Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe

Kinnaird, Jane; Keighren, Margaret; Kinsey, John A.; Eaton, Michael A. W.; Fincham, J. R. S.

doi:10.1016/0378-1119(82)90207-4

Cited by 114 publications

(45 citation statements)

References 22 publications

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“…An additional consensus sequence, PuCTPuAC, was found within and near the 3' terminus of all intervening sequences of the glucoamylase gene. An identical sequence is similarly located within intervening sequences of other filamentous ascomycetes (T. reesei [54] and N. crassa [26,67]) and is related to the consensus sequence TACTAAC found within intervening sequences of S. cerevisiae genes (30). This latter sequence has been postulated to be required for RNA splicing in S.…”

Section: Resultsmentioning

confidence: 89%

“…The intervening sequences were short (ranging from 55 to 75 bp) and were all located within protein-encoding sequences. Short intervening sequences have also been found in genes of other filamentous ascomycetes, namely, the cellobiohydrolase gene of Trichoderma reesei (54) and the glutamate dehydrogenase gene (26) and histone H3 and H4 genes (67) of Neurospora crassa. In contrast, the trp-J gene of N. crassa has been shown to lack intervening sequences (50 5' end of the glucoamylase gene was hybridized to total poly(A)+ mRNA from starchgrown cells, and S1 nuclease-resistant products were analyzed by gel electrophoresis under denatured conditions (49).…”

Section: Resultsmentioning

confidence: 99%

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Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

Nunberg¹,

Meade²,

Cole³

et al. 1984

Mol. Cell. Biol.

160

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The filamentous ascomycete Aspergillus awamori secretes large amounts of glucoamylase upon growth in medium containing starch, glucose, or a variety of hexose sugars and sugar polymers. We examined the mechanism of this carbon source-dependent regulation of glucoamylase accumulation and found a several hundredfold increase in glucoamylase mRNA in cells grown on an inducing substrate, starch, relative to cells grown on a noninducing substrate, xylose. We postulate that induction of glucoamylase synthesis is regulated transcriptionally. Comparing total mRNA from cells grown on starch and xylose, we were able to identify an inducible 2.3-kilobase mRNA-encoding glucoamylase. The glucoamylase mRNA was purified and used to identify a molecularly cloned 3.4-kilobase EcoRI fragment containing the A. awamori glucoamylase gene. Comparison of the nucleotide sequence of the 3.4-kilobase EcoRI fragment with that of the glucoamylase I mRNA (as determined from molecularly cloned cDNA) revealed the existence of four intervening sequences within the glucoamylase gene. The 5' end of the glucoamylase mRNA was mapped to several locations within a region -52 to -73 nucleotides from the translational start. Sequence and structural features of the glucoamylase gene of the filamentous ascomycete A. awamori were examined and compared with those reported in genes of other eucaryotes.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

Nunberg¹,

Meade²,

Cole³

et al. 1984

Mol. Cell. Biol.

160

View full text Add to dashboard Cite

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“…1). Transformant T-508 exhibited multiple distinct bands in Sau3A or Mbo I digests, and, based on their relative intensities, some of the bands probably T-51 4 T-520 reflect the superimposition of several copies of a common fragment. We estimate that approximately four copies of the am fragment integrated together in this transformant.…”

Section: Resultsmentioning

confidence: 98%

“…The N. crassa strain used as the transformation host (N24) had a deletion removing the entire am (glutamate dehydrogenase) gene (4) and, in place of the methylated '-'q region, had unmethylated DNA partially homologous to C-'v (5). Thus, the flank region represented the only significant region of homology between the transforming DNA and the host genome.…”

mentioning

confidence: 99%

DNA sequence duplications trigger gene inactivation in Neurospora crassa.

Selker

Garrett

1988

Proc. Natl. Acad. Sci. U.S.A.

223

130

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Transforming sequences are faithfully replicated in vegetative cells ofNeurospora but are typically subject at high frequency to sequence alterations and methylation in the period between fertilization and nuclear fusion. Previous work showed a correlation between the occurrence of these radical changes, referred to by the acronym RIP, and the presence of sequence duplications resulting from the introduced DNA. Various possible causes for the RIP process were inves-

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“…The in vitro techniques of eDNA synthesis and hybridisation to oligonucleotide probes have led to the isolation of the glucoamylase eDNA of Aspergillus niger (6) and the am gene ofN. crassa (24). The method most often used, however, has involved the screening of genomic libraries for complementation of mutants or expression in Escherichia coli or S. cerevisiae.…”

Section: R Van Heeswijck a Mig Roncero: Transformation Of Mucormentioning

confidence: 99%