Crassa DNA Research Articles

Cloning the quinic acid (qa) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK-308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.

A study of the organisation of the ribosomal ribonucleic acid gene cluster of Neurospora crassa by means of restriction endonuclease analysis and cloning in bacteriophage lambda

1. Total Neurospora crassa DNA was restricted with endonucleases and fragments carrying rRNA coding sequences were identified by hybridization with Xenopus laevis ribosomal DNA probes. 2. The repeating unit of the rRNA gene cluster was found to be 8.6 kbp, arranged in a head-to-tail fashion. 3. Digestion with Hind III yielded fragments of 3.4 kbp and 5.2 kbp and both were cloned. 4. Digestion with Eco RI yielded fragments of 2.2 kbp, 3.0 kbp and 3.4 kbp; the 3.0 kbp fragment was cloned. 5. Sequences coding for RNA (S-rRNA)1 of the smaller subribosomal particle were found (at least 90%) in the 2.2 kbp EcoRI subfragment of the 5.2 kbp Hind III fragment. 6. The coding sequences for the major RNA species (L-rRNA) of the larger subribosomal particle were located mainly (at least 95%) in the 3.4 kbp Hind III fragment. 7. For comparison, a Hind III digest of total yeast DNA was cloned and recombinants containing a 6.4 kbp rDNA fragment were isolated.

Transcription of repetitive DNA in Neurospora crassa

Repeated DNA sequences of Neurospora crassa were isolated and characterized. Approximately 10-12% of N. crassa DNA sequence were repeated, of which 7.3% were found to be transcribed in mid-log phase of mycelial growth as measured by DNA:RNA hybridization. It is suggested that part of repetitive DNA transcripts in N. crassa were mitochondrial and part were nuclear DNA. Most of the nuclear repeated DNAs, however, code for rRNA and tRNA in N. crassa.

Transcription of non-repeated DNA in Neurospora crassa

Repeated and non-repeated DNAs of Neurospora crassa mycelial cells were fractionated by hydroxyapatite chromatography and characterized separately for reassociation kinetics and thermal stability profiles of reassociated DNAs. Approx. 90 % of N. crassa DNA sequences reassociated like single-copy DNA. About 35 % of these unique sequences were found to be transcribed in mid-log phase of mycelial growth when measured by DNA · RNA hybridization at a RNA C 0t of about 90 000.