Front Matter

plaques were noted. Analysis of one of these indicated the presence of an approxi-

mately 1.3 kilobase DNA insert that was subcloned into theEcoRI site of the ex-

pression plasmid pUC8–2, generating plasmid pUC8-2.14 (Haas et al., 1991). The

nucleotide sequences of both strands of the entire cDNA insert were determined. The

fragment consisted of 1287 base pairs, had a G + C content of 45 %, and possessed a

consensus polyadenylation sequence. Furthermore, it hybridized well to totalR. de-

lemarpoly(A)+RNA, strongly suggesting its origin in that organism.

Of the six possible reading frames in the cloned cDNA, only one contained an

open reading frame of sufficient length to encode a protein in excess of 30 kDa,

the mass of the lipase as determined by biochemical studies. The actual lipase-en-

coding region within this reading frame was identified by reference to the knownN-

terminal amino acid sequence of authentic lipase. A region of the gene sequence was

identified whose predicted complementary amino acid sequence was identical to that

of the first 28 amino-terminal amino acids of the pure lipase. In fact, the predicted

amino acid sequence agreed completely with that of the purified fungal enzyme not

only for residues 1–28 for which conclusive assignments were possible from amino

acid sequence data, but also for residues 29–40 for which ‘best guess’ sequence

estimates could be made from the protein sequencing data. This identity between

the amino acid sequence predicted by the cDNA and that of the lipase located

theN-terminal end of the lipase-encoding region within the cloned cDNA. A single

strong translation termination site downstream of this site established the size of the

mature lipase as 269 amino acids. The molecular mass of the resulting predicted

polypeptide was 29.6 kDa, in agreement with the value determined biochemically

for the purified authentic fungal enzyme.

Analysis of the DNA sequence indicated that the lipase is initially synthesized as a

preproenzyme, consisting not only of the 269-amino acid mature enzyme, but also of

a 97-amino acid propeptide fused to its amino terminus, and a 26-amino acid export

signal peptide at the amino terminus of the propeptide. SinceE. colilacks the ne-

cessary proteases for processing fungal maturation signals, one would expect to be

able to detect these precursors inE. coliextracts. Accordingly, polypeptides with

molecular weights of 39.5 and 42.1 kDa, corresponding to the predicted sizes of

the corresponding pro- and prepro-lipases, were detected by immunoblotting of elec-

trophoretic gels containing whole-cell lysates ofE. coliexpressing the cloned lipase

gene.

Analysis of the amino acid sequence predicted from the lipase gene sequence

indicated the presence of the pentapeptide Glycine (Gly)-His-Ser-Leucine (Leu)-

Gly. This is of the general type Gly-X-Ser-X-Gly noted to be highly conserved

among lipases (Brenner, 1988). This sequence constitutes a portion of the catalytic

triad of lipases, the Ser being the primary catalytic residue (Brady et al., 1990). Other

than the His and Asp residues that complete the catalytic triad, the pentapeptide is the

sole highly conserved sequence common to lipolytic enzymes.

However, analysis of the gene and protein sequences made it clear that lipases

from related organisms, as well as from organisms that are less closely related,

can share substantial homology. The high levels of structural homology between

the Rd and Rm lipases (above) were reflected, though perhaps not as strongly, in

their nucleic acid and amino acid sequences. The sequences of the coding portions

of the genes for the Rd and Rm lipases were 56 % identical overall. The degrees of

.5 Cloning and characterization of an expressedR. delemarlipase gene 77