Biochimica et Biophysica Acta, 1171 (1993) 299-303
299
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 90434
Short Sequence-Paper
A yeast gene (BLH1) encodes a polypeptide with high homology
to vertebrate bleomycin hydrolase, a family member
of thiol proteinases
Ulla Magdolen a, Giinter Miiller b, Viktor Magdolen c and Wolfhard Bandlow
Institut fiir Genetik und Mikrobiologie, Miinchen (Germany), b c / o Hoechst Aktiengesellsehaft Frankfurt am Main,
Frankfurt (Germany) and " Max-Planck-lnstitut fiir Biochemie, Martinsried (Germany)
(Received 28 September 1992)
Key words: Bleomycinhydrolasegene; Antitumor drug; Cysteineproteinase; (S. cerevisiae)
We have purified bleomycin hydrolase from yeast (molecular mass 55000 Da). Using protein sequence-derived degenerate
oligonucleotide primers and amplification by polymerase chain reaction, the yeast gene BLH1 was isolated and characterized.
The deduced amino acid sequence (483 amino acids) exhibits surprisingly high homology to vertebrate bleomycin hydrolase (43%
identical residues and 22% conserved exchanges). It contains three blocks of sequences found conserved in other members of the
thiol proteinase family and thought to be associated with the catalytic centre. BLH1 is non-essential under all growth conditions
tested. However, in the presence of 3.5 mg bleomycin/ml medium wild-type cells have a slight growth advantage compared to
blhl mutant cells.
Bleomycin is a mixture of closely related glycopeptide antibiotics secreted from Streptomyces verticillus
[1]. It causes radiomimetic D N A damage in bacteria
and eukaryotes. It also inhibits growth of a number of
squamous and small cell type tumors in situ and in
vitro, although its exact mechanism of action is unknown. Due to its lack of hepatic, renal and bone
marrow toxicities, the drug is extensively used in tumor
therapy [1,2]. A more general use of the drug is,
however, primarily hampered by two drawbacks, (i) the
pulmonary toxicity [3] and (ii) the resistance of certain
cell types and tissues to the drug due to high intracellular activities of the thiol proteinase bleomycin
hydrolase [4-6]. The ubiquitous and abundant occurrence of this enzyme in all animal tissues and in
microorganisms points to an important, although as yet
unknown function. In order to study bleomycin resistance, to identify the physiological role of the proteinase and to understand why certain tumors are
refractory to therapy by the drug, it may be helpful that
we have isolated a yeast gene displaying high homology
Correspondence to: W. Bandlow, Institut fiir Genetik und Mikrobiologie, Maria-Ward-Strasse la, D-8000 Miinchen 19, Germany.
The nucleotide sequence data reported in this paper have been
submitted to the EMBL/Genbank Data Libraries under the accession number X69124.
of the encoded protein to bleomycin hydrolase from
rabbit.
We have isolated the bleomycin hydrolase protein
from yeast during a study of a cAMP receptor glycolipid-anchored to plasma membranes [7]. We have enriched the latter protein by isolating crude plasma
membranes from yeast spheroplasts by Percoll gradient
centrifugation and sequential purification of the detergent-solubilized protein by cAMP-agarose affinity
chromatograpy, molecular sieving and phenyl Sepharose lipophilic chromatography which uses the hydrophobicity of the glycolipid anchor-containing protein as
a parameter for separation. When the eluted proteins
were separated on a sodium dodecylsulfate polyacrylamide gel, one of the major bands comigrated with the
authentic, 8-N3-[32p]cAMP photoaffinity-labeled product (apparent molecular mass 54 kDa, as deduced from
electrophoretic mobility) [7]. This material was excised
from the gel, eluted and cleaved either with trypsin or
CNBr. The peptides were separated by high performance liquid chromatography. Two major fragments
(one from each cleavage) were sequenced (indicated in
Fig. 2). It turned out from the sequence data (see
below) that they obviously do not belong to the glycosyl-phosphatidylinositol-anchored cAMP-binding protein from yeast plasma membranes. Whether the enriched protein with homology to bleomycin hydrolase
binds to cAMP agarose and phenyl Sepharose directly
or interacts with the native cAMP receptor protein
300
fortuitously or in a functional complex remains to be
established in the future.
The corresponding gene was isolated by using the
polymerase chain reaction method. Since the intramolecular arrangement of the two eluted and sequenced peptides was unknown, three primers were
designed from each of them, two overlapping primers
in the one and a third one in the reverse direction.
Pairwise amplification by polymerase chain reaction
and crosswise hybridization led to the identification of
a DNA fragment, which encoded sequences from both
protein fragments. The genomic DNA clone, pPMY
11.0, subsequently isolated from a gene bank constructed from BamHI-digested yeast DNA, contained
about 11 kbp of genomic DNA and was characterized
by restriction and hybridization analyses. The region
cross-hybridizing to the amplified DNA fragment could
be narrowed down to two HindIII fragments, which
were sequenced subsequently from the central HindIII
site in both directions to yield the complete DNA
sequence of the yeast BLH1 gene (Fig. I A, B). The
nucleotide sequence analysis and the deduced primary
structure of the protein are displayed in Fig. 2. The
open reading frame comprises 483 triplets with a deduced molecular mass of the encoded protein of 55.5
Da, the calculated isoelectric point is at pH 9.58.
Structural analyses by using the algorithms of Kyte and
Doolittle, and Hopp and Woods do not reveal any
striking structural features and suggest a globular, hydrophilic protein. Southern hybridization with a labeled DNA fragment, derived from the coding region
of the BLH1 gene shows that BLH1 occurs in single
copy in the yeast genome (one example is given in Fig.
1D, lane 1). The codon bias index [8] of 0.35 points to a
relatively high expression. The DNA sequence flanking
the gene at its 5' side displays no obvious T A T A box.
However, many yeast genes are known which lack
obvious T A T A sequences. In the putative promoter
region of BLH1 a perfect consensus sequence for an
abundant DNA binding protein, Grf2p [9], is found,
immediately followed by a pyrimidine-rich sequence
(Fig. 2). Grf2p may have a role in nucleosome positioning by excluding nucleosomes from the vicinity of its
binding site. The same combination of sequence motifs
as in the BLHl-upstream region also occurs in the
promoter region of the yeast profilin gene, PFY1 [10].
Deletion of these elements in the PFY1 promoter
dramatically reduce transcription of PFY1 (H. Herrmann and W. Bandlow, unpublished results).
Data base searches using the Genetics Computer
Group (GCG) Sequence Analysis Software Package
[11] reveal significant sequence similarities to bleomycin
hydrolase, a cysteine proteinase isolated from rabbit
lung and liver. A cDNA fragment encoding the central
portion of this enzyme has been obtained from rabbit
liver and sequenced [6]. The alignment of the deduced
A
E
HH
EHE
I
II
III
H
I
I
I lOOO bp I
B
I
I
N
I
C
Jl
EH
[
Jl
E
11oo bp
4
I 500 bp
CN
b
C
I
N
""~-~--Jj
C
EH
I
E
# I~ it
N
C
2500 bp
EH
I
E
blh 1
II
CN
3500 bp
D
1
Jme
2
3
4
5
~
~
~
3.5
kbp
~
~
~
2.5
kbp
i
m
I.I
kbp
Fig. h Inactivation of the BLH1 gene. (A) Genomic restriction map
of the BLH1 locus. The arrow indicates direction of transcription.
(B) Restriction map of the sequenced region. Restriction sites relevant for gene disruption are shown. (C) Genomic organization after
integration of the linearized plasmid pPMP9-A at the BLH1 locus by
homologous recombination. Vector sequences between the duplicated BLHl-coding sequences (indicated by thick bars) are not
drawn to scale. (D) Genomic Southern blot from wild-type and
disruption mutants. Genomic DNA was isolated from the haploid
wild-type strain W303-1A (lane 1), from haploid disruption strains
(lanes 2 and 3), from heterozygous diploid strains (lanes 4 and 5) and
restricted with Ndel. The blots were probed with a digoxigeninlabeled fragment corresponding to the BLH1 fragment cloned into
pPMP9-A (indicated by thick bars in (C), C, Clal; E, EcoRI; H,
HindIlI; N, Ndel.
rabbit and yeast protein sequences is shown in Fig. 3A.
Within the region of the known rabbit sequence, the
homology is highly significant (40 S.D. units, using the
program A L I G N [12] with the mutation data matrix,
100 random runs and a penalty for a gap of 20). The
yeast sequence extends beyond the known rabbit sequence by 21 amino acids to the N-terminus and 158
residues to the C-terminus. The overlap of the two
proteins contains 43% identical amino acids and 22%
conservative exchanges at comparable positions (Fig.
301
3A). The search for protein motifs and domains reveals
that a stretch of 17 amino acids (pos. 92-108 of Blhlp)
is highly homologous to the region containing the cysteine residue conserved in the catalytic centre of eukaryotic thiol proteinases (Fig. 3B). These include calpain, plant and protozoan cystein proteinases, vertebrate lysosomal and secretory cathepsins, and two developmentally co-regulated cysteine proteinases from
the slime mold Dictyostelium discoideum. In addition,
it can be seen from Fig. 3B that Blhlp from yeast also
has significant homology to two sequence blocks of
other cysteine proteinases (which were previously unknown from the rabbit enzyme) implied to contribute
to the active centre, but conserved only in a subset of
thiol proteinases.
In order to gain some information about the physiological role of the protein, we replaced the structural
BLH1 gene with two different incomplete blhl alleles
260
GRF2
.............
C G T A T A T T C A G T G C T T T T T G A A A T T T A G A A A A T A G T C G 6 ~ T T A T T G A T T A T G C T A C A T A T G T T A T T T T G C T T T A G T A T G T G T C A A A T A G C A T G C A A T C G C C C G G T A C T G A G T G A C A C A T C CTTT'~FFCCC
C T C C C A C C A C A A A G T C A C C T G C T C T C T T A A T C A A T G G G G T T G C A T G T G G T A A T C A A T T G A T A A C G C C G G C T G A C A A G T C G C C G A C G G G C A C C C A T A A A T A A A C G A T A A A T AGCCC~'FGCT C T T T T G T T A C
ATG C T T C C T A C T T C T G T T T C C CGG T C C TTG T A T CTC AAG ACG TTT AGA AGC CAC CTT T T A CGA G C A C C A C A A A T T G T T T T A A A A A G A ATG T C C T C T T C C ATC
Met Leu Pro T h r Set Val Set Arg Set Leu Tyr Leu Lys Thr Phe Arg Set His Leu Leu Arg Ala Pro Gin lle Val Leu Lys Arg Met Set Ser Ser Ile
+103
GAT A T C A G T A A G A T C A A C T C T TGG A A C A A A G A G T T T CAA TCC G A C T T A ACC CAT C A A TTG GCA A C C A C T G T C C T T A A G A A T T A T A A T G C C G A T G A T G C A CTG
A s p Ile Ser Lys Ile Asn Ser T r p Ash Lys Glu Phe Gin Set A s p Leu Thr His Gln Leu Ala Thr T h r Val Leu Lys Ash Tyr A s n A l a A s p A s p Ala Leu
~205
TTC A A C AAG A C T A G A CTG C A A A A G CAA G A T A A C A G G G T T TTC AAC A C T G T T G T C TCT ACT GAT TCC A C T C C A G T C A C C A A C C A A A A A A G C T C T G G T A G A TGT
Leu Ash Lys Thr A r g Leu Gln LyS Gln A s p Ash Arg Val Phe Ash Thr Val Val Set Thr Asp Set Thr Pro Val Thr A s n G i n Lys Ser Set G I y A r g Cys
+307
TGG T T G T T T G C C G C T A C C A A T C A A C T G CGC TTG A A T G T T CTT TCT GAA TTG A A C T T A A A A GAA T T C C A A CTG T C C C A A G C T T A C T T G T T C ~
T A C G A T AAG
Trp Leu Phe Ala Ala Thr Ash Gln Leu A r g Leu Ash Val Leu Ser Glu Leu Ash Leu Lys Glu Phe C l u Leu Set Gln A l a T y r Leu Phe Phe T y r A S p Lys
+409
TTG G A A AAG G C C A A C T A T T T C TTG G A C CAA A T C G T C T C C TCG G C T G A T C A A G A C A T C G A C TCA C C T C T C GTG CAA T A C T T G T T A G C A G C T C C A A C A G A A GAC
Leu G l u Lys Ala Ash Tyr Phe Leu A s p Gln Ile Val Ser Set Ala A s p Gln A s p Ile Asp Ser A r g Leu Val Cln Tyr Leu Leu Ala Ala Pro Thr Glu ASp
+511
GGT G G C C A A T A C A G C A T G q ~ T TTG A A C C T A G T C A A G AAG TAT G G C C T T ATG CCC AAA G A T TTA T A C G G G G A T C T A C C T T A T T C T A C T A C T G C G T C C A G A AAA
Gly Gly Gln Tyr Ser Met Phe Leu Ash Leu Val Lys Lys Tyr Gly L e u Met Pro Lys A s p Leu Tyr Cly A s p Leu Pro Tyr Ser Thr Thr A l a Set Arg Lys
+613
TGC A A T T C T C T G TTG A C T A C T A A A C T G A G A G A A T T T G C C GAG A C T C T A AGA A C A G C T TTG AAA G A G C G T T C T G C C C A T G A T T C C A T A A T T G T C A C T C T G A G A
Trp Asn Set Leu Leu Thr Thr Lys Leu A r g Glu Phe Ala Glu Thr Leu Arg Thr Ala Leu Lys Glu Arg S~r Ala A S p Ash S~r Ile Ile Val Thr Leu Arn
primer I-A
+715
GAG C A A A T G C A A A G A G A A A T C T T C A G G T T G ATG TCG TTG TTC A T G GAC A T A C C T C C A GTG CAA C C A A A C GAG C A A T T C A C T T G G G A A T A C G T T G A C A A A GAC
Glu Gln M~t Gln Arm Clu Ii~ Phe Aro Leu Met get Leu Phe Met A s p Ile Pro Pro Val Gln Pro A s n Glu Gln Phe Thr T r p Glu T y r Val A s p Lys ASp
+817
AAG AAA A T C C A C A C T A T C A A A TCG A C T C C G T T A G A A T T T GCC T C C A A A T A C GCA A A A T T G GAC C C T T C C A C G CCA G T C T C A T T G A T C A A T G A T C C A A G A CAC
Lys Lys Ile His Thr Ile Uys Set Thr Pro Leu Glu Phe Ala Ser Lys Tyr Ala Lys Leu Asp Pro Ser Thr Pro Val Set Leu Ile A s n A s p Pro Arg His
+919
CCA T A T G G T A A A T T A A T T AAG A T C G A T C G T T T A G G A A A C GTC C T T GGC GGA G A T G C C CTG A T T T A C T T A A A T C T T G A C A A T G A A A C A C T A T C T A A A TTG G T T
Pro Tyr G l y Lys Leu Ile Lys Ile A s p Arg Leu Gly Ash Val Leu Cly Gly Asp Ala Val Ile Tyr Leu A s n Val A s p Asn C l u T h r Leu Set Lya Leu Val
+1021
Wv
GTT A A G A G A T T A C A A A A T A A C A A A G C T G T C T T T T T T GGA T C ~ C A C A C T CCA A A G T T C A T G GAC A A G A A A A C T G G T G T C A T G G A T A~'F G A A T T G T G G A A C TAT
Val Lys A r g Leu Gln ASh Asn Lys Ala Val Phe Phe Cly Set His Thr Pro Lys Phe Met A s p Lys Lys Thr Gly Val M~t A s h II~ ~ ] u Leu T r D ASh "~vr
p r i m e r 2-A
/
primer 2-C
C C T G C C A T C G G C T A T A A T ~ A C C T C A G C A A AAG G C A TCG CGT A T T AGA T A C C A T GAA A G T TTG A T G A C T C A T G C T ATG ~ G A T C A C T G G C T G C C A C G T C G A T
Pro A l a If@ G l v Tvr Ash Leu Pro C]n Gln Lvs A l a Sat Arg Ile Arg Tyr His GIu Set Leu Met Thr His A l a Met Leu Ile Thr G l y CyS His Val Asp
+1225
GAA A C G T C T A A A q~FA C C A C T T C G C T A C CGC G T T G A A A A T TCC TGG G G T A A A G A C T C C G G T A A A G A C G G A T T A T A C G T G A T G A C T C A A A A G T A C T T C G A G GAG
Clu T h r Ser Lys Leu Pro Leu A r g Tyr A r g Val Glu A S h Set Trp GIy Lys A s p Set Gly Lys A s p G l y Leu Tyr Val Met Thr G l n Lys ~ r Phe Glu Glu
+1327
TAC T G C T T T C A A A T T G T G G T C G A T A T C A A T G A A T T G C C A AAA GAG CTG G C ~ TCA AAA T T C ACC T C A G G T A A G G A A GAG C C G A T T G T C T T G C C C A T C TGG G A C
Tyr Cys Phe Gln Ile Val Val A s p Ile A S h Glu Leu Pro Lys Glu Leu Ala Set Lys Phe Thr Set Gly Ly$ G l u G l u Pro Ile Val Leu Pro Ile T r p Asp
+1429
CCA A T G G G T G C T TTG G C C A A A TAA
Pro Met G I y Ala Leu A l a Lys ***
ATAGTT TCAGCAGCTC TGATGTAGAT ACACGTATCT CGACATGTTT TATTTTTACT ATACATACAT AAAAAGAAAT AAAAAATGAT AACGTGTATA
Fig. 2. Nucleotide and deduced amino acid sequences of BLH1. The putative Orf2p-binding site is overlined, the adjacent pyrimidine-rich region
is indicated by dots. The two peptides sequenced from the purified protein are underlined. The DNA regions corresponding to the primers
effective in PCR amplification are overlined by arrows. The sequences of these degenerate oligonucleotides were: 5' CAA/GATGCAA/GC/
A G I / C G A A / G A T (I-A); 5' CCIATI/CGCI/CGGA/GTAA/GTT (2-A); 5' TGT/CTGI/CGGI/CAA/GA/GTTA/GTA (2-C). I denotes
inosine.
302
as detailed in Fig. 1C. For this purpose the coding
sequence contained between the ClaI site at pos. + 100
and the EcoRI site at pos. +615 was cloned into the
yeast integration vector pRS306 harbouring the URA3
gene as a selective marker [13] to yield clone pPMP9-A.
After cleavage of pPMP9-A with HindlII (pos. +383
of the coding region of BLH1) the resulting linear
plasmid DNA was transformed into either haploid or
diploid yeast wild-type ceils. The correct integration of
the plasmid by homologous recombination at the BLH1
locus leads to the duplication of those BLH1 DNA
sequences cloned into pRS306, the resulting two incomplete copies being separated by sequences from the
vector (Fig. 1C). The integration event was proven by
Southern blotting of genomic DNA restricted with
NdeI. The blot was probed with the BLHl-derived
sequences cloned into pPMP9-A. DNA from wild-type
cells yields a single signal corresponding to 1.1 kbp
(Fig. ID, lanes 1), whereas the haploid disruption
mutants display two signals as expected, corresponding
to 2.5 and 3.5 kbp (Fig. 1D, lanes 2 and 3) instead of
the 1.1 kbp band (cf. Fig. 1B and C). Heterozygous
diploids contain both, the wild-type and the disrupted
alleles. Consequently, all three signals can be seen with
DNA from heterozygous diploid disruption strains (Fig.
1D, lanes 4 and 5).
blhl disruption mutants were found to be viable in
the haploid state. When the growth behaviour of the
haploid mutant and the isogenic wild-type strains was
compared, no differences were observed on glucose,
galactose and glycerol media. Also long-term experiments, where cells were shifted from one carbon source
to another (eight changes of media), revealed no disadvantage for the blhl mutant strains compared to wildtype cells. In addition, heterozygous diploids sporulate
well, and the spores germinate. It must be concluded
from these results that the gene is dispensable for
vegetative and meiotic growth, cell viability and differentiation. Finally, the haploid wild-type strain (with a
functional BLH1 allele) was compared to the disruption strain with respect to growth in the presence of
bleomycin. For this purpose co-cultivation experiments
of the haploid strain W303-1A and of the isogenic
blhl mutant strain were performed: cells of both strains
pre-grown overnight in the presence of bleomycin were
mixed and incubated for 2 days in the absence of 3.5
mg bleomycin/ml medium (which leads to about 50%
growth inhibition). The ratio of the wild-type and mutant cells was monitored by replica-plating at the beginning of the experiment and after two days of growth
by testing uracil auxotrophy (URA3 has been used as
the selective marker to inactivate BLH1). The initial
A
- - -A~QI~I~:KRMSS
S DISKINSWNKEFQSDLTHQEATTVLKNYNADD~LLNKTR
HMD~LD
I CERRAT
D ~. . . . F ~ ; ~ ; A Q N V G . - . . . . . . . . . . . . . . . .
yeast B l h l p
rabbit BLM hydrolase
L O K Q D N R~
yeasl BIhlp
N T ~V S T D S T ~
H V~ P Q E G
rabbit BLM hydrolase
- - - - - Q HV~Q
yeast Blhlp
rabbit BLM hydrolase
L ; S Q A Y,L E ~ Y ;D;K L E K A N Y F E D
F~S
~V ~ ~W~V
E R C Y FF~N
yeast B l h l p
rabbit BLM hydrolase
M F L
MLVN
yeast Blhlp
rabbit BLM hydrolase
SADDS
VT LR EQMQRE
G~ T K A Ei S A T O D T M M EE
yeast Blhlp
S - ~P~EFA
P I ~.....
E F Y
rabbit BLM hydrolase
......
L~ A A T Q L
N V L S E ~N L K S~ E
I F ; S C L;N V M R L ; P F M K K ~ N I
E
Q I VS S ~D Q D
- - DSR
A FVD T~Q K K E P EbG~
L V K ~ YG L MPK D L Y G D L P Y S T T ASR
~ ~E~(~VV
KCFP~SH T ~ E ~ R
S K YAKR Q H V~P
QG~
~ ~O¥ L L A A P T E OiGG~Y
VO~ L LM N P ~NDGG~WD
S
K WN S L L T T K L R E F A E T LR T A L K E R
R MN D. L N H K MREF C I R ~R N - M VHS
~R L M S L FMD I PP VQ P NEQF~E
V V C I C L G NP P ....
E T F~W
¥ VDKDK
K I H TIK
E Y ROK D K N Y O K | G
- L D P S T P V S L INOPR
- - H P~G K~I K I DR EGN V L~GD
L F N M E D K I C F VN~P R P Q H KYN REY T VD YESNM V~R
A - - K
B
Blhlp
~
BLM hydrolase
cathepsin H
cathepsin L
cathepsin S
CP1
CP2
cruzain
papain
aLeurain
oryzain y
calpain
.l!~SC
L 133aa - - V N H ~ V ~ A
V~ 130u- - L DH'GV;~!VV~-
~
-
-
-P'V K D'OG
- T D I C~G
H C~
S!~P
A L~D~W~
S T ,T ~.is~ m.
~s~v
S T ~ ~ S TT L ~
I -
131=a- - V N H G V ~ V V , f ~ Z34a=L DI;fG I ~ ! i V Y 130aa -
L D H G V ~ V V~G -
126aa131aa132aa149aa-
L DHG V~ L V~VDB~VAAV~VNH~V~AV~V N~V~A
V~KG~Y
SV~A
-
Z2eaa
11aa - - : ~ K P ~ . S .
N W~N N~Y
F -
:1.5. . . .
~ K ~ E W ~ M ~ Y
I ~ l a a - -'VK,N~_~p~I~L H F ~ D Q ~ Y
I ZGa=- - ~ K I t ~ W ; ~ G A B W ~ E Q E Y I -
4eaa - S
Zl . . . .
I KN,~T
7aaZlaa11aa 11aa-
W~ I K~Y
T QW~E
E~Y
N~Y
- I K~S~W~A~D'WQDN~Y
- I KN ~Wi~AOWGD
NGY
- I R~ pW!~QV EWTGAWS
- I KN~'8~,GTGW~E
I -
I
I
F
F
-
Fig. 3. Comparison of the deduced amino acid sequence of BLH1 to other cysteine proteinases: (A) residues identical to the known sequence of
rabbit bleomycin hydrolase [6] at corresponding positions are shaded. (B) Alignment of Blhlp with other cysteine proteinases in the regions
containing the putative active-site cysteine, histidine and asparagine (indicated by stars) [14]. Amino acids identical to Blhlp are shaded.
Sequences were taken from [6,14-19].
303
ratio of 55% blhl mutant and 45% wild-type cells
changed after two days (i.e., 26 generations) to 27%
mutant and 73% wild-type cells, indicating a slightly
higher sensitivity of the mutant cells to the drug. In
contrast, the ratios of both types of ceils did not change
significantly in the absence of bleomycin (55%/45%
versus 56%/44% blhl/BLH1 cells). These results
make it likely that BLH1, in fact, encodes a cysteine
proteinase capable of hydrolyzing bleomycin. The lack
of a detectable phenotype in blhl mutant cells in the
absence of bleomycin as well as the only slightly increased sensitivity to bleomycin suggest that yeast may
contain other proteinases with overlapping substrate
specificities in addition to bleomycin hydrolase.
We thank E.-M. Wetekam for excellent technical
assistence. The work was supported by a grant of the
Deutsche Forschungsgemeinschaft to W.B. (Ba415/194).
References
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