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Figure 14.6 Integration of kalilo plasmid into mitochondrial genome. The letters for regions of mtDNA are arbitrary labeled by letters. AR = autonomously replicating plasmid; IS = insertion sequence of plasmid. mtDNA is linearized for purpose of illustration. Adapted from Griffiths (1995).

14.4.1.1 Linear Plasmids

The majority of N. intermedia strains collected from Kauai are senescent and were named "kalilo," Hawaiian for "dying." Some wild strains of N. crassa collected from Aarey near Mumbai, India, died in about 20 subcultures and these strains were named "maranhar," which in the Hindi language means "prone-to-death" (Court et al., 1991). Kalilo and maranhar strains contain 9- and 7- kb plasmids, respectively. The structural features of the plasmids are deduced from electron microscopy, gel electrophoresis, sensitivity to 5 and 3 exonuclease and sequencing. Both pKAL and pMAR are linear plasmids having terminal regions with inverted repeats of base sequences. A protein is bound to the 5 termini inverted repeats as indicated by resistance to 5 exonuclease digestion (Griffiths, 1995). There are two open reading frames whose amino acid sequences suggest that they encode DNA and RNA polymerases. pKAL and pMAR do not have sequence homology. Based on detection by Southern hybridization, KALILO or homologous plasmids are found in Neurosporas collected from different regions of world.

Radiolabeled DNA sequences of pKAL and pMAR hybridized only to mitochondrial DNA from senescing cultures, showing that plasmids inserted into mitochondrial genome —consistent with the maternal transmission of the plasmids DNAs (Myers et al., 1989; Court et al., 1993). The full-length plasmid, flanked by inverted terminal repeats, inserts at a single site into mitochondrial DNA. The plasmids therefore exist in mitochondria in two forms: an autonomously replicating (AR) form and as an insertion sequence (IS), which for the kalilo are denoted mtAR-kalDNA and mtlS-kalDNA, respectively. Plasmids could induce senescence by inserting into mtDNA and inactivating an indispensable gene (Figure 14.7).

14.4.1.2 Circular Plasmids

The Mauriceville strain of N. crassa and the Varkud strain of N. intermedia have circular mitochondrial plasmids, normally benign but they can mutate into variant forms and become "killer" plasmids. During continuous growth in race tubes or during sequential conidial transfers in slants, these strains showed either "stop-start" growth or senescence.

Figure 14.7 Map of wild type Neurospora crassa mitochondrial DNA. The outer circle shows the fragments produced by HindIII restriction enzyme. The inner circle shows the fragments produced by EcoRI restriction enzyme.

The variant strains had deficiencies of cytochromes aa3 and b (Akins et al., 1986). Densitometry of ethidium bromide stained gel following electrophoresis of total mtDNA shows plasmid accumulation relative to the mitochondrial DNA, i.e., they had become suppressive. DNA sequencing revealed the insertion of a mitochondrial tRNA sequence and short deletions in the plasmid DNA. As both plasmids encode reverse transcriptase, it is hypothesized that during replication, the variant forms of plasmids arise from the benign forms via an RNA intermediate and reverse transcription of full length plasmid transcript that integrates into mitochondrial DNA by homologous recombination (Akins et al., 1989).

14.4.1.3 Spread of Plasmids

Yang and Griffiths (1993) made a global survey of Neurospora species by the Southern hybridization method using a plasmid probe and demonstrated that both senescent and nonsenescent strains have mitochondrial plasmids. Although few fungi have been examined for mitochondrial plasmids, it is likely that plasmids are common in fungi. Related plasmids have been discovered in strains collected globally. This raises the interesting question as to how plasmids spread. One possibility is by hyphal contact of plasmid-harboring and plasmid-lacking strains by transient fusion and mixing of the mitochondria. To test this, a senescent kalilo strain was constructed having the nuclear marker genes nic-1 and al-2 (Griffiths et al., 1990). This was mixed with a nonsenescent strain having the marker genes ad-3B and cyh-1. This heterokaryon was senescent, demonstrating that hyphal fusion could allow the plasmid to spread in nature (horizontal transmission). In some cases, vertical transmission of plasmids has been demonstrated following limited sexual recombination (Bok and Griffiths, 1999). However, the screening of populations over a period of time did not show a change in plasmid frequency. Further investigations are required to determine how the spread of plasmids in nature is checked (Debets et al., 1995).

14.5 NUCLEAR GENE MUTANTS 14.5.1 natural death

In N. crassa, a single nuclear gene mutant obtained by ultraviolet irradiation was called natural death (nd). The mutant nd dies in 2-4 sequential transfers; therefore, to preserve nd cultures for studies, its nuclei are marked and preserved in a heterokaryon in association with the wild type. For example, the nd strain is crossed to an albino strain and the white (albino) recombinant progeny (nd al) is selected that shows the death phenotype in subcultures. The mutant nd al can, however, be indefinitely maintained in combination with nuclei having the wild type (nd+) allele and when desired recovered by plating conidia formed by the heterokaryon and selecting the white nd colonies carrying the al marker allele. After extraction, the nd homokaryon died again in 2-4 subcultures but this provides the time for sexual crosses and mycelium to be grown for DNA analysis. This technique of preserving potentially lethal mutations possible with N. crassa make it very suitable for investigation of the phenomenon of aging and death.

The sequence of 65.5 kb circular mitochondrial DNA molecule of N. crassa is known and an EcoRI restriction map is shown in Figure 14.8. On the basis of their migration in agarose gels, 11EcoRI bands can be distinguished. A comparison of the restriction digest profiles of mitochondrial DNA from nd and nd+ mycelia showed unique fragments in the nd homokaryons recovered from the heterokaryon that are not present either in the wild nd+ or in the [nd + nd+] heterokaryons (Seidel-Rogol et al., 1989; Bertrand et al., 1993). The unique EcoRI restriction fragments (Figure 14.9) from mitochondrial DNA were cloned and sequenced. Comparison of its sequence with wild mtDNA reveals that nd suffers short deletions and its nucleotide sequences are rearranged. The deleted segments are those that have a palindrome sequence, suggesting that this sequence gets recognized and is removed by specific endonucleases and the separated fragments juxtaposed. It was hypothesized that unequal crossing-over between repeat sequences result in deletions followed by recombination of distant nucleotide sequences (Figure 14.10).

14.5.2 senescent

Another nuclear gene mutant senescent, derived from a phenotypically normal heterokary-otic wild isolate by extracting nuclei in the form of uninucleate microconidia (Navaraj et al., 2000) and growing them into homokaryotic cultures (Figure 14.11), exhibited the "death"

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