Gene, 119 (1992) 175-182 0 1992 Elsevier Science Publishers
GENE
B.V. All rights reserved.
175
0378-I 119/92/$05.00
06624
Cloning, sequence homobasidiomycete (Antitubulin
drugs; benomyl;
Patrick Russo*,
and expression of a P-tubulin-encoding Schizophyllum commune exon; intron;
codon
usage; fungus;
Jarmo T. Juuti* and Marjatta
nuclear
migration;
gene
in the
polymorphism)
Raudaskoski
Department of Botany, University of Helsinki, 00170 Helsinki, Finland Received
by J.K.C.
Knowles:
3 February
1992; Revised/Accepted:
13 April/l1
May 1992; Received
at publishers:
1 June 1992
SUMMARY
The /I-tubulin (fiTub)-encoding gene (U-2) of Schizophyllum commune is the first tubulin gene isolated, cloned and sequenced from higher filamentous fungi (homobasidiomycetes). The S. commune tub-2 gene is organized into nine exons and eight introns. The introns vary from 48 to 107 nt in length, and are distributed throughout the gene. The tub-2 exons code for a protein of 445 amino acids (aa), which shows great homology with BTubs of filamentous ascomycetes, plants, and animals, but less homology with yeasts. The codon usage of tub-2 from S. commune is biased, as it is in most PTub-encoding genes of filamentous fungi. The S. commune PTub shows a conserved aa sequence in the C-terminal domain, which is suggested to interact with microtubule-associated proteins in animals. In contrast, the S. commune /ITub deviates from most known PTubs by having a Cys16’ residue, which might be significant for the insensitivity of S. commune haploid strains to the antimicrotubule drug, benomyl. In tub-2 of different haploid strains, sequence polymorphisms occur in the 5’ and 3’ flanking regions. The expression of tub-2 is high in young mycelium, which has a high number of extending apical cells, but decreases with the aging of the mycelium. No significant difference in the hybridization signal intensity for the tub-2 transcripts was recorded either during intercellular nuclear migration at early mating, or in mycelia with a mutation in the B mating-type gene. The weak signal obtained with the 3’ end of tub-2 as a probe in Southern hybridizations, under conditions of low stringency, warrants attention to the possibility that S. commune might have another PTub-encoding gene highly divergent from tub-2.
In microtubules, tubulins (Tubs) are the major protein components, and by cloning the tub genes it has been possible to analyse the expression of these genes at different
developmental stages in a wide range of eukaryotic cells including fungi. In fungi the cloning and characterization of tub genes has been restricted to euascomycetes (Orbach et al., 1986; May et al., 1987; Byrd et al., 1990; Sherwood and Somerville, 1990; Doshi et al., 1991), deuteromycetes
Correspondence fat Dr. M. Raudaskoski,
Abbreviations:
INTRODUCTION
sity of Helsinki, Tel. (358-O) *Present
Unioninkatu
Department
44, 00170 Helsinki,
of Botany,
Univer-
Finland.
ethidium
1918606; Fax (358-O) 1918656.
addresses:
versity of Rochester, Fax (716) 2712683; sinki, Arkadiankatu Fax (358-O) 1917376.
(P.R.) Department Rochester,
(J.T.J.) Department 7, 00100 Helsinki,
kb, kilobase
body; MAP, microtubule
of Biochemistry,
NY 14642, USA.
aa, amino acid(s); bp, base pair(s); BTub, b-tubulin;
bromide;
Box 607, Uni-
Tel. (716) 2756663;
of Genetics, University of HelFinland. Tel. (358-O) 1917380;
associated
or 1000 bp; mAb, monoclonal protein; nt, nucleotide(s);
phyllum; SDS, sodium dodecyl sulfate; tub, gene encoding gene encoding /ITub of S. commune; Y, pyrimidine.
EtdBr, anti-
S., Schizo-
tubulin;
tub-2,
176 (Panaccione and Hanau, 1990) and yeasts (Neff et al., 1983; Toda et al., 1984; Hiroaka et al., 1984; Schatz et al., 1986;
and animals, and describe the expression of the gene during vegetative growth and mating interaction.
Smith et al., 1988). No tub genes of the higher filamentous fungi, homobasidiomycetes, have yet been included, although this approach could lead to a better understanding of the functions of microtubules during growth and mating interactions in these fungi. In the homobasidiomycetes the extension growth of the
RESULTS
AND DISCUSSION
(a) Cloning and sequencing of the Schizophyllum commune tub-2 gene Southern analysis of BamHI digested genomic DNA
mono- and dikaryotic hyphae involves prominent changes in the constitution of the microtubule cytoskeleton at the
from the homobasidiomycete S. commune, using a chicken brain /ITub-encoding cDNA (pT2, Valenzuela et al., 1981) as the probe at 42 ’ C in 40 y0 or 50 y0 formamide/ 1“/, SDS/ 1 M NaCl/lO% dextran sulfate, revealed a 4.0-kb band and a 2.4-kb band. A Southern hybridization done in 40% formamide, using a DNA probe which included 1320 bp (from aa 13 to aa 383) of the PTub-encoding gene benA of Aspergillus nidulans (May et al., 1987) recognized only the 4.0-kb band. Therefore, it was assumed that the 2.4-kb BamHI restriction fragment of S. commune contained the 3’-end of the PTub-encoding gene, since this part was almost totally missing from the benA probe. The screening of a genomic library of S. commune made
different stages of the cell cycle (Runeberg et al., 1986; Salo et al., 1989; Raudaskoski et al., 1991). Furthermore, in the homobasidiomycete S. commune, the reciprocal nuclear exchange and migration between strains with compatible mating-type genes appears to involve polymerization and bundling of microtubules (Raudaskoski et al., 1989). In order to understand the role of microtubules in these processes at the molecular level, we have started a program of cloning, sequencing and characterizing the tub genes of S. commune. In the present paper, we report the genomic nt sequence of an S. commune /ITub-encoding tub-2 gene, compare its product with /I Tubs from other fungi, plants
A
OL3 -.-_163
322 ATG
)
141
354
378
K
H
.43 -. TAA
B
V
B PstI
start II
5
.:,
L+ 21
%....>X
*j
50
:,
55
PstI-Sal1 about 650 bp -
Fig. 1. The sequenced
fragments
and the numbers
below denote
containing
the tub-2 gene was digested with BarnHI plasmids
1137 bp
BamHI-EcoRV
397 bp
100 bp of the tub-2 gene of S. commune.
and organization
fragments,
La Jolla, CA) to produce
SalI-BamHI
the size of the fragments producing
(A) The arrows
in bp. B, BarnHI;
H, HindIII;
a 4.0-kb and a 2.4-kb fragment,
pS20 and pS 19, respectively.
For sequencing,
KpnI-Hind111
indicate
the direction
K, KpnI; V, EcoRV.
and extent of the sequenced The original
which were cloned into pBluescript and HindIII-BarnHI
fragments
IEMBL4
clone
II vectors (Stratagene, from pS20 were further
subcloned in the pBluescript II vector at the respective restriction sites. In order to sequence upstream from the !@I site the fragment KpnI-KpnI was removed from the pS20. The pBluescript II oligo primers T7, T3, and KS, and oligo primers OL-1 thru OL-5 used are denoted above their respective arrows. (B) The organization of the tub-2 gene. The numbers above indicate the length (in aa) of the exons (shaded boxes). Most introns contain about 50 nt except for the third which has 107 nt. The restriction sites used in the isolation of fragments of the tub-2 gene for Southern and Northern hybridization experiments and Stop correspond
and the size of the fragments
in bp are given. The Sal1 restriction
to the ATG and TAA codons
in A.
site is located
9 nt upstream
from the KpnI site shown in A. Start
177
La Jolla, CA), and the plasmids obtained were named pS20 and pS19, respectively (Fig. IA). The ends of both inserts were sequenced, and comparisons of the deduced aa sequences of the subclones with those known from A. ni~ufa~ (May et al., 1987), Erysiphe grumini~ (S her-wood and Somerville, 1990), chicken (VaIenzuela et al., 1981; Sullivan et al., 1985), and yeast PTub
in bacteriophage 1EMBL4 (Schuren and Wessels, 1990) using both probes under the conditions used for the Southern hybridizations, led to the isolation of five clones. All the clones contained the 4.0-kb and 2.4-kb BamHI fragments identified by the Southern hybridization experiments. The 4.0- and 2.4-kb fragments were subcloned from one of the putative clones, into the pBluescriptI1 vectors (Stratagene,
1 81
CCTTI'CCTI!C! CTCCTCCGCT CTCACAAGTC ATGTAAGTCC TPCGTCAC'I'GCATCCGACGC GCGCGACGCA GGGGTXGCG
CAAAATACGC GTGTPXCGC
CGTPIGGCGC
GTCGTGGTCG CATCGTATCA TATCCAAAX! GCCCGCTCAC GCCCGCCCGC
161 1
AGCATGCGTG AAATCGTCCA CCTCCAGACC GGCCAGGTGC GTAGTATCCC GCGAGCATCT CGCGCAACGC GCTOACGCGT M R E I VH LQT GQ-
241 12
T'IT'TCGTCCACAGTGCGGCA ACCAGAT~GAZAGCGGC -C G N Q 1
GTGCTCGGTT CGCGCCTTTG AGAGGCTAAC
TCGGTCGCGT
321 AGG%XX!AAG 'ITCTGGGAAG TCGTCTCCGA CGAGCACGGT ATCGAGGCCG ATGGTCTCTA CAAGGGCACC AACGACCAGC 17=GAK VVSD F WE EH G I E A DG LY KGT ND Q 401 43
AGCTCGAGCG CATCTCGGTC TACTACAACG AGATCGGCGC GAACAAGTAC GTGCCGCGTG CGATCCTGGT CGACTPGGAG Q LER I s v Y Y N E I GA N K Y v PR AILV D L E
481 70
CCCGGTACCA TGGACTCTGT CCGCTCCGGT CCGCTIGGTG GCCTITI'CCG CCCGGACAAC 'ITCGTCTTCG GCCAGAGCGG P G T M D S V R S G P L G G L F R P D N G Q S G F V F
561 97
TGCGGGTAAC AACTGGGCCA AGGGCCZA A GN NWA KG
641 105
ACCCGACCGC GTCCGAGGCT TCGTGACATP TGCTAAGTGC GCTlT'lTTGC AGATTACACT GAGGGCGCGG AGCTCGTTGA -HYT EGA ELVD
721 115
CGCl'GTGCTC GACG'ITGTGC GCAAGGAGGC CGAGGGTACG GACTGCC'ITC AGGG'IGCGTG TGTCGATCAC TCCGCGTATA AV L D VV R KE A EG T DCL Q-
801 132
CTCCCGGTTA ATCCCTCGAT AGGCTTCCAG ATCACCCACT CCC'ICG-GTGGTGGTACGGGC GClGGTATGG GTACTCTCCT S L G G ITH G T G AG M G T L L T F Q
881 152
GATCTCGAAG ATTCGTGAGG AATACCCCGA CCGTATGATG TGCACATTCT CTGTCGTCCC GTCTCCCAAG G'PM'CGGACA I s R IRE EY P D RM M CT F s v v P S P K v s D
961 178
CCGTCGTlGA GGTGCGTCTP TCTATCCCCA TGGCAAGGGC TATGATPCAC ATGCGA'l'ZITCAGCCGTACA ACGCGACGCT T V V E 'P Y N A T L
1041 188
CTCCGTGCAC CAGCXTGTCG AGAACTCGGA CGAGACTFTC TGCATTGATA ACGAGGCGCT CTACGACATC TGCTTCCGCA S V H ENS D E T F C I D NE AL Q L V Y D I C F R
1121 214
CTCTCAAGCT CTCCACGCCG ACGTACGGTG AC'ITGAACCA TCTCGTCTCC Tl'CGTCATGT CCGGCATTAC CACTTCGT'PG T L K L ST P T Y G D L N H L v s F VM SG I T TS L
1201 242
CGCTPCCCTG GTCAGCTtXA C'IC'IGACCTGCGCAAGCTTG C~~~CTT~~AG~ RF P GQ LN s D L RX L AVNL
1281 258
GCTCAGCACT AATGCGCGAT TAGTGCCGTT CCCGCGTC'FI!CACTTCTTCA TOACCGGC'IT CGCGCCTTTG ACTGCGCGCG =-VP F P R L H F F MT G F APL TAR
1361 277
GCAGCCAGCA GTACCGTGCC GTCACCGTGC CGGAGCTCAC GCAGCAGATG lTCGACGCCA AGAACATGAT GGCTGCCTCC G S Q Q Y R A VTV P E L T KN M M A AS Q Q M F DA
1441 304
GACCCGAGGC ACGGTCGCTA CCTCACTGTG CGTPCCATTA TGATCGTCGG CATCCCGACA TAATIAACCC GCTCCAGGTT D P R HGRY LT-V
1521 314
GCCGCCATGT ?XCGTGGCAA GGTCTCCATG AAGGAGGTCG AA M F R G X V S M K EV
1601 341
C'ITCG'ICGAGTGGATCCCGA ACAACGTCCT TGCGTCGCAG TGTGACAwG F V E w I P NNV L C D I AS Q
1681 367
TCCTCGGCAA CTCGACGGCC ATCCAGGAGC ‘XXTCAAGCG F LGN S TA L F KR 1 Q E
1761 394
DECAY FL
1841 408
GCCATCGCGC AGTPCACGGA GGCGGAGTCG AACAltXAGG ACT’TGGTlGC CGAGTACCAG CAGTACCAGG ACGCGACGGT -F TE A ES D L V A DATV NM Q E Y Q Q Y Q
1921 431
CGAGGAGGAG GGCGAGTACG AGGAGGAGGT TATCGAAGAC CAGGAGTAAG CGCACACGTA E E E GE Y E E E V I E D Q E end
2001
TTCGTCGATA TC
H
sequence
sequences
involved in splicing are singly underlined.
(Sanger
et al., 1977) by following
polymerase DDBJ
of S. commune tub-2 and deduced
from Amersham
under accession
the protocol
(RPN
No. X63372.
for sequencing
aa sequence.
Methods.
CGCCCCGCGG
A
P
R
L
R
templates
M
S
VT
CCATGTTCAA GCGCAAGGCG A MF K R K A
ACCT'FZGTCA
The starts
ACTCTXCTA N S A Y
CCTGCGCATG TCCGTCACCT
G
TGTCAGCGAC CAGTTCACCG V S D Q F T
CCCCGCTCAC
ACTTCTGCTC
and ends of the introns
The sequence was determined
double-stranded
1590)], and by using [35S]dATPrS
CC~~AG~G~G~
AGGAGCAGAT GCAGAACGTC CAGAACAAGA E EQ M Q N v Q N K
GGTACACGCA GGAGGGTATG GACGAGATGG WY TQ E G M DE M
Fig. 2. Nucleotide possibly
GTACCA'IT'ITCCCGCATCGA TCGCGTCGCG TGTCGCGCCT CTATTAGGAG
are double
underlined.
by the dideoxynucleotide
in the multiwell microtitre
(370 kBq) as a label. These sequence
plate nt sequencing
data are available
Putative
chain-termination
intron method
system [T7 DNA
from EMBL/GenBank/
178 (Neff et al., 1983) indicated that the 2.4-kb fragment indeed contained the 3’-end of the /ITub-encoding gene of S. commune, while the 4.0-kb BamHI fragment contained the 5’ end. The complete nt sequence of both strands of the
(Orbach et al., 1986) and tub-2 of ~OI~etotr~~~urn grumin~co~a (Panaccione and Hanau, 1990), but significantly higherthan that of the SC genes in S. commune in which only 33-37 codons are used (Schuren and Wessels, 1990).
gene (Fig. 2) was obtained Fig. lA, and the sequenced
No clear TATA or CAAT motifs were detected among the 163 nt sequenced upstream from the start codon ATG. In addition, no C+T-rich motif, a sequence typical for filamentous fungi promoters which either lack TATA and CAAT motifs or are highly expressed (Gurr et al., 1988), was recognized in the 5 ’ noncoding region of the tub-2. In this respect, the S. commune tub-2 5’-flanking region seems to be similar to the 5’-noncoding regions of the PTubencoding genes in the other filamentous fungi, in which
using the strategies shown gene was named tub-2.
in
(b) The structure of the rub-2 gene From the start to the stop codon, tub-2 contained a total of 1807 bp (Fig. IA, 2), and by deduction encodes a protein of 445 aa (Fig. lB, 2). The locations of the exons and introns in tub-2 of S. commute were deduced from the interruptions in aa sequence homology with the /ITubs of other filamentous fungi, yeast and chicken, and from the occurrence of the consensus sequences of conserved 5’ and 3’ splice sites of introns. The coding region is split into nine exons by eight introns, which are distributed evenly throughout the gene (Fig. IB). All the introns start with GT and end with AG and they vary in size from 48 to 57 bp except for the third intron which contains 107 bp. In the first three introns, the internal consensus sequence, YGCTAAC, which is characteristic of the 3’ end of introns in filamentous fungi (Gurr et al., 1988) is clearly recognized (Fig. 2). In the following five introns this consensus sequence is less evident, but putative sequences which resemble this consensus sequence are underlined (Fig. 2). All the trends considered to be typical of constitutive and highly expressed genes are recognized in the codon usage of the tub-2 of S. commune (cf., Gurr et al., 1988). This is biased with 43 of the 61 aa codons being used (Table I). The figure is similar to that reported for the /?tubgenes benA of A. ~idul~n~(May et al., 1987), tub-2 of Neurospora crassa TABLE
I
Codon usage of tub-2’ TTT-Phe
0
TCT-Ser
5
TAT-Tyr
0
TGT-Cys
1
TTC-Phe
23
16
0
10 0
TAC-Tyr
TTA-Leu
TCC-Ser TCA-Ser
TAA-Stop
1
TGC-Cw TGA-Stop
5 0
TTG-Leu
7
TCG-Ser
8
TAG-Stop
0
TGG-Trp
4
CTT-Leu CTC-Leu
7 16
CCT-Pro
2
CAT-His
3
CGT-Arg
8
CTA-Leu
0
CCC-Pro CCA-Pro
4 0
CAC-His CAA-Gln
6 0
CGC-Arg CGA-Arg
12 0
CTG-Leu
5
CCG-Pro
11
CAG-Gln
26
CGG-Arg
0
ATT-Be ATC-Be
4 12
ACT-Thr
7
AAT-Asn
0
AGT-Ser
0
10
ATA-Be
0
ACC-Thr ACA-Thr
1
AAC-Asn AAA-Lys
21 0
AGC-Ser AGA-Arg
3 0
18
ACG-Thr
10
AAG-Lys
16
AGG-Arg
1
ATG-Met GTT-Val
7
GCTAla
4
GAT-Asp
2
GGT-Gly
18
GTC-Val GTA-Val
23 0
GCC-Ala GCA-Ala
13 0
GAC-Asv GAA-Glu
22 5
GGC-Gly
17
CGA-Gly
0
GTG-Val
6
GCG-Ala
13
GAG-Glu
33
GGG-GIy
0
a Underlining denotes codons most frequently or nearly equally used for given aa.
their transcription signals appear to be poorly recognizable (Orbach et al., 1986; May et al., 1987; Panaccione and Hanau, 1990). The lack of recognizable transcription signals could be due to the incomplete sequencing of the 5’noncoding region of the tub-2. However, in the SC genes of the same fungus all the typical eukaryotic transcription signals were detected (Dons et al., 1984; Schuren and Wessels, 1990) within the regions comparable in size to the 163 nt sequenced. (c) Comparison of coding sequences The aa sequence of the S. commune /ITub shows over 80% homology with the PTubs of other fungi, except for the P2Tub (tube) of A. nidu~an~and yeast PTubs (Table II). The percentage of identical aa with /ITubs of plants varies from 777; to 82% (TabIe II), However, the highest homology level of S. commune PTub, 84% identical aa, is obtained with chicken $2 and c/I’7 Tubs. Stretches of more than 10 aa long with 100% homology with chicken PTubs occur throu~out the S. commune /ITub aa sequence. The same sequences are also highly conserved in the PTubencoding genes of the other filamentous fungi, plants, and animals, which implies that they are functionally conserved regions in Tub molecules. For instance, the guanine moiety of GTP is now known to bind to BTub within the sequence AILVDLEPGTMDSVR, between aa 63-77 (Linse and Mandelkow, 1988), and S. commune has 100% identity to this aa sequence in the same region. The smallest sequence of PTub polypeptide which interacts with the mAb, DMIB, raised against chicken brain /ITub (Blose et al., 1984), is located in the C-terminal region between aa 416 and 430 (Breitling and Little, 1986). This sequence is highly conserved in S. commune, other filamentous fungi, and plants (cf., ref. in Table II) which explains why DMlB cross-reacts with @Tub from so many divergent organisms including several homobasidiomy~etes (Sal0 et al., 1989). The end of the sequence, aa 422-434, has recently been shown to be involved in the binding of MAPS (Melki et al., 1991) in addition to the sequences QGEFEEEG from aa 433 to 440 (Correas et al., 1990) or EXEEEGEE from aa
179 TABLE
II
Amino acid homology
(% identity)
the BTubs of Schizophyllum
between
commune and other organisms Tubsb
ob /,
Aspergillus nidulans (6)
B1
81
Aspergillus nidulans (6)
82
15
Organism” Filamentous fungi
EpichloP typhina (1)
82
Erysiphe graminis (12)
;
82
Neurospora crassa ( 10)
B
83
Yeasts Saccharomyces cerevisiae (8)
74
Schizosaccharomyces pombe (5)
73 74
Candida albicans
(13)
Slime molds Physarum polycephalum (2)
76
P
Plants 19
Arabidopsis thaliana (10) Glycine max (3)
17
Glycine maX (3)
19
Chlamydomonas reinhardii (15)
82
Animals Gallus gallus ( 14)
82
84
Gallus gallus (7)
87
84
Rat&s norvegicus (4)
l315
84
Drosophila melanogaster (11)
82
Drosophila melanogaster (11)
81
d (1) Byrd
et al. (1990)
(2) Burland
(1987) (4) Ginzburg
et al. (1985)
(1987), (7) Monteiro
and Cleveland
penheimer (1987)
et al. (1988),
(12) Sherwood
et al. (1988),
(5) Hiraoka (1988)
(10) Orbach and Somerville
’ Sequences
were collected
son was made with GCG
(1990)
(11) Rudolph
et al.
(13) Smith et al. (1988),
et al. (1984).
from the SWPROT (Madison,
et al.
(6) May et al.
(8) Neff et al. (1983), (9) Op-
et al. (1986),
(14) Sullivan et al. (1985), (15) Youngblom
(3) Guiltinan
et al. (1984)
Databank
and compari-
WI) software.
435 to 442 (Vallee, 1990). The acidic nature of the latter sequences is thought to form the basis for the interaction of MAPS with tubulin (Vallee, 1990). In S. commune the aa sequence from 422 to 442 is highly homologous to that of animal BTubs (Paschal et al., 1989; Melki et al., 1991), and includes a high number of E residues (Fig. 2). Thus, the prerequisites for the binding of MAPS appears to exist in the PTub of S. commune although none of these proteins have yet been identified in filamentous fungi. The comparison of the aa and nt sequences of PTubs from antimicrotubule drug-sensitive and -resistant strains of filamentous ascomycetes have suggested that PTub is the target for benzimidazole-derived drugs such as carbendazim, thiabendazole, nocodazole and benomyl. In N. crassa a Phe16’+Tyr change confers resistance to a benomylsensitive strain (Orbach et al., 1986). In A. nidulans an Ala16’ -+Val change confers resistance to thiabendazole,
but causes supersensitivity to benomyl, nocodazole, and carbendazim (Jung and Oakley, 1990). In contrast, the S. commune strain 4-39, from which the sequenced tub-2 gene was isolated, shows only a 50% reduction in growth when exposed to 20 x the concentration of benomyl used in tests for resistance in A. nidulans (May et al., 1990) but the strain is supersensitive to nocodazole (M.R., unpublished). In strain 4-39, aa 165 and 167 are Cys and Phe, respectively (Fig. 2). A Cys16’ is unusual among sequenced /ITubs, but it occurs in /?2Tub (Pp2) of the slime mold (Phywum polycephalum). In this organism PaTub is synthesized in plasmodia together with /IlBTub (Burland et al., 1988; Werenskiold et al., 1988). Interestingly, the inhibition of plasmodial growth requires six times higher concentration of carbendazim than the inhibiton of amoebal growth of this organism (Burland et al., 1984). In the amoeba, p2Tub is not produced, but /?lTubs, /31A and j?lB, both with AsP’~~, are produced (Burland et al., 1988; Werenskiold et al., 1988). (d) Southern and Northern analysis When BamHI, KpnI and S&I-digested genomic DNA from commonly used S. commune haploid strains 4-39, 4-40 (Schuren and Wessels, 1990), 1792-114-10 and 684 (this laboratory) were hybridized simultaneously with two fragments of tub-2, one from the 5’ and the other from the 3’ end (Fig. lB), BamHI-digested genomic DNA displayed hybridization signals that corresponded in size to the BamHI fragments subcloned into pS 19 (2.4 kb) and pS20 (4.0 kb) except for the strain 684 (Fig. 3A). In this strain genomic DNA digested with BamHI showed a double signal at 4.0 kb but no signal at 2.4 kb. The sizes of the hybridizing fragments in KpnI-digested genomic DNA in strain 684 were also different from those in the other three strains. Sequence polymorphisms like these are known to be a common phenomenon among haploid strains in homobasidiomycetes (Wu et al., 1983). A weak additional signal to those seen in Fig. 3A was obtained when the fragment from the 3’-end of tub-2 (Fig. 1B) was used as a probe for Southern hybridization in 40% formamide and the diagnostic film was exposed overnight. Neither this signal nor any new one was obtained with the fragments from 5’-end of tub-2 (Fig. 1B) even when the formamide concentration was decreased to 25% and the exposure of the film was more than 24 h. The detection of an additional, although very weak hybridization signal with the 3’-end of tub-2 might mean that S. commune has another flub-encoding gene, in which the 5’ end is highly divergent from that of tub-2. In filamentous fungi and yeasts there are species known with two highly divergent /ITub-encoding genes (May et al., 1987; Panaccione and Hanau, 1990; Gold et al., 1991) and with only one PTub-encoding gene (Orbach et al., 1986; Neff et al.,
180
-25s - 9.4
-16s
- 6.6 -4.4
-25s
-2.3 -2.0
- 0.6
BamHI Fig 3. Southern A) Southern
hybridization
with the fragments Standard
DNA from strains 4-39
(lane l), 4-40
hybridizations
(Panels
is lower at 36 h than at 20 h, but similar in haploid
EtdBr staining
agarose
mut) and wild-type
(WT) B mating-type
gene. Germlings
hybridization
with the mutant
of a nondenaturating
72 h, while a slight decrease is seen in the wild-type germlings at 64 h. The RNA content of the samples bands, 25s and 18S, in the EtdBr stained gels (B and D) aid in the comparison of the relative amounts Northern
(C and E) the sites of rRNA bands were determined
hybridizations
by hybridizing
1984). The fragments
of tub-2 gene were labelled with [a-“P]dCTP
(3000 Ci/mmol)
germlings
D and E)
with mutant
high level of tub-2 transcripts
(B
from 48 h to
B and C: 4 pg, and D and E: 8 pg. The rRNA of RNA between the different samples. In the
with the S. commune rRNA genes from Buckner
Hybridization data not shown. Methods. DNA was extracted by the CTAB (cetyltrimethylammonium bromide) TM (DuPont) gested with restriction endonucleases, electrophoresed, transfered from agarose to GeneScreenPlus Qasba,
hy-
(lane 2) and in their mating
from basidiospore
gene show constant
DNA.
gel (B) and Northern
and their mating at both 20 and 36 h. (Panels
(E) of total RNA isolated
B mating-type
simultaneously
and KpnI-digested
agarose
strains 684 (lane 1) and 1792- 114-10 strains
of tub-2 gene. (Panel
(lane 4) hybridized
is seen in the lanes with BarnHI
B and C) EtdBr staining
(lane 3) the level of tub-2 transcripts
gel (D) and Northern
WT
of total RNA (B-E) with fragments
polymorphism
(C) of total RNA isolated from mycelia grown for 20 h and 36 h. In haploid of a nondenaturating
h
(lane 2), 684 (lane 3), and 1972-14-10
of cub-2 gene. In strain 684 restriction
phage L DNA cleaved with HindHI.
465564724864 III B mut
36h
DNA (A) and Northern
of S. c~mrnune genomic genomic
from 5’- and 3’-ends
(right margin):
bridization
Sal1
KpnI
blot of digested
123123 II 20h
by the multiprime
et al. (1988).
method (Draper and Scott, 1988), diat an alkaline pH (Chomczynski and
DNA labelling systems (Amersham
RPN.
16002). The blots were prehybridized at 42°C in a solution containing 50% formamide/lO% dextran sulfate (500 kDa)/l% SDS/l M NaCl for at least 1 h, and then hybridized in the same solution plus the denaturated probe (lo6 cpm/ml) and 100 pg/ml of denaturated salmon sperm DNA. The mycclia used for RNA isolation idly transfered tracted
by the hot-phenol
was denaturated transfering
were grown method
with formamide
and homogenized
membranes
overlying
into fine powder,
of Hoge et al. (1982), followed and formaldehyde,
complete
medium.
by ultracentrifugation
and electrophoresed
Cellophane
which was collected
in CsCl-gradient
on 1% agarose
membranes
into centrifuge
with adherent
mycelium
tubes kept in liquid nitrogen.
according
and 6% formaldehyde
to Chirgwin
were rap-
RNA was ex-
et al. (1979). The RNA
gels. Northern
blots were made by
the gels by capillary
with the 32P-labelled with a solution
action to GeneScreenPlus TM (DuPont) according to the manufacturer’s protocol for formaldehyde gels and hybridized BumHI-EcoRV subfragment of tub-2. The blots were washed twice with 2 x SSC for 5 min each time at room temperature, twice
containing
2 x SSC and 1 y0 SDS at 65°C for 30 min, and several times with 0.1 x SSC at room temperature
a total of 20 min. Under conditions at 50°C.
on cellophane
into liquid nitrogen,
of low stringency
SSC is 0.15 M NaCI/O.OlS
M Na,.citrate
the formamide
concentration
was reduced
as indicated
with constant
agitation
for
in sections a and d and the blots were washed
pH 7.6.
1983; Hiraoka et al., 1984; Smith et al., 1988; Byrd et al., 1990; Sherwood and Somerville, 1990). Northern analysis of poly(A)+-enriched RNA (not shown) and total RNA from strains 1792-114-10 (matingtype A43Ba,-&), 684 (mating-type A26B26) and their mating using, BamHI-EcoRV fragment from the 3’-end of tub-2 (Fig. 1B) as a probe (Fig. 3C), showed that tub-2 transcripts were slightly smaller than the S. commune 18s RNA
(1900 + 50 nt, Buckner et al., 1988). Similar levels of tub-2 transcripts were obtained in the haploid strains (Fig. 3C, lanes 1 and 2) and in their mating (Fig. 3C, lane 3) when RNA was isolated from 20- and 36-h-old mycelia (Fig. 3B,C). This result indicated that the involvement of microtubules in the reciprocal exchange and migration of nuclei during the mating (Raudaskoski, 1973; Raudaskoski et al., 1989) seems to take place without a change in the
181 amounts of tub-2 mRNA. Instead, a decrease in the level of tub-2 transcripts was observed in the haploid strains and
S. commune rRNA genes. This work was supported by the Academy of Finland. The technical assistance of Ms. Mar-
their mating when RNA was isolated from 36-h-old instead of 20-h-old mycelia (Fig. 3C), which suggested that the expression of tub-2 might be dependent on the age of the mycelium. Comparison of the levels of tub-2 transcripts in mycelia grown from wild-type basidiospores, with tub-2 transcripts in mycelia grown from basidiospores containing a mutation in the B mating-type gene suggested slightly higher levels of tub-2 transcripts in the latter mycelia (Fig. 3D, E). This observation will be more rigorously tested in further stud-
jukka Uuskallio is gratefully acknowledged. M.R. would like to thank Dr. J.G.H. Wessels for the opportunity to learn molecular biology techniques at his laboratory.
ies, since the mutation in the B mating-type gene is known to cause constitutive intercellular nuclear migration comparable to that which occurs during mating interactions (Koltin and Flexer, 1969).
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Mol. Cell
GENE
B.V. All rights reserved.
175
0378-I 119/92/$05.00
06624
Cloning, sequence homobasidiomycete (Antitubulin
drugs; benomyl;
Patrick Russo*,
and expression of a P-tubulin-encoding Schizophyllum commune exon; intron;
codon
usage; fungus;
Jarmo T. Juuti* and Marjatta
nuclear
migration;
gene
in the
polymorphism)
Raudaskoski
Department of Botany, University of Helsinki, 00170 Helsinki, Finland Received
by J.K.C.
Knowles:
3 February
1992; Revised/Accepted:
13 April/l1
May 1992; Received
at publishers:
1 June 1992
SUMMARY
The /I-tubulin (fiTub)-encoding gene (U-2) of Schizophyllum commune is the first tubulin gene isolated, cloned and sequenced from higher filamentous fungi (homobasidiomycetes). The S. commune tub-2 gene is organized into nine exons and eight introns. The introns vary from 48 to 107 nt in length, and are distributed throughout the gene. The tub-2 exons code for a protein of 445 amino acids (aa), which shows great homology with BTubs of filamentous ascomycetes, plants, and animals, but less homology with yeasts. The codon usage of tub-2 from S. commune is biased, as it is in most PTub-encoding genes of filamentous fungi. The S. commune PTub shows a conserved aa sequence in the C-terminal domain, which is suggested to interact with microtubule-associated proteins in animals. In contrast, the S. commune /ITub deviates from most known PTubs by having a Cys16’ residue, which might be significant for the insensitivity of S. commune haploid strains to the antimicrotubule drug, benomyl. In tub-2 of different haploid strains, sequence polymorphisms occur in the 5’ and 3’ flanking regions. The expression of tub-2 is high in young mycelium, which has a high number of extending apical cells, but decreases with the aging of the mycelium. No significant difference in the hybridization signal intensity for the tub-2 transcripts was recorded either during intercellular nuclear migration at early mating, or in mycelia with a mutation in the B mating-type gene. The weak signal obtained with the 3’ end of tub-2 as a probe in Southern hybridizations, under conditions of low stringency, warrants attention to the possibility that S. commune might have another PTub-encoding gene highly divergent from tub-2.
In microtubules, tubulins (Tubs) are the major protein components, and by cloning the tub genes it has been possible to analyse the expression of these genes at different
developmental stages in a wide range of eukaryotic cells including fungi. In fungi the cloning and characterization of tub genes has been restricted to euascomycetes (Orbach et al., 1986; May et al., 1987; Byrd et al., 1990; Sherwood and Somerville, 1990; Doshi et al., 1991), deuteromycetes
Correspondence fat Dr. M. Raudaskoski,
Abbreviations:
INTRODUCTION
sity of Helsinki, Tel. (358-O) *Present
Unioninkatu
Department
44, 00170 Helsinki,
of Botany,
Univer-
Finland.
ethidium
1918606; Fax (358-O) 1918656.
addresses:
versity of Rochester, Fax (716) 2712683; sinki, Arkadiankatu Fax (358-O) 1917376.
(P.R.) Department Rochester,
(J.T.J.) Department 7, 00100 Helsinki,
kb, kilobase
body; MAP, microtubule
of Biochemistry,
NY 14642, USA.
aa, amino acid(s); bp, base pair(s); BTub, b-tubulin;
bromide;
Box 607, Uni-
Tel. (716) 2756663;
of Genetics, University of HelFinland. Tel. (358-O) 1917380;
associated
or 1000 bp; mAb, monoclonal protein; nt, nucleotide(s);
phyllum; SDS, sodium dodecyl sulfate; tub, gene encoding gene encoding /ITub of S. commune; Y, pyrimidine.
EtdBr, anti-
S., Schizo-
tubulin;
tub-2,
176 (Panaccione and Hanau, 1990) and yeasts (Neff et al., 1983; Toda et al., 1984; Hiroaka et al., 1984; Schatz et al., 1986;
and animals, and describe the expression of the gene during vegetative growth and mating interaction.
Smith et al., 1988). No tub genes of the higher filamentous fungi, homobasidiomycetes, have yet been included, although this approach could lead to a better understanding of the functions of microtubules during growth and mating interactions in these fungi. In the homobasidiomycetes the extension growth of the
RESULTS
AND DISCUSSION
(a) Cloning and sequencing of the Schizophyllum commune tub-2 gene Southern analysis of BamHI digested genomic DNA
mono- and dikaryotic hyphae involves prominent changes in the constitution of the microtubule cytoskeleton at the
from the homobasidiomycete S. commune, using a chicken brain /ITub-encoding cDNA (pT2, Valenzuela et al., 1981) as the probe at 42 ’ C in 40 y0 or 50 y0 formamide/ 1“/, SDS/ 1 M NaCl/lO% dextran sulfate, revealed a 4.0-kb band and a 2.4-kb band. A Southern hybridization done in 40% formamide, using a DNA probe which included 1320 bp (from aa 13 to aa 383) of the PTub-encoding gene benA of Aspergillus nidulans (May et al., 1987) recognized only the 4.0-kb band. Therefore, it was assumed that the 2.4-kb BamHI restriction fragment of S. commune contained the 3’-end of the PTub-encoding gene, since this part was almost totally missing from the benA probe. The screening of a genomic library of S. commune made
different stages of the cell cycle (Runeberg et al., 1986; Salo et al., 1989; Raudaskoski et al., 1991). Furthermore, in the homobasidiomycete S. commune, the reciprocal nuclear exchange and migration between strains with compatible mating-type genes appears to involve polymerization and bundling of microtubules (Raudaskoski et al., 1989). In order to understand the role of microtubules in these processes at the molecular level, we have started a program of cloning, sequencing and characterizing the tub genes of S. commune. In the present paper, we report the genomic nt sequence of an S. commune /ITub-encoding tub-2 gene, compare its product with /I Tubs from other fungi, plants
A
OL3 -.-_163
322 ATG
)
141
354
378
K
H
.43 -. TAA
B
V
B PstI
start II
5
.:,
L+ 21
%....>X
*j
50
:,
55
PstI-Sal1 about 650 bp -
Fig. 1. The sequenced
fragments
and the numbers
below denote
containing
the tub-2 gene was digested with BarnHI plasmids
1137 bp
BamHI-EcoRV
397 bp
100 bp of the tub-2 gene of S. commune.
and organization
fragments,
La Jolla, CA) to produce
SalI-BamHI
the size of the fragments producing
(A) The arrows
in bp. B, BarnHI;
H, HindIII;
a 4.0-kb and a 2.4-kb fragment,
pS20 and pS 19, respectively.
For sequencing,
KpnI-Hind111
indicate
the direction
K, KpnI; V, EcoRV.
and extent of the sequenced The original
which were cloned into pBluescript and HindIII-BarnHI
fragments
IEMBL4
clone
II vectors (Stratagene, from pS20 were further
subcloned in the pBluescript II vector at the respective restriction sites. In order to sequence upstream from the !@I site the fragment KpnI-KpnI was removed from the pS20. The pBluescript II oligo primers T7, T3, and KS, and oligo primers OL-1 thru OL-5 used are denoted above their respective arrows. (B) The organization of the tub-2 gene. The numbers above indicate the length (in aa) of the exons (shaded boxes). Most introns contain about 50 nt except for the third which has 107 nt. The restriction sites used in the isolation of fragments of the tub-2 gene for Southern and Northern hybridization experiments and Stop correspond
and the size of the fragments
in bp are given. The Sal1 restriction
to the ATG and TAA codons
in A.
site is located
9 nt upstream
from the KpnI site shown in A. Start
177
La Jolla, CA), and the plasmids obtained were named pS20 and pS19, respectively (Fig. IA). The ends of both inserts were sequenced, and comparisons of the deduced aa sequences of the subclones with those known from A. ni~ufa~ (May et al., 1987), Erysiphe grumini~ (S her-wood and Somerville, 1990), chicken (VaIenzuela et al., 1981; Sullivan et al., 1985), and yeast PTub
in bacteriophage 1EMBL4 (Schuren and Wessels, 1990) using both probes under the conditions used for the Southern hybridizations, led to the isolation of five clones. All the clones contained the 4.0-kb and 2.4-kb BamHI fragments identified by the Southern hybridization experiments. The 4.0- and 2.4-kb fragments were subcloned from one of the putative clones, into the pBluescriptI1 vectors (Stratagene,
1 81
CCTTI'CCTI!C! CTCCTCCGCT CTCACAAGTC ATGTAAGTCC TPCGTCAC'I'GCATCCGACGC GCGCGACGCA GGGGTXGCG
CAAAATACGC GTGTPXCGC
CGTPIGGCGC
GTCGTGGTCG CATCGTATCA TATCCAAAX! GCCCGCTCAC GCCCGCCCGC
161 1
AGCATGCGTG AAATCGTCCA CCTCCAGACC GGCCAGGTGC GTAGTATCCC GCGAGCATCT CGCGCAACGC GCTOACGCGT M R E I VH LQT GQ-
241 12
T'IT'TCGTCCACAGTGCGGCA ACCAGAT~GAZAGCGGC -C G N Q 1
GTGCTCGGTT CGCGCCTTTG AGAGGCTAAC
TCGGTCGCGT
321 AGG%XX!AAG 'ITCTGGGAAG TCGTCTCCGA CGAGCACGGT ATCGAGGCCG ATGGTCTCTA CAAGGGCACC AACGACCAGC 17=GAK VVSD F WE EH G I E A DG LY KGT ND Q 401 43
AGCTCGAGCG CATCTCGGTC TACTACAACG AGATCGGCGC GAACAAGTAC GTGCCGCGTG CGATCCTGGT CGACTPGGAG Q LER I s v Y Y N E I GA N K Y v PR AILV D L E
481 70
CCCGGTACCA TGGACTCTGT CCGCTCCGGT CCGCTIGGTG GCCTITI'CCG CCCGGACAAC 'ITCGTCTTCG GCCAGAGCGG P G T M D S V R S G P L G G L F R P D N G Q S G F V F
561 97
TGCGGGTAAC AACTGGGCCA AGGGCCZA A GN NWA KG
641 105
ACCCGACCGC GTCCGAGGCT TCGTGACATP TGCTAAGTGC GCTlT'lTTGC AGATTACACT GAGGGCGCGG AGCTCGTTGA -HYT EGA ELVD
721 115
CGCl'GTGCTC GACG'ITGTGC GCAAGGAGGC CGAGGGTACG GACTGCC'ITC AGGG'IGCGTG TGTCGATCAC TCCGCGTATA AV L D VV R KE A EG T DCL Q-
801 132
CTCCCGGTTA ATCCCTCGAT AGGCTTCCAG ATCACCCACT CCC'ICG-GTGGTGGTACGGGC GClGGTATGG GTACTCTCCT S L G G ITH G T G AG M G T L L T F Q
881 152
GATCTCGAAG ATTCGTGAGG AATACCCCGA CCGTATGATG TGCACATTCT CTGTCGTCCC GTCTCCCAAG G'PM'CGGACA I s R IRE EY P D RM M CT F s v v P S P K v s D
961 178
CCGTCGTlGA GGTGCGTCTP TCTATCCCCA TGGCAAGGGC TATGATPCAC ATGCGA'l'ZITCAGCCGTACA ACGCGACGCT T V V E 'P Y N A T L
1041 188
CTCCGTGCAC CAGCXTGTCG AGAACTCGGA CGAGACTFTC TGCATTGATA ACGAGGCGCT CTACGACATC TGCTTCCGCA S V H ENS D E T F C I D NE AL Q L V Y D I C F R
1121 214
CTCTCAAGCT CTCCACGCCG ACGTACGGTG AC'ITGAACCA TCTCGTCTCC Tl'CGTCATGT CCGGCATTAC CACTTCGT'PG T L K L ST P T Y G D L N H L v s F VM SG I T TS L
1201 242
CGCTPCCCTG GTCAGCTtXA C'IC'IGACCTGCGCAAGCTTG C~~~CTT~~AG~ RF P GQ LN s D L RX L AVNL
1281 258
GCTCAGCACT AATGCGCGAT TAGTGCCGTT CCCGCGTC'FI!CACTTCTTCA TOACCGGC'IT CGCGCCTTTG ACTGCGCGCG =-VP F P R L H F F MT G F APL TAR
1361 277
GCAGCCAGCA GTACCGTGCC GTCACCGTGC CGGAGCTCAC GCAGCAGATG lTCGACGCCA AGAACATGAT GGCTGCCTCC G S Q Q Y R A VTV P E L T KN M M A AS Q Q M F DA
1441 304
GACCCGAGGC ACGGTCGCTA CCTCACTGTG CGTPCCATTA TGATCGTCGG CATCCCGACA TAATIAACCC GCTCCAGGTT D P R HGRY LT-V
1521 314
GCCGCCATGT ?XCGTGGCAA GGTCTCCATG AAGGAGGTCG AA M F R G X V S M K EV
1601 341
C'ITCG'ICGAGTGGATCCCGA ACAACGTCCT TGCGTCGCAG TGTGACAwG F V E w I P NNV L C D I AS Q
1681 367
TCCTCGGCAA CTCGACGGCC ATCCAGGAGC ‘XXTCAAGCG F LGN S TA L F KR 1 Q E
1761 394
DECAY FL
1841 408
GCCATCGCGC AGTPCACGGA GGCGGAGTCG AACAltXAGG ACT’TGGTlGC CGAGTACCAG CAGTACCAGG ACGCGACGGT -F TE A ES D L V A DATV NM Q E Y Q Q Y Q
1921 431
CGAGGAGGAG GGCGAGTACG AGGAGGAGGT TATCGAAGAC CAGGAGTAAG CGCACACGTA E E E GE Y E E E V I E D Q E end
2001
TTCGTCGATA TC
H
sequence
sequences
involved in splicing are singly underlined.
(Sanger
et al., 1977) by following
polymerase DDBJ
of S. commune tub-2 and deduced
from Amersham
under accession
the protocol
(RPN
No. X63372.
for sequencing
aa sequence.
Methods.
CGCCCCGCGG
A
P
R
L
R
templates
M
S
VT
CCATGTTCAA GCGCAAGGCG A MF K R K A
ACCT'FZGTCA
The starts
ACTCTXCTA N S A Y
CCTGCGCATG TCCGTCACCT
G
TGTCAGCGAC CAGTTCACCG V S D Q F T
CCCCGCTCAC
ACTTCTGCTC
and ends of the introns
The sequence was determined
double-stranded
1590)], and by using [35S]dATPrS
CC~~AG~G~G~
AGGAGCAGAT GCAGAACGTC CAGAACAAGA E EQ M Q N v Q N K
GGTACACGCA GGAGGGTATG GACGAGATGG WY TQ E G M DE M
Fig. 2. Nucleotide possibly
GTACCA'IT'ITCCCGCATCGA TCGCGTCGCG TGTCGCGCCT CTATTAGGAG
are double
underlined.
by the dideoxynucleotide
in the multiwell microtitre
(370 kBq) as a label. These sequence
plate nt sequencing
data are available
Putative
chain-termination
intron method
system [T7 DNA
from EMBL/GenBank/
178 (Neff et al., 1983) indicated that the 2.4-kb fragment indeed contained the 3’-end of the /ITub-encoding gene of S. commune, while the 4.0-kb BamHI fragment contained the 5’ end. The complete nt sequence of both strands of the
(Orbach et al., 1986) and tub-2 of ~OI~etotr~~~urn grumin~co~a (Panaccione and Hanau, 1990), but significantly higherthan that of the SC genes in S. commune in which only 33-37 codons are used (Schuren and Wessels, 1990).
gene (Fig. 2) was obtained Fig. lA, and the sequenced
No clear TATA or CAAT motifs were detected among the 163 nt sequenced upstream from the start codon ATG. In addition, no C+T-rich motif, a sequence typical for filamentous fungi promoters which either lack TATA and CAAT motifs or are highly expressed (Gurr et al., 1988), was recognized in the 5 ’ noncoding region of the tub-2. In this respect, the S. commune tub-2 5’-flanking region seems to be similar to the 5’-noncoding regions of the PTubencoding genes in the other filamentous fungi, in which
using the strategies shown gene was named tub-2.
in
(b) The structure of the rub-2 gene From the start to the stop codon, tub-2 contained a total of 1807 bp (Fig. IA, 2), and by deduction encodes a protein of 445 aa (Fig. lB, 2). The locations of the exons and introns in tub-2 of S. commute were deduced from the interruptions in aa sequence homology with the /ITubs of other filamentous fungi, yeast and chicken, and from the occurrence of the consensus sequences of conserved 5’ and 3’ splice sites of introns. The coding region is split into nine exons by eight introns, which are distributed evenly throughout the gene (Fig. IB). All the introns start with GT and end with AG and they vary in size from 48 to 57 bp except for the third intron which contains 107 bp. In the first three introns, the internal consensus sequence, YGCTAAC, which is characteristic of the 3’ end of introns in filamentous fungi (Gurr et al., 1988) is clearly recognized (Fig. 2). In the following five introns this consensus sequence is less evident, but putative sequences which resemble this consensus sequence are underlined (Fig. 2). All the trends considered to be typical of constitutive and highly expressed genes are recognized in the codon usage of the tub-2 of S. commune (cf., Gurr et al., 1988). This is biased with 43 of the 61 aa codons being used (Table I). The figure is similar to that reported for the /?tubgenes benA of A. ~idul~n~(May et al., 1987), tub-2 of Neurospora crassa TABLE
I
Codon usage of tub-2’ TTT-Phe
0
TCT-Ser
5
TAT-Tyr
0
TGT-Cys
1
TTC-Phe
23
16
0
10 0
TAC-Tyr
TTA-Leu
TCC-Ser TCA-Ser
TAA-Stop
1
TGC-Cw TGA-Stop
5 0
TTG-Leu
7
TCG-Ser
8
TAG-Stop
0
TGG-Trp
4
CTT-Leu CTC-Leu
7 16
CCT-Pro
2
CAT-His
3
CGT-Arg
8
CTA-Leu
0
CCC-Pro CCA-Pro
4 0
CAC-His CAA-Gln
6 0
CGC-Arg CGA-Arg
12 0
CTG-Leu
5
CCG-Pro
11
CAG-Gln
26
CGG-Arg
0
ATT-Be ATC-Be
4 12
ACT-Thr
7
AAT-Asn
0
AGT-Ser
0
10
ATA-Be
0
ACC-Thr ACA-Thr
1
AAC-Asn AAA-Lys
21 0
AGC-Ser AGA-Arg
3 0
18
ACG-Thr
10
AAG-Lys
16
AGG-Arg
1
ATG-Met GTT-Val
7
GCTAla
4
GAT-Asp
2
GGT-Gly
18
GTC-Val GTA-Val
23 0
GCC-Ala GCA-Ala
13 0
GAC-Asv GAA-Glu
22 5
GGC-Gly
17
CGA-Gly
0
GTG-Val
6
GCG-Ala
13
GAG-Glu
33
GGG-GIy
0
a Underlining denotes codons most frequently or nearly equally used for given aa.
their transcription signals appear to be poorly recognizable (Orbach et al., 1986; May et al., 1987; Panaccione and Hanau, 1990). The lack of recognizable transcription signals could be due to the incomplete sequencing of the 5’noncoding region of the tub-2. However, in the SC genes of the same fungus all the typical eukaryotic transcription signals were detected (Dons et al., 1984; Schuren and Wessels, 1990) within the regions comparable in size to the 163 nt sequenced. (c) Comparison of coding sequences The aa sequence of the S. commune /ITub shows over 80% homology with the PTubs of other fungi, except for the P2Tub (tube) of A. nidu~an~and yeast PTubs (Table II). The percentage of identical aa with /ITubs of plants varies from 777; to 82% (TabIe II), However, the highest homology level of S. commune PTub, 84% identical aa, is obtained with chicken $2 and c/I’7 Tubs. Stretches of more than 10 aa long with 100% homology with chicken PTubs occur throu~out the S. commune /ITub aa sequence. The same sequences are also highly conserved in the PTubencoding genes of the other filamentous fungi, plants, and animals, which implies that they are functionally conserved regions in Tub molecules. For instance, the guanine moiety of GTP is now known to bind to BTub within the sequence AILVDLEPGTMDSVR, between aa 63-77 (Linse and Mandelkow, 1988), and S. commune has 100% identity to this aa sequence in the same region. The smallest sequence of PTub polypeptide which interacts with the mAb, DMIB, raised against chicken brain /ITub (Blose et al., 1984), is located in the C-terminal region between aa 416 and 430 (Breitling and Little, 1986). This sequence is highly conserved in S. commune, other filamentous fungi, and plants (cf., ref. in Table II) which explains why DMlB cross-reacts with @Tub from so many divergent organisms including several homobasidiomy~etes (Sal0 et al., 1989). The end of the sequence, aa 422-434, has recently been shown to be involved in the binding of MAPS (Melki et al., 1991) in addition to the sequences QGEFEEEG from aa 433 to 440 (Correas et al., 1990) or EXEEEGEE from aa
179 TABLE
II
Amino acid homology
(% identity)
the BTubs of Schizophyllum
between
commune and other organisms Tubsb
ob /,
Aspergillus nidulans (6)
B1
81
Aspergillus nidulans (6)
82
15
Organism” Filamentous fungi
EpichloP typhina (1)
82
Erysiphe graminis (12)
;
82
Neurospora crassa ( 10)
B
83
Yeasts Saccharomyces cerevisiae (8)
74
Schizosaccharomyces pombe (5)
73 74
Candida albicans
(13)
Slime molds Physarum polycephalum (2)
76
P
Plants 19
Arabidopsis thaliana (10) Glycine max (3)
17
Glycine maX (3)
19
Chlamydomonas reinhardii (15)
82
Animals Gallus gallus ( 14)
82
84
Gallus gallus (7)
87
84
Rat&s norvegicus (4)
l315
84
Drosophila melanogaster (11)
82
Drosophila melanogaster (11)
81
d (1) Byrd
et al. (1990)
(2) Burland
(1987) (4) Ginzburg
et al. (1985)
(1987), (7) Monteiro
and Cleveland
penheimer (1987)
et al. (1988),
(12) Sherwood
et al. (1988),
(5) Hiraoka (1988)
(10) Orbach and Somerville
’ Sequences
were collected
son was made with GCG
(1990)
(11) Rudolph
et al.
(13) Smith et al. (1988),
et al. (1984).
from the SWPROT (Madison,
et al.
(6) May et al.
(8) Neff et al. (1983), (9) Op-
et al. (1986),
(14) Sullivan et al. (1985), (15) Youngblom
(3) Guiltinan
et al. (1984)
Databank
and compari-
WI) software.
435 to 442 (Vallee, 1990). The acidic nature of the latter sequences is thought to form the basis for the interaction of MAPS with tubulin (Vallee, 1990). In S. commune the aa sequence from 422 to 442 is highly homologous to that of animal BTubs (Paschal et al., 1989; Melki et al., 1991), and includes a high number of E residues (Fig. 2). Thus, the prerequisites for the binding of MAPS appears to exist in the PTub of S. commune although none of these proteins have yet been identified in filamentous fungi. The comparison of the aa and nt sequences of PTubs from antimicrotubule drug-sensitive and -resistant strains of filamentous ascomycetes have suggested that PTub is the target for benzimidazole-derived drugs such as carbendazim, thiabendazole, nocodazole and benomyl. In N. crassa a Phe16’+Tyr change confers resistance to a benomylsensitive strain (Orbach et al., 1986). In A. nidulans an Ala16’ -+Val change confers resistance to thiabendazole,
but causes supersensitivity to benomyl, nocodazole, and carbendazim (Jung and Oakley, 1990). In contrast, the S. commune strain 4-39, from which the sequenced tub-2 gene was isolated, shows only a 50% reduction in growth when exposed to 20 x the concentration of benomyl used in tests for resistance in A. nidulans (May et al., 1990) but the strain is supersensitive to nocodazole (M.R., unpublished). In strain 4-39, aa 165 and 167 are Cys and Phe, respectively (Fig. 2). A Cys16’ is unusual among sequenced /ITubs, but it occurs in /?2Tub (Pp2) of the slime mold (Phywum polycephalum). In this organism PaTub is synthesized in plasmodia together with /IlBTub (Burland et al., 1988; Werenskiold et al., 1988). Interestingly, the inhibition of plasmodial growth requires six times higher concentration of carbendazim than the inhibiton of amoebal growth of this organism (Burland et al., 1984). In the amoeba, p2Tub is not produced, but /?lTubs, /31A and j?lB, both with AsP’~~, are produced (Burland et al., 1988; Werenskiold et al., 1988). (d) Southern and Northern analysis When BamHI, KpnI and S&I-digested genomic DNA from commonly used S. commune haploid strains 4-39, 4-40 (Schuren and Wessels, 1990), 1792-114-10 and 684 (this laboratory) were hybridized simultaneously with two fragments of tub-2, one from the 5’ and the other from the 3’ end (Fig. lB), BamHI-digested genomic DNA displayed hybridization signals that corresponded in size to the BamHI fragments subcloned into pS 19 (2.4 kb) and pS20 (4.0 kb) except for the strain 684 (Fig. 3A). In this strain genomic DNA digested with BamHI showed a double signal at 4.0 kb but no signal at 2.4 kb. The sizes of the hybridizing fragments in KpnI-digested genomic DNA in strain 684 were also different from those in the other three strains. Sequence polymorphisms like these are known to be a common phenomenon among haploid strains in homobasidiomycetes (Wu et al., 1983). A weak additional signal to those seen in Fig. 3A was obtained when the fragment from the 3’-end of tub-2 (Fig. 1B) was used as a probe for Southern hybridization in 40% formamide and the diagnostic film was exposed overnight. Neither this signal nor any new one was obtained with the fragments from 5’-end of tub-2 (Fig. 1B) even when the formamide concentration was decreased to 25% and the exposure of the film was more than 24 h. The detection of an additional, although very weak hybridization signal with the 3’-end of tub-2 might mean that S. commune has another flub-encoding gene, in which the 5’ end is highly divergent from that of tub-2. In filamentous fungi and yeasts there are species known with two highly divergent /ITub-encoding genes (May et al., 1987; Panaccione and Hanau, 1990; Gold et al., 1991) and with only one PTub-encoding gene (Orbach et al., 1986; Neff et al.,
180
-25s - 9.4
-16s
- 6.6 -4.4
-25s
-2.3 -2.0
- 0.6
BamHI Fig 3. Southern A) Southern
hybridization
with the fragments Standard
DNA from strains 4-39
(lane l), 4-40
hybridizations
(Panels
is lower at 36 h than at 20 h, but similar in haploid
EtdBr staining
agarose
mut) and wild-type
(WT) B mating-type
gene. Germlings
hybridization
with the mutant
of a nondenaturating
72 h, while a slight decrease is seen in the wild-type germlings at 64 h. The RNA content of the samples bands, 25s and 18S, in the EtdBr stained gels (B and D) aid in the comparison of the relative amounts Northern
(C and E) the sites of rRNA bands were determined
hybridizations
by hybridizing
1984). The fragments
of tub-2 gene were labelled with [a-“P]dCTP
(3000 Ci/mmol)
germlings
D and E)
with mutant
high level of tub-2 transcripts
(B
from 48 h to
B and C: 4 pg, and D and E: 8 pg. The rRNA of RNA between the different samples. In the
with the S. commune rRNA genes from Buckner
Hybridization data not shown. Methods. DNA was extracted by the CTAB (cetyltrimethylammonium bromide) TM (DuPont) gested with restriction endonucleases, electrophoresed, transfered from agarose to GeneScreenPlus Qasba,
hy-
(lane 2) and in their mating
from basidiospore
gene show constant
DNA.
gel (B) and Northern
and their mating at both 20 and 36 h. (Panels
(E) of total RNA isolated
B mating-type
simultaneously
and KpnI-digested
agarose
strains 684 (lane 1) and 1792- 114-10 strains
of tub-2 gene. (Panel
(lane 4) hybridized
is seen in the lanes with BarnHI
B and C) EtdBr staining
(lane 3) the level of tub-2 transcripts
gel (D) and Northern
WT
of total RNA (B-E) with fragments
polymorphism
(C) of total RNA isolated from mycelia grown for 20 h and 36 h. In haploid of a nondenaturating
h
(lane 2), 684 (lane 3), and 1972-14-10
of cub-2 gene. In strain 684 restriction
phage L DNA cleaved with HindHI.
465564724864 III B mut
36h
DNA (A) and Northern
of S. c~mrnune genomic genomic
from 5’- and 3’-ends
(right margin):
bridization
Sal1
KpnI
blot of digested
123123 II 20h
by the multiprime
et al. (1988).
method (Draper and Scott, 1988), diat an alkaline pH (Chomczynski and
DNA labelling systems (Amersham
RPN.
16002). The blots were prehybridized at 42°C in a solution containing 50% formamide/lO% dextran sulfate (500 kDa)/l% SDS/l M NaCl for at least 1 h, and then hybridized in the same solution plus the denaturated probe (lo6 cpm/ml) and 100 pg/ml of denaturated salmon sperm DNA. The mycclia used for RNA isolation idly transfered tracted
by the hot-phenol
was denaturated transfering
were grown method
with formamide
and homogenized
membranes
overlying
into fine powder,
of Hoge et al. (1982), followed and formaldehyde,
complete
medium.
by ultracentrifugation
and electrophoresed
Cellophane
which was collected
in CsCl-gradient
on 1% agarose
membranes
into centrifuge
with adherent
mycelium
tubes kept in liquid nitrogen.
according
and 6% formaldehyde
to Chirgwin
were rap-
RNA was ex-
et al. (1979). The RNA
gels. Northern
blots were made by
the gels by capillary
with the 32P-labelled with a solution
action to GeneScreenPlus TM (DuPont) according to the manufacturer’s protocol for formaldehyde gels and hybridized BumHI-EcoRV subfragment of tub-2. The blots were washed twice with 2 x SSC for 5 min each time at room temperature, twice
containing
2 x SSC and 1 y0 SDS at 65°C for 30 min, and several times with 0.1 x SSC at room temperature
a total of 20 min. Under conditions at 50°C.
on cellophane
into liquid nitrogen,
of low stringency
SSC is 0.15 M NaCI/O.OlS
M Na,.citrate
the formamide
concentration
was reduced
as indicated
with constant
agitation
for
in sections a and d and the blots were washed
pH 7.6.
1983; Hiraoka et al., 1984; Smith et al., 1988; Byrd et al., 1990; Sherwood and Somerville, 1990). Northern analysis of poly(A)+-enriched RNA (not shown) and total RNA from strains 1792-114-10 (matingtype A43Ba,-&), 684 (mating-type A26B26) and their mating using, BamHI-EcoRV fragment from the 3’-end of tub-2 (Fig. 1B) as a probe (Fig. 3C), showed that tub-2 transcripts were slightly smaller than the S. commune 18s RNA
(1900 + 50 nt, Buckner et al., 1988). Similar levels of tub-2 transcripts were obtained in the haploid strains (Fig. 3C, lanes 1 and 2) and in their mating (Fig. 3C, lane 3) when RNA was isolated from 20- and 36-h-old mycelia (Fig. 3B,C). This result indicated that the involvement of microtubules in the reciprocal exchange and migration of nuclei during the mating (Raudaskoski, 1973; Raudaskoski et al., 1989) seems to take place without a change in the
181 amounts of tub-2 mRNA. Instead, a decrease in the level of tub-2 transcripts was observed in the haploid strains and
S. commune rRNA genes. This work was supported by the Academy of Finland. The technical assistance of Ms. Mar-
their mating when RNA was isolated from 36-h-old instead of 20-h-old mycelia (Fig. 3C), which suggested that the expression of tub-2 might be dependent on the age of the mycelium. Comparison of the levels of tub-2 transcripts in mycelia grown from wild-type basidiospores, with tub-2 transcripts in mycelia grown from basidiospores containing a mutation in the B mating-type gene suggested slightly higher levels of tub-2 transcripts in the latter mycelia (Fig. 3D, E). This observation will be more rigorously tested in further stud-
jukka Uuskallio is gratefully acknowledged. M.R. would like to thank Dr. J.G.H. Wessels for the opportunity to learn molecular biology techniques at his laboratory.
ies, since the mutation in the B mating-type gene is known to cause constitutive intercellular nuclear migration comparable to that which occurs during mating interactions (Koltin and Flexer, 1969).
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J.R.:
antibodies
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tubulin.
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DM1B.J. Buckner,
Epitope
are in-
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B., Novotny,
C.P. and Ullrich, R.C.: Organization
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4 17-424.
(e) Conclusions (1) We have cloned and sequenced from the homobasidiomycete S. commune, the tub-2 gene which possesses nine exons and eight introns. The exons code for a deduced aa sequence of 445 aa. The introns are distributed throughout the gene. The codon usage of the tub-2 gene of S. wmmune is biased as it is in BTub-encoding genes of filamentous ascomycetes and deuteromycetes. (2) A high degree of homology is evident at the aa level between tub-2 of S. commune and the PTub-encoding genes of filamentous ascomycetes and deuteromycetes, but not yeasts. The highest level of identical aa, 84”,b, is obtained with the chicken Tubs, 82 and 87, and rat Tub 815. (3) At the C-terminal region of the S. commune /ITub, the region known in animal BTubs to interact with the mAb DMlB is conserved, along with another region which has been suggested to interact in animal BTubs with MAPS. A localization of Cys lhs in the S. commune PTub could be a reason for the insensitivity of S. commune to the antimicrotubule drug benomyl. (4) Southern hybridization experiments warrant attention to the possibility that S. commune might possess another /ITub-encoding gene highly divergent from rub-2. Different haploid S. commune strains have sequence polymorphisms in the flanking regions of tub-2. The level of tub-2 transcripts appears to be high when the microtubule polymerization rate is high, as during active vegetative extension of young mycelia, but it decreases when the mycelium ages and outgrowth slows down.
Burland,
T.G., Schedl, T., Gull, K. and Dove, W.F.: Genetic
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Gen. Genct.
J.H., May, G.S. and Morris, N.R.: Two
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P. and Walden,
Transformation and Gene Expression. well, Oxford, 1988, pp. 199-261,
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Black-
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