Glucoamylases are important industrial enzymes used in the conversion of starches to syrups and in other fermentation processes. Previously, the gene encoding the major glucoamylase activity of N. crassa was characterized (Stone et al. 1993 Curr. Genet 24:205-211). Here we report the identification of a possible second glucoamylase (gla-2) that is similar to a member of a class of glucoamylases represented by the GAM1 gene of S. occidentalis (Dohmen et al. 1990 Gene 95:111-121).
In our analysis of the rco-3 gene of N. crassa we identified gla-2 through partial sequencing of the DNA adjacent to rco-3 from two sites in the clone. Homology to GAM1 was found through a BLAST search using the translated single-stranded DNA sequences. An alignment by the Wisconsin GCG program 'Bestfit' is shown in Fig. 1. gla-2 and GAM1 are sufficiently similar in the sequenced regions to suggest that gla-2 is likely a functional glucoamylase with properties similar to GAM1. The 3' end of the N. crassa gla-2 coding region was sequenced and the predicted protein has a 39 amino acid extension relative to GAM1. There is no significant sequence similarity between gla-2 and gla-1 of N. crassa.
Sequence 1
287
So LYFFSGPTPKDAIQQYVKEIGLPAFQPYWSLGYHQCRWGYDTIEKLSEVVENFKKF
: : :::: : :: :: ::::::: :: :: ::
Nc SISCTLEDLLRYHQVYQQYVGLPAMQQYWTLGFHQCRWGYSNWTVVKDVVDNFRKF
392
So NIPLETIWSDIDYMDSYKDFTYDPHRFPLDEYRKFLDELHKNNQHYVPIL
::::::::::::: :::: :: : : :: :::::
Nc GIPLETIWSDIDYMKGYRDFENDPD.FSYEEGARFLEELHKNHHMTCRLS
Sequence 2
908 958
So VTILGVGHKPKSVKFENANVDFTYKKST.....VFVTGLDKYTKDGAFSKDFTITW*
:::::: : : :: : : : : :
Nc VTILGVAEAPLGVAINSHLLGSASWSYDSEGEVLSVTELQDNFKEGGWASNWTLSW
Nc NSASNSGSSPVQGGGGRLEFSSPICSMQLLSASFLAACL*
Figure 1. The S. occidentalis (So) GAM1
sequence is numbered with respect to the published initiator Met codon. Two
regions of the N. crassa (Nc) gene were sequenced and the
predicted polypeptide regions were aligned to GAM1. Identical residues
are indicated by a colon (:). The C-termini of the proteins predicted from DNA
sequence data are indicated by an asterisk (*).
The gla-2 gene and rco-3 are present in cosmid X10:E5 of the hygromycin cosmid library constructed by M. Orbach and M. Sachs (Orbach, M. J. (1994). Gene 150, 159-162; Orbach, M. J., and Sachs, M. S. (1991). Fungal Genet. Newsl. 38, 97.). A 10 Kb XbaI fragment from this cosmid contains both rco-3 and gla-2 with the gene organization shown (Fig 2). The cosmid was mapped by RFLP analysis and confirmed by genetic mapping of rco-3 with respect to his-3 (3/65) and the mating type locus (1/65) as being near the centromere of Linkage Group I (Fig. 3). The data suggest that X10:E5 could be placed on either side of mei-3.
Figure 2. Organization of the gla-2 and rco-3 genes of N. crassa. The 10 Kb XbaI (X) fragment contains a 3.4 Kb SmaI fragment (S).
AP31a.1 MMMMOOMMMMOOMMMMMMM-MMOOOOOOOOOMOOO--M AP8v.1 MMMMOOMMMMOOMMMMOMMMMMOOOOOOOOMMOOOMMM mei-3 MMMMMOMMMMOOMMMMMMMMMMOOOOOOOOMMOOOOMM X10:E5 MMM-MOMMMMOOMM-MMMOM-M--OOOOOOOMOOOOMM un-2, his-2 MMMMOOMMMMOOMMMMMMMMMMOOOOOOOOMMOOOOMM lys-4 MMMMOOMMMMOOMMMMMMMMMMOOMOOOOOMMOOOOMM Fsr-33 MMMMOMMMMMOOMMMMMMMMMMOOMOOOOOMMOOOOMM
Figure 3. RFLP mapping of cosmid X10:E5. The entire cosmid was labeled and used to probe BamHI digested chromosomal DNA from progeny of the Oak Ridge x Mauriceville RFLP mapping cross.
The complete sequence of gla-2 will be of interest to those interested in the structure and function of glucoamylases. A plasmid (pLMN1) containing the 10 Kb XbaI fragment has been deposited in the FGSC. Please contact DJE by e-mail at dje0282@zeus.tamu.edu for DNA sequence files.
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Last modified 7/25/96 KMC