Distant upstream regulatory sequences control the level of expression of the am (GDH) locus of Neurospora crassa

Abstract

We have constructed deletions in the 5' noncoding sequences of the cloned Neurospora crassa am gene. Vectors with a truncated fragment of the am gene were used in transformation experiments to introduce the deletions into the chromosome by homologous recombination. Analysis of glutamate dehydrogenase (GDH) expression by enzyme assay and immunoblots, as well as Northern and dot blots of poly (A)+ RNA, in the deletion strains indicates that there are two upstream regulatory sequences that control the level of gene expression. The closer of these two elements (URSam alpha) is at approximately 1.4 kb upstream of the transcriptional start site. The second elements (URSam beta) is located between 2.1 and 3.2 kb upstream of the transcription start site. Deletion of either of these two elements reduces am expression to about 50% of the wild-type level. Deletion of both elements reduce am expression to from 5-16% of the wild-type level. Deletion of 1.1 kb of sequence just downstream of URSam alpha, which brings this element to within 300 bp of the transcription start site, had no effect on am expression. Likewise, deletion of 3.5 kb of sequence upstream of URSam beta had no effect on expression. None of these deletions had any effect on the expression of usg-1, a gene of unknown function that is transcribed in the same direction as the am gene, and which terminates about 3.5 kb upstream of the URSam beta element.