Note: Descriptions are shown in the official language in which they were submitted.
CA 02080482 2001-05-08
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ISOLATED PROMOTER AND TERMINATOR OF ELONGATION FACTOR EF-la
Description
The present invention relates to a promoter
region from Ashbya gossypii (= A. gossypii), to fungi
which have been genetically modified with this promoter
region, and to the use thereof.
A. gossypii is employed for the production of
vitamin BZ by fermentation. It is desirable to extend the
use of the fermentation technology available for
A. gossypii by producing protein products using genetic
engineering methods. An expression system for A. gossypii
is required for this purpose. Systems of this type have
already been described for some higher ascomycetes such
as Aspergillus niger (Rambosek and Leach, CRC Critical
Reviews in Biotechnology 6 (1987), 357-393). By contrast,
to date no experience in the area of genetic engineering
is available with the hemiascomycete A. gossypii, which
is the sole representative of its genus.
An essential component of a system for the
expression of genes which code for a required product is
the so-called promoter region which is composed of
1) a functional promoter which is indispensable for
transcription of the gene, and
2) the 5' non-coding region (between promoter and
translation start) which is necessary for transla-
tion after transcription into mRNA.
The invention relates to the promoter region of
the A. gossypii TEF gene which encodes translation
elongation factor EF-:l« (= TEF-la).
This gene is very strongly expressed and there-
fore has a very efficient promoter region.
The promoter region obtained according to the
invention has the nucleotide sequence indicated in
sequence listing No. 1. Since the limits of the
functional regions of a novel and sequenced promoter
CA 02080482 2001-05-08
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region, which have the ability to initiate transcription
and translation, can be defined well at the 3' end and
less well at the 5' end, it cannot be ruled out that the
natural promoter region of A. gossypii gene differs
slightly in length from the indicated sequence.
The invention further relates to the terminator
region of the A. gossypii TEF gene which encodes trans-
lation elongation factor EF-la (= TEF-la).
The terminator region can be used for efficient
termination of transcription.
The terminator region obtained according to the
invention has the nucleotide sequence indicated in
sequence listing No. 2, position 1513-2095. 3'-Terminal
truncations of this sequence are also suitable as trans-
cription terminator.
The terminator region can be used in conjunction
with the TEF-promoter region or with other homologous or
heterologous promoters.
The invention. further relates to fungi which
contain the above mentioned promoter region or parts
thereof and/or the above mentioned terminator region or
parts thereof.
The promoter region can be inserted, in particu-
lar, into the following fungi:
Ashbya gossypii,, species closely related to Ashbya, such
as, in particular, Eremothecium ashbyi and genera unre-
lated to Ashbya, such as, in particular, Aspergillus and
Neurospora.
The novel promoter region can be prepared
a) by cloning the gene for translation elongation
factor EF-la (TEF gene) from A. gossypii including
adjoining DNA sequences and subsequently cleaving,
b) by fusion of A. gossypii DNA fragments to an open
reading frame of a promoterless gene which is
selectable in A. gossypii, isolation of strongly
expressing transformants and subsequent selection of
the TEF promoter,
2~~~:~8~
w - 3 - O.Z. 0050/41686
c) by chemical synthesis using known methods.
The novel promoter region makes it possible,
together with suitable vector systems, to bring about
overexpression of homologous and heterologous proteins in
A. gossypii and other fungi. This may entail, for ex-
ample, constitutively enhanced expression of genes of
vitamin BZ biosynthesis, and of genes which are respons-
ible for overproduction of vitamin B2, or the overexpres-
sion and isolation of proteins which are of economic
importance. It is furthermore possible with the aid of
the novel promoter region to utilize the post-transcript-
ional modification potential (e.g. glycosilation) of
A. gossypii, which in some circumstances differs from
that of other fungi. Since it is not possible to prepare
all heterologous proteins in sufficient amounts by, the-
systems hitherto used, such as Aspergillus or '
Saccharomyces, the development of expression systems with
the efficient TEF promoter region for novel host organ-
isms (for example A. gossypii in this case) is of great
importance.
EXAMPLES
1. Isolation of the Ashbya gossypii TEF gene
DNA isolated from A. gossypii mycelium was cut
with the restriction endonucleases EcoRI and BamHI.
DNA fragments which harbor the TEF gene or parts
thereof were identified after separation of the
restriction fragments according to size in an
agarose gel electrophoresis and subsequent hybrid-
ization with a 32P-labeled heterologous TEF gene
probe. The TEF gene probe comprises nucleotides 363
to 1235 of the 1377 bp-long open reading frame of
the S. cerevisiae TEF2 gene (Schirmaier and
Philippsen, EMBO J. 3 (1984), 3311-3315). A
4.6 kb-long EcoRI fragment and a 6.4 kb-long BamHI
fragment hybridized with the heterologous TEF gene
probe. Fragments with lengths in these ranges were
eluted from from agarose gels, cloned into the
2n~~~~?
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vector pUCB (Vieira and Messing, Gene 19 (1982),
259-268) which had been cut with EcoRI or HamHI,
and
transformed into E. coli. The clones with TEF DNA
were identified by colony hybridization (Grunstein
and Hogness, Proc. Natl. Acad. Sci. USA 72 (1975),
3961-3965) using the 32P-labeled heterologous probe.
The positive clones contained either the 4.6 kb-long
EcoRI fragment or the 6.4 kb-long BamHI fragment.
The two clones overlap in a 2.1 kb region which
carries the homology with the TEF gene probe and
which was sequenced (sequence No. 2). This
2.1 kb-long fragment contains the open reading frame
of 1377 bp, 136 by of the 5'-non-coding region and
582 by of the 3'-non-coding region. Beyond the
EcoRI cleavage site, a further 278 by of the_
5'-non-coding region were determined up to a HindIII~
cleavage site. Subsequently, the promoter region
was
isolated as 403 bp-long HindIII/HincII fragment
which, besides the 379 by in front of the start
codon, also harbors the first 24 by of the open
reading frame of the TEF gene, and was employed for
the constructions of pAG-100 and pAG-101 (sequence
No. 1).
2. Plasmid constructions
a) The vector pAG-1 (Fig. 1) (deposited DSM 6010),
a derivative.of the vector pEX4, was prepared as
described by Ernst and Chan, J. Bacteriol. 163
- (1985), 8-14. pAG-1 contains a 1.7 kb Sall
fragment with the kanamycin-resistance gene,
which codes for the aminoglycoside phosphotrans
ferase (APH(3')I), of the transposon Tn903. In
the original pEX4 construct, initially the
1695 by PvuII fragment of Tn903 (Oka et al.,
J. Mol. Biol. 147 (1981), 217-226) was ligated
into a plasmid with filled-in Sall cleavage
sites. The SalI cleavage sites wars: retained in
this way, and the resistance gene can be isolated
~(!~~~J!~~~:
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as 1.7 kb SalI fragment. pAG-1 contains the
Saccharomyces cerevisiae ARS elements ARS1 and
2~ ARS and undergoes autonomous replication in
Ashbya gossypii.
b) pAG-2 (Fig. 2). The 1.7 kb SalI fragment with the
kanamycin-resistance gene was cut out of pAG-1
and inserted into the SalI cleavage site of the
S. cerevisiae E. coli shuttle vector XEp24
(Botstein et al., Gene 8 (1979), 17-24; New
England Biolabs Inc., Beverly, MA, USA, 1988-1989
Catalog, 112-113). The structure of the newly
produced plasmid -- pAG-2 - was checked by re-
striction endonuclease mapping, using the XhoI
cleavage site which is located in the 1.7 kb SalI_
fragment to check the orientation of the insert.-
pAG-2 contains the Saccharomyces cerevisiae ARS '
element 2Es ARS and undergoes autonomous replica-
tion in Ashbya gossypii.
c) pAG-100 (Fig. 3). A 403 bp-long HindIII/HincII
fragment which contains the promoter region and
the first 24 by of the open reading frame of the
gene for translation elongation factor EF-la (TEF
gene) from A. gossypii was inserted, after the
protruding ends had been filled in, into the XhoI
cleavage site of pAG-2 which is located 30 by in
the 3' direction behind the translation start of
the kanamycin-resistance gene. The orientation of
the fragment in the plasmid pAG-100 produced in
this way was checked by restriction endonuclease
mapping with HindIII. Insertion of the 403 be-
long fragment resulted in replacement of the
10 N-terminal amino acids of APH(3')I by the
first 8 amino acids of A. gossypii translation
elongation factor EF-la. Deletion or replacement
of the first 19 amino acids of APH(3')I by other
amino acids does not result in loss of activity
(Chen and Fukuhara, Gene 69 (1988), 181-192). The
(.: _.. 'v ;.,'
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sequence of the SalI fragment after insertion of
the TEF promoter region is shown in sequence
No. 2. pAG-100 contains the Saccharomyces
cerevisiae ARS element 2~ ARS and undergoes
autonomous replication in Ashbya gossypii.
d) pAG-5 (Fig. 4). The 1.7 kb fragment with the
kanamycin-resistance gene from pAG-1 was sub-
cloned into the SalI cleavage site of pBR322
(Bolivar et al., Gene 2 (1977), 95-113). The
resulting plasmid - p,TL3A - contains in the
pBR322 portion one BamHI cleavage site and one
EcoRI cleavage site so that pJL3A is decomposed
by double digestion into a 375 by and a 5688 by
fragment. The large fragment was ligated to a_
2.1 kb EcoRI/HamHI A. gossypii fragment which-
contains the open reading frame of the gene for _'
translation elongation factor EF-1a (TEF gene)
(sequence No. 1). The resulting plasmid was
called pAG-5. pAG-5 contains no Saccharomyces
cerevisiae ARS elements.
e) pAG-101 (Fig. 5). The 403 by HindIII/HincII
fragment with the promoter region and the first
24 by of the open reading frame of the TEF gene
from A. gossypii was inserted into the Xhol
cleavage site located in the open reading frame
of the kanamycin-resistance gene, as described
for the construction of pAG-100. The plasmid
- produced in this way was called pAG-101. pAG-101
contains no Saccharomyces cerevisiae ARS
elements.
f) pBRS1871 (precursor plasmid for TEF promoter-lacZ
fusion): A 3113 bp-long Pstl fragment from the
plasmid pMC1871 (Shapira et al., Gene, 25 (1983),
71-82) was cloned into the PstI cleavage site of
the plasmid pBRS+ (Short et al., Nucleic Acid
Res . , 16 ( 1988 ) , 7583-7600 ) . The fragment harbors
~~~.~~ ~w
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the open reading frame of the lacZ gene from
E. coli (Ralnins et al., EMBO J., 2 (1983), 593-
597) which lacks the first seven codons.
g) pPLl (Fig. 6). pBRS1871 was linearized at the
SmaI cleavage site in front of the lacZ gene. A
1500 by HincII fragment which harbors the TEF
promoter and adjoining sequences including the
first eight codons of the TEF gene from
A. gossypii was cloned into the linearized
plasmid. This resulted in an open reading frame
which codes for a ,B-galactosidase whose first
seven amino acids are replaced by the first eight
amino acids of the EF-la from A. gossypii. It was
possible to isolate TEF promoter-lacZ fusions
with regions of various lengths of the TEF~_
promoter from this plasmid.
h) pPL2 (Fig. 9). pBRS1871 was linearized at the
SmaI cleavage site in front of the lacZ gene. A
294 bp-long Rsal/HincII fragment which contains
parts of the TEF promoter (270 bp) and the first
eight codons of the TEF gene from A. gossypii
(24 bp) was cloned into the linearized plasmid.
i) pPL3 (Fig. 10). pBRS1871 was linearized at the
SmaI cleavage site in front of the lacZ gene. A
239 bp-long HaeIII/HincTI fragment which contains
the first eight codons of the TEF gene (24 bp)
and 215 by of the regions, located in the 5'
direction in front of the start codon, of the
non-translated region was cloned into the linear-
ized plasmid.
j) pPL4 (Fig. 11). pHRS1871 was linearized at the
SmaI cleavage site in front of the lacZ gene. A
158 bp-long EcoRI/HincII fragment which contains
the first eight codons of the TEF gene and 134 by
of the regions, located in the 5' direction in
front of the start codon, of the non-translated
region was cloned into the linearized plasmid.
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k) pAG-110 (Fig. 7). Cleavage of pPLl with XbaI and
SalI resulted in isolation of a 4600 by fragment
which harbors the fusion of the 1500 bp-long TEF
promoter fragment with the lacZ gene. After the
protruding ends had been filled in, this fragment
was cloned into the filled-in BamHI cleavage site
of pAG-100.
1) pAG-111 (Fig. 8). Cleavage of pPLl with HindIII
resulted in isolation of a 3509 bp-long fragment.
The TEF promoter region is truncated by 1100 by
in this fragment. It thus corresponds to the
promoter region which in pAG-100, pAG-101,
pAG-110 and pAG-111 controls transcription of the
6418 resistance gene. After the protruding ends.
had been filled in, the 3509 bp-long fragment was
cloned into the filled-in BamHI cleavage site of
'
pAG-100.
m) pAG-112 (Fig. 12). After cleavage of pPL2 with
XbaI and SalI, a 3392 bp-long fragment which
harbors the fusion of the 294 bp-long promoter
fragment with the lacZ gene was isolated and,
after the protruding ends had been filled in, was
inserted into the filled-in BamHI cleavage site
of the plasmid pAG-100.
n) pAG-113 (Fig. 13). After cleavage of pPL3 with
XbaI and Sall, a 3337 bp-long fragment which
harbors the fusion of the 239 bp-long promoter
fragment with the lacZ gene was isolated and,
after the protruding ends had been filled in, was
inserted into the filled-in BamHI cleavage site
of the plasinid pAG-100.
o) pAG-114 (Fig. 14). After cleavage of pPL4 with
HindIII, a 3273 bp-long fragment which harbors
the fusion of the 158 bp-long promoter fragment
with the lacZ gene was isolated and, after the
protruding ends had been filled in, was inserted
into the filled-in BamHI cleavage site of the
n
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plasmid pAG-100.
p) pAG-115 (Fig. 15). After cleavage of pBRS 1871
with BamHI, a 3069 bp-long fragment which harbors
the open reading frame of the lacZ gene with the
first seven codons of the open reading frame
being missing and no promoter fragment being
fused in front of the open reading frame was
isolated. This fragment was inserted into the
BamHI cleavage site of the plasmid pAG-100.
g) pAG-120.pBIIRS (Short et al., Nucleic Acid Res.
16 (1988), 7583-7600) was cleaved with SspI and
ScaI, and a 2084 bp-long fragment was isolated.
YEP24 (Botstein et al., Gene 8 (1979), 1?-24) was
cleaved with Scal and ClaI, and a fragment
Z5 2782 by in size was isolated. This was ligated,- .
after the protruding ends had been filled in, to .,
the 2084 bp-long ScaI/SspI fragment from pBIIRS-
so that a complete ampicillin-resistance gene was
produced again (in pBIIRS- arid YEP24, ScaI cuts
in the ampicillin-resistance gene).
r) pAG-121.pAG-100 was cut with SalI and HindIII,
and a 669 bp-long fragment which harbors part of
the 6418-resistance gene was isolated. This was
cloned into the SAlI/HindIII cut plasmid pBIISR+
(Short et al., Nucleic Acid Res. 16 (1988), 7583-
7600).
s) pAG-122.pAG-100 was cut with HindIII, and a
- 940 bp-long fragment which harbors part of the
6418-resistance gene under the control of the TES
promoter. This was inserted into the HindIII-cut
plasmid pAG-121 in such a way that a complete
6418-resistance gene was produced. Transformation
of this plasmid into E. coli permits transformant
selection on kanamycin-containing medium.
t) pAG-123.pAG-122 was cut with SalI~and BamHI, and
a 1639 bp-long fragment which harbors the G418-
resistance gene under the control. of the TEF
4'
-~ 10 - O.Z. 0050/41686
promoter was isolated. This was inserted into the
ScaI-cut plasmid pAG-120, which made selection of
E. coli transformants on kanamycin-containing
medium possible.
u) pAG-130.pBIIRS+ (Short et al., Nucleic Acid.
Res. 16 (1988), 7583-7600) was cleaved with
HindIII and HincII, and the 403 bp-long
HindIII/HincII TEF promoter fragment was
inserted.
v) pAG-131. An HaeIII/AccI fragment which is 260
by
in size and which contains 25 nucleotides of the
3' end of the TEF gene and regions adjacent
thereto in the 3' direction (terminator fragment)
was isolated from the clone which harbors the
fragment 2.1 kb in size, which contains the TEF-
'
gene, of genomic A. gossypii DNA. After the
protruding ends had been filled in, this fragment
was inserted into the plasmid pBIIRS- which had
been cleaved with HincII (Short et al., Nucleic
Acid Res. 16 (1988), 7583-7600).
w) pAG-132. pag-130 was cut with ScaI and XhoI,
and
a fragment 2248 by in size was isolated. pAG-131
was likewise cleaved with Scal and XhoI, and a
fragment 1442 by in size was isolated and was
ligated to the 2248 by fragment from pAG-103 in
such a way that a complete ampicillin-resistance
gene was produced anew.
x) M13PT. pAG-132 was cleaved with BamHI, and a
fragment which is 752 by in size and which
contains the fusion of TEF promoter fragment and
TEF terminator fragment was isolated. This was
cloned into the BamHI cleavage site of M13mp9.
y) M13PT1, M13PT2, M13PT3.
M13PT was modified by oligonucleotide-directed
mutagenesis (Rramer et al., Nucl. acid. Res. 24
(1984), 9441-9556) so as to produce an Scal
cleavage site behind the stop codon of the TEF
~ :~ ~~ ~J ~. Vie' ;
w - 11 - O.Z. 0050/41686
gene (in the terminator fragment) and an NcoI
cleavage site (M13PT1), an NsiI cleavage site
(M13PT2) or an SphI cleavage site (M13PT3) in the
start codon of the TEF gene (in the promoter
fragment) (Fig. 17).
z) pAG-201. pAG-202, pAG-203 (Fig. 18).
M13PT1, M13PT2 and M13PT3 were cleaved with
BamHI, and the fragment which is 751 by in size
and has promoter region and terminator region of
the TEF gene was isolated from the cleavage. This
TEF signal sequence was inserted into the BamHI
cleavage site of the plasmid pAG-123 to yield the
plasmid pAG-201. The same method was used to
construct the plasmid pAG-202 from M13PT2 and the
plasmid pAG-203 from M13PT3.
3. Transformation of A. gossypii with TEF promoter. '
region plasmids
The transformations were carried out in
accordance with the following scheme:
- Inoculate 200 ml of MA2 with about 1-2x10' spores
- Incubate in flasks with baffles at 27°C and
350 rpm for 32-40 h.
- Remove mycelium by filtration with suction and
wash lx in 30 ml of H20
- Determine fresh weight (about 2-3 g)
- Suspend mycelium in 30 ml of SD and incubate at
30°C in a shaker for 30 min.
- Suspend mycelium in 5-10 ml of SPEZ per g fresh
weight
- Incubate'in a water bath shaker at 30°C, check
protoplast formation under the microscope (a
degree of protoplast formation of more than 90
should be reached after 30 min.)
- Filter) protoplast suspension through glass
filter (Schott, porosity 1)
- Centrifuge filtrate for 5 min. (Sorvall
SM24 rotor, 1800 rpm)
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- Wash sediment lx in 20 ml of ST and lx in 20 ml
of STC
- Suspend protoplasts in 20 ml of STC and determine
titer in a counter
- After centrifugation, resuspend protoplasts to a
density of 4x108/ml in STC
- Add 100 u1 of protoplast suspension to DNA in a
maximum of 15 ~1 of TE and mix (amounts of DNA:
for replicating TEF promoter region plasmids: 1-
10 gig; for integrative transformation with
linearized TEF promoter region plasmids: 15-
gig)
- Incubate at room temperature for 15 min.
Cautiously add 1 ml of PTC40 and mix by inversion_
15 - Centrifuge for 5 min. (Heraeus Biofuge A,-
1500 rpm)
- Cautiously remove supernatant, and suspend
sediment in 1 ml of SMTCI
- Incubate at 27°C for 3 h., mix about every
20 45 min. by inversion
- After centrifugation, suspend sediments in 1 ml
of SM
- Mix suspension with 9 ml of SMA2 top layer and
place on SMA2 plate (20 ml of SMA2 agar per
plate)
- Incubate plates at 27°C for 18 h.
- Place 6418 layer on plates (0.54 ml of 6418 stock
solution + 0.46 ml of H20 + 6 ml of 0.5 ~ of
agarose (in HZO, preheated to 42°C))
- Incubate plates further at 27°C, transformants
are visible after 2-3 days in the case of repli-
cating plasmids, and after 3-6 days in the case
of integration
Media and solutions
Media: MA2: Peptone (Gibco casein
hydrolyzate (No. 140) . 10 g/1
Yeast extract (Gibco) . 1 g/1
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Glucose . 10 g/1
myo-Inositol . 0.3 g/1
SMA2-agar: Sorbitol . 1 M
Peptone . 10 g/1
Yeast extract . 1 g/1
Glucose . 20 g/1
myo-Inositol . 0.3 g/1
Agar (Gibco) . 12 g/1
SMA2 top layer: As SMA2 agar, 0.8 % agarose in
place of agar
Solutions: SD: 1M sorbitol; 50 mM dithiothreitol
SPEZ: 1M sorbitol; 10 mM Na phosphate
buffer pH 5.8;
10 mM EDTA; 2 mg/ml Zymolyase 20 T
(Seikagaku Kogyo Co., Tokyo) r .
ST: 1M sorbitol; 10 mM tris-C1 pH 8
STC: 1M sorbitol; 10 mM tris-Cl pH 8;
10 mM CaCl2
TE: 10 mM tris-C1; 1 mM EDTA
PTC40: 40 % (w/v.) polyethylene glycol 4000
(Merck); 10 mM tris-C1 pH 8; 10 mM
CaCl2
SMTCI: 50 % SM (see below); 50 % STC;
0.03 g/1 myo-inositol
SM: SO % 2 M sorbitol; 50 $ MA2
6418 stock solution: 2 0 m g l m 1 G 4 1 8
(Geneticin, Gibco) in
Ha0
4. Results of transformation with TEF promoter region
plasmids
The results of various transformations carried
out as in Example 3 ase compiled in Table 1. In all
the experiments, tranaformants were selected with a
6418 concentration of 0.3 mg/ml per transformation
plate. Growth of A. gossypii mycelium is completely
inhibited at this 6418 concentration. On transforma-
tion with the recombinant DNA, vectors pAG-1 and
2~~~~~~~;
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pAG-2, in which the 6418-resistance gene is under
the control of the original bacterial promoter and
not under the control of the TEF promoter region, no
transformants are produced at this concentration. In
order to obtain transformants with these recombinant
DNA vectors, the 6418 concentration must not exceed
0.1 mg/ml per transformation plate. At this concen-
tration up to 80 ~ of the colonies which appear are
not transformants.
TABLE 1: Transformation results
Experi- Plasmid DNA per Transform- Transformants
meet transf., ants per per viable
~g ~g of DNA protoplasts
1 pAG-1 10 0 0
1 pAG-2 10 0 0 -
1 pAG-100 10 10 1.2 x 10-°
2 pAG-100 0.1 10 1.6 x 10-5
3 pAG-10 0 1 3 3 . 4 x 10-'
3 pAG-101, 20 0.05 1.I x 10-5
linear
ized
with
BamHI
,
5. Results of transformation with lacZ plasmids
In order to investigate the functioning ability
of the TEF promoter further, derivatives of the
plasmid pAG-100 in which the gene for p-galactosid-
ase from E. coli (lacZ gene) is under the control of
the TEF promoter were constructed. For this, various
regions of the promoter region of the TEF gene were
fused in front of the open reading frame of the lacZ
gene, with the first seven codons of the lacZ gene
being replaced by the first eight codons of the TEF
gene. The plasmid pAG-110 harbors an approximately
1.5 kb-long HincII TEF promoter fragment in front of
the lacZ gene and the plasmid pAG-111 the 403 bp-
long HindIII/HincII TEF promoter fragment which has
already been employed for the constructions of
pAG-100 and pAG-101. The plasmid pAG-112 harbors a
~~~G~~'a%
- 15 - O.Z. 0050/41686
294 bp-long TEF promoter fragment, plasmid pAG-113
a 239 bp-long TEF promoter fragment and pAG-114 a
158 bp-long TEF promoter fragment.
In addition, pAG-115 which harbors the open
reading frame of the lacZ gene without fusion to a
promoter fragment was constructed as control plasmid.
After transformation of these plasmids into
A. gossypii, the expression of the lacZ gene was checked
using a color test. The ~-galactosidase encoded by the
lacZ gene cleaves X-Gal (5-bromo-4-chloro-3-indoyl p-D-
galactoside) to the blue dye 5-bromo-4-chloroindigo.
pAG-110, pAG-111 and pAG-112 transformants formed blue
colonies on medium which contains X-Gal (Miller,
Experiments in Molecular Genetics, Cold Spring Harbor,
New York 1972, 48) in a concentration of 100 ~g/ml. No-
blue coloration was visible in the case of transformants. '
which contained pAG-113, pAG-114 or pAG-115.
Fig. 16 shows a summary of the various TEF
promoter fragments which were fused in front of the lacZ
gene. A + represents a blue coloration of the colonies on
X-Gal-containing medium, a - represents no visible blue
coloration.
For a further investigation of ~-galactosidase
expression, the p-galactosidase activity of liquid
cultures of pAG-110, pAG-111, pAG-112, pAG-113, PAG-114
and pAG-115 transformants was determined. The mycelium
was disrupted with glass beads for this (Rose, M.;
Casadaban, M.J. and Botstein, D., Proc. Natl. Acad. Sci.
USA Vol. 78, No. 4 (1981), 2460-2464). 0.5 g of mycelium
which had grown in MA2 liquid medium containing 200 ~g/ml
G 418 was taken up in 0.1 mM Tris, pH 8.0/20 $ (vol/vol)
glycerol/1 mM DTT/1 mM PMSF and, after addition of 0.5 g
of glass beads~(diameter 0.45-0.5 mm), frozen away at
-20°C. To disrupt the mycelium it was shaken vigorously
(Vortex) at 4°C for l5,sec. 12 times. It was subsequently
centrifuged at 10000 rpm (Sorvall cooled centrifuge)
twice for 20 min. The supernatants were diluted 1:10 and
20~~~°~
- 16 - O.Z. 0050/41686
1:20, respectively, in Z buffer (0.06 M Na2HP0~/0.04 M
NaH2P0~/0.01 M KC1/0.001 M MgS04/0.05 M ~-mercaptoeth-
anol). The ~-galactosidase activity in the diluted
protein crude extracts was determined by cleavage of o-
nitrophenyl p-D-galactopyranoside (Miller, Experiments in
Molecular Genetics, Cold Spring Harbor, New York 1972,
353 ff). The enzyme.activity was related to the protein
concentration in the crude extract, which was determined
by the Bradford method (Bradford, M.M., Anal. Biochem. 72
(1976), 248-254). The results of the ~-galactosidase
activity determination are shown in Table 2. The amount
of o-nitrophenol (measured as OD4zo) liberated per minute
and mg of total protein is indicated.
TABLE 2: ~-Galactosidase expression
Plasmid Measurement p-Galactosidase activity
No. (relative units, ODazo%mg min.)
pAG-110 1 3.62
2 3.54
3 2.45
pAG-111 1 3.07
2 3.29
3 3.63
4 3.16
pAG-112 1 1.89
2 1.90
3 1.79
4 1.75
pAG-113 1 0
2 0
pAG-114 1 0
2 0
pAG-115 1 0
2 0
Sequence listing
No. 1:
Sequence type: nucleotide
Sequence length: 409 base pairs
Strandedness: single strand
r r~ ~;
- 17 - O.Z. 0050/41686
Topology: linear
Molecule type: genomic DNA
Source: A. gossypii
Properties: promoter region
AAGCTTGCCT CGTCCCNCGC GGGTCACCCG GCCAGCGACA TGGAGGCCCA GATACCCTCC 60
TTGACAGTCT TGACGTGCGC AGCTCACGGG GCATGATGTG ACTGTCGCCC GTACATTTAG 120
CCCATACATC CCCATGTATA ATCATTTGCA TCCATACATT TTGATGGCCG CGACGGCGCG 180
AAGCAAAAAT TACGGCTCCT CGCTGCAGAC CTGCGAGCAG GGAAACGCTC CCCTCAGCAG 2=~0
ACGCGTTGAA TTCTCCCCAC GGCGCGCCCC TGTAGAGAAA TATAAAAGGT TAGGATTTGC 300
CACTGAGGTT CTTCTTTCAT ATACTTCCTT TTAAAATCTT GCTAGGATAC AGTTCTCACA.360
TCACATCCGA ACATAAACAA AAATGGGTAA GGAAAAGACT CACGTTAAC 409
No. 2
Sequence of the 2.1 kb EcoRI/BamHI fragment with the open
reading frame of the TEF gene
20 3A 40 50 50
5' OdATfQ'CL~ G1CC,CrL~Ct~ ~GlC ,~'aTld~U, CGe'fbCGlf! TGCC~C1~C
70 80 90 100 110 120
crTerTCrrr c~rxrac~rc c.~rT~ errcQacca T~cac~rrcrc ~c~carc
130 140 1sa 1so 1, o lao
C9sACdIAEA GSLA~PGt39 TlAOQddAdQ dtI~C~Tfl Al.'GTTG1CL-I vdl~iTTC:dI:
190 200 210 220 ?30 240
GTCG1CTCTC ~7ldCI'C~1C TICCdCeOCt C1C:~TQ AC~Gi'f1'G6 I~GT~i'.GaC
250 260 270 280 2~0 300
AIGbGJI~CCI TCGAGidGIT CC~C~d6CbC G~'OCCG1C! TGCCrAdf,GC Ti'C~'t2Cl~C
- 310 320 330 340 3:4 360
Tack rrrn~au u°rcaacas c~cac~cac~ c~ccr~TC~.e caTacacaTr
370 3E0 390 400 410 420
xc~~rTa~ acrrarcac Ta~ucrx caccrc~crc Tc~TTCacx aracxcc~c
uo 4va 4so 460 4~0 4ao
ac~caa~a rcaa~cat r~tavcca acTrar~c cnac~vc casrrrcax
4~o soo slo 520 spa s4o
~rroabcrc ~czoccrca crr~cocr ~ra~rcr~ bccaCOCTCn s~cam~c
sso sso s7o sao s9o soo
caooc:~TCZ rcocrriac ct~ccci~c uoca"crsw x~rrcccar caacaar~Tc
6I0 620 630 6t0 6=0 660
Gl,CPGCC~CCd CTCf~Gi3~C GGCa6d~~ lCI~GGa6lC CeCCLiCrlC
670 680 690 700 710 920
IT~Cl~GC TCCC~C7d CCCTIaG~S 6~TGGCIT3eC TTCC11TCYC C~CfG~C
730 740 7so 7so 770 7ao
- 18 - O.Z. 0050/41686
No. 2 (Continuation)
'w'1'CrlCdalC~1 TC~ll1'G~CGC C7~CC1CC~1C GCt~dTGC! ~1GCGCIG GwIG~GWC
190 E00 d14 d20 830 880
ACC.uGCCTG GTGCCCTC~ GGGTaIC~bCC 1RG1'T~GG CClIC11TC~CCCd
850 860 8?0 8E0 890 900
CCrCTCaGaC CuCTC71C3a CGGTTG16~ TfGCGT~'OC IGa
910 920 934 940 950 960
GGTxTTGGEl CGQTCG6T CGGC71G1G!'C G1GJICCGCI'C TCbTCJ~GCC aCGT~TGQ'1'
970 9a0 990 1000 1010 1020
GiTIC~TTCC CCCL"11~C TAT G? CC~fC~IGIT GCa"CCaCCaG
1030 lOdO 1050 1060 1070 1080
~11TIGCiCC aGO~'C1~C 1GC1~'dClbC G~OG°f~ Cll CT;~:CGTC
1090 1100 1110 11~ 1130 lld0
~IGGlG~ C11161GG111 CCPTlOC6CT dC~llGi LC4CLL'~CC 6~lCOQ'~?
u:~o ueo u~g uav ux~ uuu
GaCI~ITCI 1C'CCT1~Q' CaTTQ°f~l'C M~CCC~C G1d181~C1C 2GC~G1?1C
1210 1ZZ0 1330 140 150 1240
TCTCGGTC! TC~Ci'OC~C b!i'OC11GTD 1GT1'CCdCGa CT!'cT~GiC
1~0 ugp u9p 1300 1310 1320
J1IGMCGbCI GadG~iGEdCiT6 CiaG~C ClIllC1'TCC1' u,IAO~~°t
1330 1340 1350 1360 1370 L3E0
C~dC4C'~Cl '~rGTCBdCT! 'fG~°OC~l~C 1M~CCi~T6? CTCinJGGC 'rPi~0.G~C
1390 1400 IdlO 140 1130 1410
ray r~r~c,~ct ccC~c~ G~cas~c ~eca~cc
1450 . 1160 1170 1110 Id90 1300
~ICnTC TTGTCAiGTC CCaC81o0CT G~?~CCTC1 CCiBGOCCOC cc~6oQ
151A L'u0 1330 3510 1550 1580
GCt~t lGa6TB~CPC liC3ri1'J~il~ Gdn'C~TGY! Zl°ClICUCa 'TCTC~Ti1'Cd
1570 1u0 1594 1600 1610 1620
bTl~'! ?ITlin?u '1~'t~?d'li't i~3'G1~! CllaA0~1'Gi ?llI~R~S
1630 1610 1650 1564 16'0 1680
T'1TT~CTCG 1GITClTCTC CCCaGaTCCC ~GtTIIIGTG C~C~6T A1T~TGC
1690 1700 1710 1724 1720 1740
crcSaBTCet, T3t3AiTOC! uaxo~?a2d C.roetcTCO~1 xrCQix~,C:a eC90G9CGr
1750 1760 17?0 1780 1190 1800
CQGTC1~C'd8 a:I'G1'C~Aa2 TT(~CC1GCCT ClI~TGCCTC Ca6G~txlGldl T11'GCZCGaC
1810 1820 1830 1840 1850 1450
~Q'GT~ C AaGGITIiCC CaTa1'GC~CP ATCGGCGG<1G allICGPTGC
1870 18E0 1890 1900 1910 1920
CACJIGOC~'rCT 1'CC'11~L~ GdCCTO~~CC ?TCCaCTGCl' IGITCICItiG T1CGCGr.TIG
1930 1940 1950 1960 190 1980
'lTaaCTTTC C1GOCCI~'! ~TOOCGCa ITIsIJSOC1T aTP60Gt'i~ GCtaCGeCl2
1990 2000 2014 ~0~0 2034 1040
CTGIOOCaIGa CGlCCl ~GGaCD00N' iTi~Tl2~ GGDC1CGG4
2050 2060 2090 x030 2090
~~1TI1'11W GCCaCCOaIC CaTGl'L'GCa? :C~Za? TG11CG~'dG GbTCC 3'
2(~~~~~
- 19 - O.Z. 0050/41686
No. 3
Sequence of the kanamxcin-resistance gene - TEF promoter
region fusion (TEF promoter region sequences underlined)
20 30 ;0 50 50
5' CTCGaC'i'C2A ATC~JC C~idCCGAd CAtTCATGG PClaMAA2C c~ITCLTu
7o ao 90 loa 1.o zzo
A4CCC1'fXbC AGCOCGC1GG Gf~d(~fGa ATACCCCP~! M2GICClaC ACdCTC~TGA
130 140 150 160 1'0 180
TG(xuCCTC 1GL121t~,bC TGICCfCI'OC Q'CGTGUGa AGC;"CTTCQ GBCPCbTACV
190 200 210 220 2;0 2a0
iGGCCfCUT CCCCC:ATG 'rCGGCGGa AaGTGaGG~ GCCICfCITC A.TCaGaGCIR
250 260 270 280 2~0 300
T~'."GP1GC! GG~ICCaGfTC fI'G31'i'Z'1'G~ A .. TGCCICG~i Ct~I'CTGCt?
310 320 330 340 350 360
'1'c~TCCwaaC l:GCCTGATC TGaT~I~Ca ~.TCac~ rICTTCGaITT ~Caa1
370 330 390 400 41A 420
GtCdCCfTGT Gu;TCUUT f~7~Cfi AC~TfGGCa ACaTAaaAIT ATaTGTCIT
470 440 450 660 4i0 4E0
GLiCUTUI 1TACafluC~ G~dTICaaC GGG'1'QnT6 aOCCaTaTfC
490 500 SIO 520 510 540 -
AJiCs~G~llaC GTQTGQ'CC
:30 560 570 580 590 600 a
610 620 630 640 6:0 660
570 6a0 690 700 710 720
730 740 750 760 770 7a0
79o aoo slo ago a3a :4o
aso aso a7o aao ago 900
910 920 93A 910 950 960
GGCCCC6aT! laaTTOCaaC ~fCG~fiOCTC lTf1'lmTCO GTayuIIGC
970 980 990 1000 1010 10x0
i9 1TCTCG~C1 ATCdlCdaT~TC C1TTCT~C Cu~3
1030 1014 1050 1060 1070 10x0
cc~caracr xrrl~crcaa as cc~cccr~c craaTCxxr racacarAc
1Q90 1100 II10 1120 1130 1110
Axcr~ca~ ~aucacocr caa~caara aZC~crrc c~am~a c~TZr~tc
II50 1150 2170 ZZaO 1190 1200
CC?lt~'CCTG aT~'GC1TC GdT,CTC1CC ICfOOGi2CC OC~6aauC IGCaTTfC3IC
1210 1210 1230 1240 1250 12b0
CrllTaGiaG aaTaTOG'TCa lTnOr.~Gaa laTaTT4'11°0 a'1'GlQCtTCLTG
1270 1ZE0 1390 1300 1310 I32A
C~61TOC 1!'1'CGiI'rOC TGrIIlGTld! ~ aCl~ilrC Ca'IiTi°CG~
1330 1340 1350 136!1 I37A 13a0
Q'C~OC OOCUl~OC ui~'11C OCfiT~T~6 A~CWQra i~ITCfI~C
1390 1400 1~10 1~120 1430 1140
carp ccxoc~ra xaacucrc rcrsucua T~arucca ~c~rx
1450 1410 1470 1410 1490 1500
s ua~aC gas rrcrcaax aruscxrar ~c
15!o lsao 133a is4o ipso ipso
a tai xo ~~~c~ta r~cxcac~ ariCCaaar
1570 15E0 1590 1600 1610 lbZO
C'!t'GCClfCC TITC~ICl~C OC~TGIG lt~~T~'! CaTTIGGlI AC~T'iR°!
~~~'~~o
- 20 - O.Z. 0050/41686
No. 3 (Continuation)
1630 16x0 1650 1660 16'0 1580
Cl~2bTC G:ITTGaTLI TC~ITI~TTC ldTIA~TTOC dG3T:~'.dT:T G~TGCTCGa2
1690 1700 1710 1720 1730 1740
GbGr~1TTC!' ~IITCaGadT? CCIT7IdTTOC TTGg3.lUQ' (~UGaGGI2 "3CCCTGACT
1750 1760 1770 1780 1,'90 1800
'fG~CGCG~CG GxGQ'tTGT TGa1T1,1TC GLICPrffCC I'G.1 .G':~d~lG C1TGG1TG
1810 1820 1830 1840 1850 1860
CCGTCTPCC C'~dCMCGCB C,~CCGTTC~ TG~GC~ dk~CTi~L ATCbCC.IdCT
1870 1880 1890 1900 I91A 1920
GOTCC~CT1 GUC~GQ CTGTC~CC GTCOCTCCCT cad C~T&
1930 1940 1950 1960 1970 1980
G OIGCt~TTCa G~tGTl2 C~CTUt~ GC~PrZTTC ~CCBt~CdCA c~TUOCGC:T
1990 2000 2010 CIO 2030 2010
aTrp'G~!' T~GTCaCG 1TC8Tt1~1CC CCTATrGGC Cl'GlCCCTGC
2050 2060 2070 2010 2090 2100
GCGCT~C1 G~ifir~iT C~TCc~ TI,C~CIWT G~~ITCflCCO TTGC~CC
Z11A
CGGl!lLGG TxlC 3~
Xeys to Figures 1 to 8
Fig. 1: Plasmid pAG-1. ARS: S. cerevisiae ARS1 sequence;
2 micron; EcoRI fragment of the S, cerevisiae
2~
plasmid with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin)
resistance; black arrow: S. cerevisiae cycl-13
promoter; black box: S. cerivisiae CYC1 termin-
ator; white arrows represent the direction
of
transcription.
Fig. 2: Plasmid pAG-2. amp: ampicillin resistance;
2 micron: EcoRI fragment of the S. cerevisiae
2~
plasmids with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin)
resistance; ORI: arigin of plasmid replication
in
E. coli; white arrows represent the direction
of
transcription.
Fig. 3: Plasmid pAG-100. amp: ampicillin resistance;
2 micron: EcoRI fragment of the S. cerevisiae
2~
plasmid with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin)
resistance; ORI: origin of plasmid replication
in
2~c~.~'~r~~~
- 21 - O.Z. 0050/41686
E. coli; black arrow: A. gossypii DNA fragment
with TEF promoter region; white arrows represent
the direction of transcription.
Fig. 4: Plasmid pAG-5. amp: ampicillin resistance; G418r:
(kanamycin) resistance; ORI: origin of plasmid
replication in E. coli; TEF: A. gossypii EcoRI/
BamHI fragment with ORF for the translation
elongation factor; white arrows represent the
direction of transcription.
Fig. 5: Plasmid pAG-101. amp: ampicillin resistance;
G418r: 6418 (kanamycin) resistance; ORI: origin
of plasmid replication in E. coli; TEF:
A. gossypii EcoRI/BamHI fragment with ORF for the
translation elongation factor; black arrow:
A. gossypii DNA fragment with TEF promoter-
region; white arrows represent the direction of '
transcription.
Fig. 6: Plasmid pPLl. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication in
E. coli; lacZ: E. coli lacZ gene; prom: 1500 by
A. gossypii DNA fragment with the TEF promoter
region.
Fig. 7: Plasmid pAG-110. 2u: EcoRI fragment of the
S. cerevisiae 2~ plasmid with replication origin;
URA3: S. cerevisiae URA3 gene; prom: 1500 by
A. gossypii DNA fragment with the TEF promoter
region; lacZ: E. coli laxZ gene; G418r: 6418
(kanamycin) resistance gene; ari: origin for
plasmid replication in E. coli; amp: ampicillin-
resistance gene; black arrow: 1500 by A. gossypii
DNA fragment with the TEF promoter region; white
arrows represent the direction of transcription.
Fig. 8: Plasmid pAG-110. 2p: EcoRI fragment of the
S. cerevisiae 2~ plasmid with replication origin;
URA3: S. cerevisiae URA3 gene; prom: 403 by
A. gossypii DNA fragment with the TEF promoter
2~~~~~~
- 22 - O.Z. 0050/41686
region; lacZ: E. coli lacZ gene; G418r: 6418
(kanamycin) resistance gene; ori: origin for
plasmid replication in E. coli; amp: ampicillin-
resistance gene; black arrow: A. gossypii DNA
fragment with the TEF promoter region; white
arrows represent the direction of transcription.
Fig. 9: Plasmid pPL2. amp: ampicillin-resistance gene;
M13+: replication origin foz single-stranded DNA
isolation; ori: origin for plasmid replication
in E. coli; lacZ; E. coli lacZ gene; prom:
294 by A. gossypii DNA fragment with a part of
the TEF promoter region (2?0 bp).
Fig. 10: Plasmid pPL3. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication--:
in E. coli; lacZ; E, coli lacZ gene; prom:.
239 by A. gossypii DNA fragment with a part of
the TEF promoter region (215 bp).
Fig. 11: Plasmid pPL4. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication
in E. coli; lacZ; E. coli lacZ gene; prom:
158 by A. gossypii DNA fragment with a part of
the TEF promoter region (134 bp).
Fig. 12: Plasmid pAG-112. 2~: EcoRI fragment of the
S. cerevisiae 2~ plasmid with replication
origin; URA3: S. cerevisiae URA3 gene; prom:
294 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin
for plasmid replication in E. coli; amp: ampi-
cillin-resistance gene; black arrow: A. gossypii
DNA fragment with the TEF promoter region; white
arrows represent the direction of transcription.
Fig. 13: Plasmid pAG-113. 2~: EcoRI fragment of the
S. cerevisiae 2;a plasmid with replication
origin; URA3: S. c~revisiae URA3 gene; prom:
- 23 - O.Z. 0050/41686
239 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin
for plasmid replication in E. coli; amp: ampi-
cillin-resistance gene; black arrow: A. gossypii
DNA fragment With the TEF promoter region; white
arrows represent the direction of transcription.
Fig. 14: Plasmid pAG-114. 2~: EcoRI fragment of the
S. cerevisiae 2~. plasmid with replication
origin; URA3: S. cerevisiae URA3 gene; prom:
158 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin
for plasmid replication in E. coli; amp: ampi-
cillin-resistance gene; black arrow: A. gossypii-
DNA fragment with the TEF promoter region; white '
arrows represent the direction of transcription.
FIg. 15: Plasmid pAG-115. 2~s: EcoRI fragment of the
S. cerevisiae 2~ plasmid with replication
origin; URA3: S. cerevisiae URA3 gene; lacZ:
E. coli lacZ gene; G418r: 6418 (kanamycin)
resistance gene; ori: origin for plasmid repli
cation .in E. coli; amp: ampicillin-resistance
gene; white arrows represent the direction of
transcription.
Fig. 16: TEF promoter fragments of the ,9-galactosidase
expression plasmids.
Fig. I7: Nucleotide sequence in the ATG region and in the
terminator region of MI3PT1, M13PT2, M13PT3,
pAG-201, pAG-202, pAG-203.
Fig. 18: Plasmid pAG-201, pAG-202, pAG-203. 2~: ECORI
fragment of the S. cerevisiae 2~ plasmid with
replication origin: prom, term: 751 by
A. gossypii DNA fragment with the TEF promoter-
terminator fusian. G418r:G418 (kanamycin)
resistance gene; ori: origin point of plasmid
replication in E. coli; amp:
~~, u~ ~; ~~ 'p. ~~ f.i
- 24 - O.Z. 0050/41686
ampicillin-resistance gene; white arrows repre-
sent the direction of transcription.
Fig. 19: Nucleotide sequence of the fusion of the
promoter and terminator of the TEF gene.
