Targeted differential display of abundantly expressed sequences ...

from P. chrysosporium, or from unspliced transcripts of
cbhI.1 and cbhI.2. Firstly, more abundant classes of transcript
may out-compete rarer classes in the PCR. Such competition
may explain the failure to detect unspliced transcripts
of cbhI.1 and cbhI.2, as the fully spliced transcripts
are more abundantly synthesised after growth on these substrates
and may thus have out-competed them (Sims et al.
1994; Birch et al. 1995). Such a bias of DDRT-PCR towards
high copy number cDNAs has been demonstrated
previously (Bertioli et al. 1995). Secondly, there may be
insufficient identity between the degenerate primers and
the CBD-encoding regions within the cellulase genes. This
is the case for the cbh1-2 gene, which contains a region
coding for SQCGGLG; this differs from equivalent sequences
chosen for the design of primers 1, 2 and 3 and,
indeed, is different to analogous regions in any other
known fungal CBD (Fig. 4). Thirdly, the CBD-encoding
regions of all sequences isolated in this study are located
at the 3′ ends of the ORF. In the case of cbhII, this region
is present at the 5′ end of the ORF. Use of a specific primer
for cbhII confirmed that cDNAs derived from this gene
were present in samples prepared after growth on Avicel,
CMC, and xylan. However, when using CBD-specific
primer 1, cbhII cDNA templates were out-competed in all
PCR reactions by templates yielding smaller amplification
products, including those from cbhI.1 and cbhI.2 in the
cases of Avicel and CMC. Although such competition may
be due simply to different levels of gene expression, the
abundance of cbhII templates relative to those from cbhI.1
and cbhI.2 may also be effected by the efficiency of cDNA
synthesis, which is primed from the 3′ end of mRNA. In
addition, the larger PCR product predicted from cbhII
cDNA may be amplified less efficiently than the small PCR
products isolated in this study. Both of these factors should
be considered when designing degenerate primers to target
a family of genes. It is thus recommended that conserved
sequences should be sought which are present at
only one location in all known genes in the family and that
the location should be as near to the 3′ end as possible.
Interestingly, partial protein sequence from one of the
EGs produced by P. chrysosporium, EG1, has revealed an
N-terminal CBD containing the sequence GQCGGIG
(Uzcategui et al. 1991 b), identical to those encoded by
equivalent nucleotides in cbhI.1, cbhI.2 and cbhII, from
which primer 1 was designed. As was the case for cbhII,
however, cDNAs coding for this gene were also not detected.
Further work will be required to identify the genes from
which the three novel cDNAs containing CBD-encoding
regions are derived. However, they may originate from
transcripts of the partially characterised cbh1-5 and
cbh1-6 (Covert et al. 1992 b) genes, or from the gene that
is predicted to code for both EG38 and EG36 (Uzcategui
et al. 1991 a). Indeed, although Northern analysis would
be required to confirm patterns of regulation for these
genes, one sequence isolated in this study, cmc1, was amplified
only from cDNA prepared after growth on CMC,
which is commonly regarded as an EG substrate. In addition,
a cellulose-binding β-glucosidase has been purified
from cellulose-degrading cultures of P. chrysosporium and
this too may contain a CBD (Lymar et al. 1995). However,
not all cellulolytic enzymes that bind to microcrystalline
cellulose contain a conventional fungal CBD; this is the
case for cellobiose dehydrogenase from P. chrysosporium
(Li et al. 1996).
In conclusion, the targeted DDRT-PCR approach described
here has been successfully applied to the isolation
of novel, abundant cDNA sequences containing regions
coding for CBDs and has also confirmed previous observations
on the regulation of known genes containing such
regions. Although not a comprehensive screen, the speed
of analysis of this method makes it attractive for an initial
study of more highly expressed genes from fungi which
contain such regions. The requirement for only small quantities
of mRNA means that a broad number of growth conditions
can easily be studied in parallel. In addition, this
approach could readily be adapted to the investigation of
other gene families, provided sufficient homology exists
between related sequences for a general probe to be developed
for the hybridisation-screening of cloned cDNAs.
Failing this, additional conserved regions should be sought
within the amplified region which can be used to design
degenerate primers for screening by nested PCR.
Acknowledgements This work was supported by grant RO361
from the Scottish Office Agriculture, Environment and Fisheries Department.
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