The Neurospora Aab-1 Gene Encodes a CCAAT Binding Protein Homologous to Yeast HAP5

The Neurospora aab-1 gene encodes a CCAAT Binding Protein Homologous to Yeast HAP5

Huaxian Chen,*,1 John W. Crabb † and John A. Kinsey* *Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160 and † W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946 Manuscript received August 5, 1997 Accepted for publication October 2, 1997

ABSTRACT The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam␣ and URSam␤. A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam␣ element. AAB appears to be composed of three components. We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone. The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD. Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned. AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat. Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins. The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus. Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels. Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation.

HE am gene of Neurospora crassa encodes the ana- tion factors. Although these factors share the common Tbolic NADP-specific glutamate dehydrogenase feature of binding to sequences that include a CCAAT (GDH). In previous reports from this laboratory we motif, they can be divided into at least three families have shown that the level of expression of the am gene CTF/NF1, C/EBP, and Hap/CP1/NFY. Each family is dependent upon two enhancer-like elements that binds preferentially to sites that vary in the sequences have been designated URSam␣ and URSam␤ (Freder- that surround the common motif. Within a family, the ick and Kinsey 1990a). Using in vitro mutagenesis and proteins share significant similarity at the amino acid targeted transformation, Frederick and Kinsey sequence level, whereas there is no apparent similarity (1990a,b) demonstrated that URSam␣ is responsible between members of different families (Dorn et al. for about 50% of the normal level of am gene expres- 1987; Chodosh et al. 1988a,b; Rupp et al. 1990; Cao et sion. Using gel retardation assays, Chen and Kinsey al. 1991; Hooft van Huijsduijnen et al. 1990). (1994) demonstrated that URSam␣ serves as a binding The HAP2/3/4/5 complex of Saccharomyces cerevisiae site for a nuclear factor designated Am Alpha Binding (Guarente et al. 1984; Hahn et al. 1988; Forsburg and protein (AAB). Purified AAB was shown to be hetero- Guarente 1989; McNabb et al. 1995) represents a fam- meric with probably three subunits (Chen and Kinsey ily of CCAAT binding proteins that includes the CP1/ 1995). In DNase I protection assays, AAB protected a NF-Y complex from humans as well as heteromeric portion of the URSam␣ element that contains a motif, complexes from several other vertebrate species and 5Ј-ACCAATAA-3Ј, that is identical to the consensus the fission yeast S. pombe. The HAP2/3/4/5 hetero- binding site for the yeast HAP2/3/4/5 heteromeric meric complex activates transcription of several genes CCAAT binding protein (Chen and Kinsey 1995). This in the respiratory system: CYC1 (major isoform of cyto- CCAAT motif was shown to be essential for binding of chrome C), HEM1 (␦-aminolevulinate synthase), COX4 AAB (Chen and Kinsey 1995). (nuclear cytochrome oxidase subunit 4), and CYT1 (cy- The CCAAT pentanucleotide motif is found in pro- tochrome C1) (Keng and Guarente 1987; Olesen and moter and enhancer elements of a large number of eu- Guarente 1990; Schneider and Guarente 1991). karyotic genes and serves as a binding site for transcrip- The HAP2, HAP3, and HAP5 subunits together form the CCAAT binding protein (McNabb et al. 1995). The Corresponding author: John A. Kinsey, Department of Microbiology, HAP4 subunit is required for activation in vivo but is Molecular Genetics and Immunology, University of Kansas Medical not required for DNA binding (Xing et al. 1993). CP1 Center, Kansas City, KS 66160. E-mail: [email protected] 1 Present address: ARB/NIAMS/NIH, 10/9N256, 10 Center Dr was identified by its binding to CCAAT containing pro- MSC 1820, Bethesda, MD 20892. moters of many genes, including the adenovirus major

Genetics 148: 123–130 ( January, 1998)

124 H. Chen, J. W. Crabb and J. A. Kinsey late promoter, the human hsp70, and the ␣-globin serum was preadsorbed with E. coli extract and with fractions (Chodosh et al. 1988b). NF-Y was independently iden- of Neurospora proteins lacking AAB-1 activity obtained dur- tified by its recognition of CCAAT-containing Y box of ing the purification of AAB. Western blot and immunoscreening of a ␭gt11 library: the major histocompatibility complex class II genes Dorn The purified protein was resolved by SDS-PAGE and electro- ( et al. 1987). These proteins were subsequently phoretically transferred to nitrocellulose. The membrane was shown to be the same. The rat homolog, CBF, activates blocked in TBST solution (10 mm Tris-HCl pH 8.0, 150 mm transcription of the ␣1(I) and ␣2(I) collagen promot- NaCl, 0.05% NP-40) containing 5% non-fat milk for 1 hr at ers (Maity et al. 1988). All of these factors share con- room temperature. The primary antibody (rabbit antiserum) was added to the above blocking solution at a final dilution of served blocks of sequence and in some cases genes 1:1000 and incubated for 1 hr. After washes with TBST, the from mammalian sources can complement yeast hap2 membrane was incubated with a 1:3000 dilution of the second or hap3 mutants (Pinkham et al. 1987; Hahn et al. 1988; antibody, goat anti-rabbit IgG-alkaline phosphatase (Bio-Rad, Forsberg and Guarente 1989; Vuorio et al. 1990; Richmond, CA) for 1 hr. The membrane was washed and de- Maity et al. 1990, 1992; Hooft van Huijsduijnen et al. veloped in color substrates provided by the manufacturer Becker (Bio-Rad, Richmond, CA). 1990; et al. 1991). The CBF protein contains ␭ Sachs Sinha An N. crassa gt11 cDNA library ( et al. 1986) was im- homologs of HAP2, HAP3, and HAP5 ( et al. munoscreened by the method of Sambrook et al. (1989). The 1995). No homolog of the HAP4 subunit has been plates were incubated at 42Њ for 4 hr and overlaid with nitro- found in mammalian systems. cellulose filters, which had been soaked with 10 mm IPTG and Here we report the cloning and sequencing of the dried. The plates were incubated for an additional 4 hr at 37Њ. The filters were then blocked and screened as described for aab-1 gene that encodes the AAB1 subunit of AAB. Western blots except that the incubation times with the pri- AAB-1 is a homolog of the recently described HAP5 mary and second antibodies was 3 and 2 hr, respectively. Ph- (McNabb et al. 1995) and CBFC (Sinha et al. 1995) sub- age from positive plaques were rescreened three times at units of the heteromeric CCAAT binding proteins from lower plaque densities until a homogeneous population of yeast and rats, respectively. Disruption of the aab-1 gene immunopositive phages was obtained. Bacteriophage ␭DNA from positive plaques was purified using a Lambda Magic results in a 50% decrease in am expression as well as Prep Kit (Promega, Madison, WI). other unrelated effects on growth and differentiation. DNA probes: Probes for the gel mobility shift assays were prepared by end-labeling (Sambrook et al. 1989) of gel-purified DNA fragments of the isolated wild-type 90 bp HindIII-SacI re- MATERIALS AND METHODS gions of the URSam␣ element (Chen and Kinsey 1995). Probes for hybridization assays were made by random primer Strains: Escherichia coli strains DH5␣, Y1090, and JM109 labeling of appropriate fragments (Feinberg and Vogel- were used for plasmid, ␭gt11 and ␭J1, and M13 propagation, stein 1984). respectively (Sambrook et al. 1989). The Neurospora crassa Screening libraries by hybridization: Plaques from an N. strains (FGSC #4411-4430) representing parents and progeny crassa ␭J1 genomic library, constructed by Orbach (Orbach of a cross between Mauriceville and an Oak Ridge strain de- et al. 1986), or the N. crassa ␭gt11 cDNA were transferred to scribed by Metzenberg et al. (1984), as well as other Oak charged nylon membranes (NEN) and screened by hybridiza- Ridge strains, were obtained from the Fungal Genetics Stock tion to 32P-labelled probe under conditions recommended by Center, University of Kansas Medical Center. Strain TEC41, the manufacturer (Dupont, Wilmington, DE). used as a recipient in targeted transformation, was from the Southern hybridization: N. crassa genomic DNA was iso- author’s collection (Cambareri and Kinsey 1994). lated by the method of Metzenberg and Baisch (1981). Amino acid sequencing: The purification of AAB protein DNA samples were digested with restriction enzymes, resolved complex has been described (Chen and Kinsey 1994). About on 1% agarose gel, and transferred onto Nytran membranes 30 ␮g of the purified protein preparation was electropho- (Schleicher and Schuell, Keene, NH). DNA was fixed to the resed in a 10% SDS-polyacrylamide gel (SDS-PAGE) and membrane by UV crosslinking. Hybridization and washes transferred to Immobilon-PSQ membrane (Millipore, Bed- were performed as described (Kinsey and Helber 1989). ford, MA) as recommended by the manufacturer. After trans- Northern hybridization: RNA was isolated as described fer, the membrane was stained for 10 min with 0.1% Coo- (Frederick and Kinsey 1990a). Two micrograms of poly(A)ϩ massie Brilliant Blue R-250 in 45% methanol, 10% acetic acid, RNA from the N. crassa wild-type strain, 74-OR23-1VA (FGSC and then destained in 45% methanol, 7% acetic acid for 10 #2489) was fractionated on 1% agarose-formaldehyde gel and min. The AAB1 band was excised. Cleavage of polypeptide transferred to a membrane. Hybridization and washes were with cyanogen bromide, trypsin digestion, peptide purifica- performed as described (Frederick and Kinsey 1990a). tion by RP-HPLC, and sequence analysis by Edman degrada- Transcript size was calculated from the migration in parallel tion were performed as previously described (Crabb et al. with synthetic RNA markers (BRL) visualized by staining with 1988; Stone et al. 1992). ethidium bromide. Preparation of anti-AAB1 serum: The purified protein was DNA sequencing: DNAs were cloned into M13mp18 and separated by SDS-PAGE. The gel was stained for 30 min with M13mp19 vectors and sequenced by the dideoxynucleotide 0.05% Coomassie Blue in distilled H2O and destained with sequencing method using Sequenase version 2 (United States distilled H2O for 1 hr. The AAB1 polypeptide band was ex- Biochemical, Cleveland, OH). cised and about 30 ␮g was recovered by electroelution as de- Gel retardation assay: Gel retardation assays were per- scribed (Hager and Burgess 1980). A rabbit was immunized formed as previously described (Chen and Kinsey 1994, using multiple intradermal injections of the eluted protein in 1995). Proteins eluted from the calf thymus DNA column Freund’s adjuvant and boosted twice at 2–3-mo intervals were used as the AAB factor. (Harlow and Lane 1988). The serum was collected 1 wk af- Transformation: Neurospora transformation was carried ter the third injection. Prior to use for immunoscreening, the out as previously described (Cambareri and Kinsey 1994). Neurospora CCAAT Binding Protein 125

RESULTS open reading frame with the capacity to encode peptides identical to sequences obtained by peptide mi- AAB1 protein purification and sequence analysis: crosequencing (Figure 2). This clone was, however, The AAB protein complex was purified from N. crassa clearly truncated at both ends. Consequently, it was crude cellular extracts by a combination of ion ex- used as a probe to screen a genomic N. crassa library change and DNA affinity chromatography as described made by Orbach (Orbach et al. 1986). Six positive (Chen and Kinsey 1995). Purified AAB was fraction- clones were identified among 1.8 ϫ 104 plaques. A 12- ated by SDS-PAGE which resolves AAB into two bands kb BamHI insert from one such phage was cloned to of apparent molecular mass of 40 and 30 kD (Figure 1). pBSϪ, yielding pAAB1. We then used a fragment from The separated proteins were electroblotted onto a genomic DNA to reprobe the cDNA library. A total of PVDF membrane and, after staining, the AAB1 band 105 plaques of the ␭gt11 library were screened with a (40 kD) was excised and incubated with cyanogen bro- 650-bp genomic DNA fragment. Four positive clones mide. The resulting cyanogen bromide peptides were were identified. One of the clones contained a 1.5-kb extracted from the membrane, digested with trypsin EcoRI insert which was ligated into pBSϪ to give the and peptide fragments purified by RPHPLC. Auto- plasmid pcAAB1–1.5. This cDNA insert contains a 3Ј mated Edman analysis of isolated peptides yielded a to- poly(A)ϩ tract. It is approximately the same length as tal of 45 amino acids of sequence from three purified the aab-1 messenger RNA (Figure 1B) and thus repre- peptides (underlined in Figure 2). sents an approximately full-length cDNA. Cloning of AAB1 cDNAs and genomic sequences: Detection of RNA transcripts by Northern blot analy- A high titer rabbit polyclonal antibody against the sis: A Northern blot analysis of wild-type N. crassa AAB-1 was produced as described in the materials poly(A)ϩ RNA was performed to determine the size of and methods. When this antiserum was used to probe transcripts from the aab1 gene, using the 0.8-kb cDNA Western blots of purified AAB, a band was seen only at insert as a hybridization probe. One major mRNA spe- the 40-kD position, suggesting that the 30-kD subunits kb was observed (Figure 1B). There were 1.5ف cies of were not antigenically related to AAB-1 (Figure 1A). also two minor species of 1.3 and 3.2 kb in length. The We used this antiserum to screen a N. crassa cDNA ex- relationship of these minor RNA species to the 1.5-kb pression library made in ␭gt11 (Sachs et al. 1986). Six transcript has not been determined. strongly reactive clones were isolated among 4.5 ϫ 105 DNA sequence determination: The complete se- plaques. The cDNA inserts in all six clones appeared quence of the aab-1 gene is shown in Figure 2. Both to be identical 0.8-kb fragments. One such fragment cDNA and genomic DNA sequences were determined. was ligated into pBSϪ (Stratagene) to form plasmid There is a single long ORF with the potential to encode pcAAB1–0.8. DNA sequence analysis confirmed an a 271 amino acid polypeptide. As indicated above, this ORF has the capacity to encode all of the peptide sequences obtained by peptide microsequencing. The peptide sequences are underlined in Figure 2. There are three short introns within the aab-1 coding sequences. The first interrupts codon 30, the second is between codons 34 and 35, and the last is between codons 269 and 270. Intron sequences are shown in lower case letters. The predicted molecular weight of a protein encoded by this ORF is 30 kD. The sequence surrounding the first AUG is a reasonable fit to the Neurospora consensus sequence (Bruchez et al. 1993) and codon usage is generally consistent with that found in other Neurospora proteins (Gurr et al. 1987). As with many Neurospora genes, there is no clear polyade- nylation signal. The 5Ј end of the longest cDNA obtained is at position 248 in Figure 2. Bruchez et al. (1993) identified a 7-bp consensus sequence (TCAT Figure 1.—AAB protein and aab-1 mRNA. (A) Western CANC) around the transcriptional start site of a num- blot and silver staining analysis of AAB factor. Purified AAB ber of Neurospora genes. There is a sequence identical factor is examined by SDS-PAGE and immunoblotting with to this at six of seven positions (TCATCATT) located rabbit antiserum against AAB1 (left) and silver staining between positions 214 and 221 of Figure 2. This might ϩ (right). (B) Analysis of mRNA of the aab-1 gene. Poly(A) represent the region of the transcriptional start site(s). RNA from the wild-type strain, OR-23-1VA, was separated by electrophoresis in a formaldehyde/agarose gel and subjected In common with many other Neurospora genes there is to Northern blotting and hybridization with 0.8 kb cDNA in- no obvious TATA box; however, interestingly there are sert from pcAAB1–0.8. three CCAAT boxes upstream of the likely start region. 126 H. Chen, J. W. Crabb and J. A. Kinsey

Comparison of the deduced amino acid sequence dues 81 and 167 is 75% (65/87) identical to the se- with the databases revealed that AAB-1 is homologous quence of HAP5 between residues 127 and 212, which to the recently described HAP5 subunit of the yeast is 72% (63/87) identical to the sequence of CBF-C be- HAP2/3/4/5 complex and to the CBF-C subunit of the tween residues 36 and 122. rat CCAAT binding protein CBF. Both subunits have Mapping of the aab1 gene: To locate the aab1 gene been shown to be essential for DNA binding of their re- on the genetic map, we searched for restriction frag- spective heteromeric complexes (McNabb et al. 1995; ment length polymorphisms (RFLP) in the genomic re- Sinha et al. 1995). All three polypeptides share a block gion of the aab1 locus in two N. crassa strains: Mau- residues in length that is highly simi- riceville (FGSC #4416) and a multiply-marked strain in 87ف of sequence lar (Figure 3). The sequence of AAB-1 between resi- an Oak Ridge background (FGSC #4411). Genomic

Figure 2.—Nucleotide sequence and open reading frame of the aab-1 gene. The nucleotides of the coding strand are shown. The numbers to the right of each line refer to the nucleotide sequence. The three double underlined sequences indicate CCAAT boxes. Introns are shown in lower case letters. The three underlined amino acid sequences correspond to those obtained by microsequencing of peptides from AAB1. The asterisk indicates the location of the polyA tract in the cDNA. The 5Ј end of the longest cDNA sequenced was at position 248. The sequence has been deposited in Genbank. The accession number is AF026550. Neurospora CCAAT Binding Protein 127

Figure 3.—Sequence comparison of AAB-1, HAP-5, and CBF-C. The sequences shown are between residues 81 and 167 for AAB-1, between residues 127 and 212 for HAP5, and between residues 36 and 122 for CBF-C. Sequences were compared using the DNAstar Megalign program. Identical residues are shown boxed.

DNA was prepared from the two strains and digested herently nonfunctional). The native aab-1 locus is with more than a dozen restriction enzymes. Southern linked to the trp-1 gene; therefore, the native copy of blots of the digested DNAs were hybridized with 32P- the aab-1 gene that might have been subjected to RIP labeled 0.8 kb cDNA from pAAB1–0.8. The data from should be found among tryptophan requiring progeny all Southern blots indicated that there is a single ge- from this cross. DNA from 19 morphologically normal nomic sequence homologous to the cDNA probe. progeny digested with several restriction enzymes RFLPs were detected with only two restriction enzymes: showed no evidence of RIP of the native aab-1 locus. SacI and XbaI. Consequently, genomic DNA from the However, among the tryptophan requiring progeny progeny of a cross between Mauriceville and an Oak there were three slow-growing morphologically altered Ridge derived strain that have been extensively used for strains. Two of these showed clear indication of RIP RFLP mapping in N. crassa (Metzenberg et al. 1984), mutation at the native locus. was digested with XbaI and probed with the 0.8 kb One of the RIP mutants, RM 94-62-93, had the cDNA. Segregation of the XbaI RFLP in the progeny am::aab-1 allele that has the aab-1 cDNA sequence re- gave a pattern identical to that of con-7 or trp-1. This in- placing the am gene and thus could not be assayed for dicates that the aab1 gene is located on the right arm of GDH; however, the other mutant, RM 94-62-100, had a linkage group III. wild-type am allele at the am locus and thus could be as- Disruption of the aab-1 gene: To confirm that AAB-1 sayed for GDH. RM 94-62-100 showed in repeated as- plays a role in the expression of the am gene, the aab-1 says a reduction in GDH activity to a level that was of that of wild-type activity. This is equivalent to %50ف -gene was disrupted by the Repeat Induced Point muta tion (RIP) process (Selker et al. 1987). To accomplish values obtained when the URSam␣ element was de- this, a copy of the 1.5-kb cDNA was placed in the target- leted. This would be expected if aab-1 encodes a pro- ing vector pAL1 (Cambareri and Kinsey 1994) and tein that is an essential component of the complex that targeted to the am locus in the recipient strain, TEC41. binds and activates URSam␣ in vivo. The morphologi- Using this system, the aab-1 cDNA was introduced as a cal mutant strain RM 94-62-100 was crossed to the wild- single copy at the am locus with a selectable marker (hy- type strain ORSa and eight tetrads were isolated. The gromycin resistance). Transformants were screened by morphological phenotype segregated 2:2 and showed Southern blots to confirm the correct integration and linkage to trp-1. A typical tetrad is shown in Figure 4. one transformant Tam::aab-1 was crossed to strain FGSC GDH assays on the eight spore cultures from one tetrad #1095, which contains the trp-1 marker. To avoid prob- showed that the specific activity of GDH in all of the of that of %50ف lems often associated with analysis of crosses of primary morphologically altered strains was transformants, progeny that were tryptophan requiring wild-type strains. Analysis of random spores from crosses and resistant to hygromycin were collected and screened involving RM 94-62-100 and its descendants routinely again by Southern blots to confirm the presence of two gave map values of about 1 cM for the interval between copies of aab-1. One such strain was crossed to an Oak aab-1 (scored as a morphological mutant) and trp-1. Ridge background strain. When strains with two copies Furthermore, the morphological phenotype has segre- of a gene go through the sexual cycle both copies of gated with RFLPs at aab-1 and reduced expression of the gene can be mutated by the RIP process (Selker et the am gene through four generations of backcrosses. al. 1987). Thus, we expected copies of the aab-1 gene to To determine if the aab-1 mutations affected CCAAT be mutated in some fraction of progeny resulting from binding activity, extracts were made from RM 94-62-93 the cross. We were particularly interested in mutated and RM 94-62-100 and used in gel mobility shift experi- copies of the native gene (the copy integrated at the am ments (Figure 5). The major CCAAT binding activity locus is a cDNA copy lacking a promoter and is thus in- seen in normal strains (lane B) was absent in both mu- 128 H. Chen, J. W. Crabb and J. A. Kinsey tant strains; however, a faint band with altered mobility Rescue of the aab-1 phenotype: To demonstrate con- is visible with extracts from RM 94-62-93 (lane C), but clusively that the pleiotropic effects were because of not in extracts of RM 94-62-100 (lane D). From these aab-1 disruption, an aab-1 strain was rescued by trans- results it would appear that RM 94-62-93 has some re- formation with a plasmid containing the cloned aab-1 sidual binding activity; whereas, RM 94-62-100 appears genomic sequences. Since aab-1 strains grow so poorly, to be a complete null for binding to the CCAAT motif the heterokaryon-assisted transformation system as found in the URSam␣ element. There is no obvious dif- described by Yamashiro et al. (1996) was used. An ference in the phenotype of the two mutant strains. mtr;aab-1 double mutant strain was constructed and Not unexpectedly, the aab-1 mutation has pleiotro- placed in a forced heterokaryon with a lys-1 strain. This pic effects on Neurospora growth and development. As heterokaryon conidiated and grew normally, allowing indicated above, all isolated aab-1 mutants have highly normal spheroplasting and transformation. Sphero- altered morphology. When grown on solid media only plasts from the heterokaryon were cotransformed with very short aerial hyphae are produced, giving the cul- an aab-1 plasmid and a plasmid, pCNS43, (Staben et ture a patchy appearance (Figure 4). Supplementation al. 1988) conferring hygromycin resistance. Transfor- with glutamate does not relieve this morphological mants were selected on hygromycin and p-fluorophen- phenotype. Conidiation of aab-1 strains is greatly re- ylalanine (the latter compound selects for homokary- duced with only sporadic single chains appearing very otic strains that have the mtr mutant gene) and late in development. In addition to the reduced aerial approximately half of the selected transformants had hyphae, growth is much slower than wild-type whether normal morphology and conidiation. After repeated the strains are grown in liquid or on solid medium. On single condial isolation to insure homokaryosis, the race tubes (Ryan et al. 1943), aab-1 strains showed er- conidiating transformants were screened by Southern ratic growth with extension rates of 0.02–0.15 mm/hr blot analysis. Each of five tested transformants had the at 25Њ. On some race tubes, aab-1 strains showed a start- resident disrupted copy of aab-1 and at least one ec- stop growth pattern in which linear extension might topic wild-type copy. GDH assays on the same transfor- halt for up to 48 hr before resuming at a slow extension mants indicated that all had regained wild-type levels of rate. This should be compared to the steady extension GDH. mm/hr for the wild-type strain and 2.5 3.4ف rates of mm/hr for a strain from which the URSam␣ element had been deleted, grown under the same conditions. Because of poor conidiation, it was difficult to compare the growth of aab-1 with wild-type in liquid cultures; however, using sheared hyphal fragments as an inocu- lum, aab-1 strains appeared to have a doubling time in excess of seven hours as compared to a 2-hr doubling time for wild-type strains grown under the same conditions. In addition to effects on growth and conidiation, aab-1 strains are female sterile; however, they are fertile as males. This complex of characteristics has segregated with aab-1 through four rounds of backcrosses.

Figure 5.—Gel mobility shift analysis with the URSam␣ DNA fragment and extracts from wild-type and aab-1 mutant Figure 4.—Segregation of the aab-1 phenotype among the strains. All lanes contain the same DNA probe. Lane A con- products of a single ascus from a cross of aab-1 mutant strain tains no protein extract. Lane B contains wild-type extract. 94-62-100 to the standard wild-type strain, ORS a (FGSC Lane C contains extract from the 94-62-93 mutant strain. 2490). The cultures shown had grown for seven days at 25Њ. Lane D contains extract from the 94-62-100 mutant strain. Neurospora CCAAT Binding Protein 129

DISCUSSION of the GDH produced by normal strains. This is equivalent to the reduction in am expression seen in strains The most difficult step of isolating clones of the aab-1 that have deletions of the CCAAT site upstream of the gene from N. crassa has been obtaining sufficient pure am locus (Frederick and Kinsey 1990a). Since the aab-1 AAB1 protein from which to produce antisera and to strains also have altered morphology and slow growth, obtain protein sequence information. Previously, we it is reasonable to ask whether the reduction in GDH purified the AAB transcription factor complex to near level is responsible for this phenotype. Clearly this is homogeneity and demonstrated that it consists of at not the case since mutants lacking either the URSam␣ least two components (Chen and Kinsey 1994). Here or the URSam␤, each of which reduce am expression by we have further purified the AAB1 component to ap- have no effect on morphology and result in ,%50ف parent homogeneity by electrophoresis. A high titer only a minor reduction in growth rate. The fact that all polyclonal antisera was prepared against the purified of the aberrant phenotypes associated with the aab-1 AAB1 subunit and used to isolate a partial cDNA clone disrupted mutant strains can be rescued by the cloned from an N. crassa cDNA expression library. Sequence aab-1 gene indicates that the aberrant phenotypes must analysis of the cDNA clone confirmed an open reading be because of reduced expression of other genes whose frame containing the deduced amino acid sequence of expression is driven by the AAB transcription factor. three peptides determined by direct Edman sequence Given that expression of a number of genes must be analysis of purified AAB1. The cDNA clone was used to affected by the aab-1 mutation, it could be argued that isolate the corresponding genomic clone, a fragment the effect on the expression of the am gene might be of which was used to isolate a cDNA that contained the indirect. Although this is a formal possibility it seems entire coding region (Figure 2). The deduced AAB1 unlikely. Mutation of the CCAAT box in URSam␣ to protein has a calculated molecular mass of 29.6 kD but CCGGT results in a reduction in am gene expression migrates in SDS-PAGE as a 40-kD protein (Figure 1). that is equivalent to deletion of the entire 90-bp ele- Such discrepancies are not uncommon and notably ment (J. A. Kinsey, unpublished results). This argues have been observed for transcription factors GCN4 that the crucial sequence in URSam␣ is the CCAAT from yeast (Hope and Struhl 1985) and NF-Y from box. Further, the CCAAT box found in URSam␣ is a mouse (Hooft van Huijsduijnen et al. 1990). Either perfect fit to the consensus sequence recognized by AAB1 migrates anomalously in SDS-PAGE, or it is post- both the yeast and mammalian homologs of AAB translationally modified. (Chodosh et al. 1988a,b) but is different from the con- Comparison of the sequence of the deduced protein sensus sequences bound by other mammalian CCAAT with sequences present in protein data bases indicated binding proteins (Chodosh et al. 1988a). Also, during that AAB-1 is homologous to HAP5 and CBF-C. The re- the experiments that resulted in the purification of gion of clear homology between these proteins covers a AAB-1, we made an extensive search for other proteins sequence of 87 residues (Figure 3). Interestingly, this that bound to URSam␣. None were found. Yeast ap- region includes all parts of CBF-C that are known to be pears to have only a single CCAAT binding protein, required for interaction with the other CBF subunits as whether this will prove true for other fungi such as well as sequences required for DNA binding (Kim et al. Neurospora is unknown. 1996). Kim et al. (1996) have also suggested that this re- Although little is known regarding the regulation of gion has homology to the histone-fold motif of histone the genes that encode CCAAT binding proteins, it is in- H2A, suggesting that the protein/DNA interaction may teresting to note that there are three CCAAT boxes in the resemble that of histones. The sequence homology be- 5Ј noncoding sequences of the aab-1 gene (double un- tween AAB-1, HAP5, and CBF-C make it clear that AAB derlined sequences in Figure 2). The first box is identical is a Neurospora CCAAT binding protein. Therefore to the HAP2/HAP3/HAP4/HAP5 consensus binding AAB-1 is almost certainly one of three essential subunits. site. The other two boxes are identical in six of the seven This suggests that the other two subunits are repre- residues in this consensus sequence. This raises the possi- sented by the doublet of bands of an apparent molecular bility that the expression of aab-1 may be autoregulated. (kD observed by Chen and Kinsey (1995 30ف mass of on denaturing PAGE gels of purified AAB protein. This work was supported by grants from the National Science When RIP was used to mutate the aab-1 gene no al- Foundation (J.A.K.) (DBM-9407082) and from the National Insti- tutes of Health ( J.C.) (EY06603). teration of aab-1 sequences was seen in morphologically normal progeny. However, 3–10% of the progeny produced in RIP crosses were morphologically altered. When these morphologically altered progeny were ex- LITERATURE CITED amined by Southern blot analysis, alterations in restric- Becker, D. M., J. D. Fikes and L. Guarente, 1991 A cDNA encod- tion sites within the aab-1 gene were clearly present in ing a human CCAAT-binding protein cloned by functional com- plementation in yeast. Proc. Natl. Acad. Sci. USA 88: 1968–1972. many cases. GDH assays indicated that the strains with Bruchez, J. J., J. Eberle and V. E. A. Russo, 1993 Regulatory se- alterations at the aab-1 locus produced only about 50% quences involved in the translation of Neurospora crassa mRNA: 130 H. Chen, J. W. Crabb and J. A. Kinsey

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