Copyright D 1986 by the Genetics Society of America

GENETICAL AND MOLECULAR ANALYSES OF QA-2 TRANSFORMANTS IN NEUROSPORA CRASSA

MARY E. CASE

Department of Genetics, University of Georgia, Athens, Georgia 30602

Manuscript received October 3 1, 1985 Revised copy accepted March 28, 1986

ABSTRACT Neurospora crassa ga-2+ transformants from five different donor DNA clones

were analyzed by genetical and molecular techniques. None of the 32 transform- ants have the qa-Z+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty- one transformants have the p - 2 + inserts a t sites not linked, or not closely linked, to the ga-2 locus in LG VII. Plasmid sequences were integrated along with the 9a-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the ga-2+ gene product) was induced at 5-100% of the wild-type-induced enzyme activity, with 24 transformants in the 5 4 0 % range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N . crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-Z+ gene was changed to qa-z-, were observed in tetrads from intercrosses between unlinked duplication-type trans- formants and in selfings of such transformants.

N Neurospora crassa the development of an efficient transformation system I for the qa-2' gene has permitted a genetic and molecular study of trans- formation events involving this gene (CASE et al. 1979; SCHWEIZER et al. 1981). On the basis of genetic analyses and DNA-DNA hybridization studies, three types of qa-2+ transformants have been detected: (1) replacement of the qa-2- gene by the qa-2+ gene, (2) linked insertion of the qa-2+ gene in the qa-2- gene region or (3) unlinked duplication in which the qa-2+ gene is inserted unlinked or very loosely linked to the qa-2 locus and met-7 in the N . crassa genome (CASE et al. 1979). In these initial studies, about 50% of the trans- formants were replacement types, and 50% were unlinked duplication types (CASE 1983). Very few linked insertion-type transformants have been re- covered. The high frequency of unlinked duplication-type transformants from qa-2+ transformants in N. crassa was unlike the results obtained in yeast (HIN- NEN, HICKS and FINK 1978) or Aspergillus (YELTON, HAMER and TIMBERLAKE 1984). In the N. crassa studies, the expression of the qa-2+ gene, whether inserted as a replacement type or unlinked duplication type, was essentially equivalent to wild type as measured by levels of inducible catabolic dehydro- quinase activity (CASE et al. 1979). The present studies involve analyses of qa-

Genetics 113: 569-587 July, 1986.

570 M. E. CASE

2+ transformants obtained from five different plasmid types used as donor DNA. These plasmids contain qa-2+ gene fragments varying in size from 1.2 to 7.2 kb. Analyses of these transformants genetically and at the molecular level have provided an opportunity to characterize the integration events and to determine the expression of the qa-2+ gene.

MATERIALS AND METHODS

Strains: Two different strains were used as recipients in the transformation experi- ments, 246-8960 1-2A (qa-2; aro-9; id) inositol-requiring and 239 (qa-2; aro-9). These qa-2 strains carry a linkage group VI1 chromosome derived from 74-OR8-la. These mutant strains lack both biosynthetic dehydroquinase (encoded by the aro-9+ gene) and catabolic dehydroquinase (encoded by the qa-2+ gene) activities and are unable to grow on a minimal medium without an aromatic amino acid supplement (CASE, HAUTALA and GILES 1977). All transformants were crossed either to a methionine-requiring albino strain, met-7 (4894); al-2 (1 5300) (Fungal Genetics Stock Center, Department of Micro- biology, University of Kansas Medical School, Kansas City) or to a pantothenic acid- requiring quinic acid-nonutilizing strain, 246-36-1 2a (qa-2; pan-2) to obtain homokar- yotic isolates. Wild type 74-OR23-1A was used as the control strain for induced cata- bolic dehydroquinase activity.

Genetic techniques: Most of the N. crassa transforniation experiments were per- formed as previously described (CASE et al. 1979; CASE 1982). In addition, five trans- formants from plasmid pRC57 obtained by the lithium acetate procedure (DHAWALE, PA~ETTA and MARZLUFF 1984) were analyzed. Crossing techniques, media composition, ascospore plating procedures and procedures for assaying catabolic dehydroquinase ac- tivity have been previously described (CASE, HAUTALA and GILES 1977). In random ascospore platings of crosses of transformants to al-2; met-7 or to 246-36-12a, ascospores were plated on an appropriately supplemented medium ( i . e . , aromatic amino acids, methionine, inositol and pantothenic acid). Individual colonies were isolated to a sup- plemented medium and, subsequently, were tested for their ability to grow on quinic acid as carbon source, as well as for their biochemical requirements to determine the phenotype of each isolate. E. coli transformation experiments were done according to DAGCERT and ERLICH (1979).

Construction of plasmids: T h e origin of the qa-2' DNA varies with the different plasmid constructs. pVK88 contains one of the original qa-2+ clones, a PstI N. crassa fragment cloned into pBR322 (ALTON et al. 1978). The qa-2+ N. crassa DNA in pVK88 is from a constitutive revertant of a qa-IS mutant 105 that carries a wild-type 74-OR8- l a linkage group VI1 chromosome. The qa-2* N. crassa DNA in the following plasmids is from wild type 74-OR23-1A. pRC57 is a Hind111 subclone of pMSK331 (SCHWEIZER et al. 1981) cloned into pBR322; pMSK338 is a BamHI subclone of pMSK331 cloned into pBR325; pMECl is a BglII-BamHI subclone of pMSK338 cloned into pBR322; pMSK374 is a single-copy PstI subclone of pMSK331 cloned into pBR325; and pF33 is a HindIII-XhoI N. c ras~a fragment cloned into pBR325 (GEEVER et al. 1983).

DNA preparation: N. crassa DNA was prepared according to BLIN and STAFFORD (1 976) o r by the procedure described for Aspergallus nidulans (YELTON, HAMER and TIMBERLAKE 1984). DNA from ordered tetrad isolates was prepared from only one of each spore pair, usually isolates 1 , 4, 5 and 7. Plasmid DNA used for the N. crassa transformation experiments was prepared by the method of ISH-HOROWEIZ and BURKE (1981).

DNA-DNA hybridization: Transfer of N. crassa DNA from agarose gels to nitrocel- lulose was as described by SOUTHERN (1975). Nick-translation were as described by RICBY et al. (1977). Either the Hind111 restriction fragment from pRC57 or the BglII- BamHl fragment from pMSK338 was purified from agarose gels and used as the 32P- labeled qa-2+ probe for hybridization on Southerns. Restriction enzymes were purchased from Bethesda Research Laboratories and were used as directed.

qU-2' TRANSFORMANTS 57 1

TABLE 1

Characteristics of plasmids containing a qa-2+ gene insert used for transformation of qa-2- amm-9- recipient strains

~~~ ~

Length of sequences qa-2+ flanking insert the qa-2+ gene" qa-2+ size in qa-2+ transform-

plasmid restriction No. of No. of ants/ Plasmid (kb) fragment base pairs 5' base pairs 3' pg DNA

P M E C ~ ~ 1.2 Bgl II-Bam HI 556 187 78 pRB57 3.2 HindIII 1410 1165 80 pMSK338 3.4 BamHI 2508 187 350 pF33 1.6 HindlII-XhoIc 465 751 346 pVK88 7.2 Pst I 2145 4568 100 pMSK374 7.2 Pst I 2145 4568 100

a The qa-2+ coding region is 52 1 bp. The E. coli pBR vehicle in pMECI, pRC57 and pVK88 is pBR322; and in pMSK338, pF33,

and pMSK374 is pBR325. The Hind111 site is in the rearranged chromosome 5' to the qa-2+ gene. Thus, pF33 contains

465 bp 5' to the qa-2' gene up to the breakpoint, plus 450 bp from the rearranged chromosome between the 465 breakpoint and the Hind111 site. Only the 465 bp are considered flanking sequences with homology to the qa-2+ gene region.

B P H 89 go-2 B x H a g P X I I , I- + f ' I-' -

:g pMEC-I 7 H pF33 '1. b-4 IKB

B c pMSK338 B

I

n pRC57 H

P 1 L pVK88 pMSK374

P

FIGURE 1.-Partial restriction map of the qa gene region of N. crassa DNA and the location of the restriction fragments containing the qa-2+ gene. Abbreviations: B, BamHI; Bg, BglII; H, HindIII; P, PstI; X, Xhol. The left XhoI site is over 4 kb to the left of the adjacent BamHI site, indicated by the breakpaint in the line. The heavier line at the left end of pF33 indicates the position of the 450-bp fragment that causes constitutive expression of the qa-2+ gene.

RESULTS

Characteristics of the donor DNA used in transformation experiments: Thirty-two qa-2+ transformants obtained from five different plasmid types were analyzed in these studies. The characteristics of the N. crassa restriction frag- ments carrying the qa-2+ gene in the five different plasmid types used as donor DNAs are described in Table 1, and the locations of these fragments with respect to the qa-2+ gene are indicated in Figure 1. The qa-2+ insert size ranges from 1.2 to 7.2 kb. In addition, the number of base pairs (bp) 5' and 3' to the qa-2+ gene are given for each fragment. Certain of the fragments, such as pF33, have only 465 bp 5' to the qa-2+ coding region, whereas those fragments derived from BamHI digests have only 187 bp 3' to the qa-2+ coding region (Table 1). Other fragments have as much as 2.5 kb 5' and 4.5 kb 3' to the qa-2' coding region. The transformation frequencies vary with the

572 M. E. CASE

TABLE 2

Summary of Southern hybridization data and of the inducible dehydroquinase activities in qu-2+ transformants

~~~ ~~~

Induced catabolic dehydroquinase No. Hybfidiza- as percentage of wild type"

and name of tion Donor DNA transformants to pBR322 5-30 30-60 60-80 100

_ _ _ ~ ~ pM EC 1 -Bgl I I-BamHI 6-(BB 1-6) 6 3 2 0 l b pRC57-HZndIIl 9-(H-1-9) 8 4 0 2 3 pMSK338-BamHI 3-(B 1-3) 3 1 2 0 0 pF33-HandIII-XhoI 6-(F 1-6) 6 0 4 2 0 pVK88, pMSK374-PstI 8-(P 1-8) 5 1 2 1 4

a Catabolic dehydroquinase activity was determined as micromoles of dehydroshikimate pro- duced per minute at 37" per milligram of protein and was expressed relative to wild-type 74A levels under inducing conditions (0.3% quinic acid as carbon source for 6 hr in minimal medium).

Linked insertion type, BBI.

donor DNA, ranging from 80 transformants/pg DNA to 350 transformants/ pg DNA (Table 1). These frequencies include both integrated and abortive transformants that occur at a ratio of about 1:9 in all cases. Abortive trans- formants form small colonies that continue to grow on the original minimal medium, but fail to grow on transfer to new minimal medium.

Mitotic stability of transformants: Before all analyses, each transformant was crossed to either met-7 or the qa-2; pan-2 strain to obtain homokaryotic isolates. These homokaryotic isolates were obtained either as random ascospore isolates or as isolates from tetrads. All 32 homokaryotic transformants were tested for mitotic stability by plating conidia on an aromatic amino acid sup- plemented medium. At least 100 colonies from each transformant were isolated and tested for their ability to grow on minimal sucrose medium and on quinic acid medium as a carbon source. None of the 32 transformants segregated to give isolates having an aromatic amino acid requirement or isolates unable to grow on quinic acid as a carbon source, indicating that all homokaryotic isolates were mitotically stable.

Genetic analyses to determine linkage of transformants: Homokaryotic isolates of the 32 original transformants were crossed to a methionine-requiring mutant (met-7), which is very tightly linked to the qa gene cluster (<0.1% recombination, M. E. CASE unpublished data), in order to determine linkage to the qa gene cluster. Tetrads were isolated from 11 crosses (BB2-6, H3, H6, F5, P1, P4 and P8) representing all plasmid types except pMSK338 (BamHI fragment). (The strain numbers are listed in Table 2.) Random asco- spore platings were made from all 32 crosses. If the transforming DNA is tightly linked to the qa cluster, then all met-7+ progeny should be able to grow on quinic acid as a carbon source; whereas, if the transforming DNA is inte- grated unlinked to the qa cluster, then only 50% of the met-7' progeny should be able to grow on quinic acid. These ratios would be expected from either random ascospore platings or tetrad analyses. In all 11 crosses, tetrad analysis indicated that 50% of the met-7+ isolates grew on quinic acid (no phenotypically

q U - 2 + TRANSFORMANTS 573

aberrant isolates, as will be described later, were detected in these crosses). In random ascospore platings of the same crosses from which the tetrads were isolated, only 20-40% of the met-? isolates were able to grow on quinic acid, whereas 50% would be expected. These results indicate that some type of inviability [or conversion (to be described later)] associated with the trans- formed strain results in a deficiency of qa-2+ met-7+ isolates in the random ascospore platings.

The results of the random ascospore platings of the crosses to met-7 indicated that one transformant, BB1 from pMEC1, was linked to met-7. All other trans- formants were located at sites unlinked to the qa gene cluster within the N . crassa genome, and these transformants were classified as unlinked duplication- type transformants. The met-7 strain also carried an albino-2 allele (located in linkage group I). T w o transformants, one from pRC57 (H4) and one from pF33 (F3), showed linkage to al-2.

Southern hybridization studies: Genomic DNA from each of the 32 trans- formants was digested with XhoI or with BglII and hybridized to two different probes: (1) pBR322 to detect the presence of plasmid sequences and (2) the qa-2+ HirldIII probe to determine the number of copies of the qa-2 gene. Representative data from six unlinked duplication-type transformants showing hybridization to the qa-2+ HindIII probe are shown in Figure 2. In Southern- blots of XhoI-digested genomic DNA, hybridization with the qa-2+ HindIII probe normally revealed two bands at 8.2 and 4.6 kb. In blots of BglII-digested genomic DNA, hybridization with the qa-2+ HindIII probe normally reveals two bands at 6.7 and 2.2 kb. The qa-2+ gene is associated with the 8.2-kb band in the XhoI digests and with the 2.2-kb band in the BglII digest. The 4.6-kb band in the XhoI digest is to the right of the qa-2+ gene, and the 6.7- kb BglII band is to the left of the qa-2+ gene. The additional bands observed in the Southern blots would correspond to integration of the donor qa-2+ DNA at other sites within the genome (Figure 2). By comparing the Southern hybridization patterns of the XhoI and BglII digests among the six different transformants, it appears that the integration events occurred at several dif- ferent chromosomal sites within the genome. However, a comparison of the two different digests of transformants F4 and F5 in lanes 4 and 5 suggests that these two transformants may represent identical, independent insertions at the same site. (These two transformants came from separate plates as dif- ferent isolates in the same transformation experiment.) Thus, to determine the number of different integration sites for the donor DNA, additional restriction digests should be made with each transformant. There is relatively little evi- dence at this time to indicate the total number of different integration sites, since it is not easy to determine without extensive restriction mapping whether the different sites detected with one plasmid fragment are the same or different from those detected with another plasmid fragment. Although the sample was not large, five transformants (H5-H9) obtained by the lithium acetate trans- formation procedure (DHAWALE, PAIETTA and MARZLUFF 1984) appeared to be similar to those obtained using glusulase (CASE et al. 1979). No differences were observed in the types of transformants obtained by both procedures, since

574 M. E. CASE

1 2 3 4 5 6 1 2 3 4 5 6

-2.2

Xho I Bg/ I I FIGURE 2.-Southern blots o f genomic DNA from six unlinked duplication-type 90-2+ trans-

formants. Lane I , H8; lane 2, H9; lanes 3-6, F1, F4. F5 and F6. These DNAs were digested with Xhol or Sglll and were hybridized with a "P-labeled 9a-2+ Hindlll probe. The faint bands at 4.6 kb in the Xholdigested DNAs are clearly visible on the original Southern blot. In the Xhol digests, the middle band in lane 1 and the upper bands in lanes 2-6 also hybridized with "P-labeled pBR322 (data not shown).

the donor DNA integrated a t a number of different sites unlinked to the qa gene cluster in the N . trussa genome.

In Southern hybridization analyses with pBR322, only four of 32 transform- ants did-not hybridize to pBR322; namely, H5, P I , P3 and P5. Since plasmid sequences are present in most of the transformants, this evidence indicates that the pBR322 sequence and the qa-2+ gene sequence were inserted into the genomic DNA during the same integration event. In addition, restriction di- gests of genomic DNA from three transformants, BBI, BB2 and BB6, indi- cated that two tandem copies of the donor plasmid DNA were inserted. One of these transformants, BBI, is a linked insertion type with a tandem dupli- cation of the plasmid DNA. Restriction digests of BBI (Figure 3) indicated that the normal 8.2-kb band present in XhoI digests is missing and is replaced by a much larger band that is greater ,than 15 kb (lanes 1 and 2). Further characteristics of this transformant will be discussed later.

Additional restriction digests of genomic DNA from two unlinked duplica- tion-type transformants, BB3 and BB4 containing a single copy of pMECI, indicated that the qa-2+ gene was intact in both of these transformants and that integration into the N . crussu genome apparently occurred by a recombi- nation event between the pBR322 sequences and the N. trussa genome. How- ever, the location of the recombination events within pBR322 in these two transformants was not identical. To determine the region of integration on the plasmid, double digests with BamHI-PvuII or with BamH1-PstI were done on BB3 and BB4 (see diagram of plasmid pMECl in Figure 4 for the expected size fragments following the double digests). If no recombination occurred within a given region on the plasmid, then a normal-sized band hybridizing to pBR322 or to the qa-2+ probe would be expected. If recombination did occur within a given region, then the expected normal-sized band would be replaced by one or more bands of differing size. In BB4 the size of the BamHI-PvuII

ga-2+ TRANSFORMANTS 575

b

-8.2

- - -4.6 W

I 2 3 4 5 6 7 8 9

pBR322 -90-2+ probe * FIGURE 3.-Southern blots o f genomic D N A from BBI, 6.5 (lanes I , 2 and 4); wild type 74A

(lanes 3 and 5); and tetrad isolated I , 4. 5 and 7 from a cross of 6.5 X mct-7. The genomic DNA in the first lane was hybridized to "P-labeled pBR322. The genomic D N A in all other lanes was hybridized with a "P-labeled ga-Z+ Hindlll probe. These DNAs were digested with XhoI (lanes 1-3, 6-9) or Hindlll (lanes 4 and 5). On Southern blots, the banding patterns of wild type 74A and the recipient strain 246-89601-2A are identical with the restriction enzymes used in these studies (data not shown).

fragment hybridizing only to pBR322 should be a 1.7-kb band, and this band is present in Figure 4, lane 6. In BB4 the size to the BamHI-PstI fragment hybridizing td the qa-2+ probe should be 2.4, and this band is present in Figure 4, lane 4. In BB4, the size of the BamHI-PvuII fragment hybridizing to pBR322 or to the ga-2+ probe should be 3.9. This band is missing, and a 5.4- kb band was observed (Figure 4, lanes 1 and 6). These data, taken together, indicate that the recombination event in BB4 occurred between the PstI and PvuII sites on pBR322. (These results have been confirmed, since this p-2+ transformant has been cloned and sequenced, M. E. CASE and R. F. GEEVER. unpublished data.) In BB3 a normal 3.9-kb band is observed in the BamHI- PvuII restriction digests hybridizing to the p - 2 + probe (Figure 4, lane 3). In BB3 the size of the BamH1-PvuII fragment hybridizing only to pBR322 was expected to be a 1.7-kb band. This band is missing and is replaced by two larger bands at 5.0 and 9.8 kb (Figure 4, lane 8). These data indicate that the recombination event in BB3 occurred between the BamHl and PvuII sites on pBR322.

qa-2+ gene expression in transformants: The p-2+ gene encodes an induc- ible enzyme, catabolic dehydroquinase (CASE, HAUTALA and GILB 1977). Thus, it appeared of considerable interest to examine what effect integration of the p - 2 + gene at various sites had on the induction of catabolic dehydro- quinase. The p-2+ gene inserts in the various plasmid donor DNAs used in the transformation experiments had differing amounts of DNA both 5' and 3' to the functional p-2+ gene (Figure 1; Table 1).

To determine the inducibility of the p-2+ gene in the various inserts, all transformants. as well as the wild-type 74A control, were grown on sucrose and transferred to quinic acid (induction conditions). The levels of induced catabolic dehydroquinase activity in transformants, summarized in Table 2. are

+

3.2-

1.4- - - 0 1 -

3.9 -2.9

!.4- M, ,

c - 3.9 L4

-1.4

I 2 3 4 5 6 7 8

8umHI 8umHI BudI 8 m H l BumHI Pvull Pst I Pvull p t t l Pvull

+q0-2+ probe- - pBR322 - B

FIGURE 4.-A. Southern blots of genomic DNA from BB3 (lanes 3 and 8); BB4 (lanes I , 4. 6 and 7); and wild type 74A (lanes 2 and 5). These DNAs were double digested with B a m H I - h I l or BamHI-fsfl. T h e genomic DNAs (in lanes 1-5) were hybridized with a '*P-labeled qa-2+ Hindlll probe and (in lanes 6-8) with "P-labeled pBR322. T h e arrows indicate the unexpected bands observed on these blots. In these double digests the qa-2+ Hindlll probe hybridizes to a 3.2-kb and a 1.4-kb fragment in the qa gene cluster in the BamHI-&I1 (lane 2) digests and to a 2.9-kb and a 1.4-kb fragment in the BamHI-fsfl digests (lane 5). B. a diagram of plasmid pMECl con- taining a Bgfll-RamHI qa-2+ insert in pBR322. This diagram indicates the restriction fragment sizes following digestion with BamHI. h l l and f s t l .

qa-2’ TRANSFORMANTS 577

given as a percentage of wild-type-induced levels. Among the transformants that lacked pBR322 sequences, one transformant, H1, had 5% of wild-type activity, whereas two other transformants, P3 and P5, had 50%, and one other transformant, P1, had 100% activity. As indicated in the table, only eight of the 32 transformants (BB1, H2, H3, H4, P1, P2, P4 and P6) had levels of enzyme activity equivalent to wild type (100% activity). One of these trans- formants, BBI, was a linked insertion type. The levels of enzyme activity in the other 24 transformants derived from pRC57 (HindIII fragment), pVK88 or pMSK374 (PstI fragment), as well as transformants derived from pMECl (BglII-BamHI fragment), pMSK338 (BamHI fragment) or pF33 (HindIII-XhoI fragment) ranged between 5 and 80% of wild type. Additional restriction digests were done on each of the transformants to determine whether there was a relationship between gene expression and the size of the functional qa- 2+ gene integrated into the genome unlinked to the qa gene cluster (data not shown). These data indicated that at least an intact BgElI-BamHI fragment was present in the transformants obtained from pMEC 1 (BglII-BamHI), pMSK338 (BamHI) and in pF33 (HindIII-XhoI); however, none of the transformants obtained from these plasmids had normal levels of enzyme activity. Three transformants obtained from pRC57 (H2, H7 and H8) and four obtained from pVK88 or pMSK374 (Pl , P2, P4 and P6) had an intact qa-2+ gene, and all of these transformants had essentially wild-type levels of enzyme activity. With the exception of the two pMECl transformants (BB3 and BB4), the precise location of the recombination site within the donor plasmid DNA in the other transformants has not been determined.

qa-2+ gene expression in pF33 transformants: Transformants obtained from pF33 are of particular interest. The qa-2’ gene in this plasmid is derived from activator-independent mutant ( q u - P i ) 158-33, which contains a chromo- somal rearrangement with one breakpoint located at position -379 in the region 5’ to the major (+1) mRNA initiation site for the qa-2+ gene (GEEVER et al. 1983). Mutant 158-33 exhibits high-level constitutive catabolic dehydro- quinase activity (ca. 45% of fully induced wild type) in the absence of an active qa-1 F+ gene product, the activator. This constitutive activity is presumably due to an “enhancer-like” element introduced by the rearrangement from else- where in the N. crassa genome and located 5‘ to the -379 breakpoint. Thus, this rearrangement promotes qa-2 transcription from a considerable distance upstream to the major (+1) mRNA initiation site. The p - 2 + region from 158- 33 was cloned as a HindIII-XhoI fragment into pBR325. This plasmid, pF33, contains the 9a-Y coding region, the normal sequences 5’ to qa-2+ up to the -465 translocation breakpoint, and a 450-bp sequence derived from the region connecting the -379 breakpoint and the HindIII site in the rearranged seg- ment. The rearrangement is the result of a reciprocal recombination event, either an inversion or translocation (GEEVER et al. 1986).

When DNA from pF33 was used to transform the qa-2- strain, the resulting unlinked duplication-type transformants were found to have constitutive levels of catabolic dehydroquinase activity between 20 and 40% of fully induced wild type when they were grown on sucrose medium (noninducing conditions).

578 M. E. CASE

These constitutive levels are comparable to the constitutive levels observed in the original 158-33 q ~ - 2 " ~ mutant. Thus, the 450-bp segment from the qa-2"' mutant 158-33 inserted into pF33 apparently contains all the sequences re- quired for high-level constitutive expression of the qa-2+ gene, whether these sequences are located 5' to the qa-2+ gene in the qa gene cluster or are 5' to a qa-2+ gene inserted unlinked to the qa gene cluster. In addition, the pF33 unlinked duplication-type transformants are induced on quinic acid to levels as high as 80% of the wild-type-induced enzyme activity (Table 2), indicating that expression of the qa-2+ gene is still regulated by the activator product of the qa- lF+ gene in the qa gene cluster (GEEVER et al. 1983).

Tetrad analysis of linked insertion-type transformant: Southern hybridi- zation analyses with the pMEC 1 transformant BB 1 BgZIII-BamHI (tetrad isolate 6.5) indicated that this transformant was the result of an insertion into the qa- 2- gene region. The XhoI restriction pattern of DNA from isolate 6.5 hybrid- ized with pBR322 or with the qa-2+ probe is shown in Figure 3 (lanes 1 and 2). The normal 8.2-kb band present in strain 74A (lane 3) is missing in isolate 6.5 (lanes 1 and 2), and a band greater than 15 kb is observed. The greater than 15-kb band is compatible with a tandem repeat of pMECl inserted into the XhoI 8.2-kb fragment. In addition, three bands are observed in the HindIII digest of isolate 6.5 (Figure 3, lane 4). None of these bands correspond to the 3.2-kb band in the 74A HindIII digest (lane 5). These data indicate that the integration of pMECl into the A? crassa genome in this transformant has altered the restriction pattern in the qa-2 gene region. As indicated earlier, this transformant has been classified as a linked insertion type. This homokar- yotic isolate was crossed to qa-2+ met-7-, and 18 tetrads were isolated. Only one of the 18 tetrads had four qa-2+ isolates. The other 17 tetrads all had a 2:2 ratio of qa-2+ met-7- and qa-2- met-7' progeny. All met-7' chromosomes expected to be qa-2+ were phenotypically now qa-2- isolates unable to grow on quinic acid as a carbon source, whereas all qa-2' met-7- isolates (like the qa-2+ met-7- parental strain) were able to grow on quinic acid. Enzyme assays showed that the phenotypically qa-2- met-7+ isolates had no dehydroquinase activity. Southern hybridization analyses with XhoI-digested genomic DNA iso- layed from one of each spore pair from one of these tetrads (Figure 3, lanes 6-9) and hybridized to the qa-2+ probe indicated that the XhoI qa-2 fragment in these qa-2- met-7+ isolates was unaltered from their qa-2+ parent. [Compare the restriction pattern of isolate 6.5 (lanes 1 and 2) with tetrad isolates 1 and 4 (lanes 6 and 7)]. If, during integration of the tandem duplication, recombi- nation occurred within one of the two qa-2+ genes on the plasmid and inacti- vated one qa-2+ gene, then the remaining qa-2+ gene may have been changed to qa-2- by gene conversion during meiosis.

Tetrad analysis of unlinked duplication-type transformants: Genetic anal- yses of transformants showed that the qa-2+ gene can be inserted at several different sites within the genome, unlinked or loosely linked to the qa gene cluster. Each unlinked duplication-type transformant should contain two copies of the qa-2 gene; one copy is a qa-2- gene in linkage group VII, and the other copy is a qa-2+ gene unlinked to the qa gene cluster. Crosses were made to

qa-2+ TRANSFORMANTS

TABLE 3

Segregation ratios of qa-2+ to qa-2- in tetrad isolates from three different types of crosses: (1) cross of unlinked duplication-type transformants by qa-2* me-7-, (2) cross between two different unlinked duplication-type transformants and (3)

selfing of unlinked duplication-type transformants

579

Segregation ratios of p - Z + to qa-2- isolates in tetrads

No. in each class Total no.

'of te- Cross type 2:2 4:O 0:4 3: 1 1:3 trads

Cross I P4a X met-TA 8 2 0 1 0 1 1 P1A X met-7-a 6 1 0 10 0 17

P IA X P4a 3 0 1 1 4 2 20

P IA X P l a 2 5 1 2 0 10 P7A X P7a 0 0 10 0 0 10

Cross 2

Cross 3

determine if the two qa-2+ genes in two transformants having different South- ern hybridization patterns had integrated into the same chromosome in closely adjacent sites or had integrated into nonhomologous chromosomes. If qa-2+ genes in different transformants had integrated into the same sites on homol- ogous chromosomes, all isolates from a cross of such strains should be qa-2'. If the qa-2+ inserts are unlinked to each other, then there should be a 3:l ratio of qa-2+ to qa-2- progeny. If the qa-2+ genes are linked to each other but separable, then some recombination frequency producing up to 25% qa- 2- progeny should be observed. Twenty tetrads were isolated from a cross of two unlinked transformants P1 (isolate number 5-2-1.4) X P4 (13-4-5.5) ob- tained from pVK88 (CASE et al. 1979). As determined by Southern hybridi- zation, the qa-2+ insert in P4 contains a pBR322 sequence, whereas the qa-2' insert in P1 does not. Data from this cross and from crosses of the transform- ants P1 and P4 to qa-2+ met-7- are given in Table 3. The latter data show that, phenotypically, the two transformant types segregated normally from crosses to qa-2+ met-7-; however, unexpected results were obtained in the cross of the two unlinked transformants with each other (Table 3). No tetrads were recovered in which all four products of meiosis were qa-2+. Unexpectedly, in 11 of 20 tetrads all four products of meiosis were qa-2- (a 0:4 segregation ratio of qa-2+ to qa-23.

In enzyme assays, none of the phenotypically qa-2- isolated from one 0:4 tetrad type had any dehydroquinase activity. (Phenotypically, mitotic sister spores of each tetrad member were alike and could not grow on quinic acid as the carbon source.) Genomic DNAs isolated from one of each spore pair of a 0:4 tetrad and from the two parental strains, P1 and P4, were digested with XhoI and hybridized with pBR322, as well as with a qa-2+ BglII-BamHI probe. The results of these Southern hybridization analyses are given in Figure 5.

580 M. E. CASE

1 2 3 4 5 6

-8.2

FIGURE 5.-Southem blots of genomic DNA from parents and tetrad progeny from a cross of two unlinked transformants. Tetrad isolates 1 , 4, 5 and 7 (lanes 1-4) and the parents, PIA (lane 5) and P4a (lane 6). These DNAs were digested with Xhol and hybridized to a "P-labeled Bglll- BumHl probe.

These data clearly show that two of the tetrad isolates (lanes 2 and 3) have a band at 6 kb that is identical to the band in the parental strain PI (lane 5). T w o tetrad isolates (lanes 3 and 4) contain a 15-kb band; this is larger than the 13-kb band found in the original parental strain P4 (lane 6). This 15-kb band still hybridizes to pBR322, as is characteristic of the 13-kb band in the parental strain P4. These two p - Z + gene inserts found in PI and P4 are apparently unlinked to each other, since both bands (6 kb from PI and the altered 15 kb from P4) segregated into the same tetrad member (Figure 5, lane 3). (This isolate contains three copies of the 9 ~ - Z gene, all of them now phenotypically @-). In addition, two tetrads were recovered in this cross with a 1:3 ratio of p - Z + to ga-2- isolates (Table 3). The 0:4 ratio of ga-2+ to p-2- was also observed in genetic analyses of tetrads from crosses between the transformant P 1 and three additional unlinked duplication-type transform- ants obtained from pVK88 (PstI) that had Southern restriction patterns dif- ferent from PI and from each other (data not shown).

Cenetic analysis of selfings of unlinked duplication-type transformants: Are the segregation patterns described above typical only of those crosses, or is this a phenomenon of the ~ u - Z + gene being inserted unlinked to the 9a gene cluster? Since tetrads with a 0:4 segregation ratio of p - Z + to p - 2 - were observed in crosses of unlinked duplication-type transformants, the next ques- tion was what type of segregation pattern would be observed if isolates from an unlinked duplication-type transformant were selfed? A cross was made be- tween Pla X PIA, and the data from ten tetrads from this cross are given in Table 3. If no stability of the p - Z + genotype is observed following meiosis, then all tetrad isolates should be g&+. In fact, five tetrads were obtained with all isolates ga-Z+. However, as in the previous intercrosses of the unlinked duplication-type transformants, unexpected tetrad types were observed .with the following ratios: two segregated with a 2:2 ratio of p - Z + to g d - , two segregated with a 3:l ratio of p-2+ to p 2 - , and one segregated with a 0:4 ratio of p - Z + to qa-2- (Table 3). When one of the 0:4 tetrads was assayed for catabolic dehydroquinase activity, no activity was detected in any isolate. Ge-

qU-2+ TRANSFORMANTS 58 1

I 2 3 4 5 6 7 8 9 O I I 1213

- 4.6

FIGURE 6.-Southern blots of genomic DNA from parents and tetrad progeny from selfings of two different unlinked duplication-type transformants. First selfing (Pla X PIA): Pla (lane 1); PIA (lane 2); tetrad isolates 1 , 4, 5 and 7 (lanes 3-6). Second selfing (P7A X P7a): P7a (lane 7); tetrad isolates 1 . 4. 5 and 7 (lanes 8-11); P7A (lane 12); wild type 74A (lane 13). The genomic DNAs were digested with XhoI and hybridized to a '*P-labeled g d ? + Hind111 probe. The dots represent the faint 4.6-kb bands that are visible on the original Southern blot.

nomic DNAs from two tetrads, one with a 2:2 ratio and the other with a 0:4 ratio, were digested with XhoI and were hybridized to qa-2+ Hind111 probe. Representative Southern blots from the 0:4 (all qa-2-) tetrad (lanes 3-6) and the parents (lanes 1 and 2) are shown in Figure 6. N o differences in the banding patterns were observed on the Southern analyses between the parents and the tetra-d progeny.

To determine whether these results were unique in crosses of this transfor- mant, PI , to other transformants and in selfings, additional tetrads were iso- lated from a selfing of another unlinked duplication-type transformant, P7a X P7A. The qa-2+ insert in this transformant also contains pBR322 sequences. All ten tetrads obtained in this cross had a 0:4 ratio of ga-2+ to qa-2- (Table 3). No qa-2+ isolates were recovered. Enzyme assays showed no catabolic de- hydroquinase activity in any isolate from two tetrads. Genomic DNAs from one tetrad were digested with XhoI and were hybridized with pBR322 and also to the qa-2+ Hind111 probe. In Figure 6, a comparison of the hybridization patterns of the tetrad isolates (lanes 8-1 1) to those of the two parental strains (lanes 7 and 12) indicated that some type of DNA sequence rearrangements had occurred during meiosis, possibly leading to the inactivation of the qa-2+ inserts in this tetrad. In the DNA from the tetrad isolates (in lanes 9 and IO), the 8.2-kb band and the 4.6-kb band present in wild type 74A (lane 13) are missing, and a much larger fragment of 13 kb is present. However, the 5.7- kb band observed in the two parents is still present in the DNA from these progeny (lanes 9 and 10) as well as in the DNA from the other two isolates from the tetrad (lanes 8 and 11). In addition, this 5.7-kb band still hybridizes to pBR322 (data not shown).

Random ascospore platings were done on selfings from five additional un- linked duplication-type transformants (two from pRC57, H5 and H6; one from pMSK338, B3; and two from pMSK374, P7 and P8). Although qa-2+ isolates were obtained in each of these crosses, the percentages of qa-2- isolates were 84%, 66%, 95%, 91% and 63%, respectively. Since these crosses represent

582 M. E. CASE

three other plasmid types in addition to pVK88 and pMSK374, the occurrence of qa-2- isolates in selfings of qa-2’ unlinked duplication-type transformants is not unique to any plasmid donor. It is a property of the inserted qa-2+ DNA responding to the qa gene system at meiosis.

DISCUSSION

Nature of the integration events: Genetic data indicated that the donor DNA in 31 of the 32 transformants in these studies was inserted unlinked to the qa gene cluster. In addition, genomic DNAs from all but four of the transformants contained vector (pBR322) sequences. These results differ from the first studies with pVK88 transformants, in which the qa-2+ gene had in- tegrated unlinked to the qa gene cluster in 17 of 28 transformants, and ge- nomic DNAs from 6 of the 17 unlinked duplication-type transformants carried pBR322 sequences (CASE et al. 1979; CASE 1983). In the present study, the recovery of a larger percentage of unlinked transformants containing plasmid DNA may be the result of differences in the chromosomal background of the donor DNA. pVK88 (PstI) N . crassa donor DNA and the recipient qa-2- strain linkage group VI1 chromosome originated from wild type 74-OR8-la, whereas all other qa-2+ fragments listed in Table 1 originated from wild type 74-OR23- 1A. Because of restriction site polymorphisms, restriction maps within the qa gene cluster differ in these two wild-type strains (R. F. GEEVER, unpublished results). These differences, although not large, might prevent or reduce the frequency of replacement-type transformants. In addition, 24 transformants in the present studies were derived from donor DNAs containing qa-2+ restriction fragments with differing numbers of base pairs, both 5’ and 3’ to the qa-2+ coding region, compared to pVK88. For a replacement event to take place, a larger number of base pairs may be required than is present in the pF33 insert (465 bp 5’ to the qa-2+ gene) or with the BglII-BamHI or BamHI inserts (187 bp 3’ to the qa-2+ gene). Although the sample was not large, five unlinked duplication-type transformants obtained from pRC57 (Hind111 fragment) by the lithium acetate transformation procedure (DHAWALE, PAIETTA and MAR- ZLUFF 1984) appeared to be no different from those obtained using glusulase (CASE et al. 1979; CASE 1982). The integration of the donor DNA into several sites within the N. crassa genome unlinked to the qa gene cluster occurred following both transformation procedures.

Southern hybridization analyses of transformants obtained with each plasmid type indicate that the donor DNAs can integrate into a number of different sites in the N. crassa genome. Do the different plasmid types integrate into the same sites or different sites? Limited genetic analyses indicated that two different unlinked duplication-type transformants derived from two different plasmid types were linked to a l - 2 in linkage group I; however, it is not known whether these transformants linked to a l - 2 are integrated into the same site or into different, but closely linked, sites. In addition, one isolate (no. 3, Figure 5) from a tetrad from the cross of two unlinked duplication-type transformants, PI X P4, contains bands at 6 and 15 kb representing the two different restric- tion fragments from the two qa-2+ parental strains. This suggests that the qa-

q&2+ TRANSFORMANTS 583

2+ DNAs in these two unlinked duplication-type transformants are not linked but that they segregated independently during meiosis. KINSEY and RAMBOSEK (1984) found in their study of am transformants that more than half were unlinked to the am locus in linkage group V. Genetic analyses of a limited sample of unlinked am transformants implicated three different integration sites within the N. crassa genome. Thus, the occurrence of unlinked integration of donor DNAs during transformation is not unique to qa-2+ transformants.

In N. crassa the large number of transformants from both the qa-2+ gene and the am+ gene in which integration of donor DNA occurs unlinked to the normal gene regions contrasts with the results obtained in yeast. Early evidence with yeast leu-2+ transformants indicated that three types of transformants were recovered, equivalent to the replacement type, linked insertion type and unlinked duplication type (HINNEN, HICKS and FINK 1978). The unlinked du- plication-type transformants occurred at a low frequency. Subsequent analysis of a subclone of the yeast plasmid, pYeleu-10 indicated that this fragment contained a repeated sequence, Tyl-17, which permitted integration of this sequence along with the leu-2+ gene at the dispersed copy sites of this repeated sequence within the yeast genome (KINGSMAN et al. 1981). Thus, the integra- tion of donor DNA in yeast transformation always appears to be the result of recombination between homologous sequences. As yet there is no evidence from Southern hybridization studies for repeated sequences in N. crassa that would explain the insertion of donor DNA unlinked to the normal gene region. Transformants of the same three integration types have been reported also for the trpC gene of A. nidulans (YELTON, HAMER and TIMBERLAKE 1984). In Aspergillus, the frequency of replacement types and linked insertion types is much higher than that observed for N. crassa, and a much lower frequency of unlinked duplication types occurs.

How does integration occur? It has been assumed that integration of donor DNA occurs by pairing followed by a single crossover event between homol- ogous N. crassa DNA sequences on the plasmid and the N. crassa chromosome. This event is analogous to the Campbell model, which has also been proposed as a mechanism for the integration of donor DNA by homologous recombi- nation into yeast transformants (HINNEN, HICKS and FINK 1978).

Evidence is presented here that recombination can occur between pBR322 sequences and the N. crassa genome. pMECl carries a 1.2-kb qa-2+ (BglII- BamHI fragment) inserted in pBR322. Restriction digests with several enzymes of two different transformants from pMEC1, BB3 and BB4, indicated that recombination did occur between the pBR322 sequences and the N . crassa genome. Different regions of pBR322 were implicated, one (BB4) between PstI and PvuIII sites and the other (BB3) between PuuII and BamHI sites.

Expression of the qa-2+ gene: The first transformants studied (obtained from pVK88) produced wild-type levels of catabolic dehydroquinase activity (CASE et al. 1979); however, only four transformants were analyzed in those studies. The present studies compared the levels of catabolic dehydroquinase activity in qa-2+ transformants obtained with five different types of donor DNAs, each containing qa-2+ restriction fragments of different sizes and dif-

584 M. E. CASE

fering numbers of base pairs both 5’ and 3’ to the p - 2 + gene. The levels of catabolic dehydroquinase activity ranged from 5 to loo%, with one-third in the 30-60% range. Only certain transformants obtained with the Hind111 frag- ment (pRC57) or the PstI fragment (pVK88 or pMSK374) had wild-type levels of enzyme activity. The low levels of catabolic dehydroquinase activity in these unlinked qa-2+ transformants are comparable to the low levels of glutamic dehydrogenase enzyme activity (5-20%) produced by am+ unlinked duplica- tion-type transformants (KINSEY and RAMBOSEK 1984). It is possible that N. crassa chromosomal sequences adjacent to the integration sites of the unlinked qa-2+ genes within the N. crassa genome or the pBR322 or pBR325 sequences may prevent maximum qa-2+ expression by “position effect” mechanisms sim- ilar to those suggested by KINSEY and RAMBOSEK (1984). KINSEY and RAM- BOSEK postulated that the low levels of glutamic dehydrogenase activity in the unlinked transformants might be due to the separation from the am+ gene of an “upstream promoter” or enhancer-like sequence during the integration event, or the result of the integration of the am+ gene into a transcriptionally inactive region, making the am promoter less accessible to RNA polymerase 11.

The present studies clearly demonstrate that p - 2 + gene expression can be influenced by sequences adjacent to the 9u-P gene insert within the N. crassa genome. As noted with pF33 transformants, certain sequences adjacent to the qa-2+ gene can affect the expression of the p - 2 + gene. A 450-bp fragment, 5’ to the breakpoint in the original 158-33 qa-2”’ mutant (GEEVER et al. 1983) and now contained in pF33, acts like an enhancer sequence in these unlinked duplication-type transformants. Thus, this 450-bp fragment still confers high- level constitutive expression of catabolic dehydroquinase enzyme activity for the 9a-Y genes in the absence of the inducer quinic acid. In addition, evidence has been reported that transforming DNA in N. crassa is methylated (BULL and WOOTTON 1984); however, what effect methylation of donor DNA in transformants would have on gene expression is not known.

Meiotic behavior of transformants: Linkage between qa-2’ insertion sites in different transformants cannot be determined by crossing two transformants and analyzing random ascospore progeny. Tetrad analyses of two different types of crosses (crosses of two unlinked duplication-type transformants with each other and selfings of unlinked duplication-type transformants) indicated that, after meiosis, a high frequency of progeny expected to be qa-2+ were now qa-2-, with or without visible changes in Southern hybridization patterns. The frequent loss of the p - 2 + phenotype in these two types of crosses of unlinked duplication-type transformants was totally unexpected.

Similar meiotic behavior was observed in crosses between the linked inser- tion-type transformant BBI and met-7. Genetic analyses of tetrads from a cross of one of the p - 2 + isolates from BB1 to met-7- showed that the 9a-P phe- notype changed to ga-2- at a high frequency after meiosis.

These gene conversion-like events observed in tetrads of unlinked duplica- tion-type and linked insertion-type transformants are not seen in the first cross of a transformant to met-7 to obtain homokaryotic isolates. The tetrad isolates

qU-2’ TRANSFORMANTS 585

in the first generation have phenotypically normal segregation patterns. These gene conversion-like events may be detected following meiosis in the next crosses of a transformant to met-7 or in selfings.

The frequency with which the qa-2+ phenotype is changed to qa-2- following meiosis is much higher than expected for conventional gene conversion events. Classical gene conversion in N. crassa occurs at a frequency of <1% in tetrad analyses of pan-2 alleles (CASE and GILES 1964). In the tetrad analyses of pan- 2 alleles, gene conversion could occur in both directions, i .e. , pan-2+ to pan- 2- or pan-2- to pan-2+. Unlike classical gene conversion, the changes described here are unidirectional, because no tetrads were observed in which the qa-2- gene was changed to qa-2+. However, high frequencies of gene conversion-like events are observed for the MAT genes in yeast (KLAR, FOGEL and LUSNAK 1979). These gene conversion events with the MAT genes occur during mitosis under the control of the HO gene, whereas classical gene conversion events normally occur during meiosis in yeast and N. crassa. There is no evidence that there are any other genes controlling the observed changes in the qa-2 phenotype in N. crassa.

One way to determine whether these changes are due to gene conversion would be to analyze transformants in which the restriction pattern of the donor DNA differed from the restriction pattern of the recipient strain. If the qa-2+ gene has been changed to qa-2- by gene conversion during meiosis, then the newly derived qa-2- strain would have the same restriction pattern as the recipient qa-2- strain. The best way to determine these possible differences would be to clone both qa-2- genes from such a transformant so that each qa- 2- gene could be analyzed independently. This type of experiment has not been done as yet. If no restriction-site differences were observed, then one should consider other possible differences at the nucleotide level. If, instead of gene conversion, a deletion or a small rearrangement in the qa-2’ nucleotide sequences has produced a qa-2- phenotype, then sequencing the cloned DNA would be required to identify the precise molecular nature of the event. Al- though these changes clearly have similarities to gene conversions, one must also consider that the alterations in size of the qa-2 sequences observed on Southern blots of tetrad isolates following meiosis may be involved in altering a qa-2+ gene to qa-2-. Whatever the mechanism, there appears to be a “cor- rection process” acting during meiosis that preferentially changes the number of functional copies of the qa-2+ gene within the genome. Whether these frequent meiotic changes are unique to the qa gene cluster or are general properties of transformants in N. crassa remains to be determined by further molecular and genetic analyses of other transformants from additional cloned N. crassa genes.

The author wishes to thank NORMAN H. GILES and ROBERT F. GEEVER for helpful discussions and for critical reading of the manuscript; PAULETTE GEEVER, ALIX WEAVER and ASHA WISE for excellent technical assistance; and COLLEEN MCELFRESH for drawing the diagrams and for typing the manuscript. This work was supported by United States Public Health Service grant GM28777 from the National Institutes of Health.

586 M . E. CASE

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Communicating editor: J. E. BOYNTON