Gene, 136(1993)313-318

0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00 313

GENE 07381

Isolation of Trichoderma reesei genes highly expressed on glucose-

containing media: characterization of the tefl gene encoding translation
elongation factor 1a
(Filamentous fungi; cloning method; abundant mRNA; gene expression; exon size)

Tiina Nakari, Edward Alatalo and Merja E. Penttila

VTTBiotechnical Laboratory, P.O. Box 202, SF-02151 Espoo. Finland

Received by J.R. Kinghorn: 16 March 1993; Revised/Accepted: 5 May/7 May 1993; Received at publishers: 1 July 1993

SUMMARY

Genes that are highly expressed on glucose-containing media were isolated from the filamentous fungus, Trichoderma
reesei. A cDNA bank was prepared from glucose-grown fungus, the bank was screened with the same cDNA as a probe,
and clones giving the strongest signal were isolated. This resulted in the isolation of previously uncharacterized genes.
Five of the genes, representing the most abundant transcripts, corresponded to l-3% of the total mRNA population
and were clearly more highly expressed than the phosphoglycerate kinase-encoding gene (pgkl ) of T. reesei. Based on
sequence homology, one of the genes was identified as tefl, encoding translation elongation factor lcl (TEF). The
T. reesei TEF is most related to the Mucor racemosus TEF3, showing an overall amino acid similarity of 85%.
Interestingly, an exon of only 2 bp seems to be present in T. reesei tefl, comprising the first 2 bp of the Gly” codon.

INTRODUCTION et al., 1993). The cbhl promoter is repressed by glucose

and is thus not suitable for protein production on glu-
The filamentous fungus Trichoderma reesei is an effi- cose-containing media. Expression on glucose would,
cient producer of cellulose-degrading enzymes. Due to however, be preferred because of the easier purification
excellent secretion capacity and cheap and easy cultiva- of the product and lower amounts of proteases produced
tion, Trichoderma is also a potent host for production of by the fungus in these conditions compared to media
heterologous proteins on a large scale. The strong pro- containing complex plant material. Thus, it would be de-
moter of the main cellulase gene cbhl of Trichoderma, sirable to isolate T. reesei promoters which are highly
encoding cellobiohydrolase 1, has been used for pro- expressed on glucose.
duction of heterologous proteins on media containing A major concern is how to select especially those genes
cellulose or its derivatives (Harkki et al., 1989; Nyyssonen that are most highly expressed in certain conditions and
for which no previous nt or aa sequence, or other protein
*Correspondence to: Dr. T. Nakari. VTT Biotechnical Laboratory, P.O.
Box 202, SF-02151 Espoo, Finland. Tel. (358-O) 4565134; Fax (358-O) data exist. Isolation of strong promoters using promoter
4552028; e-mail: Tiina.Nakari@vtt.fi probe vectors is not well applicable to filamentous fungi.
This is due to the fact that DNA is integrated in trans-
Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA
complementary to RNA: dNTP, deoxyribonucleoside triphos-
formation in multiple copies into the genome, and expres-
phate; kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading sion levels of the reporter gene are consequently affected
frame; PCR, polymerase chain reaction; pgkl, gene encoding phospho- by chromosomal location and copy number. Screening
glycerate kinase; pfu, plaque-forming unit(s); SDS, sodium dodecyl
of gene banks with labelled cDNA prepared from
sulfate; snRNP. small nuclear ribonucleoparticle; SSC, 0.15 M
NaCl/O.OlS M Na,citrate pH 7.6; T., Trichoderma; TEF. translation differentially induced mRNA populations is an estab-
elongation factor la; tefl, gene encoding TEF. lished technique to identify genes with regulated mRNA
314

abundances and to relate distinct genes to specific growth

conditions of a cell. For example, the inducible T. reesei
genes encoding cellulolytic enzymes have been isolated
by differential screening (Shoemaker et al., 1983; Teeri
et al., 1983; 1987b; Penttila et al., 1986; Saloheimo
et al., 1988).
Here we have modified the general method of
differential screening in order to isolate genes that are
abundantly expressed during growth on glucose. Because
the strategy does not rely on any preassumption as to
which genes should be isolated it can result in isolation
of genes without any homology to known protein se-
quences. This indeed turned out to be the case in this
work. However, one of the genes characterized in more
detail was found to encode the translation elongation
factor lr~ (TEF).

EXPERIMENTAL AND DISCUSSION

Fig. 1. Screening for clones highly expressed on glucose-containing

(a) Construction and screening of the glucose-induced medium from a T. reesei cDNA bank. The arrows point at clones further
cDNA bank characterized. Orientation marks of the filter are denoted with stars.

For the isolation of the glucose-induced mRNA the Methods: T. reesei strain QM9414 (Mandels et al., 1971) was grown in
a 10-l fermentor in minimal medium (Penttila et al., 1987) supplemented
T. reesei strain QM9414 (Mandels et al., 1971) was grown
with 6% glucose/5% Bacto-Peptone (Difco)/l% yeast extract (Difco).
in fermentor on medium containing glucose as a carbon Glucose feeding was started after 30 h of growth to keep the concen-
source. RNA was isolated from the phase of active growth tration above 2% for a total of 48 h after which the fungus was let to
and a cDNA bank prepared using the hZAP vector which consume the glucose. Mycelium for RNA isolation (Chirgwin et al..
1979) was harvested at 45 h of growth and the poly(A)‘RNA fraction
allows forced cloning in S-+3’ orientation and in vivo
was purified by using standard oligo(dT)-cellulose chromatography as
excision of the cloned fragment in a plasmid form (Short described in Sambrook et al. (1989). The cDNA bank was constructed
et al., 1988). It was expected that the most highly ex- from 5 ug of mRNA into phage hZAP vector according to manufactur-
pressed genes should give the highest frequency when the er’s instructions (Stratagene, La Jolla, CA, USA) using the E. coli
PLK-F’ supplied by the manufacturer as the host strain. The bank was
bank was screened with a probe prepared from this same
amplified resulting in 1.1 x 10”’ pfu/ml. The cDNA bank was plated on
cDNA. In case hybridization is performed with excess LAM plates as described by the manufacturer resulting in approxi-
target compared to probe or hybridization is not allowed mately 500 plaques per plate and transferred onto nitrocellulose filters
to proceed to completion, it is to be expected that plaques according to Sambrook et al. (1989). For the screening of the bank, a
corresponding to abundant cDNAs should also give first-strand cDNA was synthesized using [w~*P]~CTP (Teeri et al..
1987a) from 5 ug of the same mRNA that was used for preparation of
strongest signals. Screening of the bank resulted indeed
the cDNA bank and half of that was re-labelled with [a-32P]dCTP
in autoradiograms where the signals obtained were of using the Random Primed DNA Labelling Kit (Boehringer-Mannheim,
different intensities (Fig. 1). Fifty plaques giving a strong Mannheim, Germany) to increase the specific activity of the probe.
signal were purified for further analysis. Fourteen filters were hybridized with this relabelled probe in 35 ml of
5 x SSPEj5 x Denhardt’s/SO% formamide/O.l% SDS/l00 ug herring
The sequencing of the clones from the 3’ end revealed
sperm DNA/l ug poly(A) (all per ml) at 42°C for 16 h. The filters were
that some of the clones were picked in multiple copies. washed twice with 2 x SSC for 10 min at room temperature and twice
The abundancy of each specific clone in the mRNA pop- in 0.1 x SSC/O.l% SDS at 68°C for 1 h, and exposed on X-Omat film
ulation was estimated by hybridizing the cDNA bank for varying times to obtain optimal difference in signal intensity. (For
with clone specific PCR probes. The clones showing the composition of SSPE, see Sambrook et al., 1989.)

five highest frequencies (cDNA1, cDNA10, cDNA12,

cDNA15 and cDNA33) corresponded to l-3% of the somal genes from a Trichoderma h bank prepared earlier
total mRNA pool. These values are about 20-50-fold (Vanhanen et al., 1989). The chromosomal genes were
higher than that of the Trichoderma pgkl gene encoding subcloned on the basis of Southern analysis of restriction
phosphoglycerate kinase (Vanhanen et al., 1989). enzyme digestions carried out for the h clones. The com-
parison between the sequences of the cDNAs and the
(b) Characterization of the isolated clones chromosomal copies revealed that not all of the cDNAs
The cDNAs of the five most highly expressed genes comprised the complete protein coding regions and that
were used as probes to isolate the corresponding chromo- in each gene a number of introns was present especially
 315

at the 5’ end of the gene. This impaired to some extent lower stringency was used in the Southern hybridization.
the exact determination of the coding regions of the genes. Also tefl is a single copy gene. In most of the organisms
To identify the genes, the aa sequences encoded by the analysed tef constitutes a multigene family, the multiple
ORFs, and nt sequences if considered appropriate, were copies showing very high sequence homology and being
subjected to homology comparisons with data base se- located in different parts of the genome (Merrick, 1992).
quences. The only clone, cDNA33, which could be clearly
identified based on these comparisons, corresponded to (c) Expression of the isolated genes
the tefl gene encoding translation elongation factor lu. The expression levels of the clones, cDNA1, cDNA10,
More extensive sequence comparisons might lead to the cDNA12, cDNA15 and tefl, during cultivation on glu-
identification of the other genes in future. However, the cose medium in fermentor were studied by Northern blot-
current difficulties to find clear similarities to existing ting (Fig. 3). The expression of the genes cDNA1,
data bank sequencies indicate that the genes isolated cDNAl0 and cDNA12 varies according to the time point
might carry out functions which are specific for filamen- of the cultivation. Typically they are most strongly ex-
tous fungi. If this is the case, the identification of the pressed early in the cultivation. cDNA15 and tefl are the
genes by sequence comparisons is further hampered by most constitutively expressed of the genes. This is the
the limited number of genes cloned from these organisms. case with tefs in general, the major exceptions being
Southern analysis indicates that three of the five genes ageing and quiescent cells.
characterized are represented by only one copy in the The expression levels of the genes are clearly higher
genome (Fig. 2). Unexpected extra signals were obtained than that of the pgkl gene of Trichoderma. This result is
with the cDNA1 and cDNA12 specific probes suggesting consistent with the frequencies of the genes in the cDNA
existence of homologous sequences in the genome. The bank. The moderate expression level of the Trichoderma
signals are rather weak compared to those corresponding pgkl is also in accordance with our earlier results showing
to the isolated cDNA copies and thus should not signifi- unsatisfactory expression levels of proteins under the
cantly affect the estimation of the abundancy of these pgkl promoter on glucose-containing medium
transcripts in the Northern analysis, especially because (Vanhanen, 1991). Thus, it seems that pgkl is not as

h-_,
82z;o
wxaa

-5.0
-4.2
(I) -3.5
0

-2.0
-1.9
-1.6
3 - -1.4
I,
0
am -0.95

A. tef 8. cDNA1 C. cDNAl0 D. cDNA12 E. cDNA15

Fig. 2. Southern analysis of the cDNA1, cDNA10, cDNA12, cDNAl5 and tefl genes. The clone-specific probes used are indicated below each panel.
Restriction enzymes used are indicated on top of each lane. Positions of the molecular markers are indicated on the right of each panel, the
corresponding sizes (kb) being shown in the right-most margin. Unexpected signals are marked with asterisks. Methods: Chromosomal DNA from
T. reesei strain QM9414 was isolated according to Raeder and Broda (1985) and 2 ug of DNA was cleaved with indicated restriction enzymes which
had at least one known recognition sequence within the coding region and with enzymes which cut outside the known area. The cleaved DNAs were
electrophoresed on 1% agarose and transferred to Hybond N nylon filter (Amersham, Buckinghamshire, UK) and hybridized with the same cDNA-
specific PCR probes as described in Fig. 3. The hybridization conditions were as described in the legend of Fig. 1. The filters were washed twice with
2 x SSC for 5 min at room temperature, in 2 x SSC/O.l% SDS at 65°C for 1 h and in 0.1 x SSC at 42°C for 40 min, and exposed on X-Omat film.
316

Pgk

s kb

- 4.4

- 2.4

-1.4

- 0.24

Fig. 3. Northern analysis of the expression of the five most strongly expressed genes during cultivation on glucose medium in fermentor. Time points
for growth (h) and the clone specific probes used are indicated at the top. 2 pg of RNA was used in the analysis except for pgkl. where 10 pg was
used. Positions of the markers are indicated on the right margin. Methods: Fermentor cultivation procedures and isolation of the total RNA are
described in Fig. 1. The RNAs were glyoxylated according to Sambrook et al. (1989), electrophoresed on 1% agarose and transferred to Hybond N
nylon filter (Amersham. Buckinghamshire, UK) and hybridized with PCR fragments of equal length (0.3 kb), synthesized from the original cDNA
clones and the pgkl gene (Vanhanen et al., 1989) and labelled to same specific activity with [c+32P]dCTP (see legend to Fig. 1).

strongly expressed in Trichoderma as for example in (T.N., unpublished). This, together with the fact that the
Saccharomyces cereuisiae (Holland and Holland, 1978) in genes are represented by only one major copy in the
which the PGK promoter is furthermore one of the genome, gives us reason to believe that their expression
strongest promoters available for foreign protein pro- is driven by strong promoters, which can be further uti-
duction. These facts could indicate fundamental differ- lized in protein production.
ences in basic physiology between Trichoderma and yeast.
The high frequency of the clones isolated in the cDNA (d) Gene tefl of Trichoderma reesei
bank and the abundant mRNAs detected by Northern The tefl nt sequence and the aa sequence deduced from
analysis demonstrate that the method applied here can it are shown in Fig. 4. At nt level the best sequence ho-
be successfully used to isolate genes that are strongly mology to known tef sequences could be found to the
expressed in given culture conditions. This is also shown tef3 gene of the filamentous fungus Mucor racemosus
by the fact that one of the genes isolated encodes TEF (Sundstriim et al., 1987). The ORF of tefl consists of
which is in all organisms one of the most abundant cellu- 1380 bp (a protein of 460 aa and 51 kDa). The size of the
lar proteins consisting of 4-6% of the total soluble pro- tefl transcript is estimated to be approx. 1.9 kb (Fig. 2),
tein pool (Miyajima et al., 1978; Thiele et al., 1985). The a value in good agreement with the length of the ORF
five genes studied here have all, except cDNA10, signifi- and additional 5’ and 3’ noncoding regions.
cant expression levels also on media containing complex The TEF protein of T. reesei shows an extensive se-
plant material as carbon source (data not shown) indicat- quence similarity to other TEFs from different organisms.
ing that they are not especially induced by glucose. TEFs are universally conserved proteins that promote
Consequently these genes could have remained unde- the GTP-dependent binding of an aminoacyl-tRNA to
tected in conventional differential hybridization ribosomal A-site in protein synthesis. Especially con-
approaches which rely on two cDNA probes and aim at served are the N-terminal parts of the proteins containing
isolation of genes especially induced in certain conditions. the GTP-binding domain. The overall aa sequence ho-
However, the use of only one cDNA probe, as studied in mology to M. racemosus TEF3 is 85% and in the GTP-
this work, can also be applied in isolation of stage specific binding domains over 90%. If conservative substitutions
genes as shown by Judelson and Michelmore (1990), in are allowed, the overall similarity between M. racemosus
case it is to be expected that the genes are the most highly and T. reesei TEFs is even higher. Similarity to the
expressed in the specific condition. human TEF is 81%. An insertion of Asp’ is found in the
The presence of abundant mRNA for a particular gene GTP-binding domain in T. reesei TEF compared to all
can be due to an efficient promoter or good stability of other eukaryotic TEFs sequenced to date. Towards the
the mRNA, both potential factors leading to high levels C-terminus of the protein there are two more insertions
of the corresponding protein. In vivo labelling experi- when compared to the M. racemosus TEF3.
ments indicate that the transcription efficiency of the Similar to other sequenced T. reesei genes, the codon
genes isolated here is clearly higher than that of pgkl usage of the tefl gene is biased against NTA codons and,
 317

rather, C and G are preferred at the third position. As

generally observed, also amongst filamentous fungi, the
genes that are highly expressed seem to have a preferred
subset of codons (Unkles, 1992). The codon usage of tefl
is most biased among the T. reesei genes characterized
so far. This is in accordance with the codon usages of
TEFs from other organisms, which show highly biased
codon preferences (Andersson and Kurland, 1990).

(e) tefl contains an exon of two base pairs

Comparison of the cDNA and chromosomal sequences
of the tefl gene revealed a number of introns with splicing
signals suggested for filamentous fungi (Ballance, 1986;
Penttilg et al., 1986; Unkles, 1992). Surprisingly, in spite
of extensive sequence confirmations the reading frame
beyond Ile14 could not be restored by simply comparing
the cDNA and chromosomal sequences. The chromo-
somal sequence seemed to lack the two Gs of the Gly”
codon, present in the P-loop motif of all characterized
TEFs (Saraste et al., 1990). Thus, to be able to restore
the correct reading frame a short 2-bp exon at nt posi-
tions 123-124 (see Fig. 4) in the chromosomal sequence
is suggested. These nt are surrounded by consensus 5’
and 3’ splicing signals, and also consensus sequences for
the internal lariat site can be found in the adjacent in-
trons. The third nucleotide, C, of the Gly” codon is con-
sequently separated by the second intron of 67 bp. It is
possible that in isolation of the chromosomal tefl copy,
a non-functional homologue was recovered. This is, how-
ever, not likely since the two independently isolated tefl
cDNA clones and the coding region of the chromosomal
copy match the nt sequence, and show no mutations.
Exons of only a few nt in length have been reported
before. An exon of three nt is found in murine neural cell
adhesion molecule (Santoni et al., 1989). Similar to the
Fig. 4. Nucleotide and the deduced aa sequence of the tefl gene T. reesei exon, almost ideal acceptor and donor splice
(GenBank/EMBL accession No. 223012). The coding nt are given in
consensus sequences surround this exon. It is also pos-
capital letters along with the aa of the protein. Introns and flanking
regions are shown in lower-case letters. The consensus intron splicing
sible that instead of being encoded by a small exon, di-
signals are underlined. Methods: The cDNA clone isolated from the h and trinucleotides can be added to the mRNA by a RNA
bank was used as a probe to isolate the chromosomal copy of the tefl editing mechanism. Highly efficient and specific changes
gene from a genomic h bank (Vanhanen et al., 1989). Based on Southern
or additions of only a few nt have been reported to occur
analysis the chromosomal gene was subcloned into pSP73 vector
(Promega, Madison, WI, USA) as a 4.5-kb KpnI-EcoRI fragment result-
post-transcriptionally in nuclear genes, for instance in the
ing in the plasmid pEA33. Because the original cDNA clone (pcDNA33) mouse glutamate receptor gene family (Sommer et al.,
constituted only the 0.7-kb C-terminal part of the gene, the N-terminal 1991).
region of the tefl cDNA was isolated as follows. First-strand 5’ cDNA
of tefl was synthesized (Sambrook et al., 1989) from 5 pg of
poly(A)+RNA prepared as described in Fig. 1 using a primer specific
for the region 910 bp downstream from ATG (underlined, marked 1). amplification (25 cycles) consisted of denaturation at 94°C for 45 s,
The cDNA:RNA hybrids were directly subjected to PCR amplification annealing at 55°C for 30 s and polymerization at 72°C for 2 min. The
with primers (underlined, marked 1 and 2) provided with restriction resulting 0.4-kb band was purified from SeaPlaque (FMC BioProducts,
enzyme cleavage sites for cloning. The PCR reaction mixture (100 ~1) Rockland, MA, USA) agarose gel, digested appropriately and cloned
contained 3 ~1 of the first-strand cDNA synthesis reaction mixture, 10 ~1 into pSP73 vector (Promega) to obtain plasmid pTEF5’cDNA.
10 x reaction buffer (100 mM TrisHCl pH 8.8/15 mM MgClJ500 mM Sequencing was carried out from the plasmids pEA33, pcDNA33 and
KCl/l% Triton X-100)/8 l.d 2.5 mM dNTPs/lOO pmol of primers/O.5 pTEF5’cDNA by the dideoxynucleotide method using a Sequenase
unit DynaZymeTM polymerase (Finnzymes, Espoo, Finland). The Sequencing kit (US Biochemical, Cleveland, OH, USA).
318

(f ) Conclusions Mandels, M., Weber, J. and Parizek, R.: Enhanced cellulase production
by a mutant of Trichoderma uiride. Appl. Microbial. 21 (1971)
( 1) We have used a modification of the differential hy- 1522154.
bridization method to isolate genes corresponding to Merrick. W.C.: Mechanism and regulation of eukaryotic protein synthe-
abundant mRNA pools without any previous knowledge sis. Microbial. Rev. 56 (1992) 291-315.
Miyajima, A. and Kaziro, Y.: Coordination of levels of elongation
of their sequences, functions or corresponding proteins.
factors Tu, Ts and G, and ribosomal protein Sl in Escherichia coli.
This resulted in isolation of highly expressed genes but J. Biochem. 83 (1978) 453-462.
should also yield genes with mRNAs of high stability. Nyyssiinen, E., Penttila, M., Harkki, A., Saloheimo, A., Knowles, J.K.C.
(2) We have applied the method to isolate T. reesei and Kerlnen, S.: Efficient production of antibodies by the filamen-
tous fungus Trichoderma reesei. Bio/Technology 11 (1993) 591-595.
genes which are strongly expressed on glucose-containing Penttila, M.. Lehtovaara, P., Nevalainen, H., Bhikhabhai, R. and
medium. Five previously uncharacterized genes were iso- Knowles, J.: Homology between cellulase genes of Trichoderma
lated which were all more expressed than the pgkl gene. reesei: complete nucleotide sequence of the endoglucanase I gene.
Gene 45 (1986) 253-263.
(3) One of the genes isolated is encoding TEF. It
PenttilL, M.. Nevalainen, H.. Ratto. M., Salminen, E. and Knowles, J.:
shows extensive sequence homology to other TEF pro- A versatile transformation system for the cellulolytic filamentous
teins, the most similar one being M. racemosus TEF3. fungus Trichoderma reesei. Gene 61 (1987) 155-164.
The T. reesei tefl gene is unique in having a 2-bp exon, Raeder. U. and Broda, P.: Rapid preparation of DNA from filamentous
fungi. Lett. Appl. Microbial. 1 (1985) 17-20.
the shortest exon ever reported.
Saloheimo, M., Lehtovaara, P., Penttila, M., Teeri, T.T., Stahlberg, J.,
(4) The promoters of the isolated genes can be used Johansson, G., Pettersson. G., Claeyssens. M., Tomme. P. and
to produce homologous or heterologous proteins on glu- Knowles, J.K.C.: EGIII, a new endoglucanase from Trichoderma
reesei: the characterization of both gene and enzyme. Gene 63
cose-containing media by Trichoderma (T.N. and M.E.P.,
(1988) 11-21.
manuscript in preparation). Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A
Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1989.
ACKNOWLEDGEMENTS
Saraste, M., Sibbald. P.R. and Wittinghofer, A.: The P-loop: a common
motif in ATP- and GTP-binding proteins. Trends Biochem. Sci. 15
We wish to thank Armi Boman and Seija Nordberg (1990) 430-434.
for excellent technical assistance, Maija-Leena Onnela Shoemaker. S., Schweickart, V.. Ladner, M., Gelfand, D., Kwok, S.,
Myambo. K. and Innis, M.: Molecular cloning of exo-
and Michael Bailey for fermentor cultivation, Sirkka
cellobiohydrolase I derived from Trichoderma reesei strain L27.
Keranen for useful discussions and Kari Keinanen and Bio/Technology 1 (1983) 691-695.
Hans Siiderlund for comments on the manuscript. This Short, J.M., Fernandez, J.M.. Sorge, J.A. and Huse, W.D.: Lambda-
study was supported by the Foundation for Biotechnical ZAP: a bacteriophage lambda expression vector with in vivo exci-
sion properties. Nucleic Acids Res. 16 (1988) 7583-7600.
and Industrial Fermentation Research (Helsinki, Sommer, B.. Kiihler, M., Sprengel. R. and Seeburg, P.H.: RNA editing
Finland). in the brain controls a determinant of low ion flow in glutamate-
gated channels. Cell 67 (1991) 11-19.
Sundstrbm, P., Lira. L.M., Choi, D.. Linz. J.E. and Sypherd, P.S.:
REFERENCES Sequence analysis of the EF-lx gene family of Mucor racemosus.
Nucleic Acids Res. 15 (1987) 9997-10005.
Andersson. S.G.E. and Kurland, C.G.: Codon preferences in free-living Teeri, T., Salovuori. I. and Knowles, J.: The molecular cloning of the
microorganisms. Microbial. Rev. 54 (1990) 1988210. major cellulase gene from Trichoderma reesei. Bio/Technology 1
Bairoch, A.: SwissProt Protein Sequence Databank User Manual, re- (1983) 6966699.
lease 23, EMBL Data Library, Heidelberg, Germany, 1992. Teeri, T.T., Kumar, V., Lehtovaara, P. and Knowles, J.: Construction
Ballance, D.J.: Sequences important for gene expression in filamentous of cDNA libraries by blunt-end ligation: high-frequency cloning of
long cDNAs from filamentous fungi. Anal. Biochem. 164 (1987a)
fungi. Yeast 2 (1986) 229-236.
60-67.
Berges, T., Perrot, M. and Barreau, C.: Nucleotide sequence of the
Teeri, T.T., Lehtovaara, P., Kauppinen, S., Salovuori, I. and Knowles, J.:
Trichoderma reesei ura3 (OMPdecase) and ura.5 (OPRTase) genes.
Homologous domains in Trichoderma reesei cellulolytic enzymes:
Nucleic Acids Res. 18 (1990) 7183.
gene sequence and expression of cellobiohydrolase II. Gene 51
Chirgwin, J.M., Przybyla, A.E., MacDonald. R.J. and Rutter, W.J.:
(1987b) 43-52.
Isolation of biologically active ribonucleic acid from sources en-
Thiele, D., Cottrelle, P., Iborra, F., Buhler, J.-M., Sentenac, A. and
riched in ribonuclease. Biochemistry 18 (1979) 529445299.
Fromageot, P.: Elongation factor la from Saccharomyces cerevisiae.
Harkki, A., Uusitalo, J., Bailey, M., Penttila, M. and Knowles, J.K.C.:
J. Biol. Chem. 260 (1985) 3084-3089.
A novel fungal expression system: secretion of active calf chymosin
Unkles, S.E.: Gene organization in industrial filamentous fungi. In:
from the filamentous fungus Trichodermu reesei. Bio/Technology 7 Kinghorn, J.R. and Turner, G. (Eds.), Applied Molecular Genetics
(1989) 5966603. of Filamentous Fungi. Blackie, Glasgow, 1992, pp. 28853.
Holland, M.J. and Holland, J.P.: Isolation and identification of yeast Vanhanen, S.: Isolation and Characterization of Genes Involved in
messenger ribonucleic acids coding for enolase, glyceraldehyde-3- Basic Metabolism of the Filamentous Fungus Trichoderma reesei.
phosphate dehydrogenase, and phosphoglycerate kinase. Ph.D. thesis, VTT Publications no. 75, Finland, 1991.
Biochemistry 17 (1978) 4900-4907. Vanhanen, S., PenttilL, M., Lehtovaara, P. and Knowles, J.: Isolation
Judelson. H.S. and Michelmore, R.W.: Highly abundant and stage- and characterization of the 3-phosphoglycerate kinase gene (pgk)
specific mRNAs in the obligate pathogen Bremia lactucae. Mol. from the filamentous fungus Trichoderma reesei. Curr. Genet. 15
Plant-Microbe Interact. 3 (1990) 225-232. (1989) 181-186.