YEAST VOL. 12: 893-898 (1996)

Yeast Sequencing Reports

A Cundidu ulbicuns Gene Encoding a DNA Ligase, ENCARNACION ANDALUZ, GERMAN LARRIBA* AND RICHARD CALDERONE~

Departamento de Microbiologia, F Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain and ?Department of Microbiology and Immunology, Georgetown University, School of Medicine, Washington, D. C., 20007. U.S. A.

Received 21 December 1995; accepted 19 February 1996

A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharornyces cerevisiae. In this system, IMEl function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast an? bacteria was observed. The sequence has been deposited in the EMBL data bank under the Accession NumberfX95001 . f

KEY WORDS ~ Candida albicans; IMEI; CDC9; IME2; ATP-dependenf DNA ligase

INTRODUCTION

Candida albicuns is a diploid or near diploid, dimorphic yeast. Reduction in chromosome number to a haploid condition (meiosis) has not been demonstrated, i.e. a sexual cycle and con- comitant spore formation has either been lost by the organism or conditions which promote these events in the laboratory have not been elucidated. On the other hand, Saccharomyces cerevisiae is a haploid ascomycetous yeast which has two mating types designated as alpha (a) and a (Herskowitz, 1988). The sexual cycle can be induced by mixing opposite mating types which results in the forma- tion of a diploid cell (ala). The diploid cell can be propagated vegetatively in enriched media. How- ever, when a/a diploids are starved for nitrogen and in the presence of only non-fermentable carbon sources, diploid cells undergo entry into meiosis (Malone, 1990).

In diploids, the MATallMATa2 gene product acts to transcriptionally repress haploid-specific

*Corresponding author

genes, including a negative regulator of meiosis, also referred to as RMEI (regulation of meiosis; Mitchell, 1994; Mitchell and Herskowitz, 1986). The target of the RMEI gene is the IMEl gene (inducer of meiosis), which also receives nutri- tional signals, since its expression increases signifi- cantly when cells are shifted to sporulation media (Kassir et al., 1988). Another gene (IME2) appears to be under the regulatory control of IMEl (Mitchell, 1994; Mitchell et al., 1990). In imel mutants, the IME2 transcript cannot be detected; therefore, it would appear that IME2 is located after IMEl in the regulatory cascade (Mitchell, 1994). The IME2 gene product is an inducer in the sporulation cascade and also, apparently, acts as a negative regulator of IMEI,

As stated previously, C. albicans would appear to lack a meiotic cycle. However, Candida homologs of genes associated with the sexual cycle in S. cerevisiae have been reported (Liu et al., 1994; Malathi et al., 1994; Sadhu et al., 1992). For this reason, we have utilized a S. cerevisiae reporter gene construct (IME2-LA CZ)

CCC 0749-503X/96/090893-06 0 1996 by John Wiley & Sons Ltd

894

to complement an imel mutation in S. cerevisiae. In this regard, we have cloned five different fragments from C. albicans genomic DNA (EA1- EA5) which complement such a mutation in S. cerevisiae. Sequence analysis of a 4.5 kb fragment from EA5 indicates that it contains an open reading frame encoding a protein which has a highly significant homology with ATP-dependent DNA ligases of several microorganisms and animals.

E. ANDALUZ ET AL.

(Promega, Madison, WI). The subclone was subjected to exonuclease digestion with Ex0111 using the Erase-a-Base Kit (Promega). This pro- cess created a series of deletion-clones which were used for sequencing with either SP6- or T7- primers. DNA from different clones was amplified in E. coli JM109 (Promega) and two-strand sequencing was performed.

Sequencing was accomplished according to the dideoxy-chain-termination method (Sanger et al., 1977) as described in the Sequenase protocol by using [35S]dATP (Amersham Inc., Arlington Heights, Ill.) and the Sequenase Kit (USA Biochemical Corp., Cleveland, Ohio).

MATERIALS AND METHODS Yeast strains and media

S. cerevisiae AMP918 is an imel strain carrying an ime2::lacZ fusion whose genotype is imel- 12::TRP ime24::lacZ::LEU2 (Smith et al., 1990). In this system, IMEI function can be determined by the induction of the ime2::lacZ gene fusion in transformants plated on sporulation medium upon the addition of X-gal to the plates. Amplification of plasmids in Escherichia coli HB 101 was accom- plished by standard protocols. Transformation of S. cerevisiae was carried out by the lithium acetate method (Becker and Guarente, 1986). C. albicans strains 3153A, 1001 and 4918 have been used previously in our laboratories (Luna-Arias et al., 1991; Manning and Mitchell, 1980). Cells in the yeast form were obtained by growing the organism in liquid YPD (2% glucose, 2% Bacto-peptone, 1% yeast extract) at 30"C, whereas pseudohyphal growth was induced by transfer of starved yeast cells to prewarmed YPD medium (37°C) followed by incubation at the same temperature.

Plasmids and recombinant D N A methods IMEI in YEp24 was kindly provided by Dr AP

Mitchell. Four genomic libraries of C. albicans, each containing 6-10 kb fragments of a partial digest with Sau3AI in ~ 1 0 4 1 , were utilized in complementation experiments (Goshorn et al., 1992). Libraries 1 and 2 and 3 and 4, each in ~1041 , which carries the C. albicans URA3 marker, were used to transform S. cerevisiae AMP918. Five Ura3 transformants (EA1-5) were isolated, each of which gave a blue color in sporulation plates containing X-gal. Clone EA5, which contains an insert of 7.9 kb, was analysed in the present study.

Sequencing A 4.5 kb ClaIIKpnI restriction fragment of the

EA5 insert was subcloned in pGEM7f(+)

Northern analysis Total RNA from yeast or hyphae was iso-

lated from 20 ml cultures of C. albicans grown until OD,,,=0.5 as described by Cross and Tinkelenberg (1991). Northern analysis was based on the methods of Lehrach (1977) and Davis et al. (1986) and was carried out as described in Transfer and Detection Protocols (NENR Research Prod- ucts, Patent 4,455,370). Ribosomal rRNA was determined with 0.04% methylene blue in 0.5 M- sodium acetate (pH 5.2) (Sambrook et al., 1989).

Sequence analyses software DNA sequence assembly was performed using

the DM5 program (Mount and Conrad, 1986). Computer analyses of DNA and peptide sequences were performed at NCBI using the BLAST network service.

RESULTS AND DISCUSSION The complete sequence of the putative C. albicans DNA ligase-encoding gene (Ca CDC9; Figure 1) is available from EMBL data bank (Accession No. X95001). An open reading frame of 2592bp encodes a a polypeptide of 864 amino acids. There are five possible initiation codon (AUG) sites with canonical TATA boxes preceding all of them. The first one has been referred to as the start site for the numbering of nucleotides and amino acids. This ATG is preceded by a CCAAC box (double underlined) identical to that described in the UAS2 region of the CYCl gene from S. cerevisiae (Forsburg and Guarente, 1988). Two stop codons (TGATAA, underlined in Figure 1) mark the beginning of the 3'-non-coding region. Also, a perfect match to the TAG . . . TATGT . . . TTT

RHR DNA LIGASE GENE 895

-317 l~lCCATCAAlCAllllCCClAACAllAllACAlCCAAAAlAllCClllCIuAll~AACllllllCCCAClCAAAlAAl~ClClACAAClClAAA -216 C~CAACTCAlTACCAClTllCClACCACAACAlCAllCTlCAlAACACCAACCACTCAllCCCACAlAllCAAACllCCllACllllCClClCCCllACAlCACA -I08 CCAAGAGAClClClCCCAAACACAAAAAAAAlAAACCCAllClCAlCAllllCACCCllClClACAlCAllllCCCCTTATlCAlClCACCACAACAACACAGlClAC

1 ATCACAlAllllllAUCCACAlCC~ACCACCllClCCAAATCATAllAClCCAlCClTCAClllAllCAClAAACAAllCTllCACAACCl1CACCCCCllACAAAA I ~ ~ Y F L W D I R P P S P N D I T P S ~ ~ ~ L ~ ~ E L F D X ~ D C Y R X

~ ~ E S L C O F R T V T E K K A F I I K T F I N T F R T H I C N D I Y P S A

~ ~ K L I F P E K S G R I Y F I K E v A L A R L L I K ~ Y K I P K E S E D Y

~ ~ I T L H O Y N K L I O R S R R F S I D ~ K K I R D L P L P A S R I I S K

109 CACAGlClTCClCAlTllCCAAClClCACTCACAACAAACCAllCAllAlCAMAClllTAlAAAlACAllTCCAACACACAlTCCCAAlCATAlAlACCCllCACCA

217 AAAClAAlAllCCCCCAAAAClCCCCAACAATAlAllllAlCAAACAACllCCAllCCClCC~ClllTCAllAAAAlClACAAAAlCCCCAACCAAlCCCAACAClAl

325 AlCACAllACACCAllCCIuCAAAClClAlCMAGClCACCAACGllTlCCAllCAlCACAAA~ACAlACClCAlllCCCAIlCCAACCAlCAACCAll~~lTCCAAA

433 ACCACCCCAAllClGCAlAAAClCCACCAATATAClClACCCCAAAlTAAClCllClllACAlCAAllCCCAllCCAAAACClClClCACCClCACATACAlAllClT ~ ~ 6 R R P I V D K L E E I l V P O I N S S L O O L A L E K V S O C O l ~ l L

541 AAACCAClAlllGATI\AlCTAAClAlACCACAAClAAGAlCCCTlAllCAlAlAClAClTAAlAAAlCCAlClTAAClACCAlCCACACAllllllllCAACACllCC I ~ l K P L F D N L S I P E V R Y L I H I L L N K S I L l S n E R F F F N T W

548 CACCCCCAlGGAlAlIGAClCTllACCAlAlCCAAlCACllACAAAACACAllACAAllTlCAACAAATCCCCAlllCACAClACACCClClCCACllACClAlACAl ~ ~ ~ H P D C I R V F S I C N D L ~ K ~ L D ~ S ~ N P D L R L D Q L ~ L ~ I H

767 CCllClllCAAClTl~AACCACACllClCACAAACAllAACAACCACllAlAAAA~CCllClCAAAAACTlACAAACAAACCATCAAAlCCAlCClCCAl~TCACAAA 2 6 3 P C F K F K P ~ L S E R L T T S Y K l l V K K L ~ R K H E ~ D P P ~ ~ K

865 AAAllCCAACAClIACGAllCCAAAAlAAAllllACAlCCAACAGAAAAlCCAlCClG~lCCTAlCCllCllCAlAACCAlCClCATACCllCAACl111lllCACCA Z ~ ~ K F D E L C L E N K F Y I E E ~ K ~ O C O R ~ ~ L L H K ~ C O S F K F ~ S R

873 CCCllCAACCAllAClCllA1llAlACCCACAAACTlTCC~ClllGClCCCClCAClAAATlTlTCCCCCAlCClllTCCCCCAAAlAlACAAlClCYCAllllACAC ~ 5 R L K D Y S Y L Y C E S F O F C A L l K F L A H A F A C N I D S V I L D

la81 GCCOMAlOOTCGCCTATCAiTAlCACCGTAATClCATCCTTCCATT1CCAACTTlAA~AlCAClCCCAAllCAACAAACTClCCCACACT~lAChACChlCC~lCAG M I C E I V A Y O Y E R N V I L P F C l L K S L A l O E S V R O F l l 1 0 0

1188 lAlCAACAACACACAGCAlAlCCAllllYCCllClCTTlCACAllllClllllAAACCCCAAACAllTAAClAAllAlCCCllATlCTlCCCCAAAAACAlClTGAAC

1297 CGAAllCllACACCAATACCAAACACAllTCAAClTllACAlACACCCCTACCl~CllClClCCAACAlAlCCAAACACCAAlCACACAACllClCAClAClCGAlCl

1405 CACCClllAClAllC~ACAAlClACACClCAAClAlCAAAllCAlCCCllCACAMCCCACAllCCATAAAAClCAAACCACACTAlTlACAAAAAlllCClCAAAAl

1513 TTACATCllClCClCATlCCCAAATClCCACCCAlAAAAAAClCClAlAlCfCCCGAllCUAAClClAAClCAlCCClllAllAlACllllllCCACClClCCCAAT

1621 CCTAlACAAAllCACGACTlTCAlAACAlCCMACAClCAClCAlCCAAAClCCAllAAAACACAlClClCAAlCCClCCCCAAClCTlCAlCAAAlllGCAAClAAA

1729 A l l C C C A C l l l l l C C A T 7 C A T C C C * C T C * T T C O T l A C l l l l C C A A A l C A C A G C l C C l l C A A T T C A C A C l C C A C C A G G C A C C C l C l A l C C l C l G C C C l C A A C T l l A C A l

1837 AACAAlCAllClCCAAAAAlAACAGAACACAAAlClAllCAlCAAlClClCACAllACACCAClACACCCAlAlCAACCCAAAlTAlAlCAAlCAlllAAAlAAACCA

1945 C A A A C l G C A l l G G C C A A C A A A C C l C A A C C T C T C T * C T C C l C A A A l l C A A A A A A C l C A A A G T l C A A l C l C A l T l C T l l A C l C C C A l A G A C l T l l l C

2053 AllAlGAGlCAlAAAACACACCCTCACCCCCA~CTlACAACCAlCCAACAAATCAAACCAAlCCllAAACAGlAlCGlCGAAAAATTClCAATlCWllGAlCl lCCA

2181 ACPJIAC1ACCAAAlA~TCClCIlTACTCAACCACAACllCClClATCCAGlCAATAlClAAClAAACCAAllCAlllCCTCAAACCAAlClCCAlAlAlCAAlCCATT

2288 AAAACACCAlClClTCllC~~ClACAACCATATlTlAlAllTCCCAClAAAAAllCCCAlA~llllAACCAlATCCTlCAlCAATAlCCACAClCAlACAlCAlACAC

2377 CAlCCClTGAACAlTCllCl~CCC~A~CllTCTC~~~ClC~~ClTC~~C~CCllCClAAlCCAlllCAllCCCC~CAlCTlAAACCTlCCAlCTACllCllCAAACCl

~ ~ Y E ~ ~ T A Y P F F L V F O I L F L N C K D L T N Y P L F F R K N I L N

U ~ R I L R P I P N R F E V L D T R L C S S L E D I E R A I R E V V S S R C

~ E G L Y L K I V P L K Y E I D C F R N P O W I K V K P E Y L E K F C E N

~ L D L V V l C K S P A l K N S I ~ C C L K S V l D C F l I V F C T C A N

641 C I E I E E F D K I E R L ~ H C K ~ I K ~ ~ ~ ~ ~ P P E L L I K ~ ~ ~ K

677 l P T F ~ l H P S O S L V L E I R A R S I O l R A C l L Y A V G S l L H

( 1 1 3 N N H C R K I R E O K S I O E C V T L O E Y T H I K A N Y I N D L N K A

6 ~ 9 O l A L C K _ K R F P V Y S L D N L L K L K K V K V E S D L F S C l E ~ L

8 8 6 I M S D K R E A O C E V l R I E E ~ K A M V K O Y C C K l V N S V ~ L A

7 2 1 1 N Y C l n V l l E R E L P V S S O f L S K C l D L V K P I Y I I E C I

~ ~ ~ K R C C Y L O L E P Y F I F A S K N W ~ N ~ N H ~ V D O I ~ D S I I I H

7 9 3 H P L N I Y V P K L S E S E L E O L R N G F D W G D L K P W I I L F K G 2485 llClCAlTllACCTGlClCCCAAlAACTlCACCClCCAlTlAAACGCAGAAllGACAGATTCAGlCCAGCTGTCCAACATTllATCCAATCTlCClCAlAGlAAlCCC

2593 l~l~CCAllCCACACCAClAAlCCllCClCAAATlCAlAACAlClCAAACCAAAlTlClCCCCAAATCCTTAllCATAAAAAlCClCCClCllCCAGAATlCCACA 2701 TTlTCllAClCACCCATTCCllCACCCllCAAlCAACAlCAAllAlATlCCCCAlCClCACCAllACAACllTAGClCAlGllClATlTAlATGlClllGTGCGlAAC 2808 CACCACACACClCCCCAACCCAAlllCACAllC~CCTCAAlCAClACAlllCAlAAClAlCCTCClCAllC~~~lllAClATCCACACA~~~~~CClClCCCCTlAT 2917 GllCCAlACAlAllCACAAGCCC~~~CAACllACllACClAllCCAlAAlCCTAClAlCCACClAlAllCCACllCAAAllClllCAlAAAAlAAlCllACllCCACA 3025 CUCAlllACAACAACAACAAAAAAACCAACACAAAClCAAlTlClllllClCCCllCCllClAllAlllllllllllCCllllllCClCCCCCCClCCCClCAAACA

~ ~ ~ L S F Y V C C N N L R L D L K A E L R O S V E L S N I L S N V A H S N P

Figure 1. Nucleotide sequence of Cu CDC9 and deduced amino acid sequence. Upstream regulatory sequences in the 5' region include TATA boxes (underlined) and CCAAC box (double underlined). Three regions have been indicated in the open reading frame: the putative catalytic site (boxed), a DNA ligase signature I1 (underlined), and two adjacent nuclear targeting sequences (double underlined). In the non-coding 3' region, presumptive terminator codons have been underlined and sequences thought to be important for transcription termination and/or polyadenyla- tion have been indicated by heavy dots.

tripartite consensus sequence (heavy dots in Figure 1) that has been postulated to be a signal for transcription termination andlor polyadenylation in S. cerevisiae (Zaret and Sherman, 1982) appears between nucleotides 178 and 239 downstream from the first stop codon.

The program BLAST (Altschul et al., 1990) was used to identify homologous genes. The nine top homology scores were ATP-dependent DNA ligases from different origins, including viruses (Smith et al., 1989), fission yeast (CDCZ7; Johnston et al., 1986; Barker et al., 1987), human DNA ligase I (Barnes et al., 1990), baker's yeast (CDC9; Barker et al., 1985) and Desulfurolobus

ambivalens (see order in Figure 2). The region containing the active center of these ligases is shown in Figure 2. Recently, the amino acid sequence of several human DNA ligase I1 peptides, including a polypeptide that contains the active site lysine residue, has been determined (Wang et al., 1994). We have included such a stretch in Figure 2 to show that it has the highest homology to the C. albicans counterpart. A peculiarity of C. albicans ligase is the presence of Met instead of Tyr adjacent to the Lys shown to be adenylated in bovine DNA ligase (Tomkinson et al., 1991). Accordingly, the proposed active site motif of DNA ligases, Lys-(Tyr/Ala)-Asp-Gly-(Xaa)-Arg

896 E. ANDALUZ ET AL.

C. albicans 301 IEEKMDGDRMLLHKDGDSFK FPSRRLK Human ligase I1 1 SEIKYDGERVQVHK GDHFS YPSRSLK Fowlpox virus 233 VEFKYDGERIQIHKHDKNFK YPSRSLK Rabbit fibroma virus 234 AEIKYDGERVQIHKYDDVYE FYSRNLR Sch. p m b e 413 CEYKYDGERAQVHFTEDGKFYVFSRNSE Vaccinia virus 228 AEMYDGERVQVHKNNNEFA FFSRNMK Human ligase I 565 CEYKYDGQRAQIHALEGGEIFSRNQE s. cerevisiae 415 SEYKYWERAQVHLLNDGTMRIYSRNGE Desulfurolobus 258 VDYKYDGERGQIHKAGDKIF IFSRRLE ambivalens

Figure 2. Alignment of sequences from DNA ligases showing partial homology in the putative active site region. The hexa- peptide marked in boldface is the most highly conserved region in ATP-dependent DNA ligases from several origins: fowlpox virus (Accession No. S41974); rabbit fibroma virus (Accession No. U00761); Sch. pornbe (Barker et al., 1987); vaccinia virus (Smith et a!., 1989); human ligase I (Barnes et uf., 1990), S. cerevisiue (Barker et al., 1985) and Desulfurolobus ambivalens (Kletzin, 1992). The first lysine (K) of each sequence is thought to be adenylated in all of them. Gaps have been inserted to maximize homology. Additional amino acids from regions showing maximal homology are also indicated in bold type.

C. albicans 469 EGLVLK N VQLKYEIDGFRNPDWIK aba a a an* a Figure 4. Northern analysis of Cu CDC9 mRNA transcript.

(A) Total R N A from C. nlbicans 3453A yeast (Y) or pseudo- hyphal (M) form was probed with a 1.3 kb internal probe XhoI-X!zoI (from base 1720 to base 3062 of Figure 1). (B) Ribosomal RNA species were stained with niethylene blue for

Human ligase I 720 EGLMVKTLD VDATYEIAK RSENWLK

Sch. p m b e 568 EGLWKMLEGPDSHYEPSK RSRFlWLK

*** a*.

..a*.. .. . .* *** aaaaa**.*** *aaaaaa an* at.

S . cerevisiae 571 EGLWiK%LEGPESBYEPSK RSRNRLK

Human ligase I17 GTYEPGK RRIILK quantification purposes. Figure 3. Alignment of the deduced amino acid sequences from the signature I1 region of several ligases. Amino acids conserved in, at least, two ligases are dotted. Gaps have been inserted to maximize homology.

(Tomkinson et al., 1991), should be modified to Lys-(Tyr/Ala/Met)-Asp-Gly-(Xaa)-Arg. The pres- ence of tripeptide (Phe/Tyr)-Ser-Arg about 13 positions apart from the active site motif is also a common feature of each ligase.

Figure 3 shows the comparison of the amino acid sequences in the DNA ligase signature 2 region. Whereas these stretches are extremely well conserved in S. cerevisiae and Schizosaccharo- myces pornbe ligases, their similarity to the human DNA ligase I and especially to C. albicans DNA ligase is much lower. Finally, two overlapping bipartite nuclear targeting sequences (underlined in Figure 1) are observed. These fit the distinctive amino acid pattern proposed by Dingwall and Laskey (1991), i.e. two adjacent basic amino acids (Arg or Lys), followed by a spacer region of any ten amino acids, and at least three basic residues (Arg or Lys) in the five positions after the spacer region. It is noticeable that DNA ligases from S. cerevisiae and Sch. pornbe, contain similar stretches which carry only two, instead of three, basic amino acids in the last five amino acids and the same is true for DNA ligases from some poxviruses (fowlpox or

Vaccinia viruses). Although these viruses replicate in the cytoplasm of animal cells, when their DNA ligases are expressed in yeast, some enzyme must reach the nucleus since Vaccinia ligase comple- ments a cdc9 mutant from S. cerevisiae (Kerr et al., 1991). This could mean that the entry of proteins into the nucleus of C. a1bicar.r~ is more restrictive as compared with the same process in budding or fission yeast. Interestingly, human DNA ligase I carries, like its C. albicans counter- part, a canonical nuclear targeting motif.

Expression of the Ca CDC9 gene was deter- mined by using Northern analysis. As shown in Figure 4, a major mRNA species of about 3.8 kb was seen in C. albicans strain 3153A when grown as yeast (lane a) or during formation of pseudo- mycelium (lane b). A faint band of 1.8 kb was also observed in some experiments. The intensity of the latter increased in old samples and its size was even smaller than the open reading frame of Ca CDC9, suggesting that some in vitro degradation was taking place. The same was true for the other two C. albicans strains, 1001 and 4918 (not shown). Figure 4, bottom, also shows the ribosomal RNA patterns from the corresponding samples, which were used for quantification purposes.

Although our results suggest a possible relation- ship of Ca CDC9 to S. cerevisiae IMEl , at present there are insufficient data to speculate any further.

RHR DNA LIGASE GENE 897

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ACKNOWLEDGEMENTS The authors wish to thank Dr A. P. Mitchell (Institute of Cancer Research and Department of Microbiology, Columbia University) for S. cerevisiue AMP 918 and IMEl constructs. The C. ulbicans genomic libraries were kindly provided by Dr S. Scherer (Department of Microbiology, University of Minnesota School of Medicine). This research was supported in part by Public Health Service Grant PO1 A137251 to R. C. and PB-92- 524 from DGICYT to G. L. Sabatticals for E. A. and G. L. at Georgetown University were possible within the program Estancia de Profesores e inves- tigadores en el extranjero from Ministerio de Educacion y Ciencia.

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