Polyclonal antibodies to and oligonucleotides derived from N-terminal sequences of media proteins of Neurospora crassa were both used to screen the FGSC lambda-ZAP I and lambda-ZAP II cDNA libraries. Primary sequencing of the identified phagemids revealed homologies to a number of known genes. These genes are listed and the cDNA clones are available from our laboratory to interested parties.
A library screen was initiated in an attempt to identify Neurospora crassa genes that code for secreted proteins. The signal sequences and promoters from these genes were to be studied in an effort to increase the levels of secreted heterologous protein in a N. crassa production system.
Polyclonal antibodies were generated (HYI-Bio Products) using 2.5 mg of extracellular proteins contained in conditioned media (Vogels with 2% sucrose) from 6 day old N. crassa cultures. PAGE-SDS (Novex) analysis of the concentrated media revealed between 17-20 major extracellular proteins of N. crassa. Western blot analysis confirmed reactivity to the rabbit antibodies generated. Lambda-ZAP I/II libraries available from the Fungal Genetics Stock Center were screened using this polyclonal antibody. Three phagemids were isolated, purified, and partially sequenced from both ends (Table 1). Additionally, proteins from culture supernatants separated by PAGE-SDS were transferred to a PVDF membrane and five were N-terminal sequenced. A degenerate DNA probe was made to one band from a sucrose grown culture corresponding to a protein of approximately 14.4 kd. This was chosen as a candidate because no extracellular proteins of this weight have been reported to date. The probe was digogxigenin labeled and used to identify clones in the Lambda-ZAP I/II libraries. Eight resulting phagemids were isolated and partially sequenced (Table 1). All sequences were analyzed and tentatively assigned by homology using FastA in the GCG package.
Table 1. Clones from the lambda-ZAP library identified by either the antibody screening method or by oligonucleotide screening method.
Antibody
screen
_________________________________________________________
Clone Homologous Organism
sequence (GCG
acession #)
____________________________________________________________
1c Fatty acid S.cerevisiae
synthetase
(x03977)
4d Ribosomal protein K. marianus
(S53436)
11c Ribonucleotide M. auratus
reductase M2 (golden hamster)
subunit (x68127)
_______________________________________________________________
_______________________________________________________________
Oligonucleotide
screen
_______________________________________________________________
Clone Homologous Organism
sequence (GCG
acession #)
________________________________________________________________
28b Superoxide C. elegans
dismutase
(Z27080)
4a Sucrose binding Glycine max
protein (L06038) (soybean)
2a mRNA for N. crassa
mitochondrial
ADP/ATP carrier
(X00363)
26b Fructose S. pombe
1,6-bisphosphate
aldolase (D17415)
19b Photolyase N. crassa
(X58713)
13a mRNA for protein Oenothera hookeri
kinase C (evening primrose)
inhibitor
homologue (X62838)
16a N-myristoyltransfe H. capsulatum
rase (L25118)
1b mRNA for N. crassa
mitochondrial
ADP/ATP carrier (
X00363)
______________________________________________________________________
______________________________________________________________________
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Last modified 7/25/96 KMC