Identification of several novel Neurospora crassa genes in the lambda- ZAP I and lambda-ZAP II libraries

Roxanne A. Yamashita and W. Dorsey Stuart - Department of Genetics and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822

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.

Clone         Homologous                        Organism                   
              sequence (GCG                                                              
              acession #)                                                                
1c            Fatty acid                        S.cerevisiae               
4d            Ribosomal protein                 K. marianus                
11c           Ribonucleotide                    M. auratus          
              reductase M2                    (golden hamster)                   
              subunit  (x68127)                                                          


Clone         Homologous                           Organism                   
              sequence (GCG                                                              
              acession #)                                                                

28b           Superoxide                           C. elegans                 

4a            Sucrose binding                     Glycine max       
              protein (L06038)                    (soybean)                                       

2a            mRNA for                             N. crassa                  
              ADP/ATP carrier                                                            

26b           Fructose                              S. pombe                   
              aldolase  (D17415)                                                         

19b           Photolyase                            N. crassa                  

13a           mRNA for protein                    Oenothera hookeri          
              kinase C                            (evening primrose)         
              homologue (X62838)                                                         

16a           N-myristoyltransfe                   H. capsulatum              
              rase (L25118)                                                              

1b            mRNA for                              N. crassa                  
              ADP/ATP carrier (                                                          

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Last modified 7/25/96 KMC