Insertional/Tn mutagenesis in Neurospora?

Matthew Sachs msachs at ADMIN.OGI.EDU
Fri Feb 24 14:39:41 EST 1995


>Greetings. In random discussions, several of us Neurosporologists have
>remarked on the challenge presented by cloning genes in this filamentous
>fungus. This stumbling block would be dramatically reduced by insertional
>mutagenesis, but this aspect of the system remains undeveloped. Attempts in
>this lab to apply REMI in Neurospora have been unfruitful, though rumor has
>it that this approach works in "every other" fungus. Has anyone had recent
>luck or insights into using any sort of insertional or (especially)
>transposon mutagenesis in Neurospora or parallel filamentous fungi?
>Hopefully we can come up with some likely approaches and/or confer on this
>topic here and/or at the Fungal Genetics Conference next month. Any
>thoughts...?
>-Peter Margolis

Re:  Insertional mutagenesis in Neurospora:

S. Kang and R. L. Metzenberg (1993), Genetics 133(2): 193-202.
Insertional mutagenesis in Neurospora crassa: cloning and molecular
analysis of the preg+ gene controlling the activity of the transcriptional
activator NUC-1.

Abstract:
The transcriptional activator NUC-1 controls the transcription of the genes
for phosphorus acquisition enzymes, and its activity is regulated by the
negative regulatory factors, PREG and PGOV In this report, we describe the
cloning and molecular analysis of the preg+ gene. In Neurospora crassa, as
in higher eukaryotes, transformation frequently results in nonhomologous
integration of transforming DNA. Insertion of transforming DNA into host
genes mutates the gene and provides a molecular tag for cloning it. We
obtained two mutants that have an insertion in the preg+ and pgov+ genes,
respectively, among 2 x 10(5) transformants. The preg+ gene was cloned by
screening a Neurospora genomic DNA library with DNA sequences flanking the
transforming DNA of the rescued plasmid. Northern analysis showed that the
transcription of the preg+ gene is not regulated by phosphate. The
carboxy-terminal half of PREG shows strong homology with Saccharomyces
cerevisiae PHO80 whose function is analogous to that of PREG. The pregc
mutations are located in the well conserved residues which may directly
interact with the residues in the regulatory domain of NUC-1.




-----------------------------------------------------------
Matthew Sachs
Department of Chemistry, Biochemistry and Molecular Biology
Oregon Graduate Institute of Science and Technology
20000 NW Walker Road
P.O. Box 91000
Portland, OR  97291-1000
503 690-1487 Phone
503 690-1464 Fax
msachs at admin.ogi.edu





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