Copyright 0 1995 by the Genetics Society of America

MLBI, a Suppressor of m k 7 - l (a Mutant RPL4A), Is RPL4B, a Second Ribosomal Protein L4 Gene, on a Fragment of Saccharomyces Chromosome XZZ

Yasuyuki Ohtake' and Reed B. Wickner

Section on Genetics of Simple Eukaryotes, Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Manuscript received November 1, 1994 Accepted for publication February 13, 1995

ABSTRACT The mak7-1 mutant loses the killer toxinencoding MI dsRNA. MAK7 is RPL4A, one of two genes

encoding ribosomal protein L4. KRBl is a dominant suppressor of mak7-1 that is tightly centromere- linked, but not linked to centromere markers of chromosomes Z-XW. Our orthogonal field agarose gel electrophoresis analysis of chromosomal DNA from strains with KRBl shows a novel band of -250 kb. This band hybridizes with an RPL4Bspecific probe, but not an WL4A (MAK7)specific probe. The RPL4Bspecific probe also hybridizes to chromosome X I where the original RPL4B is located. KRBl is meiotically linked to this extra chromosome. Disruption of either the RPL4B gene on chromosome XII or that on the extra chromosome results in loss of the killer phenotype and a decreased concentration of free 60s subunits. Thus, the RPL4B on the extra chromosome is KRBl and is active. The extra chromosome contains chromosome X I sequence between Lambda 5345 clone (ATCC70558) and Lambda 6639 clone (ATCC71085) of Olson's Lambda library, indicating that KRBl represents a chromo- somal rearrangement involving chromosome XZZ and explaining the earlier genetic data.

T HE L A dsRNA virus of Saccharomyces cermisiae has a single 4.6-kb segment encapsidated in isometric

particles. Its (+) strand has two open reading frames (ORFs), the 5' gag encoding the major coat protein (Gag) and the 3' polencoding a multifunctional protein domain. Pol is expressed only as a Gag-Pol fusion pro- tein formed by a -1 ribosomal frameshift event in the region of overlap of the two Oms. MI is a 1.8-kb satellite dsRNA of LA, encoding the killer prepro-toxin but de- pending entirely on L A proteins for its replication and packaging (reviewed by WICKNER 1992, 1994).

Among the chromosomal genes necessary for the propagation of MI is MAK7, which is identical to RPL#A, one of two genes encoding ribosomal protein L4 ( A R E - VALO and WARNER 1990; YON et al. 1991; Y. OHTAKE and R. B. WICKNER 1995). Spontaneous dominant chromo- somal suppressors of muk7-1 were isolated at high fre- quency (WICKNER and LEIBOWITZ 1977). Their genetic analysis proved that they were all tightly centromere linked, but not linked to the centromeres of chromo- somes I-XVI, and so they were considered to define a single locus, called KRBl for killer replication bypass (WICKNER and LEIBOWITZ 1977). Since MAK7 was mapped on WII (WICKNER and LEIBOWITZ 1976), the possibility that KRBI was simply an extra chromosome WIIwas tested and excluded (WICKNER and LEIBOWITZ 1977).

Cmespondingauthur Reed B. Wickner, Bldg. 8, Room 207, National Institutes of Health, Bethesda, MD 208924830, E-mail wickner@helix.nih.gov

Ltd., Z-lSl, Ohmon-kite, Ohta-ku, Tokyo 143, Japan.

Genetics 140: 129-137 (May, 1995)

' Present address: Central Research Laboratory, Asahi Breweries,

Here we show that KRBl is a second copy of RPL4B, the other gene encoding ribosomal protein L4, on a 250-kb fragment of chromosome X I that includes the centromere and most of the left arm. Two L4 genes are necessary for propagation of MI dsRNA, although one is sufficient for cell growth.

MATERIALS AND METHODS

Strains, media, phages and plasmids: Strains of S. mevisiae used in this study are listed in Table 1. Escha'chia coli DH5a (F- @80dlacZAM15A(lacZYA-argF) U169 endl r e d l hsdRl7- (a- q+) doeR thi-1 supE44 gyrA96 relAl X-) was used for DNA manipulations. E. coli C600 (supE44 hsdR thi-1 thr-1 leuB6 lacy1 tonA21) was used as the host for lambda phage. Lambda phage clones of the OLSON library (OLSON et al. 1986; RILES et al. 1993) were obtained from ATCC, BIO 101 (CA), or kindly provided by DR. LINDA RILES at Washington University School of Medicine. The lambda phage DNAs were prepared by using Lambdasorb (Promega) following the manufactur- er's protocol. The plasmids pYRC20 (RPL4A = MAK7) (Y. OHTAKE and R. B. WICKNER 1995) and pL41 (RPL4B) (YON et al. 1991) were constructed previously (Figure 1). Deletion of the QnI fragment from pYRC20 produced pX3KX16 in which the T3 promoter is brought adjacent to the QnI site inside RPL4A. We used pYRC412 (@4B::LEU2) (Y. OHTAKE and R. B. WICKNER 1995) for gene disruption of a KRBl strain (Figure 1). The 6.7-kb HindIII fragment from pL4-1 carrying RPL4B was inserted into the HindIII site of pRS316 to con- struct pYRC416.

Genetic manipulation: The killer assay was performed as described previously (RIDLEY et al. 1984). Tetrad analysis was performed by the usual methods (MORTIMER and HAW- THORNE 1975). Yeast were transformed by the lithium acetate method (ITO et al. 1983).

Pulsefield gel electrophoresis and Southern and Northern hybridization: Yeast chromosomes were prepared as described

130 Y. Ohtake and R. B. Wickner

TABLE 1

Yeast strains

Strain Genotype

539 MATa ural mk7-1 KRBl 2473 MATa ura3 mk7-I KRBl 2785 MATa arg4 leu2 ura3-52 mak7-1 KRBl 2787 MATa h 2 ura3-52 mak7-I KRBl 2788 MATa met5 leu2 ura3-52 mak7-1 KRBl 2789 MATa arg4 leu2 ura3-52 mak7-1 KRBl s37 MATa h 2 - I met5 ?? 2907 MATa his3-200 leu2 trpl-901 ura3-52 ade2-10 YOC76 MATa his 3-200 leu2 trpl-901 ura3-52 ade2-10 rpl4A::LEU2 (Y. OHTAKE and R. B. WICKNER

YOClOl MATa his3 leu2 trpl ura3 mak7-1 KRBl YOC102 MATa his3 leu2 trpl ura3 mak7-1 KRBl rpl4B::LEU2 YOC103 MATa his3 leu2 trpl ura3 mak7-1 krbl::LEU2 YOC104 MATa his3 leu2 trpl ura3 mak7-1 KRBl rpl4B::LEU2 YOC105 MATa his3 leu2 trpl ura3 mak7-1 krbl::LEU2

1995)

K+ means the strain is a killer.

previously (SCHWARTZ and CANTOR 1984) and separated on a 1% agarose gel in 0.5X TBE buffer at 14" using a CHEF apparatus (Bio-Rad Laboratories, CA). After the gel was stained with ethidium bromide and a photograph taken, the chromosomes were blotted onto a nylon membrane as de- scribed previously (GERRING et al. 1991).

The L4 RNA probe to detect both the RPL4A and RPL4B genes was prepared by transcription with [cY-'~P]UTP and T3 RNA polymerase of Msddgested pX3KX16 (L4 probe in Fig- ure 1). This probe differs in only 23 out of 405 bp between RPL4A and RPL4R. pYRC67 was made from pYRC315 by dele- tion of the ClaI fragment fusing ZEU2with the T3 promoter. An RNA probe for IXU2 was prepared from an EcoRI digest of YRC67 by transcription using T3 RNA polymerase and [a- ' PIUTP. Lambda phage DNA was labeled by the Multi Prime labeling kit (Promega) using [CY-~'P]~CTP.

Southern hybridization using the labeled RNA probes was performed as follows. The prehybridization and hybridization were for 1 hr at 55" in 5X SSPE, 50% formamide, 0.1% SDS, 5X Denhardt's solution, and 10 pg/ml denatured salmon sperm DNA and for 15 hr in the same buffer with the labeled RNA probe, respectively. After the hybridization, the filters were washed twice with 2X SSPE and 0.1% SDS for 15 min at 55", 1 X SSPE and 0.1% SDS for 30 min at 55", and 0.1 X SSPE and 0.1% SDS for 30 min at room temperature.

E

- URobo

RPL4A X M S K H - I I pYRC2O

FIGURE 1 .-Plasmids and probes used in this study. 0, open reading frame of RPL4A and RPL4B; restriction sites shown are as follows: C,

PnI; M, Msd; S, M I ; Sp, Sphl; X, XbuI; Xh, XhoI.

H c w s SP

I I I pL4-1 ...a ".._ ...- -... .... -.. -..

RR48 Specmc pDbe &WWWCA4CA& ChI; E, EcoRI; H, HindIII; Hp, HpaI; K,

Southern hybridization using the labeled phage probes was performed as follows. The prehybridization and hybridization were for 1 hr at 65" in 6 X SSC, 5 X Denhardt's solution, 0.5% SDS and 10 pg/ml denatured salmon sperm DNA and for 15 hr in the same buffer with the labeled phage, respectively. After the hybridization, the filters were washed twice with 2X SSC and 0.1% SDS for 15 min at 65", 1 X SSC and 0.1 % SDS for 30 min at 65", and 0.1 X SSC and 0.1 % SDS for 30 min at room temperature.

Probes specific for the coding regions of RPL4A or RPL4B, respectively, were synthesized and used to detect the RPL4A or RPL4B gene, or their mRNAs separately: RPL4A CGAT- CAAGGCAACAACGTGG and RPL4B: CAATCAAAGAGA- CAACATGG.

These oligomers were labeled using T4 polynucleotide ki- nase and [y-"P]ATP. Southern hybridization and Northern hybridization using these probes were performed as follows. Prehybridization and hybridization were for 1 hr at 45" in 5X SSPE, 0.3% SDS and 10 pg/ml denatured salmon sperm DNA and for 15 hr at 45" in the same buffer with the labeled probe, respectively. After the hybridization, the filters were washed twice with 5X SSPE and 0.1% SDS for 15 min at 45", and once with 2X SSPE and 0.1% SDS for 15 min at room temperature. Polysome preparation and analysis: Polysome preparation

Plasmid

E

I lkb I

(A)EtBr staining

KRB7 Strains

8 7

KRBl Is RPL4B on a Fragment of MI 131

+ XI1

(B) L4 Probe

KRB7 Strains v) -

4 XI1

4 vlll

(C)RPUA-specific probe

KRB7 Strains

+ x11

+ Vlll

(D)RPL4B-specific probe

KRB7 Strains v) 7

+ XI1

4 vlll

+Ex Chr. - + Ex. Chr.

FIGURE 2.-KRBZ strains have a 250-kb extra chromosome that hybridizes to an RPL4Bspecific probe. (A) Ethidium bromide- stained gel of the KRBl strains. Pulse field gel electrophoresis used a pulse time of 60 sec for 15 hr and 90 sec for 8.5 hr at 200 V at 14". (B) Southern hybridization with the L4 probe hybridizing to both the RPL4A and RPL4B genes. ( C ) Southern hybridization with the RPL4A-specific probe. (D) Southern hybridization with the RPL4Rspecific probe. The extra chromosome and chromosomes MI and WII are indicated by arrows. Strains are indicated on the top of the lanes.

132 Y. Ohtake and R. B. Wickner

1 2 3 4 5

I II II I I I I I Killer + - - + - + + - + - + - - + - + + - + -

EX. Chr. + * . 0 0 e

FIGURE S.-kXBKRBI is meiotically linked to an extra chromosome. The conditions of the pulse field gel electrophoresis were the same as for Figure 2. The extra chromosome is indicated by an arrow.

and analysis were performed as described previously (Y. Ow TAKE and R. B. WICKNER 1995).

RESULTS

Mapping of RPL4B: MAK7 ( = R P I A A ) was mapped on the left arm of chromosome WIZ by classical genetic methods (WICKNER and LEIBOWITZ 1976). We also showed that the h4AK7 gene is identical to the RPL4A gene, one of the two genes encoding ribosomal protein L4 (Y. OHTAKE and R. B. WICKNER 1995). Here, we determine the location of RpI,4B, the other gene en- coding ribosomal protein L4 (AREVAI .~ and WARNER 1990; YON el al. 1991).

Southern hybridization with an RPL4A gene probe,

made by T7 RNA polymerase transcription of EcoRI- digested pYRC20, against the standard chromosomes separated by pulse field gel electrophoresis detected chromosome WIZ, and XII or N. Since RPL4A (MAK7) is on chromosome WZZ, the RPL4B gene is located on chromosome X I Z or N. The RpI,4B gene probe, made from pL4-1 by transcription with T7 RNA polymerase, hybridized with XIZor Nsupporting this conclusion. In dot blot hybridization against blots of Lambda clones of OWON'S yeast genomic library (OLSON el al. 1986; RILES el nl. 1993), the RPL4R probe hybridized with Lambda 4354 (chromosome MI) and Lambda 1398 (chromosome X) (data not shown). Together, the data suggest that the RPL4B gene is located in Lambda 4354 on the left arm of chromosome MI. This was confirmed

Killer

Ex. Chr.

KRRl Is WL4B on a Fragment of XII

6 7 8 9 10

133

I II II II II I " + + + + - - + - + " + - + - + + -

by Southern hybridization against restriction digests of the Lambda 4354 clone (data not shown). CUSICK also mapped RPL4B to the same position next to RNPl (Cu- SICK 1994). Lambda 4354 also contains the COFI gene that is 15 cM to the left of the centromere of XII (MOON et al. 1993).

KRB1 strains have a 250-kb extra chromosome hy- bridizing with an RPL4B probe: The chromosomes of KRBl strains were analyzed by pulse field gel electro- phoresis (Figure 2A). We found that five out of six KRBl strains have a 250-kb extra chromosome migrat- ing between chromosomes III and VI. We performed Southern hybridization against this blot using the L4 probe (Figure 1) that hybridizes to both RPL4A and RPL4B. We found that this probe hybridized to the extra chromosome in addition to chromosomes XII (RPL4B) and WII (RPL4A) (Figure 2B). In this and

other blots, hybridization of all probes to chromosome XIIwas weak, but definitely present on the original film. Aberrant migration and poor transfer of XII DNA in blots has often been noted by others. Next, we used the RPL4A- and RPL4Bspecific probes. The RPL4Bspecific probe hybridized with the extra chromosome and chro- mosome XII (Figure 2D), while the RPL4A-specific probe hybridized with only chromosome VIII (Figure 2C). These data indicate that the K R R I chromosome might be this extra chromosome and the KRBl gene might be a second copy of the RPL4B gene.

KRBl is meiotically linked to the extra chromosome: The KRBl gene was not linked to centromere markers of any of the known 16 chromosomes but was tightly centromere-linked by tetrad analysis (WICKNER and LEI- BOWTZ 1977; WICKNER et nl. 1983). To confirm that the extra chromosome has the same characteristics as the

134 Y. Ohtake and R. R. Wickner

A LEU2 probe

80s

,i 80s

80s

i L Y KRBl TNlOl)

K

i FIGURE 4.-The disruptants of the RPL4B gene on chromosome XII and of that on the extra chromosome lost the killer

phenotype and have a lower concentration of free 60s ribosomal subunits than the parent strain. (A) Pulse field gel electrophore- sis of the strains disrupted for RPL4B on chromosome XII or the extra chromosome. Chromosome XII (RPL4B), III (LEU2), and the extra chromosome ( K R B I = RPL4B) are indicated by arrows. The conditions of pulse field gel electrophoresis were the same as in Figure 2. The right panel is Southern hybridization using the LEU2 probe to detect the location of the disruption site. Strains are indicated on the top of the lanes. (B) Polysome profiles of rp14B disruptant strains. The killer phenotype is expressed as (+) or (-) below the polysome profile. All strains are mak7-l and carry the extra chromosome.

KRBI chromosome observed previously, we performed tetrad analysis with a cross of the mak7-1 KRBl strain 539 and the rpl4A disruptant strain YOC76 and analyzed the chromosomes of 10 tetrads by pulse field gel electro- phoresis (Figure 3). The extra chromosome was identi- fied by Southern hybridization with the RPL4Rspecific probe. As expected, all spores with the extra chromo- some showed the killer phenotype and those without it showed the nonkiller phenotype. The comparison of the killer phenotype and the trpl marker showed that

the KRBl phenotype was tightly centromere-linked (PD = 4, NPD = 6, T = 0). Thus, the KRBl chromosome is this extra chromosome.

KRBl is a second RPL4B gene: To confirm that the KRBl gene is the second W L 4 B gene on the extra chromosome, RPL4B gene disruption was performed. The mak7-1 KRBl strain YOClOl was transformed with the rpl4B::IJCUZ disruption gene and two q14B dis- ruptants on chromosome HI, and two disruptants on the extra chromosome were obtained (Figure 4A). All

KRBl Is RF’L4B on a Fragment of XI1 135

Killer: + RPLQA

+ FIGURE 5.-The second L4 gene (RPL4B) complements the mak7 (rpZ4A) disruption. The killer phenotype is shown as + or -

- below the polysome profile.

four disruptants lost the killer phenotype, suggesting that the RPL4B gene on the extra chromosome is the KRBl gene and is active. It also shows that both copies of the RPL4B gene must be active to suppress muk7-1. We previously showed that disruption of q14B in a wild- type strain results in a decrease in the amount of free 60s ribosomal subunits and a very low MI copy number (Y. OHTAKE and R. B. WICKNER 1995). Further, while the mak7-1 KRBl strain (YOC101) has a normal amount of free 60s ribosomal subunits, a decrease was observed in the krbl and q l4B disruptant strains similar to that seen in the qZ4A (mak7) disruptant strain (Figure 4B and Figure 5). We also observed that RPL4B transcripts in all disruptants decreased in comparison with the par- ent strain but did not disappear (data not shown). These data suggest that both RPL4B genes of KRBl strains are active and that the RPL4B on the extra chro- mosome is the KRBl gene. Thus, two copies of RPL4B can suppress a defect in RPL4A, but a single copy of RPL4B cannot. Since YON et al. showed that disruption of both q l 4 A and q l 4 B is a lethal event (YON et al. 1991), it is clear that one L4 gene is sufficient for growth, but two are necessary for propagation of MI dsRNA.

We confirmed this interpretation by introduction of a single copy of the RPL4B gene into the q l 4 A ( m u k 7 ) disruptant strain. As shown in Figure 5, the killer phe- notype and the concentration of the free 60s subunits

of the q l 4 A disruptant strain YOC76 were restored by the single copy plasmid pYRC416 containing the RPL4B gene, suggesting that the second RPL4B suppresses the rpl4A disruption (Figure 5). Similarly, RPL4A on a CEN plasmid restores to normal the deficiency of 60s sub- units produced by an q l4B disruption mutation.

The extra chromosome contains chromosome W se- quences: Where did the extra chromosome come from? Although many genes have been mapped, KRBl is the only gene that maps on an extra chromosome, even though its size is 250 kb. Furthermore, an extra chrome some was not observed in any other strains. Thus it is doubtfbl that it is a novel chromosome. Since the extra chromosome contains the RPL4B gene, we suspected that it arose from chromosome XI. We analyzed the extra chromosome by Southern hybridization using as probes Lambda clones of chromosome XlZfrom Olson’s Lambda library (OLSON et al. 1986; BUS et al. 1993). As shown in Figures 6 and 7, all clones used hybridized with chrome some X 4 although weakly, probably because of aberrant migration of X I or poor transfer to the filter. Lambda clones 5345 (ATCC70558) and 5379 (ATCC70559) hy- bridized with all chromosomes because they have tele mere sequences. All clones from Lambda 5345 (ATCC70558), almost at the extreme left end, to Lambda 6639 clone (ATCC71085), located to the right of the cen- tromere, hybridized with the extra chromosome. The right-most clone that hybridizes, Lambda 6639 clone, is

136

A

Y. Ohtake and R. B. Wickner

0

I

-.

near the ASP5 gene. The earlier genetic data showed that asp5 was clearly segregating 2+:2- in crosses of K R B I strains with asp5 strains. Thus, the extra chromosome does not have the ASP5 gene. This length determined by hy- bridization with Lambda clones corresponds exactly to the length observed in the pulse field gel electrophoresis (Figure 7). These results indicate that KRBl represents a chromosomal rearrangement involving chromosome X I .

DISCUSSION

KRBl was first identified as a dominant chromosomal suppressor of mak7-I, restoring the cell's ability to prop agate the MI dsRNA encoding the killer toxin (WICKNER and LEIBOWITZ 1977). Although K R B I was tightly cen- tromere-linked, no linkage could be detected with the centromeres of the known chromosomes I-XVl. The possibility that KRBI was an extra copy of one of the normal chromosomes was considered and ruled out based on the normal 2+:2- segregation of markers on chromosomes I-XVlin crosses of the type KRBl X standard marker (WICKNER and LEIBOWITZ 1977). The chromosome XII marker used was asp5. MAK7is identi- cal to RPL4A (Y. OHTAKE and R. B. WICKNER 1995),

0 50 100

FIGURE 6.-Extent of chromosome XI1 sequences on the extra chromo- some. The conditions of pulse field gel electrophoresis in B were the same as in Figure 2. For A the pulse time was 15 sec for 20 hr at 200 V at 14" to separate more clearly the extra chromosome from other chromosomes hybridizing with the probe. The chromosomes were prepared from the KRBl strain 539. The probes of A clones are expressed as ATCC number above the lanes. The ex- tra chromosome is indicated by arrows. In B, the most slowly migrating chromo- some is XU.

one of the two genes encoding ribosomal protein L4 (AREVALO and WARNER 1990; YON et dl. 1991). We found an extra chromosome of -250 kb in KRBl strains, which cosegregates with KRBl in meiotic crosses and carries a copy of RPL4B. Inactivation of either copy of RPL4B in KRBl strains results in failure to suppress the need for MAK7 (RPL4A) for propagation of M I , showing that the second RPL4B gene on the extra chro- mosome is the KRBl gene.

We found that RPL4B is on the left arm of XII and that the migration of the extra chromosome on orthog- onal field gel electrophoresis is consistent with the ex- tent of chromosome XII sequences that we have shown it contains. This suggests that the extra chromosome is linear, although we have not directly tested this point. This fragment of XII does not include asp5, explaining why it was not detected in the earlier study in which asp5 was used as the standard chromosome XII marker. In the earlier study, it was noted that KRBl arose fre- quently in most muk7-1 spore clones from crosses of the type, wild-type killer X muk7-I. That not all of the cells of the spore clone were killers shows that the extra chromosome was not inherited from one of the parents of the cross and must have arisen by an event during

150 200 250

70558 70275 70406 70194 70899 70681 70559 70280 71 166 70276 71 085 70477

70563

FIGURE 7.-The physical map of the extra chromosome. The A clones of Olson's yeast genomic library (OLSON et al. 1986; RILES et al. 1993) are indicated by lines with their ATCC number. ATCC70275 cames the RPL4B.

KRBl Is RF'L4B on a Fragment of XZZ 137

outgrowth of the spore clone. Since M I is lost from the cells of the mak7-l spore clones, and once lost, the suppression would not have been observed, the extra chromosome must have arisen frequently and early enough to appear in the few cells of the spore clone that had not yet lost MI. The nature of the event pro- ducing the extra chromosome will not be clear until the structures of its ends are determined.

Although W L 4 B is not particularly close to its centro- mere, KRBl never recombined with its centromere. This is because it is not the particular copy of RPL4B on the extra chromosome that produced the suppres- sion, just the presence of an extra copy. The extra copy of W L 4 B is present in the spore clones that get the extra chromosome, a perfectly centromere-linked event. In the earlier studies, spore viability as high as 100% was observed in crosses of the type mak7 KRBI X mak7, suggesting that the two complete chromo- somes XI1 preferentially disjoined. Crosses of the type mak7 KRBl X mak7 KRBl, which should have two com- plete copies of XZZ and two fragments, also gave high spore survival and frequently produced 2K+:2K- segre- gation, suggesting the frequent loss of one of the chro- mosome XIIfragments in the diploid.

While only one L4 gene is necessary for cell growth (AREVALO and WARNER 1990; YON et al. 1991), two cop- ies are needed for propagating MI dsRNA. Our recent data (Y. OHTAKE and R. B. WICKNER 1995) shows that the level of free 60s subunits is critical for propagation of MI dsRNA. The mRNA of the helper virus L-A lacks poly(A), and there is evidence that the 3' poly(A) SWUC- ture is involved in translation initiation at the point of 60s subunit association. Thus poly(A)- mRNA is expected to be affected more severely than the cellular poly(A) + mRNAs by a deficiency of 60s subunits.

Dr. Y. OHTAKE was supported by Asahi Breweries (Japan).

LITERATURE CITED

AREVALO, S. G., and j. R. WARNER, 1990 Ribosomal protein L4 of Saccharomyces cmm'siae: the gene and its protein. Nucleic Acids Res. 18: 1447-1449.

CUSICK, M. E., 1994 RNP1, a new ribonucleoprotein gene of the yeast Saccharomyces meuisiae. Nucleic Acids Res. 22: 869-877.

GERRING, S. L., C. CONNELLY and P. HIETER, 1991 Positional map ping of genes by chromosome blotting and chromosome frag- mentation, pp. 57-76 in Guide to Yeast Genetics and Molecular Biology, Vol. 194, edited by C. GUTHIUE and G. R. FINK. Academic Press, San Diego.

ITO, H., Y. FUKUDA, K. MURATA and A. KIMuRA, 1983 Transforma- tion of intact yeast cells treated with alkali cations. J. Bacteriol.

MOON, A. L., P. A. JANMEY, K. A. LOUIE and D. G. DRUBIN, 1993 Cofilin is an essential component of the yeast cortical cytoskele- ton. J. Cell. Biol. 120: 421-435.

MORTIMER, R. K., and D. C. HAWTHORNE, 1975 Genetic mapping in yeast, pp. 221-233 in Yeast Cells, edited by D. M. PRESCOTT. Academic Press, New York.

OHTAKE, Y., and R. B. WICKNER, 1995 Yeast virus propagation de- pends critically on free 60s ribosomal subunit concentration. Mol. Cell. Biol. (in press).

OLSON, M. V., J. E. DUTCHIK, M. Y. GRAHAM, G. M. BRODEUR, C. HELMS et al., 1986 Randomclone strategy for genomic restric- tion mapping in yeast. Proc. Natl. Acad. Sci. USA 83 7826-7830.

RIDLEY, S. P., S. S. SOMMER and R. B. WICKNER, 1984 Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by LA-HN and confer cold sensitivity in the presence of M and LA-HN. Mol. Cell. Biol. 4: 761-770.

RILES, L., J. E. DUTCHIK, A. BAKTHA, B. K. MCCAULEY, E. C. THAER et aZ., 1993 Physical maps of the six smallest chromosomes of

ics 134: 81-150. Saccharomyces cerevisiaeat a resolution of 2.6 kilobase pairs. Genet-

SCHWARTZ, D. C., and C. R. CANTOR, 1984 Separation of yeast chrc-

153: 163-168.

mosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37: 67-75.

WICKNER, R. B., 1992 Double-stranded and single-stranded RNA vi- ruses of Saccharomyces cereuisiae. Annu. Rev. Microblol. 46: 347- 375.

WICKNER, R. B., 1994 Viruses of yeasts, fungi and parasitic microor- ganisms, pp. in Fields Virology, Ed. 3, edited by B. N. FIELDS, D. M. KNIPE, P. M. HOWLEY. Raven Press, New York (in press).

WICKNER, R. B., F. BOUTELET and F. HILGER, 1983 Evidence for a new chromosome in Saccharomyces cerevisiae. Mol. Cell. Biol.

WICKNER, R. B., and M. J. LEIBOWITZ, 1976 Chromosomal genes essential for replication of a double-stranded RNA plasmid of Saccharomyces mevzsiae: the killer character of yeast. J. Mol. Biol.

WICKNER, R. B., and M. J. LEIBOWITZ, 1977 Dominant chromosomal mutation bypassing chromosomal genes needed for killer RNA plasmid replication in yeast. Genetics 87: 453-469.

YON, J., A. GIALONGO and M. FRIED, 1991 The organization and expression of the Saccharomyces m i s i a e L4 ribosomal protein genes and their identification as the homologues of the mamma- lian ribosomal protein gene L7a. Mol. Gen. Genet. 227: 72-80.

Communicating editor: E. JONES

3: 415-420.

105: 427-443.