660 670 080 690 CGAAGCGCTT A T C T C T C C G C T C C e A G C T C C A T T T C C C A T A TCTTGGACCC A T C T T G A C C C A T C T T C C A C C A T G G T T A T C T T T C T C T T G A A A C T C A G

710 720 730 760 750 760 770 780 790 AAAAGCGACT CGCGATGCCA GCATGATAGG GCCGGCCTGG ACCATTCACC AGCACCAGCA GTCGGCCCTG A T C C C T A T T C G A G T G A C G A C A G C A T G

810 azo 830 8 L O 850 160 870 380 890 A C T C C A T C C A TTCAACCGAC GGCCCCGACT CCGTCATCCC CAACTCCAAC CCCAAGAABA CCGTCCGACA G A G A G T C C G C T T G C T G G C T A G G C A T C

- GACCCGCGAG

910 GGCCTGATCC

920 930 A C G G C G C C G C C T T T T T T T G G

960 cc TCAACGAG

950 AAGATTCCCG

910 TGCTGTTGGC

970 G T T T A T C C T G

980 G G T C T T C A G C

990 ATGCGC

1010 1020 1030 1060 1050 1060 1070 1080 1090 CATCTTGGCG GGTGTGGTCA C T C C T C C T C T G A T C A T A T C A AGCTCGCTGA GCCTGCCGTC C G A T C T C C A G C A G T A T C T C G T T T C G A C C T C G C T T A T

1110 1120 1130 1140 1150 1160 1170 1180 I190 TGCGGGCTGC TGTCGATGGT CCAGATAACG CGATTTCATA TCTACAAGAC ACCGTAAGTT AGCTTCATGT CGGAGTGGCT AGCCATAAGA ATCAAG

These results show that heterologous integration is not random in A. nidulans. The 2-thioxanthine technique could have detected integration events at nine well characterized genes. Not only does the amdS gene integrate into just one of these loci (uapA), but it also seems to integrate in a very specific manner. All 2- thioxanthine-resistant transformants were nearly identical and all rescued plasmids and meiotic excision events (T1.l and T19.2) were, within the limits of restriction and Southern blot analyses, identical. The integration-excision events between the uapA and amdS sequences seem to be the result of a specific recombination pathway. In fact, we know that the sequence CTTTTTTTGG is present 3' to the amdS stop codon (CORRICK, TWOMEY and HYNES 1987) and within the uapA open reading frame (position +260 from the ATG) (Figures 5 and 7). This sequence is between the CZaI site within the amdS open reading frame and the XbaI site distal to the termination codon (Figures 4 and 5). The CZaT site is conserved and the XbaI site is lost in the plasmid pAN5O 1. Thus, this sequence might be involved in the integration and excision events. Sequencing through the borders of the amdS/uapA integration (T19.3) and excision (pAN501) products would allow the detailed characterization of this event.

The above observation agrees with previous results (TILBURN 1988) which suggested that transformants comprised distinct classes with respect to the locations of heterologous integration events. Thus, it is not always possible to use selective techniques to isolate specific inactivation events. However, we were partic- ularly fortunate in obtaining integration events in the uapA gene. Because uapA mutants are leaky, due to the presence of at least one other permease able to incorporate uric acid into the cell (ARST and SCAZZOC- CHIO 1975), uapA cloning by direct complementation would have been extremely laborious. We have ob-

FIGURE 7.-Nucleotide sequence of the uapA 5' upstream region and part of the uapA open reading frame. The start codon is indicated. The region duplicated in uap-100 (419- 557) is underlined and its borders are indicated with arrows. The possible sequence involved in uapA inactivation is also underlined (932-941).

served only one other case of heterologous integration into a known gene: the inactivation of the W A gene at very low frequency on several independent occasions using different transforming plasmids. However, integration events at W A do not show the uniformity found in amdS/uapA heterologous recombination. These results will be reported elsewhere (J. TILBURN and C. SCAZZOCCHIO, unpublished results). It is possible that integration into W A depends on special fea- ture of the target sequence, at the level of primary or secondary structure, rather than on limited sequence identity.

The excised sequences from appropriate transformants allowed us to obtain a genomic uapA clone. This in turn was used to characterize a cis-acting mutation with an interesting phenotype (uap-100). The most striking characteristic of this mutation is that, while maintaining a strict dependence on the uaY and areA gene products, it increases the level of the uapA transcript 8-fold. This increase is greater than that found with uptake studies (ARST and SCAZZOCCHIO 1975; SCAZZOCCHIO and ARST 1978). This can most easily be explained by assuming a limited capacity to trans- late uapA mRNA, which may or may not be linked to a limited capacity to incorporate uupA permease molecules in the membrane. The other interesting fea- tures of uap-100 are that it bypasses the need for induction and leads to uapA expression in an areAIO2 background. How does a 139-bp duplication result in this phenotype? It is possible that the uap-IO0 duplication includes the areA or the uaY binding sites or both. A duplication of the areA binding site could account for the suppression of the areA102 phenotype. The phenotype of this and other areA mutations im- plies that the binding sites for areA are not unique, but that different sites respond differentially to mod- ified areA gene products (ARST and COVE 1973; ARST and SCAZZOCCHIO 1975; AL-TAHO, SEALY-LEWIS and

T h e uapA Permease of A. nidulans 349

WT uap-700 WT uap-100

NI I NI I NI I NI I

a- 21kb

1

FIGURE 8.-Northern and dot blot analyses of the uapA transcript in a wild-type and a uap-100 background. Poly(A)+ RNA was extracted in every case from 1 mg of total RNA as described in MATERIALS AND METHODS. The whole preparation of poly(A)+ RNA was loaded onto a 1 % agarose gel. The yield from different extrac- tions was not uniform and varied between 2 (for WT, NI and 1) and 10 ag (for uap-100, NI and I). After fractionation, the mRNAs were transferred onto nitrocellulose which was hybridized with a uapA-specific M 13 single-stranded probe (containing the Bglll-PstI 1.8-kbp fragment shown in Figure 5) as described in MATERIALS AND METHODS. (A) shows a 6-hr exposure and (B) a 60-hr exposure of the autoradiogram at -80". The size of the uapA-specific mRNA is indicated. The membrane was then dehybridized and probed with plasmid pSF5 which detects the actin mRNA. The actin controls reflected the differences in loading (results not shown). Taking into account these differences, the uap-100 mutation results in a -5-fold increase of noninduced uapA mRNA and an -8-fold increase of the induced uapA mRNA. A more accurate estimate was obtained by dot blots shown in (C). The mRNAs were loaded in the same order as in Figure 8B. The densitometry readings, normalized against the actin concentrations and giving an arbitrary value of 1 to the WT NI, were 2.5, 5 and 20 for WT 1, uap-100 NI and uap-IO0 I , respectively. W T indicates the wild-type strain (biA1); uap-100 indicates an isogenic strain carrying this mutation (biAI uap-100). N I indicates mycelium grown under non-inducing conditions and 1 indicates mycelium grown under inducing conditions (see MATERIALS AND METHODS).

SCAZZOCCHIO 1984). If the areA102 mutation results in a drastic loss of binding specific for the uapA- upstream areA binding site, a duplication of this site might restore efficient binding through by establish- ing a co-operative effect. A similar cooperative effect might explain why strains carrying the uap-100 mutation strongly express the uapA gene in the absence of induction while still requiring the presence of the uaY gene product. In other words, the uap-100 dupli-

cation might increase the affinity of the uaY binding site for the unliganded form of the uaY gene product, as already proposed by SCAZZOCCHIO and ARST (1 978).

We thank G. FAUGERON for communicating the sequence replacement procedure prior to publication, VICKY GAVRIAS for crit- ically reading the manuscript and colleagues who provided plasmids and gene libraries (see MATERIALS AND METHODS). This work was supported by the C.N.R.S. G.D. was supported by an E.E.C. grant ("Action").

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Communicating editor: R. L. METZENBERG

a gene coding for the uric acid-xanthine permease of aspergillus

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