Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa

Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.     

Mutation at the am locus of Neurospora crassa

Forty-eight new mutations at the am locus of Neurospora crassa have been characterized. Nineteen mutations were induced by UV; of these, eight were missense, two were frameshifts, two were nonsense, three were deletions and four were unidentified. Twenty-nine mutations were induced with nitrous acid; of these, twenty-one were missense, three were frameshifts, one was nonsense, two were deletions and one was genetically unstable.     

The control of allelic recombination at histidine loci in Neurospora crassa

The gene rec-1⁺ which reduces allelic recombination at the his-1 locus by a factor of between 15 and 30 has no effect upon allelic recombination at the his-2, his-3, his-5, his-6 and his-7 loci. Other genes controlling recombination at two of these loci, namely rec-x at his-2 and rec-w at his-3, have been found. There is a strong possibility that rec-x may be identical with rec-3, so far known to regulate recombination only at the am-1 locus. It is probable that the stocks used all carry a rec⁺ gene which regulates recombination at the his-6 locus, since all prototroph frequencies are low, but no regulatory gene active at the his-5 and his-7 loci.     

Use of fluorescent protein to analyse recombination at three loci in Neurospora crassa

We have inserted a histone H1-GFP fusion gene adjacent to three loci on different chromosomes of Neurospora crassa and made mating pairs in which a wild type version of GFP is crossed to one with a mutation in the 5' end of GFP. The loci are his-3, am and his-5, chosen because recombination mechanisms appear to differ between his-3 and am, and because crossing over adjacent to his-5, like his-3, is regulated by rec-2. At his-3, the frequencies of crossing over between GFP and the centromere and of conversion of 5'GFP to GFP(+) are comparable to those obtained by classical recombination assays, as is the effect of rec-2 on these frequencies, suggesting that our system does not alter the process of recombination. At each locus we have obtained sufficient data, on both gene conversion and crossing over, to be able to assess the effect of deletion of any gene involved in recombination. In addition, crosses between a GFP(+) strain and one with normal sequence at all three loci have been used to measure the interval to the centromere and to show that GFP experiences gene conversion with this system. Since any gene expressed in meiosis is silenced in Neurospora if hemizygous, any of our GFP(+) strains can be used as a quick screen to determine if a gene deleted by the Neurospora Genome Project is involved in crossing over or gene conversion.     

Biochemical analysis of isoallelic series at the al- i locus of Neurospora crassa

The neutral carotenoids of 3 phenotypically distinct albino-1 (al- i) strains, a wild type, 2 heterokaryons containing 2 al- i, alleles and 1 heterokaryon containing al- i+al-2 markers were analyzed. All al- i strains and the al- i heterokaryons contained large amounts of phytoene and only traces of higher carotenoids such as -carotene and lycopene which are responsible for the phenotypic variation at this locus (from pure white to lemon yellow). The biochemical lesion for al- i mutants affects phytoene dehydrogenase and enzyme leakiness accounts for the gene polymorphism. There is no evidence for interallelic complementation at the al- i locus.     

Two kinds of "recombination nodules" in Neurospora crassa

Two morphological types of recombination nodules, termed early and late, are recognized in Neurospora crassa. Eighty nuclei at different substages were used to determine numbers of nodules per nucleus, distribution of nodules along the nucleolus-organizing chromosome, and distribution of nodules among the two largest chromosomes. Early nodules appear at the synaptonemal complex at early zygotene and increase in number during zygotene until a dramatic reduction occurs at zygotene-pachytene transition. Thereafter early nodules are steadily eliminated until they disappear by diplotene. Late nodules are also present during zygotene. Their number doubles at the zygotene-pachytene transition and stays at this level until diplotene. The total number of nodules is rather constant through zygotene and pachytene. Distribution of bivalents with 0, 1, 2, etc. nodules follows a Poisson distribution at zygotene, but not at pachytene, where variance is less than the mean, indicating positive interference. Nodules are distributed nonrandomly along the nucleolus-organizer bivalent. The pattern differs slightly in nuclei of different origin. Nuclei with unusual synaptonemal complexes sustain normal levels of recombination by having the same amount of nodules as normal nuclei. In abnormal nuclei nodules are preferentially associated with normal segments. It is proposed that early nodules do not participate in any form of recombination but have a role in finding an appropriate site for a crossing-over event. Morphological change to the late type indicates that the site has been reached and the exchange event can be mediated by the late nodule.