Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The origin and structural features of a newly-detected plasmid in a deviant Neurospora intermedia strain Xu, Yan 1998

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1998-0148.pdf [ 4.92MB ]
Metadata
JSON: 831-1.0088437.json
JSON-LD: 831-1.0088437-ld.json
RDF/XML (Pretty): 831-1.0088437-rdf.xml
RDF/JSON: 831-1.0088437-rdf.json
Turtle: 831-1.0088437-turtle.txt
N-Triples: 831-1.0088437-rdf-ntriples.txt
Original Record: 831-1.0088437-source.json
Full Text
831-1.0088437-fulltext.txt
Citation
831-1.0088437.ris

Full Text

THE ORIGIN AND STRUCTURAL FEATURES OF A NEWLY-DETECTED PLASMID IN A DEVIANT NEUROSPORA INTERMEDIA STRAIN by Y A N X U B.Sc. Anhui University 1986 M . S c . The University of Science and Technology of China 1991 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Botany ) We accept this as conforming to the required standard T H E U N I V E R I T Y OF B R I T I S H C O L U M B I A March 1998 © Y A N X U , 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^?0TA/y Y The University of British Columbia Vancouver, Canada Date A ^ g . 7 DE-6 (2/88) 11 Abstract During a survey of mitochondrial D N A plasmids in natural populations of Neurospora intermedia and Neurospora crassa. many new mitochondrial plasmids, both linear and circular, were found. Also, new circular and linear plasmids appeared to arise spontaneously from existing plasmids. A new mitochondrial linear D N A plasmid has been detected in Neurospora intermedia Harbin strain 3983M-7.0 which is a derivative of Neurospora intermedia Harbin strain 3983M. The origin of this new plasmid was studied and its complete nucleotide sequence was determined. This new D N A element is a mitochondrial linear plasmid. It is not derived from the nuclear or mitochondrial genome. It is related to a linear plasmid in Neurospora intermedia Harbin strain 3983. This newly-detected plasmid, named Har-7.0, is 7.0 kilobase pairs (kb) in length. It carries perfect terminal inverted repeats (TIR) of 347 base pairs (bp). Extending inward from the terminal repeats are two long open reading frames with similarity to D N A and R N A polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of the senescence-inducing plasmid maranhar, originally described in Neurospora crassa. Overall the two plasmids have identical genetic organization and are clearly homologous at the sequence level. ORF1 of this newly-detected 7.0 kb plasmid is 2691 bp in size and ORF2 is 3108 bp. In ORF1 there is just mono-nucleotide (1-nt) substitution and mono-nucleotide insertion relative to marDNA. In ORF2 there are several substitutions (up to 4-nt) and small insertions (up to 4-nt). The distribution of maranhar plasmid in different species, N . crassa andN. intermedia, could be due to horizontal transfer or gene flow between these two. Table of contents Abstract List of tables List o f figures Introduction Neurospora life cycle D N A plasmids in eukaryotes Ki l l e r plasmids in Kluyveromyces lactis Plasmids SI and S2 of Zea mays Plasmids in filamentous fungi Objectives of these studies Materials and methods Strains Media and growth conditions D N A isolation Enzyme digestion and gel electrophoresis Cloning of the mitochondrial plasmid Labelling nucleic acid Probes Southern blot analysis Dot-blot hybridization iv Page P C R reaction 18 D N A sequencing 19 Computer analysis of D N A sequence 20 Chapter one. The origin of the newly-detected 7.0 kb plasmid of deviant N . Intermedia Harbin strain 3983M-7.0 21 Introduction 21 Results 23 Discussion 25 Chapter two. The structural features of the Har-7.0 plasmid. 43 Introduction 43 Results ' 4 5 General sequence organization of the Har-7.0 plasmid D N A . 45 Sequence similarity between Har-7.0 and maranhar. 47 ORF1 48 O R F 2 48 Terminal inverted repeats 49 The intergenic region 50 The 5'terminal nucleotides 50 Sequence similarity between Har-7.0 and Har-L 50 Discussion - ' 51 Summary 58 Literature cited , 82 V List of tables Page Chapter one Table 1 - 1. The plasmid content of strain 3983 and its derivatives. 22 Table '1 - 2. The transmission of maternal mitochondrial plasmids to ascospore progeny from crosses. 30 Chapter two Table 2 - 1. The marDNA-homologous plasmids with size similar to maranhar in a sample of 19 natural isolates of N . intermedia. 44 Table 2 - 2. Size (bp) of domains of maranhar and Har-7.0 plasmid. 46 Table 2 - 3. Quantity of changed residues. 47 Table 2 - 4. Quantity of deleted residues. 47 Table 2 - 5. Quantity of added residues. 48 Table 2 - 6. The changed amino acids in O R F 1 . 48 Table 2 - 7. The changed amino acids in ORJF2. 49 List of figures Page Chapter one Figure 1 - 1 A The hew plasmid Har-7.0 replacing the original Har-L detected in mitochondria of a culture 3983M-7.0 derived from strain 3983M. 31 Figure 1 - 1 B Filter of the gel probed with Har-L plasmid. 31 Figure 1 -2 Linearity o f the Har-7.0 plasmid in strain 3983M-7.0. 33 Figure 1-3 Southern hybridization of the different parts of Har-7.0 to the three Zhis i plasmids. 35 Figure 1 - 4 Comparison of restriction maps of the plasmids Har-7.0 and maranhar. 38 Figure 1-5 The restriction map of Har-L plasmid. 40 Figure 1-7 The P C R fragment amplified from D N A in mitochondria of strain 3983M-7.0. 41 Chapter two Figure 2 - 1 The nucleotide sequence of the top strand (5' to 3') of Har-7.0. 60 Figure 2 - 2 The features of the 7050-bp Har-7.0 plasmid. 47 Figure 2 - 3 The alignment of the sequences of Har-7.0 and maranhar, Har-7.0 and Har-L. 66 Figure 2 - 4 The sequence alignment of ORF1 of Har-7.0 and maranhar. 74 Figure 2 - 5 The sequence of alignment of O R F 2 of Har-7.0 and maranhar. 76 Figure 2 - 6 The sequence alignment of TIRs of Har-7.0 and maranhar. 78 Figure 2 - 7 The sequence alignment of the intergenic regions of Har-7.0 and maranhar. 79 Figure 2 - 8 The comparison of the conserved motifs in plasmid-encoded R N A polymerases of maranhar and Har-7.0. 81 1 Introduction Neurospora life cycle The genus Neurospora belongs to the class Ascomycetes of the kingdom Fungi. The ascomycetes are the largest class of fungi and provide most of the species that have been widely used in genetics. Neurospora shows a mycelial form of cellular organization in which hyphae have partial septa that delineate hyphal compartments. Within each compartment there are several nuclei. The septa have central pores through which the nuclei and cytoplasm can pass thus uniting the various hyphal compartments into a continuous protoplasmic system. This form of cellular organization is referred to as coenocytic. The life cycles of N . crassa. N . sitophila and N . intermedia involve both sexual and asexual propagation (reviewed by Beadle 1945). These species are all heterothallic and consequently require fusion of opposite mating types in order to complete the life cycle. Mating type is. determined by a mating type locus which is located on Linkage Group 1 (Perkins et al. 1982). The two mating types, designated A and a, are determined by codominant alleles. On suitable crossing medium, either mating type is capable of producing female sexual structures, protoperithecia. Protoperithecia consist of coiled filaments of specialized hyphae which become surrounded by a thick layer of hyphae. The coiled filaments are destined to become ascogenous hyphae. From each filament a sexual hypha, the trichogyne, is produced, which grows towards and fuses with a fertilizing cell o f the opposite mating type. The male cells may be either vegetative hyphae, or asexual spores called macroconidia or the less abundant microconidia. It has been shown that trichogyne growth and localization of the male fertilizing cell is a chemotactic response 2 initiated by the presence of a pheromone released by the male fertilizing cell, whose growth is inhibited by female pheromone (Bistis 1983, 1986). After fusion, the nucleus from the male cell is transferred through the trichogyne into the ascogenous hyphae. The paternal and maternal nuclei undergo a number of synchronous mitotic divisions to form a small mass of dikaryotic ascogenous hyphae. A t the same time the protoperithecium enlarges and becomes blackened with melanin and eventually forms the mature fruiting body, the perithecium. Karyogamy eventually occurs in the penultimate hyphal compartments of the ascogenous hyphae. Immediately after karyogamy, meiosis occurs and the four products of meiosis undergo a round of mitosis to give a total of eight nuclei which form the ascospores. A t maturity the asci elongate and eject their spores through the ostiole of the perithecium. The ascospores germinate under high temperatures, 60°C, and form mycelia. On vegetative medium, aerial mycelium is formed and conidia are produced through mitosis. The conidia can become airborne and give rise to new colonies that continue the life cycle. DNA plasmids in eukaryotes Plasmids are small extragenomic D N A molecules that can reproduce inside l iving cells. They replicate separately from the genome, but some can integrate covalently into the genome and replicate as part of genomic D N A . Plasmids were originally discovered in bacteria, but later analogous molecules were found in eukaryotes. The first plasmid detected in a eukaryote was found in baker's yeast. Saccharomyces cerevisiae (Sinclair et al. 1967). This plasmid, named '2u\ turned out to be circular and located within the nucleus. Subsequently many plasmids have been recorded in fungi, few in plants, and none in animals. Virtually all the plasmids discovered so far in filamentous fungi are mitochondrial. There are basic two types: circular and linear plasmids, reviewed by Nargang (1985), Esser et al. (1986), Meinhardt et al. (1990), Fecikova (1992), Kempken (1995) and Griffiths (1995). 3 A l l circular plasmids with different sizes except Mauriceville and Varkud probably replicate autonomously by a rolling circular mechanism used in the replication of circular elements in prokaryotes, and some of them have concatameric structures of the basic unit (Maleszka 1992). Linear plasmids are found in different sizes ranging from 7 to 9 kb. They possess terminal inverted repeats with terminal 5' bound proteins, with one exception in which the plasmid has a terminal hairpin loop (Miyashita et al. 1990). They exist as monomers: no multimer of their basic unit has been proven, even through such structures have been suggested. These plasmids replicate autonomously, presumably using their 5' terminal proteins as primers, as do adenovirus and bacteriophage <f>29 (Salas 1988, Sakaguchi 1990). So far, no particular function or phenotype can be associated with most of these plasmids. Almost all o f these plasmids have been found in mitochondria (Meinhardt et al. 1990), some in chloroplasts (Turmel et al. 1986) and some in cytosols (Stam et al. 1986). Some locations are still unknown, but no linear plasmid has been proven to be within the nucleus. Plasmids residing in the nucleus are transmitted to sexual progeny from either male or female parents. Plasmids in the cytoplasm are passed to sexual progeny when plasmid-containing strains behave as females. However leakage of paternal cytoplasm has been detected (May and Taylor 1989, Erickson et al. 1989, Yang and Griffiths 1993b). Generally, fungal plasmids are stable through asexual and sexual reproduction. Furthermore, plasmids are transmissible by heterokaryosis and by incompatible cell contact. (Griffiths et al. 1990, Collins and Saville 1990). It has been demonstrated that some laboratory strains of Neurospora carry suppressor alleles that eliminate the kalilo plasmid (Griffiths et al. 1992). In natural isolates of Neurospora there is considerable variation in suppressive ability (Yang and Griffiths 1993c). 4 Some plasmids are widely dispersed both within and between species (Yang and Griffiths 1993a, Arganoza et al. 1994). Also plasmid distribution is apparently random (Arganoza et al. 1994) suggesting that plasmids are freely mobile in natural fungal populations. There is only one case of intergeneric distribution of plasmids in fungi, and that is the kalilo plasmid which is found in Neurospora and Gelasinospora (Wei et al. 1996). Ki l l e r plasmids of Kluyveromyces lactis A wi ld type strain of the yeast Kluyveromyces lactis was found to harbor two linear plasmids: K l (8.9 kb) and K 2 (13.4 kb) (Gunge et al 1981, Wesoloski et al. 1982 a, b). Cells lacking these elements are killed by the plasmid-containing strains. The plasmids are thus the genetic basis of the killer phenotype. It has been found by mutagenesis that K l encodes the killer toxin, while K 2 seems to be responsible for maintenance of the plasmids. Loss of K l resulted in toxin-sensitive non-killers and K l has never been seen without K 2 (Niwa et al. 1981, Wesoloski et al. 1982c). These plasmids were found to reside in the cytoplasm outside mitochondria (Stam et al. 1986). Transformation of these plasmids into Saccharomyces cerevisiae confers the killer phenotype (Gunge et al. 1982, Gunge and Sakaguchi 1981). Plasmids are stably inherited through vegetative propagation only in rho° strains (containing no mitochondrial D N A ) of S. cerevisiae. suggesting genetic incompatibility between the plasmids and the m t D N A (Gunge and Yamane 1984). Transfer of the plasmids and expression of the killer phenotype has also been possible in S. kluyveri and Candida pseudotropicalis (Sugisaki et al. 1985). The K l and K 2 plasmids share no homology at the D N A level, even in the terminal repeats which are 202 bp and 184 bp respectively (Sor et al. 1983). Each plasmid has 5' bound terminal proteins, which are different between the two plasmids (Kikuchi et al. 1984). Sequence and transcription analysis revealed that the K l plasmid encodes four ORFs , whereas ten ORFs have been 5 identified for K 2 (Hishinuma et al. 1984, Stark et al. 1984, Sor and Fukuhara 1985, Tommasino et al. 1988). Two of the ORFs of K l encode for the toxin (Stark and Boyel .1986), one encodes a resistance factor (Tokunaga et al. 1987), and one most probably encodes a D N A polymerase (Jung et al. 1987). One of the ten K 2 ORFs probably encodes a D N A polymerase (Tommasino et al. 1988), and a second O R F encodes an R N A polymerase (Wilson and Meacock 1988). It has been shown that the K l and K 2 plasmids replicate like adenovirus (Fujimura al. 1988). Plasmids SI and S2 of Zea mays Two linear mitochondrial plasmids SI and S2 have been associated with cytoplasmic male sterile (cms) S lines of Zea mays (Pring et al. 1977). Each SI and S2 plasmid has 5' bound terminal proteins and terminal inverted repeats (Kemble and Thompson 1982, K i m et al. 1982). They share identical TIRs, and a common sequence of 1462 bp at one terminus (Paillard et al. 1985). Besides plasmids free in the mitochondria, there are integrated copies in the m t D N A of the cms-S line (Lonsdale et al. 1981, Schardl et al. 1984). Schardl et al. (1984) demonstrated that the plasmids can recombine with homologous regions of m t D N A , leading to linearization of the m t D N A genome. Spontaneous reversion to fertility is normally accompanied by the recirculization of the linearized m t D N A and loss of the plasmids, but small deletions can be observed in the integrated copies of the plasmids (Schardl et al. 1985). However, loss of the plasmids during sterility reversion is under nuclear control; it is not an S-cytoplasm trait (Escote et al. 1985). In some cases, free plasmids are present in cytoplasmic revertants (Escote-Carlson et al. 1988). Consequently these plasmids are not the causative agent of cms, which is further evident from the fact that similar plasmids (R plasmids) exist in fertile lines (Weissinger et al. 1982, Levings et al. 1984). R l and R2 share sequence homology at the D N A level with the S plasmids, which themselves are considered to have come into existence by recombination between R l and R2. 6 Sequence analysis of the S1 plasmid revealed an O R F homologous to a D N A polymerase gene, whereas S2 shares homology with an R N A polymerase gene (Kuzmin and Levchenko 1987, Sederoff et al. 1986, Kuzmin et al 1988, Oeser 1988). Plasmids in filamentous fungi Plasmids are a common feature of filamentous fungi, where they show considerable diversity (Esser et al. 1986, Meinhardt et al. 1990, Yang and Griffiths 1993a, Arganoza et al. 1994). The origin and evolution of eukaryotic plasmids generally has been the subject of much research effort. Many authors (Natvig et al. 1984; Taylor et al. 1985; Rohe et al. 1991; Kempken et al. 1992; Wang and Lambowitz 1993, for example) have considered the origins and phylogenetic relationships of fungal plasmids. Others have considered structural rearrangements of plasmids and their interaction with mitochondrial D N A (Bertrand et al. 1985, Akins et al. 1988, Myers et al. 1989, Court et al. 1991, Nargang et al. 1992, Vierula and Bertrand 1992, Oeser et al. 1993, Yang and Griffiths 1993a, Hermanns and Osiewacz 1994, Hermanns et al. 1995). The linear k a l D N A and m a r D N A cause death of the host (Bertrand et al. 1985, Myers et al. 1989, Court et al. 1991). Some circular plasmids interact with the mitochondrial genome at the transcript level, and reverse transcription results in molecular hybrids (Akins et al. 1988). Some plasmids share a homologous region with m t D N A . For example, the Labelle circular plasmid bears a 1.6 kb region that is also found in m t D N A (Nargang et al. 1992). Most Neurospora strains do not carry the LaBel le plasmid (Nargang et al. 1992, Arganoza et al. 1994), but all m tDNAs of all Neurospora species carry the homologous region. The direction of transfer of this D N A historically, whether from m t D N A to plasmid or from plasmid to m t D N A , is still uncertain. In other filamentous fungi virtually all the natural plasmids discovered have been linear (these are listed in Kempken 1995). However, these show many of the transactions described above for the linear plasmids of Neurospora. For example recombination with m t D N A has been found in Agaricus species 7 (Robison et al. 1991), Claviceps purpurea (Tudzynski and Esser 1986, Oeser et al. 1993) and Podospora anserina (Hermanns and Osiewacz 1992, Hermanns et al. 1995). Therefore previous studies on plasmids in filamentous fungi have shown that they can not only spontaneously generate variability in their own genomes, which is undoubtedly important for their own evolutionary flexibility, but that they can recombine with the mitochondrial genome, and that these types of recombinations seem to have been important in the evolutionary histories of several well-studied fungi. The plasmids of Neurospora are among the best studied in fungi. Worldwide sampling of natural isolates has been performed, and these isolates constitute the basis for plasmid studies in the genus. Neurospora plasmids may be linear or circular, and they fall into at least six homology groups (Yang and Griffiths 1993a, Arganoza et al. 1994). Most of these homology groups are distributed across more than one species of Neurospora (Natvig et al. 1984, Taylor et al. 1985, Yang and Griffiths 1993a, Arganoza et al. 1994, Marcinko-Kuehn et al. 1994) and even across genera (Wei et al. 1996). Within a homology group there is both structural variation and nucleotide substitution. Homologous circular plasmids can be found in different species and distant geographical locations (Natvig et al. 1984; Nargang 1985; Nargang et al. 1993, LaBel le , and Griffiths and Yang 1995, Harbin-1.). One example of homologous linear plasmids is the kalilo plasmids of N . intermedia (Chan et al. 1991), N . tetrasperma (Marcinko-Kuehn et al. 1994), and Gelasinospora species (Wei et al. 1996). Change in plasmid sequence have been observed in culture. Derivatives of the linear kalilo plasmid (ka lDNA) of Neurospora have been observed to form spontaneously during routine culturing. Similar derivatives have been observed for the maranhar plasmid and the m a r D N A -homologous plasmids (Yang 1994). Giant derivatives, similar-sized 'sibling' plasmids, nested short forms (Yang and Griffiths 1993a), and hairpins and deletions bearing only the terminal inverted repeats (Vierula and Bertrand 1992) have all been detected as spontaneous variants arising by unknown molecular mechanisms. This type of generation of new plasmids is from 8 parental linear types to new linear types. Also, the derivation of new circular plasmids from parental circular types have been observed. A small circular plasmid has been proved to stem from 0.9 kb segment of Harbin-1 in a vegetative culture of N . intermedia strain 3983 (Griffiths and Yang 1995). Modified plasmids of the circular plasmids Varkud and Mauriceville have been observed repeatedly in other studies (Akins et al. 1986, 1989). Surprisingly, two new linear plasmids Har-L and Har-L ' in strain 3983M of N . intermedia have been derived from combination of the circular plasmid Harbin-1 and a marDNA-homologous linear plasmid in strain 3983 of N . intermedia. Therefore, mitochondrial plasmids evolve constantly over time. Only the linear kalilo and maranhar plasmids have a readily observable effect on their hosts. The first fungal phenotype shown to be produced by an extragenomic plasmid is senescence in Neurospora (reviewed by Griffiths 1992). The two linear plasmids kalilo and maranhar aggressively insert into mitochondrial D N A , associating with abnormal mitochondrial physiology and ultimately to death of the culture. Also the circular Mauriceville plasmid of Neurospora occasionally recombines with mitochondrial D N A in a variety of ways leading to senescence (Akins et al. 1986). None of the natural plasmids examined to date are derived from the Neurospora mitochondrial genome. Most commonly the natural plasmids encountered in fungi are of the linear type. The natural circular type seems to have been found almost exclusively in Neurospora (Griffiths et al. 1995). The circular plasmids that have been characterized range in size from 0.9 to 5.3 kb. They all exist as a series of one or more monomer units joined in a head-to-tail fashion. Based on hybridization studies, the circular plasmids were placed into one of the homology groups, Mauriceville, LaBelle, F i j i , Java, M B 1 , V S and Harbin-2 (Natvig et al. 1984, Nargang 1985, Saville and Collins 1990, Griffiths and Yang 1995). 9 The complete D N A sequences of five circular (Mauriceville, Varkud, LaBelle , F i j i and V S ) (Akins et al. 1988, Pande et al. 1989, L i and Nargang 19,93, Saville and Collins 1990) and two linear, kalilo and maranhar, (Chan et al. 1991, Court and Bertrand 1992) mitochondrial plasmids are known. The overall structures of linear plasmids kalilo and maranhar are typical of most other linear plasmids discovered to date. The general type of structure has been termed an 'invertron' (Sakaguchi 1990). The characteristics are as follows. First, there is a terminal inverted repeat, whose size is characteristic of the individual plasmid. Second, at the terminus at each end there is a protein bound to the 5' nucleotide. Third, starting within the terminal repeats there are two large non-overlapping open reading frames running on opposite strands towards the middle of the plasmid. These reading frames are open only i f mitochondrial codon usage is assumed. Smaller ORFs are present in other frames, but these are probably insignificant. The presumptive amino acid sequences of the ORFs suggest in one case a viral-type D N A polymerase, and in the other case an R N A polymerase similar to those of bacteriophages and yeast mitochondria (Chan et al. 1991, Court and Bertrand 1992). These ORFs are both transcribed (Vickery and Griffiths 1993, Court and Bertrand 1993). Fourth, there is an intergenic region between the ORFs. N o function has been proposed for this region, although it presumably contains transcription termination signals. In all cases except V S (Saville and Collins 1990), sequencing studies have revealed long ORFs that occupy a large portion of the coding capacity of the plasmids. These ORFs encode polymerases that are presumably involved in replication or transcription of the plasmids. The highly similar Mauriceville and Varkud plasmids both give rise to abundant unit length transcripts that carry the information for expression of the long O R F . These plasmids encode reverse transcriptases that function in the replication of the plasmids (Nargang et al. 1984, Akins et al. 1988, Kuiper and Lambowitz 1988, Wang and Lambowitz 1992 & 1993, Kennell and Lambowitz 1994, Lambowitz et al. 1995). The Mauriceville reverse transcriptase is unusual in that it is capable of initiating c D N A synthesis directly opposite the 3'-terminal nucleotide of the template R N A . It has been suggested that the enzyme may be a primitive 10 reverse transcriptase related to those that first evolved from an RNA-dependent R N A polymerase (Wang et al. 1992). Curiously, a variable ratio of Varkud transcripts contain a 1.2 kb leader sequence that is derived from the 5' end of the mitochondrial small r R N A . A number of possible mechanisms for the synthesis of this unusual transcript have been discussed (Akins et al. 1989). The LaBel le and F i j i plasmids also give rise to unit length transcripts but these are not abundantly expressed. The LaBelle plasmid O R F was originally thought to be related to reverse transcriptases, but it has now been shown that the plasmid encodes a DNA-dependent D N A polymerase with motifs characteristic of the B family of D N A polymerases (Pande et al. 1989, Schulte and Lambowitz 1991, L i and Nargang 1993). The Fi j i plasmid encodes a D N A polymerase with almost 50% amino acid identity to that of LaBelle. The polymerases from these two plasmids are unusual in that they contain the amino acid motif 'thr-thr-asp' in place of 'asp-thr-asp', which is thought to be important for the activity of the B family polymerases ( L i and Nargang 1993). The V S plasmid does not encode a polymerase and is apparently dependent on the Mauriceville / Varkud enzyme for its replication (Saville and Coll ins 1990). V S R N A possesses a self-splicing activity that may be used to generate monomeric V S R N A s from multimeric R N A s transcribed from multimeric versions of V S D N A . The genetic organization and nucleotide sequence of Neurospora circular plasmids has led to the suggestion that they are related to mitochondrial introns and mobile genetic elements (Nargang et al. 1984, Lambowitz et al. 1985, Lambowitz 1989). M u c h of the observed behavior of the different plasmids supports this view. The Mauriceville plasmid was first described by Collins et al. (1981), who established the monomer length and demonstrated the presence of circular concatamers of up to six repeats. They also showed that the plasmid produced an approximately -full-length transcript. Nargang et al. (1984) sequenced the 11 plasmid and found structural features reminiscent of introns. First, the O R F showed a codon usage similar to that of fungal mt D N A introns. Second, there were D N A sequences characteristic of the conserved elements E , P, Q, R, E ' , and S, which interact to promote splicing in group I mitochondrial D N A introns. They were all in correct position and alignment. This suggested that Mauriceville is an intron progenitor, that it is an excised intron that can replicate independently,, or that is has recombined with part of an intron. Miche l and Lang (1985) also showed that the Mauriceville O R F contained seven conserved blocks of amino acids that are shared with four different group II introns. These observations, together with the existence of a full-length transcript, gave rise to the idea that the plasmid might be a mobile intron, capable of insertion by reverse transcription, a property also shared by retrotransposons (Lambowitz 1989). To test the idea A k i n et al (1986, 1989) did a series of experiments. Their findings suggest insertion through an R N A intermediate. It was also noted that the splice junctions were not those expected for either group I or group II introns, so the plasmid inserts were not acting as introns. The distribution of plasmids may be explained in part by the ability of plasmids to transfer between strains during unstable vegetative fusions (Collins and Saville 1990, Griffiths et al. 1990). Linear maranhar D N A was originally found in the several field-collected strains of N . crassa from India which are prone to precocious senescence and death (Court et al. 1991). In these isolates, senescence is induced by the integration of the 7.0 kb linear mitochondrial plasmids (marDNA) into the mitochondrial genomes (Court et al. 1991). These isolates were named as the Maranhar strains of N . crassa. The maranhar is a Sanskrit word meaning moribund. The m a r D N A is not derived from the m t D N A nor the nuclear genome of Neurospora. The nucleotide sequence of m a r D N A was determined by Court and Bertrand (1992). The plasmid is 7052-base pairs in length and has perfect terminal inverted repeats of 349 bp. Each D N A strand contains a long open reading frame using the Neurospora mitochondrial genetic code (Heckman et al. 1980). It begins within the TIR and extends toward the center of the plasmid. 12 ORF-1 codes for a single-subunit R N A polymerase. The ORF-2 product may be a B-type D N A polymerase. A separate coding sequence for the terminal protein could not be identified; however, the D N A polymerase of maranhar has an amino-terminal extension which probably comprises at least the part of the terminal protein of the maranhar plasmid. The maranhar plasmid can integrate into m t D N A by a mechanism that generates the full-length plasmid insertion sequences which invariably are flanked by very long inverted repeats of m t D N A (Court et al. 1991). The study of fungal plasmids can uncover a wealth of diverse molecular processes that are relevant not only to the plasmids themselves but also to the properties of D N A in general. Plasmids represent easily studied examples of a mysterious class of D N A known as parasitic or selfish D N A , a type of D N A that seems to exist only for the purpose of existing. This class of D N A includes plasmids, introns, transposons, and viruses. Analysis of plasmids has shown possible areas of connection between these different types, leading to new insights into their evolution. Most DNA-even genomic DNA-cou ld be considered selfish, so the study of the plasmids can provide clues about primitive genomes. 13 Objectives of these studies Originally, the studies were focused on the origin of a newly-detected iinear mitochondrial plasmid Har-7.0 in a deviant strain 3983M-7.0 of N . intermedia. Such studies might provide some knowledge about the diversification and evolution of fungal plasmids. The whole sequence analysis of this new plasmid may provide not only functional analysis for itself but also functional comparison with related plasmids. Incentives for this studies were three-fold. First, the general structure of this plasmid was of interest. Is it a linear plasmid? Does it have 5' terminal proteins? Is it a mitochondrial plasmid? Second, the origin of this plasmid was of interest. Was it derived from the Har-L in strain 3983M or other plasmids? Is it related to any other plasmid in Neurospora? Third, the structural features of this plasmid were of interest, since the restriction map of this plasmid is similar to that of maranhar in Maranhar strain of N . crassa. Maranhar is a senescence-causing plasmid: what is the sequence difference between these two plasmids and is it related to its function ? 14 Materials and methods Strains Escherichia coli strain D H 5 a was bought from Bethesda Research Laboratories, B R L . The natural isolate Harbin strain 3983 of N . intermedia was obtained from the Fungal Genetics Stock Center ( F G S C stock number 3983), Department of Microbiology, University of Kansas Medical School, Kansas City, Kansas. It was collected from Harbin, China. Its mating type is a. Media and growth conditions A single colony of Escherichia coli D H 5 a strain was inoculated in 3-5 m L L B broth and shaken at 50 rpm for 16 hours. Cultures were harvested by spinning for 20 seconds in 1ml eppendorf tubes. Vegetative culturing of Neurospora was performed exclusively on Vogel's minimal medium containing 2% glucose (Vogel, 1956) at room temperature. Serial subcultures were made in 10 x 75 mm tubes. Subculturing was performed as described by Griffiths and Bertrand (1984). For the growth of mycelium for nucleic acid isolation, conidia were prepared by suspending in 8-10 ml of sterile distilled water, then this suspension was added to liquid Vogel's medium 15 (approximately 10°" conidia/ml). The liquid Vogel's medium was inoculated and shaken at 200 rpm for 16-40 hours. Cultures were harvested by suction filtration through Whatman #1 filters in Buchner funnels. D N A Isolation Plasmid D N A in Escherichia coli strain D H 5 a was isolated according to the method modified from Greene (1988) and Bimboim (1983). D N A from mitochondria was isolated according to the small scale m t D N A method of Myers (1988), with addition of a proteinase K digestion prior to phenol/chloroform precipitation of protein. A l l procedures were carried out at 0-4°C, unless otherwise noted. One hundred and fifty microlitres of liquid culture was harvested and stored on ice until needed. The mycelial pellet was ground with a half volume of acid-washed sand ( B H L ) and suspended in 30 ml D N A isolation buffer (44 m M sucrose, 50 m M T r i s - H C l , p H 7.6, 1 m M E D T A ) . The suspension was centrifuged at 2 krpm for 5 minutes in an SS-34 rotor. The supernatant was transferred to a tube then centrifuged for 15 minutes at 15 krpm in an SS-34 rotor to pellet the mitochondria. The mitochondrial pellet was suspended in 3.5 ml 70% sucrose in T 1 0 E ] (10 m M Tr i s -HCl , p H 7.6, 1 m M E D T A ) , and layered with 1 ml 44% sucrose in TJQEI . The flotation gradients were centrifuged at 45 krpm for 1 hour in an SW50.1 rotor. Mitochondria were collected from the interface beneath the 44% sucrose step gradient layer, and diluted to 3 ml with 2 ml T 2 0 o E i (200 m M T r i s - H C l , p H 7.6, 1 m M E D T A ) in microfuge tubes. The tubes were centrifuged at 10 krpm for 10 minutes to pellet the mitochondria. The mitochondrial pellets were pooled and resuspended in 350 ul of a solution of T^QOE], and 40 ul of 20% SDS was added to lyse the mitochondria, followed by the addition of 20 ul of a 10 mg/ml solution of proteinase K ( B R L ) . D N A solution was incubated at 37°C overnight. Protein was extracted by the addition of a half volume of Tris-H C l saturated phenol, and a half volume of chloroform/isoamyl alcohol (24:1). Tubes were mixed by inversion and centrifuged for 15 minutes at 10 krpm. The aqueous phase was 16 collected and this step was repeated once. Nucleic acids were precipitated from the aqueous phase by the addition of 2.5 volumes of ethanol containing 200 m M ammonium acetate, and incubated overnight at -20°C. The nucleic acids were pelleted and washed with 70% ethanol. The m t D N A was resuspended in 100-150 ul T 1 0 E , (pH 8.0), with the addition of 5 ul of 20 mg/ml RNase A ( S I G M A ) . R N A was digested at 55°C for an hour. D N A concentration was determined by U V absorption at 260 nm wavelength. Typically, the yield was 10-20 ug of m t D N A per 150 ml of liquid culture. Enzyme digestion and gel electrophoresis Restriction enzyme ( B R L ) digestion of D N A was as described by the manufacturer. Restriction digestion of 2 ug D N A was carried out for an hour at 37°C. Digestion of 2 ug D N A with Lambda exonuclease ( B R L ) was performed by incubating for an hour at 37°C in a solution of 6 7 m M glycine K O H , p H 9.4, 2.5 m M MgCf_, 50 ug/ml B S A and 10 units of Lambda exonuclease. Digestion of 2 ug D N A with exonuclease III ( B R L ) was for an hour at 37°C in a solution of 50 m M Tr i s -HCl , p H 7.5, 5 m M M g C l 2 , 5 m M D T T , 50 ug/ml B S A and 50 units of exonuclease III. 3.5 ul of 6x loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 40% sucrose in H 2 0 ) was added to the digestion reaction. Samples were loaded into wells of 0.8% agarose gels and separated by size at 40-60 volts for 16 hours. The buffer for gel electrophoresis was l x T A E (40 m M Tris acetate, p H 7.6, 2 m M E D T A ) . Gels were stained with ethidium bromide (0.5 ug/ml) for 20-30 minutes and rinsed in water for 5-10 minutes, then photographed (using Polaroid 57 film) under short wave U V illumination. Cloning of the mitochondrial plasmid Vector pUC18 ( B R L ) was used for cloning according to the supplier's instructions. EcoR I, Hind III, X b a I and Pst I were used for digestion of the vector and Harbin strain 3983M-7.0 17 mtDNA including Har-7.0 plasmid separately. A restriction endonuclease digestion of the vector was ligated to the same restriction endonuclease digestion of mtDNA. The recombinant bacterial plasmids were transformed into Escherichia coli DH5a competent cells (BRL) according to the supplier's instruction. Labeling nucleic acid Oligolabelling was carried out according to the protocol from Pharmacia. At total of 25-50 ng of D N A to be labelled was used in 36 ul of dH20..The D N A solution was denatured by heating for 2-3 minutes in a water bath at 95-100°C, then placing on ice for 2 minutes. 10 ul of reagent mix (dNTP) and 1 ul (10 units) of Klenow fragment was added. 3 ul (30 uCi) of 3 2 P - d C T P (3000 Ci/mmol) was added and mixed. The labelling reaction was carried out at room temperature for 1-16 hours. Usually 1/3 to 1/2 of the 32p w a s incorporated into the labelled D N A . Probes The pUC18 clones of Har-7.0 1.2 kb and 4.5 kb Hind III (BRL) fragments, 2.3 kb and 3.0 kb EcoR I (BRL) fragments and 1.9 kb Xba I (BRL) (see Har-7.0 map in Fig. 1-4) were extracted from Escherichia coli DH5a and linearized by Kpn I (BRL) for use as a probe. Southern blot analysis Southern blot analysis was performed essentially as described by Southern (1975). D N A separated by gel electrophoresis was denatured for 30-45 minutes in an alkaline solution of 0.5 N NaOH, 1.5 M NaCl, and then neutralized for 45 minutes in 1 M Tris-HCl, 3.0 M NaCl. D N A was transferred to Hybond filter (BRL) with 2x SSC (lx = 0.15 M NaCl, 10 m M 18 sodium citrate, p H 7.0) for 24 hours. After transfer, the filters were baked at 80-90°C for 2-3 hours. D N A fragments were detected by hybridization to 32-p_ i a r j e l l ed probes. Filters were pre-hybridized for 2-16 hours at 42°C with a hybridization solution of 40% deionized formamide, 1% SDS, l x Denhardt's solution (lOOx = 2% B S A , 2% P V P , 2% ficoll), 1 M N a C l , and 0.4 mg/ml denatured herring sperm D N A (Bertrand 1985). Hybridizations were carried out in the same solution with the addition of labelled probe to 10^ cpm/ml. Hybridization was for 16-24 hours at 42°C. After hybridization, filters were washed in 2x SSC at room temperature for 5 minutes, twice in 2x SSC, 0.5% SDS at 65°C for an hour. After air drying, blots were wrapped in Saran Wrap and exposed to Kodak X-Omat R P fi lm for the appropriate time. Dot-blot hybridization A single colony of Escherichia coli D H 5 a strain was inoculated in 1 ml L B broth and was incubated for 16 hours. The culture was harvested and suspended in a solution of 0.2N N a O H and 1% SDS. The suspension was transferred to hybond filter ( B R L ) through a dot-blot apparatus (BioRad). The filter was saturated with an alkaline solution of 0.5 N N a O H and 1.5 M N a C l for 5 minutes at room temperature to denature D N A , and then neutralized for 5 minutes in a solution of 1 M Tr i s -HCl p H 8.0 and 3.0 N a C l . After air drying, the filter was baked at 80-90°C for two hours. The subsequent procedure was the same as for the Southern blot hybridization. P C R reaction P C R amplification was carried out using the PerKin-Elmer Cetus D N A Thermal Cycler and P C R Reagent K i t ( B R L ) . D N A from mitochondria of strain 3983M was used as template for 19 PCR reaction. Within 0.5 ml PCR tube the appropriate amount of template DNA, 10 ul 10X reaction buffer, 5 ul primer 1:5' A A G A A T T A A G C G G G G A A A G 3' complementing primer binding sites, 3' TTCTTAATTCGCCCCTTTC 5', at the 3' ends of the target sequence on the 3' to 5' strand (20 pmol/ul), 5 ul primer 2: 5' TAAAGCCGAGTCAGGATGA 3' complementing primer binding sites, 3' ATTTCGGCTCAGTCCTACT 5' at the 3' ends of the target sequence on the 5' to 3' strand (20 pmol/ul), 10 ul 2 m M mix, 0.5 ul Tag D N A polymerase (5 U/ul) and appropriate amount of d d H 2 0 were added to make total volume of 100 ul. The above mix in the 0.5 ml PCR tube was incubated in the D N A Thermal Cycler using the fol lowing parameters: Thermocycle file: Segment 1: 94°C 30 sec. Segment 2: 47.5°C 30 sec. Segment 3: 72°C 30 sec. Total 30 cycles. Soak fi le: 4°C. After the PCR amplification the reaction was run in agarose gel (1.5%) for 1.5 hours at 80 V to see i f there was any expected PCR fragment present under U V light in agarose gel. D N A sequencing D N A sequence was determined using an automated sequencing system (ABI 3 73A D N A Sequencer). The Taq DyeDeoxy™ terminator Cycle Sequencing Ki t (Applied Biosystems) was used for sequencing reactions. Universal primers were used for sequencing the recombinant plasmids containing inserted fragments: the 1.2 kb and 4.5 kb Hind I I I fragments, 2.3 kb and 3.0 kb EcoR I fragments and 1.9 kb Xba I fragment of Har-7.0 plasmid. The new primers were designed as primers for the remainder of the cloned 20 fragments. When the sequence of all cloned fragments was determined, the sequences close to the 3' and 5' terminal regions of the whole clone fragments were employed to design primers that could be used to prime polymerization outwards towards the Har-7.0 plasmid ends. Total D N A from mitochondria (mtDNA and plasmid D N A ) was purified and used as a template for sequencing both termini. From the first round of such sequencing, secondary primers were designed from the 3' terminal regions of the sequence. This outward walking process was repeated until no more sequence could be obtained, and this was presumed to be the terminus of the plasmid. A l l segments were sequenced at least twice and conflicts were resolved by further sequencing. Standard D N A fragments were included in every sequencing run and results from these showed that the sequencing procedure was 98-99% accurate. Computer analysis The D N A sequence was analyzed using Assembly L I G N , MacVector and B L A S T N (Altschul et al. 1990) computer programs. 21 Chapter one The origin of the newly-detected 7.0 kb plasmid of deviant N. intermedia Harbin strain 3983M-7.0 Introduction The study is based on the senescent strain 3983 of N . intermedia from Harbin, China. The plasmid content of the senescent Harbin strain 3983 of N . intermedia changed in the process of subculturing. Previous studies on this strain (Yang and Griffiths 1993a, b) showed that it contained a variety of plasmids, listed in Table 1-1. The linear 1.4 kb and 4.0 kb plasmids were not evident in the present study; either they are absent or present at levels that cannot be detected by staining with ethidium bromide. There are five prominent plasmids, three linear and two circular. The three prominent linear plasmids in strain 3983 are plainly visible on a stained gel. These plasmids, of 7, 8, and 9 kb, are referred as " Zhis i" plasmids after a Chinese word for dying. Previous studies showed that all three Zhis i plasmids hybridize to a probe consisting of the senescence plasmid maranhar (marDNA; Yang and Griffiths 1993a). The two circular plasmids in strain 3983 were named Harbin-1 and Harbin-2 (Har-1 and Har-2; Yang and Griffiths 1993a). Generally, circular plasmids are not visible on stained gels and appear as complex ladders in Southern hybridizations. In one subculture, labelled 3983M, the three Zhis i plasmids had apparently disappeared and were replaced by one smaller prominent linear plasmid of 5.5 kb, which was named Har-L, and one less prominent linear type Har -L ' 22 (5.3 kb). In addition, a small new 0.9 kb circular plasmid with a different ladder pattern was sometimes found in strain 3983M. Hybridization studies showed the new circular 0.9 kb plasmid comprised part or parts of Har-L plasmid alone and suggested that the new plasmid Har-L in strain 3983M is formed from a combination of linear Zhisi plasmids.and the circular Har-1 plasmid present in the mitochondria of strain 3983. L o w levels of the Har-1 plasmid are still present in strain 3983M (Griffiths and Yang 1995). The plasmid content of strain 3983M is listed in Table 1-1. Table 1-1: The plasmid contents of 3983 strain and its derivatives. The linear plasmids are listed by size in kb except the Har-7.0. Har-1, Har-2 and Har-0.9 are circular plasmids. Strain Plasmids 3983 9.0, 8.0, 7.0,4.0, 1.4, Har-1, Har-2 . . . ' 3983M 5.5, 5.3 (low copy#), 4.0, 1.4, Har-2, Har-0.9, Har-1 a t l o w l e v e l 3983M-7.0 Har-7.0, 4.0, 1.4, Har-2, Har-0.9, Har-1 a t l o w l e v b l In one out of 20 conidial isolates of 3983M, labelled 3983M-7.0 the 5.5 kb Har-L and 5.3 kb Har -L ' had disappeared and was replaced by one prominent linear plasmid of 7.0 kb, which was named Har-7.0 (Fig. 1-1 A ) . The plasmid content of strain 3983M-7.0 is listed in Table 1-1. The origin of this new 7.0 kb linear plasmid was of interest. The derivation of this plasmid might be relevant to the mechanism of plasmid diversification and evolution in general. This chapter describes the study on the origin and structure of this newly-detected mitochondrial plasmid (Har-7.0) and shows that the Har-7.0 plasmid in this aberrant 3983M-7.0 strain is probably similar to the original 7.0 kb Zhisi plasmid of strain 3983. The copy number of the original 7.0 kb Zhisi plasmid or its derivative fell to undetectable value in the 23 aberrant 3983M. Then the copy number of this plasmid rose to the normal detectable value in the aberrant strain 3983M-7.0. Results Mitochondrial linear plasmids appear as distinct bands in electrophoretic gels. Therefore this new 7.0 kb band was likely to be a linear plasmid. To confirm its linearity and 5'-bound protein, D N A from the mitochondria of strain 3983M-7.0 digested with 5' exonuclease or 3' exonuclease after proteinase K treatment and D N A from the mitochondria of strain 3983M-7.0 extracted without proteinase K treatment or with proteinase K treatment were run in agarose gel (Fig. 1-2). A complete digestion of 7.0 kb D N A was observed with 3' exonuclease and the 7.0 kb D N A was still present after 5' exonuclease digestion. Without the proteinase K treatment of the mitochondrial D N A extract there was no clear 7.0 kb D N A band present in the gel and after proteinase K treatment there was clear 7.0 kb D N A band present in the gel. Results indicated that this 7.0 kb D N A element was sensitive to 3' degradation, but resistant to 5' degradation and present after proteinase K treatment. So this new 7.0 kb D N A element is a linear plasmid and has a 5'-bound protein (Sakaguchi 1990). The Har-7.0 plasmid did not hybridize to nuclear D N A or mitochondrial D N A (data not shown). It hybridized to the Har-L plasmid of strain 3983M very weakly (Fig. 1-1B). But it strongly hybridized to the three 7.0 kb, 8.0 kb and 9.0 kb Zhisi plasmids of strain 3983 (Fig. 1-3). Based on the results of digestion and hybridizations, the restriction map of this plasmid was constructed (Fig. 1-4). Comparing the restriction maps of Har-L (Fig. 1-5) and Har-7.0 , it is obvious that they do not share common restriction sites. 24 But the restriction map of this plasmid is very similar to that of maranhar plasmid in Maranhar strain of N. crassa (Fig. 1-4). Since the restriction maps of three Zhisi plasmids are not known the direct comparison of restriction maps of Har-7.0 and three Zhisi plasmids can not be made. But these three Zhisi plasmids are marDNA-homologous. Therefore, this new plasmid might be related to the three Zhisi plasmids, somehow. A n attempt was made to find out i f there is any relationship o f the Har-7.0 plasmid with the three Zhisi plasmids, which might be possibly present at very low copy number in strain 3983M. To define the regions of three Zhisi plasmids homologous to Har-7.0 plasmid of strain 3983M-7.0, a series of hybridizations was performed. The 1.2 kb and 4.5 kb Hind III fragments, 3.0 kb and 2.3 kb EcoR I fragments and 2.0 kb Pst I fragment of Har-7.0 were used as probes. The Southern hybridizations of the intact D N A from mitochondria of strain 3983 were performed using these probes. The 1.2 kb and 4.5 kb Hind III fragments, 3.0 kb and 2.3 kb EcoR I fragments and 2.0 kb Pst I fragment of Har-7.0 all strongly hybridize to the three Zhisi plasmids of strain 3983 (Fig. 1-3). Based on these hybridizations it is known that the different regions of Har-7.0 plasmid are homologous to all three Zhisi plasmids. Therefore, it is not clear whether Har-7.0 is derived in whole or in part from the three Zhis i plasmids or part (s) of those. But this new Har-7.0 plasmid of strain 3983M-7.0 may be the same plasmid as the 7.0 kb Zhisi plasmid of strain 3983. The idea is proposed that the copy number of the 7.0 kb plasmid in strain 3983M might have reduced to undetectable level and then it rose again to the normal copy numbers in strain 3983M-7.0. It is already known that there is no detectable 7.0 kb marDNA-homologous plasmid in mitochondria from strain 3983M using Southern hybridization (Yang and Griffiths 1993a). To confirm the above idea a P C R reaction has to be performed using D N A from mitochondria of strain 3983M. After the complete sequence of Har-7.0 plasmid was obtained the P C R primer pair only for the Har-7.0 plasmid was designed according to the OLIGO™4.0 (a software system developed for primer selection by Cetus Co.). It was attempted to design the primer pair to amplify the 25. 7.0 kb plasmid probably present in strain 3983M but not any regions of the Har-L plasmid in strain 3983M or any regions of Zhisi plasmids 8.0 kb, 9.0 kb probably present in strain 3983M. The sequences of the 8.0 kb, 9.0 kb plasmids are not known. But these two plasmids contain marDNA-homologous regions (Yang and Griffiths 1993a).The proposed marDNA-homologous regions of the Zhisi 8.0 kb and 9.0 kb plasmids are shown in F ig . 1-6 (Griffiths and Yang 1995). Therefore, this primer pair was designed to amplify the Har-7.0 excluding primer pairs for Har-L plasmid and marDNA using OLIGO™4.0 since the sequences of Har-L and m a r D N A are known. The positive control and negative control for P C R reaction were also carried out. The template D N A for the positive control was D N A from mitochondria of strain 3983M-7.0. Template D N A was not added in the negative control in order to indicate any possible contaminations during the procedure of the P C R reaction. The expected 0.5 kb P C R fragment was found in the P C R reaction using the primer pair (Fig. 1-7). This probably indicated that the 7.0 kb plasmid of strain 3983 still exists in strain 3983M at very low copy number undetectable using Southern hybridization. According to this study it might be suggested that this 7.0 kb plasmid probably recovered to normal copy number in strain 3983M-7.0. This turns out that the Har-7.0 plasmid of strain 3983M is probably the same plasmid as the 7.0 kb Zhisi plasmid of strain 3983 or the mutated Zhisi 7.0 kb plasmid since it is not sure i f there is any mutation in the 7.0 kb plasmid during the process. Discussion In this study a 7.0 kb linear plasmid of the N . intermedia Harbin strain 3983M-7.0 was detected. This plasmid is not derived from the nuclear or the mitochondrial genome. It is not derived from other plasmids as is the Har-L plasmid of strain 3983M (Griffiths and Yang 1995). It might be similar to the 7.0 kb Zhisi plasmid of strain 3983. The copy number of the 7.0 kb Zhis i plasmid or its derivative was most likely reduced to an undetectable level in 26 strain 3983M and then increased its copy number to normal levels in strain 3983M-7.0. The derivative strain 3983M-7.0 was detected from one out of 20 conidial isolates of strain 3983M. When the 7.0 kb plasmid in strain 3983M-7.0 was detected, the Har-L plasmid of strain 3983M had disappeared. But the reason that the Har-L plasmid of strain 3983M was lost in strain 3983M-7.0 is unknown. These two plasmids do not show strong cross hybridization in Southern hybridization. Therefore, they do not share high degree of homology at sequence level. Since the plasmid is small, 5.5 kb, and has limited coding sequence, it almost certainly relies upon the host genome for essential functions concerning replication and expression. The loss of Har-L plasmid may be an example of nuclear function affecting the replication and expression of a plasmid. Mitochondrial plasmid suppressors have been demonstrated for the first time in natural isolates of N . intermedia (Yang and Griffiths 1993c). The suppressors are plasmid specific, located in nuclear genomes, and normally show Mendelian segregation during meiosis. The plasmid suppressor could either eliminate plasmids or reduce copy number to barely detectable levels. Nuclear heterogeneity in natural isolate strain 3983 of N . intermedia has been observed (Yang 1994). Strain 3983, 3983M and its derivative 3983M-7.0 might have different nuclear genotypes regarding their support of mitochondrial plasmids. The nuclei with suppressor mutations, Har-L plasmid suppressor, might be present together with normal nuclei in 3983M heterokaryotic mycelia. Somatic segregation of these two types of nuclei results in different support of mitochondrial plasmids, without the Har-L plasmid or with the Har-L plasmid. These nuclei in mycelia could result from spontaneous mutations during their vegetative propagation. This kind of somatic segregation was also proposed to explain the rejuvenated growth of a senescent Neurospora strain (Yang 1994). Preliminary genetic studies of the new senescent strains suggest that senescence is probably due to abnormal nuclei with lethal mutations present together with normal nuclei in heterokaryotic mycelia (Yang 1994). Somatic segregation of these two types of nuclei results in both normal growth 27 and vegetative death. Successful rescue of the very sick isolates of the senescent Neurospora strain by repeated plating and selecting of conidia suggests nuclear heterogeneity in these senescent strains (Yang 1994). The loss or low copy number of the three Zhisi plasmids in strain 3983M could be due to the superior replication of the new plasmid Har-L over the previous three Zhisi plasmids (Griffiths and Yang 1995). Therefore, the loss of Har-L plasmid in strain 3983M-7.0 could be due to the Har-L plasmid suppressor in strain 3983M-7.0. Meanwhile, an assumption must be made that the Har-L plasmid suppressor could also suppress the replication, transcription or translation of the Zhisi 8.0 kb and 9.0 kb plasmids but not 7.0 kb plasmid. It turns out that the copy number of the Zhis i 7.0 kb plasmid or its derivative in strain 3983M-7.0 rose to normal copy number without Har-L present. Another possibility is that the Har-L suppressor might only suppress the expression of the Har-L plasmid but not the expression of the 8.0 kb and 9.0 kb plasmids, but the expression of Zhisi 8.0 kb and 9.0 kb plasmids in strain 3983 of N . intermedia might depend on a function (e.g. plasmid-encoded D N A polymerase) of the 7.0 kb Zhis i plasmid. The Har-7.0, somehow, might be mutated in strain 3983M-7.0 relative to the 7.0 kb Zhis i plasmid in strain 3983 and can not support the expression of the 8.0 kb and 9.0 kb Zhisi plasmids any more. The dependence of one plasmid on another has been seen in the V S plasmid. The V S plasmid does not encode a polymerase and is apparently dependent on the Mauriceville/Varkud enzyme for its replication (Saville and Collins 1990). The possibility can't be ruled out that the reappearance of 7.0 kb plasmid in strain 3983M-7.0 could be due to the cytoplasmic segregation (Yang 1991). The mitochondria in 3983M strain might be heterogeneous, some containing the Har-L, some containing the 7.0 kb plasmid at very low copy number. When conidia are formed with the mitochondria containing the 7.0 kb plasmid from their parent cytoplasm mostly containing Har-L, the derivative strain with mitochondria containing the 7.0 kb plasmid without Har-L plasmid forms. 28 In routine propagation of plasmid-containing strains generally all subcultures show the plasmid in more or less constant amounts. (Senescence plasmids are an exception to this rule.) In the few cases where asexual ceils have been examined by isolating single conidial isolates the majority of these isolates shows the plasmids, but occasionally a plasmid-free isolate w i l l be found that might represent some kind of suppressor mutation or cytoplasmic segregation (Yang 1991). The plasmid loss could occur during either sexual reproduction leading to ascospore formation, or asexual reproduction leading to conidiation. For example, in culturing Ka l i lo strains of Neurospora occasional nonsenescent conidia are produced (Griffiths and Bertrand 1984). Also occasional ascospores lack plasmids, but tests on these have shown no evidence of suppressors (Yang and Griffiths unpublished results), so some type of cytoplasmic segregation is inferred. N o studies on regulation of plasmid copy number have been attempted. But the variation of the three Zhisi plasmid copy numbers between different isolates of N . intermedia Harbin strain 3983 has been observed (Yang 1994). The copy number of the 7.0 kb marDNA-homologous plasmid in ascospores from the cross N . intermedia 1940 a x 1766 A appeared to be reduced relative to the copy number of this plasmid in female parent. A n d also a new 7.0 kb linear plasmid, homologous to the original 9.0 kb plasmid of female strain N . intermedia 3977, arose spontaneously in ascospore progeny from cross 3977 x 3709 (Yang 1994). During vegetative growth the plasmids in 3983M-7.0 are relatively stable. The transmission of the Har-7.0 plasmid in strain 3983M-7.0 during the sexual cycle is shown in Table 1-2 (Yang and Griffiths, unpublished data). But the stability of the Har-7.0 plasmid in 3983M-7.0 during the sexual cycle is different from that of 7.0 kb plasmid in strain 3983 (Yang 1994, see table 1-2). These different natural isolates of N . intermedia used as paternal parents were from a variety of geographical locations. During the sexual cycle the three Zhis i plasmids either all existed in the progeny or were all lost together. The loss of the three Zhis i plasmids of 3983 strain during the sexual cycle could be caused by plasmid suppression (Yang and • 29 Griffiths 1993c). The loss of Har-7.0 plasmid of 3983M-7.0 in almost all crosses suggests Har-7.0 might be incomplete or defective in some way that makes its passage through the cross impossible.. Or it is possible that the transmission of Har-7.0 plasmid through the sexual cycle might rely on the existence of other two Zhisi plasmids. But the m a r D N A in Maranhar strain of. N . crassa is maternally inherited (Court 1991). The difference between the inheritance of the Har-7.0 plasmid in the 3983M-7.0 strain of N . intermedia and. that of m a r D N A in Maranhar strain of N . crassa could be due to the different genetic background of the different parental strains or different structural features between these two plasmids. i 30 o co O o co ca Vi T 3 "a, a o -S3 o o ca 15 <4H O c o co a CO CN •8 3 cn -o CU Vi & c/T C<-H o c ca bO C 03 0) co O O OX) o g o o g 'c3 i i "3 co B P a o CD 2 § <D £ <D o | ^ CD j§ O o "3 -5 ^ 'co i2 ^ ca ca ca ca ca ca ca m CN ca o d <+H o > CN ca -2 U ca CO O ICS IK -t-» CO o u I -~ * — ' t - t - ^ ON 31 Figure 1-1A The new plasmid Har-7.0 replaced the original Har-L detected in mitochondria of a culture 3983M-7.0 derived from strain 3983M in one out of 20 conidial isolates A , D N A in the EtBr-stained gel. Lane 0, 1 kb ladder. Lane 1, 2, 4-20, D N A of strain 3983M. Lane 3, D N A of strain 3983M-7.0 B , filter of the gel probed with the Har-L plasmid 33 Figure 1-2 Linearity of the Har-7.0 plasmid in strain 39.83M-7.0 D N A from strain 3983M-7.0 was extracted without proteinase K treatment or with proteinase K treatment and was digested with 3' or 5' exonuclease after proteinase K treatment. Lane 1, D N A without proteinase K treatment Lane 2, undigested D N A with proteinase K treatment Lane 3, D N A digested with 3' exonuclease after proteinase K treatment Lane 4, D N A digested with 5' exonuclease after proteinase K treatment m t D N A 7.0 kb -35 Figure 1-3 The Southern hybridization of the different parts of Har-7.0 to the three Zhis i plasmids D N A from mitochondria of strain 3983 Figure 1-3 (1st): Lane 3983, D N A from mitochondria of strain 3983 Lane 1.2 kb Hind III insert, the linearized recombinant D N A containing p U C 19 vector and 1.2 kb Hind III fragment of Har-7.0 Lane 4.5 kb Hind III insert, the linearized recombinant D N A containing p U G 19 vector and 4.5 kb Hind III fragment of Har-7.0 Lane 2.3 kb E c o R l insert, the linearized recombinant D N A containing p U C 19 vector and 2.3 kb EcoR I fragment of Har-7.0 Lane 3.0 kb EcoR I insert, the linearized recombinant D N A containing p U C 19 vector and 3.0 kb EcoR I fragment of Har-7.0 Lane 2.0 kb Pst I insert, the linearized recombinant D N A containing p U C 19 vector and 2.0 kb Pst I fragment of Har-7.0 Figure 1-3 (2nd): A , filter of the gel containing lane 1.2 kb Hind III insert, 3983 and 3983 probed with the cloned 1.2 kb Hind III fragment of Har-7.0 B , filter of the gel containing lane 4.5 kb Hind III insert, 3983 and 3983 probed with the cloned 4.5 kb Hind III fragment of Har-7.0 C , filter of the gel containing lane 2.3 kb EcoR I insert, 3983 and 3983 probed with the cloned 2.3 kb EcoR I fragment of Har-7.0 D , filter of the gel containing lane 3.0 kb EcoR I insert, 3983 and 3983 probed with the cloned 3.0 kb EcoR I fragment of Har-7.0 E , filter of the gel containing lane 2.0 kb Pst I insert, 3983 and 3983 probed with the cloned 2.0 kb Pst I fragment of Har-7.0 -vl CO CD b o b O" O" C7 • i i i i i | | 3983 mtDNA > 4 * MJffiffiijL 3983 mtDNA 1.2 kb Hindlll Insert 3983 mtDNA QJ 3983 mtDNA 4.5 kb Hindlll Insert 3983 mtDNA O 3983 mtDNA f 2.3 kb EcoRI Insert 3983 mtDNA O 3983 mtDNA 3.0 kb EcoRI Insert 3983 mtDNA |TI 3983 mtDNA 2.0 kb Pstl Insert 38 Figure 1-4 Comparison of restriction maps of maranhar plasmid (Court et al. 1991) from N. crassa and Har-7.0 plasmid from N. intermedia. maranhar Hind III EcoR I Bgl II Xba I "Pst I Hinc II Harbin-7.0 0 . 6 1 . 2 1 . 3 4 . 5 3 . 0 2 . 3 0 8 Hind III 0 6 EcoR I 1 . 7 4 . 0 3 . 8 1 . 9 1 . 5 Bgl II 0 9 Xba I 2 . 2 2 . 0 2 . 8 Pst I Ikb EcoR V Kpn gure 1-5 Restriction map of Har-L (Griffiths and Yang 1995). Harbin-L 0.15 1 1.7 2.3 1.2 | o 15 Bgl II 5.0 0.5 EcoR I 1.4 4.1 EcoR V 5.5 Hind III 0.4 1.5 4.0 5.1 Kpnl Pst I 0.2 5.1 0.2 Xba I Ikb 41 Figure 1-6 The P C R fragment amplied from D N A in mitochondria of strain 3983M. Lane 0, 1 kb ladder Lane 1, 2, the P C R fragments amplied from D N A in mitochondria of strain 3983M-7.0 as a positive control Lane 3, 4, the P C R fragments amplied from D N A in mitochondria of strain 3983M Lane 5, negative control 0 1 2 3 4 5 V 0.5 kb -43 Chapter two The structural features of Har-7.0 plasmid Introduction The sequences of the Har-L and marDNA are already known (Griffiths and Yang 1995, Court et al. 1992). The interest in the structure of the newly-detected Har-7.0 plasmid stems from the finding that it weakly hybridized to the Har-L plasmid in the Harbin strain 3983M of N . intermedia in Southern hybridizations and the restriction map of Har-7.0 is similar to that of the m a r D N A in the Maranhar strain of N . crassa (Chapter 1 & Court et al. 1992). The Harbin strain 3983M-7.0 of N . intermedia and the Maranhar strain of N . crassa are both senescent strains and contain similar linear plasmids. Furthermore, ma rDNA in Maranhar strains of N . crassa integrates covalently into the mitochondrial D N A as the form IS-marDNA (Court et al. 1991). The inserted form of m t D N A rapidly predominates over the wi ld type m t D N A , resulting in abnormal mitochondrial function, senescence and death (reviewed by Griffiths 1992). But preliminary study showed that Har-7.0 plasmid seemed not to integrate into m t D N A ( X . Yang, unpublished data). So the structure of the Har-7.0 plasmid was of interest. The sequence of this plasmid may provide some clue about the origin of this plasmid. A n d the sequence comparison between this plasmid and maranhar may have some explanation on the different functions of these two plasmids. 44 M a r D N A hybridized with eleven linear plasmids with sizes similar to m a r D N A in a sample of 19 natural isolates of N . intermedia (Yang and Griffiths 1993 a). The m a r D N A was originally found in N . crassa. but these marDNA-homologous plasmids are all in N _ intermedia. These plasmid species are as follows (Yang and Griffiths 1993 a): Table 2 -1. The marDNA-homologous plasmids with size similar to maranhar in a sample of 19 natural isolates of N . intermedia Strain# Mar-homologous plasmid N / S Origin Neurospora intermedia 1940 7.0 kb S LaBelle, U S A 3336 7.0 kb S Monte Alegre, Brazi l 3983 7.0 kb, 8 kb and 9 kb S Harbin, China 3983M Har-L, Har-L2 S 3983 Derivative 3983M-7.0 Har-7.0 S 3983M Derivative 3986 7.2 kb and 7.0 kb N Shengyang, China 4853 7.0 kb S Wau, N e w Guinea P27 8.6 kb N Manila, Philippines 3350 7.2 kb N Piracunuga, Brazi l 3709 7.5 kb N Salinas, Puerto N = nonsenescent; S= senescent The m a r D N A or the marDNA-homologous plasmid or part of it are found in both N . crassa and N . intermedia and in diverse geographical locations. These hybridizations do not show whether the marDNA-homologous plasmids are substantially identical or share only some homologous regions. Based on previous studies (Yang 1994), these marDNA-homologous 45 plasmids do not possess the insertion activities of the marDNA, because no marDNA-homologous fragment was found in the mitochondrial genome of strains carrying these plasmids, and there was no substantial change of D N A of the strains. Therefore the senescence of these strains is probably not associated with the insertion of these marDNA-homologous plasmids like the senescence of the Maranhar strains. The geographical distribution of the mar-homologous plasmids suggests a relatively ancient origin. The interspecific distribution indicates either the plasmid could migrate through gene flow since N . crassa and N . intermedia w i l l cross or the plasmid is in some respect infectious. Horizontal transfer of fungal D N A is important in both process and study of fungal evolution. Two questions have arisen. The first is the degree of homology between the Har-L and the Har-7.0 and between the Har-7.0 and the marDNA. The second is the structural difference between the Har-7.0 and the marDNA. Since the N . intermedia and N . crassa both belong to the genus Neurospora. the full D N A sequence of the Har-7.0 plasmid would provide an interesting evolutionary comparison with the marDNA sequence. Therefore the plasmid was fully sequenced. Results General sequence organization of Har-7.0 plasmid D N A The complete nucleotide sequence of the 5' to 3' strand of the Har-7.0 plasmid is presented in F ig . 2-1. Only this strand was sequenced. The Har-7.0 plasmid sequence shows remarkable similarity to that of the m a r D N A in the Maranhar strain of N . crassa except some substitutions (up to 5-nt), small insertions (up to 6-nt) and small deletion (up to 5-nt) relative to m a r D N A . The sizes of the various regions of the plasmid are shown in Table 2-2, together 46 with comparison with marDNA. The plasmid is 7050 bp in length, 2 bp shorter than m a r D N A (7052 bp), explaining the same size of Har-7.0 and maranhar in agarose gel. The Har-7.0 D N A sequence shows perfect terminal inverted repeats of 347 bp. The repeats run from nucleotide position 1 to 347 and 6704 to 7050 (This numbering uses the same plasmid orientation as that used by Court et al. 1992). Table 2-2 Size (bp) of domains of maranhar and Har-7.0 plasmids Plasmid Length T I R a O R F l b IGS C O R F 2 b Har-7.0 7050 347 2691 669 3108 m a r D N A 7052 349 2691 716 3069 a TIR , terminal inverted repeat b O R F , open reading frame C IGS, intergenic sequence There are two large non-overlapping open reading frames, on opposite strands and running from just inside the terminal repeats towards the center. ORP1 runs from position 292 to 2982, and O R F 2 from 3652 to 6759. The ORFs overlap with the terminal repeats in both plasmids, as shown in Fig . 2-1. Their sizes and orientation are similar to those of marDNA. The general sequence organization of the Har-7.0 plasmid is typical of fungal linear plasmid 'invertrons' (Sakaguchi 1990). The features of the plasmid are presented in Fig . 2-2 below. 47 O 0RF1 896aa ORI-2 1u3baa o Fig . 2-2. The features of the 7050-bp Har-7.0 plasmid. The plasmid and its TIRs are represented by thick lines and hatched regions, respectively. Open circles indicate the terminal proteins, which are bound to the 5' nucleotides of the linear plasmid D N A . The locations and orientations of the two large ORFs are indicated by open arrows. Sequence similarity between Har-7.0 and maranhar Overall, the Har-7.0 plasmid has an A T content of 72.5%, which is similar to that of m a r D N A (72.3%, Court and Bertrand 1992) and k a l D N A (70%, Chan et al 1991), through the TIRs are less AT- r i ch (60.1%) like that of marDNA (60.5%). A n alignment is shown in F ig . 2-3. A high level of nucleotide similarity is seen. The quantity of changed , deleted and added residues relative to m a r D N A are shown in Table 2-3, 2-4 and 2-5. Table 2-3. Quantity of changed residues. A T C G A 17 4 5 T 16 11 4 C 2 6 6 G 5 5 8 Table 2-4. Quantity of deleted residues. A T C G 4 5 1 5 48 Table 2-5. Quantity o f added residues. A T C G 4 5 3 0 ORF1 ORF1 has 2691 bp, the same length as the equivalent O R F in marDNA. ORF1 encodes a presumptive protein of 896 amino acids, which should encode R N A polymerase by homology to m a r D N A (Court and Bertrand 1993). The ORF1 sequences of the two plasmids are 98.96% similar at the D N A level and 98.44% similar at the amino acid level. The sequence alignment is shown in Fig . 2-4. A l l of the amino acid differences are substitutions and no insertions or deletions. The changed amino acids are.shown in Table 2-6. Table 2-6. The changed amino acids in O R F 1 . Ala, A Leu, L Trp Arg, R Lys, K Asn Asn, N 3 Lys Met, M Asp, D Phe, F Leu Cys, C Pro, P Gin, Q Ser, S Thr Glu, E Thr, T lie Gly, G Trp, W His, H Tyr, Y Ser lie, 1 Thr, Leu Val, V Ala O R F 2 O R F 2 has 3108 bp, 42 bp larger than the equivalent O R F of marDNA. The difference is accounted for by the 36 bp of extra coding sequence at the end of the O R F 2 and in addition there is a two-amino acid insertion in Har-7.0 D N A . The ORF2 D N A sequence shows a 49 98.2% similarity to marDNA. The Har-7.0 0 R F 2 codes for a presumptive D N A polymerase protein of 1035 amino acids, which by comparison with the equivalent m a r D N A O R F codes for a D N A polymerase. The alignment with the marDNA ORF2 is shown in F ig . 2-5. There is 96.69% amino acid similarity. The changed amino acids are shown in Table 2-7. Table 2-7. The changed amino acids in ORF2 . Ala, A Thr, Pro Leu, L lie Arg, R Lys, K 3 Asn, Arg Asn, N Thr, Ile, Lys Met, M Thr Asp, D Phe, F Leu, Ile Cys, C Ser Pro, P Gin, Q Ser, S Thr Glu, E Gin Thr, T Arg Gly, G Trp, W His, H Tyr, Y His, Asn Ile, 1 Leu Val, V Glu, Leu Terminal inverted repeats Sequencing primers based on the sequences of the two outermost edges of the unique region produced identical sequences for the proximal several hundred bases of the termini, showing that most likely the plasmid has terminal inverted repeats. From this point on, sequencing primers could not have distinguished between identical terminal structures. The end cloning of the terminal fragments was not tried due to the previous unsuccess of similar linear plasmid (Wei et al. 1996). Hence it was not possible conclusively to demonstrate the identity of the repeats distal to this point even through the sequences obtained for both ends were the same. However, most likely the termini are identical inverted repeats. The terminal inverted repeats are 347 bp long, 2 bp shorter than those of marDNA, which are 349 bp. The alignment is presented in Fig . 2-6. According to the alignment these two terminal inverted repeats show great similarity except four 1 -nt substitutions in the inner and middle parts and 50 one 1-nt addition and one 5-nt substitution and four 1-nt substitution at the very end (terminal). There is a 96.26% similarity at D N A level. The intergenic region The intergenic region is 669 bp, 47 bp shorter than the equivalent m a r D N A region which is 716 bp. A n alignment is shown in Fig . 2-7. A high level of nucleotide similarity is seen, with the exception of a small block of nucleotide deletion (42nt) relative to m a r D N A at one end of the intergenic region next to the ORF2 and some substitutions and small length deletions (up to 3-nt) and 1-nt insertions. The 5' terminal nucleotides The 5' nucleotides of Har-7.0 plasmid are T. This is uncommon to the linear mitochondrial plasmids although the 5' nucleotides of G e l - K a l D N A are also T. The 5' nucleotides of all other characterized mitochondrial linear plasmids are purines (G: p C l K l , Oeser and Tudzynski 1989; pFSC2 Fusarium solani. Samac and Leong 1989; kalilo, Chan et al. 1991; maranhar, Court 1992. A : p F S C l of F. solani. Samac and Leong 1989; S - l , Paillard et al. 1985; S-2, Levings and Sederoff 1983; 11.3-kb plasmid of Brassica. Turpen et al. .1987). The 5' nucleotides of numerous bacteriophages are also purines (A: <b29, (j)15, M 2 Y , G A C p - 1 , Escarmis et al. 1984. G : P R D 1 , Savilahti and Bamford 1986; Gerendasy and Ito 1987). Sequence similarity between Har-7.0 and Har-L The alignment of Har-7.0 and Har-L nucleotide sequences has shown that there is a 170 bp common nucleotide sequence region shared by both plasmids. This is shown in F ig . 2-3. This common region starts from the 5' end to number 170 nucleotide in the central part of terminal 51 inverted repeats of Har-7.0 plasmid. This result is consistant with the idea that the Har-L plasmid contains marDNA-homologous region (Griffiths and Yang 1995). Discussion In the Harbin 3983M-7.0 derivative strain of N . intermedia, a 7.0 kb linear plasmid, named Har-7.0, was detected that has same size as marDNA. The studies on full sequencing showed that this plasmid is almost identical to the marDNA originally isolated from several field-collected strains of N . crassa (Court 1992). General structural features are very similar to those of marDNA. There are terminal inverted repeats, and two major ORFs starting just inside the inverted repeats and running towards the center of the plasmid. There is a short intergenic region. Both 5' ends are resistant to exonuclease digestion, presumably due to the presence of a covalently bound protein (Vierula et al. 1990). The nucleotide similarity is 98.55% over all the region, over 98% in the ORFs. The sequence accuracy is 98%-99% (see materials and methods). So the sequence error is l % - 2 % . Therefore, these two plasmids are still pretty similar. But these two are not identical due to the different restriction maps. These features make it virtually certain that the two plasmids are related by descent from a common ancestral plasmid. This kind of similarity is also seen in Ge l -ka lDNA; k a l D N A (Wei et al. 1996) and L A - k a l D N A (Marceko-Kuehn et al. 1994)! The similarity of k a l D N A and L A -k a l D N A was judged by restriction endonuclease site mapping and the sequences of the terminal inverted repeats. But the Har-7.0 plasmid is much more similar to m a r D N A than G e l - k a l D N A is to k a l D N A according to the nucleotide sequence alignments. Such a situation contrasts with plasmids that show similar organization but are only functionally related. For example the k a l D N A , marDNA, pAls and many other linear plasmids are organized along very similar lines, and show conserved blocks of amino acids in the open reading frames (Meinhardt et al. 1990, Hermanns and Osiewacz 1992, Kempken et al. 1992), but do not 52 cross-hybridize at the D N A level. In such plasmids the conserved amino acids suggest relatedness to viral polymerases, but i f this is true the relatedness is no longer manifested at the D N A level. This kind of similarity might reflect functional constraints on nucleic acid processing enzymes (analogy) rather than relatedness (homology). The Har-7.0 and m a r D N A plasmids from N . intermedia and N . crassa are almost identical: the variation was due to nucleotide substitutions, small length mutations and sequence errors beyond the scope of the current instrumentation's accuracy (see Materials and Methods). These two virtually identical plasmids were found in different species from different locations. The distribution o f the homologous plasmids in nature and the presence of these identical plasmids in different species supported the hypothesis that these plasmids could be transmitted between isolates independently of their host mitochondrial genomes (Taylor et al. 1985). This was also shown for a circular plasmid in N . crassa 516 (Roanoke, L A ) , N . intermedia 435 (Fiji) and N . tetrasperma 2510 (Hanalei, H A ) (Taylor et al. 1985). The similarity of this circular plasmid was judged by D N A - D N A hybridization and restriction endonuclease site mapping. Horizontal transmission can be inferred for the plasmids Har-7.0 and m a r D N A in different Neurospora species. Laboratory experiments have shown that horizontal transmission can occur within a species and between species. Horizontal transmission of kalilo and other plasmids has been demonstrated between compatible and incompatible strains of the same species of Neurospora (Griffiths et al. 1990, Collins and Saville 1990, Debets et al. 1994). Transmission of the kalilo and Hanalei-2 plasmids from N . intermedia to N . crassa has also been shown (Griffiths et al. 1990). Even transmission between fungal genera has been demonstrated. Kempken (1995) has transferred the Ascobolus immersus linear plasmid pAI2' to Podospora anserina. although the plasmid is unstable and gradually disappears in its new host. There is only one case of intergeneric distribution of plasmids in fungi, and that is the kalilo plasmid, which is found in Neurospora and Gelasinospora species (Wei et al. 1996). It seems that interspecific and intergeneric 53 incompatibility can be overcome. The presence of these identical plasmids in different species also could be explained by gene flow since N . intermedia and N . crassa often cross readily in the laboratory (Perkins et al. 1976). But the horizontal transfer mechanism is preferred since some sexually-isolated Neurospora still contain many plasmids. Curiously the Har-7.0 and m a r D N A plasmids were not found at the same location. The Har-7.0 plasmid of N . intermedia was found in Harbin, China and marDNA plasmid of N . crassa was found in India. There seems to be no sufficient proximity to potentially allow contact and transfer. The distribution of the m a r D N A plasmid family is disjunct shown in table 2-1. This distribution pattern could reflect incomplete sampling. Alternatively it could be a significant biological pattern reflecting sites of evolutionary origin, selection, host suppressor mutation (Griffiths et al. 1992, Yang and Griffiths 1993c) or dispersal. Another possible factor might be historical patterns of trade and settlement that could have influenced distribution patterns by transporting spores on produce such as sugar cane. The mar-homologous plasmids are more commonly encountered than the plasmids hybridizing to kalilo in natural population samples (Yang and Griffiths 1993a). The sample size might be too small to come to any conclusions. The comparison of the virulences of the marDNA-homologous plasmids and k a l D N A -homologous plasmids might provide some explanation for this kind distribution. If the marDNA-homologous plasmids are not so virulent as the kalDNA-homologous plasmids both the host and the plasmid might not be destroyed and the general incidence of the plasmids in population could be maintained. Alternatively, horizontal transmission of marDNA-homologous plasmids in N. intermedia might be easier transferred than that of kalilo-homologous plasmids. But the marDNA-homologous plasmids are less commonly encountered than the Harbin-1/ LaBelle plasmid in natural population samples (Yang and Griffiths 1993a).The marDNA-homologous plasmids are linear and the Harbin-1 /LaBelle plasmid is circular. Hanalei-2 circular mitochondrial plasmids can transfer more efficiently to other strains through compatible and incompatible heterokaryon formation (Griffiths et al. 1990, Co l l in and Saville 1990 and Debets et al. 1994). It might be conceivable that the 54 Harbin- 1/LaBelle plasmid can transfer more efficiently than the linear marDNA-homologous plasmids between natural isolates. Both strands of Har-7.0 plasmid were translated by computer using MacVector programme, in all reading frames, using the Neurospora mitochondrial genetic code (Heckman et al. 1980) in which T G A is not a stop codon but encodes tryptophan. The ORFs of m a r D N A are known to be transcribed (Court and Bertrand 1993) and the same is true for the ORFs of k a l D N A (Vickery and Griffiths 1993). Candidate proteins were noted in Maranhar strains, but no translational products have been demonstrated in either case. The comparison of the conserved motifs in plasmid-encoded R N A polymerases of m a r D N A and the Har-7.0 is shown in F ig . 2-8. Eight of the nine motifs of the putative R N A polymerase (ORF1) of the Har-7.0 are exactly the same as that of m a r D N A (Court et al. 1992). There are three amino-acid substitutions within motif IV. In addition to the overall homology to known D N A polymerase, the O R F 2 product of Har-7.0 contains the exactly same highly conserved motifs as that of m a r D N A which are characteristic of the proof-reading (Bernad et al. 1989) and polymerization (Bernad et al. 1987, Jung et al. 1987) domains of B-type D N A polymerases, although there are 22 changed amino acids in O R F 2 (Table 2-7). B-type D N A polymerases; possessing the three consensus segments, 1, 2, and 3, which are characteristic of this family of polymerases; are encoded by linear bacteriophages (Escarmis and Salas 1982, Savilahti and Bamford 1987, Salas 1991, Yoshikawa and Ito 1982) and other linear mitochondrial plasmids (Chan et al. 1991, Kempken et al. 1989, Oeser and Tudzynski 1989, Paillard et al. 1985, Robison et al. 1991, Roch et al. 1991, Court et al. 1993). Therefore the putative D N A polymerase of Har-7.0 plasmid is also likely to be a terminal-protein-primed B-type D N A polymerase. A l l the changed amino acids in O R F 2 are outside the proof-reading and polymerization domains. This means the fundamental functions of the putative D N A polymerase are very well 55 conserved. Furthermore the consistently spaced serine, tyrosine, lysine and asparagine ( S K Y N ) residues thought to represent part of a terminal protein sequence (Oeser and Tudzynski 1989, Chan et al. 1991) is also present in Har-7.0 plasmid O R F 2. There are two 1-aa substitution between these spaced amino acids. There are 12 extra amino-acids at the carboxy-terminus relative to that of ma rDNA O R F 2 because of the T A T codon which encode lysine in Har-7.0 instead of the T A G stop codon in marDNA. This is curious in the view of conservation of the rest of the ORFs . Similar situation is seen in G e l - k a l D N A O R F 2 relative to k a l D N A O R F 2 (Wei et al. 1996). The D N A polymerase and R N A polymerase ORFs in Har-7.0, Gel-kalilo (Wei et al. 1996), maranhar (Court et al. 1992), kalilo (Chan et al. 1991) and p C L K l (Oeser and Tudzynski 1989) start in the TIRs of their respective plasmid, suggesting that a single promoter sequence, which is identical for both genes, may be found in the TIRs of each of these genetic elements. The 5' start site of both ORFs of maranhar map very close to the ends of the plasmid within the repeats, at around position 50 (Court and Bertrand 1993). Upstream of the start site, a 23-bp " promoter " region showed 10 matches to the comparable region of the Claviceps plasmid p C L K l (Gessner-Uhlrich 1994). The kalilo ORFs also have a common start site close to the end of the plasmid, at around position 101 (Vickery and Griffiths 1993). However, the kalilo presumptive promoter region did not resemble that of maranhar. The Har-7.0 and m a r D N A have almost identical imperfect dyad symmetry except one substitution in the terminal sequences of the TIR, which is thought to be the downstream of the promoter region. The promoter regions of these two plasmids are almost identical. The 3' ends of both kalilo and maranhar map to the intergenic region. L ike the intergenic region of m a r D N A there are several repeats in both direct and inverted orientation within the intergenic region of Har-7.0 plasmid. The arrangement whereby transcripts and ORFs start in the terminal inverted repeat has also been found in several linear plasmids in other fungi [for example, in Claviceps (Gessner-Uhlrich 1994) and Podospora (Hermanns 1994) species]. 56 The terminal nucleotides of the plasmids are likely to be the origin of replication and, in the case of the Neurospora kalilo and maranhar plasmids, sites of integration into m t D N A . But a previous study (Court et al. 1992) suggest that there is no evolutionary conservation in the nucleotide sequences and palindromic regions between the terminal nucleotides of maranhar plasmid and those of kalilo plasmid even through these two plasmids show the similar insertion behavior. But the mode of kalilo and maranhar insertion is quite novel, having never previously been observed in any other system, eukaryotic or prokaryotic. Both IS-k a l D N A (the inserted form of kalilo) and IS-marDNA (the inserted form of maranhar) are found flanked by long inverted repeats of the m t D N A , formed from the D N A to one side of the insertion point. The reciprocal of this product (containing the other flanking sequence) has not been demonstrated. The insertion mechanism must be different in the two plasmids. A R - k a l D N A (the free form of kalilo) inserts by matching 5 bp from anywhere within the last 20 bp or so at its terminus with an identical quintet in the m t D N A (Chan et al. 1991). Presumably, some kind of crossover event then forms a recombinant molecule. Crossing-over leads to an almost full-length kalilo molecule flanked by the m t D N A to one side of the recombination point (Court et al. 1991). Maranhar integrates as a full-length copy, the insertion process has no need for a 5 bp match of terminus and the target sites in the m t D N A (Court et al., 1991). Homologous recombination is not responsible for the integration of maranhar plasmid. Previous studies on the kalilo plasmids of N . intermedia (Chan et al. 1991), N . tetrasperma (He 1995 & Marcinka-Kuehn 1994) have shown that the >L tetrasperma form ( L A - k a l D N A ) is almost identical to the N . intermedia form (ka lDNA) , differing only by a 60-bp deletion and a 13-bp insertion of unknown origin at the same position in the center of the inverted repeats. This modification might account for the lack of insertion of the L A - k a l D N A plasmid into the m t D N A in N . tetrasperma. The Gelasinospora plasmid (Gel -ka lDNA) is clearly homologous to the k a l D N A and L A - k a l D N A . Its ORFs are virtually the same as those i n the Neurospora plasmids; although there are small mutations, 57 the continuity of the reading frames is preserved. However, the intergenic region and the terminal repeats show numerous small and large mutations, which might prevent its insertion. Similarly the small mutations, including deletions, insertions and substitutions in the intergenic region and the terminal repeats of Har-7.0 plasmid, relative to those of marDNA, might explain the lack of insertion of Har-7.0 plasmid into m t D N A in N . intermedia. But the possibility can not be ruled out that the nuclear suppressors might be involved. The suppressors may inhibit plasmid insertion. (Griffiths et al. 1992). A t one end of Har-L, beginning at position 207 right after the TIR, there is a section of 170 bp with 100% similarity to the termini of the Har-7.0 plasmid. This segment is not present at the other end of Har-L. This segment is the only marDNA-homologous region of the Har-L probably derived from a Zhisi plasmid. This further supports that the 5.5 kb Har-L is partly from a Zhis i plasmid which is marDNA-homologous (Griffiths and Yang 1995). Without the sequences of the Zhis i plasmids the comparisons of the sequences of the Har-7.0 and the Zhis i plasmids can not be carried out. Therefore, it is not sure i f the Har-7.0 is identical to the 7.0 kb Zhisi plasmid or not. A n d the sourse of the extra material of the 8.0 kb and 9.0 kb Zhis i plasmid can not be determined. 58 Summary After the investigation of this newly-detected plasmid it is found that this new plasmid in a deviant Harbin strain of N. intermedia is a linear mitochondrial plasmid with structural features almost identical to maranhar plasmid originally found in Maranhar strain of N . crassa. This new plasmid Har-7.0 in N. intermedia Harbin strain 3983M-7.0 is related to Zhis i 7.0 plasmid in N. intermedia Harbin strain 3983. It is probably the same as 7.0 kb Zhis i plasmid. The mechanism of the evolvement of this new plasmid might provide some clue about plasmid evolution in nature. Many similarities existed between maranhar and Har-7.0. Plasmid size was identical, the restriction enzyme map was pretty similar on the basis of fragment patterns from 5 enzymes, and both plasmids were resistant to 5' exonuclease digestion. The identity with maranhar was remarkable at D N A sequence. The nucleotide similarity is 98.55% over all the region. It was the first time a maranhar plasmid had been confirmed in a fungal strain N. intermedia other than N. crassa. The distribution of identical plasmids in these two species could be explained by horizontal transfer or gene flow. . The horizontal transfer of plasmids has been demonstrated. N_ intermedia and N. crassa may cross. This w i i l allow plasmid transfer between these two species. But the horizontal transfer mechanism is preferred. 59 More study is needed on the distribution of marDNA-homologous plasmids. The more extensive survey of related plasmids might find more marDNA-homologous plasmids in other species of Neurospora or another genera like kalilo plasmid. Plasmids from different species or genera could become usual markers for studying host evolution. Further studies on the replication, transcription and translation of Har-7.0 plasmid could be carried out. Are the terminal nucleotides of the plasmid the origin of replication? Do small deletion and nucleotide substitutions the terminal repeats effect the transmission of this plasmid during the sexual cycle ? Are the ORFs transcribed like the ORFs of m a r D N A and k a l D N A ? Are there any candidate proteins for translational products like in Maranhar strain ? Do the nucleotide substitutions at the terminal repeats effect the integration of this linear plasmid making it avirulent ? 60 Figure 2-1. The nucleotide sequence of the 5' to 3' strand of Har-7.0 plasmid. The t e r m i n a l i n v e r t e d repeats are underlined.The amino-acid sequences of the 0RF1 and ORF2 are p r e s e n t e d below the n u c l e o t i d e sequence. This strand of the n u c l e o t i d e sequence i s the sense-strand f o r ORF1 and the antisense-strand f o r ORF2.The p r i m a r y sequences s t a r t at the f i r s t methionine i n each sequence. P o t e n t i a l t r a n s l a t i o n i n i t i a t i o n codon (ATG) and termination codons (TAA and TAG) are bold. 1 T G G G G G G A G T A C A T A A A T C C C T A C T T T A T A A A A T G A A C A T C C C C C T T A T A G A G G A A A T A G 6 1 C C T A T A T A A C A C T A T G A C T G A T C A T T A G A T C A G T C T A T A G T G T T T T A C T A T T T C C T C A A C 1 2 1 C T A T A A C A T G C G T T T T A C C T T T C C C G C T T A T G C G G C T C T T G A C A G A G T T G G G G C A G A T C C 1 8 1 A A A G A C T A C A G T A C G C A C A T C T C C A G A C A G A T C C G G G A G C G A A G C C T A C T A T T A C C T G T C 2 4 1 A A G A G G A G A G C T G A A A A A T A G T A G G A A T C G G G G T C A C A G A A T A T A T T T T T A A T 6 A T T A A A 0 R F 1 > M I K 3 0 1 T T T T A T T T T A C C A C A T T T C A C A T G A A A T T T C C T G C A C G T T T A T T T T C A A C T T C A T T C A C A F Y E T T F H M K F P A R L F S T S F T 3 6 1 T T G A A T A A T A C G A A T A T A A T A A A T G A A A A A G A T G T T A A A T A T A T T A C T A A A A A C T T C T T A L N N T N I I N E K D V K Y I T K N F L 4 2 1 T T A G A A A A T T C A A A T G G T T T T A A T T T A A T T A A A A A T A T A A T T A A T T C T G A T G A A A C A T C T L E N S, N G F N L I K N I I N S D E T S 4 8 1 G A A G T T A A A C A A A A G A A A A T T G A A G T T G A A T T A A A T A A T A T A T G G C A T A C T G A A A T A A C T E V K Q K K I E V E L N N I W H T E I T 5 4 1 G A C A T T T T A C A G A A A A A A A G A A G C T T A G G G T T G G A T G C A A T A G G A A C T T C T A T T T T A G C T D I L Q K K R S L G L D A I G T S I L A 6 0 1 A A A G A T T T T C A T A A T T T A A T A G G A G A T A T A G A G A A T T T T A T T A A T A A A G G A A G A A C A A A T K D F H N L I G D I E N F I N K G R T N • 6 6 1 A A G T T A C C A G G C G T T G A A T A C T T G A A A C C A T C A T T A A T T A T T T C T A T T G T C T T A G G A A A A K L P G V E Y L K P S L I I S I V L G K 7 2 1 G T T A T T C C T T T C A G T C T T A G A C A T T C A G A T A T T T T A A A C C A A C C T A C T C A T T C T T T A T T T V I P F S L R H S D I L N Q P T H S L F 7 8 1 G C A G A A A T A G G A A A A A C A T T A A A A T A T C A A T C T A T T T T T G A A T T A C A T C A T A G A A T T G G T A E I G K T L K Y Q S I F E L H H R I G 8 4 1 C T T A T T C A A A A T C G T G T T G A A G A A A T T A A A A A T T C T A C T G A A A A A A A A T T A G T A T C T G A A L I Q N R V E E I K N S T E K K L V S E 9 0 1 T T T A A T A A A T T A A C C A A A G T A C T A G A T G A A T A T A A A A A C A C T T T A A A A A A C T T A G A A G A A F N K L T K V L D E Y K . N T L K N L E E 961. TTAAGCGGGG AAAGTATTAT AAAAGTAGGA L S G E S I I K V G 1021 AGTGAATTTT ACTCTCTCGA AGAACAAATT S E ' F Y S L E E Q I 1081 TTACCTAAAA ATAAATTAAA TACATTAATT L P K N K L N T L I 1141 CTCCCAATGA TTATACCACC ACTTGAATGA L P M I I P P L E W 12 01 TATGGTGGAA CTATACTTAA TAATAAACAC Y G G T I L N N K H 12 61' GAGAATTCAG ATGCAAATGA TATGACATAT E N S D A N D M T Y 1321 AAATCAAAAA TAC CATATAT TATTAATTTA K S K I P Y I I N L 13 81 TTTATTAATC GTGATAAAAA AGATAATGTT F I N R D K K D N V 1441 GCTTTATTGG GTGAGTATAT GAAGGATCGT A L L G E Y M K D R 15 01 CATAATAGTA AATTTCTTTA•TCATTCTTCT H N S K F L Y H S S 1561 GTTAAGGAGT TCTATATGAC GGTATTTATA V K E F Y M T V . F I 1621 TGTGCGCTTA ACATACAAGG TGGGGAATTG C A L N I Q G G E L . 1681 CAGAAATTAA ATGATATTGG ATTAAAAGCT Q K L N D I G L K A 1741 CTTGATAAAA GATCTAAAGA AGAAAGATTG . L D K R S K ' E . E R L 1801 ATTGATATAG ACAATTATGA AATATGAAGA I D I D N Y E I W R 1861 TGTGCCTTGG AGTTGAAAGG GTATAAAGAG C A L E L K G Y K E 1921 CTTATGGATG CAACATGTAA TGGATTACAA L M D A T C N G L Q 1981 TTAGCAGAAA AAGTTAATTT ACTTAAGAGT L A ' E . K V N L L K S 2041 TCTGAAGTAA TTCCTCATAT AAAACAAGAA S E V I P H I K Q E 2101 ACAAACCTTG AGCGTATAAA TGTAGAGAGA T N L E R I N V E R TTAGGATTTA CAGCACTTTT AAGTGAAACT L G F T A L L S E T ATAGCTAAAA ATAAATCAAT AAGATATATA I A K N K S I R Y I AATAATATAA CATTAATGGA TACAATTGAA N N I T L M D T I E AAAATTGATG ATAATGAGAA AATTATTGAA K I ' D D N E K I I E AGGATTAGAC CATTAAGAAC AAAATCAGTA R I R P L R T K ' S V AATAAAGAGT TAGTTGATGC CGTTAACTTT N K E L V D A V N F AAACTTTTAG ATTTTATTAC TAGAGATGAA K L L D F I T R D E ATTATTTATA AACATATTCA TCCTGACTCG I I Y . K H I H P D S AAAAACCCAA AGATTAGTGA AATAACGACT K N P K I S E I T T ATAATTTCAA TAGCAAAATT AATGAAAGAT I I S I A K L M K D GATTGAAGAG GAAGATTTTA TACTTCAAGT D W R . G R F Y T S S GCTAGAAGTT TACTTCTATT TAAAGAAGGA A R S L L L ' F K E G TTAAAAATAT ATACTGCAAA TGCTTTCGGT L K I Y T A N A F G GATTGAGTCG AACAAAATTT GCATAAAATT D W V E Q N L H K I GAAGCTGATG AACCATTACT ATTTTTAGCC E A D E P L L F L A GACCCCAATT TTATTTCACA TTTACCAATT D P N F I S H L . P I CATTTAAGTG CAATGGTAAA TGATTTTGTT H L S A M V N D F V ACTGAAAATG ATAATCCTAG GGATTTATAT T E N D ' N P R D L Y ATTTTAGAAG CATCAAAATC GTACGAACAC I L E A S K S Y E H TGTCTAGTTA AAAGGGGACT TATGACTATT C L V K R G L M T I 2 1 6 1 A C A T A T G G A G C A A C T G A A A G A G G A A T T T A T T Y G A T E R G I Y 2 2 2 1 G A T G A A T G A A A C A A A A C T G C A G G G C T T C A T D E W N K T A G L H 2 2 8 1 A A A G A T G T T G T A T T T A C T C A A A A G A A T A T A K D V V F T Q K N I 2 3 4 1 T T A T T T A A A A T T C A T C C T A A T T T A A A T A C T L F K I H - P N L N T 2 4 0 1 G T A T T A T G T G A A T T A G A T T T A C C T G T T A A T V L C E L D L P V N 2 4 6 1 C A A A A A T A T A A T A A A T T T A C T A A A T A T A A T Q K Y N K F T K Y N 2 5 2 1 A A A T T A G T A T T A A G A A A A G C A G A T A C T A C T K L V L R K A D T T 2 5 8 1 T T T A T A C C C A A T T T T G T T C A C T C A A T G G A T F I P N F V H S M D 2 6 4 1 A T A A G A G A T G A A G G A C G C A A A A T T A A C T T T I R D E G . R K I N F 2 7 0 1 G C A A A T G A T A C T G C A T G A C T A T C A T G A T A T A N D T A W L S W Y 2 7 6 1 G A T T C T A G T T T T T T A A G A A G A T T T C A T A A T D S S F L R R F H N 2 8 2 1 G G A G A A G A T G C T C A T T T T G A T A A A G A T G G T G E D A H F ' D K D G 2 8 8 1 A C A G A T A T T C C T G A T C G T A T T G A T A T A C C T T D I P D R I D I P 2 9 4 1 A T A T A T A A A G A A A T T C T T C A T T C T G A G T A T I Y K E I L H S E Y 3 0 0 1 A A A T A A T A T A A A T A T A G T G G G A T T T A A C A A 3 0 6 1 T T T T T T A A A G G G A A T A C A C A A A T T T G T G T A 3 1 2 1 A G T A A A T A C A C A T G T A A T A A C A A A G T A A T G 3 1 8 1 A C C C A G G A T C T A C T T C C T C T C A A A A T C T T A 3 2 4 1 T A C C T A T A T T T A A T A C T G T T A A G T A T A T A A 3 3 0 1 A C C A A A A T T A C A C C T G T C C A G C T C G T G A C A 3 3 6 1 A A G G G A G A A A A A T A A A A A A A A A A G G G A A T G 3 4 2 1 T A C A C A A A T C T T T T G T G T A T T C C T T T T T T T 3 4 8 1 G T A T A C C T T T T T T A A A G T A T A C A C A A A T A C 3 5 4 1 T C A A G G T T A A C C C A A A T A A C A T T T A T A A T A G A T C A A A T T G T A T C T A A A T T C T T T C A A A A A D Q I V S K F F Q K T T T G T A T G T A T A G A T T C G G A T A T A G C T C C A F V C I D S D I A P C T T C T T T G G A G T A A A A T T A T T T A T A A T T C T L L W S K I I Y N S C T A A T G G T A T A T T T T A A T A G T A T A G T T A A A L M . V Y F N S I V K T G A G T A A C T C C T T A T G G T T T A G T A A T A C A A W V T P Y G L V I Q G A A A C A A C T T A T G T T G C A T C T A A A C G A T A T E T T Y V A S K R Y T C T A T T T C T A A G C A G A A A C A A A T A C A A G C G S I S K Q K Q I Q A G G A A G T A A T A T T G T T T T A T T A A T A A A A A C A G S N . I V L L I K T G C A A G T A T T C A T G A T T G T T T T G C A A C T C A T A S I H D C F A T H G T A A A G C A A A G T T T T A T A C G T A T T T A T T C T V K Q S F I R I Y S T A T A T C C A A T T A A G A A T C C A A G C T A A A T A T Y I Q L R I Q A K Y G T A T T A A C C C A T G T A A C T A T T G A T A A T G G T V L T H V T I D N G A A A C C T C T T G T A G T A A A T A A A G A T A A T A A A K P L V V . N K D N K T T T A T T A A C T A A A T G A T A A A T A A T G A T T G T F I N * > A A A A G G G A C A A C A C A A A T A G T G T A T A C C C T T T C C C T T T T T T T T T A A T T A T T A T A C G G T T A T T T G T T T A T G A T A G G T A G T A C G T G C A T T G C T A T T C T A C T G A A G G T T A A A T C A T A G A A A A A T T T A T A A T T A T A A T A T T T A A T A T T A A A T A A A G T A C T G T A A C G A G A T A G G T A A A A C C C T C G C A A A A C C T G C A T T C C C A T A A A A A A T A G G G A T T C T T T T T T A A A G G T A T A C A C A A A T A C T G T T G T G T A T A T C T T A T A C C G A A A A A G T A T T A A T A T T A A A C T A A A T A A C A A T C A T T A C C T C A T 3 6 0 1 A T T G T A A A T A A T G G T G T G A G G A A A A T A A A A 3 6 6 1 T T T T A G A T A A T C A T A T A T A A T A T G A T T A T A K L Y D Y I I H N Y 3 7 2 1 A G T T T C A C C C A C A T C T G A T A C C A A G G C T T T T E G V D S V L A K 3 7 8 1 T A A T G G T G A A G T A T T A A C T C A T T T A C C T A A L P S T N V W K G L 3 8 4 1 C G A T A T A A G A T T A T A T T T T T G A T A T T T A A C S I L N Y K Q Y K V 3 9 0 1 T C A T C T T T C T T G A A A T A G T A C C C T A C T T T C W R E Q F L - V R S E 3 9 6 1 A T C T A G A T T A T G T G T G G T A T T A T C T T T G T T D L N H T T N . D K N 4 0 2 1 T T T A C C A C C A A A A T C T A A T A G A T A T A A T T T K G G F D L L Y L K 4 0 8 1 T T G T C C G T T A T A T T C A G C T T T A A A T T T C C C Q G N Y E A K F K G ' 4 1 4 1 T G G T T T T T C C A C A A A T A T T G A A T C A G T A T C P K E V F I S D T D 4 2 0 1 C A T C A G A A T T C T A G A T C A T G A T G C T G T T G C M L I R S W S A T A 4 2 6 1 T C C A T C A T C T T T C T C T C C A T C T A G G T A A G A G D D K E G D L Y S 4 3 2 1 A C A C A A T A C A G G A C A T G G T T T T T T A T C G T A C L V P C P K K D Y 4 3 8 1 A A A T T C G T G A A T A A C G T T A T T A G T T A A T T G F E H I V N N T L Q 4 4 4 1 T T T A A T T T C A G C G G C G C T A T C A T T C A T A C C K I E A A S D N M G 4 5 0 1 T T T A G C C A T C T G T T T C T T T G A A C C T T T A G T K A M Q K K S G K T 4 5 6 1 A T A T T C C T T A A A A G G A T C A T C T C T T T T T T C Y E K F P D D R K E 4 6 2 1 A T A T C C G T A C T C A A C T G C T A A T T T T A A T T C Y G Y E V A . L K L E 4 6 8 1 A A T G G G A A A A A T T A A T T T T T G T A C T C C A T T I P F I L K Q . V G N 4 7 4 1 A T T T A T T G C T G G A G T A A T A A T T T T T G C T C T N I A P T I I K A R T G T A A T A T T A G T T G T T A T A C T T T A G A A T A C * ' F V T T T A T T A T A A T G T A T T C T T T T T C T A T A T C A K N Y H I R K R Y W T G A A A T T A C C . T C A A A A T T T T C G T T T A T A C A S I V E F N E N I C A G A G T A T A G T T T T T C A C G T T T A T C A T A T C C S Y L K E R K D Y G T G T A A C T G T T C C T A A T T C C A A G C T A C G T C C T V T G L E L S R G T C C A T T A T A C A G T G C T T C G A A A T C A T T A A T G N Y L A E F D N I T T T A G T T A T T C C T T T A C A T T T A A T T T C T A A K T I G K C K I E L T C C T G A T A T G A A A A T T G C A C G T T T A A T T A A G S I F I A R K I L A C A T C C T T C T C C A A T A A A A G C A C T A T C T A A C G E G I F A S D L A G T A T A T G C T G A A T T A A T T A T A T G T T T A T A T Y A S N I I H K Y T G C A G C A A T G G A T G T A G A G T T T A T G A T A A A A A I S T S N I I F T A A A A G T T C G T A A T T T T T T T C T G A T T G T G C L L E Y N K E S Q A T C T C A C G T A A T G T T T A T C A T C A T C T A C T T C R V Y H K D D D V E A A T A T T A T C T A A T T C A T T A G T A G T T A G C A T I N D L E N T T L M T G T T C T A C C A T A T A A A G T G T T C A A A A G C A A T R G Y L T N L L L A T T A T C T T T A A T T G A G G C A A A A T G C T C A A T N D K I S A F H E I G A A A A C G T A A C T C T C A A G T A C T T C T A T T T T F V Y S E L V E I K T T C A G A A A A A T A T C A C C C T G T T C A T T C A C C E S F Y W G T W E G G A C T T T A A C T C T G C A A G G T A A T A C T G G A A T V K V R C P L V P I T A C A A A T C C A A A T A T T T C A T T T A A G T T T T T V F G F I E N L N K 4 8 0 1 A G A A A A A G T A T G G A C T G G T A T A C C A A C A G G S F T H V P I G V P 4 8 6 1 A C T A T T A A A A T C A A A T G A G A A A A T A T T A T T S N F D F S F I N N 4 9 2 1 A C C G A A A T A A G C A G C T C T A A C G G C T C T T T C G F Y A A R V A R E . 4 9 8 1 T T T A G G A G T T A A T T T T T T C T C C T C T T T T T T K P T L K K E E K K 5 0 4 1 A G T A G T T T C A T T T T C T T C T T C T T C T T C T A C T T E N E E E E E V 5 1 0 1 T A A G A A G G C C A A T G C A G A T G C G G T T T T A A C L F A L A S A T K V 5 1 6 1 T G T A T T A T T A C T C A T T T C C A T C A T T A A T T G T N N S M E M M L Q 5 2 2 1 G A T A A T G G T T T C T T T T C T C G T C G A T C A T T T I I T E K R T S W K 52 81 T G T A A T C A T T T C A C C T T T T T T G G G A T C A A T T I M E G K K P D I 5 3 4 1 A T A T T C T A A A T T A T C T T T A T T A A C A A A T T T Y E L . N D K N V F K 5 4 0 1 A T T A T G A T C T T T A G C T A A T T T A T C T A G T G A N H D K A L K D L S 5 4 6 1 T G T T A T A G T A T G C C G C T C A G C T T T T C C T C C T I T H R E A K G G 5 5 2 1 T T T T A T T G A A A G A A T A T C A A G A T C T T T A G A K I S L I D L D K S 5 5 8 1 T A A T A T T T T G A T A A T A A A A T T A A T A T C A A A L I K I I F N I D F 5 6 4 1 T T T G T C A T A T T T T A A C A T A T C T T T T A T A C A K D Y K L M D K I C 57 01 A T C A G A A A T G T A A T A A G T A A G A G A T T T A T T D S I Y Y T L S K N 57 61 C A T C G T T G A A T C A C C A T T C C C A G T A G G A A C M T S , D G N G T P V 5 8 2 1 T T T T T T A T C T T G T C T T T T A T C T C T T T T C G T K K D Q R K D R K T 5 8 8 1 T T C A A C A T T G A C T A A A G T A T T A T C A T A G T A E V N V L T N D Y Y 5 9 4 1 T C T T T T A A G A C C A T C A C C A T G C T T A A C A T C R K L G D G H K V D C A T T G G C A T C A T C A T T G C A G T A G G G T A A A G M P M M M A T P Y L T A T A A T T G G A A T A A A A A T T T C A T T A C G T C C I I P I F I E N R G A A G T C T T C C T T T T A G T T T A G G T A A A A A A T A L R G K L K P L F Y G T C A A A C A A T G A T A A A A T G T T A T T A G G T G A D F L S L I N N P S T T C A T T T G G T A A A A A T A A C G T T C T A T A A A C E . N P L F L T R Y V T C T A G T A A T A T T T A T T C T A A A A G T T G A A T A R T I N I R F T S Y A T A T A A A G C C T T T A T A T C T T T T T C T A A G T A Y L A K I D K E L Y A T T A G T A T A C A T A G C A G C T C A C T C A A A T A G N T Y M A A W E F L A T A A T A C T C A T A A T C A G G T A T T A A T C C A A C Y Y E Y D P I L G V G T A A G G A A A T T T C C C T T T T T T A G T A A T G A T Y P F K G K K T i l T C C T G G T A G G A G T C T A C A T G A A T C G G C A A T G P L L R C S D A I T T T T T T T T T A G G T T C A A A T T T A T A T G A A A T K K K P E F K Y S I T A T T A T T T T T T C T A C A A C A A A T T C T T G A A C I I K E V V F E Q V T T T G G A A A A A T T A T G G C A A T A T A C T G T A T G K S F N H C Y V T H T G C T A G T A A C A T T T C A C G T T G A G A A A T A A A A L L M E R Q . S I F T C C A T C A T A A A A T C C A C A A G C A T A A G C A A T G D Y F G C A Y A I T T G A A A T G T T T C A A T A T C A A A T G C T A A T A T Q F T E I D F A L I T G G C T C T A T A T T T C C A A C T T T T T G A G G T G T P E I N G V K Q P T A T A A T A A T A C A T A T T A T C A T T A A T T C A A T G Y Y Y M N D N I W H T T C A A A C T G G T A C A A T A A T C T A T T A T C A C G E F Q Y L L R N D R 6 0 0 1 T G A A A T A A C A T C T A T A A G A T G A T T T A C A C C A T C T T T G A T A A T T T T C A C T T T A A G A A T A A C S I V D I L H N V G D K I I K V K L I V 6 0 6 1 A T T T C C A T T A T C A A A A A T A C G A A T G T T A T T T T C T T T A T C A T G T T T T G T T T C A A C A C C A T A N G N D F I R I N N E K D H K T E V G Y 6 1 2 1 A A A T T T A A G A T C C A T T G T G T G T G G A A C A A A T T C T G G T C T A G A A A G T T T A G A C T T T C C T T T F > K L D M T H P V F E P R S L K S K G K 6 1 8 1 A G G A A T A G T A G T G T G T A T A T T A G T T T T T T C T T T T G C T A T A G G A T G A A A A A C T G G A T T T T T P I T T H I N T K E K A I P H F V ' P N K 6 2 4 1 A A C T T T A A A A T T A A G C T T T C T T A A T C T T A T A A A A A G T T T A T C A A C T G G C T C T A A A T A G T A V K F N L K R L R I F L K D V P E L Y Y 6 3 0 1 T T T T T C A G A A A G A A A A A T A A T T C T A T C A T A A A T T T G T T G T A A G A T T A T A A G T G G A T C A G T K E S L F I I R D Y I Q Q L I I L P D T 6 3 6 1 A T T A A A A A T G A C T A A A A A G T G T C T G T C T A C T G A T A T A T T A G T A T T A C T A T C A A C A T T T C N F I V L F H R D V S I N ' T N S D V N R 6 4 2 1 A A G T A T A A C C C C A A G T G C G T A T G T A A A A T T T T T A T C T A A T T T C A T G A A T A G T T G A C G T A G L I V G L A Y T F N K D L K M F L Q R L 6 4 8 1 T A A T T C A A C A T T A A T A T T G A A T T C A T T A A T A T T T A A A A G T G A T T T T A A A T T A A A A T C A T T L E V N I N F E N I N L L S K L N F D N 6 5 4 1 T T T T T C A T A T T C A C C A A T G T T A C T T A A A A T C A T A T G T T T A G G T T C G G T A G T A C T T T C T C A K E Y E G I N S L I M H K P E T T S E W 6 6 0 1 T T T T A A T T T A G G G T T T T C T T T C T T A A A A T C T T T C A A A T A T T T A A T T T G A T T T T G A A C T T C K L K P N E K K F D K L Y K I Q N Q V E 6 6 6 1 A T T T G A A G A A T T T T G T T C A A T A T T G G C T C G A T T T C C A C T C G T G G A A A A T A A A C G T G C A G G N S S N Q E I N A R N G S T S F L R A P 6 7 2 1 A A A T T T C A T G T G A A A T G T G G T A A A A T A A A A T T T A A T C A T T A A A A A T A T A T T C T G T G A C C C F K M H F T T F Y F K I M < O R F 2 6 7 8 1 C G A T T C C T A C T A T T T T T C A G C T C T C C T C T T G A C A G G T A A T A G T A G G C T T C G C T C C C G G A T 6 8 4 1 C T G T C T G G A G A T G T G C G T A C T G T A G T C T T T G G A T C T G C C C C A A C T C T G T C A A G A G C C G C A 6 9 0 1 T A A G C G G G A A A G G T A A A A C G C A T G T T A T A G G T T G A G G A A A T A G T A A A A C A C T A T A G A C T G 6 9 6 1 A T C T A A T G A T C A G T C A T A G T G T T A T A T A G G C T A T T T C C T C T A T A A G G G G G A T G T T C A T T T 7 0 2 1 T A T A A A G T A G G G A T T T A T G T A C T C C C C C C A 66 Figure 2-3. The alignment of the sequences of Har-7.0 and maranhar and Har-L ( Court et a l . 1992, Yang and G r i f f i t h s ) The stars represent the consensus sequence r e l a t i v e to Har-7.0 plasmid. The horizontal lines represent the deleted residues. 7 K b 1 T G G G G G G A G T A C A T A A A T C C C T A C T T T A T A A A A T G A A C A T C C C C C T T A T A G A G G A A A T A G _ * * * * * * * T C G ( 3 Q * * * * G * * * *rp* * * *rp* * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * jja^—L 206 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 61 C C T A T A T A A C A C T A T G A C T G A T C A T T A G A T C A G T C T A T A G T G T T T T A C T A T T T C C T C A A C Mc^IT * * * * * * * * * * * * * * * * * Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p H^iL" —Li * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 121 C T A T A A C A T G C G T T T T A C C T T T C C C G C T T A T G C G G C T C T T G A C A G A G T T G G G G C A G A T C C ] V [ a r Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * H ^ r - — L * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 375 7 K b 181 A A A G A C T A C A G T A C G C A C A T C T C C A G A C A G A T C C G G G A G C G A A G C C T A C T A T T A C C T G T C Max" * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 2 4 1 A A G A G G A G A G C T G A A A A A T A G T A G G A A T C G G G G T C A C A G A A T A T A T T T T T A A T G A T T A A A J y [ a r * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * 7 K b 3 01 T T T T A T T T T A C C A C A T T T C A C A T G A A A T T T C C T G C A C G T T T A T T T T C A A C T T C A T T C A C A J V ^ X " * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * £ * * * * * * * * * * * * 7 K b 3 61 T T G A A T A A T A C G A A T A T A A T A A A T G A A A A A G A T G T T A A A T A T A T T A C T A A A A A C T T C T T A ^ C U T * * * * * * * * * * * * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 4 2 1 T T A G A A A A T T C A A A T G G T T T T A A T T T A A T T A A A A A T A T A A T T A A T T C T G A T G A A A C A T C T Mar" * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 481 G A A G T T A A A C A A A A G A A A A T T G A A G T T G A A T T A A A T A A T A T A T G G C A T A C T G A A A T A A C T J ^ g ^ * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * 7 K b 541 G A C A T T T T A C A G A A A A A A A G A A G C T T A G G G T T G G A T G C A A T A G G A A C T T C T A T T T T A G C T MSX" * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Q * * * * * * * * * * * * * 7 K b 6 01 A A A G A T T T T C A T A A T T T A A T A G G A G A T A T A G A G A A T T T T A T T A A T A A A G G A A G A A C A A A T Ma3T * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 67 7 K b 6 6 1 A A G T T A C C A G G C G T T G A A T A C T T G A A A C C A T C A T T A A T T A T T T C T A T T G T C T T A G G A A A A j / j a r * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 7 2 1 G T T A T T C C T T T C A G T C T T A G A C A T T C A G A T A T T T T A A A C C A A C C T A C T C A T T C T T T A T T T jyj^-j-- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 7 81 G C A G A A A T A G G A A A A A C A T T A A A A T A T C A A T C T A T T T T T G A A T T A C A T C A . T A G A A T T G G T ]y[£-j~ * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K B 8 4 1 C T T A T T C A A A A T C G T G T T G A A G A A A T T A A A . A A T T C T A C T G A A A A A A A A T T A G T A T C T G A A J V J Q J - * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ^ * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 9 0 1 T T T A A T A A A T T A A C C A A A G T A C T A G A T G A A T A T A A A A A C A C T T T A A A A A A C T T A G A A G A A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 9 6 1 T T A A G C G G G G A A A G T A T T A T A A A A G T A G G A T T A G G A T T T A C A G C A C T T T T A A G T G A A A C T jyj^-^. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 1 0 2 1 A G T G A A T T T T A C T C T C T C G A A G A A C A A A T T A T A G C T A A A A A T A A A T C A A T A A G A T A T A T A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 1 0 8 1 T T A C C T A A A A A T A A A T T A A A T A C A T T A A T T A A T A A T A T A A C A T T A A T G G A T A C A A T T G A A ^ * * * * * * * * * * * * * * * * * * * ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 1 1 4 1 C T C C C A A T G A T T A T A C C A C C A C T T G A A T G A A A A A T T G A T G A T A A T G A G A A A A T T A T T G A A Mar" ********** ********** ********** ********** ********** ********** 7 K b 12 01 T A T G G T G G A A C T A T A C T T A A T A A T A A A C A C A G G A T T A G A C C A T T A A G A A C A A A A T C A G T A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 12 61 G A G A A T T C A G A T G C A A A T G A T A T G A C A T A T A A T A A A G A G T T A G T T G A T G C C G T T A A C T T T J ^ Q ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 1 3 2 1 A A A T C A A A A A T A C C A T A T A T T A T T A A T T T A A A A C T T T T A G A T T T T A T T A C T A G A G A T G A A J ^ Z " * * * * * * * * * * * * * * * . * * * * £ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 1 3 8 1 T T T A T T A A T C G T G A T A A A A A A G A T A A T G T T A T T A T T T A T A A A C A T A T T C A T C C T G A C T C G MaX" ********** ********** ********** ********** ********** ********** 7 K b 1 4 4 1 G C T T T A T T G G G T G A G T A T A T G A A G G A T C G T A A A A A C C C A A A G A T T A G T G A A A T A A C G A C T * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 15 01 C A T A A T A G T A A A T T T C T T T A T C A T T C T T C T A T A A T T T C A A T A G C A A A A T T A A T G A A A G A T ]\^-jT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 15 61 G T T A A G G A G T T C T A T A T G A C G G T A T T T A T A G A T T G A A G A G G A A G A T T T T A T A C T T C A A G T 68 Mar ********** ********** ********** ********** ********** *********£ 7Kb 1621 TGTGCGCTTA ACATACAAGG TGGGGAATTG GCTAGAAGTT TACTTCTATT TAAAGAAGGA Mar ********** ********** ********** ********** ********** ********** 7Kb 1681 CAGAAATTAA ATGATATTGG ATTAAAAGCT TTAAAAATAT ATACTGCAAA TGCTTTCGGT . Mar ********** ********** ********** ********** ********** ********** 7Kb 1741 CTTGATAAAA GATCTAAAGA AGAAAGATTG GATTGAGTCG AACAAAATTT GCATAAAATT Mar ********** ********** ********** ********** ********** ********** 7Kb 1801 ATTGATATAG ACAATTATGA AATATGAAGA GAAGCTGATG AACCATTACT ATTTTTAGCC Mar ********** ********** ********** ********** ********** ********** 7Kb 18 61 TGTGCCTTGG AGTTGAAAGG GTATAAAGAG GACCCCAATT TTATTTCACA TTTACCAATT •^ jg^  * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 1921 CTTATGGATG CAACATGTAA TGGATTACAA CATTTAAGTG CAATGGTAAA TGATTTTGTT Mar ********** ********** ********** ********** ********** ********** 7Kb 1981 TTAGCAGAAA AAGTTAATTT ACTTAAGAGT ACTGAAAATG ATAATCCTAG GGATTTATAT M^£" * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 2 041 TCTGAAGTAA TTCCTCATAT AAAACAAGAA ATTTTAGAAG CATCAAAATC GTACGAACAC jyjg^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 2101 ACAAACCTTG AGCGTATAAA TGTAGAGAGA TGTCTAGTTA AAAGGGGACT TATGACTATT Mar ********** ********** ********** ********** ********** ********** 7Kb 2161 ACATATGGAG CAACTGAAAG AGGAATTTAT GATCAAATTG TATCTAAATT CTTTCAAAAA Mar ********** ********** ********** ********** ********** ********** 7Kb 2221 GATGAATGAA ACAAAACTGC AGGGCTTCAT TTTGTATGTA TAGATTCGGA TATAGCTCCA J 4 A R * * * * * * * * * * * * * * * * * * * I J l * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * 7Kb 22 81 AAAGATGTTG TATTTACTCA AAAGAATATA CTTCTTTGGA GTAAAATTAT TTATAATTCT Mar ********** ********** ********** * * * * * * * J J I * * * * * * * * * * * * * * * * * * * * * * 7Kb 2 341 TTATTTAAAA TTCATCCTAA TTTAAATACT CTAATGGTAT ATTTTAATAG TATAGTTAAA Mar ********** ********** ********** *•* ******** ********** ********** 7Kb 2401 GTATTATGTG AATTAGATTT ACCTGTTAAT TGAGTAACTC CTTATGGTTT AGTAATACAA Mar ********** ********** ********** ********** ********** ********** 7Kb 2461 CAAAAATATA ATAAATTTAC TAAATATAAT GAAACAACTT ATGTTGCATC TAAACGATAT Mar ********** ********** ********** ********** ********** ********** 69 7Kb 2 521 AAATTAGTAT TAAGAAAAGC AGATACTACT TCTATTTCTA AGCAGAAACA AATACAAGCG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 2581 TTTATACCCA ATTTTGTTCA CTCAATGGAT GGAAGTAATA TTGTTTTATT AATAAAAACA JyJar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * 7Kb 2 6.41 ATAAGAGATG AAGGACGCAA AATTAACTTT GCAAGTATTC ATGATTGTTT TGCAACTCAT jyj^£. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 2 7 01 GCAAATGATA CTGCATGACT ATCATGATAT GTAAAGCAAA GTTTTATACG TATTTATTCT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 27 61 GATTCTAGTT TTTTAAGAAG ATTTCATAAT TATATCCAAT TAAGAATCCA AGCTAAATAT Mar - * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 2 821 GGAGAAGATG CTCATTTTGA TAAAGATGGT GTATTAACCC ATGTAACTAT TGATAATGGT Max ********** ********** ********** ********** ********** ********** 7Kb 2 881 ACAGATATTC CTGATCGTAT TGATATACCT AAACCTCTTG TAGTAAATAA AGATAATAAA MaiT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 2 941 ATATATAAAG AAATTCTTCA TTCTGAGTAT TTTATTAACT AAATGATAAA TAATGATTGT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3 0 01 AAATAATATA AATATAGTGG GATTTAACAA AAAAGGGACA ACACAAATAG TGTATACCCT * * * * * * * * * * * * * * * * * * * * * * * * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3061 TTTTTTAAAG GGAATACACA AATTTGTGTA TTCCCTTTTT TTTT AAT TATTATACGG Ma3T * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p r j T ^ * * * * * * * * * * * * . * 7Kb 3118 TTAAGTAAAT ACACATGTAA TAACAAAGTA ATGTTTGTTT ATGATAGGTA GTACGTGCAT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 317 8 TGCACCCAGG ATCTACTTCC TCTCAAAATC TTATATTCTA CTGAAGGTTA AATCATAGAA M^IT * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 323 8 AAATACCTAT ATTTAATACT GTTAAGTATA TAATTTATAA TTATAATATT TAATATTAAA ********** ********** ********** ********** ********** ********** 7Kb 3298 TAAACCAAAA TTACACCTGT CCAGCTCGTG ACAAGTACTG TAACGAGATA GGTAAAACCC Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3 3 58 TCGAAGGGAG AAAAATAAAA AAAAAAGGGA ATGCAAAACC TGCATTCCCA T AAAAAA Mar * * * * * * * * * * * * * * * * * * * * * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * £ j ^ * * * * * * 7Kb 3 415 TAGGGATACA CAAATCTTTT GTGTATTCCT TTTTTTTTCT TTTTT-AAAG GTATACACAA 70 * * * * ^ * * * * * * * * r p * * * * * * * * * * * * * * * ^ * * * * * * * *'P(2 * * * * * r p * * * * * * r p * * * * * * * 7Kb 3 47 4 ATACTGTGTA T A C C — T T T T TTAAAGTATA CACAAATACT GTGTATATCT TATACCGAAA MaX" * * * * * * * * * * * * * * r p r p * * * * * * * * * * * * * * * . * * * * * * * * * * * * * * * * £ * * * * * * Q * * * * * 7Kb 3 532 AAGTATTAAT CAAGGTTAAC CCAAATAACA TTTATAATAT ATTAAACTAA ATAACAATCA Max" *_******** ********** *_******** ********** ********** ********** 7Kb 3 592 TTACCTCATA TTGTAAATAA TGGTGTGAGG AAAATAAAAT GTAATATTAG TTGTTATACT Ma-£- ********** ********** ********** ********** ********** ********** 7Kb 3 652 TTAGAATACT TTTAGATAAT CATATATA-A TAT GA TTATATTTAT TATAATGTAT * * * * * * * * * * * * * * * * * * * * * * * * * * Q * Q Q * G G G G G * G * * * * * * * * * * 7Kb 37 06 TCTTTTTCTA TATCAAGTTT CACCCACATC TGATACCAAG GCTTTTGAAA TTACCTCAAA MaX" ********** * * * * * * £ * * * ********** ********** ********** ********** 7Kb 37 66 ATTTTCGTTT ATACATAATG GTGAAGTATT AACTCATTTA CCTAAAGAGT ATAGTTTTTC * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3 82 6 A C G T T T A T C A T A T C C C G A T A T A A G A T T A T A T T T T T G A T A T T T A A C T G T A A C T G T T C C T A A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3 88 6 T T C C A A G C T A C G T C C T C A T C T T T C T T G A A A T A G T A C C C T A C T T T C T C C A T T A T A C A G T G C * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3 946 TTCGAAATCA TTAATATCTA GATTATGTGT GGTATTATCT TTGTTTTTAG TTATTCCTTT Jjjg^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 4006 ACATTTAATT TCTAATTTAC CACCAAAATC TAATAGATAT AATTTTCCTG ATATGAAAAT Max" ********** ********** ********** ********** ********** ********** 7Kb 4066 TGCACGTTTA ATTAATTGTC CGTTATATTC AGCTTTAAAT TTCCCACATC CTTCTCCAAT Mar" ********** ********** ********** ********** ********** ********** 7Kb 412 6 AAAAGCACTA TCTAATGGTT TTTCCACAAA TATTGAATCA GTATCAGTAT ATGCTGAATT Mar- ********** ********** ********** ********** ********** ********** 7Kb 4186 AATTATATGT TTATACATCA GAATTCTAGA TCATGATGCT GTTGCTGCAG CAATGGATGT Max" ********** ********** ********** ********** ********** ********** 7Kb 42 46 AGAGTTTATG ATAAATCCAT CATCTTTCTC TCCATCTAGG TAAGATAAAA GTTCGTAATT Mar" ********** ********** ********** ********** ********** ********** 7Kb 43 0 6 TTTTTCTGAT TGTGCACACA ATACAGGACA TGGTTTTTTA TCGTATCTCA CGTAATGTTT Mar ********** ********** ********** ********** ********** ********** 7 1 7Kb 43 66 ATCATCATCT ACTTCAAATT CGTGAATAAC GTTATTAGTT AATTGAATAT TATCTAATTC J^ g^  ********** ********** ******* * * * ********** ********** ********** 7Kb 442 6 ATTAGTAGTT AGCATTTTAA TTTCAGCGGC GCTATCATTC ATACCTGTTC TACCATATAA Mar ********** ********** ********** ********** ********** Q* ******** 7Kb 4486 AGTGTTCAAA AGCAATTTAG CCATCTGTTT CTTTGAACCT TTAGTATTAT CTTTAATTGA Mar ********** ********** ********** ********** ********** ********** 7Kb 4 546 GGCAAAATGC TCAATATATT CCTTAAAAGG ATCATCTCTT TTTTCGAAAA CGTAACTCTC Mar ********** ********** ********** ********** ********** ********** 7Kb 4606 AAGTACTTCT ATTTTATATC CGTACTCAAC TGCTAATTTT AATTCTTCAG AAAAATATCA Mar ********** ********** ********** ********** ********** ********** 7Kb 4666 CCCTGTTCAT TCACCAATGG GAAAAATTAA TTTTTGTACT CCATTGACTT TAACTCTGCA Mar ********** ********** ********** ********** ********** ********** 7Kb 472 6 AGGTAATACT GGAATATTTA TTGCTGGAGT AATAATTTTT GCTCTTACAA ATCCAAATAT Mar ********** ********** ********** ********** ********** ********** 7Kb 47 86 TTCATTTAAG TTTTTAGAAA AAGTATGGAC TGGTATACCA ACAGGCATTG GCATCATCAT Mar ********** ******£*** ********** ********** ********** ********** 7 Kb 4 84 6 TGCAGTAGGG TAAAGAC TAT TAAAATCAAA TGAGAAAATA TTATTTATAA TTGGAATAAA Mar Q* ******** ********** ********** ********** ********** ********** 7Kb 4906 AATTTCATTA CGTCCACCGA AATAAGCAGC TCTAACGGCT CTTTCAAGTC TTCCTTTTAG Mar ********** ********** ********** *** * * ***** ********** ********** 7Kb 4966. TTTAGGTAAA AAATATTTAG GAGTTAATTT TTTCTCCTCT TTTTTGTCAA ACAATGATAA J/[^ £. ********** ********** ********** ********** ********** ********** 7Kb 5 02 6 AATGTTATTA GGTGAAGTAG TTTCATTTTC TTCTTCTTCT TCTACTTCAT TTGGTAAAAA Mar ********** ******RP*** ********** ****** *^** * *** ********** 7Kb 508 6 TAACGTTCTA TAAACTAAGA AGGCCAATGC AGATGCGGTT TTAACTCTAG TAATATTTAT ]V[Qr * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . * * * * * * * * * * * * * * * * * * * * 7Kb 5146 TCTAAAAGTT GAATATGTAT TATTACTCAT TTCCATCATT AATTGATATA AAGCCTTTAT Mar ********** ********** ********** ********** ********** ********** 7Kb 52 0 6 ATCTTTTTCT AAGTAGATAA TGGTTTCTTT TCTCGTCGAT CATTTATTAG TATACATAGC Mar ********** **^******* **********,****^***** ********** ********** i 7Kb 52 66 AGCTCACTCA AATAGTGTAA TCATTTCACC TTTTTTGGGA TCAATATAAT ACTCATAATC 72 Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 532 6 AGGTATTAAT CCAACATATT CTAAATTATC TTTATTAACA AATTTGTAAG GAAATTTCCC Mar * * * * * A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *******rp** 7Kb 53 86 TTTTTTAGTA ATGATATTAT GATCTTTAGC TAATTTATCT AGTGATCCTG GTAGGAGTCT Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ^ * * * * * G * * * * * * * * * * * 7Kb 5446 ACATGAATCG GCAATTGTTA TAGTATGCCG CTCAGCTTTT CCTCCTTTTT TTTTAGGTTC Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *,* * * * * * * * * * * 7Kb 5506 AAATTTATAT GAAATTTTTA TTGAAAGAAT ATCAAGATCT TTAGATATTA TTTTTTCTAC Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 5566 AACAAATTCT TGAACTAATA TTTTGATAAT AAAATTAATA TCAAATTTGG AAAAATTATG Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 5 62 6 GCAATATACT GTATGTTTGT CATATTTTAA CATATCTTTT ATACATGCTA- GTAACATTTC Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 5 686 ACGTTGAGAA ATAAAATCAG AAATGTAATA AGTAAGAGAT TTATTTCCAT CATAAAATCC Mar * * * * * * * * * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * 7Kb 57 46 ACAAGCATAA GCAATCATCG TTGAATCACC ATTCCCAGTA GGAACTTGAA ATGTTTCAAT s Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 5806 ATCAAATGCT AATATTTTTT TATCTTGTCT TTTATCTCTT TTCGTTGGCT CTATATTTCC Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * Q * * * * * * * * * * * * * * * Q * * * * * * * * * * * * ^ * * 7Kb 58 66 AACTTTTTGA GGTGTTTCAA CATTGACTAA AGTATTATCA TAGTAATAAT AATACATATT Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 592 6 ATCATTAATT CAATGTCTTT TAAGACCATC ACCATGCTTA ACATCTTCAA ACTGGTACAA Mar * * * * * * * * * * * * * * ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 59 8 6 TAATCTATTA TCACGTGAAA TAACATCTAT AAGATGATTT ACACCATCTT TGATAATTTT Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ^ * * * * * * * * * * * * * * * * * * * * * 7Kb 6046 CACTTTAAGA ATAACATTTC CATTATCAAA AATACGAATG TTATTTTCTT TATCATGTTT Mar * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * 7Kb 6106 TGTTTCAACA CCATAAAATT TAAGATCCAT TGTGTGTGGA ACAAATTCTG GTCTAGAAAG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Q* * * * * * * * * 7Kb 6166 TTTAGACTTT CCTTTAGGAA TAGTAGTGTG TATATTAGTT TTTTCTTTTG CTATAGGATG jyj^-jT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 73 7Kb 622 6 AAAAACTGGA TTTTTAACTT TAAAATTAAG CTTTCTTAAT CTTATAAAAA GTTTATCAAC Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * 7Kb 6.2 8 6 TGGCTCTAAA TAGTATTTTT CAGAAAGAAA AATAATTCTA TCATAAATTT GTTGTAAGAT Mar * * **^** * * * ********** ********** ********** ********** ********** 7Kb 634 6 TATAAGTGGA TCAGTATTAA AAATGACTAA AAAGTGTCTG TCTACTGATA TATTAGTATT Mar * * * * * £ * * * * * * * * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 640 6 ACTATCAACA TTTCTAAGTA TAACCCCAAG TGCGTATGTA AAATTTTTAT CTAATTTCAT Mar ********** ********** ********** ********** ********** ********** 7Kb 646 6 GAATAGTTGA CGTAGTAATT CAACATTAAT ATTGAATTCA TTAATATTTA AAAGTGATTT Mar* ********** ********** ********** ********** ********** ********** 7Kb 652 6 TAAATTAAAA TCATTTTTTT CATATTCACC AATGTTACTT AAAATCATAT GTTTAGGTTC Mar ********** ********** ********** ********** ********** ********** 7Kb 65 8 6 GGTAGTACTT TCTCATTTTA ATTTAGGGTT TTCTTTCTTA AAATCTTTCA AATATTTAAT Mar ********** *****^**^* ********** ********** ********** ********** 7Kb 6 646 TTGATTTTGA ACTTCATTTG AAGAATTTTG TTCAATATTG GCTCGATTTC CACTCGTGGA j ^ ^ - j f * * * * * * * * * r p * * * * * * * * * * * * * * * * * * * Q * * * * * 7 ^ * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 6706 A A A T A A A C G T G C A G G A A A T T T C A T G T G A A A T G T G G T A A A A T A A A A T T T A A T C A T T A A A A A J^g^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 6766 TATATTCTGT GACCCCGATT CCTACTATTT TTCAGCTCTC CTCTTGACAG GTAATAGTAG Mar A********* ********** ********** ********** ********** ********** 7Kb 6826 GCTTCGCTCC CGGATCTGTC TGGAGATGTG CGTACTGTAG TCTTTGGATC TGCCCCAACT M ^ £ ~ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . 7Kb 6886 CTGTCAAGAG CCGCATAAGC GGGAAAGGTA AAACGCATGT TATAGGTTGA GGAAATAGTA j^gj-- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *(~J7-Y* * * * * * * * * * * * * * 7Kb 6946 A A A C A C T A T A G A C T G A T C T A A T G A T C A G T C A T A G T G T T A T A T A G G C T A T T T C C T C T A T A A J^gj- * * * * * * * * * * * * * * * * * * * * * * * * * * * Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 K b 7 0 0 6 G G G G G A T G T T C A T T T T A T A A A G T A G G G A T T T A T G T A C T C C C C C C A £4g2T * * * * * * * * * * * * * 7 ^ * * * / \ * * * * 7 ^ * * * *Q * * * * C C C G A * * * * * * * _ Figure 2-4. The sequence alignment of ORFl of Har-7.0 and maranhar. The stars represent the consensus sequence. ORFl> 7Kb MIKFYFTTFH MKFPARLFST SFTLNNTNII NEKDVKYITK NFLLENSNGF NLIKNIINSD Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb ETSEVKQKKI EVELNNIWHT. EITDILQKKR SLGLDAIGTS ILAKDFHNLI GDIENFINKG Mar *******]\j** * * * * * * * * * * . * * * * * * * * * * * * * * * * * * g * * * * * * * * * * * * * * * * * * * * * 7KB RTNKLPGVEY L K P S L I I S I V LGKVIPFSLR HSDILNQPTH S L F A E I G K T L KYQSIFELHH MAR * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * p * * * * * ' * * * * * * * * j * * * * * * * * * * * 7KB RIGLIQNRVE EIKNSTEKKL VSEFNKLTKV LDEYKNTLKN L E E L S G E S I I KVGLGFTALL Mair * * * * * * * * * * * * * * Y * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb S E T S E F Y S L E EQIIAKNKSI RYILPKNKLN TLINNITLMD T I E L P M I I P P LEWKIDDNEK Mar * * * * * * * * * * * * * * * * * * * * * * * J * * * * * J ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7KB I I E Y G G T I L N NKHRIRPLRT KSVENSDAND MTYNKELVDA VNFKSKIPYI I N L K L L D F I T Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *********rp * * * * * * * * * * 7Kb RDEFINRDKK DNVIIYKHIH PDSALLGEYM KDRKNPKISE ITTHNSKFLY H S S I I S I A K L Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb MKDVKEFYMT VFIDWRGRFY TSSCALNIQG GELARSLLLF KEGQKLNDIG LKALKIYTAN Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb AFGLDKRSKE ERLDWVEQNL HKIIDIDNYE IWREADEPLL F L A C A L E L K G YKEDPNFISH Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ^ j * * * * * * * * 7KB LPILMDATCN GLQHLSAMVN DFVLAEKVNL LKSTENDNPR DLYSEVIPHI KQEILEASKS Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . * * * * * * * * * * 7Kb YEHTNLERIN VERCLVKRGL MTITYGATER GIYDQIVSKF FQKDEWNKTA GLHFVCIDSD Mar ********** ********** ********** ********** * * * * * * * * * Y ********** 7Kb I A P K D W F T Q . KNILLWSKII YNSLFKIHPN LNTLMVYFNS IVKVLCELDL PVNWVTPYGL Mar ********** *****JJ**** ********** ********** ********** ********** 7Kb VIQQKYNKFT KYNETTYVAS KRYKLVLRKA DTTSISKQKQ IQAFIPNFVH SMDGSNIVLL Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb IKTIRDEGRK INFASIHDCF ATHANDTAWL SWYVKQSFIR IYSDSSFLRR FHNYIQLRIQ Ma.ir * N * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb AKYGEDAHFD KDGVLTHVTI DNGTDIPDRI DIPKPLWNK DNKIYKEILH SEYFIN *> Max" * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 76 Figure 2-5. The sequence alignment of 0RF2 of Har-7.0 and maranhar. The stars represent the consensus sequence.The horizontal lines represent the deleted amino-acids. 7Kb FVKLYDYIIH NYKNYHIRKR YWTEGVDSVL AKSIVEFNEN ICLPSTNVWK GLSYLKERKD Mar * * * * * * * * * * g * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb YGSILNYKQY KVTVTGLELS RGWREQFLVR SEGNYLAEFD NIDLNHTTND KNKTIGKCKI Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb ELKGGFDLLY LKGSIFIARK ILQGNYEAKF KGCGEGIFAS DLPKEVFISD TDTYASNIIH Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb KYMLIRSWSA TAAAISTSNI IFGDDKEGDL YSLLEYNKES QACLVPCPKK DYRVYHKDDD J^ Q^ ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb VEFEHIVNNT LQINDLENTT LMKIEAASDN MGTRGYLTNL LLKAMQKKSG KTNDKISAFH Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . * * * * * * * * * 7Kb EIYEKFPDDR KEFVYSELVE IKYGYEVALK LEESFYWGTW EGIPFILKQV GNVKVRCPLV Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb P I N I A P T I I K ARVFGFIENL NKSFTHVPIG VPMPMMMATP YLSNFDFSFI N N I I P I F I E N Ma.2r * * * * * * * * * * * * * * * * * * * * * * Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb RGGFYAARVA RELRGKLKPL FYKPTLKKEE KKDFLSLINN PSTTENEEEE EVENPLFLTR Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * j ^ * * * * * * * * * * * * j * * 7Kb YVLFALASAT KVRTINIRFT SYTNNSMEMM LQYLAKIDKE L Y I I T E K R T S WKNTYMAAWE * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *JyJ* * * * * * * * * * * 7Kb FLTIMEGKKP DIYYEYDPIL GVYELNDKNV FKYPFKGKKT IINHDKALKD LSGPLLRCSD Mar * * * * * * * * * * * * * * * * * * ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb AITITHREAK GGKKKPEFKY S I K I S L I D L D K S I I K E W F E Q V L I K I I F N I DFKSFNHCYV Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb THKDYKLMDK ICALLMERQS IFDSIYYTLS KNGDYFGCAY AIMTSDGNGT PVQFTEIDFA Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * j ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb LIKKDQRKDR KTPEINGVKQ PTEVNVLTND YYYYYMNDNI WHRKLGDGHK VDEFQYLLRN 7 7 Ma. IT * * * * * * r p * * * * ^ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * y * * * * * * * * * * * * * * * * * * 7Kb DRSIVDILHN VGDKIIKVKL IVNGNDFIRI NNEKDHKTEV GYFKLDMTHP VFEPRSLKSK Max * * * * * * * * * Y * * * * * * * * * * * * £ * * * * * * * * ^ * * * * * * * * * * * * * * * * * * * * * ^ * * * * * * 7 K b GKPITTHINT KEKAIPHFVP NKVKFNLKRL RIFLKDVPEL Y Y K E S L F I I R D Y I Q Q L I I L P Ma.3t * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * j £ * * * * * * * y * * * * * * * * * * * * * * * * * * * y * 7Kb DTNFIVLFHR DVSINTNSDV NRLIVGLAYT FNKDLKMFLQ RLLEVNINFE NINLLSKLNF Max" * * * *^j* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *.* * * * * * * * * * * * * * * * * * 7Kb DNKEYEGINS LIMHKPETTS EWKLKPNEKK FDKLYKIQNQ VENSSNQEIN ARNGSTSFLR Mar" * * * * * * * * * * * * * * * * * * * * **j^p****** * * * * * * * * * * * * * * * * j £ * p * * * * * * * * * * * 7Kb APFKMHFTTF YFKIM Max" * * * * * * * * * * * * * * * Figure 2-6. The sequence alignment of TIR of Har-7.0 and maranhar. The s t a r s represent the consensus sequence.The h o r i z o n t a l l i n e s represent the deleted residues. 7Kb 1 TGGGGGGAGT ACATAAATCC CTACTTTATA AAATGAACAT CCCCCTTATA GAGGAAATAG Mar _*******rpQ QQQ****Q** **rp****rp** *rp * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 61 C C TATATAAC ACTATGACTG ATCAT TAGAT CAGTCTATAG TGTTTTACTA TTTCCTCAAC Mar * * * * * * * * * * * * * * * * * Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *********rp 7Kb 121 CTATAACATG CGTTTTACCT TTCCCGCTTA TGCGGCTCTT GACAGAGTTG GGGCAGATCC Ma.ir Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 Kb 181 AAAGACTACA GTACGCACAT CTCCAGACAG ATCCGGGAGC GAAGCCTACT ATTACCTGTC Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 2 41 AAGAGGAGAG CTGAAAAATA GTAGGAATCG GGGTCACAGA ATATATTTTT AATGATTAAA Mar * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * i p * * * * * * * * * * * * * * * 7Kb , 3 01 T T T T A T T T T A CCACATTTCA CATGAAATTT CCTGCACGTT TATTTTC 79 Figure 2-7. The.sequence alignment of the i n t e r g e n i c regions of Har,-7.0 and maranhar. The s t a r s represent the consensus sequence. The h o r i z o n t a l l i n e s represent the deleted residues.The numbers at. the l e f t i n d i c a t e the p o s i t i o n s of the f i r s t residues i n that l i n e i n the whole nucleotide- sequence-of the Har-7.0 plasmid. 7Kb ATGATAAA TAATGATTGT Mai" * * * * * * * * * * * * * * * * * * 7Kb 3 0 01 AAATAATATA AATATAGTGG GATTTAACAA AAAAGGGACA ACACAAATAG TGTATACCCT Max" * * * * * * * * * * * * * * * * * * * * * * * * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3 061 TTTTTTAAAG GGAATACACA AATTTGTGTA T T C C C T T T T T T T T T AAT TATTATACGG Max" * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * r p r p ^ * * * * * * * * * * * * * 7Kb 3118 TTAAGTAAAT ACACATGTAA TAACAAAGTA ATGTTTGTTT ATGATAGGTA GTACGTGCAT i Mai" * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 317 8 TGCACCCAGG ATCTACTTCC TCTCAAAATC TTATATTCTA CTGAAGGTTA AATCATAGAA Mai" * * * * * * * * r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 323 8 AAATACCTAT ATTTAATACT GTTAAGTATA TAATTTATAA TTATAATATT TAATATTAAA Mai" * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 Kb 3298 TAAACCAAAA TTACACCTGT CCAGCTCGTG ACAAGT.ACTG TAAC GAGATA GGTAAAACCC Max- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3 358 TCGAAGGGAG AAAAATAAAA AAAAAAGGGA ATGCAAAACC TGCATTCCCA T AAAAAA Max" * * * * * * * * * * * * * * * * * * * * * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * ^ ^ * * * * * * 7Kb 3415 TAGGGATACA CAAATCTTTT GTGTATTCCT T T T T T T T T C T T T T T T - A A A G GTATACACAA Max" * * * * ^ * * * * * * * * < p * * * * * * * * * * * * * * * ^ * * * * * * * * r - p Q * * * * * f p * * * * * * r p * * * * * * * 7Kb 347 4 ATACTGTGTA T A C C — T T T T TTAAAGTATA CACAAATACT GTGTATATCT TATACCGAAA Max" * * * * * * * * * * * * * * r p r p * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * £ * * * * * * Q * * * * * 7Kb 3 532 AAGTATTAAT CAAGGTTAAC CCAAATAACA TTTATAATAT ATTAAACTAA ATAACAATCA Max" * * * * * * * * * * * * * * * * * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 35 92 TTACCTCATA TTGTAAATAA TGGTGTGAGG AAAATAAAAT GTAATATTAG TTGTTATACT Max" . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7Kb 3652 -• 80 Mar TTAGAATACT TTTAGATAAT CATATAGACG CCTGGGGGGG CT o -a u t-l •a <+-t o 0 0 cu oo c3 O ft T3 CU o o a CU oo " a c o a -a cu t CU 0 0 C o o CU o Cl o oo O H a o o cu H oo I tN CU l-H .g O PQ w bfl ,oo bp ' a cu tl—1 0 0 +-> oo X> rd rd rd rd LO m Cn—Cn Q — Q > J — J J H « — « J — J a—a EH ft-w> CO «—« Cn—Pu D — Q > — > J — J rd rd LTI rd rd in ro «—« M—ca a—a Q — Q Q — Q I—I H « « s—s ca—ca j — j ft—ft ft ft 2 — 2 J — J W H rd rd o co Si rd rd o U rd 2 a—a > < — > H «—« o—or o—a H 1—I > — > J J o—o > H >H ft ft EH EH > — > S 3 rd rd rd rd in in co co 0 — CD > H — > H EH EH 1— I — I — I EH EH s—s J — J C D — C D OS—Pi « — « > — > J — J C J — C J Pi—Pi ca—ca rd rd rd rd tN . CNJ I—I H > > ra—ca CO— w >H >H j — j a — Q ft—ft a—a Q — a a—a w — p a EH EH o o — c o « — « J — J J — « — « W — H > — > Pn—Cn Q — Q co—co • J — J a—rn o—o j — j C D — C D a—a C J — C J EH EH <—< a — Q 2 — 2 J — J M M ft ft J — J rd rd rd rd ro ro H r H c a — w J H H 3 = 1 Pn—Pn J — J J — J ft—ft H — H pa—pa P U — P i 3 — 3 43 a—a i—i—i—i Cn—Pn > H — X ca—ca c o — c o a—a J — J H 1—I ca—ca «—« > H > H H H « « a—a D — Q ^—« J — J ft—ft «—« ft—ft H H D — Q I—I M Pi—Cel D — Q ft—ft M — H Q — Q E H — E H C D — C D a—a Q — O rd rd in rd rd in Q — Q c o — c o > H — > H H 1—I Pi Pi M 1—I Cn—Cn c o — c o o—a « — K >—> > H > H 3 — 2 c o — c o . s—s <—< EH EH a—a E H - - E H <--< Cn- -Cn U - - C J Q - — Q a— -a H — — M c o - -co <--< Pn- -Pn a- -a M — — M -« P U - - P U rd rd rd rd (N CM rH H a--a c o - -co C D - - C D Q - - Q 2 - - 2 c o - -co Cn- -Cn a--a ft-ft Cn- -Cn <- -< O--a hH — — M o- -a «--« o--o «--« c o - -co rd rd rd rd 0 0 oo CM CN Xi U 2 rd r- 2 82 Literature cited Akins R A , Kel ley R L and Lambowitz A M 1986. Mitochondrial plasmids of Neurospora: integration into mitochondrial D N A and evidence for reverse transcription in mitochondria. Ce l l 47: 505-516 Akins R A , Grand D M , Stohl L L , Bottorff D A , Nargang F E and Lambowitz A M 1988. Nucleotide sequence of the Varkud mitochondrial plasmid of Neurospora and synthesis of a hybrid transcript with a 5' leader derived from mitochondrial R N A . J. M o l . B i o l . 204: 1-25 Akins R A , Kelley R L and Lambowitz A M 1989. Characterization of mutant mitochondrial plasmids of Neurospora species that have incorporated t R N A s by reverse transcription. M o l . Ce l l . B i o l . 9: 678-691 Almasan A and Mishra N C 1990. Characterization of a novel plasmid-like element in Neurospora crassa derived mostly from the mitochondrial D N A . Nucleic A c i d Res. 18: 5871-5877 Arganoza M T, M i n J, H u Z , Akins R A 1994. Distribution of seven homology groups of mitochondrial plasmids in Neurospora: evidence for widespread mobility between species in nature. Curr Genet 26: 62-73 Beadle G W 1945. Genetics and metabolism in Neurospora. Physiol. Rev. 25: 643-663 Bedinger P, de Hostos E L , Leon P and Walbot V 1986. Cloning and characterization of a linear 2.3 kb mitochondrial plasmid in maize. M o l . Gen. Genet. 205: 206-212 Bertrand H , Chan B S-S and Griffiths A J F 1985. Insertion of a foreign nucleotide sequence into mitochondrial D N A causes senescence in Neurospora intermedia. Ce l l 41: 877-884 Bertrand H and Griffiths A J F 1989. Linear plasmids that integrate into mitochondrial D N A in Neurospora. Genome 31: 155-159 Bertrand Ff, Griffiths A J F , Court D A and Cheng C K 1986. A n extrachromosomal plasmid is the etiological precursor of k a l D N A insertion sequences in the mitochondrial chromosome of senescent Neurospora. Cel l 47: 829-837 Bistis G N 1983. Evidence for diffusible, mating type-specific trichogyne attractants in Neurospora crassa. Exper. M y c o l . 7: 292-295 Bistis G N 1986. Autolysis and development of Neurospora crassa. Fungal Genetics Newsletter 33: 15 B r o d a P 1979. Plasmids. W H Freeman, New York 83 Caten C E 1972. Vegetative incompatibility and cytoplasmic infection in fungi. J. Gen. Microbiol . 72: 221-229 Chan B S-S, Court D A , Vierula P J and Bertrand H 1991. The kalilo linear senescence-inducing plasmid of Neurospora is an invertron and encodes D N A and R N A polymerases. Curr. Genet. 20: 225-237 Chevangeon J and Digbeau S 1960. U n second facteur cytoplasmique infectant chez Pestalozzia annulata. C.R. Hebd. Seances Acad. Sci. Ser. D 251: 2247-2249 Chevangeon J and Lefort C 1960. Sur l'apparition regulier d'un "mutant": infectant chez un champignon du genre Pestalozzia. C.R. Hebd. Seances Acad. Sci. Ser. D 250: 22247-2249 Coll ins R A and Lambowitz A M 1983. Structural variations and optional introns in the mitochondrial D N A s of Neurospora strains isolated from nature. Plasmid 9: 53-70 Coll ins R A and Saville B J 1990. Independent transfer of mitochondrial chromosomes and . plasmids during unstable vegetable fusion in Neurospora. Nature 345: 177-179 Coll ins R. A . , Stohl L . L . , Cole M . D . and Lambowitz A . M . 1981. Characterization of a novel plasmid D N A found in mitochondria of Neurospora crassa. Ce l l 24:443-452. Court D A and Bertrand H 1992. Genetic organization and structural features of the senescence inducing linear mitochondrial plasmid maranhar of Neurospora crassa. Curr. Genet. 22:385-397 Court D A and Bertrand H 1993. Expression of the open reading frames of a senescence-inducing linear mitochondrial plasmid of Neurospora crassa. Plasmid 30: 51-66 Court D A , Griffiths A J F, Kraus S R, Russell A J and Bertrand H 1991. A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India. Curr. Genet. 19: 129-137 Davis R H and de Serres F J 1970. Genetic and microbiological techniques for Neurospora crassa. Methods Enzymol. 17A: 79-143 Debets F , Yang X and Griffiths A J F 1994. Vegetative incompatibility in Neurospora: its effects on horizontal transfer of mitochondrial plasmids and senescence in natural populations. Curr. Genet. 26 1994. 113-119 Debets F, Yang X and Griffiths A J F 1995. The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia Curr. Genet. 29 1995.44-49. Dujon A and Belcour L 1989. Mitochondrial D N A instabilities and rearrangements in yeasts 84 and fungi. In Mobi le D N A , ed. D E Berg, and M M Howe. Washington D C : American Society for Microbiology Erickson L , Kemble R and Swanson E 1989. The Brassica mitochondrial plasmid can be sexually transmitted. Pollen transfer of a cytoplasmic genetic element. M o l . Gen. Genet. 218:419-422. Escote L J, Gabay-Laughnan S J and Laughnan J R 1985. Cytoplasmic reversion to fertility in cms-S maize need not involved loss of linear mitochondrial plasmids. Plasmid 14: 264-267 Escote-Carlson L J, Gabay-Laughnan S J and Laughnan J R 1988. Reorganization of mitochondrial genomes of cytoplasmic revertants in cms-S inbred line W F 9 in maize. Theor. A p p l . Genet. 75: 659-667 Esser K , Kuck U , Lang-Hinrichs C, Lemke P, Osiewacz H D , Stahl U and Tudzynski P 1986. Plasmids of eukaryotes. Fundamentals and applications. Springer, N e w York Fec ikovaH. 1992 Mitochondrial plasmids. Biologia 47:507-514. Fleming R and Person C O 1978. Disease control through the use of multilines: a theoretical contribution. Phytopathology 68: 1230-1233 Fleming R and Person C O 1982. The consequences of polygenic determination of resistance and aggressiveness in nonspecific host-parasite relationships. Can. J. Plant Pathol. 4: 89-96 Fujimura H , Yamada T, Hishinuma F and Gunge N 1988. D N A replication in vivo of linear D N A killer plasmids p G K L l and p G K L 2 in Saccharomyces cerevisiae. F E M S Microbiol . Lett. 49: 441-444 Gilbert W and Dressier D 1968. D N A replication: the rolling circle model. Co ld Spring Harbour, Symp. Quant. B i o l . 33: 473-478 Gil lham N W 1978. Organelle Heredity. Raven Press, New York Griffiths A J A 1992. Fungal senescence. Annu. Rev. Genet. 26: 351-372 Griffiths A J F and Bertrand H 1984. Unstable cytoplasms in Hawaiian strains of Neurospora intermedia. Curr. Genet. 8: 387-398 Griffiths A J F and Bertrand H 1986. Expression of senescence in Neurospora intermedia. Can. J. Genet. Cytol . 28: 459-467 Griffiths A J F , Kraus S R and Bertrand H 1986. Expression of senescence in Neurospora intermedia. Can. J. Genet. Cytol . 28: 459-467 85 Griffiths A J F, Kraus S R, Barton R, Court D A , Myers C J and Bertrand H 1990. -Heterokaryotic transmission of senescence plasmid D N A in Neurospora. Curr. Genet. 17: 139-145 Griffiths A J F , Yang X , Barton R and Myers C J 1992. Suppression of cytoplasmic senescence in Neurospora. Curr. Genet; 21: 479-484 Griffiths A J F 1995. Natural plasmids of filamentous fungi. Microbiol . Rev., Dec. 1995, p. 673-685 Griffiths A J F and Yang X 1993. Senescence in natural populations of Neurospora intermedia. M y c o l . Res. 97 (11). 1993. 1379-1387. Griffiths A J F and Yang X 1995. Recombination between heterologous linear and circular mitochondrial plasmids in the fungus Neurospora. M o l . Gen. Genetics 249 ( 1 ). 1995.25-36 Gunge N and Sakaguchi K 1981. Intergeneric transfer of deoxyribonucleic acid killer plasmids, p G K L l and p G K L 2 , from Kluyveromyces lactis into Saccharomyces _ cerevisiae by cell fusion. J. Bacteriol. 147: 155-160 Gunge N , Murata K and Sakaguchi K 1982. Transformation of Saccharomyces cerevisiae with linear D N A killer plasmids from Kluyveromyces lactis. J. Bacteriol. 151: 462-464 Gunge N , Tamaru A , Ozawa F and Sakaguchi K 1981. Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character. J. Bacteriol. 145:382-390 Gunge N and Yamane C 1984. Incompatibility of linear D N A killer plasmids p G K L l and p G K L 2 from Kluyveromyces lactis with mitochondrial D N A from Saccharomyces cerevisiae. J. Bacteriol. 159: 533-539 Hamilton W D , Axelrod R and Tanese R 1990. Sexual reproduction as an adaptation to resist parasites (a review). Proc. Natl. Acad. Sci. U S A 87: 3566-3573 Hermanns J, Asseburg A and Osiewacz H D 1995. Evidence for giant linear plasmids in the ascomycete Podospora anserina. Curr. Genet. 27(4). 379-386 Hermanns J and Osiewacz H D 1992. The linear mitochondrial plasmid p A L 2 - l of a long-lived Podospora anserina mutant is an invertron encoding a D N A and R N A polymerase. Curr. Genet. 22:491-500. Hermanns J and Osiewacz H D 1994. Three mitochondrial unassigned open reading frames of Podospora anserina represent remanants of a viral type R N A polymerase gene. 86 Curr. Genet. 25: 150-157 Hickey D H and Rose M R 1988. The Evolution of Sex, pp. 161-175, edited by R E Michod and B R Levin. Sinauer Associates, Sunderland, Mass. Hishinuma H , Nakamura K , Hiraj K , Nishizawa R, Gunge N and Maeda T 1984. Coning and nucleotide sequences of the linear D N A killer plasmids from yeast. Nucleic A c i d Res. 12:7581-7597 Jung G , Leavit M C and Ito J 1987. Yeast killer plasmid p G K L l encodes a D N A polymerase belonging to the family B D N A polymerases. Nucleic A c i d Res. 15: 9088 Kawano, S, Takano H , M o r i K and Kuroiwa T 1991. A mitochondrial plasmid that promotes mitochondrial fusion in Physarum polycephalum. Protoplasma 160: 167-169 Kemble R J and Mans R J 1983. Examination of the mitochondrial genome of revertant progeny from S-cms maize with cloned S - l and S-2 hybridization probes. J. M o l . A p p l . Genet. 2:161-171 Kemble R J and Thompson R D 1982. SI and S2, the linear mitochondrial D N A s present in a male-sterile line of maize, possess terminal attached proteins. Nucleic A c i d Res. 10:8181-8190 Kemken F, Meinhardt F and Esser K 1989. In organello replication and viral affinity of linear, extrachromosomal D N A of the ascomycete Ascobolus immersus. M o l . Gen. Genet. 218:523-530 . , . Kemken F . 1995. Plasmid D N A in mycelial fungi, p. 169-187. In U . Kuck ( ed.) , The Mycota. II. Genetics and biotechnology. Springer-Verlag K G , Heidelberg, Germany. Kemken F, Hermanns J and Osiewacz 1992 Evolution of linear plasmids. J. M o l . Evo l . 35: 502-513. Kennell J C , Wang E and Lambowitz A M . 1994. The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo. M o l . Ce l l . B i o l . 14:3094-3107. K ikuch i Y , Hirai K and Hishinuma F 1984. The yeast linear D N A killer plasmids, p G K L l and p G K L 2 , possess terminally attached proteins. Nucleic A c i d Res. 12: 5685-5692 K i m B D , Mans R J, Conde M F and Levings III C S 1982. Physical mapping of homologous segments of mitochondrial episomes from S male-sterile maize. Plasmid 7: 1-14 Konz C, Sumegi J, Udvardy A , Racsmany M and Dudits D 1981. Cloning of m t D N A fragments homologous to mitochondrial S2 plasmid-like D N A in maize. M o l . Gen. ( 87 Genet. 183: 449-458 KuckTJ 1989. Mitochondrial D N A rearrangements in Podospora anerina. Exp. M y c o l . 13: 111-120 Kuiper M T R and de Vries H 1985. A recombinant plasmid carrying the mitochondrial plasmid sequence of Neurospora intermedia LaBelle yields new plasmid derivatives in Neurospora crassa transformants. Curr. Genet. 9: 471-477 Kuiper M T R and Lambowitz A M 1988. A novel reverse transcriptase activity associated with mitochondrial plasmids of Neurospora. Cel l 55: 693-704 Kuzmin E V and Levchenko I V 1987. SI plasmid from cms-S maize mitochondria encodes a viral type D N A polymerase. Nucleic A c i d Res. 15: 6758 Kuzmin E V , Levchenko I V and Zaitseva G N 1988. S2 plasmid from cms-S-maize mitochondria potentially encodes a specific R N A polymerase. Nucleic A c i d Res. 16: 4177 Lambowitz A M 1989. Infectious introns. Cel l 56: 323-326 Lambowitz A M , Akins R A , Garriga G , Henderson M , Kubelik A and Maloney K A 1985. Mitochondrial introns and mitochondrial plasmids of Neurospora. In: Quagliariello E, Slater E C, Palmeieri F, Saccone C and Kroon A M . Achievements and perspectives in mitochondrial research,'Vol.11: Biogenesis, Amsterdam: Elsevier Science Publishers pp. 237-247 Lazarus C M , Earl A J, Turner G and Kuntzel H 1980. Amplification of a mitochondrial D N A sequence in the cytoplasmically inherited 'ragged' mutant of Aspergillus amstelodami. Eur. J. Biochem. 106: 633-641 Lazarus C M and Kuntzel H 1981. Anatomy of amplified mitochondrial D N A in 'ragged' mutants of Aspergillus amstelodami: excision points within protein genes and a common 215 bp segment containing a possible origin of replication. Curr. Genet. 4: 99-107 Leving III C S , Sederoff R R, H u W W L and Timothy D H 1984. In: Citerri O, Owe L III (eds ) Structure and function of plant genome. Life Sciences 63, Plenum, N e w York, pp 363-371. L i Q and Nargang F E 1993. Two Neurospora mitochondrial plasmids encode D N A polymerases containing motifs characteristic of family B D N A polymerases but lack the sequence asp-thr-asp. Proc. Natl . Acad. Sci. U S A 90: 4299-4303 Lindberg G D 1959. A transmissible disease of Helminthosporium victoriae. Phytopathology 49: 29-32 88 Lonsdale D M , Thompson R D and Hodge T P 1981. The integrated forms of the SI and S2 D N A elements of maize sterile mitochondrial D N A are flanked by a large repeated sequence. Nucleic A c i d Res. 9: 3657-3668 Maheshwari R, Pandit A and Kannan B 1994. Senescence in strains of Neurospora from southern India. Fungal Genetics Newsletter. Maleszka R 1992. Electrophoretic profiles of mitochondrial plasmids in Neurospora suggest they replicate by a rolling circle mechanism. Biochem. Biophys. Res. Commun. 186: 1669-1673 Mannella C A and Lambowitz A 1978. Interaction of wi ld type and poky mitochondrial D N A in heterokaryons of Neurospora. Biochem. Biophys. Res. Commun. 80: 673-679 Marcou D 1961. Notion de longevite et nature cytoplasmique du determinant de senescence chez quelques champignons. Ann . Sci. Nat. Bot. 11: 653-764 Mather K and Jinks J L 1958. Cytoplasm in sexual reproduction. Nature (American) 182: 1188-1190 M a y G and Taylor J W 1989. Independent transfer of mitochondrial plasmids in Neurospora crassa. Nature 339: 320-322 Marcenko-Kuehn M , Yang X , Debets F arid Griffiths A J F 1994. A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma. Curr. Genet. 26 (4). 1994. 336-343 Meinhardt F, Kempken J and Esser K 1990. Linear plasmids among eukaryotes: Fundamentals and applications. Curr. Genet. 17: 89-95 Miche l F and Lang F 1985. Mitochondrial class II introns encode proteins related to reverse transcriptases of retroviruses. Nature ( London) 316:641-643. Mitchel l M B and Mitchell H K 1952. A case of maternal inheritance in Neurospora crassa. Proc.Natl. Acad. Sci. U S A 38: 442-449 Miyashita S, Hirochitka H , Ikeda J and Hashiba T 1990. Linear plasmid D N A s of the plant pathogenic fungus Rhizoctoina solani with unique terminal structures. M o l . Gen. Genet. 220: 165-171 Myers C J, Griffiths A J F and Bertrand H 1989. Linear kalilo D N A is a Neurospora mitochondrial plasmid that integrates into the mitochondrial D N A . M o l . Gen. Genet. 220:113-120 Nagy F, Torok I and Mal iga P 1981. Extensive rearrangements in the mitochondrial D N A in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana. M o l . Gen. Genet. 89 183:437-439 Nargang F E 1985. Fungal mitochondrial plasmids. Exp. M y c o l . 9: 285-293 Nargang F E , Be l l J B , Stohl L L , and Lambowitz A M 1984. The D N A sequence and genetic organization of a Neurospora mitochondrial plasmid suggest a relationship to introns and mobile elements. Cel l 38: 441-458 Nargang F E , Pande S, Kennell J C , Akins R A and Lambowitz A M 1992. Evidence that a 1.6 kilobase region of Neurospora m t D N A was derived by insertion of part of the LaBel le mitochondrial plasmid. Nucleic A c i d Res. 20: 1101-1108 Natvig D O, May G and Taylor J W 1984. Distribution and evolutionary significance of mitochondrial plasmids in Neurospora species. J. Bacteriol. 159: 288-293 Newcombe K D and Griffiths A J F 1972. Adjustable platforms for collecting asci. Neurospora Newsletter 20: 2 N i w a O, Sakaguchi K and Gunge N 1981. Curing of the killer deoxyribonucleic acid plasmids of Kluyveromyces lactis. J. Bacteriol. 148: 988-990 Oeser B 1988. S2 plasmid from Zea mays encodes a specific R N A polymerase: an alternative alignment. Nucleic Acids Res. 16: 8729 Oeser B , Rogmann-Backwinkel P and Tudzynski P 1993. Interaction between mitochondrial D N A and mitochondrial plasmids in Claviceps purpurea: analysis of plasmid-homologous sequences upstream of the L r R N A gene. Curr. Genet. 23:315-322. Oeser B and Tudzynski P 1989. The linear mitochondrial plasmid p C I K l of the phytopathogenic fungus Claviceps purpurea may code for a D N A polymerase and an R N A polymerase. M o l . Gen.' Genet. 217: 132-140 Osiewacz H D and Esser K 1984. The mitochondrial plasmid of Podospora anserina: a mobile intron of a mitochondrial gene. Curr. Genet. 8: 299-305 Osiewacz H D , Hermanns J, Marcou D , Triffi M and Esser K 1989. Mitochondrial D N A rearrangements are correlated with a delayed amplification of the mobile intron ( p l D N A ) in a long-lived mutant of Podospora anserina. Mutat. Res. 219: 9-15 Paillard M , Sederoff and Levings III C S L 1985. Nucleotide sequence of the S- l mitochondrial D N A from the S cytoplasm of maize. E M B O J. 4: 1125-1128 Pande S, Lemire E G and Nargang F E 1989. The mitochondrial plasmid from Neurospora intermedia strain LaBel le - lb contains a long open reading frame with blocks of amino acids characteristic of reverse transcriptases and related proteins. Nucleic Acids Res. 17: 2023-2042 90 Pandit A , Kannan B and Maheshwari R 1994. Presence of nuclei carrying recessive lethal gene in a wild-isolate of Neurospora. Fungal Genetics Newsletter. Perkins D D 1974. The manifestitation of chromosome rearrangements in unordered asci of Neurospora. Genetics 77: 459-489 Perkins D D , Radford A , Newmeyer D and Bjorkman M 1982. Chromosomal loci o f Neurospora crassa. Fungal Genetics Newsletter 33: 15 Perkins D D and Turner B C 1988. Neurospora from natural populations: toward the population biology of a haploid eukaryote. Exp. M y c o l . 12:91-131 Perkins D D , Turner B C and Barry E G 1976. Strains of Neurospora collected from nature. Evolution 30: 281-313 Pring D R, Leving C S, H u W W L and Timothy D H 1977. Unique D N A associated with mitochondria in the "S" type cytoplasm of male-sterile maize. Proc. Natl . Acad. Sci . U S A Rieck A , Griffiths A J F and Bertrand H 1982. Mitochondrial variants of Neurospora intermedia from nature. Can. J. Genet. Cytol . 24: 741-759 Robison M M R, Royer J C and Horgen P A 1991. Homology between mitochondrial D N A of Agaricus bisporus and an internal portion of a linear mitochondrial plasmid of Agaricus bitorquis. Curr. Genet. 19: 495-502 Rohe M , Schrage K and Meinhardt F 1991. The linear plasmid p M C 3 - 2 from Morchella conica is structurally related to adenoviruses. Curr. Genet. 21:173-176 Sakaguchi K 1990. Invertron, a class of structurally and functionally related genetic elements that includes linear D N A plasmids, transposable elements, and genomes of adeno-type viruses Microbiol . Rev., Mar. 1990. p.66-74 Salas M 1988. Initiation of D N A replication by primer proteins: bacteriophage d>29 and its relatives. Curr. Topics Microbiol . Immunol. 136: 71-88 Samac D A and Leong S A . 1989 Chraracterization of the termini of linear plasmids from Nectria-haematococca and their use in construction of an autonomously replicating transformation vector. Curr. Genet. 16(3). 1989. 187-194. Saville B J and Collins R A 1990. A site-specific self-cleavage reaction performed by a novel R N A in Neurospora mitochondria. Cel l 61: 685-696 Schardl C L , Lonsdale D M , Pring D R and Rose K R 1984. Linearization of maize mitochondrial chromosomes by recombination with linear episomes. Nature 310: 292- 296 91 Schardl C L , Pring D R and Lonsdale D M 1985. Mitochondrial D N A rearrangements associated with fertile revertants of S-type male-sterile maize. Cel l 43: 361-368 Schulte U and Lambowitz A M 1991. The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel D N A polymerase that may be derived from reverse transcriptase. M o l . Cel l . B i o l . 11: 1439-1458 Sederoff R R, Ronld P, Bedinger P, R iv in C, Walbot V , Bland M and Levings III C R 1986. Maize mitochondrial plasmid S - l sequences share homology with chloroplast gene psbA. Genetics 113: 469-482 Seidel-Rogol B , K i n g J and Bertrand H 1989. Unstable mitochondrial D N A in natural-death mutants of Neurospora crassa. M o l . Cel . B i o l . 9: 4259-4264 Sheng T 1951. A gene that causes natural death in Neurospora crassa. Genetics 36: 199-212 Sinclair J H , Stevens B J, Sanghavi P and Rabinowitz K 1967. Mitochondrial-satellite and circular D N A filaments in yeast. Science 156: 1234-1239 Sor F and Fukuhara H 1985. Structure of a linear plasmids of the yeast Kluyveromyces lactis: compact organization of the killer genome. Nucleic A c i d Res. 9: 147-155 Sor F , Wesolowski M and Fukuara H 1983. Inverted terminal repetitions of the two linear D N A associated with the killer character of the yeast Kluyveromyces lactis. Nucleic A c i d R e s . l l : 5037-5044 Southern E M 1975. Detection of specific sequences among D N A fragments separated by gel electrophoresis. J. M o l . B i o l . 98: 503-517 Stahl U , Kuck U , Tudzynski P and Esser K 1980. Characterization and cloning of plasmid-like D N A of the ascomycete Podospora anserina. M o l . Gen. Genet. 178: 639-646 Stam J C, Kwakman J, Meijer M and Stuitje A R 1986. Efficient isolation of the linear D N A killer plasmids Kluyveromyces lactis: evidence for location and expression in the cytoplasm and characterization of their terminally bound proteins. Nucleic A c i d Res. 14:6871-6884 Stark M J A and Boyd A 1986. The killer toxin Kluyveromyces lactis: characterization of the toxin subunits and of the genes which encode them. E M B O J. 5: 1995-2002 Stark M J R, Mileham A J, Romanos M A and Boyd A 1984. Nucleotide sequence and transcription analysis of a linear D N A plasmid associated with the killer character of the yeast Kluyveromyces lactis. Nucleic A c i d Res. 12: 6011 -6030 92 Stillman B W 1983. The replication of adenovirus D N A with purified proteins. Ce l l 35:7-9 Sugisaki Y , Gunge N , Sakaguchi K , Yamasaki M and Tamura G 1985. Transfer of D N A killer plasmids from Kluyveromyces lactis to Kluyveromyce fragilis and Candida pseudotropicalis. J. Bacteriol. 164: 1373-1375 Taylor J W , Smolich B D and M a y G 1985. A n evolutionary comparison of homologous mitochondrial plasmid D N A s from three Neurospora species. M o l Gen. Genet. 201: 161-167 Tokunaga M , Wada N and Hishinuma F 1987. Expression and identification of immunity determinants on linear D N A killer plasmid p G K L l and p G K L 2 in Kluyveromyces lactis. Nucleic A c i d Res. 15: 1031-1046 Tommasino M , Ricc i S and Galeotti C L 1988. Genome organization of the killer plasmid p G K L 2 from Kluyveromyces lactis. Nucleic A c i d Res. 16: 5863-5877 Tudzynski P and Esser K 1986. Extrachromosomal genetics of Claviceps purpurea. Curr. Genet. 10:463-467. Turker M S, Domenico J M and Cummings D J 1987a. Excision-amplification of mitochondrial D N A during senescence in Podospora anserina. A potential role for an 11 base-pair consensus sequence in the excision process. J. M o l . B i o l . 198: 171-185 Turmel M , Bellemare G , Lee R W and Lemieux C 1986. A linear D N A molecule of 5.9 kilobase-pairs is highly homologous to the chloroplast D N A in the green alga Chlamydomonas moewasii. Plant M o l . B i o l . 6: 313-319 Turner B C and Perkins D D 1979. Spore killer, a chromosomal factor in Neurospora that kills meiotic products not containing it. Genetics 93: 587-606 Vestvaber D and Schatz G 1989. DNA-protein conjugates can enter mitochondria via the protein import pathway. Nature 338: 170-172 Vickery D and Griffiths A J F 1993. Transcription of the kalilo linear senescence plasmid from Neurospora intermedia. Plasmid 29: 180-192 Vierula J P and Bertrand H 1992. A deletion derivative of the kalilo senescence plasmid forms hairpin and duplex D N A structures in the mitochondria of Neurospora. M o l . Gen. Genet. 234: 361-368 Vierula P J , Cheng C K , Court D A and Bertrand H 1990. The kalilo senescence plasmid of Neurospora intermedia has covalent-linked 5' terminal proteins. Curr. Genet. 17: 195-201 Vogel FI J 1956. A convenient medium for Neurospora. Microbiol . Genet. Cel l . 13: 42-43 93 Wang H , Kennel J C , Kuiper M T R, Sabourin J R, Saldanha R and Lambowitz A M 1992a. The Mauriceville plasmid forms hairpin and duplex D N A structures in the mitochondria of Neurospora. M o l . Gen. Genet. 234: 361-368 Wang H , Kennel J C , Kuiper M T R, Sabourin J R, Saldanha R and Lambowitz A M 1992b. The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins c D N A synthesis at the 3' end of template R N A . M o l . Ce l l . B i o l . 12:5131-5144. Wang H and Lambowitz A M 1993. The Mauriceville plasmid reverse transcriptase can initiate c D N A synthesis de novo and may be related to reverse transcriptase and D N A polymerase progenitor. Cel l 75:1071-1081 Wei Y , Yang X , Griffiths A J F 1996 Structure of a Gelasinospora linear plasmid closely related to the kalilo plasmid of Neurospora intermedia Curr Genet 29: 150-158 Weissinger A K , Timothy D H , Levings III C S, H u W W L and Goodman M M 1982. Unique plasmid-like mitochondrial D N A s from indigenous maize races of Latin America. Proc; Natl . Acad. Sci. U S A 79: 1-5 Wesolowski M , Alger i A and Fukuhara H 1982a. Ki l l e r D N A plasmids of the yeast Kluyveromyces lactis. III. Plasmid recombination. Curr. Genet. 5: 205-208 Wesolowski M , Alger i A , Goffrini P and Fukuhara H 1982b. Ki l l e r D N A plasmids of the yeast Kluyveromyces lactis. I. Mutations affecting the killer phenotype. Curr. Genet. 5: 191-197 Wesolowski M , Dumazert P and Fukuhara H 1982c. Ki l l e r D N A plasmids of the yeast Kluyveromyces lactis. II. Restriction endonuclease maps. Curr. Genet. 5: 199-203 Wilson D W and Meacock P A 1988. Extranuclear gene expression in yeast: evidence for a plasmid encoded R N A polymerase of unique structure. Nucleic Acids Res. 16: 8097-8112 Yang X and Griffiths A J F 1993a. Plasmid diversity in senescent and nonsenescent strains of Neurospora. M o l ; Gen. Genetics 237 (1-2). 177-186. Yang X and Griffiths A J F 1993b. Male transmission of linear plasmids and mitochondrial D N A in the fungus Neurospora. Genetics 134 (4). 1993. 1055-1062 Yang X and Griffiths A J F 1993c. Plasmid suppressors active in the sexual cycle of Neurospora intermedia. Genetics 135 (4). 1993. 993-1002 ' 94 Yang X 1991. Senescence in Neurospora. M.Sc thesis. The University of British Columbia, BC, Canada 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0088437/manifest

Comment

Related Items