Abstract:
Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae
and was used for amplification of a chitin synthase fragment by the polymerase
chain reaction method. The primers used in the amplification corresponded to two
widely conserved amino acid regions found in chitin synthases of many fimgi.
Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately)
as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a
candidate for chitin synthase because most amplified regions from other fimgi so far
exhibited similar sizes (600-750 bp). The selected fragment was extracted from the
gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were
designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS
was digested by several restriction enzymes and was found to contain BamHl and
HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open
reading frame of 200 amino acids. The derived polypeptide was compared with
other related sequences from the EMBL database (Heidelberg, Germany) and was
matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The
closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus
oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR
reaction showed a strong signal at the fragment selected for cloning and weaker
signals at the other two fragments. Southern hybridization with partially digested
Phascolomyces articulosus genomic DNA showed a single band. The amino acid
sequence was compared with sequences from other chitin synthase gene classes
using the CLUSTALW program. The chitin synthase fragment from Phascolomyces
articulosus was initially grouped in class n along with chitin synthase fragments
from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the
same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS
revealed conservation of intron positions that are characteristic of chitin synthase
gene fragments of zygomycetous fungi.
