Genetical and biochemical characterization of a novel mutantof neurospora crassa showing senescence phenotype

Abstract

Thesis submitted for the degree of Doctor of Philosophy Indian Institute of Science, Bangalore, India

Title: Genetical and Biochemical Characterization of a Novel Mutant of Neurospora crassa Showing Senescence Phenotype Author: A. Navaraj

Introduction Fungi are composed of cylindrical filaments, or hyphae, which are branched and divided into segments by transverse walls. A fungal colony is a radially expanding system of hyphae showing unlimited growth. The immortal nature of fungal colonies is reflected in their ability to be propagated continuously by transferring asexual spores (conidia) or small amounts of vegetative mycelium to fresh nutrient agar medium.

Derivation of a Novel Senescent Strain of Neurospora It was previously thought that fungal colonies growing in natural habitats are genetically pure (homokaryotic), i.e., the nuclei in the multinucleate mycelium of an “individual” colony are all identical. However, recessive mutations can arise in nature and remain masked by wild-type nuclei, resulting in heterokaryotic mycelia.

To test this, a wild isolate of Neurospora intermedia collected from burnt sugarcane was resolved into component nuclear types by plating uninucleate microconidia. Of the 159 homokaryotic colonies derived, one culture, Maddur 1991-10110 (place/year of collection/original isolate number/homokaryotic derivative number), senesced during subculturing. The production of aerial hyphae and conidiophores decreased, and the surface mycelium ceased growth upon further subculturing. Supplementation of growth media with yeast extract, peptone, casamino acids, or yeast nucleic acid hydrolysate did not improve growth.

The senescence character was introgressed into N. crassa through nine backcrosses to the wild-type strain and preserved as a heterokaryon with aml ad-3B nuclei (inactive mating-type allele and ad-3B as a forcing marker). The sen mutant gene was recovered when desired by crossing (sen + aml ad-3B) heterokaryon to the wild-type N. crassa (Oak Ridge strain).

Genetic Characterization of the sen Mutant Crosses between the senescent strain and wild-type N. crassa showed 1:1 segregation of senescent and wild-type characters in ascospores, proving that a single nuclear gene controls senescence. This gene was named the senescent (sen) gene.

Linkage analysis using the “alcoy” tester strain indicated linkage to cot-1, suggesting location in linkage group IV or V. Further crosses ruled out group IV, and mapping placed sen ~4 map units right of his-1 in linkage group V. Gene order was determined as:

cyh-2 - his-1 - sen - al-3 - pab-1

Complementation tests showed that sen is not allelic to nearby loci (un-11, un-19, mus-12), confirming identification of a new nuclear gene associated with death phenotype in Neurospora.

Biochemical Characterization of the sen Mutant To test similarity with apoptosis in animals, genomic DNA from the sen mutant was analyzed. Unlike apoptotic DNA fragmentation, the sen mutant did not show oligonucleosomal laddering, suggesting a distinct mechanism of death.

Temperature strongly influenced senescence: at 34°C, death occurred within 2 subcultures, while at 26°C, death was delayed to 6-9 subcultures.

Respiration in the sen Mutant At 34°C, respiration of glucose-stimulated conidia was cyanide-insensitive, indicating cytochrome deficiency. Oxygen uptake was inhibited by salicyl hydroxamic acid (SHAM), confirming an alternate oxidase pathway. At 26°C, early subcultures showed cyanide-sensitive respiration (wild-type-like), but later subcultures became cyanide-insensitive, suggesting progressive cytochrome deficiency.

Spectral analysis confirmed absence of cytochrome c at 34°C. Complementation tests with cyt-12-12 (cytochrome c structural gene mutant) and cyt-2-1 (cytochrome c heme lyase mutant) showed sen is not allelic to these loci.

Thus, the sen mutation does not prevent cytochrome synthesis but interferes with transport or assembly of cytochromes in mitochondria in a temperature-sensitive manner.

Hypothetical Model of Senescence In Neurospora, at least seven protein components of the Translocase of the Outer Membrane (TOM) mediate recognition, insertion, and translocation of precursor proteins across the mitochondrial outer membrane.

It is hypothesized that SEN encodes a nuclear protein component of the TOM receptor complex. The mutant SEN protein assumes different conformations depending on temperature:

At 34°C, abnormal conformation prevents binding of precursor proteins, blocking essential protein import and hastening death.

At 26°C, partial native-like conformation allows delayed death, as functional TOM complexes are initially present but diluted out by abnormal mitochondria during growth.

This model explains the temperature-sensitive senescence phenotype observed in the sen mutant.