Cloning of the rDNA repeat unit: An EcoRI fragment spanning the entire nontranscribed spacer region of Neurospora crassa wild type strain 74A

Cloning of the rDNA repeat unit: An EcoRI fragment spanning the entire nontranscribed spacer region of Neurospora crassa wild type strain 74A This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol31/iss1/3 Burnes, P.A., J-H. Kinnaird and J.R.S Fincham* The nonsense mutant aml7, is suppressible by ssu-1 by tyrosine insertion in residue 313 of NADPNeurospora crassa suppressors act on amber. specific glutamate dehydrogenase. It can revert to either Leu313 or Tyr313, consistent with the nonsense codon being either amber (UAG) or ochre (UAA) (Seale et al., 1976 Genetics 86: 261-274). DNA sequencing of the wild type gene (Kinnaird and Fincham, 1983 Gene 26 : 253-260) shows codon 313 to be CAG (glutamine). Furthermore, we find that am17 is induced to revert with-nitroquinoline oxide (NQO), a mutagen reported to be specific for G-C base paires (Prakash et al., 1974 J. Mol. Biol. 85: 51-65). We conclude that the nonsense codon,in am17 is amber (UAG). Since known Neurospora supersuppressors all suppress the same set of mutants (Seale, 1976 MGG 148: 105-108) they must all suppress amber. There is no evidence as yet for ochreor UGA-suppressing mutations in Neurospora. Given the very selective codon usage found so far in strongly and constitutively transcribed Neurospora genes (reviewed in Kinnaird and Fincham, 1983), UAA and UGA nonsense mutants would in any case be expected to be much less frequent than UAG in such genes. With no condons with A in the 3' position , only tyrosine (UAE) can mutate by single base-pair substitution to UAA and only tryptophan (UGG) or cysteine (UG~) to UGA; UAG, on the other hand, can arise from the abundant glutamate (GAG), glutamine (CAG) and lysine (AAG) codons. Department of Genetic, University of Edinburgh, King's Building, Edinburgh EH9 3JN, Scotland. *(Present address: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, England.) Chambers, C., R.J. Crouch and S.K. Dutta Cloning of the rDNA repeat unit: An EcoRI fragment spanning the entire nontranscribed spacer region of Neurospora crassa wild type strain 74A. iable Nontranscribed spacer regions are known to be varbetween species, and thus may be used to identify species differences of the genus Neurospora. This led us to clone this region from a known 74A wild type N. crassa strain. pMF2, a plasmid containing the coding sequences for 17S-5.8S-26S rRNA with additional flank ing sequences, was constructed by Free et al. in 1979. We used their clone as a probe to clone adjacent sequence. Initial cloning experiments were conducted using pBR325. A clone of 3400 bp containing most of the nontranscribed spacer region of Neurospora crassa rDNA EcoRI site. The clone may include the promoter, as well sequences was inserted into the chloramphenicol gene as the termination sites, for transcription of rRNA 17S, 5.8S and 26S in Neurospora crassa 74A wild type. To achieve this, plasmid pBR325 was restricted with EcoRI and then treated with bacterial alkaline phosphatase (BAP) at 65° C for 1h. The preparation was then shaken with an equal amount of phenol saturated with 0.1 M Tris-HCl, pH 9.0, followed by the addition of an equal volume of chloroform, shaken again, and then centrifuged at 15,000 rpm for five min. The upper aqueous solution was removed, brought to 1M with NaCl, EtOH precipitated in dry ice for 15 min, then centrifuged again. The pellet was vacuum dried and resuspended in distilled water. Total nuclear DNA isolated from wild-type N. crassa strain 74A (FGSC #987) was R E S T R I C T I O N M A P O F p B R 3 2 5 -PG I --EC&l restricted with EcoRI. The N. crassa nuclear EcoRIdigested DNA was subjected to electrophoresis on 0.7% agarose gels and the band which carresponded to the nontranscribed spacer sequences was cut out of the gels, electroeluted, and run over a DE-52 column. This DNA was ligated with the BAP-treated EcoRI-digested pBR325 DNA in a ration of 1O:1 to lOO:1, and then incubated at 4° C for 1 to 2 days. Escherichia coli strain LE 392 was used for the transformation experiments.

transcribed spacer region of Neurospora crassa wild type strain 74A.

iable
Nontranscribed spacer regions are known to be varbetween species, and thus may be used to identify species differences of the genus Neurospora. This led us to clone this region from a known 74A wild type N. crassa strain. pMF2, a plasmid containing the coding sequences for 17S-5.8S-26S rRNA with additional flank ing sequences, was constructed by Free et al. in 1979. We used their clone as a probe to clone adjacent sequence.
Initial cloning experiments were conducted using pBR325.
A clone of 3400 bp containing most of the nontranscribed spacer region of Neurospora crassa rDNA EcoRI site. The clone may include the promoter, as well sequences was inserted into the chloramphenicol gene as the termination sites, for transcription of rRNA 17S, 5.8S and 26S in Neurospora crassa 74A wild type.
To achieve this, plasmid pBR325 was restricted with EcoRI and then treated with bacterial alkaline phosphatase (BAP) at 65° C for 1h.
The preparation was then shaken with an equal amount of phenol saturated with 0.1 M Tris-HCl, pH 9.0, followed by the addition of an equal volume of chloroform, shaken again, and then centrifuged at 15,000 rpm for five min. The upper aqueous solution was removed, brought to 1M with NaCl EtOH precipitated in dry ice for 15 min, then centrifuged again. The pellet was vacuum dried and resuspended in distilled water. Total nuclear DNA isolated from wild-type N. crassa strain 74A (FGSC #987) was

R E S T R I C T I O N M A P O F p B R 3 2 5 -PG I --EC&l
restricted with EcoRI. The N. crassa nuclear EcoRIdigested DNA was subjected to electrophoresis on 0.7% agarose gels and the band which carresponded to the nontranscribed spacer sequences was cut out of the gels, electroeluted, and run over a DE-52 column. This DNA was ligated with the BAP-treated EcoRI-digested pBR325 DNA in a ration of 1O:1 to lOO:1, and then incubated at 4° C for 1 to 2 days. Escherichia coli strain LE 392 was used for the transformation experiments. The colonies whose DNA generated the appropriate restriction fragment sizes were rechecked and the DNA blotted onto nitrocellulose paper, then hybridized to an rDNA 32P-labeled probe. This probe contained approximately 600 bps in common,with the spacer region which was being cloned.

rDNA OF N.crassa
The probe was derived from pMF2 DNA (Free et al. 137: 1219-1226, 1979). When the plasmid pMF2 DNA is restricted with Pst I two fragments are generated -a 4.36 kb fragment (the cloning vector, p8R322) and a 6 kb fragment (the Neurospora rDNA insert), The 6 kb fragment contains the entire coding regions for 17S to 5.8S to 26S rRNA and additional sequences approximately 1 kb which are portions of the spacer regions.
The EcoRI fragment inserted into the EcoRI site of pBR325 is approximately 3.4 kb in size and contains mostly the nontranscribed spacer region, plus external transcribed spacer region, and additional coding sequences approximately 150 bps near the 3' end of 26S rDNA.
The desired clone contains the 3.4 kb insert, as well as the 6 kb piece of pBR325 DNA. When this DNA is restricted it generates the following fragment sizes: using Hind III → 5.5 kb, 2.9 kb, 900 bp; using Pst I → 4.9 kb, 2.8 kb, 1.6 kb; using EcoRI→ 5.9 kb, 3.4 kb; using BamHI → 9.3 kb; using XhoI → 8.9 kb, 400 bp; and using SmaI 9.3 kb.