Targeted differential display of abundantly expressed sequences ...

A number of PCR-based methods for rapidly comparing
profiles of gene expression have been reported. One
such method, differential display (DDRT-PCR), involves
the random amplification of sequences from cDNA populations
of interest using short oligonucleotide primers in
combination with primers which anneal to the polyA tail
of the cDNA; amplification products are then directly compared
on polyacrylamide gels (Liang and Pardee 1992).
Here, the DDRT-PCR approach has been modified to
screen specifically for sequences containing regions encoding
CBDs from P. chrysosporium, after growth on three
different carbon (C) sources. In this case, the short primers
used for DDRT-PCR are replaced by longer degenerate
primers designed to anneal to a conserved region of the
CBD-encoding sequence, allowing the annealing temperature
of the PCR reaction to be elevated, and thus to be
more specific. CBDs are present in the majority of fungal
cellulases studied to-date and have also been reported in a
xylanase from H. insolens (Dalbøge and Heldt-Hansen
1994), a β mannanase (Stålbrand et al. 1995) and an acetyl
xylan esterase (Margolles-Clark et al. 1996) from T. reesei.
Materials and methods
Organism and culture media. P. chrysosporium ME446 (ATCC
34541) was maintained on 2% (w/v) slopes malt-extract agar. The
culture medium was a modified Vogel’s medium as described previously
(Tempelaars et al. 1994), containing 0.2% (w/v) of a carbon
source from Avicel (microcrystalline cellulose), carboxymethyl cellulose
(CMC) (amorphous cellulose) or oatspelt arabinoxylan.
DNA manipulations. DNA was extracted from P. chrysosporium as
described by Raeder and Broda (1985). Southern blotting was onto
Hybond N membrane (Amersham) and hybridisation was performed
using the low-stringency conditions described in Sambrook et al.
(1989). All techniques referred to below were according to the
manufacturers’ recommendations. Colony hybridisations were made
on Hybond C extra membrane (Amersham). PCR-amplified DNA
fragments were purified for cloning using the Promega Wizard PCR
Preps kit. PCR-amplified DNA was cloned using the pGEM-T vector
cloning kit (Promega) and transformed into Stratagene Ultracompetent
Epicurian coli XL2-blue MRF′ cells. Plasmid preparations
were made using the Qiagen Plasmid Miniprep kit. Sequencing of
cloned PCR products was performed using the ABI PRISM Dye Terminator
cycle sequencing kit of Perkin Elmer. The 32 P-radiolabelled
probe DNA was prepared using the Random Primed Labelling kit of
Pharmacia.
RNA extraction and cDNA synthesis. P. chrysosporium was inoculated
into liquid culture as previously described and the mycelium
was harvested after 4-days stationary incubation at 37°C (Tempelaars
et al. 1994). The mycelium was ground under liquid nitrogen
and RNA extracted using the Qiagen RNeasy kit. Poly (A) + mRNA
was purified from this with a Dynal’s Dynabeads mRNA extraction
kit. cDNA was synthesised from mRNA with the Pharmacia First-
Strand cDNA synthesis kit, using the NotI primer supplied with the
kit.
Design of PCR primers and RT-PCR conditions for targeted differential
display. Figure 4 shows an alignment of the amino-acid sequences
of 30 previously published fungal CBD regions. From amino-acid
positions 2–8, 25 of these vary only at position 7, involving
sequences GQCGGI/N/QG. Three degenerate oligonucleotide primers
were designed to anneal to DNA sequences coding for these regions.
Primer 1 (5′-GGNCAGTGCGGNGGNATPyGG-3′) anneals
to sequences coding for GQCGGIG, primer 2 (5′-GGNCAGTGC
GGNGGNCAGGG-3′) anneals to sequences coding for GQCGGQG,
and primer 3 (5′-GGNCAGTGCGGNGGNAAPyGG-3′) anneals to
sequences coding for GQCGGNG. On the basis of codon usage in
known P. chrysosporium genes, the codon CAG was employed for
amino-acid Q. Each of these primers was used independently in
DDRT-PCR reactions with a primer which anneals to the poly-A tail
(5′-ATTCGCGGCCGCAGGAT 15 ), which is derived from the Pharmacia
NotI dT primer used for cDNA synthesis. Primer 4
(5′-GCACTGCGAGTAGTA-3′) was used in combination with primer
1 to PCR-amplify the region coding for the CBD from the cbhI.1
gene of P. chrysosporium, cloned in 3E2D (Sims et al. 1994). The
cbhII upstream primer, 5′-CCTCAGCCCTTACTACGC-3′, was as
used for RT-PCR in Tempelaars et al. (1994) with an annealing temperature
of 55°C. To prevent primers 1–3 acting as RAPD primers,
generating PCR products in the absence of any other primer, an annealing
temperature of 65°C was employed. The RT-PCR conditions
were: one cycle of 94°C for 1 min, 65°C for 1 min, 72°C for 2 min;
30 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 1.5 min, and a
cycle of 72°C for 10 min.
Results
Isolation of cDNA sequences which hybridise
to the CBD-encoding region of cbhI.1
from P. chrysosporium ME446
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P. chrysosporium was grown for 4 days at 37°C in medium
containing either Avicel, CMC, or oatspelt arabinoxylan.
The rationale for choosing these C sources is that Avicel
is commonly regarded as an exocellulase substrate, CMC
as an endocellulase substrate, while xylan is the major component
of hemicellulose; the expression of different components
of the lignocellulolytic system has been shown to
occur after growth on each (Broda et al. 1995). cDNA was
synthesised from poly (A) + mRNA prepared from mycelia
grown on each medium and, in each case, 50 ng was
used in PCR reactions containing either CBD primer 1, 2
or 3, each in combination with an oligo-dT primer which
anneals specifically to the poly-A tail of cDNA.
Initially, however, to test whether, under all conditions,
intact mRNA had been extracted and converted to cDNA,
RT-PCR was performed using a primer which anneals to
the cbhII gene of P. chrysosporium in combination with
the oligo-dT primer. This cellulase gene is expressed after
growth on Avicel, CMC, and xylan (Tempelaars et al. 1994)
and thus cDNA derived from it should be detected in each
sample. This proved to be the case, and a PCR product of
a size expected from cbhII cDNA using these primers, approximately
1.4 kb, was generated from each cDNA sample,
whereas no such product was generated from genomic
DNA (Fig. 1).
To test whether the products amplified, using CBD
primers 1, 2 or 3 with the oligo-dT primer, contained sequences
which share homology with a CBD-encoding region,
each population of PCR products was separated by
gel electrophoresis. These were then Southern blotted and
hybridised, using low-stringency conditions, to a probe derived
from the P. chrysosporium cbhI.1 gene. To prepare