inactivation of the uma-1 gene by repeat-induced point mutation

Copyright 0 1996 by the Genetics Society of America

The Vacuolar ATPase of Neurospora crmsa Is Indispensable: Inactivation of the uma-1 Gene by Repeat-Induced Point Mutation

Tracy L. Ferea and Barry J. Bowman

Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064 Manuscript received January 5, 1996

Accepted for publication February 12, 1996

ABSTRACT To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which

encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that - - the vacuolar ATPase is essential in Neurospora crassa.

T HE vacuolar ATPase is a proton pump found in all eukaryotes (FORGAC 1989; BOWMAN et al. 1992).

Located throughout the endomembrane system, the enzyme generates an electrochemical gradient across organellar membranes. Transport of many small mole- cules is dependent on the energy stored in the electrochemical gradient. Thus, the vacuolar ATPase plays a major role in establishing the molecular contents of organelles such as endosomes, vacuoles or lysosomes, regulated secretory vesicles, and the trans Golgi net- work.

The vacuolar ATPase is a complex enzyme consisting of at least 10 subunits (ANRAKU et al. 1992; BOWMAN et al. 1992; WIECZOREK 1992; GRAHAM et al. 1995; SUPEKOVA et al. 1995). The catalytic subunit is encoded by the vma-1 gene and has been characterized in many organisms (BOWMAN et al. 1988; ZIMNIAK et al. 1988; HIRATA et al. 1990; KANE et al. 1990; PUOPOLO et al. 1991; PENG et al. 1994). This 67-kD protein appears to be the site of ATP hydrolysis. Inactivation of the catalytic subunit in Saccharomyces cereuisiae results in the complete loss of vacuolar ATPase activity (HIRATA et al. 1990; NOUMI et al. 1991; KLIONSW et al. 1992).

The phenotype of cells lacking a functional vacuolar ATPase has been examined only in S. cereuisiae. Surpris- ingly, the vacuolar ATPase is not essential for viability in this organism. Null strains, however, are unable to grow on nonfermentable carbon sources, indicating that the mutation causes a defect in oxidative phosphorylation. This finding suggested to us that the vacuolar

Cmesponding author Tracy Ferea, Department of Biology, Sins- heimer Laboratories, University of California, Santa Cruz, 95064. E-mail: [email protected]

Genetics 149: 147-154 (May, 1996)

ATPase might be essential in obligate aerobes such as Neurospora crassa.

Genes in N. crassa can be selectively inactivated by introducing an extra copy of the gene into the haploid genome (SELKER and GARRETT 1988; SELKER 1990). During asexual vegetative growth N. crassa tol- erates gene duplications. However, during the sexual cycle before karyogamy, the genome is scanned for duplicated regions. When duplications are detected, multiple point mutations are introduced into both the endogenous and ectopic copies of the gene. This phenomenon has been named RIP, for repeat-induced point mutation.

The haploid nature of N. crassa and the lack of auton- omously replicating plasmids have made analysis of essential genes difficult. Although RIP is a powerful tool for selective inactivation of a gene, its use has been limited because inactivation of essential genes is lethal. One way around this problem has been to use genetic methods to generate heterokaryotic strains that have wild-type nuclei and nuclei containing an inactivated gene (METZENBERG and GROTELUESCHEN 1992; HARK- NESS et al. 1994). The inability to isolate from such heterokaryons a homokaryotic strain carrying the inactivated gene has been used as evidence that a gene is essential. We have used the vma-1 gene to develop a method that we called “RIP & Rescue.” This method generates a homokaryotic strain that can be used to assess the phenotype of a strain carrying a gene inactivated by RIP. RIP & Rescue may be generally applicable to the analysis of essential genes.

MATERIALS AND METHODS Plasmids: For our initial experiments we attempted to dis-

rupt the vma-1 gene by homologous replacement. Using

148 T. L. Ferea and B. J. Bowman

TABLE 1

Plasmids and N. crassa strains used

Plasmids Fragments in pBluescript SK+

pTF2 pTF3

vma-1 : : am (see Figure I ) vma-1 fused to hph (see Figure 1)

Strains MT Description Phenotype ~~

5883 A am132 AI' Am- Hyg' ~~ ~ ~~~~~

TF2-5 A 5883/pTF2 Al' Am' Hygs TF2-5-4 A Fl of TF2-5 X AA1 Al+ Am' Hygs TF2-13 A 5883/pTF2 AI+ A m + HygS TF2-134 A FI of TF2-13 X AAl Al' Am' Hygs AA1 a amnz al-3 AI- Am- HygS TF3-13 a AAl/pTFS Al- An- Hyg'

vma-1RR1 A F1 of TF2-134 X TF3-20 Al' Am- Hyg' vma-1RR2 A F1 of TF2-13-4 X TF3-20 AI+ An- Hyg' 30 A Fl of TF2-134 X TF3-20 AI' Am- HygR

TF3-20 a AAl/pTF3 Al- Am- Hyg'

methods previously described (SAMBROOK et al. 1989), we constructed the plasmid pTF2, which contained a fragment of the uma-l gene (BOWMAN et al. 1988, accession #J03955) interrupted with the am gene (see Figure 1 and Table 1). First, a plasmid containing a 4.4kb BamHI fragment of the vma-1 gene in the vector pBluescript SKf (Stratagene, La Jolla, CA) was linearized by cutting with BglII. A 2.7-kb BamHI fragment containing the functional am gene was then inserted into the uma-1 coding region. am encodes the NADP-specific glutamate dehydrogenase (JSINNAIRD and FINCHAM 1983), a marker selectable in transformation of An- strains. The 2.7-kb am fragment originated from pJR2 (KINSwand RAMBOSEK 1984).

To rescue strains with mutations in the vma-1 gene, we constructed the plasmid pTF3, which contained a complete functional copy of the uma-1 gene. A 6kb SphI-EcoRVfragment (Figure 1) was inserted into the plasmid pCSN43, which had been digested with the same enzymes. pCSN43 has the selectable marker hph, which encodes hygromycin phosphotransferase, inserted between the Aspergillus nidulans trpCpro- moter and terminator, and cloned into pBluescript SK+ ( STABEN et al. 1989).

N. crmsa strains: Plasmid pTF2 was introduced into the N. crassa amly2 strain (#5883, Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS) by transformation, using the procedure of VOILMER and YANCW~KY (1986) as modified by ROYER and YAMASHIRO (1992). Strain #5883 has a 9-kb deletion of the region encoding glutamate dehydrogenase. Thus, the a m gene of the integrated plasmid can be used as a selectable marker without being subjected to RIP.

pTF2 transformants were selected on plates containing Vo- gel's minimal medium, 2% sorbose, 0.05% fructose, 0.05% glucose, and 0.02% inositol (FIGS) (ROYER and YAMASHIRO 1992). Top agar with 0.2 mM glycine was used to select for A m + transformants. Two primary transformants, strains TF2- 5 and TF2-13, contained a single ectopic copy of plasmid DNA as determined by Southern hybridization. Further analysis suggested these transformants were heterokaryotic, i.e., contained some untransformed nuclei. To obtain homokaryons both transformed strains were crossed to strain (amlsn, al-3). Two progeny, strains TF2-5-4 and TF2-13-4, were selected that had the A m + , Al+ phenotype and had inherited the integrated pTF2 plasmid. Analysis of these strains by Southern blots and hybridization showed no evidence of mutation as

assessed by changes in the size of NluIII restriction fragments from the vma-1 gene. NlaIII, which cleaves the palindrome CATG, was used because the cytosine of the dinucleotide CpA is the most frequent target of RIP (SELKER 1990). The region of the vma-1 gene that was probed contained nine NlaIII sites.

Plasmid pTF3 was introduced into host strain AA1 (amlgp, al-3). The al-3 mutation causes an albino phenotype and is closely linked (six map units) to the vma-1 locus on linkage group V (SISTA et al. 1994). For selection of pTF3 transformants both top and bottom agar were supplemented with alanine (0.2. mg/ml), and the bottom agar contained hygromycin B (150 pg/ml, Boehringer Mannheim, Indianapolis, IN). Strain TF3-20 was isolated by streaking conidia on selective medium (FIGS plates with hygromycin B, 150 pg/ml) and then isolating single colonies. Four rounds of isolation were performed.

For standard genetic analyses, i.e., crossing strains and analyzing progeny, the procedures of DAVIS and DE S E W S (1970) were used.

Scoring A m + and Al' phenotypes: Progeny were grown on FIGS medium supplemented with either 0.2 mM glycine or 0.2 mg/ml alanine. An- progeny grew on medium supplemented with alanine, but not on the medium supplemented with glycine. The Al' phenotype was scored after 3-5 days in 1 ml culture tubes. In some experiments the number of AI+ and Al- colonies were counted on plates incubated for 2 days at 30°, followed by 2-3 days at room temperature in constant light.

Analysis of transformants: To test for homologous integration of pTF2 at the uma-l locus, DNA was digested with BglII; Southern blots were prepared and hybridized to a radiola- beled 1.6-kb BglII-BamHI fragment containing most of the vma-1 coding region (see Figure 1 ) . PCR was also used to screen for homologous integration. We selected oligonucleotide-primers that would amplify a 2.1-kb fragment if the pTF2 plasmid had integrated at the endogenous vma-l locus. The sequence of one of the primers was derived from the 3' end of the am gene (GTTGCTGGATCACACTCG) and the other (TGATGCATGCGCTGTCTG) from just beyond the end of the protein coding region of the vma-1 gene.

DNAanalysis: DNAwas extracted by the method of OAKI.EY et al. (1987) or ARONSON et al. (1994). Radioactive DNA fragments were prepared using the random priming kit of Phar- macia (Piscataway, NJ). PCR was performed according to man-

A. Z f rn

region of the ma-1 gene in pTFZ. showing position of am insert

E

region of the ma-1 gene in pTF3

B.

BamHl

BamHl Sphl

W ' l

11.9 kb

vma- 1

BamHI/Botll ECORV

Fl(;l:RE l.--Constn~tion of plasmids containing the umn-I gene. (A) The DNA fragments inserted into plasmids pTF2 and pTF3 are shown with identical regions of the umn-1 gene overlapping. The arrows indicate the direction of transcrip tion i n the proteincoding region (lightly stippled box). This region is tnmcated at thc 3' cbnd in pTF'2. Rrgions that flank thc umn-1 coding region are shown i n darker stipple. (R) The complete pTF2 and pTF3 plasmids have the inserts shown in A cloned into the plasmids Rluescript SK' and pCSN4.7, respectively. pCSN43 is tlerivrd Irom Rluescript SK+ antl also contains the hph gene.

ufacturers' protocols using either Tnq polymerase or the Expand Long Template I'CR System (Roehringer "ann- heim). Direct sequencing of PCR products was done w i t h the (:ircum\'ent (exo ) sequencing kit (New England Riolahs, Reverlv, MA). RIP & Rescue: To generate a viable stnin that lacked a

functional vmn-1 gene ;\I the endogenous locus, two strains carning an ectopic copy of the vmcr-f gene were crossed (see Figure 2 ) . The strain 'TF2-13-4 was crossed to the strain TF.S 20. Progeny were sclectctl on FIGS medium supplemented with alanine and hygromycin R and then screened by analysis of Southern blots, as described in RESIITS.

The sequence of the vmn-1 gene was determined in two of the strains that appeared to contain mutations. The Expand PCR kit (Boehringrr Mannheim) w a s r~sctl to amplify sclec- tively the endogenous copy of the umn-1 gene. The 5' oligonucleotide ~(;<;,\T(;TC;<:(C/T)r\C.C;XC(:(;AG was used w i t h the 3' oligonucleotide T<;~T~(~TG(;(;<TTGT(,TG to generate a 5 k l ) PCR product. This product WAS either usc~l for direct sequencing or reamplified with the sequencing primers and then directly sequenced.

ATPase assay \'acuolar membranes were isolated and vacuolar :VIPasr activity \VAS assayed as described (DSCHIIM and RO\VMMAN 1995).

RESULTS

Inactivation of the ma-Z gene by RIP To generate a strain o f ,V. cv-msn that lacked a functional vacuolar

TABLE 2

Analpis of progeny from crosses in which one parent has a duplication of the ma-1 gene

No. of A I A No. of A I Rltio Cross progeny progcny :\I ' /..\I

TFL-5-4 X Ail 133 100 0.75 TF2-I .W X A+\ 1 I(i5 1 3 1 0.79 TF-13 X 5883 233 163 I .4J

ATPase, wc focuscd on the inactivation of the rrmn-1 gene, which encodes the catalytic subunit (Ro\v\L\s d fd. 1988). Initially, we attempted lo replace the vmn-1 gene with a disrupted copy. '\! ~ r n . ~ strain #.X83 ( a m l y 2 ) , which has a deletion of the o m gene, was transformed with plasmid pTF2 (Figure l ) , which contains a 4.4-kb fragment of the 7~mn-1 gene interrupted w i t h a functional um gene. One hundred thirtysix individual Am' transformants were analyzed bv Southcrn hyhrid- intion and/or PCR as described in \I.ATF.RIAIS hSI)

METHODS. We found no evidence of a homologoas integration into the vmn-1 gene. h described in \lr\TF.RlhlS

AND METHODS, 05'0 of these transformants \ w " used to obtain homokaryotic strains that had a single pTF2 plasmid integrated at an ectopic site.

These strains, TF!-5-4 antl TF2-1-34, were crossed with strain AAl (omly2 , nl-3). If the vmn-1 gene were inactivated bv RIP and were essential for \iihili?, we predicted that AI'' progeny would be underrepresented because of the close linkage of the nl-3+ and vmn-1 loci. For both crosses AI' progeny were underrepresented by 21-25% when comparcd to the AI- progenv (Table 2). A similar experiment was performed in which the ectopic copy of the vmn-1 gene was in the nl-3- parent ( M I ) instead of in the nl-3' parent (strain #5883) of the cross. The strain TFSl3 was constructed by trans- forming strain AAl with pTF3. Strain TFSlS was then crossed to strain #5883 (nmls2, n1-3+). Albino progeny were underrepresented bv 30% when compared to the AI' progeny (Table 2). Together these cxperimcnts showed that a region of chromosome 5 from the parent carrying a duplication of the vmn-1 gene was untlerrep resented in the progenv, regardless of whether the pa- rental strain was AI or AI'. Although not conclusive, the data suggested that the vmn-1 gene was being RlPcd in 20-30% of the nuclei and was essential for viahilit?,. These experiments also indicated RIP did not occur in most of the nuclei carrying a duplication, suggesting it would be feasible to introduce unaltered functional ectopic copies of the vmn-1 gene into the progeny.

RIP & Rescue: To generate viable strains with null mutations in the endogenous copy of the vmn-1 gene, we developed a method called "RIP k Rescue." RIP k Rescue was performed with two strains, both of which carried a duplication o f the vmn-1 gene (Figure 2).

1.50 T. 1.. Ferea and R . J . Bowman

RlPing strain TF2-13-4

["][Z][".] m - 7 + . wed to a w m-I+. wed to 81.3~ m-W', liirked to a/*

lh'ked 10 hph ( P m ) link to hph (pTn) RJtedtohph ( P m )

RlPed 6 Rescued

FIGI'RE 2.--Diagram of the RIP & Rescue procedure. The genotypes of the two parent strains are shown in boxes. AI' progeny able to grow on hvgromycin were selected. The possible genotypes of these progeny are shown below the parent... assuming that progenv without a functional vmn-1 gene wollld not be viahle. Note that some of the progeny could also inherit the integrated pTFY2 plasmid. which is not shown.

Strain TF2-1.3-4, designated the RIPing strain, carried an extra nonfunctional copv of the vma-1 gene. Strain TF3-20, designated the Rescue strain, carried an ectopic functional copv of the vmn-1 gene linked to the hvgro- mvcin phosphotransferase gene. The RIPing strain and the Rescue strain were crossed to induce the RIP process.

The strategy was to screen for progeny in which RIPing had occurred in the nuclei of the RIl'ing strain, inactivating the endogenous copv of the vmn-1 gene (Figure 2). Although this event tvaq expected to he lethal, -30% of the progeny should also inherit, by independent mort- ment, the functional ectopic copv of the vmn-1 gene from the Rescue parent. The phenotype of such progeny would he AI', hvvomvcin-resistant (HVC).

The RIP & Rcscue procedure was feasible because the RIP process is not 100% efficient and because both the endogenous and ectopic copies of the vmn-1 gene frequently came through the cross with no mutations. We selected 52 AI'; Hvg' progeny from the cross. DNA was prepared from these strains, digested with the restriction endonuclease NlnIII, and analvzed by the method of Southern. A 1.6-kb RflI-BnmHI probe to the coding region of the vma-1 gene was used to diagnose loss of restriction sites. Most of the progeny selected as AI'; Hyg' showed the same size restriction fragments as the transformed parent.. . Six of the 32 strains showed larger NlnIII restriction fragment5 than the parents (Figure S ) , indicating that the vma-1 gene had been RIPed. It was not possible to determine from these data whether the new fragments were derived from the endogenous or the ectopic copv of the vmn-1 gene.

Sequence analysis of the mutated yma-I genes: To determine precisely the nature of the mutations, we analyzed the sequence of the endogenous copy of the

.

' I 16 17 2 19 20 21 22 23 0 7 24 25 26 2728 29 30 31 32

E x c ! u r j c u

I-

FIGURE 3.-Southern blot% of genomic DNA digested with NlnllI. DNA from 32 AI+ Hyg' progeny from the cross of TF2- 1.34 and TF3-20 are shown, along with the parent strains and the untransformed .5883 strain (omlJ.) . The strains in lanes 2. 8, 2'2. and 30 and those designated \ma-lRRI and m a - 1RR2 appear t o have lost some .Vlnlll restriction sites. The four prominent bands seen in a l l lanes range in size from 190 to 500 bp.

vmn-1 gene. PCR was performed to amplie selectively a 5 k b region containing the endogenous vmn-1 locus from two strains, designated vma-1 RRl and vma-1 RR2 (Figure 3). As a control, the vmn-1 gene of the untrans-

Analysis of an Essential Gene

0 kb 1 kb 2 kb 3 kb 4 kb 5 kb

Barn Bp ' Barn

I vma-7 protein coding region ' Barn ' reglon duplicated in

151

Bg l Barn region sequenced in vma-1RRZ I I II 11111 111 llll Ill1 I I I1 I I I I

FIGLIR~, 4.-Sites of mutations in the vma-1 gene. The endogenous .oma-l gene is compared to the region duplicated in the plasmid pTF2, with identical regions shown as overlapping. The region of the endogenous gene that was sequenced in the strains vma-IKK1 and vrna-lRR2 is also shown with each nucleotide change indicated by a vertical line. The arrows indicate the sice ofa non- sense mutation found in both strains.

' +

Ihrmccl strain #5883 was also analyzed. Direct sequencing of amplified DNA from vrna-1KR1 and vma-lRR2 showed multiple nucleotide changes within the endogenous gene (Figure 4). vma-lRR1 had 42 changes, while vma-l RK2 had 34 changes. The alteration of the nucleotide sequence resulted in multiple amino acid substitu- tions, as well as the introduction of a stop codon after Lys129 in both strains.

Analysis of the sequences showed two features of the nucleotide changes introduced by RIP. In the coding strand every change was a G to A transition, and these transitions resulted in a loss of %lo% of the G-C base pairs in the region recognized as duplicated (see below). Analysis of the context of nucleotide changes showed results similar to those reported by SELKIX (1990) with 74% of the nucleotide changes occurring in the CpA context.

The second interesting feature was that changes occurred on only one side of the am interruption. The experiment had been performed with a plasmid in which the duplicated region was divided into two pieces, separated by the urn gene (Figure 4). In the region sequenced all the changes in the 71m.a-1 gene were located 3' to the RglII site, the site in which the am gene was inserted in pTF2. We sequenced 804 bp 5' to tht: BgLI site and 142 bp beyond the 3' end of'the duplication and found no changes. It appeared that in these two strains only the region 3' t o the BglH site was recognized as duplicated.

We wanted to determine if the region 5' to the am gene insertion ofpTF2 had integrated into the genome. The absence o f this fragment in the transformants would explain the lack of RIPing in this region. A South- ern blot probed with a 2.2-kb S$hI-Rglll fragment of the uma-1 gene indicated the region 5' of the am gene was duplicated in the TF2-13 transformant (data not shown).

Phenotype of vma-1 null strains: We prepared vacuolar membranes From the vma-1RR1 and vma-1RR2 slrains and measured the specific activity of the vacuolar ATPase. In both strains the ATPase activity was essen-

tially the same as in the wild-type controls (data not shown). To determine if a functional wrna-1 gene was necessary for cell viability, further genetic experiments were performed. vma-IRK1 and vma-lRR2 (both Al') were crossed to the AI- strain AAl (Figure 5). If the vmcl-1 gene is essential, two predictions could be made about the viable progeny resulting from the cross. First, the ratio of Al+ to A l p should be -1:2 because only those Al' progeny that inherit a functional ectopic copy of the uma-1 gene by independent assortment should be viable (Figure 5). The Alp progeny should be viable because they have inherited a functional copy of the vrna-1 gene at the endogenous locus. A few crossovers are expected between the vma-1 and al-3 genes, but these do not significantly affect the prediction. Results of these crosses are shown in Table 3. Among the progeny of vma-lRK1 and vma-lRR2 the ratio of AI+ to AI" was -1:2. A s a control, strain 30 (see lane 30 in Figure 3 ) was also crossed with strain AA1. Strain 30 appeared to be RIPcd in the ectopic copy of the vma-1 gene rather than in the endogenous uma-1 gene (data not shown). Among the progeny from strain 30, Al~- and AI+ phenotypes were equally represented (Table 3 ) , as predicted if the endogenous urnn-1 gene was functional.

The second prediction from the experiment was that only those Al+ progeny that inherited the ectopic copy of the ?,ma-1 gene would be viable; therefore, they should all be HygR. Only half of the Al- progeny would be expected to be Hy$ because they do not require the ectopic copy of the vma-1 gene for viability. When tested for hygromycin resistance, all of the 25 Al' progeny tested were HygR (Table 4), while five of the 12 Alp

progeny tested were HygR. These results indicated that a lethal mutation was tightly linked to the al-3 locus and that this mutation could be complemented by pro- viding a functional ectopic copy of the uma-1 gene.

S . cereuisine is typically grown on a more enriched medium than N. cl-assa. It was possible that if grown on complete medium, vacuolar ATPase null strains of N. cra~sn would be viable, like S . ctler~isiae (NEISON and NELSON 1990). Spores from the crosses Mna-lRKl and

152

vma-I RRI or vma-1 RR2

T. L. Ferea and B . J . Bowman

I FIGLIRE !%-Testing the phenotype of the vma-

lRRl and vma-1RR2 strains. The vma-lRRI and vma-lRR2 strains were crossed to strain M l . Pos- sihle genotypes of‘ the progeny are shown. The endogenous linked vmn-I and 01-3 genes are shown in the unfilled boxes. The functional vmn- I gene linked to the hph gene, ectopically integrated in the genome, is shown in the filled box.

I “I ma-7+ u \ -”z+)

inviable?

vma-lRR2 to AAl did not show an increase in viable AI’ progeny when germinated on FIGS medium supplemented with 0.2 mg yeast extract per ml (data not shown).

DISCUSSION

Using a genetic procedure, we have generated the first vacuolar ATPase mutants in an organism other than S. cPrPuisiaP. Our results strongly suggest that the vacuolar ATPase is essential for viability. These results contrast those obtained with S. cmvisiae, which indicated vacuolar ATPase null strains were viable under normal growth conditions (NELSON and NELSON 1990;

We hypothesize that the reason for this difference is a fundamental difference in the metabolism of the two organisms. S. cm-misiaP, a facultative anaerobe, is viable without vacuolar ATPase activity as long as it is grown on a fermentable carbon source. This suggests that oxidative phosphorylation is disrupted in vacuolar ATPase null strains, perhaps due to uncoupling of the mito- chondria.

The connection between inactivation of the vacuolar ATPase and loss of mitochondrial function is probably explained by changes in cytosolic calcium (TANIDO et al. 1995). In both S. cmmisiae and N. crnssa the calcium concentration in the vacuole is at least 100-fold higher than in the cytosol (CRAMMER and DAVIS 1984; MILLER et al. 1990; OHYA et al. 1991). In cells without a functional

OIWA Pl a/. 1991).

vacuolar ATPase, the concentration of calcium in the cytosol rises significantly and is likely to disrupt im- portant metabolic processes (OHYA et al. 1991; HA- IACHMI and EILAM 1993). Disruption of normal mitochondrial function would be lethal in an obligate aerobic organism such as N. crassa. We are currently placing the vma-1 gene under the control of a regulatable promoter. These studies may lead to a better un- derstanding of the physiological role of the vacuolar ATPase.

To determine the phenotype of strains with an inactivated vacuolar ATPase, we developed a procedure that we have named RIP & Rescue. With this method it was possible to mutate the endogenous copy of an essential gene by RIP and still recover viable progeny. BV independent assortment the progeny inherited a functional ectopic copy of the gene from the Rescue parent. The effectiveness of the method of RIP & Rescue has been shown by both genetic and molecular criteria using the vma-1 gene. This method should be generally applicable to the analysis of other essential genes.

One other method for analyzing essential genes in N. crmsa has been reported (METZENRERG and GROTE- I.UESCHEN 1992; HARKNESS et al. 1994). The method called “sheltered RIP” employed a different strategy to construct strains that contain both a functional copy and a RIPed copy of an essential gene. This method used the mei-2 allele to obtain disomic ascospores. These disomics break down into heterokaryons with two types of nuclei, those with a mutated endogeneous gene

Analysis of an Essential Gene 153

TABLE 3

Effect of inactivation of the vmml gene on viability of progeny

NO. o rN+ NO. Ratio Cross progeny progeny AI+/”

vma-1RR1 X AAl 30 55 0.55 vma-1RR2 X AAl 29 61 0.48 Strain 30 X AA1 65 70 0.93

and those with a normal endogenous gene. Inability to recover a homokaryotic strain carrying the mutated gene indicates that the RIPed gene is essential.

Strains generated by the RIP & Rescue procedure are homokaryotic and therefore readily amenable to genetic and molecular analysis. Because RIP & Rescue strains carry the complementing copy of the gene at an ectopic locus, PCR can be used to amplify specifically the endogenous gene for sequence analysis. Using homokaryotic strains also simplifies genetic analysis. With heterokaryons skewing of nuclear ratios can make the outcome of a cross difficult to predict. In the homokaryotic strains generated by RIP & Rescue an inactivated essential gene is predicted to behave as a simple reces- sive lethal allele.

The null-strains generated by the RIP & Rescue procedure should be powerful tools for the analysis of essential genes. A simple genetic cross can replace the functional Rescue copy with a different functional copy. It should be possible to investigate functional copies of the gene, which have been altered by site directed mutagenesis or are controlled by a regulatable promoter.

Our experiments may also shed some light on the poorly understood RIPing process (SELKER 1990). We constructed a plasmid in which the uma-1 gene was interrupted with the am gene. Transformation with this plasmid generated two duplicated regions susceptible to RIP, separated by 2.7 kb of unique sequence. Even though the duplicated regions were in close proximity, it seems that only one paired with the endogenous copy of the gene during the RIPing process. Sequence analysis of the endogenous uma-1 gene showed that all the mutations occurred in the 1.6 kb duplicated region 3’ of the am gene insertion in the pTF2 plasmid (Figure 4). One might expect the smaller 1.6-kb duplicated region (downstream of the am gene) to be less likely to RIP than the larger 2.8-kb duplicated region (upstream of the am gene). However, in both of the strains analyzed, only the smaller duplicated region was mutated. The simplest explanation of these data is that a single RIPing event caused multiple mutations during pairing in a single region. This is supported by the polarized nature of the nucleotide changes.

GM-28703 and GM-08123. This work was supported by U.S. Public Health Services grants

TABLE 4

Cosegregation of the Al+ phenotype and hygromycin resistance

Phenotype No. of progeny

AI+ HygK 25 AI’ HygS 0 AI- HygK 5 AI- HygS 7

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Communicating editor: R. H. DAVIS

inactivation of the uma-1 gene by repeat-induced point mutation

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