John R. S. Fincham

The complete nucleotide sequence of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene

The complete nucleotide sequence of a 2.7-kb genomic fragment, containing the Neurospora crassa am [NADP-specific glutamate dehydrogenase (GDH)] gene, has been determined. The transcription initiation and polyadenylation sites have been defined by S1 mapping. There are at least four initiation sites between 35 and 60 bases downstream of a TATAAA sequence. The single polyadenylation site is immediately downstream of a six-nucleotide sequence which is present in the corresponding position in the N. crassa gene (pl) coding for the imported proteolipid component of mitochondrial ATP synthase [Viebrock et al. Embo J. 1 (1982) 565-571] the initiation sites also show some sequence similarity between am and pl. There are two introns, of 66 and 61 bases respectively; they interrupt sequences that are conserved (highly so in the case of the second intron) between Neurospora and Escherichia coli. As well as showing typical consensus splicing sequences, the two introns show some striking internal similarities to each other. The am gene resembles the pl gene in its strongly selective use of codons (pyrimidines preferred to purines and guanine to adenine in third positions). The coding sequence here reported requires four corrections to be made in the previously published amino acid sequence of Neurospora GDH.

Frameshift mutations affecting the N-terminal sequence of Neurospora NADP-specific glutamate dehydrogenase

Abstract A series of ultraviolet light-induced revertants from the mutant am6, mapping at the left-hand (“N-terminal”) end of the structural gene for NADP-specific glutamate dehydrogenase, have been shown to have amino acid substitutions in the N-terminal tryptic peptide. Only a few were found to have the wild-type sequence; the great majority had the replacement Ser5 → Pro and most had a further altered sequence extending one, two, three or four residues to the left. The most extensively altered revertant had a sequence with the extra residue Met at the N-terminus: Met-Leu-Thr-Phe-Pro-Pro- instead of the normal sequence N-acetyl-Ser-Asn-Leu-Pro-Ser-. The results are interpreted as meaning that am6 is a frameshift mutant, with the insertion of a base in the Ser5 codon, and that the revertants are all deletions at various positions to the left. Most of the revertants can be explained as single-base deletions, but some appear to have arisen by a more complex type of event. One revertant is a four-base deletion. The longest double-frameshifted sequence, on the basis of the simplest hypothesis as to its origin, defines the first 17 bases of the messenger RNA coding sequence. The altered sequences do not appear to affect the enzyme activity, except that they do, to different extents depending on the sequence, affect its sensitivity to heat.

The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects--more mutants and a summary.

A further series of mutant am alleles, encoding potentially active NADP-specific glutamate dehydrogenase (GDH) and capable of complementation in heterocaryons, have been characterized with respect to both GDH properties and DNA sequence changes. Several mutants previously studied, and some of their same-site or second-site revertants, have also been sequenced for the first time. We present a summary of what is known of the properties of all am mutants that have been defined at the sequence level.

The interpretation of gene conversion data from ordered eight-spored asci

Currently favoured models postulate that gene conversion is due to the correction of mis-matches in heteroduplex DNA. If heteroduplex is formed reciprocally on both chromatids participating in recombination, the mis-matches due to a heterozygous site will be different on the two chromatids, and there will be four correction probabilities to be taken into account. It is shown that, given the frequencies of the five different kinds of aberrant ascus ratios, it is possible to calculate four alternative sets of values for the four correction probabilities and the total number of asci in which heteroduplex is formed. These four solutions reduce in effect to two when there are no other markers distinguishing the two chromatids. With the aid of flanking markers and the assumption that heteroduplex formation is chemically polarized, it is possible, in principle, to choose one best solution. The method has been applied to the five one-point crosses in Sordaria fimicola from which most data are available. The data from four different mutants crossed to wild type are compatible with a restricted model in which the correction frequencies, from mutant to wild and from wild to mutant, are the same on both chromatids. In the case of the fifth mutant the data are not consistent with this restricted model, and indicate different correction frequencies in the two chromatids.

Alternative modes of mRNA processing in a 3′ splice site mutant of Neurospora crassa

SummaryThe am8 mutant of Neurospora crassa is shown to have a double base-pair change, GTG for the normal TAG, at the 3′ end of the second intron of the am (NADP-specific glutamate dehydrogenase, GDH) gene. The greater part of the mutant am transcript accumulates as two fragments hybridising to probes for sequences respectively upstream and downstream of the 5′ end of the intron. Two processed transcripts approximating to normal full length mRNA were identified. In one the second intron was intact; in the other the second intron was spliced out through the use of an AAG sequence, 20 base-pairs into the third exon, as a 3′ acceptor site. A GAG sequence, only four base-pairs downstream from the normal acceptor site, does not appear to be used.