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Characterization of viruses causing lysis of a toxic bloom-forming alga, Heterosigma akashiwo (Raphidophyseae) Tai, Vera 2002

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CHARACTERIZATION OF VIRUSES CAUSING LYSIS OF A TOXIC B L O O M -FORMING A L G A , HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) by V E R A TAI BSc. (Hon.) University of New Brunswick, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE i n THE F A C U L T Y OF G R A D U A T E STUDIES Department of Botany We accept this thesis as conforming to the required standard The University of British Columbia October 2001 © VeraTai , ,2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT Two viruses, HaRNAV and HaNIV, infecting the toxic bloom-forming alga Heterosigma akashiwo (Hada) Hada ex Hada et Chihara were characterized. HaRNAV is a 25 nm, single-stranded R N A virus with a genome size of approximately 9100 nucleotides. The virus particle has a lipid component as indicated by its sensitivity to chloroform and contains at least 5 structural proteins ranging in apparent size from 24 to 34 kDa. This is the first report of a single-stranded RNA virus infecting a phytoplankton. HaNIV is a 50 nm, double-stranded D N A virus with a 38.5 kb genome. HaNIV particles are also sensitive to chloroform and the capsid contains a major 38 kDa protein and a less abundant 20.5 kDa protein. HaRNAV and HaNIV caused lysis of a different range of strains, but both predominantly caused lysis of strains from the Northeast Pacific. Because different viruses infected different host strains, the complexity of virus-host interactions in the environment is demonstrated. Over 90 % of the HaNIV genome was sequenced and is represented as two contigs, 17 765 and 15 858 base pairs in length. Open reading frames identified from the sequences had similarities with terminase, lysozyme, recombination, and structural proteins of bacteriophages from the Myoviridae, Podoviridae, and Siphoviridae. The homology of HaNIV ORFs with proteins from diverse bacteriophages suggests that the evolution of HaNIV involved numerous horizontal gene transfer events. HaNIV was also shown to be latent. Latency may be an important survival strategy for phytoplankton viruses. Irrefutable evidence proving that the host of HaNIV particles is Heterosigma akashiwo and not a bacterium, however, is imperative before the ecological and evolutionary implications can be determined. iii T A B L E O F C O N T E N T S Abstract i i Table of Contents i i i List of Tables v List of Figures vi Acknowledgements viii Chapter 1. Introduction to the bloom dynamics and harmful effects of Heterosigma akashiwo 1 1.1. Harmful Algal Blooms, a Definition 1 1.2. Harmful Effects of Heterosigma akashiwo 3 1.3. Bloom Dynamics of Heterosigma akashiwo 4 1.4. Viruses in Marine Environments 8 1.5. Literature Cited 11 Chapter 2. Characterization of HaRNAV and HaNIV, novel single-stranded R N A and double-stranded D N A viruses causing lysis of Heterosigma akashiwo 19 2.1. Introduction 19 2.2. Materials and Methods 22 2.2.1. Host cells 22 2.2.3. Virus isolates 22 2.2.4. Growth conditions 23 2.2.5. Amplification and purification of viruses 23 2.2.6. Transmission electron microscopy 25 2.2.7. Chloroform sensitivity 25 2.2.8. Structural proteins 26 2.2.9. Nucleic acid extraction and analysis 26 2.2.10. Hybridization assay 28 2.3. Results 29 2.3.1. Morphology 29 2.3.2. Chloroform sensitivity 31 2.3.3. Structural proteins 32 2.3.4. Nucleic acid type and genome size 33 2.3.5. Hybridization 35 2.3.6. Host range 36 2.4. Discussion 36 2.4.1. HaRNAV 38 2.4.2. HaNIV 40 2.4.3. Host range and ecological significance 42 2.5. Literature Cited 45 iv Chapter 3. Genomic sequences from HaNIV show homology to bacteriophage genes 55 3.1. Introduction 55 3.2. Materials and Methods 57 3.2.1. Construction of HaNIV clone library 57 3.2.2. Sequencing and assembly of contigs 58 3.2.3. Sequence analysis 59 3.2.4. Detection of the HaNIV genome in the host, Heterosigma akashiwo 61 3.3. Results 62 3.3.1. Sequence analysis 62 3.3.2. Phylogenetic analysis 67 3.3.3. Analysis of the host genome 68 3.4. Discussion 72 3.4.1. ORF 16A and 18A possibly encode terminase subunits 72 3.4.2. ORF 18B shares similarities with phage lysozymes 73 3.4.3. ORF IB is homologous withNinB sequences 74 3.4.4. ORF 21A may be a structural protein 75 3.4.5. Phylogeny 76 3.4.6. Algal virus or bacteriophage? 78 3.4.6. HaNIV may be latent 81 3.5. Literature Cited 83 Appendix A . Sequence of HaNIV contig A 92 Appendix B. Sequence of HaNIV contig B 98 V L I S T O F T A B L E S Table 2.1. Sensitivity of various Heterosigma akashiwo strains to HaNIV and HaRNAV. + indicates the virus caused cell lysis, - indicates no lysis compared to a control culture, * indicates a discrepancy from the result reported by Lawrence et al. (2001). NEPCC 522 was the strain used for virus isolation. 37 Table 3.1. GenBank accession numbers for protein sequences used for phylogenetic analyses. * indicates that the assigned function for the protein is putative. 60 Table 3.2. ORFs predicted from HaNIV sequence. The letters A and B denote the contig from which the ORF was identified. The coordinates describe the location of the ORF from the first nucleotide of the first start codon with a ribosome binding site (RBS) to the last nucleotide of a stop codon. If a RBS was not detected, then the ORF is described from the first encountered start codon, regardless of a RBS. The reading frame is shown in parentheses. * indicates a GTG start codon, otherwise it is A T G . The length of the translated ORF is in amino acids (aa). Matches show significant matches (P < 0.005) for the appropriate ORF. For the highest ranking match, GenBank accession numbers for the protein database precede the description of the protein and e-values and identities are provided. Identities show the percentage of identical amino acids in the homologous region (length shown in parentheses). 65 vi LIST OF FIGURES Figure 1.1. Bloom dynamics of Heterosigma akashiwo. Initiation, growth, and termination phases are described in the text. 5 Figure 1.2. Potential role of viruses in bloom dynamics. (A) Viruses are rapidly transmitted through a dense monospecific population compared to (B), a population of low density. Rapid, species-specific virus transmission might explain the sudden dissipation of Heterosigma akashiwo blooms. 7 Figure 2.1. Electron micrographs of virus particles causing lysis of Heterosigma akashiwo. A) HaNIV particle negatively stained with U A . B) HaRNAV from the lower band in the sucrose gradient, stained with PTA. Scale bars = 50 nm. 30 Figure 2.2. Growth of Heterosigma akashiwo in the presence of (A) HaNIV and (B) HaRNAV with and without chloroform. For both graphs, open symbols indicate chloroform treatments with the appropriate virus added (O) and without (A). Solid symbols indicate control treatments (no chloroform) with virus added (•) and without (A). The arrow indicates when viruses were added to the cell cultures. In each case, values are the mean ± SD, n = 8. 31 Figure 2.3. Structural proteins of Heterosigma akashiwo viruses. Proteins obtained from purified HaNIV (lane 1) and HaRNAV from the upper band (lane 2) and lower band (lane 3) of the sucrose gradient. Molecular weight markers (lane 4). The single 18 kDa capsid protein from tobacco mosaic virus was used as a control (lane 5). The proteins were stained with Coomassie Blue. 32 Figure 2.4. Restriction enzyme digests of HaNIV DNA. HaNIV D N A was digested with Hind III (lane 1) and Hinc II (lane 2). Bacteriophage X D N A digested with Hind III (lane 3) was used as a molecular weight marker. 33 Figure 2.5. Genomic R N A isolated from HaRNAV particles (lane 3). dsRNA from bacteriophage (p6 (lane 1) and ssRNA from T M V (lane 2) were used as controls. R N A standards were run in lane 4. 34 Figure 2.6. Hybridization of HaNIV D N A with D N A from A) non-infected Heterosigma akashiwo, (B) HaNIV infected H. akashiwo, (C) a resistant strain of H. akashiwo, and (D) unlabeled HaNIV. D N A was blotted onto the membrane in serial dilutions. 35 vii Figure 3.1. Location of HaNIV ORFs along contigs A and B. Black arrows indicate ORFs with homologies to proteins in GenBank. The arrowhead indicates the direction of transcription. 64 Figure 3.2. Unrooted parsimony tree of viral L S U terminases and HaNIV ORF 18A. The consensus tree is shown. Numbers at branch nodes indicate percentage bootstrap support from 100 replicates. Values less than 50 % are not shown. Any significant support from the neighbor-joining analysis is shown in parentheses. 69 Figure 3.3. Unrooted parsimony tree of lysozyme amino acid sequences and HaNIV ORF 18B. The consensus tree is shown. Numbers at branch nodes indicate percentage bootstrap support from 100 replicates. Values less than 50 % are not shown. Any significant support from the neighbor-joining analysis is shown in parentheses. 70 Figure 3.4. PCR analysis of D N A from infected and non-infected Heterosigma akashiwo. PCR products with HaNIV primers using template D N A from non-infected H akashiwo (lane 1), infected H. akashiwo (lane 2), and HaNIV particles used as a positive control (lane 4). D N A was not amplified from D N A isolated from a resistant strain of H akashiwo (lane 3). The negative control was run in lane 5 and molecular weight markers in lane 6. 71 V l l l A C K N O W L E D G E M E N T S I would like to thank my supervisor, Dr. Curtis Suttle, for providing me with a challenging and rewarding place to work for my Master's degree. And along with my committee members, Dr. Max (F.J.R.) Taylor and Dr. Jim Hudson, thank you for supporting my research and providing critical and helpful advice when asked. My research would not have been successful without the help and advice of Amy Chan, Dr. Janice Lawrence, Steven Short, and Dr. Andrew Lang. Thank you Amy, in addition to managing the lab, for helping me get my project off the ground. Thank you . Janice for many insightful conversations, improving my culturing techniques, and taking the time to work with me on the electron microscope. Thank you Steve for helping me with cloning techniques, PCR, sequence analysis, and all things molecular. And for the helpful suggestions made during the late stages of my thesis, thank you Andrew. I would like to thank Elaine Humphries and the E M lab at U B C for the use of their facilities. Thank you to the group at the Nucleic Acid Protein Services (NAPS) lab at U B C for their prompt and helpful service. For funding, thank you to the Natural Sciences and Engineering Research Council (NSERC). Finally, thank you to those who have made the lab an enjoyable place work and helped me out whenever needed. Thanks to Sean Brigden, Andre Comeau, and Alice Ortmann. Thank you to Tanya St. John for being an endless source of friendship and curing my soccer itch. And thanks to Alex Culley, Cindy Frederickson, Nicole McLearn, Karen Reid, Maarjte Verschuur, Eric Chu, George Huang, Michelle Lin, Cydney Nielsen, Nicole Radziminski, Nathan Taylor, Darrell Yip, and Derek Yuen. 1 CHAPTER 1. Introduction to the bloom dynamics and harmful effects of Heterosigma akashiwo Harmful algal blooms (HABs) have occurred at least since biblical times based on a passage in Exodus ".. .all the waters that were in the river were turned to blood. And the fish that were in the river died, and the river stank, and the Egyptians could not drink the water of the river" (Exodus 7: 20-21). These blooms of phytoplankton are noted for their striking colour, hence they are more commonly referred to as red tides, and their toxic effects to humans and aquatic animals. Of the estimated 3400-4100 species of phytoplankton, about 300 have been reported to form blooms including diatoms, dinoflagellates, silicoflagellates, prymnesiophytes, and raphidophytes, but only 60-80 of these are known to be harmful or toxic (Sournia 1995). Included in the group of harmful bloom-forming phytoplankton is a member of the Raphidophyceae, Heterosigma akashiwo (Hada) Hada ex Hada et Chihara, the subject of this thesis. Harmful Algal Blooms, a Definition What constitutes a harmful algal bloom is not strictly defined and is better understood when broken down into two parts: what is "harmful" and what is a "bloom". Traditionally, phytoplankton are considered to be harmful if they threaten public health or aquaculture industries, but they also have negative in situ ecological effects, such as allelopathy, on organisms not directly related to human interests (Smayda 1997). Public 2 health usually is threatened by toxin-producing phytoplankton. Shell-fish poisoning is the most common cause due to the accumulation of toxins in shell-fish as they feed on phytoplankton. In the Pacific Northwest, paralytic shell-fish poisoning (PSP), caused by saxitoxins produced by dinoflagellates from the genus Alexandrium, amnesic shell-fish poisoning (ASP), caused by domoic acid produced by diatoms from the genus Pseudo-nitzschia, and diarrhetic shell-fish poisoning (DSP), caused by okadaic acid produced by dinoflagellates from the genus Dinophysis, are of concern (Taylor et al. 1994, Horner et al. 1997). Although harmless to the shell-fish, the presence of these toxins renders them poisonous to humans and forces the closure of shell-fish farms. Harmful phytoplankton species also adversely affect fish aquaculture. Fish mortalities due to phytoplankton result from several mechanisms: physical damage to the gills, by the barbed spines of Chaetoceros spp. for example; asphyxiation due to oxygen depletion following blooms, such as with Ceratium species; gas-bubble trauma caused by extreme oxygen supersaturation of seawater from algal photosynthesis; and chemical toxicity due to the release of toxins, including brevetoxins, hemolysins, cytotoxins, or ammonia produced by species such as Karenia brevis, Gyrodinium aureolum, Chrysochromulina polylepsis, and Noctiluca scintillans (Black 1991, Smayda 1997). Blooms are usually linked to harmful phytoplankton events because dense populations, in sum, have more cells to cause physical damage, use more oxygen, or produce a greater abundance of toxins. A toxic event and the occurrence of a bloom, however, are not mutually dependent. Ingestion of only a few cells of a toxic phytoplankton can result in the death of fish larvae (Smayda 1997). In addition, not all blooms are harmful. Blooms are often described as significant increases in population 3 size measured as abundance (i.e. cells-L"1). Defining "significant increases in population size", however, is problematic because maximal, potential bloom levels can vary depending on geography, season, and species (Smayda 1997). Nor is it useful terminology because toxicity can result from high or low biomass bloom events and high or low cell abundances (Smayda 1997). Despite these conceptual difficulties, monitoring the presence of harmful species, the abundance of toxins, and understanding the factors that regulate the occurrence, toxicity, and bloom dynamics of specific species are important strategies for mitigating the negative effects of phytoplankton. Harmful Effects oi Heterosigma akashiwo Heterosigma akashiwo is considered a harmful phytoplankton species because of its devastating impact on finfish aquaculture. A member of the Raphidophyceae, H. akashiwo is a 10-25 |jm, yellow-brown, oval-shaped, unicellular flagellate species that lacks a cell wall (Honjo 1993, Taylor et al. 1994, Smayda 1998). This organism has also been referred to as Heterosigma carterae (Hulbert) Taylor and has been mistaken for Olisthodiscus luteus Carter in some publications (Taylor 1992, Throndsen 1996). H. akashiwo forms blooms that cause fish kills of cultured salmon, yellow-tail, and red sea bream in coastal seas of temperate and sub-arctic areas of both Northern and Southern hemispheres (Chang et al. 1990, Honjo 1993, Taylor and Haigh 1993). In Japan, damage caused by H. akashiwo blooms has amounted to 2 billion yen (about $25 million) over a period of 16 years (1972-1987) (Honjo 1993). In British Columbia, from 1986 to 1991, H. akashiwo was responsible for losses of greater than $12 million to the finfish aquaculture industry (Taylor 1993, Taylor and Horner 1994). It is not known how Heterosigma akashiwo causes fish mortalities. The production of reactive oxygen species such as superoxide and hydroxyl radicals resulting in damage to fish gill lamellae has been suggested (Yang et al. 1995). H. akashiwo is also believed to produce low amounts of neurotoxins that are chemically similar to brevetoxins produced by Karenia brevis (Khan et al. 1997). This result is consistent with an earlier study by Black et al. (1991) who proposed that the death of juvenile chinook salmon {Oncorhynchus tshawytscha) exposed to a H. akashiwo bloom was likely due to a toxic agent rather than deoxygenation, oxygen supersaturation, or physical gill damage. In addition, it was found that H. akashiwo was toxic only when bacteria were present in culture experiments with salmonids (Carrasquero-Verde 1999). The specific mechanism of H. akashiwo fish toxicity remains unclear, however, and further research is required. Bloom Dynamics oi Heterosigma akashiwo Blooms of Heterosigma akashiwo occur in cycles of initiation, growth, and termination (Fig. 1.1). The blooms originate from an over-wintering population of non-motile vegetative cells and cysts in ocean sediments (Yamochi 1984, Honjo 1993). The blooms are initiated by the release of motile cells from the resting stages triggered by a rise in temperature above 10°C (Taylor and Haigh 1993, Smayda 1998, Imai and Itakura 1999). Once released, for successful bloom initiation, the motile cells must overcome competition from other species and obtain sufficient nutrients for cell growth. Because 5 Figure 1.1. Bloom dynamics of Heterosigma akashiwo. Initiation, growth, and termination phases are described in the text. H akashiwo cysts can germinate under low-light conditions, H. akashiwo has an advantage over competing species such as the diatom Skelotonema costatum which prefers light for germination (Smayda 1998, Imai and Itakura 1999). H. akashiwo is also known to produce allelopathic substances which inhibit the growth of competitors and deter grazers (Pratt 1966, Verity and Stoecker 1982, Egloff 1986, Uye and Takamatsu 1990). For growth, Heterosigma akashiwo requires inorganic nutrients such as phosphate and nitrate, vitamins, and trace metals, especially iron and manganese (Honjo 1993, Smayda 1998). As a flagellated cell, H akashiwo obtains nutrients from deeper waters at night and photosynthesizes at the surface during the day through diurnal vertical 6 migrations (Watanabe et al. 1988). The daily upward and downward migrations allow H. akashiwo to take advantage of stratified environments where nutrients limit growth in the euphotic zone, but are at higher concentrations at depth (Honjo 1993, Smayda 1997, Smayda 1998). Stratification due to fresh water run-off from rivers may also trigger blooms through the introduction of iron and other micronutrients into coastal systems (Honjo 1993, Taylor and Haigh 1993). Once a bloom of Heterosigma akashiwo has developed, it can last several days (Nagasaki et al. 1996) to several months (Taylor and Haigh 1993). The blooms appear as red-brown discolourations of the water and can reach magnitudes of 200 million cells-L"1 (Taylor et al. 1994). Not all blooms reach harmful levels, however. A preliminary estimate suggests that fish will begin to die at 12 million cells-L"1, but this value varies depending on the species, size of the fish, and environmental factors (Taylor et al. 1994). The dissipation or termination of Heterosigma akashiwo blooms often occurs very suddenly and may be triggered by several factors. Physical factors include the break-down of stratification by wind-induced turbulence (Taylor and Haigh 1993). Nutrient limitation may also be involved (Smayda 1998). Increasing evidence, however, indicates that biological factors, such as bacteria and viruses, can have a significant role in H. akashiwo bloom termination (Nagasaki et al 1994a, b, Kim et al. 1998). As pathogens, bacteria and viruses can kill or cause lysis of H. akashiwo resulting in a reduction of H. akashiwo cells and the disintegration and dissipation of the bloom. In addition, viruses are typically species or strain specific and would be rapidly transmitted through a dense monospecific population as in bloom situations (Fig. 1.2). Although the specific triggers are not known, during bloom termination, motile H. akashiwo cells cease vertical 7 migrations, form cysts or remain as non-motile vegetative cells, and sink, coming to rest in the sediments (Itakura et al. 1996). As described above, Heterosigma akashiwo bloom dynamics are characterized by several phases: initiation, rapid and exponential growth, maintenance, and termination. The factors that regulate the length, perseverance, severity, and dissipation of H. akashiwo blooms, however, are not well understood. Figure 1.2. Potential role of viruses in bloom dynamics. (A) Viruses are rapidly transmitted through a dense monospecific population compared to (B), a population of low density. Rapid, species-specific virus transmission might explain the sudden dissipation of Heterosigma akashiwo blooms. 8 Viruses in marine environments There is considerable interest in understanding the factors regulating H. akashiwo bloom dynamics because of the damaging effects of H. akashiwo blooms to aquaculture fish. Viruses are an important factor to consider because, in addition to the possibility of directly causing bloom termination by cell lysis, they may also influence bloom distribution, length, and severity. Viruses may affect other ecological processes as well such as nutrient cycling (Gobler et al. 1997, Wilhelm and Suttle 1999), primary productivity, community structure, and genetic transfer (Suttle et al. 1990, Bratbak et al. 1994). Until recently, viruses in aquatic environments were believed to be low in abundance and ecologically unimportant (Wiggins and Alexander 1985). These estimates were based on culture assays with various bacterial hosts. Thus, there is little historical data regarding marine phytoplankton viruses. Most reports of phytoplankton viruses result from incidental observations of virus-like particles inside cells using transmission electron microscopy (reviewed in van Etten et al. 1991). Total direct virus counts, however, based on transmission electron and epifluorescence microscopy, have revealed that viruses generally range from 106-109-mr1 in aquatic environments and are frequently 10 fold higher than the bacterial abundance (Bergh et al. 1989, Proctor and Fuhrman 1990, Suttle et al. 1990, Bratbak et al. 1994). These estimates are 103-107 times higher than those based on culture assays (Bergh et al. 1989). Although in aquatic environments most viruses are bacteriophages (viruses infecting bacteria), viruses infecting eukaryotic phytoplankton are also a component of 9 the aquatic community. To date, eukaryotic phytoplankton viruses have been isolated which infect Aureococcus anophagefferens (Gastrich et al. 1998), Chrysochromulina sp. (Suttle and Chan 1995), Heterosigma akashiwo (Nagasaki and Yamaguchi 1997, Lawrence et al. 2001), Micromonas pusilla (Mayer and Taylor 1979, Waters and Chan 1982, Cottrell and Suttle 1991), and Phaeocystispouchetii (Jacobsen et al. 1996). Viruses have also been implicated in the bloom termination of Emiliania huxleyi (Bratbak et al. 1993, Bratbak et al. 1996). Two viruses have been isolated and described which infect Heterosigma akashiwo. In Japan, a large, 200 nm H. akashiwo virus (HaV) was isolated (Nagasaki and Yamaguchi 1997). This virus is very similar to viruses found replicating in cells isolated during the termination of a H. akashiwo bloom (Nagasaki 1994a, b). In British Columbia, a small, 30 nm virus infecting Heterosigma akashiwo was isolated from the Strait of Georgia (Lawrence et al. 2001). This virus, named HaNIV for Heterosigma akashiwo nuclear inclusion virus, differs from HaV. In addition to size, HaNIV forms crystalline arrays as it assembles inside the nucleus, as opposed to the cytoplasmic assembly of HaV (Lawrence et al. 2001). These significant differences indicate that HaNIV is unrelated to HaV. Other studies have shown that isolates of H. akashiwo viruses vary in the range of host strains that they infect (Nagasaki and Yamaguchi 1998, Tarutani et al. 2000). Together, this evidence indicates diversity among H. akashiwo viruses, yet other than T E M studies and studies investigating the impact of these viruses on the host, there is little information about the viruses themselves and their ecological role, particularly in bloom dynamics. 10 This thesis examines two unrelated viruses isolated from the Strait of Georgia, British Columbia that cause lysis of Heterosigma akashiwo. In contrast to previous studies that examined the effects of H. akashiwo viruses on their host, the viruses themselves are the targets of investigation. Chapter 2 describes the characterization of the two viruses, HaRNAV and HaNIV. The characterization of HaRNAV has resulted in the discovery of the first single-stranded R N A (ssRNA) phytoplankton virus. The name HaRNAV, meaning Heterosigma akashiwo R N A virus, is derived from the nature of its genome. In addition, the characterization of HaNIV demonstrates the diversity of double-stranded D N A (dsDNA) algal viruses. Chapter 3 provides the genome sequence of HaNIV. The genome sequence was not completed, but is presented as two contigs, 17 780 and 15 858 base pairs in length. These sequences represent over 90 % of the genome. The sequence has revealed open reading frames from HaNIV that are homologous with genes from diverse bacterial viruses, thus providing evidence of horizontal gene transfer. It was also demonstrated that HaNIV is latent, i.e. the virus does not replicate immediately upon infection of its host, but its genome is integrated into the host's D N A or carried extrachromosomally. The characterization of Heterosigma akashiwo viruses will hopefully result in a better understanding of the factors regulating H. akashiwo blooms. Virus characterization can lead to the development of new tools and techniques for monitoring the effects of viruses in the natural environment. In addition, this thesis has demonstrated the diversity of viruses in marine environments and the need for further exploration of marine microbes. 11 LITERATURE CITED Bergh 0, Borsheim K Y , Bratbak G, Heldal M (1989) High abundance of viruses found in aquatic environments. Nature. 340: 467-468 Black E A , Whyte JNC, Bagshaw JW, Ginther N G (1991) The effects of Heterosigma akashiwo on juvenile Oncorhynchus tshawytscha and its implications for fish culture. J. Appl. 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Ecol. 14: 109-112 15 Pratt D M (1966) Competition between Skeletonema costatum and Olisthodiscus luteus in Narragansett Bay and in culture. Limnol. Oceanogr. 11: 447-455 Proctor L M , Fuhrman JA (1990) Viral mortality of marine bacteria and cyanobacteria. Nature. 343: 60-62 Smayda TJ (1997) What is a bloom? A commentary. Limnol. Oceanogr. 42: 1132-1136 Smayda TJ (1998) Ecophysiology and bloom dynamics of Heterosigma akashiwo (Raphidophyceae). In: Anderson D M , Cembella A D , Hallegraeff G M (eds) Physiological Ecology of Harmful Algal Blooms. Springer-Verlag Berlin Heidelberg, p 113-131 Sournia A (1995) Red tide and toxic marine phytoplankton of the world ocean: an inquiry into biodiversity. In: Lassus P, Arzul G, Erard E, Gentien P, Marcaillou C (eds) Harmful Marine Algal Blooms. Proceedings of the 6 t h International Conference on Toxic Marine Phytoplankton. Lavoisier, pi03-112 Suttle CA, Chan A M (1995) Viruses infecting the marine Prymnesiophyte Chrysochromulina spp.: isolation, preliminary characterization and natural abundance. Mar. Ecol. Prog. Ser. 118:275-282 16 Suttle CA, Chan A M , Cottrell M T (1990) Infection of phytoplankton by viruses and reduction of primary productivity. Nature. 347: 467-469 Tarutani K, Nagasaki K, Yamaguchi M (2000) Viral impacts on total abundance and clonal composition of the harmful bloom-forming phytoplankton Heterosigma akashiwo. Appl. Environ. Microbiol. 66: 4916-4920 Taylor FJR (1992) The taxonomy of harmful marine phytoplankton. G. Bot. Ital. 126: 209-219 Taylor FJR (1993) Current problems with harmful phytoplankton blooms in British Columbia waters. In: Smayda TJ, Shimizu Y (eds) Toxic Phytoplankton Blooms in the Sea. Elsevier, Amsterdam, p 699-703 Taylor FJR, Haigh R (1993) The ecology of fish-killing blooms of the chloromonad flagellate Heterosigma in the Strait of Georgia and adjacent waters. In: Smayda TJ, Shimizu Y (eds) Toxic Phytoplankton Blooms in the Sea. Elsevier, Amsterdam, p 705-710 Taylor FJR, Haigh R, Sutherland TF (1994) Phytoplankton ecology of Sechelt Inlet, a fjord system on the British Columbia coast. II. Potentially harmful species. Mar. - Ecol. Prog. Ser. 103: 151-164 17 Taylor FJR, Horner R A (1994) Red tides and other problems with harmful algal blooms in Pacific Northwest coastal waters. In: Review of the marine environment and biota of Strait of Georgia, Puget Sound and Juan de Fuca Strait. Can. Fish. Aquat. Sci. Tech. Rep. pi75-185 Throndsen J (1996) Note on the taxonomy of Heterosigma akashiwo (Raphidophyceae). Phycologia. 35: 367 Uye S, Takamatsu K (1990) Feeding interactions between planktonic copepods and red-tide flagellates from Japanese coastal waters. Mar. Ecol. Prog. Ser. 59: 97-107 van Etten JL, Lane L C , Meints R H (1991) Viruses and virus like particles of eukaryotic algae. Microbiol. Rev. 55: 586-620 Verity PG, Stoecker D (1982) Effects of Olisthodiscus luteus on the growth and abundance of tintinnids. Mar. Biol. 72: 79-87 Watanabe M , Kohata K , Kunigi M (1988) Phosphate accumulation and metabolism by Heterosigma akashiwo (Raphidophyceae) during diel vertical migration in a stratified microcosm. J. Phycol. 24:22-28 18 Waters RE, Chan A T (1982) Micromonaspusilla virus: the virus growth cycle and associated physiological events within the host cells; host range mutation. J. Gen. Virol. 63:199-206 Wiggins B A , Alexander M (1985) Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems. Appl. Environ. Microbiol. 49: 19-23 Wilhelm SW, Suttle C A (1999) Viruses and nutrient cycles in the sea. Bioscience. 49:781-788 Yamochi (1984) Mechanisms for outbreak of Heterosigma akashiwo red tide in Osaka Bay, Japan. Part 3. Release of vegetative cells from bottom mud. J.Ocean Soc. Jpn. 40: 343-348 Yang CA, Albright LJ , Yousif A N (1995) Oygen-radical-mediated effects of the toxic phytoplankter Heterosigma carterae on juvenile rainbow trout Oncorhynchus mykiss. Dis. Aquat. Org. 23: 101-108 19 CHAPTER 2. Characterization of HaRNAV and HaNIV, novel single-stranded RNA and double-stranded DNA viruses causing lysis of Heterosigma akashiwo INTRODUCTION Viruses generally consist of a nucleic acid genome of either D N A or R N A encapsidated by a protein shell. Viruses are unable to perform any metabolic functions on their own. Thus, in order to express genes and replicate, they must infect a host organism and take over the cellular machinery. Although simple in structure, these entities can cause illness and sometimes death of their host. For unicellular organisms, such as bacteria and phytoplankton, a lytic viral infection, in which progeny viruses are produced and released by cell lysis, always results in cell death. There are greater than 4000 known virus species (Murphy et al. 1995) infecting a diversity of organisms including archaeabacteria, bacteria, fungi, plants, insects, and animals. A virus species is defined as "a polythetic [based on multiple criteria] class of viruses that constitutes a replicating lineage and occupies a particular ecological niche" (Mayo 1996). The actual number of viruses is undoubtedly much higher and it is generally believed that there are several viruses specific for every species of living organism (Fenner 1974, Fuhrman 1999). Viruses have been observed in almost every group of algae, but few of these viruses have been isolated and characterized. 20 Viruses can be distinguished from one another based on several physical, antigenic, and biological properties. Primarily, viruses are characterized by the morphology of the viral particle and the nature of the viral genome (Fenner 1974). Other characteristics used to describe viruses include the mass, density, and stability of the virion, the number and size of structural and non-structural proteins, the presence of lipids or carbohydrates, genome organization, the site of replication, assembly, and maturation, host range, mode of transmission, and geographic distribution (Murphy 1996). These characteristics are used as a basis for virus classification. The classification of viruses follows a hierarchical scheme and is governed by the International Committee for the Taxonomy of Viruses (ICTV). Virus species are grouped into genera, families, and occasionally orders based on certain common characters such as biochemical composition, replication strategy, and nature of the particle structure. This classification scheme was used initially as an organizational system and, unlike the classification of organisms, was not meant to imply any phylogenetic relationships. However, information based on genome sequence data and similarities in gene order and arrangement has provided the means for phylogenetic analyses. Three orders of viruses have recently been established to reflect phylogeny. They are the Caudovirales - the tailed bacteriophage group (Maniloff and Ackermann 1998), the Mononegavirales - a negative stranded ssRNA virus group (Pringle 1991), and the Nidovirales - a positive stranded ssRNA virus group (Cavanagh 1997). Viral isolates that infect phytoplankton are typically large (100-200 nm) double-stranded D N A viruses. A number of these isolates, infecting Micromonas pusilla (Mayer and Taylor 1979, Waters and Chan 1982, Cottrell and Suttle 1991), Chrysochromulina 21 spp. (Suttle and Chan 1995), and the brown alga Ectocarpus siliculosis (Miiller et al. 1990), have been included in the virus family Phycodnaviridae (Pringle 1999). This family was established based on viruses that infect an exsymbiotic Chlorella-Wke, alga (van Etten 1995, van Etten and Meints 1999). A D N A polymerase gene-based phylogeny suggests that the Phycodnaviridae is monophyletic and most closely related to herpes viruses (Chen and Suttle 1996). However, the Phycodnaviridae shares structural and genomic features with the iridoviruses, vaccinia virus (a pox virus), and the unclassified African Swine Fever virus (van Etten and Meints 1999). Other viruses infecting eukaryotic phytoplankton, including Aureococcus anophagefferens (Gastrich et al. 1998), Emiliania huxleyi (Bratbak et al. 1993, Bratbak et al. 1996), Heterosigma akashiwo (Nagasaki and Yamaguchi 1997), and Phaeocystis pouchetii (Jacobsen et al. 1996), have not been assigned to a family but are morphologically similar to the Phycodnaviridae. In addition to Phycodnaviridae-like particles, other types of viruses have been reported which infect microalgae. Milligan and Cosper (1994) and Garry et al. (1998) described a phage-like particle that infects Aureococcus anophagefferens, although some concern has been expressed that this may be a contaminating bacteriophage (virus infecting bacteria) (Suttle 2000). More recently, Lawrence et al. (2001) have described a virus (HaNIV) that infects Heterosigma akashiwo which is much smaller (30 nm) than viruses belonging to the Phycodnaviridae and forms crystalline arrays in the nucleus. This chapter provides further characterization of HaNIV in terms of morphology, structure, and genome type. As well, a new virus, Heterosigma akashiwo R N A virus (HaRNAV), is described. It is the first single-stranded (ss) R N A virus reported to cause lysis of phytoplankton. The characterization of HaNIV and HaRNAV provided 22 information to compare the two novel viruses with known eukaryotic algal viruses and other virus families. M A T E R I A L S A N D M E T H O D S Host cells. Heterosigma akashiwo (NEPCC 522), obtained from the Northeast Pacific Culture Collection at the University of British Columbia, was used for the isolation and amplification of the viruses. This strain was treated with 500 p-gml"1 ampicillin, 500 plgml"1 kanamycin, and 100 Jig-mi"1 gentamicin or 500 ag-ml"1 ampicillin, 250 ag-ml"1 streptomycin, and 500 ag-ml"1 kanamycin in an attempt to render the cultures axenic (Cottrell and Suttle 1993). Only the latter treatment was successful. To test the host range of the viruses, fourteen additional strains of Heterosigma akashiwo (Table 2.1) were tested as potential hosts. In addition to the 12 strains used by Lawrence et al. (2001), two strains, H93616 and H94608 (Nagasaki and Yamaguchi 1998), were tested in this study. Virus isolates. Viruses were isolated and cloned as described previously (Suttle et al. 1991). Briefly, 200 L water samples were collected and filtered to remove plankton and most bacteria, but leaving the virus-size fraction. The filtered sea-water was concentrated using an Amicon spiral wound ultrafiltration cartridge with a 30 kDa cut-off. The concentrated virus-size fraction was added in a dilution series to exponentially-growing cultures of Heterosigma akashiwo. Samples positive for lytic agents of H. akashiwo caused cell lysis or a decrease in cell growth. Clonal isolates from these positive samples were obtained by repeatedly diluting the lytic agents to extinction. 23 Inoculating an exponentially growing culture of H. akashiwo with the end-point dilution resulted in the replication and amplification of a single lytic agent of H. akashiwo. The amplified lytic agents (i.e. cell lysates) were filtered through a 0.22 |im pore-size G V Durapore® filter (Millipore) and stored at 4 °C. HaNIV was isolated in 1997 from water collected in Discovery Passage, BC, Canada (Lawrence et al. 2001). HaRNAV was isolated from the Fraser River Plume (49° 04.749'N X 123° 23.076'W) in the Strait of Georgia, BC on August 22, 1996. The isolate originated from a 200 L water sample collected at the chlorophyll maximum (depth of 6.7 m). Growth conditions. Experiments for initial T E M characterization, chloroform sensitivity assay, and host range study were conducted with 5 ml cultures of exponentially growing Heterosigma akashiwo. The experiments were performed in triplicate, with the exception of the chloroform sensitivity assay, which was done in replicates of eight. A l l cultures were grown in F/2 enriched seawater (Guillard 1975) (30 °/00) supplemented with 10 nM sodium selenite. The cells were incubated at 20 °C with a lightdark cycle of 14:10 using 260 or 200 ujriol-m'V 1 photo synthetically active radiation. Daily measurements of in vivo chlorophyll fluorescence (Turner Designs fluorometer) were used as a measure of cell growth. In each assay 1 % or 0.1 % (v/v) of a 0.22 Jim filtered cell lysate containing the viruses was added to the host cell culture. A 90 % decrease in fluorescence compared to control cultures was considered an indicator of cell lysis. Amplification and purification of viruses. Ten to fifteen 800 ml or 2 L cultures of Heterosigma akashiwo were grown exponentially in 1 L Erlenmeyer or 2.5 L low form 24 culture flasks (Fisher), respectively. Growth was monitored by in vivo fluorescence of subsamples of the cultures. After 5-7 days, 0.1 % (v/v) of a 0.22 um filtered cell lysate was added to each flask and incubated until cell lysis occurred within 7 days. The lysates from each culture were pooled, filtered, ultrafiltered (30 kD cut-off), and concentrated as described by Cottrell and Suttle (1991) with the following modifications. The retentate following ultrafiltration was centrifuged at 4100 X g for 30 min to remove remaining bacteria and the supernatant centrifuged for 5 h at 108 000 X g to pellet the viruses. The virus pellet was softened and resuspended in approximately 75 p:l of the supernatant. The resuspended pellets were pooled and incubated with 1 lI-nT1 DNase I and 0.1 U-^T 1 RNase A for 3 h at room temperature to remove nucleic acids not contained within virus particles. Sucrose gradients were used to purify the virus particles. For HaNIV, sucrose step gradients (10, 20, 30, and 40 %) were prepared in F/2 + Se medium and left to linearize overnight at 4 °C. Sucrose gradients from 5 to 40 % were made up in 50 m M Tris (pH 7.6) for HaRNAV. The virus suspensions were layered over the linear sucrose gradients (200 - 300 jj.1 of viruses per gradient) and centrifuged at 50 000 X g in an SW-40 Beckman rotor for 1 h to purify HaNIV or 3 h for HaRNAV. Bands were visualized by shining a light directly over the sucrose gradient and removed by inserting a 23-gauge needle with a syringe into the tube just below the band. For HaNIV, there was a single band at 3.3 cm from the top, whereas the HaRNAV preparation resulted in bands at 2.2 and 4.0 cm. The purified viruses (~ 1 ml) were placed in a 1.5 ml screw-capped microcentrifuge tube. The tube was topped up with 0.22 | im filtered F/2 + Se or 50 m M Tris (pH 7.6), floated in a 14 ml centrifuge tube filled with water, spun for 3 h at 115 000 25 X g (SW-40 rotor), the supernatant removed, and the procedure was repeated. The virus pellets were softened with 50 | i l of supernatant at 4 °C and resuspended in 100 | l l media or 50 m M Tris. To confirm that a lytic agent of Heterosigma akashiwo was purified, the viruses were diluted 1000-fold and added back to exponentially growing cultures of H. akashiwo. Transmission electron microscopy. For initial characterization by T E M , 0.22 [im filtered lysates were spun for 3 h at 115 000 X g onto carbon and formvar coated, glow-discharged, 400-mesh copper T E M grids. The grids were stained with 1 % uranyl acetate (UA) (w/v) for 20 sec and excess stain was wicked off with filter paper. Purified virus particles provided a cleaner sample with less debris for T E M characterization. In addition, the purification of the particles was confirmed by comparing the morphology of these particles to particles from cell lysates before and after virus purification. The purified virus particles were prepared for T E M by placing 10 ul of the purified virus sample on top of a T E M grid. The viruses were left to adsorb to the grid for 30 min at room temperature. The virus drop was removed with filter paper and the grid was stained with either 1 % U A or 2 % phosphotungstic acid (PTA) (pH 5.5) as described above. The grids were viewed with a Zeiss 1 OC-transmission electron microscope at an accelerating voltage of 80 kV. Sizes of virus particles were determined from negatives using a scale lupe calibrated with a 2160 waffle-grating replica. Chloroform sensitivity. An aliquot (500 |il) of 0.22 um filtered lysate was vigorously shaken with an equal volume of chloroform for 5 min. Chloroform was removed by centrifugation at 4100 X g for 5 min and the aqueous phase containing the 26 viruses was recovered and left for 6 h at room temperature to let any remaining chloroform evaporate. The same procedure was completed with 500 ji l of media as a control. The chloroform-treated viruses, chloroform-treated media, and non-treated viruses were added to exponentially growing Heterosigma akashiwo cells as described above. Structural proteins. Purified virus particles (3-10 jil) were heated for 5 min at 100 °C. A 12 % SDS/polyacrylamide separating gel with a 4 % stacking gel was prepared following the protocol of Ausubel et al. (1992). The samples were electrophoresed at 10 mA through the stacking gel and then at 15 mA through the separating gel. Tobacco mosaic virus (TMV) was used as a control. The gel was fixed with 50 % v/v methanol and 10 % v/v acetic acid and stained overnight with 0.5 % Coomassie Blue. Protein molecular weight standards (Gibco BRL) ranging from 14 300 to 200 000 M W were used for size calibration and the gel was analyzed with Gel-Pro analyzer software (version 3.1.00.00 for Windows 95.NT, Media Cybernetics). Nucleic acid extraction and analysis. Purified HaNIV (500(il) was incubated for 15 min at 68 °C with 0.1 % (w/v) SDS and 5 m M EDTA. The lysed particles were extracted once with an equal volume of phenol, twice with an equal volume of phenol: chloroform (1:1), and once with an equal volume of chloroform. The nucleic acids were precipitated with 2 volumes of 100 % ethanol and incubated at -20 °C overnight. This preparation was spun for 25 min at 17 500 X g, the supernatent removed, and the nucleic acid pellet washed with 70 % ethanol. The pellet was air-dried, resuspended in water, and stored at - 20 °C. 27 Nucleic acids from HaRNAV were obtained using the QIAamp Viral R N A kit (Qiagen) following the manufacturer's protocol and stored at - 20 °C. Nucleic acid concentration was quantified by absorbance at 260 nm. Nucleic acid type was determined by digesting the viral nucleic acids with 0.07 (ig-lif1 DNase I in 0.1X SSC (IX = 0.15 M N a C l and 0.015 MNa 3citrate-2H 20, pH 7.0), 0.07 ug-ul"1 RNase A in 0.01X SSC, and 0.07 UguT 1 RNase A in 2X SSC. Double stranded (ds) R N A is not digested by RNase A under high salt conditions (Semancik et al. 1973). Bacteriophage X dsDNA, bacteriophage 96 dsRNA, and T M V ssRNA were used as controls. The nucleic acids were electrophoresed through a 0.75 % agarose gel, stained with 0.5 pLg-lil"1 ethidium bromide, and visualized with a U V transilluminator (Fisher). The genome size of HaNIV was estimated by digesting 0.7 p,g of genomic D N A with the restriction enzymes Hind III (1 Uul~') and Hinc II (2 UuT 1 ) at 37 °C for 3 h using the manufacturer's (New England Biolabs) recommended buffers. The digested D N A was electrophoresed and visualized as described above. X D N A digested with Hind III (Gibco BRL) was used to calibrate the size of the digested fragments and the size of the bands was determined using Gel-Pro Analyzer software. For HaRNAV, the whole genomic R N A was run on a 0.75 % denaturing formaldehyde gel alongside a 0.24-9.5 kb R N A ladder (Gibco BRL), bacteriophage 96 dsRNA genome, and T M V ssRNA genome. The R N A samples and formaldehyde gel were prepared and run following standard protocols (Sambrook et al. 1989). The genomic R N A from HaRNAV particles was analyzed to determine if the genome was poly-A tailed. HaRNAV R N A was run through an Oligotex™ mRNA column (Qiagen) following the manufacturer's protocols. These columns isolate poly-A 28 tailed RNAs. A l l other nucleic acids are washed through the column. The poly-A tailed RNAs are then eluted from the column with water. Nucleic acids that were washed through the column were re-isolated using the QIAamp Viral R N A kit (Qiagen) following the manufacturer's protocol. A l l nucleic acids were examined on a 0.75 % formaldehyde gel alongside R N A size standards as described above. Hybridization assay. Exponentially growing Heterosigma akashiwo cultures were infected with 1 % (v/v) of an HaNIV 0.22 filtered lysate and harvested 6 days post infection, before the cells lysed. Cells from infected and non-infected cultures were pelleted by centrifugation (1500 X g for 10 min), resuspended with media, and centrifuged again. The cells were resuspended in 400 | l l of water and lysed in a solution containing 0.1 M Tris, pH 8, 0.05 M EDTA, 0.2 M NaCl, and 1 % SDS (Saunders 1993). D N A was extracted using a standard phenol and chloroform: isoamyl alcohol protocol, similar to the one described for HaNIV, precipitated with ethanol, and resuspended in water. Dilutions of D N A from infected and non-infected H akashiwo, ranging from 30 ng to 10 pg, were dot-blotted (VacuDot-VS, American Bionetics) onto a nylon membrane. HaNIV D N A and H akashiwo D N A from a strain resistant to HaNIV (NEPCC 522 treated with ampicillin, streptomycin, and kanamycin) were also blotted onto the membrane as positive and negative controls, respectively. The membrane was dried for 2 h at 80 °C and the D N A was cross-linked with U V light (UV Crosslinker, Fisher) to the membrane. A biotinylated probe was constructed from HaNIV D N A by nick translation (BioNick™ Labeling System) following the manufacturer's protocol (Gibco\BRL). Hybridization was conducted at 42 °C overnight in 35 X 300 mm tubes (Robbins 29 Scientific). The hybridization solution contained 25 % formamide, 6X SSPE (1.08 M NaCl, 60 m M NaH 2 P0 4 , 60 m M EDTA, pH 7.4), 5X Denhardt's solution (0.1 % w/v Ficoll, 0.1 % w/v polyvinylpyrrolidone, 0.1 % w/v BSA), 1 % SDS, and 100 ng-ml"1 probe. The membrane was washed and the probe was detected by chemiluminescence using the PhotoGene Nucleic Acid Detection System (Gibco/BRL) following the manufacturer's protocols. The only modification to the protocol was that the initial wash was conducted at 50 °C rather than 65 °C. The signal was visualized by exposing the membrane to BioMax Light film (Kodak) for 30 min. RESULTS Based on T E M observations, purified HaNIV particles were indistinguishable from those found in cell lysates before purification. HaNIV is 50 nm in diameter and hexagonal in shape (Fig. 2.1 A) suggesting icosahedral symmetry. The HaNIV particles have densely staining circular cores and an extension at one vertex of the capsid that extends 13 nm from the edge of the core. The five other vertices are 7 nm away from the edge of the core. The extension was not visible in all HaNIV particles, likely depending on the orientation of the'particle. Enveloped particles were not observed. The purified particles were able to cause lysis of exponentially growing cultures of Heterosigma akashiwo and virus-like particles (VLPs) of the same morphology were recovered from the cell lysate (data not shown). For HaRNAV, the virion is 25 nm in diameter and polygonal in shape (Fig. 2.IB). The three dimensional particle is probably icosahedral, but appeared more spherical in 30 shape than HaNIV. The particles did not appear be enveloped. The particles were negatively stained, but the stain also completely penetrated some particles suggesting that these particles may be "empty" and devoid of nucleic acids (Hemida and Murant 1989, Hemida et al. 1989). From the sucrose gradient, bands at 2.2 and 4.0 cm contained morphologically similar VLPs (data not shown). However, only particles from the lower band consistently caused lysis of Heterosigma akashiwo (data not shown) suggesting that viruses in the top band were incompletely assembled. Purified particles, VLPs from cells infected with purified VLPs, and VLPs from cell lysates before purification were indistinguishable by T E M . Figure 2.1. Electron micrographs of virus particles causing lysis of Heterosigma akashiwo. A) HaNIV particle negatively stained with U A . B) HaRNAV from the lower band in the sucrose gradient, stained with PTA. Scale bars = 50 nm. 31 Chloroform sensitivity After inoculation with chloroform-treated HaNIV and HaRNAV, H. akashiwo " cultures continued to increase in fluorescence while cultures inoculated with untreated viruses rapidly decreased in flurorescence (Fig. 2.2) indicating that both HaRNAV and HaNIV are chloroform sensitive. Figure 2.2. Growth of Heterosigma akashiwo in the presence of (A) HaNIV and (B) HaRNAV with and without chloroform. For both graphs, open symbols indicate chloroform treatments with the appropriate virus added (O) and without ( A ) . Solid symbols indicate control treatments (no chloroform) with virus added (•) and without (A). The arrow indicates when viruses were added to the cell cultures. In each case, values are the mean ± SD, n = 8. 32 Structural proteins The size and number of structural proteins in the virus particles were determined by polyacrylamide gel electrophoresis (PAGE). HaNIV particles contained at least two structural proteins (Fig. 2.3, lane 1). A 38.0 kDa protein was stained more intensely than a second 20.5 kDa protein. HaRNAV contains at least five proteins, 33.9, 29.0, 26.1, 24.6, and 24.0 kDa (Fig. 2.3, lane 3). Virus particles collected from the upper band in the sucrose gradient had an additional 39.0 kDa protein (Fig. 2.3, lane 2). H Z B SE « ^ 53 S H k D a 43.0 29.0 18.4 14.3 Figure 2.3. Structural proteins of Heterosigma akashiwo viruses. Proteins obtained from purified HaNIV (lane 1) and HaRNAV from the upper band (lane 2) and lower band (lane 3) of the sucrose gradient. Molecular weight markers (lane 4). The single 18 kDa capsid protein from tobacco mosaic virus was used as a control (lane 5). The proteins were stained with Coomassie Blue. 33 Nucleic acid type and genome size Nucleic acids from HaNIV were not digested by RNase A, but were digested with DNase I (results not shown) indicating that the HaNIV genome is double-stranded DNA. The HaNIV genome was digested into 12 and 15 fragments by Hind III and Hinc II, respectively (Fig. 2.4). After estimating the size of the fragments, the genome of HaNIV was determined to be approximately 37 000 base pairs (bp) based on the Hind III digest and 40 000 bp based on Hinc II. Averaging these estimates results in an approximate genome size of 38 500 bp. kbp 23.1 4.4 9.4 6.6 2.3 2.0 0.6 Figure 2.4. Restriction enzyme digests of HaNIV DNA. HaNIV D N A was digested with Hind III (lane 1) and Hinc II (lane 2). Bacteriophage X D N A digested with Hind III (lane 3) was used as a molecular weight marker. 34 Nucleic acids from HaRNAV were digested by RNase A in 0.0 IX and 2X SSC, but not with DNase A (results not shown). This result indicates that the genome of HaRNAV is single-stranded RNA. Based on gel electrophoresis, the length of the HaRNAV genome was estimated to be approximately 9100 nucleotides (Fig. 2.5). The genomic RNA was not retained by the poly-A tail purification column, but was recovered from the wash (data not shown). Therefore, the HaRNAV genome does not contain a poly-A tail. a Z si 03 X kb 9.49 7.46 4.40 2.37 Figure 2.5. Genomic RNA isolated from HaRNAV particles (lane 3). dsRNA from bacteriophage (p6 (lane 1) and ssRNA from T M V (lane 2) were used as controls. RNA standards were run in lane 4. 35 Hybridization HaNIV D N A hybridized to Heterosigma akashiwo D N A from infected cells, but not to D N A from non-infected cells (Fig. 2.6). Amount of D N A blotted (ng) 30 10 3 1 0.3 0.1 0.03 0.01 B O c D c Figure 2.6. Hybridization of HaNIV D N A with D N A from A) non-infected Heterosigma akashiwo, (B) HaNIV infected H. akashiwo, (C) a resistant strain of H. akashiwo, and (D) unlabeled HaNIV. D N A was blotted onto the membrane in serial dilutions. 36 Host range Host range results of HaNIV and HaRNAV are reported in Table 2.1. HaNIV caused lysis of 6 of the 7 strains of Heterosigma akashiwo from the Northeast Pacific, the resistant strain being NWFSC 503 (Table 2.1). HaNIV was unable to cause lysis of the strains from the Japanese or Northwest Atlantic coasts. HaRNAV caused lysis of 2 strains of Heterosigma akashiwo (H93616, H94608) from Japan and 3 strains (NEPCC 102, NEPCC 522, NEPCC 764) from the Northeast Pacific (Table 2.1). Japanese strains NIES 4 and NIES 293, the Northeast Pacific strains NEPCC 278, NEPCC 625, NWFSC 500, and NWFSC 503, as well as all strains from the Northwest Atlantic coast were resistant to HaRNAV. DISCUSSION A number of significant findings are reported in this study. Demonstrated, for the first time, is the existence of a single-stranded (ss) R N A virus (HaRNAV) that infects a microalga. As well, the partial characterization of this virus and a novel double-stranded (ds) D N A virus (HaNIV) has been provided. The characterization of HaRNAV and HaNIV describes two types of viruses that have never before been reported for algal species. Both these viruses infect Heterosigma akashiwo, an ecologically and economically important toxic bloom-forming alga. Therefore, there are at least 3 different viruses that infect and cause lysis of this organism. These results and their implications are discussed in detail below. Table 2.1. Sensitivity of various Heterosigma akashiwo strains to HaNIV and HaRNAV. + indicates the virus caused cell lysis, - indicates no lysis compared to a control culture, * indicates a discrepancy from the result reported by Lawrence et al. (2001). NEPCC 522 was the strain used for virus isolation. Strain Origin of Isolation HaNIV HaRNAV Northwest Pacific NEPCC 102 English Bay, BC, Canada + + NEPCC 278 Marietta, WA, USA +* -NEPCC 522 Jericho Beach, BC, Canada + + NEPCC 625 Genoa Bay, BC, Canada + -NEPCC 764 Kyoquot Sound, BC, Canada + + NWFSC 500 Kalalock, WA, USA +* -NWFSC 503 Manchester, W A , USA - -Atlantic NEPCC 560 Long Island Sound, N Y , USA -NEPCC 630 Long Island Sound, N Y , USA - -C C M P 525 Long Island Sound, N Y , USA - -U T E X L B 2005 York River, Gloucester Point, V A , USA - -Japanese NIES 4 (type strain) Fukuyama Bay, Hiroshima, Japan - -NIES 293 Onagawa Bay, Miyagi, Japan - -H93616 Hiroshima Bay, Japan - + H94608 Hiroshima Bay, Japan - + 38 H a R N A V . HaRNAV is the first ssRNA virus found to infect phytoplankton. Another ssRNA virus infects the green alga Char a corallina (Gibbs et al. 1975, Skotnicki et al. 1976), however, it is rod shaped with similarities to the furoviruses (van Etten et al. 1991). HaRNAV is polyhedral and is more similar in size and shape to plant viruses belonging to the families Bromoviridae, Comoviridae, Sequiviridae, and Tombusviridae, the invertebrate virus families Nodaviridae and Tetraviridae, the vertebrate virus family Picornaviridae, and the bacteria viruses of the Leviviridae (Murphy et al. 1995). However, except for the Sequiviridae and Picornaviridae, HaRNAV differs from these virus families in genome size and the number of structural proteins. Compared to HaRNAV, which has a single 9100 nucleotide genome and at least 5 structural proteins, these virus families typically have a smaller (5.5 kb for Tetraviridae, 4-4.7 kb for Tombusviridae, 3.5-4.3 kb for Leviviridae) or segmented (Bromoviridae, Comoviridae, Nodaviridae) genomes and have one (Bromoviridae, Leviviridae, Tetraviridae, Tombusviridae), two (Comoviridae), or three (Nodaviridae, Tetraviridae) capsid proteins (Murphy etal. 1995). HaRNAV is most similar to the Sequiviridae. Viruses belonging to the Sequiviridae are 30 nm in diameter, have a genome size ranging from 9-12 kb, and have at least 3 capsid proteins ranging from 22.5 to 34 kDa (Hemida and Murant 1989, Hemida et al. 1989, Reavy et al. 1993). In addition, like HaRNAV, the genome of some sequiviruses is not polyadenylated (Turnbull-Ross et al. 1992, Mayo et al. 1995). It is not known if sequiviruses are sensitive to organic solvents like HaRNAV, but lipids have not been reported for these viruses. Sensitivity to chloroform and other organic solvents is an indicator of a lipid component in the structure of the virus (Fenner et al. 1974). 39 Viruses belonging to the families Flaviviridae and Togaviridae are sensitive to organic solvents and have lipid envelopes in addition to having a similar genome size to HaRNAV (Murphy et al. 1995). These viruses, however, are larger (40-60 nm for Flaviviridae and 70 nm for Togaviridae) than HaRNAV. Although HaRNAV was sensitive to chloroform, a lipid envelope was not obvious in T E M observations of HaRNAV particles (Fig. 2.IB). The lipid component may reside inside the protein shell, like viruses belonging to the Phycodnaviridae (van Etten and Meints 1999). Viruses of the dsDNA family Corticoviridae are also sensitive to organic solvents, but do not have an envelope. Instead, a lipid bilayer forms between the inner and outer shell of the virus particles (Silbert et al. 1969, Ackermann 1995). The double-band pattern in the sucrose gradients of HaRNAV has been observed in other systems, including anthriscus yellows virus (AYV) belonging to the Sequiviridae (Hemida and Murant 1989, Hemida et al. 1989). The A Y V particles from the upper zone of the sucrose gradient were shown to be "empty" protein shells. Our results suggest a similar explanation for the upper band of particles from HaRNAV. The structural similarity of the particles from the upper and lower bands, based on PAGE, indicates that they are particles of the same virus (Fig. 2.3). However, because particles from the upper band were non-infectious, consistently penetrated by the negative stain, and because nucleic acids could not be isolated from them (data not shown), these particles are considered to be incompletely assembled HaRNAV. The additional 39.0 kDa protein obtained from particles in the upper band may represent an immature protein that needs to be cleaved to complete the particle or may be a scaffolding protein. 40 Although HaRNAV shares many characteristics with the Sequiviridae, further analysis, especially of the genome organization, is necessary to determine the relation of HaRNAV to known families of ssRNA viruses. Compared to known algal viruses, however, HaRNAV is unique. HaNIV. HaNIV, although a dsDNA virus, does not resemble previously characterised algal viruses including a Phycodnaviridae-Yike virus infecting Heterosigma akashiwo isolated from Japan (Nagasaki and Yamaguchi 1997). HaNIV is a polyhedral, 50 nm virus with a 38.5 kb dsDNA genome and at least 2 structural proteins. The virus is also known to assemble within the nucleus (Lawrence et al. 2001). In contrast, other dsDNA algal viruses isolated and characterised are 100-200 nm icosahedral viruses assembling in the cytoplasm with 100-380 kb genomes (van Etten and Meints 1999). In addition to differing from known algal viruses, HaNIV is also unique among all known dsDNA viruses. Few icosahedral dsDNA viruses are smaller than 60 nm and possess a small number of structural proteins. Although it is common for R N A viruses to have one or two structural proteins, most icosahedral dsDNA viruses rarely contain fewer than 10 proteins. The Papovaviridae, a family of viruses infecting vertebrates, is structurally similar to HaNIV. These viruses are icosahedral, 40-55 nm in diameter and contain 3 structural proteins (Frisque et al. 1995). As with HaNIV, these viruses also assemble in the nucleus and form crystalline arrays (Howatson 1973). However, because the genome of papovaviruses is only about 5 kb (Frisque et al. 1995) it is unlikely that HaNIV is related to the Papovaviridae. 41 Viruses with a similar dsDNA genome size to HaNIV include vertebrate viruses belonging to the Adenoviridae and bacteriophages of the Podoviridae. Adenovirus particles are typically 80-110 nm in diameter with fibres protruding from the capsid and contain approximately 40 different proteins (Russell et al. 1995). These viruses are also known to assemble in the nucleus and form crystalline arrays of progeny virus particles or protein subunits (Home 1973, Russell et al. 1995). Podoviruses are around 60 nm in diameter, contain 12 proteins, and have a small tail (Ackermann and Dubow 1995). Despite having some similarities with known dsDNA virus families, HaNIV differs significantly from these families in either genome size or structure. In addition, HaNIV is chloroform sensitive whereas most small, icosahedral, dsDNA viruses are resistant to chloroform. Sequence analysis of the HaNIV genome is needed to further assess the relationship of HaNIV to other viruses. The size of the free virus particles of HaNIV differs from the intracellular particles described by Lawrence et al. (2001). Intracellularly, the particles are ca. 30 nm in diameter and are organized into crystalline arrays. In contrast, extracellular HaNIV particles are 50 nm in diameter. Because of the size difference between the intracellular and extracellular particles, it was important to demonstrate that the purified virus particles were the same as those observed intracellularly. This was shown by hybridizing genomic D N A from purified HaNIV particles to D N A from infected H. akashiwo cells (Fig. 2.6). The difference in size between intracellular and extracellular HaNIV is likely because the intracellular particles are probably not mature viruses. The particles could be empty capsids awaiting final completion, in which the particles would expand in size 42 before they are released from the cell, as occurs with T4 phages (Black et al. 1994). The particles could also be excess viral proteins formed into paracrystalline masses, as observed with adenovirus infection (Henry et al. 1971, Russell et al. 1995). Host range and ecological significance. The characterisation of HaNIV and HaRNAV demonstrates the diversity of viruses infecting microalgae. This diversity has significant implications for our understanding of virus-host interactions in the environment and the effect of these interactions in bloom dynamics and other ecological processes. To assess the interactions of HaNIV and HaRNAV with Heterosigma akashiwo, a host range study, which tests the specificity of the viruses' pathogenic effect, was conducted. Similar to other viruses that infect Heterosigma akashiwo (Nagasaki and Yamaguchi 1998, Nagasaki et al. 1999, Tarutani et al. 2000) and other microalgae (Sahlsten 1998), HaNIV and HaRNAV are strain-specific. We tested the viruses against host strains from distant geographic areas (Japan, the Northeast Pacific, and the Northwest Atlantic) unlike previous host range studies (Nagasaki and Yamaguchi 1998, Nagasaki et al. 1999, Tarutani et al. 2000) that tested host strains from the same geographic area. Although HaNIV and HaRNAV are unrelated viruses, both were isolated from the Strait of Georgia, BC and mostly caused lysis of host strains from the Northeast Pacific, the same geographical location as their isolation. Both viruses were unable cause lysis of H akashiwo strains originating from the Northwest Atlantic and HaNIV caused lysis of host strains from the Northeast Pacific only. The host range of HaNIV was similar to that reported by Lawrence et al. (2001) except for two discrepancies (Table 2.1). In the present study, Heterosigma akashiwo 43 strains NWFSC 500 and NEPCC 278, both isolated from Washington State, lysed when challenged with HaNIV and the lytic effect was propagated. The reason for the differences in host range observed between the present study and that of Lawrence et al. (2001) are unknown. HaRNAV caused lysis of fewer Heterosigma akashiwo strains from the Northeast Pacific than HaNIV, but was able to lyse two strains from Japan. Its pathogenic effect, however, was restricted to Pacific host strains. Nagasaki et al. (1999) did not find a relationship between the location of virus isolation and the isolation location of susceptible host strains. In other words, the area of host or virus isolation was not a good predictor of host range. However, virus isolates were tested only against strains isolated from various areas of Japan; therefore, their result does not exclude the possibility of large-scale geographic patterns affecting host range. On a smaller spatial scale, the variation in host specificity of HaNIV and HaRNAV demonstrates the complexity of virus-host interactions in the natural environment. Although HaNIV and HaRNAV were originally isolated off the same strain of Heterosigma akashiwo, each virus infected a different subset of host strains. Therefore, in a given geographical area with a diversity of host strains, multiple viruses likely co-exist, each with a different range of host specificity. This virus community may collectively determine the composition of host strains or the strains that are able to bloom in that area. Tarutani et al. (2000) have shown that H. akashiwo cells differ in virus susceptibility as a bloom progresses. Cells isolated during the bloom were most 44 susceptible to viral lysis, while cells isolated as the bloom collapsed were more resistant. This result suggests that the composition of H. akashiwo strains changes throughout a bloom and the virus community may be regulating this change. As suggested in many other studies (Suttle et al. 1990, Waterbury and Valois 1993, Hennes et al. 1995, Fuhrman 1999, van Hannen et al. 1999, Wommack and Colwell 2000), viruses may be an important factor in determining aquatic microbial community composition, not only among species, but intra-specifically as well. The discovery of HaNIV and HaRNAV demonstrates the diversity of the virus community in the Strait of Georgia, BC and the diversity of viruses infecting a single isolate of Heterosigma akashiwo. The characterization of these two novel H. akashiwo viruses has shown that these viruses are unrelated to each other, to a H. akashiwo virus isolated from Japan (Nagasaki et al. 1994a, b, Nagasaki and Yamaguchi 1997), or to other known algal viruses. The diversity of H. akashiwo viruses and the variation in their host ranges suggests that in the natural environment the interaction of diverse viruses with diverse host strains is complex. In order to understand the role of viruses in the bloom dynamics oiH. akashiwo or any other phytoplankton species, we must understand the composition of the virus community, the variation of the community in time and space, and the interaction of the individual viruses with their hosts. 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Sequence data provides information on genome organization, coding and regulatory regions, and the types of proteins and enzymes produced by the virus. The information is also invaluable for phylogenetic analyses, evolutionary studies, and understanding the process of viral infection and replication. Because viruses have relatively small genomes compared to other living organisms, many complete genome sequences have been determined and they are increasingly easier to obtain with our growing technological capabilities. The generation of sequence data has greatly advanced our understanding of evolutionary relationships among viruses. Single gene phylogenies, often based on sequence data from polymerase or capsid genes, have been useful for inferring relationships among both D N A (for example, Chen and Suttle 1996, Knopf 1998, Tidona et al. 1998) and R N A viruses (for example, Goldbach and deHaan 1994, Ward 1993). Complete genome sequences, however, are an obviously better data set for evolutionary analysis. Rather than inferring the evolution of a virus based on one or two conserved genes, with a complete virus genome sequence, the evolution of the genome as a whole can be examined at the nucleotide level. The evolution of viruses can be assessed based 56 on genome organization, gene order, and the occurrence of gene modules in addition to individual gene sequences. Among the bacteriophages for example, gene order is more conserved than nucleotide or amino acid sequence (Ackermann 1998) and through comparative genomics the mosaic nature of phage genomes (Lucchini et al. 1999, Juhala et al. 2000, Mediavilla et al. 2000, vander Byl and Kropinski 2000, Desiere et al. 2001) is revealed. Regions of clear homology are interspersed with unrelated regions, therefore demonstrating that phages have likely evolved by extensive horizontal gene transfer and the exchange of gene cassettes from a common ancestral pool of genes (Hendrix et al. 1999). Most complete genome sequences have been obtained from well-studied animal, plant, and bacteria viruses and few sequences have been reported for viruses from marine environments. The complete genome sequence has been obtained for a marine virus infecting a Roseobacter isolate (Roseophage SIOl) (Rohwer et al. 2000). Roseophage SIOl was identified as a member of the Podoviridae phage family based on physical characteristics and also shared genetic similarities with the podoviruses T3 and T7. Other than this study, however, complete genome sequences have not been determined for marine viruses and only a few phylogenetic studies have been conducted (Chen and Suttle 1996, Lee etal. 1998). As a contribution to the growing number of complete viral genome sequences and to further our understanding of marine and algal virus diversity and evolution, the genome sequence of HaNIV, a virus causing lysis of Heterosigma akashiwo, was determined. This is the first genome sequence reported for a virus with a phytoplankton host. Over 90 % of the genome represented in two contigs was sequenced. The 57 annotation of these sequences, the function of putative open reading frames (ORFs), and the genetic relationships of HaNIV to other viruses are discussed in this chapter. The sequence information was also used to show that HaNIV might be a latent virus. M A T E R I A L S A N D M E T H O D S HaNIV particles were amplified from cultures of Heterosigma akashiwo inoculated with the virus, concentrated by ultracentrifugation, and purified by 10 to 40 % sucrose gradients as described in the previous chapter. Genomic D N A from purified HaNIV particles was obtained, also described in Chapter 2, using a standard phenol: chloroform extraction method and precipitating the D N A with ethanol. Construction of HaNIV clone library. The genomic D N A was digested with either the restriction enzymes Hind III or Hinc II (New England Biolabs). p G E M -3Zf (+) plasmid vectors (Promega) were also linearized by cutting with either of the same restriction enzymes. The ends of the linearized plasmid D N A were dephosphorylated with calf intestinal alkaline phosphatase (Gibco/BRL). The digested HaNIV D N A fragments were ligated with T4 D N A ligase (Gibco/BRL) into the linearized, dephosphorylated plasmid vectors. The recombinant plasmids were transformed into Epicurian Coli® X L 1-Blue supercompetent cells (Stratagene) or Library efficiency® DH5a™ competent cells (Gibco/BRL). Transformants were selected by growing the cells on Luria-Bertani (LB) agar plates containing 100 llgml" 1 ampicillin and 100 (ll of 2 % 5-bromo-4-chloro-3-inodlyl-P-D-galactopyranisode (X-gal) and 20 p:l of 20 mg-ml"1 isopropyl-l-thio-(3-D-galactopyrano-side (IPTG) spread onto the hardened plates. 58 Transformed cells with recombinant plasmids formed white colony forming units (CFUs) whereas cells containing plasmids without an insert formed blue CFUs. Individual white CFUs were picked for overnight cultures grown in L B liquid media containing 100 (ig-mf1 ampicillin. Plasmid D N A was obtained from the overnight cultures by alkaline lysis following standard protocols (Sambrook et al. 1989) or with a QiaPrep® Spin Miniprep Kit (Qiagen). The presence and size of the inserted D N A fragment in the plasmid D N A was confirmed by digesting the recombinant plasmid with the same restriction enzyme used to linearize the plasmid. Thus, the inserted fragment was cut out of the plasmid D N A . The digested D N A was run on a 0.8 % agarose gel, stained with ethidium bromide, and visualized with an U V transilluminator. The E. coli clones were identified based on the size of the fragment carried by the plasmid vector. D N A from plasmids with different sized inserts were chosen for sequencing. Sequencing and assembly of contigs. Sequencing reactions were performed by thermal cycling using ABI PRISM BigDye Terminators (PE Applied Biosystems, Inc.). Following the reaction, excess dye terminators were removed with Centri-Sep columns (Princeton Separations Inc.) following the manufacturer's protocols. The sequence was obtained by automated sequencing using ABI 373 Stretch or ABI Prism 277 sequencers (Nucleic Acid Protein Services (NAPS) facility at the University of British Columbia). Initially, the inserted D N A fragment was sequenced from the plasmid D N A in forward and reverse directions by using sequencing primers (-21M13 and M13R from NAPS) complementary to opposite sides of the multiple cloning site on the p G E M plasmid. Subsequent sequencing of the insert was accomplished by designing sequencing primers 59 from the ends of the sequence information previously obtained (i.e. by primer walking). Sequences were analyzed and assembled into contiguous sequences (contigs) by eye using BioEdit, version 5.0.7 (Hall 1999, available from the web at http://www.mbio. ncsu.edu/BioEdit/bioedit.html). Many sequences could not be aligned to one another following fragment cloning and sequencing because not all the fragments were cloned. To obtain overlapping sequence and to resolve gaps in the sequence, PCR primers were designed from the ends of the contigs. Using HaNIV genomic D N A as the template and different pairs of primers for PCR reactions, D N A was amplified when primers annealed to adjacent locations on the genome. The PCR products were cloned and sequenced or sequenced directly as described above. Sequence analysis. Open reading frames (ORFs) were identified from the genomic sequence by translating in all six reading frames using any start codon, but ending at stop codons (BioEdit). Potential start codons (ATG or GTG) and ribosome binding sites (a series of As and Gs, 1-10 nucleotides preceding the start codon) were identified by eye. Translations greater than 75 amino acids were identified as putative ORFs. PSI(\|/)-BLAST (Altschul et al. 1997, http://www.ncbi.nlm.nih.gov/BLAST) was used to search for possible homologies of HaNIV ORFs to known protein sequences in GenBank. Phylogenetic trees were calculated for HaNIV ORFs homologous to lysozymes and the large subunit of terminase. HaNIV ORFs were aligned to protein sequences from bacterial phages, prophages, herpesviruses, and vertebrates obtained from GenBank (Table 3.1) using Clustal W (Thompson et al. 1994, available with BioEdit). The 60 Table 3.1. GenBank accession numbers for protein sequences used for phylogenetic analyses. * indicates that the assigned function for the protein is putative. Virus or species L S U terminase Lysozyme Myoviridae Enterobacteriophage T4 NP 049776 NP 049736 Enterobacteriophage PI P27753 CAA61013 Enterobacteriophage P2 NP_046758 NP 046765 Enterobacteriophage M u (p.) AAF01099 Podoviridae Enterobacteriophage T3 P10310 P20331 Enterobacteriophage T7 P03694 P00806 Enterobacteriophage P22 AAF75044 AAF75040 Bacteriophage PS34 CAA09706 * Bacteriophage PS119 CAA09710 Siphoviridae Bacteriophage lambda (A,) NP_040581 NP_040645 Bacteriophage 21 AAA32340 AAA32350 Bacteriophage HK022 AAF30354 * NP 037696 Bacteriophage HK97 AAF31098 * AAF31145 Bacteriophage HK620 AAK28891 * NP_112069 * Unclassified Bacteriophage APSE-1 NP_050979 * AAF03956 * Prophages In Xyella fastidiosa (1) AAF83318 * In Xyella fastidiosa (2) AAF84373 * In Xyella fastidiosa (3) AAF85113 * In Escherichia coli (1) BAB38385 * CP-933V in Escherichia coli AAG57223 * DLP12 in Escherichia coli P78285 * In Haemophilus influenzae P44184 * Herpesviruses HSV11 P04295 HSV7 AAC40780 Vertebrates Gallus gallus (chicken) 1LYZ Anser anser (goose) P00718 61 alignments were replicated 100 times by bootstrapping and analyzed using the Neighbor-joining and Parsimony algorithms found in Phylip (Phylogenetic Inference Package; version 3.5c; distributed by J. Felsenstein at http://evolution.genetics.washington.edu/ phylip.html). Neighbor-joining trees were calculated from a distance matrix obtained with the protein distance algorithm of Phylip. Trees were viewed using TreeView, version 1.6.5 (Page 1996, http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Detection of the HaNIV genome in the host, Heterosigma akashiwo. D N A was obtained from Heterosigma akashiwo cells infected and non-infected with HaNIV as described in the previous chapter. D N A from a strain of H. akashiwo resistant to HaNIV was also obtained. Cells from infected and non-infected cultures were pelleted by centrifugation (1500 X g for 10 min), resuspended with media, and centrifuged again. D N A was extracted using a standard phenol and chloroform: isoamyl alcohol protocol, precipitated with ethanol, and resuspended in water. D N A from infected and non-infected cells were used as templates for PCR reactions with HaNIV-specific primers. The primers used for PCR amplification were Hc l3AF (5'-CAC A T C GCC C A T GAT TCA) and Hcl3R (5'-TGA A G C A G G TCG A T C TAG) and annealed to positions 2412 and 2999, respectively on contig B (Fig. 3.1, Appendix B). HaNIV D N A was used as a control. Aliquots of the PCR products were analyzed on a 1.5 % agarose gel, stained with ethidium bromide, and visualized with a U V transilluminator. The remaining PCR products were cleaned with the QiaQuick® PCR purification kit following the manufacturer's protocol and sequenced directly as described above. 62 R E S U L T S Following the cloning procedure, ten of the thirteen Hind III fragments were cloned and three fragments, approximately 2000, 1000, and 500 bp in length, were not. Because the Hinc II digestion resulted in blunt ended fragments (Hind III digestion left 5' overhangs), the ligation of Hinc II fragments with the vector was not as successful as with Hind III fragments. Thus, only ten of the fifteen fragments were cloned. The two largest fragments and three fragments ranging from 1500 to 2000 bp are missing from the clone library. Once the cloned fragments were sequenced, overlapping regions were found between sequences from Hind III and Hinc II fragments. Eight contigs resulted from this process. A combination of PCR and direct sequencing successfully identified sequences not represented in the clone library. The eight contigs were aligned into two longer sequences 17 765 and 15 858 bp in length and are identified as contigs A and B, respectively. Due to time constraints the genome sequence of HaNIV was not completed. Direct sequencing by primer walking is continuing in order to align contigs A and B and to sequence the ends of the genome. Although the genome sequence is incomplete, the sequence of contigs A and B, representing approximately 94 % of the genome, was analyzed and annotated for this chapter. Sequence analysis. The GC content of contigs A and B were 52.01 % and 51.48 %, respectively. From contigs A and B, 76 putative ORFs greater than 75 amino acids in length were identified. A l l ORFs were numbered sequentially along with the letters A or B to 63 denote the contig from which they were identified. The location and orientation of the ORFs are diagrammed in Figure 3.1 and their coordinates on the contigs are listed in Table 3.2. The translated amino acid sequences of the putative ORFs were submitted to the PSI-BLAST program at the National Center for Biotechnology Information (NCBI) for the identification of homologs. Similarities were found to lysozymes and the small subunit (SSU) and large subunit (LSU) of terminases from bacteriophages and prophages (Table 3.2). ORF 16A was homologous to the terminase small subunit from bacteriophage HK620. ORF 18A, located directly downstream of ORF 16A, encoded a protein that was most similar to the terminase B (LSU) protein from enterobacteriophage PI. Other hits to ORF 18A included a hypothetical protein from a prophage region of Haemophilus influenzae and the C-terminus of putative L S U terminases from bacteriophages HK620 and APSE-1. ORF 18B was most similar to a lysozyme sequence from a prophage region of Xyella fastidiosa. This ORF also showed homology to lysozymes from bacteriophages PS119 and P22 and to putative lysozymes from bacteriophage PS34 (gpl9) and prophages of Escherichia coli. ORF IB was similar to the NinB proteins of bacteriophage 21, phage lambda, phage P22, and gp61 of phage HK97. The homologous region spanned the entire length of ORF IB, but did not include the first 50 amino acids of known NinB amino acid sequences. Homologies were also detected to predicted proteins of unknown function from the bacteria Mesorhizobium loti (ORF 7 A) and Legionella pneumophila (ORF 21 A) (Table 3.2). Re-iterating the PSI-BLAST search with ORF 21A and the unknown protein 64 65 Table 3.2. ORFs predicted from HaNIV sequence. The letters A and B denote the contig from which the ORF was identified. The coordinates describe the location of the ORF from the first nucleotide of the first start codon with a ribosome binding site (RBS) to the last nucleotide of a stop codon. If a RBS was not detected, then the ORF is described from the first encountered start codon, regardless of a RBS. The reading frame is shown in parentheses. * indicates a GTG start codon, otherwise it is A T G . The length of the translated ORF is in amino acids (aa). Matches show significant matches (P < 0.005) for the appropriate ORF. For the highest ranking match, GenBank accession numbers for the protein database precede the description of the protein and e-values and identities are provided. Identities show the percentage of identical amino acids in the homologous region (length shown in parentheses). O R F Coordinates Length (aa) RBS Matches Psi-Blast e-value Identity (%) Contig A 1A 446 - 670 (2) 75 -2A 670- 1359(1) 230 + 3A 1409 - 1777 (2) 123 + 4A 1768-2127(1)* 120 -5A 1946-2212(2) 89 -6A 2093 - 2344 (-2) 84 -7A 2127 -2531 (3) 135 + BAB50597, unknown protein, Mesorhizobium loti 2 x 10"7 30(132) 8A 2624 - 3409 (2) 256 + 9A 2906 - 3448 (-2) 181 -10A 3012 -3281 (-3)* 90 -11A 3413 -4024 (2) 204 + 12A 4425 - 4886 (3) 154 + 13A 4568 - 4879 (-2) 104 -14A 5004 - 5405 (3) 134 + 15A 5108 - 5569 (-2)* 130 -6 X 10"12 16A 5415 - 5867 (3) 151 AF335538, Terminase small subunit, bacteriophage HK 620 34 (134) 17A 5560 - 5811 (1)* 84 -18A 5870 - 7345 (2) * 492 P27753, Terminase B protein, bacteriophage PI; also matches hypothetical protein HIM 10, Haemophilus influenzae; PI8, bacteriophage APSE-1; terminase large subunit, bacteriophage HK620 6X 10'9 25 (303) 19A 7272 - 7796 (3) 175 + 20A 7799 - 8099 (2) 97 + 21A 8213 -9793 (2) 527 CAC33475, unknown protein in Legionella pneumophila serogroup 1, 30kb instable genetic element 4 X 10"5 19 (379) 22A 10104 - 10766 (3) 221 + 23A 10785 - 11792 (3) 336 + 66 Table 3.2. continued O R F Coordinates Length RBS Matches (aa) 24A 11808 - 12260 (3) 151 + 25A 11836 -12201 (1) * 122 + 26A 12263 -12622 (-2) * 120 -27A 12468 -13124 (3)* 219 + 28A 12537 -12902 (-1)* 122 + 29A 13120- 15543 (1)* 808 -30A 13148- 13405 (2) 86 -31A 14566- 14889 (-3) 108 -32A 15550 - 16131 (1) 194 -33A 16138- 17763 (1) * 542 + 34A 16166- 16636(2) 157 + 35A 16272 -16493 (-1) 74 + 36A 16694 -16921 (2) 76 -37A 16968 -17195 (-1) * 76 -Contig B IB 184-423 (1) * 80 -Psi-Blast e-value Identity (%) 2B 423 -893 (3) 157 + 3B 640- 903 (1) 88 -4B 811 -1116 (-1) 102 -5B 893 -1702 (2) 270 + 6B 1926 -2153 (3) * 76 + 7B 2182 -2436 (1) 85 + 8B 2364 - 2843 (3) 160 + 9B 3179 -3415 (2) 79 + 10B 3732 - 3986 (3) 85 + 11B 4083 - 4778 (3) 232 + 12B 4828 - 5205 (1) 126 + 13B 5397 - 5678 (3) 94 + 14B 5653 - 6051 (-1) 129 + 15B 5989 -6411 (1) 141 -16B 6033 -6281 (-2) 83 -17B 6166 -6525 (-1) 120 -18B 6480 - 7490 (-2) 337 + 19B 6836 - 7099 (2) * 88 -20B 7465 - 8769 (-1) 435 + 21B 7481 -7768(2) * 96 -22B 7888 -8241 (1) 118 -23B 8351 - 8641 (-3)* 97 -24B 8730-9164 (-2) 145 + 25B 8743 - 8985 (1) 81 -CAB39988, NinB protein, bacteriophage 21; also matches gp61, bacteriophage HK97; NinB, bacteriophage phage lambda; NinB, bacteriophage P22 9X 10" 44 (75) AAF84373, Phage-related lysozyme of Xyella fastidiosa; also matches lysozyme (gpl9) of bacteriophages PS119, PS34, and P22 8 X 10"' 33 (127) 67 Table 3.2. continued O R F Coordinates Length RBS Matches Psi-Blast Identity (aa) e-value (%) 26B 9168-9587 (-2) 140 + 27B 9605 - 12346 (-3) 914 + 28B 10560- 10793 (3) 75 -29B 10993 - 11232 (-1) 80 -30B 11291 - 11521 (2) 77 -31B 12517 - 14022 (-1) 512 -32B 12937 - 13599(1) 221 -33B 13038 - 13328 (-2) * 97 -34B 13475 - 13699 (2) 75 + 35B 13728 - 13953 (1) 76 -36B 13974- 14324 (3)* 117 -37B 14031 - 15704 (-2) 558 -38B 14254- 14499(1) 82 -39B 14991 - 15407 (3) * 139 -40B 15019 - 15507 (1)* 163 -from L. pneumophila resulted in significant hits to the head-tail joining protein of bacteriophages T7, phiYe03-12, and T3. Phylogenetic analyses. Phylogenetic analyses were conducted with HaNIV ORFs homologous to L S U terminases and lysozymes. Generally, the parsimony analyses gave better, more robust results with stronger bootstrap support than neighbor-joining, therefore, for each analysis, the parsimony tree is shown (Figs. 3.2 and 3.3). The poor neighbor-joining results occurred because many of the distances calculated between the operational taxonomic units (OTUs) were infinite. Based on the parsimony analysis of ORF 18A with L S U terminases from bacteriophages, prophages, and herpesviruses, ORF 18A grouped strongly with the L S U terminases from bacteriophages PI and P22 (90 % bootstrap support) (Fig. 3.2). The herpesvirus terminases grouped strongly together (100 %) and in a cluster including L S U 68 terminases from bacteriophage lambda, 21, and a prophage in Xyella fastidiosa (96 %). Bacteriophage 21 and lambda paired together (100 %), as did T3 and T7, and HK022 and HK97. The association of terminases from HK620, APSE-1, and a prophage region in Haemophilus influenzae was also supported (87 %). The analysis of lysozyme sequences (Fig. 3.3) included lysozymes from chicken {Gallus gallus) and goose {Anser anser) in addition to viral ones. The chicken and goose lysozymes grouped within the phage lysozymes although strong relationships were not found. ORF 18B grouped with prophage lysozymes in Xyella fastidiosa (83 %). Lysozymes from the siphoviruses lambda, HK022, HK97, and HK620 grouped together with strong support (100 %) with the P2 lysozyme as a sister to this group (100 %). The fifth siphovirus lysozyme included in the analysis, from phage 21, grouped with the lysozyme from prophage DLP12 in Escherichia coli on the opposite end of the tree (99 %). Lysozymes from phage 21 and DLP12 clustered with those from PS119 and PS34 (100 %). Other strongly supported groups were lysozymes from T3 and T7 (100 %) and APSE-1 andP22(100%). Analysis of the host genome. PCR with HaNIV-specific primers successfully amplified the expected 600 bp product from the D N A of infected and non-infected Heterosigma akashiwo (Fig. 3.4). Amplification was consistently stronger with template from infected cells. The sequences of these products were identical to HaNIV sequence (data not shown). 69 prophage, H. influenzae APSE-1 HK620 HaNIV, ORF 18A P22 HSV7 HSV11 prophage (1), X. fastidiosa lambda phage 21 Figure 3.2. Unrooted parsimony tree of viral L S U terminases and HaNIV ORF 18A. The consensus tree is shown. Numbers at branch nodes indicate percentage bootstrap support from 100 replicates. Values less than 50 % are not shown. Any significant support from the neighbor-joining analysis is shown in parentheses. 70 HK620 HK97 Figure 3.3. Unrooted parsimony tree of lysozyme amino acid sequences and HaNIV ORF 18B. The consensus tree is shown. Numbers at branch nodes indicate percentage bootstrap support from 100 replicates. Values less than 50 % are not shown. Any significant support from the neighbor-joining analysis is shown in parentheses. 71 "3 £ 1 2 1 z 5 a $ c3 * .3 © g > a B s s 9 ^ + fc + « « b p 2000 1500 600 Figure 3.4. PCR analysis of D N A from infected and non-infected Heterosigma akashiwo. PCR products with HaNIV primers using template D N A from non-infected H. akashiwo (lane 1), infected H. akashiwo (lane 2), and HaNIV particles used as a positive control (lane 4). D N A was not amplified from DNA isolated from a resistant strain of//, akashiwo (lane 3). The negative control was run in lane 5 and molecular weight markers in lane 6. 72 DISCUSSION By using the genome sequence of HaNIV, potential open reading frames were identified and compared to genes of other viruses. HaNIV ORFs were similar to several known bacteriophage proteins and these similarities provide insights into the evolution and biology of HaNIV. O R F 16A and 18A possibly encode terminase subunits. The translated sequences of ORFs 16A and 18A were homologous to the small and large subunit of phage terminase enzymes, respectively. Similar to phage genomes (Skorupski et al. 1992, Loessner et al. 2000, van der Byl and Kropinski 2000, Desiere et al. 2001), the SSU ORF was located upstream of the L S U ORF. The terminase (or pacase) enzyme complex is responsible for cutting D N A concatemers and packaging D N A into viral capsids (reviewed in Black 1989). Concatemers are tandem arrays of mature phage D N A covalently joined end to end. Bacteriophages use various strategies to ensure that a full complement of genetic information from the concatemer is packaged into each progeny prohead. For X, T3, and T7 phages, terminase cuts the D N A concatemers at specific D N A sequences (for lambda these are called cos sites), thus the mature D N A in each virion has identical ends (Hohn 1983). For T4 phages, the cleavage of D N A concatemers does not depend on sequence. The initial cut is random and the second cut occurs when the virion head is completely filled with D N A ("headful" cleavage) (Streisinger et al. 1967). This results in virions with terminally redundant, circularly permuted DNA. For PI and P22 phages, the initial cut in the D N A concatemer occurs at a specific sequence, designatedpac, but the second cut is non-specific and occurs when the virion is filled 73 (Tye et al. 1974). Packaging into successive proheads continues processively from this second cut in a headful manner. This results in viral genomes that have non-random terminally redundant circularly permuted ends. The presence of terminase-like ORFs in the HaNIV genome suggests that D N A packaging of the genome may be similar to the strategies of known bacteriophages. ORFs 16A and 18A were most similar to the SSU terminase of HK620 (a siphovirus) and terminase B (LSU) of coliphage PI (a myovirus), respectively, therefore HaNIV may have a D N A packaging strategy similar to these two viruses. The function of the HK620 and PI terminase complexes cannot be compared, however, because the D N A packaging strategy of HK620 is not known. Because the terminase complex of HaNIV has components of terminases similar to two different viruses, it would be interesting to test the functional complementation of the three terminases. In addition, determining the ends of the HaNIV genome will be useful for inferring a D N A packaging mechanism for HaNIV. O R F 18B shares similarities with phage lysozymes. The lysozyme amino acid sequences of many phages were homologous to HaNIV ORF 18B. Lysozymes are responsible for degradation of the host cell wall late in replication so that progeny phages can be released. ORF 18B was most similar to putative lysozymes from prophages in Xyella fastidiosa clone 9a5c. X. fastidiosa is a bacterium responsible for various diseases in economically important plants. Knowledge of prophages in the genome of X. fastidiosa was evident following analysis of its complete genome sequence (Simpson et al. 2000). In addition to prophage lysozymes, ORF 18B was homologous to lysozymes 74 encoded by gene 19 of the bacteriophages PS119, PS34, and P22, all members of the Podoviridae. There are several genes responsible for cell lysis during the late stages of phage replication. The lysis enzymes of P22 (gpl3, gpl5, and gpl9) function analogously to those of bacteriophage X (proteins S, Rz, and R, respectively) despite the absence of noticeable amino acid sequence similarities (Fig. 3.3, Rennell and Poteete 1985, Casjens et al. 1989). For P22, and similarly with A,, pores in the inner membrane are formed by gpl3 through which gpl9, the main lysozyme, can exit. The lysozymes produced by gene 19, also known as muramidases (X produces transglycosylases), degrade the peptidoglycan (or murein) layer. The function of gpl5 is not completely understood, but it is thought to complete peptidoglycan degradation or disrupt cell membranes in the presence of divalent cations (Casjens et al. 1989). Heterosigma akashiwo does not have a rigid cell wall like bacteria, but it does have a glycocalyx surrounding its cell membrane. The glycocalyx is formed by mucus secretion and is composed of acidic, complex carbohydrates (presumable of hyaluronic acid) and a neutral protein-carbohydrate complex of glycoproteins (Honjo 1993). It may be possible that ORF 18B is a lysozyme responsible for degrading the glycocalyx during lytic viral replication. O R F IB is homologous with NinB sequences. The Nin region of bacteriophage X is a region of eight, mostly unknown ORFs between the D N A replication gene P and the regulator of late transcription, gene Q (Hendrix et al. 1983). The region is so named because of the N-independent (Nin) phenotype that results from the nin5 deletion mutation which covers this region. The product of gene N is responsible for activating 75 early gene expression by antagonizing transcription termination and is required for X to grow lytically (Hendrix et al. 1983). In pN- mutants, an additional nin5 mutation allows lytic X replication because of the deletion of a transcription terminator (Hendrix et al. 1983). Thus, early genes are transcribed without a functional N gene, i.e. an N -independent phenotype. HaNIV ORF IB is homologous to NinB (also called orf) of X and to similar proteins from bacteriophage 21, HK97, P22, HK620, 933W, VT2-Sa, and prophages from Escherichia coli. Most of these proteins are similar in length, 145-148 amino acids (aa) long, with the exception of the proteins from HK620 (136 aa), VT2-Sa (181 aa), and the considerably shorter ORF IB from HaNIV (80 aa). The X NinB is one of the few nin proteins for which a function has been inferred. It is a trans-acting factor involved in X recombination (Sawitzke and Stahl 1994). In addition, Juhala et al. (2000) propose that the function of the entire nin region, is to provide a collection of genes enabling a phage to adapt to a particular ecological niche. This is an interesting hypothesis to pursue considering that HaNIV is a marine virus and other viruses with nin proteins are enterobacteriophages. However, ORFs surrounding ORF IB did not show homology to other nin proteins and further analysis is necessary in order to determine the function of this region. O R F 21A may be a structural protein. ORF 21A was most homologous to an unknown protein from an unstable genetic element of Legionella pneumophila, a human bacterial pathogen (Luneberg 2001). Both of these ORFs showed homology to the head-tail joining proteins of bacteriophages T7, cpYe03-12, and T3, all Podoviridae. Morphologically, HaNIV resembles podoviruses, but has a small extension at one apex of 76 the capsid instead of a distinct tail (see Chapter 2, Fig. 2.1 A). Because ORF 21A has homologies with head-tail joining proteins, ORF 21A may encode a structural protein involved with its capsid extension. The head-tail joining protein of T3 is 58.6 kDa (Beck et al. 1989) and it is expected that ORF 21A encodes a protein of similar size. However, a structural protein of this size was not detected from HaNIV particles (see Chapter 2, Fig. 2.3) likely because the protein is a minor component. Another potential structural similarity between HaNIV and podoviruses involves the major capsid protein. The major stuctural protein of HaNIV is 38 kDa and, although podovirus particles contain about 12 proteins ranging from 13 to 150 kDa, they also have a major capsid protein of 38 kDa (Ackermann and Dubow 1995). Unfortunately, the ORF encoding the major capsid protein could not be identified from the HaNIV genome sequences. Identifying the amino acid sequences of the HaNIV capsid proteins will be required to confirm any structural similarities between HaNIV and podoviruses. Phylogeny. HaNIV encodes several putative proteins that are homologous to bacteriophage proteins. However, the phages that encode these proteins do not belong to a single group, but span a diversity of bacteriophages. HaNIV ORFs 16A, 18A, 21 A , IB, and 18B showed the greatest similarity to proteins from a siphovirus (HK620), a myovirus (PI), podoviruses (T3 and T7), a siphovirus (phage 21), and prophages (in Xyella fastidiosa), respectively. Much like the genome of most phages, the HaNIV genome is probably a mosaic of genes with different evolutionary origins. The phylogenetic analyses performed in the present study were used to demonstrate the mosaicism of HaNIV and other viruses. In each analysis, proteins from the same phages (myoviruses T4, PI, and P2, podoviruses T3, T7, P22, and APSE-1, 77 siphoviruses lambda, 21, HK97, HK022 and HK620 and HaNIV) were included. In general, viruses belonging to the same family did not group together. Podoviruses T3 and T7 and siphoviruses HK97 and HK022 were the only pairs of viruses to consistently branch together. In addition, aside from these pairings, branches supported in one analysis, were not by the other. The lack of sequence homology among proteins from similar phages despite having analogous gene function is well recognized (Ackermann 1998). For example, as in Fig. 3.2 and other studies (Smith et al. 1999, Desiere et al. 2001), some bacteriophage terminases are more similar to herpesviruses than to other phages belonging to the same family. Lysozymes from T4 and P22 are known to share similar 3-dimensional structures and enzymatic functions to animal and plant lysozymes, but have variable amino acid sequences (Weaver et al. 1985, Holm and Sander 1994). It has also been shown that the lysozymes of T4, P22, and A, are interchangeable in complementation assays even though they have different enzymatic activities and primary structure (Rennell and Poteete 1985). The difference in amino acid sequences among proteins with analogous functions could be due either to horizontal gene transfer or the evolution of the protein from an ancestral gene originating prior to the divergence of prokaryotes and eukaryotes (Grutter et al. 1983, Ackermann et al. 1995, Hendrix 1999, Smith et al. 1999). Although some bacteriophage genes may be ancestral, horizontal gene transfer, involving the exchange of modules or blocks of genes between viruses, has been described as the major mode of bacteriophage evolution (Ackermann 1998). This mode of evolution was recognized because of the genetic mosaicism observed in phage genomes (Hendrix et al. 1999). Thus, due to the extent of horizontal gene transfer among 78 viruses, phylogenetic analyses of protein sequences can be used to infer evolutionary relationships among viruses, but not their origin. Instead of sequence comparisons, genome organization, gene order, and protein conformation and function may be better indicators of viral evolution. These types of analyses are currently driving our knowledge of the evolution of viruses. For HaNIV, because the function of most of the ORFs is unknown, gene order and organization could not be detected. Of the ORFs for which a function was putatively determined, a structural gene (ORF 21 A) followed the terminase genes (ORF 16A and 18A). This gene organization is also found in many bacteriophages (Pfister et al. 1998, Desiere et al. 1999, Lucchini et al. 1999, Juhala et al. 2000). The genomes of most bacteriophages are organized into gene clusters of related function (Ackermann 1998). These clusters include packaging, head morphogenesis, tail morphogenesis, lysis, lysogeny, and replication genes. To determine if the HaNIV genome is similarly organized, the function of the putative HaNIV ORFs must be identified. Algal virus or bacteriophage? The sequence of the HaNIV genome revealed homologies that were unexpected for a virus infecting a eukaryotic phytoplankton. Five ORFs showed homology to proteins from bacterial prophages and bacteriophages. Because the virus replicates within a eukaryotic host, it was expected that HaNIV ORFs would be homologous to genes from viruses infecting eukaryotes. There are two possible explanations for this result. HaNIV is a virus of Heterosigma akashiwo, but carries genes reminiscent of prokaryotic phages. Alternatively, the virus may infect a bacterium and not H. akashiwo. 79 The homologies of HaNIV ORFs with phage proteins is not completely surprising considering the prevalence of horizontal gene transfer in the evolution of viruses. As Hendrix et al. (1999) explain, all viruses have access to a global pool of gene sequences and the extent of horizontal gene transfer between viruses of phylogenetically distant hosts depends on the number of individual steps through "phylogenetic space" required to bring a particular sequence and virus together. Hendrix et al. (1999) were referring exclusively to bacteriophages, but the presence of diverse phage-like genes indicating the mosaicism of the HaNIV genome suggests that, if Heterosigma akashiwo is the host of HaNIV, horizontal gene transfer may be much more extensive than previously considered. In addition, because the similarities between HaNIV and bacteriophage genes are still recognizable, the gene sequences are either highly conserved or the gene transfer occurred relatively recently and the "steps" through "phylogenetic space" must have been very large. Common ancestry could also explain the sequence homology between HaNIV and bacteriophages. Homology due to common ancestry between viruses of phylogenetically distant hosts has been demonstrated previously. Between animal and bacterial viruses, the homology was not recognized through sequence similiarities, however, but phenoptypically through the solved structures of capsid proteins (Benson et al. 1999). The homology in protein structure between animal and bacterial viruses is believed to be due to common ancestry (Hendrix 1999). Homologies, both morphological and genetic, are also noted between viruses from the two domains of prokaryotes, the Bacteria and Archea, and are probably best explained as the result of common ancestry (Pfister et al. 1998, Hendrix 1999). 80 The alternative explanation for the phage homology, that the isolated particles of HaNIV are bacteriophages, cannot be completely discounted. HaNIV particles were not isolated from an axenic culture of Heterosigma akashiwo, so it is possible that bacteriophages were isolated inadvertently. Although the hybridization assay discussed in the previous chapter indicates that infection is associated with H. akashiwo cells, there could have been contaminating bacteria. Thus, i f the HaNIV particles are bacteriophages, then the bacterial host must have a physical and physiological connection with H. akashiwo. There must be a physical relationship because the infected bacteria must have pelleted with H. akashiwo during the host D N A isolation procedure of the hybridization assay. The centrifuge spin was designed to pellet H. akashiwo cells and leave bacterial cells in the supernatant, but bacteria may have been caught in the thick glycocalyx of H. akashiwo. Secondly, there must be a physiological relationship because virus inoculation results in the lysis of H. akashiwo. Therefore, a viral infection of the bacteria must negatively affect the growth of H. akashiwo. Unfortunately, ecological relationships between bacteria and phytoplankton, especially symbiotic or obligate, are poorly understood. The few studies relating bacteria and H. akashiwo concern algicidal bacteria (Kim et al. 1998, Yoshinaga et al. 1998) and the association of bacteria with H. akashiwo toxicity (Carrasquero-Verde 1999). Further evidence showing that HaNIV is a virus of H. akashiwo will have to be obtained from more complex techniques such as in situ hybridization with transmission electron microscopy or in situ PCR. Despite the ambiguity of the host, however, the homology of HaNIV ORFs to phage enzymes provides insights into the replication process and evolution of this virus. 81 HaNIV may be latent. The PCR and sequence analysis of non-infected Heterosigma akashiwo D N A indicated that at least a portion of the HaNIV genome was present (Fig. 3.4). This result suggests that HaNIV is latent, but it is not known if the genome is integrated into the host's genome or if it is extrachromosomal. A n integrase gene, which encodes an enzyme responsible for integrating phage genomes such as A,'s into its host, was not detected from the HaNIV genome but the gene may have diverged from the X-type integrases. The discovery of a latent virus that can cause lysis of Heterosigma akashiwo has important ecological implications. If viral infection is an important factor in the bloom termination of H akashiwo (Nagasaki et al. 1994a, b), progeny viruses would be released in the water column. These particles would be subject to numerous processes causing the loss and decay of viral infectivity. The decay of viral infectivity is caused mostly by solar radiation, but virus particles are also lost due to protozoan grazing and attachment with particulates (Suttle and Chen 1992, Gonzalez and Suttle 1993). Because of these mechanisms that effectively eliminate viral infectivity, other strategies must be in place to ensure the virus' survival. Latency may be a survival mechanism for viruses when host abundance is low (Steward and Levin 1984) such as following the termination of a bloom or during the winter. The prevalence of latency has been investigated for marine bacteria (Jiang and Paul 1994, Jiang and Paul 1996, Weinbauer and Suttle 1996, Weinbauer and Suttle 1999). Latency, however, has not been studied in phytoplankton. If HaNIV is latent,ithis virus, together with Heterosigma akashiwo, may provide a system to further investigate the impact of latency in viral and phytoplankton ecology. 82 In summary, two main results were obtained from the use of HaNIV genome sequence data. Putative HaNIV ORFs showed homology to bacteriophage genes suggesting an evolutionary connection between HaNIV and bacteriophages. The sequence information was also used to demonstrate that HaNIV might be latent. The full implications of this study, however, await further data. Although over 90 % of the genome has been sequenced, the entire genome sequence should be completed to rigorously analyze gene order, genome organization, and regulatory regions. 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Sequence of HaNIV contig A 10 20 30 40 50 GTCGACACAT GCCTAAAAAT ACTTGGCCAG CTTAATCTTG ACGATCTTAC GCCAGATGAA 60 ATCGCCAGTT CGTGCAGCAT TGTTCTGAAC ACGGCGCGGG CGCGCATTTC CAACATGAAA 120 GACGTTGGGT TCGTCAAAGC GTCAGGCCGC AAGCGTGAAA CCGACGCTGG CAGTGAAGCA 180 GATGTAATTT CAATCACAGA CGCGGGCCGT TCGTATCTCG CGCAAGCAAG GAGAACGGCA 240 TGAAGGTTGC TGAACTAACA ACGAGCCAAG AAATTGACGG AAAGCTGCGC CTCTCGAAAG 300 GCTATCGAGA ACTCGGCGGC GTCGATCGCA TTGATCTCAT ACAAGACTTC ATTGGCTTGC 3 60 TGACTGAGTT GCACAAAATG GAAATGGCTG ACGTTTGGGG GCATGACTAT GAGCCTCACA 420 AGGTTTGCCG TGATGACATT TACGGATGGG AAACCTCTGG CGCTGGTCGC CGTTTAAAGG 480 ATGGTGGCGC AGCATGACCC CCACAAACAA GGACTGGGAC ATGATGGTCA CAGCCCAAGA 54 0 GGCTGCTGAG ATTGCCCGCA AGGCCGACAA CCTACGCTTA CCCCGCGACC CTTACTATGC 600 CCTCAAAGTC CTTGAGGCCC GCGTGGCTGA AATCGCAAGC AAGCGCACGA TGACAAAGGG 660 GCGCGTGTCA TGAGTGCGCC CAAGCCACCC GCAATGCTGG TCTTTATAGA CGCCTATCGA 720 GGCGGAACTG AGGGAATGCC ATCAAAGTAC CGTGGGCCAT ACTTTGATCT ACTGTGCGCC 780 CTGTTCGATG CTGGCGGAGA CATTCTCAAC AAGCCTGACG TGTTGGCCCG TGCTGCACGT 840 ATAGAGCCGC GATCGTTCAA GAAGTTTTGG GATGATTGGA GCCATAAGTT TGTCATCTCA 900 GGCGACCGCG TAAGTCACCA TAAAGTTGAT GAACAATTAC TGAAATACAG CAAAAAGGTT 960 GCAGTTTTGC AGCAAAACGG TCGCAAAGGT GGCAAGAAAA CACAAAGAAA ACAGGGTGAG 102 0 GCTAAAGCAA TTGCTAAAGC AACCCAACCC AACGGGTTGG GAACCCCTTC TTACGAAGGG 1080 GGTTCCCTAA CCCTAGCACC TGAAGGCGCT GCAGCCCTTG AAGTGCTGCG CGCCACATGT 1140 GCCGAAAGTG GGCATCAAAC CGCAGAACTC TCAAAACTCC AGACGGCATT GACCGGGTAC 12 00 GAAAATGGCA AACTATTTGT CGGTGATCTT TGGACCCGTG ACACGTTCAC CGAACGCCTT 12 60 GGGCCTCAGC TAAGGGCGGC GAAGCTTGAG CTGGCTGTTC AGCGCGCACC CGTTGAAAGC 1320 AAATCCCCAA ACCTAACCGC AATCGCGGGG GGGAAGTTTT GATCATATTA ACGCAACAAT 1380 TTCTTTAACC AACCGTGGGG AGTAAACAAT GAGTTTCAAG AAAGATACAA TCAACAAGGC 14 4 0 GTTAGTCGCT GCGTGGAAAG AGGGCGGGGT TCTGTATCCG CTCAGCAAGA TCAAAGGGCC 1500 GCAAGGTGGC TATGGTTTGA CCCGGACGCG ATATTTCGTG ATGGCGTATT TGCAGGAGTG 1560 CGGCTGGTCT ATGCCACGCA TAGCTAAAGC GCTTGGGTTG AAAAATCATA CAAGTGTTTT 1620 GTACGGATTG CGTCGCGCGC ATGGTCATGA CGGTAAGCTT CCATATCTTC GTCAACCCTT 1680 GTGGAATAAA GAACTATTCA AGAACAGGGT TGCAGAAGAC TTCCAGCCAG TCGTTCACGA 17 4 0 AGACGGCGCG TTCACTAACG GACGGGGGTG GGCAGCATGA CCCCGCAAAC AGTCACAAAC 1800 ATACTGAGCC GCGCAGGTAA AGAAACCTGT ATGCAGATAG CGTCTGATCT AGGCTTGAGT 18 60 GAGTACGAAG TTCGGGCGGT CATCCAAGGC AAAGACCCGA AAGCGGTACG CGGTCGCAAG 1920 CGTTACGAGA TGAGCAGCGG CGCACATGCG CTTTACCAGT CTTGGACGCG AACGGTTAAG 1980 CCGGAAACGG GCCGCTGTGT CTATTTCATC CCAAAAGAAG GCCGCGTTTG CGGAGCCAAG 204 0 TGCAAGGGCC AGCGCTGCGA GGATCACCCG CAGACTGTTG CCAGAACAAT TAATATCGGC 2100 GCGAAAACTG GCGAGAGGTT ATCAGCATGA AACTTTCAAC TATGCTCCTA TCAATCCCGC 2160 TCATTGTCGT GCCATTGGCG CTGTACGCGA TGGCGCTTTC GTTCGGCGCA ATTGATGACG 2220 GCTTGCTTTC ATGGACAACG CCAGCAGGTG ACGTGACATC GATCCGCCGT TCTGACTGCA 2280 TCATCTTGAT CGCCATATTC TGCCTCATAA TCGAGGTGGT CAAAGCAACT AGCATGAAGG 2 34 0 CCATTGCGAT GGTCGATCAT TTGCTATCGG TCGCGGCCCT GCTTGTCATG GCTTTGTTTT 24 00 ACTTCAACCT CTCAGGCTTT GGCTCTGCAT CGTTTCTGAC ACTTGTATTC TTGCAAGCCT 24 60 TCGATGTGGT TGCGGGCGTT TGGGTAAGCA TCCGCGTTGC GCGCCGTGAC TTCTCAATAC 2520 CGGGGGCGCT TTAAATGATC TCCCTCATCG CCTTTGGCAT CATAGCCACC CTTACGCTCT 2580 GTTACCGCGT CTACAATCGC CATGACCACA CCTTCAATCC GAATGAGAGT GAGACAGAAG 2 64 0 AATGACTGAT TACACAAAGA TAGAAGCATA TGGACCATTA CGCTTGCGTT CAGGTAGTGG 27 00 CGGCTCATGG GATAATGGGC TGTGCGTTAT GGAAACTGTT CACTGGCTTT CAGGGGCAAA 27 60 AGATGCGGGC GAGACACCTA CGGACAGCCC TGAATGCGCT TGTCCTGTGC TTGGCGCTTT 2820 TGCAATTCGG TTGAATGACA GCCTGAATGA CGAAAATCGT CAGAATCTAT TACCACTAGC 2880 GCTGCCATTA ACAGGCACTC GCTCAAAAGA GCATGAAAAA GCGCGAGCTG AGTTTTTGGT 2 94 0 TATGGCGGTG ATCAATCGGA TATTGCCGCA GGTTTTTGTA AAGCGTTGGC CAGAACACGC 3000 TAAAGCGATG CGTGAGGCAG AAACGCTGCA AGAAGCACGA GAGGTGTGCC TTTCGGCCAA 3060 93 AA7AAGATGCT TCTGCTGATG CTGCTTATGC TTATGCTGCT GCTGATGCTG.CTGCTGATGC 3120 TGCTGATGCT GTTTATGCTG CTTATGCTGC CGCCGCCGCC GACGCCCACG CCGATGCTGC 3180 TTATGCTGCT GCTTCTTATG CTGCTGATGC TGCTTCTGCT GCTTCTTATG CTGCTGCTGC 32 4 0 TGATGCCCGC CAATCCATTT TCCTTCAATG CATACCAATC TTGAAAGAAG CTATTGAGCT 3300 TGGACCGAAC GCGGCAGACC AGTGGCCAGA GTTTGAGTCT CGTGCGCGTG AGCTTGGCGA 3360 GTATGTCGCC GCTCCTTGCC ACACCACACA CAAAAATCAG GAGGCCGAAT AGATGACTAA 3420 GACCATAGCT ATTCAGCGTG CCTATCATGC GAAGGATCGC GGTCTAAGCC ATGCAGAGCT 34 80 TGTGCAGAGT GAGGCTGAGA GATTGGCCTT GAACAATGGC GATGCAGCCG AAGCCCAGCG 354 0 CCGACGTAAT GCGGCGATCG ATCTAGCCAA CAAGCGCAAG CAGCCCGCTG ACATATTCTT 3600 GCTGCCAGTG TGGCTTATGC GAGACCTAGG CGGACCTAGA GACGCGATAG CCTATCTTAT 3660 TAAGCAGAGA GAGCTAACGG TCATCCACGG GCGCGCAGCC TTATCCCTTC GTGAACATAC 37 20 TGAGGGCGTT ACAATCTCGA TCAGCGGAAA TGGTGAGTTT GGTGTCTTCG TCGAAGGTGG 37 8 0 TCAGGATGGC GGTATGGAGG CCCATTGCGA TAAGCGCTTT GTTGGTATTC AGGCGTGGCG 384 0 GCTAGCTTTA GGCGCGGTTG ATCCGGTCAC GGTTAAGGCT GTTCAGAGAG TTATTAACGG 3900 TCACGCTGGG CTAAAGCAGG CGAGACGGTT GATTGGCGGC AGAGACCAGA CAGCGCACAA 3960 CATGTTGAAG GATAATCTAC ACAAAGCGCT AGATGCTGCG ACAGCCTATC TAGGGGCTGG 4020 GGCTTGACCG GAACCGCAAA CGGGTATGTA AGTGCTAAAT CGCATTGTTG CGCCGCGAGC 4 080 GCTGATGCTT CCATACAGCC ACCAGACTCT CCTACAACAA CCAAGCGCCG GGTTCCTCCT 414 0 GCTTTGGCGT ATTAGTTTTA TGTCTGGTGG CGACAACTTT CAAAGGAAGA TTCAATGGGA 4200 CCTGAAGACA TTCACCCAAG CCTAAAGAGC TTGCCGCTTG ATGGGCAAAA GCCAGAGCAT 4 2 60 ATTCAGATTA TGATGAAGTT TGCATCTGAT TTTATGTTTC GTGTTGAAAG CACTCTGCCG 4 320 CCTAGCCGGG AGCGCTCCTT GGCTATGACT AAAATTGAAG AGGCGGCAAT GTGGGCGAAC 4 380 AAAGCCATTA GCAGAAGCTA GAATTATTTA ACTTTCAAAA CGGAGTGGGG AATATGATGG 4 4 40 ATTCAGAAAA AGTAATGGAA TTGGCGCAAG CTGTTAGCGA TGCTAATGCG CGCCTCGATG 4 500 CTGCCGATGA CATGCAAAAG CAGGCATACC AAGATTCATG TGTAGCAGAG GAAGCTTTGC 4 5 60 TCGACTATCT TAGTAGCGTT GTCACGAAAG CCGCGTCTGC GCCTGCCGAA GAGATCGAGA 4 620 CGGATGGGTC AGCCCATGTC ACCATCGATC CGGTAACTGT AATCGACGCA ACAGGCGATA 4 680 CAGACGTTTT GAGCGGTATC GGCACAAGTA TGAGTGTTGG TCTTGTGGAC GAAGGCGCGG 474 0 ATGCTGATCC AGATTCACCA GTCATAGGCT CATTCTCTGA AGTATCAGAC GAAGTTGTTC 4800 GCGCTGCGGG TGGTGATCCT GTCTCGCGCG TTGATGCGCT TAATACTGCC GCGCAAATCG 4 8 60 TGCCAGACTT CGGACCCATT GAAAGCTAAC AAGTTCTCGG CAGGCAGTGC CTCCTAAAAA 4 920 TGCACAGGTC GGCGTGGTTC ACGTACCCCA CCCAGCCCCG CGCCGCCTGC TTGTCGAGAT 4 980 TCAATCAAAC AGAGGATTAA ACCATGACGC TGGATATAGA TAAAACAGTA GACACTGTAG 504 0 ACCGCAACAC AAGGCGATTG CTTGCTGTAA CGCTGGGCAT CCTCTTCTTT CTGTTTGCGA 5100 GTACCTTTGG CGCAAAAACG CTAGGCGCTC CGACTATTCA GCTCGGCATG TCATTCCTTG 5160 CTGGCGTCAT CGCGCTTTAT ACCTGGTACG CCGCACAGAA AGACAACCGC GACTTTGCAT 5220 CTGGCCGCAT TAGCGCGATC GTTACATCGA CGGCTAAAGA CATTGTGGCC ACTGGTGTTG 52 80 CTATCATCAT CATGGCGGCT GTTGCGTGGT TCACGAAGGT CTCGTCATTC GGCGCAGTCA 534 0 TTGCAGGATC TATCGGCGTT CTTGGTGCAT GGTGGATTGG TCGCCGTCTT TGGGTAGAGT 54 00 TTAAGTAGCG CGCTATGACA AAAAAGCCCT CTCCCCCCAC ACGTAAGACA GAGAAAGTCA 54 60 TAGACACTGT ACTGTCCCGC GTCGCTGCGG GTGAAAGCAT TGTCGCTATA TGTTCAGACG 5520 CTGACATGCC ATCGCAAAAG TCTTGGTATA AATGGCTGAG TGAAGACACT GGTGATTTGG 5580 GTGAGAGATA CGTGCGCGCG CGGGACGTTC AGGCCGACGC CATCTTTGAC GAAATTCTGC 564 0 AAATCGCGGA CGATGCTGCT AACGACTATA TGGAGCGAAC GGGCGAAGGC AATGAGGGCT 57 00 GGCAGTTGCA TGGTGAGCAT GTTCAGCGGT CTAGGTTGCG GATTGATGCG CGTAAGTGGA 57 60 TGGCTGGCAA GCTGGCTCCG AAGAAGTACG GTGACAAGAT TGCGGTTGAA CTGACGGACT 5820 CGCTGTCAGA TCGTTTTGCG CGCGCCATGA ATAGGGATAA GGCCGAGTAG TGGCTTTGAC 5880 GGCGACTGAT GACAAGATCA TCAGCAAGGC AATAGATTGT CGGTTTGATC CTGGTGCGTG 5940 GGCAAGATTT GCTTACGATT GGGGGCATGG CGATTTAGAG AAGCATAAAG GCCCGCGTAA 6000 CTGGCAGGAT GAAGTTAACG GCGCGATCCG TGAGCATTTA TCCAATGATG AGACAAGGTT 60 60 TCAGCCGCTT CAACTGGCGG TGGCGTCTGG TCACGGTATT GGGAAGTCGG CGCAGATGGG 6120 CATGTTGTCC AATTGGGCGA TGTCCTGTTG GCCGCAATGT CGTATCGTGA CGACTGCGAA 6180 CACTGAGAAC CAGTTGAGGA CAAAGACCAG CCCTGAAATC GGCAAGTGGT TTAAGCGATC 62 4 0 AATAACTGGT CACTGGTTTG ACGTTCAGAC GATGGGCGTG AAGGCCAAGG ATGATCCAGA 6300 GAATTGGCGT CAGGACTTCG TGCCTTGGTC AGAGCATAAC ACGGAAGCCT TTGCGGGCCT 6360 GCACAATGAG GGCCGTATCA TTCTTTTGAT GTTTGATGAG GGGTCAAACA TTCACGAGAA 6420 GGTTTGGGAG GTTGCCGAGG GCGCGCTGAC CGATGAGAAT ACGGTCATCA TCTGGATCGT 64 80 94 GTTCGGAAAC CCAACGCGTG GGTCTGGTCG CTTCAGGGAG TGCTTTAGAC GTTTTAAGCA 6540 TCGATGGAAG GGCTGGCAGA TTGATAGCCG GACAGTGCTG GGAACAAACA AGGATTACCT 6600 TAATCAAATG GTTGAGGATT ACGGCGTTGA TAGCGACATT GTGAAGGTTC GCGTTCGCGG 6660 ACAATTTCCA AGCCAATCAG CCAAGCAATT TATTAGTGTT GAAGACGTGC ATGCGGCAAG 6720 GTCGCGCCAT TTGGAGAAAG ATCAGTACAA GTTTGCGCCT GTTATAATCG GCGTTGACCC 67 80 GGCGTGGACG GGTGACGACA TGATGGAGAT CATGCTTCGT CAGGGCAACT ACTCTAAAAG 684 0 CCTAAGAACA ATACCGCGTA ATGATAATGA TTTTGAAGTC GCGCAGATCG TGGCCATGCT 6900 TCAGGATGAG CATAAAGCTG ATGCGGTCTT TGTTGATGCT GGGTATGGGA CGGGCATCGT 6960 GTCAGCGGGC CGGACAATGG GCCGCTCTTG GCGGTTGATT TGGTTCAGCA GCAAGCCAAC 7 020 TGAGCCTGCT TATGTGAACA AGCGCGCTGA AATGTGGGGC CGCATGAAGC AATGGCTCAA 7 080 GGATGGCGGC TCAATCGACT TACAAGACCA AGGGCTTTAT GACGACTTGA TTGGCCCTGA 7140 GACGGTCATT CCAAAGTCAG GCAATTTTGA CGGCAAGATA CAGCTTGAAA GCAAAGAGGA 7 2 00 CATGAAGGCG CGTGGCATTC CATCGCCTAA CCGTGGTGAT GCGTTGGCTT TGACCTTTGC 72 60 AGAACTGGTC CATGCGAAGG CGCGTATTCA CCCACACAGA CAGGAGCGTG GCAATGTTCG 7320 ACAGTATGAC CCTTACGCCG ACGCTTGATC GGCTAGAGTT TCGCAATCTG AGCGCCTACG 7 380 AGTTTCTTGA TGTTGCACTG CCCCTGTTGG CGCTGCATCG TGAGGAGCTG GCGACAGACA 7440 AAGAGAAGAT GATCCTTAAC CCGGACCATG AGAGTTATCT TCGTATGGAT GACATGGGCC 7 500 TATTGATGGT CATCGGGGCT TATCGTGGTG AAGAATTGGT TGGCTATAGC GTCAGCATCG 7 560 TCACCAAAAA CCTGCACTAT TCTGATCTGA AGGTATGCCA GAACGATGTT TTGTTTCTGG 7 62 0 TTGAGCCTGA GCGCAAGGGA TCGGCTGGCC TTCGTTTAAT CCGAGAGACT GAGCGTCAGG 7 680 CTTTGGACTG GGGGGCAGAG ATCATGCTTT GGCACGCAAA GCCTGGAACA AATCTTGACA 77 40 AGCTTATGCC GCGTCTAGGT GCGAGCGTTC AAGACATTAT CTGGAAGGTG GATTTGTGAT 7800 GGCATTTTCA GCAATCGGCG GAACTGTTCT TGGTGGCATT TACCAAGGTG AAAAGAGCCG 7 8 60 TAAGGCGCAA AAGAAGGCAC AGCGGAAAGC CGCCAGTGAT GCCGAAGCAG CGCGTCGGCA 7 920 GGCTGACCGC GAGTTTAATC GTGCTAATCA AAAGTCGCCT AATGTGGCGG CTCTAACGAA 7 980 GCGCAACAAG GCATCTTCTA AGTCTGGCGT TTCAGGCACA TTCCTTACGG GCAATCAGGG 804 0 TGCTCCGACA AATTCGGGCA TGTTGGGCCG TACCACATTG TTAGGCGGTT AATTGGCTTA 8100 TAGTGATGCA CCGCGCGGCG GTCTAACCAA AACATCCCGT TACGGGGCGC TACAGACAGC 8160 CAGATCGCCG TGGCTTAGCC ATTGGGCTGA GCTGGCTGAC CGTATCGATC CTATGGCTGT 8220 TCGTATTGAG AAGAGTGACC GCAATCGTGG CGAGCGTTCG CGTGGCAAGA TTATTGACAA 8280 TACGCCGACG CTGGCTTTGC AGACGCTGAA GGCCGGGCTT ATGGCGGGCA TGACCAGCCC 834 0 CGCGCGTCCG TGGCTTCGTT TGTCTCTAGC AGATCATGAT CTAATGGAGT TCGAGCCTGT 84 00 TAAGGAATGG CTTTCAGGCG AGACCAAGCG CATCTTGCGT GTGTTTCATC GTTCTAACAC 8 4 60 ATATTTGATG CTGCATCAGA TGTATGGCGA CCTTGGGCTG GGTGGTTCAG GCTGCGCGAT 852 0 TGCGATGGAT AGCTATGAGA AGGTCGTCAC TCATTACAGC GCGCCGCTTG GTGAGTTTTG 8580 TCTGGCGGGT GATTTTCTTG GCACGGTGAA CACTGTTGGT CGCCGATTTG AGAAGACGGT 8 64 0 GGGCGAAGTT GTTGGTGAGT TCGGTTACAA GAACGTGTCG CAAACCGTTC GCTCACAGTA 87 00 CGATAATGGT AACTATCAAT CGCCTGTGGC GGTCATTCAC ATGATTGAGC CACGCCGAGA 87 60 GCGTGACACA AGCAAGATGG ATAGTCGCAA TAAGGCTTTT TCATCCTGCT ATTACGAAGA 8820 GCAAAAGGAT GGTATTGACC CAACAGTCTT ACGTGAGAGT GGTTATGATG ATTTTCCCGC 8880 GCTTTGTCCG CGCTGGGACC GTCGCTCTGG TGATATTTAC GGCACAAGCC CTGCTATGAT 894 0 GGCGCTGGGC GACATTAAGC AGCTTAATCA CGAGCAGCTA ATGAAGGGTA CGGCAATTGA 9000 ATACAAGGTT AAGCCGCCTG TAAACGTGCC TACGTCTTTA GAGCATACGC CGCTGGACAT 9060 GTTGCCGGGT GGCCGTGTGT TCTCGGATAT GTCCGGGCCT AACTCGCGCA TTCAATCGCT 9120 TTGGGAGGTT AATCTTGATC TCGGTGACTT GCGTGAAGAC ATTTACGACA TTCGCCAGCG 9180 AATTGATTCA GCCTTCTTTA AGGACGTGTT CCGCATGTTT GAGGGTGGTC CGGGCGTAAA 92 4 0 GACTGCGACA GAGGTTGCAG AGCGTCACGA AGAAAAAATG CTGATGCTTG GCCCTGTTCT 9300 GGAGCGCTTG CATGGCGAAC TGCTAAGGCC GTTGGTTGAG AATACATTCT CGCGGATGAT 9360 TGATACGGGC ATAATTGCGC CGCCGCCACC AGAGCTTGAT GATGTTGAGT TGAATGTGGA 9420 GTTCACGTCT GTTCTAGCCC AAGCCCAGCA AGCGATTGGG ACAGACGCGG TAGATCGTTA 94 80 CGTAAATACG ATTGGCGTAG TTGCCCAGCT TAAGCCGGGT GTTTTGGATA AGTTTGATGA 954 0 AGACGTGTGG GCTGATCAGT ATGCCGATGC GTTGGGTGTC GATCCTAAAA TGATTATCGG 9600 GAACGAGCGT GTTGGCTTTA TTCGCGAGGC TCGCGCTGAA CAAGAGCGTA AGCAGGCGGC 9660 GATGGATTCC GCTGCGCAGC AAGCGTCAGT CGCAAAAGAC CTTGGCGCGG TCGATACTGG 97 20 AAATGGTGGC AATATGGCGA TGGACGTTAT GAACCAATTC GCAGGCTACG GCTCGCCATC 97 80 AGGTACTGAA GTTTGACCGA TAGCTGGTTA GGGGCCGAAG ACATCGGCAA GCGTCGCCAA 984 0 GAGATAACAG AAGAACAGGC GCAGAAGCGT CAGCAATTGC TTGATGATAT GCGTTGGGTG 9900 CTTGGGGATG CGCGTGGTCA GCGTGTGATC GTCAACATGC TGGAGCATTG CCGCCTAAGC 9960 GTGAGCAACT TCACCGGAAA CTCAGAAACG TTCAAGCTGG AAGGTAAGCG CGAGGTCGCC 10020 CTGCAAATGC TGGCTTGGAT CAAACAAGCC TCACCAGAGG AGGCGCGGAC AATCGTTGCA 1008 0 AGTCTATTCG TGGGAGAAGA CACATGACCG AGAACACTAC AGAATCGTTG CTGACAGCAG 1014 0 CCACAGACAC CACCGAAAGC ACTGTCACAT CGGAAGCGGA TTCTAATCAG GAGCAGGCAA 10200 CTGCAACTGA AGAGACCGCA CCAGCCGAAA CCAAAGCAGA GGATGAGGTT AAAGCCAAAG 102 60 AGGCGGAGAC CGAAACCAAA GCAGAGGACG GCGACACAGC GGCTGATGAC AAGCCTGTTG 10320 AATACACTTT TGAGCTTCCA GATGGCGTCG AAATGGCCCC TGAAACACTG GAAGGTTTGA 1038 0 AGGGTATCGC GGGTGAGTTA AAGCTTGATC AGGACGGCGG ACAAAAGCTT GCGAGCTTTG 104 4 0 GCGCTGAGAT GTTTCAAAAA TGGGAAACCG CACAGGCTGA TGCCATTGAG ACCGTAAAGT 10500 CTGAATGGGC TGAGGTTACC AAAGCAGACA AGGAGATTGG CGGAGCAAAC TTCGACAAGA 10560 ACCTTGGTAT TGCCAAGTCA GCTTTGGAAA AGTTCGGAAC CCCTGAATTG AAATCATTCT 10 620 TGGATGAGAC AGGGCTGGGA AATCACCCTG AGATCAACCG CTTGTTTTGG AAAATGGGCA 10 680 ACCAGATTTC CGATGACACT TTGGTTGCAG GCGATCCTGC ACCGACCGCG CGGCTCACGC 10740 GAGCTGAACG GCTTTACGGC ACAAACTAAA CAAGGAGCCT ATAGATGGCG ACTCTCTCCA 10800 CAACACACCC AACGCTATTA GACTTGGCTA AACGTCAAGA CCCTGATGGC AAAATCGCAA 10860 CTGTTGCTGA AATCCTGTCA GAGACGAATG AAATTCTTGA TGACATGATC TGGCTTGAGT 10920 GCAATGACGG CACGTCTCAT CAGACAACCG TTCGCACAGG TCTGCCAGAA GGCACATGGC 1098 0 GCAAGATTTA CGGCGGCGTT CAGCCTTCTA AATCGACCAC CGCAAAGATC AAAGATTCAA 110 4 0 CTGGCATGCT TGAAGCGTAT GCTGAAGTTG ATAAAGCTTT GGCCGATCTA AACAGCAATA 1110 0 CGTCAGCCTT CCGCCTATCT GAAAGCACGG CGCACCTTGA GGGGATGAAC CAGCAACTTG 11160 CTGAAACAAT CTTCTATGGT GATGTTGCCA CCTACGCGGA GCGCTTCACA GGTCTAGCCC 11220 CACGTTACTC GACTAAGGCG TCAGGTGAGC AATCGCAGAA CATTATTCTT CAGGGTGGTG 11280 TGACGCCAGA CGCTGCGGAT AATTCGTCAA TCTATCTGAT TGTGTGGGAC CCAAACAAAG 1134 0 TGCATGGCCT TTATCCTAAA GGCTCAATGG CTGGCCTTCA CCATGAAGAC AAGGGTCAGG 114 00 TCACAATTGA GAACCTGAAC GGCGACGGTG GACGAATGGA AGCTTACCGT GACCACTTCC 114 60 GTTGGGATTG TGGCATCTGC GTGCGTGACT GGCGTTATGT CGTTCGCATT CAGTATAATG 11520 ACGAAGACCT GACAGGTGAT GCGGCTTCTG GTCCTGACCT AATCGATCTG ATGGCGGAAG 11580 CTTGTGACCA AGTTCCTAAC CTCAACGGGC GTCCGGCTTG GTATATGAAC AAGCGCGCGA 11640 TGTCTTACCT TCGCCGTCAA GTTCGTTCGT CAACGTCTGG CTCTACACTG AGCCGTGAAA 11700 ACTATAATGG CAAGATGACA ACCATGTTTG AAGGCATTCC GTGCCCACGC GTGGATGCGC 117 60 TTCTTCGTAC CGAAACTGGC ATTGCTACAT CTTAAGGGAA TAAGACTATG ATTTTAGACA 11820 CACGAACAGA GTTCTGTGAC GCTGAAGCGC TCAACACGGG CGGCGCAGGC TCATATCTTG 11880 TCGGTGACGT TATCGACACA GGCGGGGATG GTATCAACTT TGTTGATGAC CTTTACTTGG 1194 0 TCATTAAGGT TGAAACGACT GCCACATCAG GCGGCTCTGC AACGGGCCAG TTCCACCTTG 12000 TATCGGATGC GGGCGCAGCT ATCGCGACTG ATGGCTCTGC AACTTATCAT TACTCAACAT 120 60 CTGCAATTGC AGTGGCAACG CTTGTTGCTG GCTATCAGGT TTGCGCTATT CGCCTGCCTG 12120 CTGGTGAGTA TGAACGCTAT GTTGGTGTTT TGCAGACGAC AGGCACGGCG GCATTTACGG 12180 CGGGCAAGAT TGACGCCTTC CTGACACCAA ACATTGATCA GTGGAAAGCA TTCGCTGACG 1224 0 CGCCAAACGC TTCAGTAGTT TAAGGGGACC CTGAATGTCT GATAATATGC AACAAAGAAA 12300 AGTTGTCGCG AAAGGCTACT ACCCGGTTCC GGGTAGTGGC TGGCGCGTCT TCAATCCCGG 12360 TGAAGATATT TACGTCCCTG CCGGGTTCAA GGCTAAGTGG CTTGAGCCAT CTGGCTATGA 12420 CAAAAAGAAG CGGGCCGTAA AGGCTGCTCC ATTGAAAGCG CCAGTAAGTG ACGACAGCGC 12480 TTGATATTAG CAACGTGGCG CTGTCCCTGA TTGGGCAGAA CGTCACGATT GCATCAATCA 1254 0 ATCCTCCGGA GCAAAGTGTC GAAGCAGAGA AATGCGCTCT GTTTTGGCCG GGTGTGCGTG 12600 ACCAGATATT GGCTGCGCAC ACTTGGAGCT TTGCGATTGA GCGGCAAAGC CTTGCGCTTT 12660 TGGGCGTAAC ACCGCCTGCG CCGTTTCAAT ATGCCTATGC TTATCCGGGG GATTGCATCA 12720 GGTTCATCGG TGTGCGCAAT CCAAGCGCCA CTGATGACTT GGTGAATGAT GATTGCCGTC 127 80 TTGGCTCCGA AGATGGGGCA AGGGCGATCT ATACGAATAC AGAGAACGCT GTAGGTGTTT 1284 0 TCATTAAGCG GCAGTCTGAG GCAACACTGT ACGGGCCGAG CATGGTCGTT GCAGCGCAGC 12 900 ACTTGATGGC GTCCCGCCTA GCGGGTCCGA TTATTCAGGG TGTGCCGGGG ATGCAAGTCG 12 960 CTAAGGAAAT GCTTCAGCTA TACATAGAGT TTTTGACGAT AGCGAAACGC GAGGATGCTG 1302 0 CACAATCCCG GCTTCGCAAT ACAGGGGATA ATGACAAGTA TAAAGCTGGA CAGCTTGCCG 13080 CGCGCGGCGC GAATGGCGTA ACGGAGACCC CTATTATCCG TGGCTAAACT TTATACGCGA 1314 0 TCTTTTAATG GCGGCATTAT CTCCCCTGAA ATGTGGGGTC GCATTGATGA CCTTAAATCG 13200 AACACGGGGC TGGCTGTCTG TCATAACTTT GTGGTCTTGC CACAAGGGCC AGTCTCAACC 132 60 CGAACAGGCT TTCGCTTTGT ACGAGAGGTC AAGGATAGCA CGAAGTTTAC CCGGCTTATT 1332 0 CCCTTTCGCT TTTCTGCGAC GCAGACAGTT ATGCTGGAGT TTGGCGAGGC ATATATCCGG 13380 TTCCATAGCT TTGCAGCAAC GGTTTTGACG CCAACGACGG GCGTTGATGC TTTTGATGCA 134 4 0 GGGTTAACTT ACGACCAAGG CGATATTGTT ACCGAAAGCA GCAAGACGTG GTACGCGGTT 13500 AGCGACGTAC CAACGGGCCA TACGCCAAGC TCAAACGAAT ATGGCTCTGC TCCAGTCGTT 13560 TCTGCAACAT GGTCTCTTAC TAGCGCCGGG AATGCGTCAA TTCCATCTGG TTATACGTTC 13620 TACGGCACTT CATTGCCGGA AGATGTCGGA GTTGGTGACA AGGCGGCAAT ATCCAGAACA 13680 ACTTACACGT TTGATGAGGG CGCTGGCGGC AGCTTAGAAC CGGACGTGGT TCTTGTGGAA 137 4 0 AGCACGGTTT ATGATGCTTA TGATGGCGTC GCTAGTTCTG CGTCAGCGGG TTACTGGTAC 13800 GAGTTGGGGA TAGTTTATGA GATACCATCC CCTTATGCCG AATCCGATTT GGCGGATATT 138 60 CATTATGCGC AATCAGGTGA CATTTTAACG CTGACCCATC CCCGGTATCC GGTTCAGGAA 13920 TTGCGTCGGT ATGGTGTGAC CAATTGGACA CTGAGCGCCT CTGTGTTTGG GTCAACACTG 13980 AGCGCTCCAA CAATATCAAG CGTAACGCCA GTTACGGCGA CTAGCCCATC AGACACGCAA 14 040 AGCTATAGCT ATGTCGCGAC GAATGTCACT GACGACCAGA TTGATGAGAG TGTTGCAAGC 14100 GCGGTTGTGA GCGCAACAAA CCAGCTTTTC GATACGGGTG CTAAAAATAC TATTACATTT 14160 GGGGACGCGG AGCGACGCAA CGTCTATAAA CTAAGCGGTG GCCTTTATGG TTATATCGGG 1422 0 CAGACAACGA CCACGACGCT TGTGGATGAC AATATTGCGG CTGACATTAG CCGCGCGCCT 14280 CCTGAAAACC AGACGCCGTT TGCAAGTGAT TATCCTCGAA CGGTCGCATA TTTTGAGCAA 14 340 AGGAAGTGCT TTGCAGGCAC GTCAGGACTA ACTCAAACCT TGTGGCTGAC AAAGACGGGG 14 400 ACAGAGGCAA ACCTAGATTA TTCTATTCCT GTGCGATCTG ATGATGCTAT CTCGATCAAG 14 4 60 TTGGCATCTT TGGAAAGTTC GACAATCCGT CACGCGGTTC CTGTTGGTGA TCTAATTCTT 14520 CTGACGGATA GCGCTGAGTG GAGAATTTTT GCGACGGGCG ATTCAGCTTT GACGCCGACA 14 580 AGTATTGCTG CACGGGTTCA GGGCGGCATC GGAGCTAGCA ATGTTCAGCC TGTTATTGTT 14 640 GGCCGCAATG TCGTTTATGC GGCAGCGCGG GGCGGTCACA TGAGAGCGCT TGGATTTGAG 147 00 TTTGACGCTA ACTCCTATGT CTCGGTTGAT TTGAGCCTTA GAGCGGCGCA CCTGTTTGAT 147 60 TATAAGACGA TCAAGGATAT GGCTTACGGC AAGGGGCCAG TCCCTATTGT TTGGGCAGTC 14 820 TCCAGCGATG GGCAATTGCT TGGTGTCACG CTTGTTCCAG AGCAACAGGT TTATGCTTGG 14 880 CATACACATG CCACAGAAAA CGGCGTGATT GAAAGCTGCG CGGTGATTGA AGAGGGCGAT 14 940 GACTCTATCC TCTACTGCGT TATTCAGCGT GAAATTGATG GCGCGTCAGT TCGTTACGTT 15000 GAGGCAATGG CTAGTAGGTT CTTTGCTAAT GCGGATGATT ATATCGGTCT AGATAGTTGC 15060 ATCAGCTATA ACGGAACTTC AACGAAAAGC TTCAGCAACC TCGATCATCT TGAAGGTGAA 15120 ACAGTTTATG CTCTAGTTGA TGCAGCGGTG CAGGGGCCGT TTACGGTATC GTCTGGCGCT 15180 GTAACAATTA GCGAGGCGGG CGAAAAGGTC CACATTGGCA AACAGATTAA GGCGAGAATG 15240 CAGACGCTTC CAATGGTTGC CGAGGTCGAA GCCTATGCGC AAGCTACGGT TAAGAATGTA 15300 AATGAAGTCA CATTAAGAAC GTATCGATCT GGTCGCTTCT TTGCGGGCGA AAGCCTAGAT 15360 AAGCTGTATG AATCCAAGAC GCGGACTGTT GAGAGTTTTG GCGAAGCGCC TGCGCTCATT 154 20 AGCAAAGAGA CCGACCTTCG TATCGGAACA TCGTGGGCTA AGTCTGGACA AGTTATTGTT 15480 CAAACCGTTG ACCCGCTTCC TGTGACAGTT TCGGCTATCA CGCTTCATTG TCAGTTTGGG 15540 GGTTAATCTA TGGCGATTGC ATCGACAGCA ATGGGCGCTG CCGGAACGGC AAGTAATACA 15600 GTTGGCGCAT ATTATAGTGC GCTAGGTCAA AAGTCGTCTT TGAAGTTCCA AGCCACACTG 15660 GCAGAGATAA ACGCAAAAGC GGCTGAAAGT GATGCGCGCC TATCCTTGGA TCGCGGCGAA 1572 0 AAGATGGAGC AAAAAGCCGC TCTTGATACC GCCGCTTTGA AGAGTGACCA GCGTGTCGCT 157 80 TATGGCTCAA GCGGGATAGA TTTAGGGTCT GACACAGCGG TTCAGGTTTT AACCTCGACA 1584 0 GATGTGCTTG GAGAGATGGA TCGCAACCAG ATTAAAGCCA ATGCATTACG CGAAGCGTGG 15900 GGTCATCGCA CGCAGGCGGT TAATGACCGC AATAGCGCTC TTATGAGCAG AGCAACGGCT 15960 GACGGCATCA GCCCTATGAT GAATGCAGCC ACAACGTTTC TGACAGGCGC GGGACAAGTT 16020 GCACAACAAT ACTACACAGC CAAAGAGTCT GGTGCGCTCC AAACCAGTAA AGAAAATTGG 16080 GCGAAGTATG GAGACAAGGC TAAGTCGTTC TTGTCTGGCT TGAAGGGGTT TTAATCAGTG 1614 0 AGAGTTCCTG CAAGACGTTC ATTTGATGTA CTACCCGGAG GCGGAAGCGC GGGACGGTTA 16200 TCTGATCCGG GATCAGGCCG TCTCGCAAGC ATTCAGGGCC GTGAGCAACA GCAATTTGGG 162 60 CAAACGCTTC AAGGGCTTGG CGCGGCCTTG GGGCAAGAAG TTGAAAAACA ACAATACAAG 1632 0 ATCAACCAAG CCCGCGTCCG TGAAACAGCT TTGCAGTTCC GTGCCGACAT GGCGGAAGCT 16380 GAAAAAGAGT ATTCAGAGTT TAAGGGCGCG GAATTGGTGG CGGGTACGCG TCCCGTCATG 16440 CGCGATATTG AAGAGCGCTT GTCTAAACGC CGCAATGAAT TGCTTGAAGG CATATCATCC 16500 CCGGCGGCTA AAGAGGCATT TGGCGCGACA GCGGATGAGA TGTTTGCTAG GTGGTCAGAA 16560 CGGGCTGGCA GGTATGAGGC GGAGCAGACT GATTTTTATA TTGAACAGCA GCGTGATGGC 16620 ATTATTCAGG CTTCTTTAGA AACAGCGATT AGTCGCCCAG AAACGCGGGG CGAAAGCCTC 16680 TTAAACGCGA ACAATGTTTT GCGTGAGAAG TTTGAAGATC AAGGGTTTGA TGGTGAGCGG 16740 97 CTGGATCAAA AAGCGCGTGC AGCGATGGGC CTGGATGCCG ATCGAATTGA TGATGCGCAA TCGGCGGATG TTGCAGAGGC GATGGGCACC GTGAACGCCA AGGCGGCGGA TGTCTGGGAT AAAATGGCGG GCATGATCGA AAACCCTGAA ACGCTAAAAG CGCGCGATAC GGCGGCAGAG GCGTGGGAAA GTCTTGAGCA GGGTGGTTTG CAGCTAGGAC CGCAAACACG GATACAGATG GATGATCCGA AGCGCGAGAC GGATTTGCAA GCCGGTGATG CCGAAGGTGC TTTGAGCTTC GGTGATTATA AAAGCCTTCG CACCAAGGTT GTTAAGAGTG CGCGCAGCTT AACGCAGGTC AACGAAGGTA ACAAAGGGGG GCTACTTCTG CGTGAGCATG GCAAGGAGCC TTCTGATGAG GCTAAAATCA AGGTGACACG TCCTTGGTGG ATTGAAGAGA TTGGCGAGGT GCCGGAGCGG AACACCGATA AAATTACTAC GATTTCCCAA ATGAC GAGCTTTTGT CGGGTCAGAT TAGTATCTTG 16800 GAGGTTGTTG ACGCAGCGCG GGGTATGGTC 168 60 GCGGTTAAGA AGAAAGCCTT TACACGCGAT 16920 GTCGCCGAAG GTGACTATGG CACGTCTTTG 1698 0 ATGCAAGAGG CGGTTAAGTC TCGGCTGAAT 1704 0 GCGCAAGCGA CGCGAGATGC CACGGTCACG 17100 GTGGATGCGC TGCCGCGTGA TGTCTGGGAG 17160 CGCACATGGG AGGAAGCGCA GGCGAAACGC 1722 0 ACTTACAATG ACGTATACAG CTTTCTTGAT 17 28 0 CTGAATGGCA ACGCTGATAA GCTTTCAGAG 17 34 0 AGTTCTGCGA TGGATGATGA CGGTGCAACG 174 00 ATCACAAATG CCTTAGATCG TTCAGGCGTG 17 4 60 GCCTATGACC ATGCGGCGCA GCAGTATCAG 17 520 TGGCGTGAAA AAACGATTGA GCGCCTTGCG 17580 TTTAATGACA CGACAGGCGG GGCGTTTGAG 17 64 0 CATGTGCAGG CGGTTTTAAT GGCGTTCGGA 17700 GACAAATTGA ATACATCTTA CTCGATGGCG 17760 17765 98 APPENDIX B. Sequence of HaNIV contig B 10 20 30 40 50 AAGCTTATCA GCGTTGCCAT TCAGGAAGCT CAAAGCACCT TCGGCATCAC CGGCATCAAG 60 AAAGCTGTAT ACGTCATTGT AAGTTTGCAA ATCCGTCTCG CGCTTCGGAT CATCGCGTTT 120 CGCCTGCGCT TCCTCCCATG TGCGCATCTG TATCCGTGTT TGCGGTCCTA GCTGCTGACG 180 GACGTGGCGC AGCAACATCA ATATCATGGC CTATGGCTTA CGCCGGAAGA CTGGAAGTTG 240 ATATTCTTGG CCGCGCTGGA TCAAGAGCTT CGTGTTGTGC CTAATCTTAA CAATAACGGA 300 TTTGTTCAGC TTGGCCGCTC ATCTTCAAAA CTAAGCGTCG AAGAGATGAC TGCGCTAATG 3 60 GATCTGATCG AGGCGTACTC CGCGCAAAAC GAAATTACAT TACATGACGG GAAAGAGGCC 4 20 GCATGACCCT CCCTGCCCCC GCCCTCAAAC GCCTACAAAG CGCGCTCTAC GGACAGAAAC 480 CACGCTGCAT TAAGCATGAT GTCCCGTATG AGACAATCAA AGCAGATGTA TTGCTGGCAA 54 0 AGGCTGACCG CAACGAGGCG GGGCAGTACC TTTGCATGAC GTGTGCCGGG CCTATGAGCT 600 TTCGCGCTGA GATGGTTCCG AATAAAGCAA CCGTGGGCCA TGTTCTCGCG CTGTCAACAA 660 GTACGACAAC CCCCGATGGC CATAAATACC AAGATCACCC CGGCCACGTT CTTAGTAACT 720 GCGGACTGGA ATGCTGGAAA TGCAATCAGG CTGACAATTG CACCACCGTC GCCAAGACGA 7 80 ACGCCACCGC CCGTAACTTC GCTGTCGTCA AAGGCAGACC CAAAAGCGGC AAAATACAAT 840 CACGAAACCA ATGGCCCACT GGCCGTAAGA TACAATCGAG AGGATTTGGA AGATGAGCGC 900 TTCTAGCTTG TTATTTGGCC CCGCTAATCC AGGCGCCCGC GTGCTTGCAT GGTTTTCGTG 960 TGGTGCAGCC AGCGCCGTCG CTGCTAAACT AGCGGTTAAA AAGTATGGCG ACCGCTGCGA 102 0 GGTATTGTAT TGCGACACCT TCGCACATGA ACATCCAGAC AATCGCCGCT TCTTCGATGA 108 0 TTGCGAAAAG TGGATAGGGC GAGAGATCAA AGTCATTGGG TCGGATAATT ACGAAGACAT 114 0 TTACGATGTT TTCGAGCGTA CTGGCTGGCT TGTGGGCGTG GGTGGTGCGC GTTGCACTGT 12 00 CGAGCTAAAG AAGAACGTGC GCCGCGCGTA TCAGCGGCCA GATGACATAC ACATTTTTGG 12 60 TTATACGCCA GACGAGCAGA ATCGGGCTGA CAGGCTCACC CATTCAAGCC CTGAGCTTTT 132 0 CTGCGAGTTT CCGCTGATCG ATCAAAAAAT TAGCAAGTCT GACTGCCATA GTCTTTTGAA 1380 CATTGCAGGC ATCGAGCAGC CCGCAATGTA TCGCATGGGT TACAGAAACA ATAATTGTAT 14 4 0 TGGCTGCGTG AAAGGTCAGG CGGGCTACTG GAACAAGATC AGAGTAGACT TTCCAGAAAT 1500 TTTCGAGCGC ATGTCAAAAG TCGAGCGCAA CCTAGGCGCG GCCATCTGTA AAAAAGAAGG 1560 AAAGCACGAG GACGGGACGC GCTGGCGAGA GGTCGTTTAT CTGGATGAGC TAGACCCCAA 1620 ACAGGGCCGA TATGAGCAAG AAGCAGACAT TGACTGCGGC GTTCTGTGTA TTGGCGCTGA 1680 TACGCTTAAA AAGTTCACCC CATGACCCAC CAAACACTAT ACGCCATAGG AGAATAATAT 17 4 0 GACTCTACTA TCAATAATAA CATGGCCTTT CCGCGCGTGG TGGAAAGCTG TCAACAAAGC 1800 CAGCGCTCCC GATCCTGATG ATATGGTTAG CGGCTGGGAT TCGTTTGTGT GGGTAGAGCG 18 60 TACCACTCAT CCCACGCAAC AACACAAAGG ACAAAACTTA TGAGTGACGT GGGTGAGGAT 1920 ATGCTGTGAC TAATCATGTA AAGAGATTAG CGCTGGCTAT AGCTGGCAGT AAGTACGACA 1980 GGCCAGAAGC AGCGCTTCCA AGACTAACAC CAGAGCAATT GCAGAGCCAG ATACGCGCGT 2040 TCTCACCTGA CGCTGTAGCT GTGCTTAAAG AGCTAAGAGT GATTGCCGCT GAGGATGGTC 2100 GCAGAGACAC TAATGACTTG ATCCGCATTT TTATAGACGA AATGTTGAAA TATTAGGCTG 2160 TTCAAAACCT ATGGAGAAAC CATGACAAAT TTTTTAATTT TCGGCTGTAT AGTGACAGCT 2220 GTAATCACTG TAGTAGTTGC TGCTTTGTTG GGAAAGAGCG CAGACTATTA TTATTTTAAC 2280 CGATTTGAAT CACCAAAAGG ACTTACCACA GCTGACTGCA TTGATCAGTA CTACAACAGG 234 0 TTGGCCCAAG CCAAAGATGC AGCATGGATA GGCGGCGGTC TGTTTGCACT TCAAGCTTTC 2400 ATGATTTTGG CCACATCGCC CATGATTCAG GGGCAATAAT GTTCAATAAG CTTTTAGTCA 24 60 TTTTTCTGAT TTTGGTTCTA TCTGTAATGC CTGCTTCTGC TGATTCCCGG TCTGAGATAC 2520 AAACAATGAT AGACGCAGCC GCAGAGCGTT ATAGCATGGA GCCTGTGAGG TTTCGTGTTG 2580 TCGAGCCTGA TCACAAGCTG TTTAAGCGAG ACCCAGCCAT AGCCAAGGCT GTGCGCTGGC 2 64 0 GGGACGGAGA CGAAGCCATT TACATTGTAT CAACGCGAGC AAAAAGCCAG TCCGCCCTCT 27 00 TGGTGGTTAT AGACCATGAA ATGTCACACC TTAAAGCGTG GCGTGTGCAT GGCGAGAATA 27 60 TCAAAGTTCA CGGCCATCAA TGGAAGTCCA CTTGTCGAAA GTATTCCGTA CTTGGTTCAC 2820 GTGCGTGTTA TAAGTCATTT CAATAATAGT ATCCCCGTCG AGCAAAGCTT AAACTAGATG 2880 CCTATCATCT TTAGCGTCAA ATACACACGC GCCGCATCAG GCAACCCACG CGAGCCGAAC 294 0 CAGTTTGACA CGGTTGCCGC TGAATAGTTT TTGCCAGTGA TAGCGCCAAG CTCGCGCGCT 3000 99 AGATCGACCT GCTTCATTTT GAGTGCGTCA AGCGCCTGCT TTGCTTCTTC GTTTGTCATG 3060 CGTCGTATAT AGCGCTGATG CATTTATTTT GCAAGCGCGC AAGTTTTTTA CACTTTAGGG 3120 CTTGCGCAAG TGCGCAAGGT GGACTAGATT GAACTCATAG ACAGAACGGA GATATGCAAT 3180 GAGTAGCAAT AAATCAGAAT GGATTAAACT AATGCGCCCA TTGGTAGACG GTCACGCCAC 324 0 ACAAAAAATG GTAAGTGCAT TCGACCAGCT TACGTCGCTT ATGTTTTCGG CTGGCGAGAT 3300 TGACTTTGAT GAGTTTATCG CAACCCAGTC GGTTATATCA ACGGCGCGCG ACATCGCTCG 3360 CCAATTTAAC TCCATAGGCC AAGAAGGTCG GTTTTTGAGA GAAAAGGATG CCGCATAATG 3420 ATTGCCAAGG AGATCGAAGA GATTTACCCG CGCGAGTTTT TGGACGCTTT AACGGACGCA 34 80 GTCGCAACAA TGGGTGATGA GTATGATCTT GATACGCCAG AGAACTGGGT GGTTTTTCTT 354 0 TTCGATGCCG TCAATGAAGC GGGCTACTGC TTCGCTAATA AGGAAACAAT AGCCCGCTCT 3600 ATACGCTGAT TTGCTACGCT GGCTCAGCTA GCCTCGATAG GGCCGAAGCC TCTCACGGAG 3660 TATATATCTA CCCCACCCGC CCCGTCCAGA GCAATCTGGC GGGTTAAGGG GTAGATAGAG 3720 GATAATGATG GATGACAAAA CAGACATGGA ATATTCGCAT TCAGGGTTAT GGAACCTTTG 37 80 ATTTTGAAGG AACCGAGCAA GAGGCTGAAG AAATGCGTAA GCACAAGGCA AAATGGGAGG 384 0 GAGGTTCTGG AATGAAGTGG CGAAAAGACA ATCAGACAGA ACTTGACCGC CTGACGGAAG 3900 AAATGGCTGA TATTTTCGAC AATGGCGAAG GTGTGCCGCG CGAGCTTTTT ATGAAGCGCG 3 9 60 CCGCTCTTAT TAGGTCGCAA TCAAGCTAGC TTAGCCCGCC CCGCCTAGCC TAGCTAGCGG 4 020 GGTTTCGGGG TGAGAGGCGC TGTATCAGAG CGCCCCTCGA TAGCCACGAA AGGATCAAAC 4080 CAATGGCTAA TAAACTAAGT ACAGCAATCT GCTATACGAT TGCAATAGCC TTCACAGCCT 414 0 GTCTGGCGTC ACTGTCAGCG GGTCACTGGA TCATCCAGCT TGAAGGCCAG CCGAACTGGG 4200 TCACTGCTTT TGTGGTTGTC GGTTCGGTCT GCGTCTCGCT ACTGGTTGCC GTATGTGCAT 42 60 TCCTAGCGGG TCGCGGGTTC AGCAAGGTTC TGCTAGGTGC GGCGTTCGTT TTCTTGATCG 4320 CTGACACATA TCAGAACGGG CTAGGATACC AGACGGTGAC AAGCCTGACG GTATCGGGTG 4 38 0 AAGTCGAAGC GGCTCAAGGT CGCCTTAGCA ACGCTCAGAT CGCGCTACAA AGCATTGTAT 4 44 0 TGCCTGAACG TACATGTCTC TGCCCAAAGA CGAAGGCCGC CGATCTGGCG CAATATAAAG 4500 CAAAGCGGGT TACACCACTG GCAGAGATTG CAAAAGCGGA AGCTGCGTTA GCCAAGCTGA 4 560 ACACCCCGAA AGCCTCTGAC GCCGCTGTTA TGGCTGTCAT GCTCGCTCTA CAGATCGCAA 4 62 0 ACGCGCTTCT GTTCAGTGCA TTAGGTCGTG GAAGTCGGAG CAAGCCTGTA GAGGCTGTTC 4 680 AGGCTGAAAC AAAACGGACT GGCCCTACGC TAGTCTACAC AGCACCGGCA TTGACCGATT 47 4 0 ACGAGCGTAA GGCTCTTGAG GTCGCAAGCG CCGGAGCATA ACCTAGGCCC TGCGTTTCGG 4800 CGTGGGGCTT TTTTATGGAG AGAGAATATG GATAAACGAG ATTCCTTGTC GCAGCCAGTT 4 8 60 CAGCCTGCCG CAGATGATGG CAATGGTGTT CTAAGATTCA AAGAAAACAG CATCGTAAGG 4 920 TATTTGCTCG ACGCAGGTGG ATTAGACCTA AATGACATTG CAATAGCCAA GAGTGACGCT 4 980 CCAAAGGAAG ATTGGCAGCA GTTTCATCAG CTAATTGGGT ATTCTGTTTC AGGCATACCT 5040 AGCGCTGATG ATATACTGCA TCAGGCGGTA GAGGAGATGA TTAATAATGG AGGGGATTGG 5100 CGAGACGCCA AGATTGCGGC GCTTCAGAAA AGGTTAGATG CCATTAAGTT GCATATGAGG 5160 GACGCCGTGT CTGAGGTTTT CGAGATACAT CCTGACGTCC TCCCCTAATC CCCCAGCCTA 5220 ACCTCTGCCT TTTTAGCGCG GGCTAATCAA CACTAAGGAA AGAGATATGG ACTGGATTGA 5280 AAGCATCCTC AAAAAGATGA GCGATCGACA GCTTTTTTGG GCACAAGCAG CATGTATGGG 534 0 AGCGCTAGTT TTCTTGTTTG TAATTGCTCT GGCTAATCCA CACTAAGGAG AGAGATATGA 54 00 GCGTTAAAAT AACTGGGCCT GTCGCCATCG ACACAGGCGA TTTTGGGAAG TTTGCCCTGA 54 60 TAATTGGTGA AACAGGCTAC AAGTTCCCGG CTCATTACAA GTCAATATGC CCTTGGACGC 5520 ATGAGGCAGA CGTTGAAGAT TATTTGCGAT TCCTTATTGA TAAAGCTAGA GCAGAGGGCA 5580 TCCAGATAAA GGAAGTGCCT AGTTCAGAGG TCTCTGCTCT ATCTGTTGTC GTTCCCGCAT 5 64 0 CGCGCAAGCC TAATCAACAC TCGCCACAAG CTGACCATTA GATTGATCGA TAGGCGGATA 57 00 CTTGCTTCCG CCCCAATCAT CCACAACAAA CGCCTTTTGC TCCATGAACG GAACGCAGGC 57 60 ATTGTCGATA TTATGGACAT ACCGCTCAAT TCGGGTCTGG CTTTCATAAG TGCAGGCTGG 5820 CCCATCAAAG CGCGCATTGC ACGAAGCGAG GTAAGAAACG TTTATCCTCT GCGCGCAGGC 5880 CGTTCGGCCC GCCTCGGCAT ATTGTAGCTG GGTGCTGCCC CATTCGTAGA ACCGCCAGAT 594 0 GCCGAACAGG ATAGCAATGC CTAGCGCAAG CAGAGCGCCG TTTATGAAAT GGGTTACGTC 6000 GACGGGTTTA GGCCCGCGCC TGATTTTGCT TACCCGCATC GCGCATCCCA TGCCGCCCAA 6060 ATTACGGCGA CGTATTCTTG TGCAAAGGCT GGCAGAGATT CAAACTTAGT CGCTGACATG 6120 GTGTTCGGCG TTAGATCGAC GCAGACCGCT TGCTCAACTT CGCTAGCTGT TTCCGTCAGA 6180 ACTGATGAAT CGTTCGCGGA GACGCTCTGG CAGCTCGCCA GTATCACGAA CACGCCTGCG 62 4 0 AACAATATTA ACGGCGTTAG TAACTTGTGC AGATTCTTCA TCGAGGTTTT CCCTTACCTG 6300 TTCCTGTACT CTACGAGATT TAGCTTCAGC GCGGGCTTTG GCTTGTCGCC TGCCTCTGGC 6360 CTCATGGTGT TCGGCCTGCC ATTTGTAAGC CACCAAAACG AGACCAACGG TTAAGACGAT 6420 100 CTGCGCCCAT CCCAACAAAT TAGATCAGGC CGATCGACAA ACGCCTGCCA CTGTCTTCAA GGAACCCGTT CGATGGTTCA AATAGATCAT ACGTCATCAT ACTTCCGCCA TTCATCCACT GTGTAGCCTT TGCGTTTCGT AGATAGCCGA AGTGTGCCAT TCATCATCAC ATGCCTTCAG CTCGGCAATA GTCTACCTTG TTCGTCTGTG TTTGAACGCG AGCCTTGTTC TTTGCGGCGA AGCCTCTGAC CGCGATCATT CTGGTCAGTG GAATTTAACC CTTAATGTCG CTTTGCCCTG AAGGTTGTCG CGCAATCTGG CACCTGCTCA GAGAACATGA TAGCCAATGC TGTCCGAACG CCCTGACCAA CATAAAGGTA CCAATGTTCT CCCAGCCATT TGGCGGATCA AGCTCTAAGC TTATCAAGCC TCAAGTGATT GTAAGCTGCG AGCTGCTGCT CAATTCATTT ATTTGGACCT ACCGTCTCGC CGCGCCTCTA TCAACGGGTG GTTTGCGCTT TACTCTGGCG ATCACTGAAT CGCCATTTCG TTAGTGTCGC TAACAACAGA TTGTTATTGG CCGTAGCTTC CAAGAACGGT ACGAGACAAC CACCAATCTC TCCGCTTTAC TGCTGGCGCG GGTCTGCGGT CTTCAACGCT CCCAATCGAC TGCGCCTGCG GGCGCGCAAT TGGCTTTAGT TTAGCTTGAC GCAACAGGTC CTTCCCAGCG CAAGCGTTTT TAATCAGAAA GGTTTATCTG TTCGGGTCTT ATCAAGCCCG TTCTTGGCGA CAAGACGGAA CTGCGTGTTT CTGTCAGCAA TCAATCAGGA GTAGGATCAG GCGTCCACTT AATATTTGCT CGCTCTTTTG ATTTCCGCTG CCTTCAAGCA ACGGCAGTGC CCATAGGACA GCCCGGCTTC ACAGCGCCGC ATAGCGTCCC AAGCTAGTCG TGTTTCATAA CGGGTCTTGC GGAAGGCCCG CTTGCTCTTC GCTCATCAAT GTTTCCATTT TTTTTTTAAG CCTTTTTAAA TAGCGTGGAG TGTTTCCAAG CTTCCTGAAC GCTTGGCTTG GATCGCGGAA GCATCACGCG TGAATCCACC CAAAACCGCA CACAAACGCG GCCCTCACCA CACTACGCTT CAACGAAGTT GTCTTCGCCA TAGCGTAACA AGGCTCAACT TGTGCCAGTC TTGCGGTTCA TGTAAGCGGT TGTCAGCGGC GCTCTCTGGC ATTCAGCGCC ATAAAGGCCG GACTTCATCT CTCGCCAAAA TTTGATCAGG TTGACCTTCT TGTAGTGCCT ACCCGTCGCA GCTAGCCAGC CGCCATTCAA TGGCTGGTGA CCTTTTTGAG TTATGTCGGC GCAAGCTTTG CGGCGGCCTT AGTGAGCATG AGGCCCAGAA GCCCATGCAG TGTCGCCTTC GCTCTATGCC CGTCATCGGC TTAGCGGGTC AAGCAATCTT CTTTGCTGTA TGACGGACGT TGAACAGCGC CAGCTTGAAA AAAAGTAAGC TTGACGCAAA ATACCCCTAA AATCGCAACA CACCGGGTTG AAGCACACGA CATTTCGTTA TTCAATCAAG CGCTTCAATC AGGATAATCC GCTTTCATGC ATCAGAATAC ATGCTCTTGC AACACCCTCC AACGATCTTT TGCAGACAGG GTAGTCTGCA TCAAGCAACT ATTTGATCGC TTCGTGGGCG CCGGGGTCTG GAAGGCGGCG CGCTCCCGCT AATACCCACA GCGCCAACGG TGCGTTTTAG GGGTTTTCGA ATCTTCTTGC GTTTCAGGCG TCATAGCCAA GCGCGCCAAT AGAACCTTCT TCAGACCGCT TTGTAAAGAA CGCGCAAAGG TGATAGGTAA GCGCCAATCG TCATCGTAAG GGCGCTAATT AACGTCTGGC ATCTACTTCA CATGGTGATA TGACCACTTG AGCCGTGACT CTTGCCAGCC TGCTGCGTAC CGCCCCCACA GGCGCTAGCG AACCTCGATC TGCAATGCGA CAGATCATCG AGTCTGCCGC CTCAGCCGGA TCGCTCCATT GCAAAGCCAT GACAACGCCA TGATAGTTCT GACACCGACA CCAGCGCCCA GTAAACATAC AACGCAATAA ACTTTACTCT AGACGAAGGC AGCTCGCGCG CGAACCTCAA TCAGCCTTTA CCACCATCAG AGTCTGCGCT AGCTTCAGAA AGCTCAACTG AGACCCTCAC TCAACAATGC TAGGCAACAT TGTTTCGGAC GGAAGACATC CGCGCTTAAG TGGCAAGCGT TTCTGTAGGC AAGAATGGGG GCCATTTACC GAATTGTCTC CAACCGCTGC CGTAACCCTT TATAGTCAAC CGCTAATGCG TTGGGTCGTA GCCGCATTGC CAAGACCGAG TTCCACCCGA TATTGATCAG AAGACCCGAA CTGGGGCAAG TTGAGCTACC TTACAACGCC ATTCTGTCCG GTGTCAGCAT AGTGTTTCAG CGCCTGACAA GCATACTCTA GTATTTGCAG TCGATTTTGT TGCACGCGCG AAAATAGCAG CGGGCAGCAA ATCCACATAT GCTGTCGCGA GAGTTAGCGT TTAAGCTTAC TTTAACCGAT GACGCCGTTT TGGCCAGAAG GTTGCGAGCA ACTAGCGCAA AAGCCCTCCG GCAAGGCCAA AACTTGTCAC GAATGAACCC TTAAAATTGG TTGGCTTGCC GCTCTTCTTC GCTTGTAGAG ACTTGAGCAG GCCAGCCATT CCATCTAGTC CAAAGTCTTT AGCGCGATTT GCACAGCATC CTACTTTACG GATCAACATA ACTGGTCTCT TTTTCATTGC GTAAGTATCG AGAGATTGAC GGCGGTTCCA ATAAAACGCA CTGTAATCAT ATACATGTAA TTTCGGGTAG GGTTCCGGCA GCCACGCTTA TGCCTCAATC CTCAAGTGAT ATCGTCATCG AACATCCATT CCATAACAAG ATAGACGTAT CTTTGTGAGC CGCTGCCGCC CTCAGTCCGA GGGGGCGATA AAGCGCTTCA AAAGCCTTCA CGTCTTCATC GTCTCACAAC TGGCGATTTC TGTCACCGCT CATATCGCAA CGCTTTGCCG TTGGGTCTTT GACCAGCGCC CGGTCTTAAG AAAGCGGGTC CGCCATTGGC CATCCTCCAG CGTCGTTCTG CCTCAATGCT TAATGTCCGC CGACAAAGCC GGATGCCACC ACACAGTCCA GTGCTTGACG TAGCAAGCCA GAAGCCACGT GATTGCAGCG TGTTGTCCAA ATCTGGTCCA ATCCCGGCGC CCACCATGCA GTCCGCCTCT TTGCTCAAAT CCCCGTGATC TGCGATGCCC CGCTTCTGAA AGGCTTTACC CAGGGTGGAC GACTCTACCC AGCGGCGCGA TTATCCCTCC TTTGCGCCGT CACGGAACAC ATCGGGCCAT TACACAAGAC 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 101 CTACTCCAGC GCCACCCGTC TTCTCTGCGT ACCGAAGCGT GATAGACGTT AGGGTCATGT 9900 CCTCAGGTAT AGCCCACGTA ATTTGGGTCT TGCCCGTTAC TGCAAAGCTG TAAGCATATG 9960 GCCCGCCGTC ACCATATGTT TGTGAAGTAT CGAGGGCAGT CAGCGTAGGC CCGGTTATCA 10020 CGGCATTAGC AAGAAGTGGG AAAGAGCGCG CGCCAATAGC CCCTCGCGGC GCAATTTGAG 1008 0 CCTGCGCCAG AACGGCGGCG TAGACCCACG GACCAAGATA GTTGCCTGGA TGATTGTAGC 1014 0 CAGACCCAGC CCCATCCCCT AGCGCGAACA ACTGACGATC TTTGACTTGT GGCGTATAAG 10200 CGCGCGGACG CGCATACAGC ATTTTTACCG CCGTGACGAA AGCTGCGCCA CTCGCATCTG 10260 TAACCTGTGG TGTAAACCAG CCGCCGTAGC GATAAAGAAC CTCCGAAAAC AGGGGAGCTA 10320 CCGTCTGCCC AAAACCATTT CCAGTTGTAC TTAGCCAGAC TGAAATACGG TTGCCGCTAA 10380 TTTCTATATA TAGCTCAACA TCGGATGCAA ACCATGTACC CTGTTGCAAG TTTTGATAAT 104 4 0 TGTCAGCCGC TCCAGCTGGA CCTGACGATA AGCGCCAGCG AATGTTTCCG CCCGCTGTCT 10500 TAGAGATACG CAACCAATCG AGCGGACCTG CGCCGACCTG AATAACGACT GAAGCATCAG 105 60 GGTCTGTTGT GTCATCAACT AATTCTGACA CGTCCAAAGA TACCCGATAC AAGCAATCAA 10620 TGACTTCTTT TGAGCCTGAC CAAACGTCAG AGACCTCTGT GATTGTCTCT TCGTCATCAA 10680 TTAAGATGCT ATCAACTTCA TCATACCCGT CGCGTACCGC AACAAACTGC CGCCCAAAAT 10740 CGAGACAAGG AATATTGAGA CGGTTCGCGT ATGAGCGAAC CAGACCGCAC GCCTGATCTC 108 00 GCGTGTCCTG CTCGGATCGA ACACCGTATT GACTGTCTGT CGGGTCAAGA GCTGGTCTGA 10860 CGCATGTCGC TAAAACAGAT TGTGGCAGCG TTTCCAGATT GCTAGCCCAG CTTATAGCTG 10920 CGTCTAAATC GTCTGGATCA AGCGTAGCTC CATGGTTCAT GCCAAACGAA AATATAATAA 10980 TATCGGGACT TAGTTGCTCA ACCAAGTCTT GCCATGACTG AGTTCCATCA GTCCACCACT 11040 CAACCCCTGA CGGCTGATTA CCGCTACCGG GCGCAGCAGC ATTAAAGTTA CTGTATGTTT 11100 TGCCGCCAAT AGCGCGGTTA TAAAATGTAA AACCACCCTG TCCAAGTTGC TCAGATAACC 11160 ATTGTTCAAT CGCGGCCGAC AACATATCAC CGCGCCCGTT CGCATTGGCG AGATAAGTCG 11220 AAATGGAGTC ACCCACCAGC ACAACAACCG GGTTTTCAGT AAACGTGGTT ATAGAGGTGT 112 80 TTTTGGGGGT ATGCTGGCCT GCATCAAGCG GAATGACCTG CTTTCGATAA ATTCCAGTGA 1134 0 GCGCGCCACT ACCAAAGAAA CTAGCGTTTG CGGTGGCTTC AATATTCGGC GCATCTGTCG 114 00 TGTAATCGTT ATCTATGATG ATGCGGTCAG ATTGCGCCGA CGCCGCCTCA ATCGCGGTTT 114 60 TGACAGCGCT GCCCGTCGCG CCTGTCGCAA CATATCGCGC CAAGTTAATT TCACCTGTGT 11520 CTAGATAGCG AAAAGCGCCC ACTGTGCCGG ACTTGTCGCT AGTGAGCGTA AACCAGACAC 1158 0 CGCCATCACC TTCGCTAGAT GTAACCTCAT CGGCGCTGAC ATTAGATGAC TGGTCACCTG 11640 CTTCCCATAC GCACAAGCCT TCGCCGCCAT CACCCGTTGC GTCACGGCCT GACAATATAA 117 00 AACCCTGCCC AGCAACAGGC GTATAGGTTA AGCCTCGCAA CTCCGTCTTC GTCTTAACCG 117 60 CCGGCGCAGC CCAATTTATT GCGCTAGCAA GAGTTCCGTC TTCAATCGCC CCGGAAAGCG 11820 TAACAATCGC GGCAGCAATC CCCGAAGTAA GGATAACCTC CGCATCAATA TCAGAGACAG 11880 CGGAAACGTC CGCAATGAAT GGGCTTACCG CCGCAATGGC GTCCAAAAGG CCCGCCGCAG 11940 GAGTGTCGCC AACAGGCAGA AGTGCCGCAC GGGCGATCTT GTCATTCAAC TGCTGTATTT 12000 GCAGCGTCGT TCGGTCGCGC GCATCATTTA GAACAGTCGG GTAAAAGCCC CCTGCGTTTG 12060 TGAATGCAGC TTCTTGCGTG TAGTCAACGT CAGAAACAAT AAAGATTGTC TGTGTCGCCG 12120 TTGGCGCTGT AACCATTGTC ACATTACCGC CCGGACTTGC CTCTTGGTCC GCGTTTAAAG 12180 TGACTGTGTA ATTCGTTGTA ATCGTCAGAA CAGTTTGAAC ATCAGACTCC TCACGGATAA 122 4 0 CTTGCAAATC CGCCTGAGAA AAACATTTAA ACGAAAAGGA AAATACAGTC GTTGAGCCGT 12300 TACCCGTGAA AGGTCCGGCC AGCGCGCTCT GCGTGGCTAT TGTCATGTTT TAGATTCCCG 12 360 TTGGTGTAGA GGGCGCGGAA ATGCCGCTTA GTGAAAAGTC GATTGAACTT AGTTCAGATT 12 42 0 CAATCTGATC AGCCTTTAGG CCAAGATACG CAACTGACGC TGCGAACAGG ATAAAAAACA 12480 AAACAATCCA AATACGAATC TCGCGCTTAG ACATCAGTTA TCGTCATCCG GTCCAGTAAT 1254 0 AAGAGCCTCA TAAAGCGTAG GATCATCATC TTCCGCAAGC GTTTTTTCAA TTCGAGCGAT 12 600 CTGCAAGGTT CCAGGAATGC CAAACAAAAG ACCTAGCCCA GTCCCCGCCG CGCGCACGCC 12 660 CGACCGAACC GCCTCTGCAT CCAGCTCGCC^CTCTGCAAAG TAATCATGGC CAGCAGAAAA 12720 GAAGCGCTCA GCCGCCTGTG ACAAATCTGA CCCGGCCTGC AATGACGAGG GGCCACTGTC 127 80 ATATTTGCTA AAGAATGGCG ACAAACCAGG AACCATGCCT ATGACGTTGC GCTGCATCAG 1284 0 CATCAGTGTG TTCTGCCAGT ATGGATCTTC TTCGTCTCCG AACCCGTCTC GGATTAATGC 12 900 CTCAACCATG GACGCTGCGA CATTCAATAA AATGAAATGC TTAAACACCT GAAACACGCG 12 960 ATTAAAGTTT TGCGCCTTTG TGATGTCATT TCGAGCGATA CCGTACATGT TCGAGACATA 13020 GCCCCACATG AAGGTCAACA GTCGCTCCAT CTCCGGGCCA CCCTGCAAAA CAGACTGATC 1308 0 AAGCTCCAAT CCAGAGCCTT GCGTGGTCGC AACCGCCCGA TCAGCCTCAA GGATCGCCTC 1314 0 GCGCTCGCTT TTACCCTGAT CAAGCGCCTG GTTATAAATC CCCATCCAGA GCGGCCCAGA 13200 AACACTGTGC TTCTCAATAA AGGCCATGAG CCAATAACCA GCGCCCTGAA TCTTGCCGAA 132 60 102 CCGGCTCTGC ACGCTCACGC CGGGCCAAGC AATAACCGTC CAAAGCCACC GCGCCGTGTG ACCTTGCGTG AGACGCAGGC AGTCAGATCG ATTGGTCGAG CTGGCGCTCA GCCGCGATAT CCCCGTTTCA ATGTCTTGCG GCCGCCATAT CCCATATTCA GGCGCATTAA TTCAGACGAT GGTGTTTCAA AATGGCCAGC AGTGATGGAT GCCAGCTCTT AGCTTGCTCA CGACGAAGCG TCACCGGACT GCCCCTGGCA GGGTCAAGCT TCACGCGCCT CGCAAGGCAT CGCGCCGTTA CGGCGTTCGG GCCAATGCAT ATTGCTTCAG TTCTTCACGG ATCTCATCTG TCCGACGTGT TCACCAAACG TCCTCGACGC ATATGCTTTT TACTTTTCAA TACTGACCTT CGCGCTTCCG CACTGGCGGA AAATATGCCT ACCGCGCTTT ATGAAAACCG CCATAATACG TGGAAGTAAG GAGGCATTCA CCGACAACAG CGCGTGATGG TCGCGCATGT AGACGCAAAG GCCCGCGTTG AGGTCGGTAT GTCCCGAACT CGATATTCGA CTGCATCCCA CGCCATTCAT GCGCAACCGA CGCGCCTGTT CCGTCATGTC ATGCCTCTAT GCATAAACAA TGCGCGTTTC GCGCCTCAGC GGTTTTTCAC GCAGGTCATT GTGCATCTGC GGCTCATCAG TTCGATGCGC TAAACGCCCG CATCCCACTT TCGCATATGT CTTCCCGGCG TGCGCTCGGC CCTCAATCGT TCTCACGCAC CCGACGCAAA TCAGCTTCGT CAGCCAGGGC GCTCAGCTTC TAAACAGCTT CCTGTTCTGG GCGCCATGTC GCTCTAGGCT AACTATCGAA CAAAGCTT TCCTAATCCG ACTTTTCGTT CAGCAATTCC AGATTGGCGC CCCGTCCCGT AGCGTTTCAA CCTCTCGGAT AAATGTCATT GGCGTTCTAC CCATACCATT TGCGCATAAA TCGTCTGAAC CCGCAGACGC GTGACGCTGC CGACGGATCA CAAGACTTGG CAATTCGCCT CTCGCGGCGC AATCTTGCCA GGCATCAAAA GCGCTTTTCC ACGGAAGGCT CGCATCGCGA GCCCGCAACA AAACTCATTC CTCGCGTATT CGCAAACTTG GCGCGCCATC GCCGCGTGCT GCCGGGCTTA AATAGCCACC TTCCATCATC GCCATCGCGA CATAACTTCC ACCCATCTCT ACCGGCTGGC CTTATATTCT TTCCTTATAA GTCACGAGCA CGACATGATA AATTTCGGCC GCGCAACTGG CGCCGGTCTA CTCATGCGCC AATCGCCTTC ACGCGCGACC AAGAATGGCC TCGGATCAGG AATTTCCTCT AGAGGATCGC CGTGACCTGA ATTCTGTTGG GACGCCCTGC TCCGCGATCA AGATGACGCC ATCTGCCCAC TCAAGATGTT GAGACCAAAC CTAAAGCGAA TGCGTGATTT GCGTTGCGAT ACCTTGCCGC TTGCGCAACG TTGCCCGTAT TGCATGGCTT AACTCTCTGA ACTGCGCTCG AGAGCCTTGA TTCGTGATAT GCAAGGAAGC TCATGCTGCA GCCGCCCGTA ATGACCTTAT AATGGAATAG CGAATGGTCG AGCATGTCGC GGCTTCGGCA GCCGCAACCA GAGTGTGCGG GGCCATTCTG GCCTTTTTCT ACAGCCCCGG CTCTGCATAA TCAGTCGCAA CCCCTTAAAC AGCTCAGCGC TCGCGGTTAA GATGCCTTGA GGCGCAAATC GTTGTTACGT TTCTCAAACG AATGGTTTCA TGCAACAGGC TCAATATGCT CGCTCTTTCA TTCCATTCTT TATTTCAACG TCGACTTTTT ATCGTGTTTA CTGCCAACAT GGTCACGGTT CATTGCCATC TGTGCTTTTT CCTGTAGCGC CTTGCAGCGC TGCTTGTAAA AGTTTTGCGT CTTCGCTTTG ATTGCTCCAA GCAGCACAAC TAACATCGCC AGGCAGGCGC GATTAGCCTT GCATGGCTTG GCGCCTTCTT CGATCGGCAG CTTGCTTAGC GCTCATTGAA CGTATTTATC CTTTCGCCAC TATCAGGATG CATCCAGTGA CCATCTGCGC CGCGGCGCTT CCTTGCGGGC CCTTCGAAAG GCATCCGATC GGCGGTAGAC TTGGTGCGAC TCGTGTTCTC TCATGCTGAA CATAACGCGG TCAAACCAAG CATTGTCAGC GATACTCTTT GAGACACACG CAAGAATTAC AAAACCCGCG CAATCACGTC GATAATAGCC TAGGTGAAAC GCAAGTCCAA CGCTTCAATC TTGATCGGTC TTCAATCACC CAGAATGTCC CTGGCGGAGC CTCGATCAGG ATTCTCTAAC AATCTCATCA CTGACGCCCC GGTCATGCGC AGAATAGCCC TGCATTCTGC GATCTTCTTC CTCAATCTCT ATGCGTATAG GTGGAGATTA ATCATTGATC AGCCTTTTCA CACCGCTTCC GCCCATGATC CGCCTTAAAC TTCACCATTC GCGGCCACCC ACGCACCATG CACTTCCGCC GTGTTTCGCA CGTGCCGACA AAAGATGTCG ATCCGTAATC GCCTTCGCGC 13320 13380 13440 13500 13560 13620 13680 13740 13800 13860 13920 13980 14040 14100 14160 14220 14280 14340 14400 14460 14520 14580 14640 14700 14760 14820 14880 14940 15000 15060 15120 15180 15240 15300 15360 15420 15480 15540 15600 15660 15720 15780 15840 15858 

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