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The characterization of atm-1 in Caenorhabditis elegans Huang, Chin 2011

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The Characterization of atm-1 in Caenorhabditis elegans  by  Chin Huang  B.Sc. Honours, Simon Fraser University, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     June 2011  © Chin Huang, 2011  ii Abstract Loss of function of ATM (ataxia-telangiectasia, mutated) was discovered to be the genetic cause of the human disorder Ataxia-Telangiectasia (A-T).  A-T is a rare, autosomal recessive human disorder that presents with multiple symptoms, including ataxia, telangiectasia, neurodegeneration, immunodeficiency, radiosensitivity, genomic instability, and a predisposition to developing cancer.  The predicted ATM orthologue in Caenorhabditis elegans (C. elegans) is the gene atm-1.  C. elegans is a model system that is easily amendable to molecular and genetic research.  In this thesis, I have characterized the structure of atm-1 and examined its mutant phenotype.  The predicted gene model for atm-1 was smaller than known orthologues.  Using cDNA analysis and sequencing, I have shown that the three gene predictions atm-1, K10E9.1, and F56C11.4 make up the complete coding region.  I observed differences from the original prediction including unpredicted splice sites and exonic sequences.  A single atm-1 mutant allele, gk186, exists.  I have shown that the gk186 deletion produces two alternative transcripts, one of which retains the protein kinase domain.  Thus, in contrast to previous expectations that this mutation generated a loss of function phenotype, it is likely that it produces a protein with kinase function.  The phenotype of the mutant is radiation sensitivity, as expected, but is not as sensitive as brd-1, another loss of function mutant in the same double strand break repair pathway.  This result is consistent with partial function retention of atm-1.  In addition, mutant atm-1 showed chromosomal instability in X-chromosome loss and subsequent sterility.  Some of the mutational events were captured as lethals using the eT1 balancer system documenting a mutator phenotype of atm-1.  Characterization of atm-1 gene function in C. elegans may provide additional information about its function in other organisms including humans.      iii Table of Contents Abstract .................................................................................................................................... ii Table of Contents ................................................................................................................... iii List of Tables .......................................................................................................................... vi List of Figures ........................................................................................................................ vii List of Abbreviations ........................................................................................................... viii Acknowledgements ................................................................................................................ ix Dedication ................................................................................................................................ x 1. Introduction ......................................................................................................................... 1 1.1 Introduction to Ataxia-Telangiectasia ................................................................................... 1 1.1.1 The molecular cloning of the Ataxia-Telangiectasia mutation - ATM ............................. 1 1.1.2 The importance of studying ATM .................................................................................... 2 1.2 Double-strand break repair .................................................................................................... 2 1.3 Introduction to splicing and alternative splicing ................................................................... 2 1.4 Nonsense mediated decay is a quality control mechanism for mRNA ................................. 3 1.5 National Center for Biotechnology Information ................................................................... 3 1.6 Caenorhabditis elegans ......................................................................................................... 4 1.6.1 The genomic translocation, eT1, is a genetic balancer system ......................................... 5 1.6.2 Curated C. elegans data are available at WormBase.org, an online database .................. 6 1.6.3 atm-1 in C. elegans ........................................................................................................... 7 1.7 Thesis Objectives: characterizing the C. elegans ATM orthologue, atm-1 .......................... 8 2. Materials and Methods ..................................................................................................... 14 2.1 Nematode maintenance, culturing, and mating ................................................................... 14 2.2 Generation of complementary DNA (cDNA) library ......................................................... 14 2.3 Sequencing and sequence alignment ................................................................................... 15 2.4 Single worm DNA extraction ............................................................................................. 15 2.5 Primer design and polymerase chain reaction (PCR).......................................................... 15 2.6 atm-1(gk186) outcross and gk186 deletion detection by PCR ............................................ 16 2.7 Ionizing radiation sensitivity assay ..................................................................................... 16 2.8 DAPI staining ...................................................................................................................... 17  iv 2.9 Quantifying genomic instability .......................................................................................... 17 2.10 Creating atm-1(gk186);eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177) ................................. 17 3. Results ................................................................................................................................ 22 3.1 Molecular characterization of the wildtype atm-1 gene ...................................................... 22 3.1.1 The predicted atm-1 gene is much smaller than other ATM homologs.......................... 22 3.1.2 K10E9.1 and F56C11.4 are uncharacterized gene predictions upstream of atm-1 ......... 22 3.1.3 F56C11.4, K10E9.1, and atm-1 gene predictions produce one single mRNA transcript….  ................................................................................................................................. 23 3.1.4 cDNA PCR products support a larger atm-1 gene model ............................................... 24 3.1.5 Sequencing reveals the nucleotide composition of the larger atm-1 gene model with novel unpredicted changes ........................................................................................................... 24 3.1.6 The extended gene model of atm-1 does not reveal any additional protein domains ..... 25 3.1.7 Section summary ............................................................................................................ 25 3.2 Molecular characterization of the atm-1 mutant strain, VC381 .......................................... 38 3.2.1 atm-1(gk186) is a deletion that does not prevent transcription ....................................... 38 3.2.2 atm-1(gk186) produces two alternative transcripts near the deletion region .................. 38 3.2.3 The atm-1(gk186) alternative transcripts do not skip exon 22 and utilize novel unpredicted splice sites ................................................................................................................ 39 3.2.4 The gk186 transcripts are predicted to produce distinct products ................................... 39 3.2.5 Section summary ............................................................................................................ 39 3.3 Phenotypic characterization of VC381 ............................................................................... 46 3.3.1 Mutant atm-1(gk186) does not have any obvious phenotypes........................................ 46 3.3.2 atm-1(gk186) is sensitive to double strand breaks caused by ionizing radiation ............ 46 3.3.3 atm-1(gk186) spontaneously produces a dominant high incidence of male (Him) phenotype and eventually becomes sterile ................................................................................... 47 3.3.4 Him and sterile atm-1(gk186) strains have a reduced number of diakinetic chromosomes. .............................................................................................................................. 47 3.3.5 Quantifying independent atm-1(gk186) lines indicate inherited genomic instability ..... 47 3.3.6 atm-1(gk186) produces lethal mutations that can be captured by the eT1 balancer system.. ........................................................................................................................................ 48 3.3.7 The forward mutation frequency of atm-1(gk186) is similar to the general mutator dog-1 ………… ....................................................................................................................................... 48 3.3.8 Section summary ............................................................................................................ 48  v 4. Discussion........................................................................................................................... 57 4.1 The extended gene model requires further refinement........................................................ 57 4.2 The alternative transcripts of atm-1(gk186) may have residual ATM-1 function .............. 58 4.3 Function conservation of atm-1(gk186) phenotypes ........................................................... 58 5. Conclusions ........................................................................................................................ 61 Bibliography .......................................................................................................................... 62 Appendices ............................................................................................................................. 65 Appendix A : WS205 exonic sequences .......................................................................................... 65 A.1 F56C11.4 ........................................................................................................................ 65 A.2 K10E9.1 .......................................................................................................................... 65 A.3 atm-1 ............................................................................................................................... 67 Appendix B : Full contig of the extended gene model ..................................................................... 68 B.1 atm-1 full contig ............................................................................................................. 68 Appendix C : atm-1(gk186) transcripts ............................................................................................ 71 C.1 atm-1.a ............................................................................................................................ 71 C.2 atm-1.b ............................................................................................................................ 73 Appendix D : atm-1 protein translations .......................................................................................... 77 D.1 atm-1 ............................................................................................................................... 77 D.2 atm-1.a ............................................................................................................................ 78 D.3 atm-1.b ............................................................................................................................ 79   vi List of Tables Table 1.1  All possible progeny genotypes combinations for a self-fertilizing eT1 heterozygous hermaphrodite ................................................................................................... 12 Table 2.1  List of primers created and used. ........................................................................... 19 Table 2.2  A list of strains used and created. .......................................................................... 20 Table 3.1  cDNA PCR primer combinations and expected product sizes. ............................. 34 Table 3.2  Summary of novel changes for the atm-1 gene model .......................................... 35 Table 3.3  Summary of changes in the atm-1(gk186) transcripts ........................................... 44 Table 3.4  VC381 Characteristics at 20°C. ............................................................................. 50 Table 3.5  List of obtained atm-1 Him strains with their associated diakinetic chromosome numbers VIA DAPI. ............................................................................................................... 53 Table 3.6  The progeny of atm-1(gk186) fluctuates or decreases as a function of generations................................................................................................................................................. 55 Table 3.7  Summary of atm-1; dpy-18/eT1(III);unc-46/eT1(V) screen. ................................. 56   vii List of Figures Figure 1.1  Different mechanisms of alternative splicing ......................................................... 9 Figure 1.2  One arm of the C. elegans hermaphrodite germline ............................................. 10 Figure 1.3  eT1 balancer system ............................................................................................. 11 Figure 1.4  atm-1 gene model as depicted on WormBase (WS205) ....................................... 13 Figure 2.1  Agarose gel of wildtype (WT) and atm-1(gk186) PCR products using the poison primer technique. .................................................................................................................... 21 Figure 3.1  HomoloGene comparison between known ATM homologues ............................ 26 Figure 3.2  CCD result for the atm-1 649AA query ............................................................... 27 Figure 3.3  Genomic region 183,000bp to 209,000bp of chromosome I on wormbase.org (WS205) .................................................................................................................................. 28 Figure 3.4  Genomic region 183,000bp to 209,000bp of chromosome I with the three ESTs that support linkage of the three gene models ........................................................................ 29 Figure 3.5  yk220a.5 also suggests linkage between K10E9.1 and atm-1 .............................. 30 Figure 3.6  F-K and K-A Junction primers design .................................................................. 31 Figure 3.7  cDNA PCR results using the F-K and K-A Junction primers .............................. 32 Figure 3.8  Wildtype cDNA primer design and products ....................................................... 33 Figure 3.9  Comparison between the predicted gene models and the extended gene model .. 36 Figure 3.10  CDD prediction of the extended atm-1 protein sequence .................................. 37 Figure 3.11  atm-1 cDNA primer design and products ........................................................... 41 Figure 3.12  Comparison between atm-1 and wildtype cDNA products ................................ 42 Figure 3.13  Abbreviated gene models for atm-1 ................................................................... 43 Figure 3.14  Conserved Domains Database depiction of the two atm-1 translations ............. 45 Figure 3.15  The atm-1 mutant is sensitive to IR .................................................................... 51 Figure 3.16  atm-1 mutants have reduced number of diakinetic bodies ................................. 52 Figure 3.17  atm-1(gk186) mutants have genomic stability defects ....................................... 54   viii List of Abbreviations  A-T (Ataxia-Telangiectasia)  ATM (Ataxia-Telangiectasia, Mutated) PI3K (Phosphatidylinositol 3-kinase) PIKK (PI3K- Related protein kinase) DSBs (Double strand breaks) DDR (DNA damage response) IR (Ionizing radiation) NHEJ (Non-homologous end joining) HR (Homologous recombination) ESTs (Expressed sequence tags) cDNA (Complementary DNA) mRNA (messenger RNA) NCBI (National Center for Biotechnology Information) CDD (Conserved Domain Database) Chr (Chromosome) Dpy (Dumpy) Unc (Uncoordinated) pre-mRNA (primary mRNA) NMD (Nonsense mediated decay) PTC (Premature termination codon) EJC (Exon-junction complexes) TMP (4,5’,8-trimethylpsoralen) UV (Ultraviolet) RNAi (RNA interference) HU (Hydroxyurea) EMS (Ethyl methanesulfonate) SAGE (Serial analysis of gene expression) RACE (Rapid amplification of cDNA ends) Grays (Gy)  ix Acknowledgements  My deepest gratitude goes to Dr. Ann Rose.  She has been an extraordinary supervisor, mentor, and most importantly, friend.  Thank you for supporting me through the highs and the lows.  I would like to thank to my lab mates Dr. Nigel O’Neil, Dr. Martin Jones, Jessica McLellan, George Chung, Sanja Tarailo, Shir Hazir, Zoe Lohn, and Domena Tu for insightful contributions and moral support.  As well, thanks to my collaborators Dr. David Baillie and Shu Yi Chua.  Dr. Allan Mah for feedback and comments.  I’d also like to thank my committee members, Dr. Jan Friedman and Dr. Terry Snutch, for their curiosity, criticism, and input in the project.  Thank you Mathias, Candice, and Courtney.  You’ve helped more than you could ever imagine.  And lastly, my family for not trying to understand science but appreciating the hard work that it entails.     x Dedication  This thesis, all my hard work, and all my accomplishments are dedicated to my grandmother.   1 1.     Introduction 1.1 Introduction to Ataxia-Telangiectasia Ataxia-Telangiectasia (A-T) is a rare, autosomal recessive human disorder that presents with multiple symptoms [1-4].  Suspicion and diagnosis of A-T occurs at an early age when ataxia, lack of motor control, hinders the child’s ability to walk normally.  Ataxia progressively worsens and eventually immobilizes the child [4].  Telangiectasia, which is the constant dilation of the blood vessels of the eyes and face, ensues within the first decade of life [4].  In addition to the visible features, A-T patients have varying degrees of progressive neurodegeneration, immunodeficiency, abnormal or absent thymus, insulin-resistant diabetes, and are susceptible to developing cancer [4].   On a cellular level, their cells are radiosensitive, have cell-cycle checkpoint defects, and display chromosomal instability [1-5]. These chromosome defects are visible cytologically and are characterized by shortened telomeres, chromosomal rearrangements, and telomere fusions [5-9]. 1.1.1 The molecular cloning of the Ataxia-Telangiectasia mutation - ATM ATM (Ataxia-Telangiectasia, Mutated), was discovered by positional cloning, to be the genetic cause of A-T [10, 11].  ATM is a member of the phosphatidylinositol 3-kinase (PI3K) – related protein kinase (PIKK) family.  The PIKK proteins functions as serine/threonine protein kinases [2, 3, 5, 12, 13].  In response to double-strand breaks (DSBs) in DNA, inactive ATM dimers localize to the break and disassociate into active monomers.  Activation of ATM is facilitated by the MRE11-RAD50-NBS1 complex [2, 12-15].  The DNA-associated and active ATM then subsequently phosphorylates up- and down-stream targets that amplify the damage signal and initiate specific cell-cycle checkpoints, the DNA damage response, and the apoptosis pathways to appropriately address the DSB [2, 13].  The current understanding of activation and signal transduction pathways of ATM are often reviewed in the literature [2, 3, 12, 13].      2 1.1.2 The importance of studying ATM  The study of ATM is important because of the severe clinical manifestations of A-T patients.   Moreover, ATM appears to be involved in a wide range of biological processes.  Studying ATM has improved our fundamental understanding of intracellular signalling transduction and control.  It has shed light on how molecules are able to “sense” specific cellular situations and facilitate post translational modifications in a spatial and temporal manner to elicit specific responses.  ATM has also provided insight into how misregulation of signalling pathways, such as those involved in the DNA damage response (DDR) and cell-cycle checkpoints, can lead to tumorigenesis [16, 17].  Importantly, ATM facilitates DSB repair, a process essential for genomic stability and immunoglobulin generation, therefore this work will contribute to our understanding of cancer and immunity [2, 13]. 1.2 Double-strand break repair DSBs are complete breaks in DNA that can occur naturally, like those required for proper meiosis, or from external sources, such as ionizing radiation (IR) [18, 19].  As a result of improper DNA repair, DSBs can lead to genomic lesions including deletions, translocations, and fusions.  These large scale genomic events can also lead to genomic instability which is a hallmark of cancer [16, 17].  DSBs are typically repaired in one of two ways: non-homologous end joining (NHEJ) or homologous recombination (HR) [18, 19].  During NHEJ, the nucleotides at the ends of the DSB are processed before ligation.  This process can result in gains or losses of nucleotides at the break point resulting in mutations.  HR on the other hand is a more accurate form of repair because it takes advantage of homologous sequences as template, typically from a sister chromatid, to direct repair.   1.3 Introduction to splicing and alternative splicing Splicing is the process of removing introns from primary mRNA (pre-mRNA) transcripts to concatenate coding exons to ensure proper translation of the protein.  The splicing machinery (spliceosome) is a large nucleoprotein complex comprised of five small nuclear RNAs and about 150 proteins.  The spliceosome recognizes splice site sequences at the exon-intron boundaries and catalyzes the removal of introns while joining the adjacent  3 exons.  Accessory splicing proteins aid in splicing by recognizing enhancer and suppressor sites associated with their particular splice site [20].   Alternative splicing is the processing of a single pre-mRNAs to generate different coding forms (isoforms) of mRNA [20, 21].  This allows a single gene to produce variants of a protein with different functions.  There are several mechanisms by which alternative splicing can occur (Figure 1.1).  The most common form of AS is exon skipping which is the exclusion of an exon [21].  The two flanking exons of the excluded exon are then spliced together.  Another type of alternative splicing is the usage of alternative 5’ and 3’ splice sites that are not typically recognized for generating the native transcript.  Other possibilities of alternative splicing include intron retention, mutually exclusive exons, alternative 5’ starts, alternative 3’ poly adenylation sites, and trans-splicing from two different mRNAs (not shown in Figure 1.1) [21, 22].  Because of alternative splicing, the number of proteins types produced in an organism is much larger than the number of genes in the genome [21]. 1.4 Nonsense mediated decay is a quality control mechanism for mRNA Nonsense medicated decay (NMD) is a surveillance process that recognizes premature termination codons (PTCs) in mRNA and initiates degradation of the aberrant transcript.  NMD is important because translation of aberrant transcripts results in truncated proteins that could disrupt normal protein function; dysfunctional proteins with a dominant-negative function or a deleterious gain-of-function.  Current studies suggest a model that requires the aberrant transcript be initially translated by the ribosomal complex.  The first pass of translation by the ribosomal complex displaces exon-junction complexes (EJC) laid down during pre-mRNA splicing.  If a PTC is present, then EJC downstream of the PTC will not be displaced from the pre-mRNA and the presence of EJC signals other factors to initiate degradation of the aberrant transcript [23].   1.5 National Center for Biotechnology Information The National Center for Biotechnology Information (NCBI) is a central database that houses raw data and provides tools to help scientists access and utilize the information in a user friendly way.  Information is constantly being added and NCBI is constantly updating its  4 website and providing new ways for accessing and interpreting new datasets [24].  One tool that is provided is HomoloGene.  This tool automatically identifies and compares homologous and paralogous genes identified in all the sequenced eukaryotes in their database.  HomoloGene reports also contain other information that is gathered through their other tools and resources.  The Conserved Domain Database (CDD) is another tool that takes all the protein domains from NCBI databases and consolidates them into one searchable database.  The Conserved Domain Search allows users to input protein sequences which are then compared to the CDD to identify known protein domains within the query sequence.  1.6 Caenorhabditis elegans The microscopic nematode, Caenorhabditis elegans (C. elegans), was first proposed as a model organism by Sidney Brenner to understand mammalian nervous systems in a biologically relevant but simplified organism [25].  Since then, work with C. elegans has produced a complete high quality genomic sequence, a complete cell lineage map, and a large community dedicated to the use of C. elegans as a genetically tractable model organism.  Some of the advantages of C. elegans as a model system include: a short three and half day life cycle, a relatively simple and transparent morphology, its ability to be easily amended to genetic and high throughput technologies, its informative phenotypes, and its ease of cultivation in the laboratory [26, 27].  Another advantage of C. elegans is that its genes are highly conserved with those of higher eukaryotes, including humans.  Thus genetic and molecular studies can have a direct impact on the understanding of human biology and disease [28].   C. elegans are typically hermaphroditic, capable of propagating the species in a clonal fashion, and capable of producing approximately 300 progeny through self-fertilization.  Males occur naturally at an approximately 0.1% frequency and are hemizygous for the X chromosome as a result of X nondisjunction or loss [29].  Importantly, the presence of male C. elegans allows for sexual reproduction and genetic manipulation through genetic crosses.  Developmentally, worms hatch from eggs and develop through four larval stages (L1 through L4) before becoming adults.  Interestingly, the hermaphrodite gonad produces oocytes in a production-line fashion starting with mitotically dividing cells originating at the distal end of  5 the gonad (Figure 1.2).  The cells then progress into the transition zone where these cells become meiotic.  Mature oocytes are fertilized by sperm as they pass through the spermatheca and are then expelled through the vulva [25-27]. 1.6.1 The genomic translocation, eT1, is a genetic balancer system  One of the advantages of working with C. elegans is the existence of a well characterized system that can be used to study mutations.  eT1 is a stable reciprocal translocation of the C. elegans chromosomes (Chr) III and V [30-32].  Physically, the left portion of Chr III is attached to the left portion of Chr V and the right portion of Chr III is attached to the right portion of Chr V; named eT1(III) and eT1(V), respectively (Figure 1.3).  eT1(III) pairs with the normal Chr III and eT1(V) pairs with the normal Chr V during synapsis.  The regions lacking homology during synapsis are recombinationally suppressed.  These include the translocated regions of eT1(III), eT1(V), the right portion of the normal Chr III, and the left portion of normal Chr V.  The eT1 balancer system is maintained as a heterozygote and contains the mutations dpy-18 and unc-46 in the recombinationally suppressed regions of the normal chromosomes (Figure 1.3).  These visible markers allow the different chromosomes to be followed by observing the phenotypes of the progeny produced by a self-fertilizing eT1 heterozygote.  Heterozygous eT1 worms produce a ratio of 4:1:1 wildtype, to Dumpy Uncoordinated (DpyUnc), to Unc-36 worms.  Unc-36 worms are eT1 homozygotes because the breakpoint in eT1 disrupts unc-36 on Chr III.  A  large portion of the eT1 progeny arrest as embryos due to aneuploidy (10/16) (Table 2.1).   The eT1 system is useful because it has been used to capture lethal mutation events that occur within the recombination suppressed regions of normal Chr III and Chr V.  These mutagenesis screens have been used to obtain and characterize mutational events in addition to determining forward mutation frequencies [30, 31, 33, 34].  Mutations in essential genes located on Chr III or Chr V are easily identified because the mutations will cause lethality to progeny homozygous for the normal chromosomes, which are marked with dpy-18 and unc-46.  Therefore, failure of worms to produce progeny with DpyUnc worms indicates that a lethal mutation has occurred and been captured.  The eT1 balancer also facilitates the  6 maintenance of mutations because they are recombinationally suppressed in their respective regions of Chr III or V. 1.6.2 Curated C. elegans data are available at WormBase.org, an online database The nearly complete sequence of C. elegans was published in 1998 by The C. elegans Sequencing Consortium, and the final gaps of the approximate 100 megabase genome filled in 2002 [35, 36].  The sequencing data are available at WormBase.org, a public database that aims to provide the scientific community with a curated resource based on published literature.  In addition to the complete genomic sequence, WormBase.org also contains gene models, gene expression patterns, numerous other resources and tools, and the complete genomic data corresponding to four other Caenorhabditis species for comparative genomics [37]. The accuracy and integrity of the information on WormBase is achieved by integrating published data with bioinformatically generated information.  An example of this is the curation of predicted gene models with the use of Expressed Sequence Tags (ESTs).  First, programs like GeneFinder use known characteristics of gene structure to scan the genomic sequence and predict open reading frames (protein coding genes).  Data such as ESTs can then be used to validate the predicted models.  ESTs are short sequence reads obtained from the ends of vectors cloned with complementary DNA (cDNA).  cDNAs are reverse transcribed from messenger RNA (mRNA), and thus sequencing obtained from the cDNA inserts represents exon sequences from genes and can support predicted protein coding gene models.  Because only the 5’ and 3’ ends are sequenced, the ESTs represent the starts and stops of protein coding genes, while the remainder of the model is inferred.  Sequencing the full cDNA would identify the complete gene structure [38].  A recent large scale example of curating gene models can be seen in the modENCODE project for C. elegans [39].  Because WormBase is periodically updated, every 5th data release is frozen to allow researchers to cite and reproduce research without having to worry about slight changes in annotation or sequence [37].   7 1.6.3 atm-1 in C. elegans atm-1 is a predicted homolog of ATM in C. elegans [40].  Based on the WormBase version WS205, atm-1 produces a 5,431 base pair (bp) primary RNA, which is spliced into a 1,950bp mRNA.  The atm-1 mRNA is predicted to encode for a 649 amino acid (AA) protein.  On the C. elegans physical map, atm-1 is located on Chr I from 203,037bp to 208,467bp (Figure 1.4).  One mutant strain exists for atm-1, named VC381.  VC381 was created by the C. elegans Gene Knockout Consortium using the mutagen 4,5’,8-trimethylpsoralen (TMP) activated by ultraviolet light (UV) [41].  The result is a 548bp deletion in atm-1 that removes the 3’ splice site of the 1st intron named gk186 (Figure 1.4).  Because the 3’ intron splice site is lost in atm-1(gk186), exon 2 lacks the required splice recognition sequences that are required for correct splicing.  The lost splicing signal would most likely result in exon skipping of exon 2, which means splicing occurs from exon 1 to exon 3.  If this is the case, the resulting transcript would result in a frameshift mutation producing multiple PTC.  The protein would be truncated and missing the functional kinase domain.  This would likely lead to a non-functional ATM-1 protein [42]. There have been numerous studies involving atm-1, either with the gk186 allele or the use of RNA interference (RNAi) [42-46].  Garcia-Muse and Boulton found that atm-1(RNAi) did not elicit cell cycle arrest or recruit ATL-1 when replication fork stalling was induced VIA hydroxyurea (HU), indicating that atm-1 does not participate in replication fork stalling resolution [44].  The authors also found that after a dose of 75 Grays (Gy) of IR, atm-1 did not prevent ATL-1 or RPA-1 formation, indicating that atm-1 is not responsible for recruiting these proteins to DSBs.  After 75Gy IR, atm-1(RNAi) mutants were unable to induce cell cycle arrest or apoptosis, which contrasts wildtype mitotic and pachytene germ cells respectively, indicating that atm-1 is required for proper DNA damage, cell cycle checkpoint, and apoptosis response to DSBs.  Weidhaas et al (2006) showed that atm-1(gk186) mutants had an increased abnormal vulva phenotype when subjected to 400Gy IR treatment, suggesting that atm-1 is involved in DSB response in the authors’ model of preventing reproductive cell death [46].  Stergiou et al (2007) demonstrated that atm-1(gk186) mutants had defective apoptosis induction at lower doses of IR treatment (15Gy and 30Gy), which is consistent with Garcia-Muse and Boulton [42].  However, at a high dose (120Gy), atm- 8 1(gk186) resembled wildtype worms in apoptosis induction, suggesting that other repair pathways may compensate or aid in DSB repair at high IR dosages.  Stergiou et al also report that atm-1(gk186) mutants are sensitive to UV-C radiation.  A study by Lee et al (2010), consistent with Garica-Muse and Boulton and Stergiou et al, found that atm-1(gk186) is defective in arresting mitotic germ cells in mutant worms post 75Gy IR treatment [45].  Also consistent with Garcia-Muse and Boulton, mutant atm-1 worms were not sensitive to HU treatment. A conflicting result from Bailly et al showed that L4 atm-1(gk186) mutants were not sensitive to IR.  However, L1 worms were sensitive to IR treatment, suggesting differences in function for ATM-1 in meiotic verses mitotic germ cells, respectively [43]. 1.7 Thesis Objectives: characterizing the C. elegans ATM orthologue, atm-1 These studies involving atm-1 have shed light on its function in C. elegans.  However, these studies do not focus specifically on the mechanism of ATM-1 but instead on other proteins.  Thus, the purpose of my thesis is to provide further characterization of atm-1.  My study will provide a foundation for current and future studies to build upon.  In my thesis, I aim to provide characterization of atm-1(gk186) by studying it molecularly and by examining its phenotype(s).  First, I address the validity of the current wildtype atm-1 gene model.  Then, I provide molecular characterization of the mutant atm-1(gk186) allele and the consequences of the gk186 deletion on atm-1 transcription and translation.  Finally, I characterize atm-1(gk186) in the context of IR sensitivity in meiotic germ cells, genomic instability, and its effects on mutation rates.    9  Figure 1.1  Different mechanisms of alternative splicing.  Constitutive exons are shown in blue.  Alternatively spliced regions are in purple.  Solid lines indicates introns.  Splicing is indicated by dotted lines.  Adapted from [21]. *NOTE* Purple segment in Intron retention indicates the inclusion of an intron, now considered an exon.   10     Figure 1.2  One arm of the C. elegans hermaphrodite germline.  DTC, distal tip cell; Mitotic, mitotically proliferating region, Tz, transition zone; Sp, spermathecum containing sperm; V, vulva at the uterus containing embryos.  Figure adapted from [44].  11   Figure 1.3  eT1 balancer system.  A phenotypically wildtype eT1 worm with a heterozygous genotype is shown.  Blue represents Chr III sequences.  Red represents Chr V sequences.  During synapsis, sequences of Chr III right and Chr V left lack homologous sequences for homologous recombination to occur.  Normal chromosomes are marked with visible markers, dpy-18 for Chr III and unc-46 for Chr V.  unc-36 on Chr III is disrupted by the eT1 translocation resulting in an Unc phenotype for eT1 homozygotes.  Homozygous normal Chr III and V worms appear Dpy Unc.  Based on data from [31].   12  Table 1.1  All possible progeny genotypes combinations for a self-fertilizing eT1 heterozygous hermaphrodite. Blue represents Chr III sequences.  Red represents Chr V sequences.  Each genotype is labelled with its corresponding phenotype.  The surviving progeny has a 4:1:1 wildtype to DpyUnc to Unc (unc-36) ratio.  10/16 embryos arrest because of aneuploidy. Based on data from [31].   13      Figure 1.4  atm-1 gene model as depicted in WormBase (WS205).  The deletion in the gk186 allele is indicated by a red bar below the gene model. Modified from www.wormbase.org  14 2.     Materials and Methods 2.1 Nematode maintenance, culturing, and mating C. elegans strains were maintained and cultured at 20°C on Petri plates containing standard nematode growth media (NGM) streaked with Escherichia coli OP50 as outlined by Brenner [25].  Worms were observed on a WiLD Heerbrugg M3 light microscope.  Mating between strains was conducted at a 3:1 hermaphrodite to male ratio overnight on mating plates, and mated hermaphrodites were individually re-plated the following day. 2.2 Generation of complementary DNA (cDNA) library 10 saturated 35mm plates containing mixed stage worms for each strain were collected by washing with M9 buffer for total RNA isolation.  The worms were pelleted by centrifugation.  0.5mL of packed worms were resuspended in  2mL of Trizol (Invitrogen, Catalog Number 15596-018).  The worm/Trizol mix was vortexed, subjected to freeze-thaw, and incubated in a 37°C water bath.  Nucleic acids were isolated by adding 1mL chloroform followed by centrifugation and isolation of the supernatant.  1mL isopropanol was added to the supernatant, mixed, and re-centrifugated.  The nucleic acid pellet was washed with 0.5mL 75% ethanol and allowed to dry.  The pellet was resuspended with DEPC-treated water.  RNA quality was estimated by running through a 1% agarose gel.  The nucleic acids were treated with DNase using the Fermentus DNase I, RNase-free kit and protocol (#EN0521).  DNase was removed with phenol-chloroform followed by chloroform to isolate total RNA.  RNA was precipitated overnight at three volumes of 100% ethanol to sample ratio with the addition of a 0.3M final concentration sodium acetate.  Following centrifugation and removal of supernatant, the RNA pellet was washed with 0.5mL 75% ethanol and resuspended in DEPC-treated water.  cDNA was generated using the RevertAid H Minus M-MuLV Reverse Transcriptase kit from Fermentas using poly-dT primers (#EP0451).  The quality and concentration of the total RNA and cDNA were measured using a NanoDrop spectrophotometer.    15 2.3 Sequencing and sequence alignment PCR Products that needed to be sequenced were first gel purified using QIAGEN’s QIAquick Gel Extraction Kit and then eluted in double distilled water.  Sequencing was performed by NAPS (located in the NCE building, UBC).  Sequences were aligned manually to sequences obtained from WormBase (WS205) using BioEdit.  Full contigs of my sequencing reactions are located in Appendix A and B.   2.4 Single worm DNA extraction Single adult worms were individually placed in 5μL of lysis buffer (10mM Tris-HCl, 50mM KCl , 2.5mM MgCb, 0.45% NP40, 0.45% Tween20, 0.01% gelatin, 100μg/ml Proteinase K).  Tubes were freeze thawed twice in liquid nitrogen and incubated at 56°C for 1 hour, then 95°C for 15 minutes to inactivate the proteinase K.  Temperature cycling was done in a 96 well Eppendorf Mastercycler Gradient thermocycler. 2.5 Primer design and polymerase chain reaction (PCR) Primers were designed using the Primer3 website interface http://frodo.wi.mit.edu/primer3/ and are listed in Table 2.1.  PCR was conducted on a Dyad DNA engine machine using the appropriate temperatures for the type of polymerase and for the annealing temperatures of the primers.  Gel electrophoresis resolution of DNA products was done in a 1% agarose gel with ethidium bromide in 1x TAE buffer. cDNA PCRs were conducted using New England BioLabs Inc Phusion High-Fidelity DNA Polymerase to ensure high fidelity products for sequencing.  Suggested Phusion protocol for PCR cycling was used: 98°C denaturing, 72°C elongation at 45 seconds/kb, primer specific annealing temperature (between 56-60°C)  and cycled for 34 cycles.  Phusion HF 5x buffer was used. 10mM stock dNTP solution was mixed from 100mM stock solutions of each dNTP bought from Applied Biological Materials (abm) Inc.  A 20μL reaction consisted of 4μL 5x HF buffer, 0.5μL 10mM dNTPs stock solution, 0.4μL of each primer, 0.2μL of 1/10 dilution of cDNA library, 0.1μL Phusion polymerase, and 14.4μL of double distilled water.  16 2.6 atm-1(gk186) outcross and gk186 deletion detection by PCR VC381, atm-1(gk186), was outcrossed to wild type C. elegans males (strain – VC2010) to remove as many second site mutations induced by the TMP/UV treatment that was used to generate gk186.  Two F1’s from the above cross were individually plated and 10 F2’s (5 from each F1) were subsequently individually plated.  F2’s were allowed to lay eggs before being single worm lysed.  After confirmation of an F2 homozygous for gk186, the outcross was repeated for a total of 10 outcrosses.  The final 10th outcrossed worm was used for all subsequent phenotypic characterizations. (Table 2.2) The gk186 deletion was detected using PCR with the poison primer technique [47].  The forward primer (AAATTCGATTTTTCGCGA), reverse primer (CAATTGACGCAATTTGCA), and poison primer (TCACTCCGACGTCCGTTT), were used to detect the deletion.  The poison primer is designed from sequences that are located in the gk186 deletion region.  Therefore in wildtype worms, the poison primer is able to anneal and produce the smaller PCR product.  gk186 removes the sequences required for the poison primer to anneal to thus only the forward and reverse primers are used to produce PCR product in the mutant strain. The wildtype genotype produces a 956bp PCR product whereas the mutant gk186 deletion strain produces a larger 1233bp product (Figure 2.1). This PCR was done using Taq Plus DNA Polymerase (abm).  The abm Taq suggested protocol was followed: 94°C denaturation, 72°C elongation at 1 minute/kb (2 minutes total), 58°C annealing, and cycled for 34 cycles.  2.5μL of 10x abm PCR buffer, 1μL 50mM MgCl2, 0.5μL dNTP stock solution (abm), 0.4μL of each primer (forward, reverse, and poison primer), 0.1μL Taq polymerase, and 14.7μL of double distilled water was added to the 5μL lysis containing F2 worms for the PCR reaction. 2.7 Ionizing radiation sensitivity assay For each strain, 30 adult Po’s were plated on 3 plates (10 worms per plate) and allowed to lay eggs for 2 hours giving approximately 50 eggs per plate.  These worms were synchronized to 1 day old adults and subjected to different IR dosages.  IR was applied by the TORREX 150D X-Ray Inspection System.  In the machine, the uncovered plates were  17 placed on tray 7.  The settings were 5mA at 145kV which administers 15Gy in 135 seconds.  For 0Gy, worms were still placed in the machine uncovered but not subjected to IR.  30 and 45Gy were subjected to 270s and 405s of IR, respectively.  After IR, worms were allowed to rest in 20°C for 20 hours.  10 plates with 3 worms each were plated for each dose and a 5 hour brood was collected.  The number of unhatched eggs and adults were scored 24 and 72 hours post brood collection.   % survival was calculated by dividing the total number of adults by the total progeny (adults + unhatched eggs).  Standard error was used for calculating statistical error.   2.8 DAPI staining 20-30 adult worms for each strain were picked onto a watch glass containing 5μL of M9 buffer.  250μL of 150mM DAPI in 96% ethanol was added and allowed to evaporate in the dark at room temperature for 1-2 hours.  500μL of M9 was used to destain the worms for 2 hours in room temperature.  Worms were mounted on a microscope slide atop a 3% agarose pad.  Diakinetic chromosomes were observed in oocytes located before the spermatheca using a Zeiss Axioscope fluorescent microscope with 40x objective using OpenLab software. 2.9 Quantifying genomic instability 20 atm-1(gk186) L4 worms were individually plated and the subsequent progeny scored for total progeny and the presence of males.  A single L4 worm from each line was plated to propagate the line.  This was repeated for 20 generations.  2.10 Creating atm-1(gk186);eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177) atm-1(gk186) males were crossed to homozygotes eT1 hermaphrodites (derived from BC2200).  F1 males (genotype atm-1/+;eT1(III)/+;eT1(V)/+)  were then crossed to dpy-18(e364);unc-46(e177) hermaphrodites (also derived from BC2200).  Progeny from this cross were individually plated and allowed to self and produce progeny.  The progeny was screened for the presence of Unc-36 worms indicating that the eT1 chromosomes have been retained (eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177)).  Every parental giving progeny with some Unc-36 had their DNA extracted and genotyped by PCR for the gk186 deletion.   18 Heterozygous atm-1(gk186) with eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177) were obtained and allowed to self again.  The progeny of these were genotyped again to identify and obtain a strain homozygous for atm-1(gk186) (genotype: atm-1(gk186);eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177))(Table 2.2).    19 Table 2.1  List of primers created and used. (#) indicates primers used for sequencing.  (*) indicates primers designed by S.Y. Chua (Baillie Laboratory, Simon Fraser University). Name Forward or Reverse Sequence Annealing T (°C) a * Forward ATCTGAAACACGCGATAGCTC 59.49 b #* Forward ACAATCAGGTGCCATGGATAG 59.83 c #* Reverse CAAGTGCCACGTCTCTTCTTC 60.04 d #* Forward GAAGAAGAGACGTGGCACTTG 59.99 e # Forward ATACGATGCGGCAATAAACC 59.82 f # Reverse ACCCGACGATACAGCTTTCC 61.40 g # Forward TCTGTATTGGCCAACTGTCG 59.72 h #* Reverse TACTCGCCGAAACTTCTTCTG 59.64 I * Reverse TCGGAAATTTGTCATCGTTTC 59.93 j #* Reverse CGCGTTCAATTTATGTTTTCC 59.49 seq1 # Reverse TCCAAATCCATTTCCATAAGC 58.89 seq2 # Reverse CAACTCCGAACTCTCTCATCG 60.00 seq3 # Reverse CGAGCTGATTGTCAAGATTCG 60.92 seq4 # Forward CCAATCATATTTGCGAAAACC 59.31 seq5 # Forward AGGCGAGAAACATCTACATCG 59.35 seq6 # Forward ATGGGTGATGAAGATGAGACG 59.94 seq7 # Forward ACCGAATTGACAGATCACAGG 59.98 seq8 #  Forward GTCTGTATTGGCCAACTGTCG 60.57 seq9 # Forward GTCGAAGCTGTGGATAAGTGC 59.90 seq10 # Reverse CATCGTTTCCACGTGATTAGG 60.37 seq11 # Forward CCCGTTAGACACTGAATGTGG 60.42     20 Table 2.2  A list of strains used and created. Name Genotype Created by: KR4831 atm-1(gk185)*5 (outcrossed 5 times) Jim Huang KR4941 atm-1(gk186)*10 (outcrossed 10 times) Jim Huang KR5059 atm-1(gk186);eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177) (Isolate 1) Jim Huang KR5060 atm-1(gk186);eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177) (Isolate 2) Jim Huang KR4948 atm-1(gk186)(h2680) Jim Huang KR4947 atm-1(gk186)(h2681) Jim Huang KR4952 atm-1(gk186)(h2682) Jim Huang KR5104 atm-1(gk186)(h2683) Jim Huang KR5105 atm-1(gk186)(h2684) Jim Huang KR5106 atm-1(gk186)(h2685) Jim Huang VC381 atm-1(gk186) Genome Knockout Center VC2010 N2 Wildtype Genome Knockout Center VC655 brd-1(gk297) Genome Knockout Center BC2200 eT1(III)/dpy-18(e364);eT1(V)/unc-46(e177) Dr. Robert Johnsen      21       Figure 2.1  Agarose gel of wildtype (WT) and atm-1(gk186) PCR products using the poison primer technique.  22 3.     Results 3.1 Molecular characterization of the wildtype atm-1 gene 3.1.1 The predicted atm-1 gene is much smaller than other ATM homologs C. elegans atm-1 is predicted to produce a 649AA polypeptide (WormBase, WS205), whereas other orthologues have at least 2,700AA (Figure 3.1)[40]. Evident from this comparison, between ATM-1 and other known orthologues, is that ATM-1 is shorter and resembles the C-terminal ends of other ATMs (Figure 3.1).  The summary of nematode ATM-1 on NCBI’s CDD reveals that it contains the PI3Kc_like and FATC domains like other ATM homologues (Figure 3.2) [2, 13, 48].  However, ATM-1 is lacking domains known to be in other ATMs, including the FAT and TAN domains that are typically N-terminal of the kinase domain [48, 49].  Also evident from this comparison is that much of the N-terminal portion of ATMs appears to be species specific and lacks any known domains.  Because of evolutionary divergence between ATM homologs, the domains may be less conserved and thus not recognizable through bioinformatic domain analysis and sequence comparisons.  For example, the TAN domain is not present in the Drosophila melanogaster prediction but present in the Saccharomyces cerevisiae prediction, and the FAT domain is only predicted for the human prediction (Figure 3.1).  These structural differences between the C .elegans ATM-1 and other known ATMs bring into question the accuracy of the atm-1 prediction.  3.1.2 K10E9.1 and F56C11.4 are uncharacterized gene predictions upstream of atm-1  A mis-annotated N-terminal extension might be the reason why atm-1 does not resemble other known ATMs.  To address whether the N-terminal portion of atm-1 might be mis-annotated, I analyzed the genomic region upstream of atm-1 for evidence of an extended atm-1 gene model.  Directly upstream of atm-1 are two predicted genes that are uncharacterized: K10E9.1 and F56C11.4 (Figure 3.3).  K10E9.1 is directly upstream of atm-1 and is predicted to produce a large 12,836bp primary transcript.  The coding sequence spans 16 exons, which corresponds to 4,356 nucleotides (nt) and results in an 1,451AA protein.  Upstream of K10E9.1 is F56C11.4.  F56C11.4 is predicted to be an 847bp transcript  23 encoding 3 exons totaling 690nt which translates into a 229AA protein.  The close proximity and location of these two predictions suggested that K10E9.1 and F56C11.4 could be the mis-annotated N-terminal extension of atm-1.  In fact, three ESTs also support the idea of a larger atm-1 gene model (Figure 3.4).  Each EST has a 5’ and 3’ sequence read from that specific clone and is represented by .5 and .3 at the end of the read names.  EST clone yk1279h03 suggests that F56C11.4 and atm-1 are expressed as one mRNA.  Likewise, clone yk175d02 suggests K10E9.1 and atm-1 are expressed as a single mRNA.  A closer inspection through a BLAST search of yk220a2.5 reveals that it too suggests K10E9.1 and atm-1 are expressed as a single mRNA because a small portion of the .5 EST read aligns to the end of the K10E9.1 gene prediction (Figure 3.5).  Overall, these three ESTs that overlap with the atm-1 gene prediction suggest that atm-1, K10E9.1, and F56C11.4 may form a single transcript. 3.1.3 F56C11.4, K10E9.1, and atm-1 gene predictions produce one single mRNA transcript  To provide support for a single, large transcript comprised of the merging of the three gene predictions, I created a wildtype (N2) cDNA library.  To probe the coding sequences, I designed PCR primers that span my predicted junctions between adjacent gene models. Two sets of primers were designed: one pair between F56C11.4 and K10E9.1 (F-K Junction), and the other between K10E9.1 and atm-1 (K-A Junction)(Figure 3.6, Table 2.1).  If the three gene models do in fact produce a single transcript, then splicing of the exons between the gene models will produce one continuous template.  This single transcript will allow the PCR reaction to proceed and produce the predicted products.  Conversely if the three gene models are indeed separate genes, then the genes will not be spliced together.  In this case, the primers will not be able to amplify any PCR product.  Panel “A” in Figure 3.7 shows the result of the PCR reaction for the F-K Junction.  The arrow indicates a product close to the expected size of 674bp.  Panel “B” shows the result of the PCR reaction for the K-A Junction.  Again, the arrow indicates a product roughly of expected size of 682bp (Figure 3.7).  My results indicate that both primer sets produce PCR products.  These experiments support my prediction of a larger gene model, a merger of atm-1, K10E9.1 and F56C11.4 gene predictions, for the ATM homologue in C. elegans.  24 3.1.4 cDNA PCR products support a larger atm-1 gene model  To understand the molecular structure of the merged gene model of atm-1, my collaborator Shu Yi Chua (Simon Fraser University, British Columbia) and I created primers to amplify products from the cDNA library spanning the entire new gene model (Figure 3.8A, Table 2.1).  These primers successfully amplified products which overlapped each other and are roughly of predicted size indicating that the individual gene models represent the larger gene model well (Figure 3.8B, Table 3.1).  Lanes 1 and 2 from Figure 3.8B also support the previous F-K Junction result and lanes 3 and 4 support the previous K-A Junction result.  I observed no evidence for alternative transcription based on the single bands seen in each lane.  3.1.5 Sequencing reveals the nucleotide composition of the larger atm-1 gene model with novel unpredicted changes  The above atm-1 cDNA PCR products obtained were sequenced.  The sequencing resulted in between 1- to 6-fold coverage of the entire atm-1 gene model with an average of 2-fold coverage.  The exact exonic sequence of the entire atm-1 gene model and the intron-exon splice sites can be described by comparing the transcript sequence to the genomic sequence (Appendix B).  The majority of the sequence aligns exactly with the predicted models of the three genes: F56C11.4, K10E9.1, and atm-1.  Comparing both the contiguous cDNA sequence and the genomic sequence from WormBase (WS205), I was able to determine the differences between the predicted gene models and my merged atm-1 gene model (Table 3.2).  First, a novel 5’ splice site occurs at the F-K boundary.  This unpredicted splice site occurs at the predicted TAG stop of F56C11.4 and disrupts the stop codon.  Interestingly, this terminal exon of F56C11.4 does not splice to the predicted first exon of the K10E9.1 prediction.  This last exon of F56C11.4 splices to a novel exon I discovered, located 433nt upstream of the predicted first exon of K10E9.1. It contains 163nt and utilizes novel splice sites also previously unpredicted.  This exon also explains the size discrepancy seen in Figure 3.7A.  The F-K Junction PCR product’s size appears to be closer to 900nt which is close to the sum of the prediction (674bp) and the novel exon (163bp).  This novel exon splices to the predicted first exon of K10E9.1.  However, another interesting discovery I have  25 made is that this first exon has unpredicted exonic sequences added to both the 5’ and 3’ ends of the predicted exon.  Nine exonic nucleotides are attached to the 5’ end and 42nt are added to the 3’ end.  These extensions on the predicted first exon of K10E9.1 are also accompanied by novel splice sites.  Finally, splicing between the K10E9.1 and atm-1 predictions is described.  The novel 5’ splice site removes 42nt from the end of the last predicted K10E9.1 exon and the novel 3’ splice site removes 30nt from the beginning of the first predicted atm-1 exon.  Overall, I have refined my extended atm-1 gene model by discovering and annotating novel splice sites, a new exon, and additional coding sequences.  The entire 7,137nt coding segment for my extended atm-1 cDNA is in frame.  The cDNA encodes for 2,378AA which is much larger than the 649AA initially predicted.  My newly revised gene model of atm-1 depicting the changes is illustrated in Figure 3.9B. 3.1.6 The extended gene model of atm-1 does not reveal any additional protein domains  The revised 2,378AA ATM-1 sequence was reanalyzed through the CDD.  The results from this analysis only revealed the PI3K_c and FATC domains (Figure 3.10).  The TAN and FAT domains remain unpredicted.  The extended atm-1 gene model is most similar to the Drosophila melanogaster HomoloGene prediction (Figure 3.1).  Again, the lack of predicted domains in the extended atm-1 gene model could be a result of the chosen bioinformatic analyses, the current knowledge of these domains, and/or species divergence. 3.1.7 Section summary I have discovered that the ATM homolog in C. elegans has been mis-annotated and is in fact represented by three gene predictions: F56C11.4, K10E9.1, and atm-1.  Sequencing of PCR products spanning the three gene models reveals the exact nucleotide composition of the gene model and novel changes that were unpredicted.  The revised 2,378AA ATM-1 protein is now more similar to other known ATMs than the previously predicted 649AA protein. 26  Figure 3.1  HomoloGene comparison between known ATM homologues.  The C. elegans prediction resembles the C-termini of other ATMs in other organisms.    Modified from NBCI HomoloGene.  (http://www.ncbi.nlm.nih.gov/)   27     Figure 3.2  CCD result for the atm-1 649AA query.  PI3Kc_like and FATC domains are predicted.  The small size of ATM-1 is the most likely reason why the FAT and TAN domains are not predicted (see  Figure 3.1). (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml)   28    Figure 3.3  Genomic region 183,000bp to 209,000bp of chromosome I on wormbase.org (WS205).  F56C11.4 and K10E9.1 are directly upstream of atm-1 (Y48G1BL.2).  Modified from wormbase.org (WS205).  29    Figure 3.4  Genomic region 183,000bp to 209,000bp of chromosome I with the three ESTs that support linkage of the three gene models.  The dotted lines infer that there are sequences between the two end reads that should be present within the full cDNA.  Modified from wormbase.org (WS205).   30  Figure 3.5  yk220a.5 also suggests linkage between K10E9.1 and atm-1.  A. BLAST search of yk220a2.5.  Teal “Hits” represent homology between the EST read and the genome.  The red circle indicates a portion of the sequence read located within the K10E9.1 prediction.  This indicates where the clone read starts.  The start is continuous with the remainder of the read which aligns with atm-1 suggesting a transcript spanning across these two gene models.  B. The BLAST search sequence homology between the EST and the genome.  Modified from wormbase.org (WS205).  31    Figure 3.6  F-K and K-A Junction primers design.  Black arrows indicate the rough locations of the F-K primers (b + j).  Red arrows indicate the approximate locations of the K-A primers (e + f).  Successful PCR products are amplified only if the three gene models produce a continuous transcript.  If the upstream genes are not extensions of atm-1, the PCR reaction will fail to produce product.  32    Figure 3.7  cDNA PCR results using the F-K and K-A Junction primers (b+j for F-K Junction and e+f for K-A Junction; Table 2.1).  Black arrows indicate product of roughly expected size for each reaction. A.  The F-K Junction product (predicted size: 674bp).  B. The K-A Junction product (predicted size: 682bp).  33  Figure 3.8  Wildtype cDNA primer design and products.  A. Primers designed for amplifying overlapping PCR products from the wildtype cDNA library for the extended gene model.  Primer sequences and exact location indicated in Table 2.1.  Black arrows indicate forward primers and red arrows indicate reverse primers.  B.  Agarose gel with each lane representing products from a primer combination. Lane: 1. a+c, 2. b+c, 3. d+f, 4. e+f, 5. g+h 6. g+i.  Expected product sizes given in Table 3.1.    34 Table 3.1  cDNA PCR primer combinations and expected product sizes. Primer sequences listed in Table 2.1. Lane in Figure 3.8B Primers used Expected size based on predictions 1 a + c  2,495bp 2 b + c  2,176bp 3  d + f  2,859bp 4  e + f   682bp 5  g + h  1,703bp 6  g + i  1,212bp    35 Table 3.2  Summary of novel changes for the atm-1 gene model. Underlined sequences indicate splice sites. Location of Observed Change Type Of Observation WormBase Prediction Observed Sequence from cDNA Sequencing F56C11.4 and K10E9.1 junction Novel Splicing F56C11.4: AAAGAATAG(STOP)TGCTGGA    F56C11.4: AAAGAATA(SPLICE)   5’ intron splice sequence: TA//GTGCTGGA  433nt upstream of K10E9.1 Novel Exon and Novel Splicing Genomic Sequence (not predicted to be exonic): ATTACAGTTGGTGTCATTACCGAAAATTCTC GGTCGTCCACATTCTCTCATATATCGTACATC GTTGGGGTCTCGAAGCGAGAGATTTCATTCT TCGTGAGATTTTCGAGCTCACTGGTAGTTTGT CCAACTTGATATCAGTGGCTCATAAGGATGG AGAACAGTCCAAGGTTTGAGT  Novel 163nt exon   3’ intron splice sequence: ATTACAG//TT  5’ intron splice sequence: AG//GTTTGAGT  First exon of K10E9.1 Additional Exonic Sequences and Novel Splicing First Exon:  TTTTCAGAAATGCATAATG(START) CGTTTGATT…                                                                                                               …TTGAATTGACCG(SPLICE)GTGATAGCAGTA GTAAGAAGAAGGACGATGCCACGTTTGATAG TGCGTAA  5’ 9nt addition to exon: AAATGCATA(EXON)   3’ intron splice sequence: TTTTCAG//AA  3’ 42nt addition to exon: (EXON)GTGATAGCAGTAGT AAGAAGAAGGACGATGCCACGTTTGATA  5’ intron splice sequence: TA//GTGCGTAA  K10E9.1 and atm-1 junction Novel Splicing K10E9.1: AGGTACATGCGGTCGGGTCTTG CAGCGAAATAATGCATTTTTAA(STOP) 5’ intron splice site: AG//GTACATGC  (Removes 42nt including the stop codon)  atm-1: ATG(START)AAACATTACAAAAAAA TTATTCATCAGAG 3’ intron splice sequence: TCATCAG//AG  (Removes   30nt including the start codon)   36     Figure 3.9  Comparison between the predicted gene models and the extended gene model.  A. Wormbase.org predicted gene models of F56C11.4, K10E9.1, and atm-1.  B. The revised extended gene model for atm-1 merging F56C11.4, K10E9.1, and atm-1.  The green highlights indicate novel atm-1 coding sequences (Table 3.2). 7,137nt coding, 2,378AA  37    Figure 3.10  CDD prediction of the extended atm-1 protein sequence.  Only the PI3K_c and FATC domains are identified through bioinformatic prediction.  Estimated locations of where the TAN and FAT domains should be predicted are indicated.  Modified from Conserved Domains Database. http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml   38 3.2 Molecular characterization of the atm-1 mutant strain, VC381 3.2.1 atm-1(gk186) is a deletion that does not prevent transcription   The atm-1 mutation, gk186, is a deletion that removes the 3’ intron splice site for the 22nd exon of the revised atm-1 gene model (Figure 3.9).  This mutant strain, named VC381, is thought to have null ATM-1 function because exon 22 lacks the required sequence information for correct splicing.  This will most likely result in exon skipping of exon 22 resulting in the splicing of exon 21 to exon 23.  This transcript is predicted to produce a frameshift mutation resulting in a truncated protein lacking the kinase domain and null ATM-1 function [21].  Another argument for VC381 to have null ATM-1 function is through degradation of the atm-1 mRNA due to NMD resulting from the multiple PTC that would be present in the mRNA if exon 22 was skipped [23].  To gain insight to which mechanism is utilized (exon skipping or NMD), I generated a cDNA library from the mutant strain VC381.  From this cDNA library, I probed for atm-1 transcripts. I used the same primers that were used to characterize wildtype atm-1 (Table 3.1).  The atm-1 transcripts produced in VC381 are stable cDNAs, and can be used for PCR analysis (Figure 3.11).  Consistent with the predictions, the PCR products encompassing the deletion region are shorter than their PCR products from wildtype worms (Figure 3.11, Lanes 5 and 6).  These transcripts suggest that the atm-1 mRNAs are not immediately degraded via NMD. 3.2.2 atm-1(gk186) produces two alternative transcripts near the deletion region  An unexpected result from the atm-1(gk186) cDNA PCRs was the production of two PCR products from primers that encompassed the deletion (Figure 3.11, Lanes 5 and 6).  I verified these bands by running the PCR products at a lower concentration to reduce visual saturation (Figure 3.12).  In comparison to wildtype cDNA PCR products amplified with the same primers, it is obvious that the gk186 allele is a deletion that generates two alternative transcripts (Figure 3.12).      39 3.2.3 The atm-1(gk186) alternative transcripts do not skip exon 22 and utilize novel unpredicted splice sites  To further understand the molecular nature of these transcripts, the two gk186 transcripts were purified and sequenced.   To ensure sequence fidelity, 2-fold coverage was obtained for each transcript, which I have designated as atm-1.a and atm-1.b for the top and bottom bands (larger and smaller transcript), respectively (Appendix C).  Surprisingly, both transcripts did not skip the full exon affected by the deletion.  Sequencing of atm-1.a revealed the use of a novel 3’ intron splice site within the affected exon (exon 22)(Figure 3.13B).  The nucleotide sequence and changes are summarized in Table 3.3.  atm-1.b incorporates the previous novel 3’ intron splice seen in atm-1.a but also utilizes a novel 5’ splice site within the previous exon (exon 21) (Figure 3.13C, Table 3.3). 3.2.4 The gk186 transcripts are predicted to produce distinct products To study the consequences of these new transcripts, the full atm-1.a and atm-1.b transcripts were translated (Appendix D).  Multiple stop codons appear when atm-1.a is translated with the first stop occurring quickly after the new splice site.  This results in a truncation of the protein at residue 1843.  The effect of this truncation is the removal of the catalytic PI3K domain (Figure 3.14A).  Presumably, this protein would lead to a kinase-null mutant.  Interestingly, translation of atm-1.b reveals that the effect of the two novel spice sites results in an in-frame translated protein.  The full atm-1.b transcript is predicted to be a 2,266AA polypeptide, which leads to a protein that is 112AA residues shorter than the wildtype polypeptide and would maintain the catalytic PI3K domain at the C-terminal end (Figure 3.14B).  The implications of this transcript with the preserved kinase domain will be addressed in the Discussion. 3.2.5 Section summary The atm-1 gk186 allele is a deletion that results in unexpected consequences for the transcription and translation of atm-1.  The deletion affects splicing but not by exon-skipping as expected.  Stable atm-1 transcripts are detected in the transcriptome corresponding to two alternative transcripts.  These transcripts arise as a result of novel splice sites in the vicinity  40 of the deletion.  The larger atm-1.a transcript uses a novel splice site within exon 22.  This splice variant results in multiple PTC, the first stop truncating the protein immediately after the new splice site.  The smaller atm-1.b transcript uses two novel splice sites; one splice site within exon 21 and the other within exon 22.  This transcript translates in-frame with 112AA lost relative to its wildtype atm-1 counterpart.  The atm-1.b transcript is predicted to have an intact kinase domain.     41  Figure 3.11  atm-1 cDNA primer design and products.  A.  Primers designed for amplifying overlapping PCR products from the wildtype cDNA library for the extended gene model.  Primer sequences are listed in Table 2.1.  Black arrows indicate forward primers and red arrows indicate reverse primers.  B. Agarose gel with products amplified from VC381 cDNA library for overlapping products of the extended gene model shown in Figure 3.9B.  Each lane represents products from a primer combination. Lane: 1. a+c, 2. b+c, 3. d+f, 4. e+f, 5. g+h 6. g+i.  Expected product sizes are given in Table 3.1. (*) Expected sizes of products in lanes 5 and 6 are 1,279bp and 788bp respectively if exon-skipping of the 22nd exon occurs. (*) Lanes 5 and 6 on this gel are flipped horizontally to be visually consistent with other figures     42     Figure 3.12  Comparison between atm-1 and wildtype cDNA products using two primer sets; g + h (Lanes 5), and g+i (Lanes 6).  Double bands are produced for both primer sets when product is amplified from the atm-1 cDNA library.  Single bands are produced from the wildtype cDNA library when using the same primer sets.    43    Figure 3.13  Abbreviated gene models for atm-1. (A) wildtype, (B) atm-1.a and (C) atm-1.b transcripts.  The first stop encountered during translation for atm-1.a is indicated with a “1st STOP”.  (*) Indicates location of the novel splice sites used in splicing for the two transcripts.  44 Table 3.3  Summary of changes in the atm-1(gk186) transcripts.  Exon designation based on the extended gene model (Figure 3.9B)     Transcript 5’ Intron Splice Sequence  : Location of change 3’ Intron Splice Sequence  : Location of change Transcript Consequence atm-1.a AG/TATGCATC (Wildtype)  :end of 21st exon ATCACAG/AT  :within the 22nd exon Frameshift, multiple stop codons.  Truncated protein lacking PI3K domain  atm-1.b GG/GTGAGAAA  :within the 21st exon ATCACAG/AT  :within the 22nd exon In-frame splicing. 112AA removed but maintains the PI3K domain  45   Figure 3.14  Conserved Domains Database depiction of the two atm-1 translations. A. atm-1.a translation reveals multiple stop codons, the first located at residue 1843 (Indicated by a red line and *) effectively truncating the protein product without the kinase domain (Red X).  B. atm-1.b translation reveals a deletion of 112 AA starting at residue 1802 (Indicated by a red line and **).  This protein maintains its PI3K_c (labelled PIKKc_ATM) and FATC domains but is 2266AA long (Indicated by a red circle).  Estimated locations of where the TAN and FAT domains should be predicted are indicated.  atm-1.b’s FAT domain may be affected by the deletion.  Modified from Conserved Domains Database. http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml   46 3.3 Phenotypic characterization of VC381 3.3.1 Mutant atm-1(gk186) does not have any obvious phenotypes  Before any phenotypic characterizations were conducted, the mutant strain VC381 was outcrossed to our laboratory wildtype strain, VC2010, to remove any background mutations left by the TMP/UV treatment.  The outcrossing was done ten times and the homozygous gk186 deletion was reisolated and confirmed using a poison primer PCR technique (Materials and Methods [47]).  The 10th outcross (Strain: KR4941) was subsequently used for all phenotypic characterizations (Table 2.2).  Morphologically, atm-1(gk186) appears wildtype and does not immediately exhibit any obvious phenotypes.  However, brood analysis revealed that it has an average brood size of 206.6 (compared to 300 for wildtype) at 20°C and produces males at a 0.15% frequency (compared to 0.1% for wildtype [29]) (Table 3.4).  3.3.2 atm-1(gk186) is sensitive to double strand breaks caused by ionizing radiation  The ATM protein in other species is known to respond to DSB damage (Introduction).  The sensitivity to DSBs of the C. elegans gene atm-1(gk186) was assessed using a protocol Youds et al established in the Rose lab (Youds, PhD Thesis 2007, University of British Columbia)[50].  This assay measures the percent survival of progeny of a parental hermaphrodite post ionizing radiation (IR).  The rest period post IR allowed me to collect a specific brood and measure the impact of DSBs on the meiotic pachytene cells during IR treatment [43, 50].  Along with atm-1, I assayed VC2010 wildtype as a negative control and brd-1 as the positive control.  brd-1, which is one half of a heterodimer with brc-1, is required for homologous recombination (HR) repair of DSBs therefore worms deficient in brd-1 are unable to repair DSBs via HR and the eggs arrest as embryos.  The pooled results of three biologically replicate experiments are shown in Figure 3.15.  At the highest dose (45Gy) tested, wildtype worms had a survival rate around 90%.  The same dose on brd-1 is nearly lethal.  atm-1 mutants are also sensitive to IR, although not as much as brd-1.  Overall, the results indicate that atm-1 is sensitive to IR.   47 3.3.3 atm-1(gk186) spontaneously produces a dominant high incidence of male (Him) phenotype and eventually becomes sterile  Maintaining atm-1(gk186) worms through generations has yielded an unexpected result.  atm-1 normally produces a low frequency (0.3%) of males (Table 2.2).  However, over several generations, the frequency of males increases and the overall fecundity of the worms was negatively affected (data not shown).  The increased male frequency, referred to as the Him phenotype is dominant and persists until the strain becomes sterile (data not shown).  Multiple atm-1(gk186) Him strains were isolated and frozen (Table 2.2).   3.3.4 Him and sterile atm-1(gk186) strains have a reduced number of diakinetic chromosomes  Dominant Him strains have been characterized before [51].  Many of these Him strains yield translocations between the X chromosome and an autosome.  More recently, Ahmed and Hodgkin have identified another mutant (mrt-2) that results in X:autosome fusions [52].  To understand how the Him phenotype arises in my strains, I visualized the chromosomes using DAPI staining.  Normal wildtype and freshly outcrossed atm-1 worms have the normal complement of 6 pairs seen as 6 distinct spots when visualized using DAPI (Figure 3.16A).  In the dominant Him atm-1(gk186) worms, the DAPI staining revealed a reduced number of diakinetic chromosomes.  The different Him strains collected in the lab show examples of worms with 5, 4, and 3 pairs of diakinetic chromosomes (Figure 3.16B, C, and D respectively, and summarized in Table 3.5).  3.3.5 Quantifying independent atm-1(gk186) lines indicate inherited genomic instability  Chromosomal fusions, translocations, Hims (X nondisjunction), and sterility are all indicators of genomic instability [16, 51, 52].  To gain insight and to quantify the genomic instability defect in atm-1 mutants, I individually plated 20 L4 atm-1(gk186) worms.  The progeny of each worm was scored and a subsequent generation for each line was propagated through a single L4 offspring.  This was repeated for 20 generations and the results are summarized in Figure 3.17 and Table 3.6.  The results indicate that atm-1 worms do display genomic instability.  Through 20 generations, 16 of 20 lines have become sterile.  Various  48 outcomes are observed as the independent lines can become only sterile, Him, or first become Him and then sterile.  These results also point to a model in which heritable mutations that confer Him and sterility are generated in an atm-1(gk186) background.  The progeny numbers for each generation of different lines are also unpredictable with some gradually decreasing in progeny numbers where other lines have fluctuating progeny numbers (Table 3.6).  3.3.6 atm-1(gk186) produces lethal mutations that can be captured by the eT1 balancer system  In order to capture mutational events induced by atm-1(gk186) for further analysis, I created atm-1; dpy-18/eT1(III);unc-46/eT1(V)  which uses the eT1 balancer system  in an atm-1(gk186) background to try and capture mutational events that lead to lethality (Materials and Methods)(Table 2.2).  With the help of Dr. Ann Rose, 1139 F2’s were plated and their F3 progeny screened for the lack of DpyUncs.  A total of 11 independent lines lacking DpyUncs were obtained after rescreening potential positives (Table 3.7).  Thus, the mutational events created in the atm-1(gk186) background are amenable to capture by the eT1 balancer. 3.3.7 The forward mutation frequency of atm-1(gk186) is similar to the general mutator dog-1  Our mutation screen revealed a forward mutation frequency of 0.97% (from the observation of 11 captured mutations in 1139 lines; Table 3.7).  This is a very similar mutation rate to that of dog-1 (0.9%) which is responsible for resolving G track secondary structures during replication [34].  My 0.97% is similar to 500 Roentgens of gamma radiation (0.96%), less than 0.004M ethyl methanesulfonate (EMS) (1.5%), less than 0.11% formaldehyde treatment (1.58%), less than 120J/m2 UV exposure (3.1%), and greater than the spontaneous rate of 0.06% of the eT1 balancer [30, 31, 33].  3.3.8 Section summary The atm-1 deletion strain, VC381, appears morphologically wildtype.  However, the atm-1(gk186) allele demonstrates genomic instability and is sensitive to DSBs caused by IR.  The observed genomic instability events appear to be rearrangements that reduce the chromosome number but maintain a full complement of genomic information for normal development.   49 Moreover, the atm-1(gk186); eT1 balancer system was able to capture lethal mutations.  Using this system we observed a forward mutation rate of 0.96%, which is similar to dog-1 which acts as a general mutator due to its role in resolving G tract secondary structure in DNA and produces a variety of mutational events [34].   50 Table 3.4  VC381 characteristics at 20°C (*Data from Plates 6-10 done by Jessica McLellan). Plate # Total Brood Total Males  1  193  0  2  154  0  3  203  0  4  186  1  5  196  1  6*  227  0  7*  237  0  8*  214  0  9*  238  0  10*  218  1     Totals 2066 3 Percent Males Average  206.6 0.3 0.15% Standard Error 8.17 0.15 0.07%  NOTE: 1 sterile was omitted from the progeny count scored by Jessica McLellan (Rose Lab, University of British Columbia).    51  Figure 3.15  The atm-1 mutant is sensitive to IR.  The graph shows normalized percent survival for wildtype, brd-1, and atm-1, at three IR dosages: 0, 30, and 45 Gy.  Standard error bars are indicated.  These results were pooled from three biologically replicated experiments.  97.05% 89.46% 40.97% 2.54% 100.00% 90.13% 59.96% 0.00%10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%100.00%0 5 10 15 20 25 30 35 40 45Survival (%) Dose (Gy) Normalized Percent Survival of Various IR Dosages WildtypeBrd-1Atm-1 52   Figure 3.16  atm-1 mutants have reduced number of diakinetic bodies.  DAPI Staining of oocytes in the hermaphrodite gonad arm.  Examples of various chromosome bodies observed: A. 6 bodies seen in progenitor strain atm-1(gk186), B. 5 bodies seen in atm-1(gk186;h2681), and C. 4 bodies seen in atm-1(gk186;h2680), D. 3 bodies seen in atm-1(gk186;h2685)Table 3.5.  53 . Table 3.5  List of obtained atm-1(gk186) Him strains with their associated diakinetic chromosome numbers VIA DAPI. atm-1(gk186) strain allele # of Diakinetic Chromosomes Observed h2680 4 h2681 5 h2682 5 h2683 5 h2684 5 h2685 3  54   Figure 3.17  atm-1(gk186) mutants have genomic stability defects.  20 independent lines propagated over 20 generations.  The generation at which the line became Him and/or sterile are indicated with a squiggly hook (  ) and X, respectively.  55 Table 3.6  The progeny of atm-1(gk186) fluctuates or decreases as a function of generations.  Total progeny counts for the 20 independent atm-1(gk186) lines for 20 generations as seen in Figure 3.17.  ( - ) indicates sterility. Line F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 1 108 116 142 144 167 4 37 11 12 5 -          2 282 144 147 160 218 113 69 87 -            3 245 -                   4 110 49 41 24 78 91 48 135 160 49 97 -         5 63 1 -                  6 126 37 60 80 37 30 6 -             7 79 138 144 202 126 107 61 79 -            8 246 133 89 78 172 10 -              9 136 77 125 130 230 61 114 37 103 183 88 113 -        10 187 6 40 59 98 38 37 31 45 81 107 43 113 148 166 41 107 181 53 77 11 262 116 187 158 206 206 154 187 59 138 18 20 23 -       12 118 58 79 74 84 20 -              13 225 83 146 116 131 107 96 77 35 38 31 93 123 168 298 158 165 111 58 1 14 141 16 12 25 38 -               15 16 30 32 29 24 14 -              16 84 83 128 130 159 203 51 43 94 135 83 23 61 67 -      17 240 125 95 44 125 57 119 115 37 68 51 60 20 3 -      18 212 111 178 152 67 60 83 5 8 58 7 -         19 108 151 189 121 95 232 187 117 144 61 37 104 4 90 155 47 46 82 24 29 20 91 61 107 31 44 86 85 60 40 81 74 40 50 33 34 14 9 100 33 24     56 Table 3.7  Summary of atm-1; dpy-18/eT1(III);unc-46/eT1(V) screen. Number of F2’s Set Up Number of Plates Lacking Dpy Uncs Number of Steriles Numbers of eT1s Total of Lethals After Re-screening Forward Mutation Rate = Number of mutations / Total screen (non- steriles and non-eT1s) * 100 1180 44 28 13 11 11 / (1180-28-13) * 100 = 0.97%   57 4.     Discussion 4.1 The extended gene model requires further refinement Revealing the structure and function of ATM is an important step in understanding the human disease A-T and requires a model organism that is capable of providing both aspects.  Here, I have established that C. elegans can be used as a model for ATM by characterizing the nematode orthologue, atm-1.  I have characterized the gene structure and documented the phenotype of the deletion mutant.  Identifying the correct gene structure for any given gene is significant because it allows future molecular and genetic experiment to be designed and carried out correctly.  I show that the predicted atm-1 gene model lacked N-terminal sequences that are poorly conserved.  The reinterpretation merges the atm-1 gene prediction with the K10E9.1 and F56C11.4 predictions, producing a predicted protein that is similar to the Drosophila ATM (Figure 3.1).  The size of the reinterpreted gene model is now more comparable to other known ATMs.  In addition to the size difference, previous unpredicted exonic sequences were found and may be important for protein function. I sequenced the cDNA for the reinterpreted atm-1 gene.  Unlike other RNA-seq techniques (EST, cDNA-seq, RNA-seq, serial analysis of gene expression (SAGE)) I designed gene specific primers to achieve this.  Because of the primer design, my products do not amplify the end of genes.  These sequences require 5’ and 3’ RACE (rapid amplification of cDNA ends) to identify the atm-1 start and stop sequences.  Thus, in principle, my extended gene structure is incomplete at the 5’ and 3’ ends and requires end sequences.  However, there are new data sets that could be data-mined through WormBase to potentially identify the end sequences of atm-1 if there is enough coverage [28, 37].  These un-biased approaches could potentially identify any alternative starts or poly-adenylation signals. Although the gene structure for atm-1 appears complete, the still unrecognized TAN and FAT domains in the atm-1 CDD prediction are a bit of a concern.  Because roughly two thirds of the protein is species specific, prediction programs like the one used in CDD may not recognize these domains in the atm-1 sequence.  These domains may have to be extrapolated first using other Caenorhabditis species to see which regions are most  58 conserved and then aligned to higher eukaryotes.  These domains would be important to identify as they could be regulatory domains that could define ATM-1’s spatial and temporal control and would be of interest for understanding its role in the DNA damage response. 4.2 The alternative transcripts of atm-1(gk186) may have residual ATM-1 function Stergiou et al have reported a transcript that has a “cryptic” splice site within exon 2 (of the original prediction)[42].  However, the authors fail to provide the sequence for this transcript but based on its translation, assume a functionally null protein from the multiple PTC seen.  Here, I first show that NMD does not immediately degrade the atm-1(gk186) transcripts.  Secondly, I provide the sequence for this “cryptic” splice site in exon 2, which is exon 22 in the extended gene model.  Comparing my transcript sequence and Stergiou et al’s transcript sequence would be the only way to verify that they are the same.  The transcript containing this “cryptic” splice site is named atm-1.a in my report, and it does indeed produce multiple PTC when translated.  However, I also provide sequence for a second transcript, atm-1.b, which is also a result of the gk186 deletion and has not been reported in the literature.  atm-1.b utilizes two alternative splice sites that translate in-frame.  112AA is deleted from the resulting transcript.  This protein retains its PI3K domain and thus cannot be assumed to be functionally null.  atm-1.b places doubt on whether atm-1(gk186) is a true knockout or a mere knockdown.  The possibility of atm-1.b producing a neomorph would have to be experimentally tested as well.  This new information would require further experimentation to identify the protein consequence of gk186 and may require re-evaluation of the current knowledge regarding atm-1.  Regardless, the phenotypes observed for atm-1(gk186) are genuine but may not confer true atm-1 knockout function. 4.3 Function conservation of atm-1(gk186) phenotypes  atm-1(gk186) is sensitive to IR but not as much as brd-1.  This may be due to functional redundancy of the related gene atl-1, the human ATR orthologue [17, 44].  Another explanation could be the presence of functional ATM-1 produced from the atm-1.b transcript described previously.  The IR sensitivity experiment conducted is most similar to the L4 IR sensitivity done by Bailly et al [43].  The results Bailley et al obtained conflict with mine as they do not observe IR sensitivity for atm-1(gk186).   Experimental factors such  59 as the brood size collected and the rest period may explain the discrepancy (i.e., 5h vs 12h brood, 20h and 24h rest period post IR).  It seems unlikely that there would be no effect for IR on meiotic cells as Stergiou et al and Garcia-Muse and Boulton both show a defect in apoptosis induction, which would result in oocytes with unrepaired DSBs [42, 44].  These unrepaired breaks would presumably lead to some form of damage, causing arrested embryos.  The indicators of genomic instability in atm-1 mutant worms are consistent with what is reported for human A-T cell lines.  In human cell lines, reduced telomere length, increased rate of telomere shortening, and telomere fusions are observed [7-9].  In both fission and budding yeast, a role for preventing telomere fusions and chromosome translocations has been documented [53, 54].  The Drosophila orthologue, tefu, also prevents telomere fusions [55].  Here, I have observed that some atm-1 mutants eventually become Him.  Him strains have been documented before and have been shown to be X:autosome rearrangements [51].  X:autosome telomere fusions, which also have a Him phenotype, have also been documented in mrt-2 mutants [52].  These worms show a reduced number of diakinetic chromosomes when DAPI stained, which is consistent with my mutant atm-1 strains.  Thus, it is likely that the atm-1 mutants I have collected could be X:autosome telomere fusions.  This would have to be confirmed through experiments such as oligo array comparative genomic hybridization (oaCGH), but would be consistent with ATM function and C. elegans Him strains.  A telomere function for atm-1 would be novel for C. elegans. ATM-1’s function in preventing telomere fusions does not appear to be specific for the X chromosome.  Some of the independent lines in the genomic instability assay become sterile without becoming Him (Figure 3.17).  This suggests that autosome:autosome events might be occurring leading to sterility.  These strains along could also be further characterized using oaCGH to understand the nature of the sterility and may provide insight to the function of atm-1 in promoting genomic stability, possibly through telomere maintenance.   The use of eT1 to try and capture lethal mutations caused in an atm-1 background was successful.  The forward mutation rate for atm-1(gk186) was calculated to be 0.96%, which  60 is comparable to another DDR gene, dog-1, which acts to resolve G tract secondary structure during replication [50].  This low rate is similar to low dosages of other mutagens (EMS, gamma radiation, and formaldehyde)[30, 31, 33].  These mutations would have had to occur on Chr. III right and Chr. V left in order to be captured by the eT1 balancer system, and thus these rates realistically represent only 1/6th of the genome.  It would also be interesting to understand the nature of these captured mutations to see if mutations such as insertions, deletions, and inversions also occur in an atm-1 background. If we consider each of the 16 independent lines that became Him/sterile a mutational event, then 16 events occurred through 230 generations, which is the sum of all the generations lived by all 20 lines (Figure 3.17).  This translates into a 7% forward mutation rate, which is comparable to the atm-1(gk186); eT1 balancer rate of 5.8% (6x 0.96%).  This suggests that atm-1 functions ubiquitously throughout the genome to promote genomic stability.  Thus, although not an essential gene, atm-1 clearly has a significant role in maintaining genomic stability by responding to DSBs and possible telomere maintenance function in C. elegans.    61 5.     Conclusions Here I have shown that the C. elegans ATM orthologue, atm-1, has been mis-annotated.  The reinterpreted gene model for atm-1 is a merger of the F56C11.4, K10E9.1, and atm-1 gene predictions.  The gk186 deletion for atm-1 produces two alternative transcripts, atm-1.a and atm-1.b.  The former translates into multiple PTC, while the ladder translates into a product that has 112AA removed but retains the PI3K domain.  This product may have kinase function.  atm-1 is sensitive to IR and displays indicators of genomic instability.  Lethal mutational events were captured through the eT1 balancer system.  Future work on atm-1 in C. elegans may provide insight into the DDR and intracellular signalling and control.  Perhaps the most significant contribution to understanding atm-1, and ATM in general, would be discovering novel therapeutic targets for ATM driven cancers.  ATM interactions for synthetic lethality could lead to targeted therapy for appropriate cancers.  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Genes Dev, 2004. 18(15): p. 1850-61.     65 Appendices Appendix A  : WS205 exonic sequences A.1 F56C11.4 ATGAGCGTTACTCAGCTAAAAAATCTGAAACACGCGATAGCTCAGCTTCTGGAGTGGGATGGAACGAAAACGGCGAGAAAGAAAATCGTCGACGAGGTGGTTCTTCTCTATCACGCTCTGGGAGCGGAGGCATTAAGTGAGGATAACCAGGAGATATATGATTTGTATGATTTATCTGCACGTATATTCAACTTAGCTAAGAAAGAAATCGAAGAAGCCAACCAACAATTCGAAAAAGAACGAAAAAAGGGCACAAGACGAAGCGAAAAACCAGTCCCAACTCCACTTTTCGAGCTATCAATACAACATTTAAAACGTTGCTGTCAGCAGGGAATCGATCACAATCAGGTGCCATGGATAGCATATTGTCTGAAACTACTGGAATTTCCAATCACAATCACCGAAAAATCGATCGAAAACGAGATTTCCAACGTGCTCCTATTGAGCTCCAACGCCTCACAGCTCCATTGGGCCGAGCATGCTCATTTGAGCAGCTTATGGAAATGGATTTGGAGCCGTGTCGAGACCGCCGATATTGGGGCACTCGCCATGAGAAATTATATGGAATTGGCGGCGAACTTGCTGGAAAACGTGGATTACGTGGTTTTCGAGAAGTCGCCTATTGACCTGATGGCGAAAGTGATGGGAACACTGAAGAAAAGTGTGGAAATGGGAAATCCCAAAGAATAG A.2 K10E9.1 ATGCGTTTGATTGATGATCTCGTGAAGCTCGCCATGATCGAAACCGTACACGGCCACCGTACCATGAACGAAGTGACACGTGGAAATATTCAAAAACTCGTGAAAACCGGAATCCAAGAGTCTCTGAAATCGGCGCACCGAAATTTCTCAAGGAGTTCGACATTTTCGATTTCCGAAGAATGCGTGAGATATCTGACGAGATGGTTGTTGGCCGAACGAAGACTTGAACAGCCGTCTGCGGCTATGAATGAATCGTTTGAATTGACCGGCCTTATCGATCTCTCCTTTGGCTCGACGATTTCCGGAAAACATAAATTGAACGCGTGGAATGGTGTAATGCAAATCCTGAATGAGCTCCTAAAAAGTCGACGACTCGAACTTCAAGTCACTGAAAAAATCGTGACAATCCTCTGGGAAAAGCGAAAATCCTACACAACGGAGCCACTCCGTACTGTGTTCTGCTCCATTCTCTCCACAGTCGTCTGCCAGGCCGATGTTCGATTCGGTCATCGGAAAGTGCCGACAATCGACTCGATTCTCAAATATTCGCTGTCTCTAATGCCAAATGTCGCATCTCTTCCCAGTGCCGCTGCGTTGACCGAAACGATTGTCAGATTCAGGACAGTATCACGAGAGGGTCTCCGTAACACGTGGGATACCGTATCCCGAACTAGCTCCGGCTCATTTGAAGTTGTTCGGCTGATTTCGGCGTTGATCTCCGTTACGGAATTTGATGAGAATTCGAGATTCGCCAACGATGAGAGAGTTCGGAGTTGGAGTTTTCGAAAAGACATAATCGAATGGGTATTGCTGGATCCGAATGCACATAGTCACAAATTACTCTATCAGTTGTGCCAGTATCATCCAACGTATTGTTATGAATCAGAAGCTTCTTCTAGTGACGACTCCCTGCTTCAAACTCTGAAACTATGTAAATTAGCTTGCTCTCCAGCTCCCCCATCGGCTCCAAAAGCCCTCCGACCACTCGAAGCTTCAATTGAAGAGATTGTGAGATATGTGCATGATAAGCTCAAGAGCATTCTCGCGACTGAAATCACTCTGCCTGCATTTGTGCTCTGCCACGAATTTGCTCTGAAGTATCCGGATAGATCTTATGAGTTTAATAAAATGTACAAAAAGCTCTACCAAATCATGGAAGATCAAGAAGAAGACGAGTTTCTCCAATCAGCTCGCCATTTCTCAAAATGGCCTCAAAATCTGACACTACCAATACAAAAACAGACAATAAATTGCATGGCTGTCTTTTTCGAAGCGAATCTTGACAATCAGCTCGTCGATCTCTGTCAGTGGAGTGACCGACGAAAAGTGCTTGTCGAGATGCTCGCCGAGCTGGCCGCCACAAGATCTGAAATTCGAGATAAACTTCAAAAATCGATGCCGTTCAACAAATTCGTCAAGGAGTGTATAATGGAGAATCGCGGTGATTTGTATGAAATGACAAAGAGATTTGAGAAATATT 66 CGTTTTTGCTCTCGATTCGGAATTTAATTGTTACTAGGATGATTATAACAAACGAAGCCGCCCGACTCCTAGGAGATGGTGAAACAATCAGTGAAACCGATATCTTCATAATCGAAAAGCGTACTCTTTCCACGTGTATTCGTAATGTGTCCGAAGGAAAAGAGTTGAGCGGCTACACACTGGACCCCTATACGGTAGCTGCCAACGTGCACAATGTGCATTTCGATCACATAAACGTCGAAATCTATCTGGAATTGCTGAAAAAGTCGCCATTTTTCGCTCAGAACATTGTACGCCACTTGTTACGGCAGAATGGAAAAGAAGCAGAAGAAGAGACGTGGCACTTGCATGCCACTGTGCTGAAAATTGTGATGAAAGATGAAAAGTTGCTGGCGGTATGCGTGGCCACAATTCCAAATATGGTTCGATATCTCAAAGTCTATCAGATTCATTTCAGTCCGAAATCCAACGCGGCGAAGTTCTTACACCTCGACATGGAATCGATTTCCCACTGCCAATCATATTTGCGAAAACCAACAAAATCATCCAATCTGATCACGGCCGCCAACTTTTTGACACTTTTCGGATGTGAAAAGCGCACGTGGAAGCGCCCGATTCTCAGATTTTGGAGCATTTTCAAGCAGCAACCGGCTATGTGTTGCGAGAAATTGCTCATTTTTGCTGAAGAATGTGTCGAACTTGGCCTGAACCACCGAATCGCTTGTCTTTTACGCGCACTGACAACCAGTGAATTCTGCCGAAAAGCTCTATGTGATGAATATCTGAAAATCGCGTTTCAGCTGACTTATCGATCGATTTTTCTGATTTTAAGCAAAAATGAGTGCAGACCAGAAATTGTGGAGCTCTGCGATGACATGAATCTTCGGTACGATCTTCTCCAGCACCAGATCAAACATGTCGCGGCTCACCATTTGGAGCACTTTGAGCGTTTCGAAACGAAAATCGCATTTTCTGTTGAGAAATTTCTGAAATCTGGAATTGATGGAATCGATTTTGAAGATTTAGGATTAGTCGAGTTTTATAAGCAGTTGAACGAAAATTTGACAGAAGACGCGATCAGGAGCAATGAGGCGAGAAACATCTACATCGTCGACATTCTCTCAACAATTTGGCTCCAGCTCCCCTCAATTCGTCCTCAAATTCTACCGATTCTCGCCCGCTTCAAGCACATTTCCCCAGCGTGGACTAATTTCCCACAGCCGCCTCATATTTCGACAAACGAGAAATCATTTCTTCAACATCTACGATTTCATCTTTATCTAAAAATGATGAATATCTCGAAATCCATGACGCAAGGCGAGTATGCCACGTGTATTATGATGCTCCTGACGAGCTACGACTCGAGCCATTTCGTTGCGGATTTGATAGAGAAAAAGCAGCTGGGAAAGCTGAAATTGCAGCAGAGAAGGAATGTTTTGTGCATTTTGAGCCGACTTTTGAAAGATCAAGCTGTGATGGGTGATGAAGATGAGACGATTATCGATCCGATTCTTTTTAAGGCAATCACCAAAGCTTCCGCCGTTTTTGAAGACACCGCCGCGTGCATCGTACCATTTTTGTTCAAAATTTGTGTGGATTTCAAGGGAAAATACGATAAATGTGTGATAAATTTGCTGGGATGTCTTAAGGGAGTTAATGCAGAAGACGAAATAGTAGTCCGCTGCCTTGCCGAATGCGTCGACTCCATTGGACTCAACGTGATTGCTCGTTACGAACGCCTGAACATTGAGACTCATTCGGAATTCGGTGTGAAATGGTTCTTCAAATTATCCCGTCTCTTTCTGAAACATGGATTTACTACGCATTCCTTCGCGATTGCGAATATATTATTTGACCGACTTTCGGCGAGAAAACGAAATACAATGATGATAGATCGTACGAGTTTAGACCGAATTGACAGATCACAGGAACTTATCAATCTTTTGGTGGAGATCTATGTGGCCGAGGGTAACTCGGTAGCGTTGTCCTCTCTGCCACCTGCGGTACAAAATAGACCGGATGTTCGGCAGGTTATGAATAAGAGTTCAAAGGAATGGTTGAAATTGCTGTCTTCGAATCAAATGGACTCGTGGGAGTTGACTATTGTGCAGTGGATGTGTGGCATCCAATTCAATGCAATCACCGGCGATAAATACCTGAATTCAATTCTTCGTTGCAATTTCAACGAGTACACCAAAAAAATCGATTCCCCATTAAAATTCGTCTATTTTCAACTATTCCATCTTTCGACGAGCACTCTGGAAATCGAAGAAGCCATTTCTAGCATGCCGTTGGCTCCAACAATCGATCAAATGCGGCTTATGATTATAGCCAACGCGACGGCAAGCTTCGAGCCACAATCCGTTGAAGAGCACGTTGTTCGAGCTGTTCGAGAGCTCCGAGAGACGTCAAATCGGCGAAAATCCGGTGGAAATGTGAAGGGAATTAATGAAAAAACAACGAGAATGGTGAAACTGGCCGAGATGCTCACCGAGAATAAAGCATACGATGCGGCAATAAACCTGCTCGACACGTGGGAGCACGAGTGTCTCCAATGGACATCTGTCGCTGCCGAATCAATCGATATCGATTTAATTCGAATCTGCAAACAACACGTGACGTGTCGGTCGGGAGATCCAAGAATGGCGGACATAAATCTACGAACAATGCATCCACGTGTCCCGGTGATGAGTGACCTGGCGATTGCCGAATGGTCACTCGCGTTGAGCAAGATTACA 67 ATTGAATATCGGAATGATATGGAAGAGGGTATTCGGATTTTGGAATTTGGATGCAAACATTTACAGAATAAGGATTCTGTAGAGACGAGGTTAAAGGTACATGCGGTCGGGTCTTGCAGCGAAATAATGCATTTTTAA A.3 atm-1 ATGAAACATTACAAAAAAATTATTCATCAGGTTCTCCTAAAACTCCATTCCGTCTGTATTGGCCAACTGTCGAAACTCGAAGAATATCGCGAAACGCGTACCTACCGTATGAAGCAGCAGGCGGTCACCGCATTCGAACAACAAATTCAAAATTCGTGTCGAACCAGTCTGGCACGTGGCAATTCGGGTGACGAATGGACGAAAAAAACGGTGCAACGGGTGAGAAAAGAGCATCAGTTTGAGAAGAATGATTTGGAAAAAGTGGATAATTCGTTGAATTCGGCGGCCCGGAAAGCTGTATCGTCGGGTTTTGATGCACTTTTATGCATCAGCCAACTGGAAGACGACGACGAAGCGATCCGCGCTTCATCTCTCATAATATTTCCATTAATCGATGTGATCTACAAATACGAAACGGACGTCGGAGTGATCGCCTTGCTCAAAGAGCACACCAAATCGAAGCTCCCGTCGAAGCTGTGGATAAGTGCCACCTCACACATTGCCTCTAAGTGCTTCTCCATCGAAAAATCGCAAATCACGAGACACTTATCACAGATTCTGTGTCATCTCATCTATGACTACCCGTATCACGTTTTGCACACAATTTTGATGTATGATGACGAGAAGAACGCTTCCAAGGTGAAAGGCTTCTTGAAAACGATATTTGACGCGCGAGCTGACCAACGGGATTCGTCGAAGCTTAAGGAGATTGTGATTACTATTCGTGAGGCTCACCAGGCTTATAGAGAAATTGCAATGCTTGACGTGAGAGGAAACGTTCGGATACAACGTGTTGAAATAAACGGAAAAACGATGTACCGATGGCCACATGATTTGAAGATTTTCAAGTGCAAATTGCGTCAATTGCCGATTCCCACCATTTCGCAAAAGATTGGTTGCCCGGGTGACTACTCGACGACTGACCTAATCACGTGGAAACGATGGAAAGATGTATTCACAATTGCCGACGGAATTTCCACTCCAAAAATCTGGGAAATTGAGGGTAGCGATGGAAAATGGTATAAAACTGTGTGGAAGAAGGACGACGTACGACAGGATGTGCTTGTTGAGCAAATGTTTGATGTTACGAATAATATGTTGGAGAAGGCGATGCTTCGGACGTATAATGTTGTCCCGTTAGACACTGAATGTGGAGTTATAGAGTTTTGTGGTGGAACTGTTAGCTTAAAAGAAGTTATGTGTGGTGTGACACGAGAAGGCGGTCTCCACCGGGAGTTCAATTCAGAAGAAGTTTCGGCGAGTAAAGTGTCGTCAATGATGAGACAAGTACAGACAGAGTCCACAGAGACACGACGACAAGTTTTTGTGGAGATTTGTCAGCAGTATTCTCCAGTTTTTAGACACTTCTTCTACACCAACTTTTCGACGGCGCAGATTTGGCGGCAAAAAATCATAAATTACAGGCAGAGCTTGGCCACGTGGAGTATTGTGTGCTACATCGTTGGCCTCGGCGATCGACACGCGTCGAATATTCTATTCGATCAGAAATTGTGCACATTTGTGCATATCGATTTGGGAATGATTTTGGAGTATAGTAAACGAACGTTGCCAGTTCCCGAACAAGTTCCATTCCGTATAACTCGAGATGTGCTCGACCCGATTCTGATTGAAGGAATCGAGAATGGACAGTTGGCTGAGGAATGTACGCAGATTATGGAGAAATTGAAGGAAAATGGAAAGGTAATCCTCGGTGTCGCCTCTGCTCTACTCCGCGAAACGATGACAAATTTCCGAGAAGCCGAACAGGCTGCCGGCCGCCCGTCCTACATTTCTGAAATGGCCATCGGACGGCTTCGAGAAAAGCTACGGGGCACCGATGACGGTGTGACGGCCCAATCGTCGAATCTTCAAATTCGGCGACTTTTACGAGAAGCTACGAGTGCTGACAATTTATCGCGAATGTTCTGCGGATGGATGCCGTTTTTGTAG    68 Appendix B  : Full contig of the extended gene model B.1 atm-1 full contig ATGAGCGTTACTCAGCTAAAAAATCTGAAACACGCGATAGCTCAGCTTCTGGAGTGGGATGGAACGAAAACGGCGAGAAAGAAAATCGTCGACGAGGTGGTTCTTCTCTATCACGCTCTGGGAGCGGAGGCATTAAGTGAGGATAACCAGGAGATATATGATTTGTATGATTTATCTGCACGTATATTCAACTTAGCTAAGAAAGAAATCGAAGAAGCCAACCAACAATTCGAAAAAGAACGAAAAAAGGGCACAAGACGAAGCGAAAAACCAGTCCCAACTCCACTTTTCGAGCTATCAATACAACATTTAAAACGTTGCTGTCAGCAGGGAATCGATCACAATCAGGTGCCATGGATAGCATATTGTCTGAAACTACTGGAATTTCCAATCACAATCACCGAAAAATCGATCGAAAACGAGATTTCCAACGTGCTCCTATTGAGCTCCAACGCCTCACAGCTCCATTGGGCCGAGCATGCTCATTTGAGCAGCTTATGGAAATGGATTTGGAGCCGTGTCGAGACCGCCGATATTGGGGCACTCGCCATGAGAAATTATATGGAATTGGCGGCGAACTTGCTGGAAAACGTGGATTACGTGGTTTTCGAGAAGTCGCCTATTGACCTGATGGCGAAAGTGATGGGAACACTGAAGAAAAGTGTGGAAATGGGAAATCCCAAAGAATATTGGTGTCATTACCGAAAATTCTCGGTCGTCCACATTCTCTCATATATCGTACATCGTTGGGGTCTCGAAGCGAGAGATTTCATTCTTCGTGAGATTTTCGAGCTCACTGGTAGTTTGTCCAACTTGATATCAGTGGCTCATAAGGATGGAGAACAGTCCAAGAAATGCATAATGCGTTTGATTGATGATCTCGTGAAGCTCGCCATGATCGAAACCGTACACGGCCACCGTACCATGAACGAAGTGACACGTGGAAATATTCAAAAACTCGTGAAAACCGGAATCCAAGAGTCTCTGAAATCGGCGCACCGAAATTTCTCAAGGAGTTCGACATTTTCGATTTCCGAAGAATGCGTGAGATATCTGACGAGATGGTTGTTGGCCGAACGAAGACTTGAACAGCCGTCTGCGGCTATGAATGAATCGTTTGAATTGACCGGTGATAGCAGTAGTAAGAAGAAGGACGATGCCACGTTTGATAGCCTTATCGATCTCTCCTTTGGCTCGACGATTTCCGGAAAACATAAATTGAACGCGTGGAATGGTGTAATGCAAATCCTGAATGAGCTCCTAAAAAGTCGACGACTCGAACTTCAAGTCACTGAAAAAATCGTGACAATCCTCTGGGAAAAGCGAAAATCCTACACAACGGAGCCACTCCGTACTGTGTTCTGCTCCATTCTCTCCACAGTCGTCTGCCAGGCCGATGTTCGATTCGGTCATCGGAAAGTGCCGACAATCGACTCGATTCTCAAATATTCGCTGTCTCTAATGCCAAATGTCGCATCTCTTCCCAGTGCCGCTGCGTTGACCGAAACGATTGTCAGATTCAGGACAGTATCACGAGAGGGTCTCCGTAACACGTGGGATACCGTATCCCGAACTAGCTCCGGCTCATTTGAAGTTGTTCGGCTGATTTCGGCGTTGATCTCCGTTACGGAATTTGATGAGAATTCGAGATTCGCCAACGATGAGAGAGTTCGGAGTTGGAGTTTTCGAAAAGACATAATCGAATGGGTATTGCTGGATCCGAATGCACATAGTCACAAATTACTCTATCAGTTGTGCCAGTATCATCCAACGTATTGTTATGAATCAGAAGCTTCTTCTAGTGACGACTCCCTGCTTCAAACTCTGAAACTATGTAAATTAGCTTGCTCTCCAGCTCCCCCATCGGCTCCAAAAGCCCTCCGACCACTCGAAGCTTCAATTGAAGAGATTGTGAGATATGTGCATGATAAGCTCAAGAGCATTCTCGCGACTGAAATCACTCTGCCTGCATTTGTGCTCTGCCACGAATTTGCTCTGAAGTATCCGGATAGATCTTATGAGTTTAATAAAATGTACAAAAAGCTCTACCAAATCATGGAAGATCAAGAAGAAGACGAGTTTCTCCAATCAGCTCGCCATTTCTCAAAATGGCCTCAAAATCTGACACTACCAATACAAAAACAGACAATAAATTGCATGGCTGTCTTTTTCGAAGCGAATCTTGACAATCAGCTCGTCGATCTCTGTCAGTGGAGTGACCGACGAAAAGTGCTTGTCGAGATGCTCGCCGAGCTGGCCGCCACAAGATCTGAAATTCGAGATAAACTTCAAAAATCGATGCCGTTCAACAAATTCGTCAAGGAGTGTATAATGGAGAATCGCGGTGATTTGTATGAAATGACAAAGAGATTTGAGAAATATTCGTTTTTGCTCTCGATTCGGAATTTAATTGTTACTAGGATGATTATAACAAACGAAGCCGCCCGACTCCTAGGAGATGGTGAAACAATCAGTGAAACCGATATCTTCATAATCGAAAAGCGTACTCTTTCCACGTGTATTCGTAATGTGTCCGAAGGAAAAGAGTTGAGCGGCTACACACTGGACCCCTATACGGTAGCTGCCAACG 69 TGCACAATGTGCATTTCGATCACATAAACGTCGAAATCTATCTGGAATTGCTGAAAAAGTCGCCATTTTTCGCTCAGAACATTGTACGCCACTTGTTACGGCAGAATGGAAAAGAAGCAGAAGAAGAGACGTGGCACTTGCATGCCACTGTGCTGAAAATTGTGATGAAAGATGAAAAGTTGCTGGCGGTATGCGTGGCCACAATTCCAAATATGGTTCGATATCTCAAAGTCTATCAGATTCATTTCAGTCCGAAATCCAACGCGGCGAAGTTCTTACACCTCGACATGGAATCGATTTCCCACTGCCAATCATATTTGCGAAAACCAACAAAATCATCCAATCTGATCACGGCCGCCAACTTTTTGACACTTTTCGGATGTGAAAAGCGCACGTGGAAGCGCCCGATTCTCAGATTTTGGAGCATTTTCAAGCAGCAACCGGCTATGTGTTGCGAGAAATTGCTCATTTTTGCTGAAGAATGTGTCGAACTTGGCCTGAACCACCGAATCGCTTGTCTTTTACGCGCACTGACAACCAGTGAATTCTGCCGAAAAGCTCTATGTGATGAATATCTGAAAATCGCGTTTCAGCTGACTTATCGATCGATTTTTCTGATTTTAAGCAAAAATGAGTGCAGACCAGAAATTGTGGAGCTCTGCGATGACATGAATCTTCGGTACGATCTTCTCCAGCACCAGATCAAACATGTCGCGGCTCACCATTTGGAGCACTTTGAGCGTTTCGAAACGAAAATCGCATTTTCTGTTGAGAAATTTCTGAAATCTGGAATTGATGGAATCGATTTTGAAGATTTAGGATTAGTCGAGTTTTATAAGCAGTTGAACGAAAATTTGACAGAAGACGCGATCAGGAGCAATGAGGCGAGAAACATCTACATCGTCGACATTCTCTCAACAATTTGGCTCCAGCTCCCCTCAATTCGTCCTCAAATTCTACCGATTCTCGCCCGCTTCAAGCACATTTCCCCAGCGTGGACTAATTTCCCACAGCCGCCTCATATTTCGACAAACGAGAAATCATTTCTTCAACATCTACGATTTCATCTTTATCTAAAAATGATGAATATCTCGAAATCCATGACGCAAGGCGAGTATGCCACGTGTATTATGATGCTCCTGACGAGCTACGACTCGAGCCATTTCGTTGCGGATTTGATAGAGAAAAAGCAGCTGGGAAAGCTGAAATTGCAGCAGAGAAGGAATGTTTTGTGCATTTTGAGCCGACTTTTGAAAGATCAAGCTGTGATGGGTGATGAAGATGAGACGATTATCGATCCGATTCTTTTTAAGGCAATCACCAAAGCTTCCGCCGTTTTTGAAGACACCGCCGCGTGCATCGTACCATTTTTGTTCAAAATTTGTGTGGATTTCAAGGGAAAATACGATAAATGTGTGATAAATTTGCTGGGATGTCTTAAGGGAGTTAATGCAGAAGACGAAATAGTAGTCCGCTGCCTTGCCGAATGCGTCGACTCCATTGGACTCAACGTGATTGCTCGTTACGAACGCCTGAACATTGAGACTCATTCGGAATTCGGTGTGAAATGGTTCTTCAAATTATCCCGTCTCTTTCTGAAACATGGATTTACTACGCATTCCTTCGCGATTGCGAATATATTATTTGACCGACTTTCGGCGAGAAAACGAAATACAATGATGATAGATCGTACGAGTTTAGACCGAATTGACAGATCACAGGAACTTATCAATCTTTTGGTGGAGATCTATGTGGCCGAGGGTAACTCGGTAGCGTTGTCCTCTCTGCCACCTGCGGTACAAAATAGACCGGATGTTCGGCAGGTTATGAATAAGAGTTCAAAGGAATGGTTGAAATTGCTGTCTTCGAATCAAATGGACTCGTGGGAGTTGACTATTGTGCAGTGGATGTGTGGCATCCAATTCAATGCAATCACCGGCGATAAATACCTGAATTCAATTCTTCGTTGCAATTTCAACGAGTACACCAAAAAAATCGATTCCCCATTAAAATTCGTCTATTTTCAACTATTCCATCTTTCGACGAGCACTCTGGAAATCGAAGAAGCCATTTCTAGCATGCCGTTGGCTCCAACAATCGATCAAATGCGGCTTATGATTATAGCCAACGCGACGGCAAGCTTCGAGCCACAATCCGTTGAAGAGCACGTTGTTCGAGCTGTTCGAGAGCTCCGAGAGACGTCAAATCGGCGAAAATCCGGTGGAAATGTGAAGGGAATTAATGAAAAAACAACGAGAATGGTGAAACTGGCCGAGATGCTCACCGAGAATAAAGCATACGATGCGGCAATAAACCTGCTCGACACGTGGGAGCACGAGTGTCTCCAATGGACATCTGTCGCTGCCGAATCAATCGATATCGATTTAATTCGAATCTGCAAACAACACGTGACGTGTCGGTCGGGAGATCCAAGAATGGCGGACATAAATCTACGAACAATGCATCCACGTGTCCCGGTGATGAGTGACCTGGCGATTGCCGAATGGTCACTCGCGTTGAGCAAGATTACAATTGAATATCGGAATGATATGGAAGAGGGTATTCGGATTTTGGAATTTGGATGCAAACATTTACAGAATAAGGATTCTGTAGAGACGAGGTTAAAGGTTCTCCTAAAACTCCATTCCGTCTGTATTGGCCAACTGTCGAAACTCGAAGAATATCGCGAAACGCGTACCTACCGTATGAAGCAGCAGGCGGTCACCGCATTCGAACAACAAATTCAAAATTCGTGTCGAACCAGTCTGGCACGTGGCAATTCGGGTGACGAATGGACGAAAAAAACGGTGCA 70 ACGGGTGAGAAAAGAGCATCAGTTTGAGAAGAATGATTTGGAAAAAGTGGATAATTCGTTGAATTCGGCGGCCCGGAAAGCTGTATCGTCGGGTTTTGATGCACTTTTATGCATCAGCCAACTGGAAGACGACGACGAAGCGATCCGCGCTTCATCTCTCATAATATTTCCATTAATCGATGTGATCTACAAATACGAAACGGACGTCGGAGTGATCGCCTTGCTCAAAGAGCACACCAAATCGAAGCTCCCGTCGAAGCTGTGGATAAGTGCCACCTCACACATTGCCTCTAAGTGCTTCTCCATCGAAAAATCGCAAATCACGAGACACTTATCACAGATTCTGTGTCATCTCATCTATGACTACCCGTATCACGTTTTGCACACAATTTTGATGTATGATGACGAGAAGAACGCTTCCAAGGTGAAAGGCTTCTTGAAAACGATATTTGACGCGCGAGCTGACCAACGGGATTCGTCGAAGCTTAAGGAGATTGTGATTACTATTCGTGAGGCTCACCAGGCTTATAGAGAAATTGCAATGCTTGACGTGAGAGGAAACGTTCGGATACAACGTGTTGAAATAAACGGAAAAACGATGTACCGATGGCCACATGATTTGAAGATTTTCAAGTGCAAATTGCGTCAATTGCCGATTCCCACCATTTCGCAAAAGATTGGTTGCCCGGGTGACTACTCGACGACTGACCTAATCACGTGGAAACGATGGAAAGATGTATTCACAATTGCCGACGGAATTTCCACTCCAAAAATCTGGGAAATTGAGGGTAGCGATGGAAAATGGTATAAAACTGTGTGGAAGAAGGACGACGTACGACAGGATGTGCTTGTTGAGCAAATGTTTGATGTTACGAATAATATGTTGGAGAAGGCGATGCTTCGGACGTATAATGTTGTCCCGTTAGACACTGAATGTGGAGTTATAGAGTTTTGTGGTGGAACTGTTAGCTTAAAAGAAGTTATGTGTGGTGTGACACGAGAAGGCGGTCTCCACCGGGAGTTCAATTCAGAAGAAGTTTCGGCGAGTAAAGTGTCGTCAATGATGAGACAAGTACAGACAGAGTCCACAGAGACACGACGACAAGTTTTTGTGGAGATTTGTCAGCAGTATTCTCCAGTTTTTAGACACTTCTTCTACACCAACTTTTCGACGGCGCAGATTTGGCGGCAAAAAATCATAAATTACAGGCAGAGCTTGGCCACGTGGAGTATTGTGTGCTACATCGTTGGCCTCGGCGATCGACACGCGTCGAATATTCTATTCGATCAGAAATTGTGCACATTTGTGCATATCGATTTGGGAATGATTTTGGAGTATAGTAAACGAACGTTGCCAGTTCCCGAACAAGTTCCATTCCGTATAACTCGAGATGTGCTCGACCCGATTCTGATTGAAGGAATCGAGAATGGACAGTTGGCTGAGGAATGTACGCAGATTATGGAGAAATTGAAGGAAAATGGAAAGGTAATCCTCGGTGTCGCCTCTGCTCTACTCCGCGAAACGATGACAAATTTCCGAGAAGCCGAACAGGCTGCCGGCCGCCCGTCCTACATTTCTGAAATGGCCATCGGACGGCTTCGAGAAAAGCTACGGGGCACCGATGACGGTGTGACGGCCCAATCGTCGAATCTTCAAATTCGGCGACTTTTACGAGAAGCTACGAGTGCTGACAATTTATCGCGAATGTTCTGCGGATGGATGCCGTTTTTGTAG    71 Appendix C  : atm-1(gk186) transcripts C.1 atm-1.a   ATGAGCGTTACTCAGCTAAAAAATCTGAAACACGCGATAGCTCAGCTTCTGGAGTGGGATGGAACGAAAACGGCGAGAAAGAAAATCGTCGACGAGGTGGTTCTTCTCTATCACGCTCTGGGAGCGGAGGCATTAAGTGAGGATAACCAGGAGATATATGATTTGTATGATTTATCTGCACGTATATTCAACTTAGCTAAGAAAGAAATCGAAGAAGCCAACCAACAATTCGAAAAAGAACGAAAAAAGGGCACAAGACGAAGCGAAAAACCAGTCCCAACTCCACTTTTCGAGCTATCAATACAACATTTAAAACGTTGCTGTCAGCAGGGAATCGATCACAATCAGGTGCCATGGATAGCATATTGTCTGAAACTACTGGAATTTCCAATCACAATCACCGAAAAATCGATCGAAAACGAGATTTCCAACGTGCTCCTATTGAGCTCCAACGCCTCACAGCTCCATTGGGCCGAGCATGCTCATTTGAGCAGCTTATGGAAATGGATTTGGAGCCGTGTCGAGACCGCCGATATTGGGGCACTCGCCATGAGAAATTATATGGAATTGGCGGCGAACTTGCTGGAAAACGTGGATTACGTGGTTTTCGAGAAGTCGCCTATTGACCTGATGGCGAAAGTGATGGGAACACTGAAGAAAAGTGTGGAAATGGGAAATCCCAAAGAATATTGGTGTCATTACCGAAAATTCTCGGTCGTCCACATTCTCTCATATATCGTACATCGTTGGGGTCTCGAAGCGAGAGATTTCATTCTTCGTGAGATTTTCGAGCTCACTGGTAGTTTGTCCAACTTGATATCAGTGGCTCATAAGGATGGAGAACAGTCCAAGAAATGCATAATGCGTTTGATTGATGATCTCGTGAAGCTCGCCATGATCGAAACCGTACACGGCCACCGTACCATGAACGAAGTGACACGTGGAAATATTCAAAAACTCGTGAAAACCGGAATCCAAGAGTCTCTGAAATCGGCGCACCGAAATTTCTCAAGGAGTTCGACATTTTCGATTTCCGAAGAATGCGTGAGATATCTGACGAGATGGTTGTTGGCCGAACGAAGACTTGAACAGCCGTCTGCGGCTATGAATGAATCGTTTGAATTGACCGGTGATAGCAGTAGTAAGAAGAAGGACGATGCCACGTTTGATAGCCTTATCGATCTCTCCTTTGGCTCGACGATTTCCGGAAAACATAAATTGAACGCGTGGAATGGTGTAATGCAAATCCTGAATGAGCTCCTAAAAAGTCGACGACTCGAACTTCAAGTCACTGAAAAAATCGTGACAATCCTCTGGGAAAAGCGAAAATCCTACACAACGGAGCCACTCCGTACTGTGTTCTGCTCCATTCTCTCCACAGTCGTCTGCCAGGCCGATGTTCGATTCGGTCATCGGAAAGTGCCGACAATCGACTCGATTCTCAAATATTCGCTGTCTCTAATGCCAAATGTCGCATCTCTTCCCAGTGCCGCTGCGTTGACCGAAACGATTGTCAGATTCAGGACAGTATCACGAGAGGGTCTCCGTAACACGTGGGATACCGTATCCCGAACTAGCTCCGGCTCATTTGAAGTTGTTCGGCTGATTTCGGCGTTGATCTCCGTTACGGAATTTGATGAGAATTCGAGATTCGCCAACGATGAGAGAGTTCGGAGTTGGAGTTTTCGAAAAGACATAATCGAATGGGTATTGCTGGATCCGAATGCACATAGTCACAAATTACTCTATCAGTTGTGCCAGTATCATCCAACGTATTGTTATGAATCAGAAGCTTCTTCTAGTGACGACTCCCTGCTTCAAACTCTGAAACTATGTAAATTAGCTTGCTCTCCAGCTCCCCCATCGGCTCCAAAAGCCCTCCGACCACTCGAAGCTTCAATTGAAGAGATTGTGAGATATGTGCATGATAAGCTCAAGAGCATTCTCGCGACTGAAATCACTCTGCCTGCATTTGTGCTCTGCCACGAATTTGCTCTGAAGTATCCGGATAGATCTTATGAGTTTAATAAAATGTACAAAAAGCTCTACCAAATCATGGAAGATCAAGAAGAAGACGAGTTTCTCCAATCAGCTCGCCATTTCTCAAAATGGCCTCAAAATCTGACACTACCAATACAAAAACAGACAATAAATTGCATGGCTGTCTTTTTCGAAGCGAATCTTGACAATCAGCTCGTCGATCTCTGTCAGTGGAGTGACCGACGAAAAGTGCTTGTCGAGATGCTCGCCGAGCTGGCCGCCACAAGATCTGAAATTCGAGATAAACTTCAAAAATCGATGCCGTTCAACAAATTCGTCAAGGAGTGTATAATGGAGAATCGCGGTGATTTGTATGAAATGACAAAGAGATTTGAGAAATATTCGTTTTTGCTCTCGATTCGGAATTTAATTGTTACTAGGATGATTATAACAAACGAAGCCGCCCGACTCCTAGGAGATGGTGAAACAATCAGTGAAACCGATATCTTCATAATCGAAAAGCGTACTCTTTCCACGTGTATTCGTAATGTGTCCGAAGGAAAAGAGTTGAGCGGCTACACACTGGACCCCTATACGGTAGCTGCCAACG 72 TGCACAATGTGCATTTCGATCACATAAACGTCGAAATCTATCTGGAATTGCTGAAAAAGTCGCCATTTTTCGCTCAGAACATTGTACGCCACTTGTTACGGCAGAATGGAAAAGAAGCAGAAGAAGAGACGTGGCACTTGCATGCCACTGTGCTGAAAATTGTGATGAAAGATGAAAAGTTGCTGGCGGTATGCGTGGCCACAATTCCAAATATGGTTCGATATCTCAAAGTCTATCAGATTCATTTCAGTCCGAAATCCAACGCGGCGAAGTTCTTACACCTCGACATGGAATCGATTTCCCACTGCCAATCATATTTGCGAAAACCAACAAAATCATCCAATCTGATCACGGCCGCCAACTTTTTGACACTTTTCGGATGTGAAAAGCGCACGTGGAAGCGCCCGATTCTCAGATTTTGGAGCATTTTCAAGCAGCAACCGGCTATGTGTTGCGAGAAATTGCTCATTTTTGCTGAAGAATGTGTCGAACTTGGCCTGAACCACCGAATCGCTTGTCTTTTACGCGCACTGACAACCAGTGAATTCTGCCGAAAAGCTCTATGTGATGAATATCTGAAAATCGCGTTTCAGCTGACTTATCGATCGATTTTTCTGATTTTAAGCAAAAATGAGTGCAGACCAGAAATTGTGGAGCTCTGCGATGACATGAATCTTCGGTACGATCTTCTCCAGCACCAGATCAAACATGTCGCGGCTCACCATTTGGAGCACTTTGAGCGTTTCGAAACGAAAATCGCATTTTCTGTTGAGAAATTTCTGAAATCTGGAATTGATGGAATCGATTTTGAAGATTTAGGATTAGTCGAGTTTTATAAGCAGTTGAACGAAAATTTGACAGAAGACGCGATCAGGAGCAATGAGGCGAGAAACATCTACATCGTCGACATTCTCTCAACAATTTGGCTCCAGCTCCCCTCAATTCGTCCTCAAATTCTACCGATTCTCGCCCGCTTCAAGCACATTTCCCCAGCGTGGACTAATTTCCCACAGCCGCCTCATATTTCGACAAACGAGAAATCATTTCTTCAACATCTACGATTTCATCTTTATCTAAAAATGATGAATATCTCGAAATCCATGACGCAAGGCGAGTATGCCACGTGTATTATGATGCTCCTGACGAGCTACGACTCGAGCCATTTCGTTGCGGATTTGATAGAGAAAAAGCAGCTGGGAAAGCTGAAATTGCAGCAGAGAAGGAATGTTTTGTGCATTTTGAGCCGACTTTTGAAAGATCAAGCTGTGATGGGTGATGAAGATGAGACGATTATCGATCCGATTCTTTTTAAGGCAATCACCAAAGCTTCCGCCGTTTTTGAAGACACCGCCGCGTGCATCGTACCATTTTTGTTCAAAATTTGTGTGGATTTCAAGGGAAAATACGATAAATGTGTGATAAATTTGCTGGGATGTCTTAAGGGAGTTAATGCAGAAGACGAAATAGTAGTCCGCTGCCTTGCCGAATGCGTCGACTCCATTGGACTCAACGTGATTGCTCGTTACGAACGCCTGAACATTGAGACTCATTCGGAATTCGGTGTGAAATGGTTCTTCAAATTATCCCGTCTCTTTCTGAAACATGGATTTACTACGCATTCCTTCGCGATTGCGAATATATTATTTGACCGACTTTCGGCGAGAAAACGAAATACAATGATGATAGATCGTACGAGTTTAGACCGAATTGACAGATCACAGGAACTTATCAATCTTTTGGTGGAGATCTATGTGGCCGAGGGTAACTCGGTAGCGTTGTCCTCTCTGCCACCTGCGGTACAAAATAGACCGGATGTTCGGCAGGTTATGAATAAGAGTTCAAAGGAATGGTTGAAATTGCTGTCTTCGAATCAAATGGACTCGTGGGAGTTGACTATTGTGCAGTGGATGTGTGGCATCCAATTCAATGCAATCACCGGCGATAAATACCTGAATTCAATTCTTCGTTGCAATTTCAACGAGTACACCAAAAAAATCGATTCCCCATTAAAATTCGTCTATTTTCAACTATTCCATCTTTCGACGAGCACTCTGGAAATCGAAGAAGCCATTTCTAGCATGCCGTTGGCTCCAACAATCGATCAAATGCGGCTTATGATTATAGCCAACGCGACGGCAAGCTTCGAGCCACAATCCGTTGAAGAGCACGTTGTTCGAGCTGTTCGAGAGCTCCGAGAGACGTCAAATCGGCGAAAATCCGGTGGAAATGTGAAGGGAATTAATGAAAAAACAACGAGAATGGTGAAACTGGCCGAGATGCTCACCGAGAATAAAGCATACGATGCGGCAATAAACCTGCTCGACACGTGGGAGCACGAGTGTCTCCAATGGACATCTGTCGCTGCCGAATCAATCGATATCGATTTAATTCGAATCTGCAAACAACACGTGACGTGTCGGTCGGGAGATCCAAGAATGGCGGACATAAATCTACGAACAATGCATCCACGTGTCCCGGTGATGAGTGACCTGGCGATTGCCGAATGGTCACTCGCGTTGAGCAAGATTACAATTGAATATCGGAATGATATGGAAGAGGGTATTCGGATTTTGGAATTTGGATGCAAACATTTACAGAATAAGGATTCTGTAGAGACGAGGTTAAAGGTTCTCCTAAAACTCCATTCCGTCTGTATTGGCCAACTGTCGAAACTCGAAGAATATCGCGAAACGCGTACCTACCGTATGAAGCAGCAGGCGGTCACCGCATTCGAACAACAAATTCAAAATTCGTGTCGAACCAGTCTGGCACGTGGCAATTCGGGTGACGAATGGACGAAAAAAACGGTGCA 73 ACGGGTGAGAAAAGAGCATCAGTTTGAGAAGAATGATTTGGAAAAAGTGGATAATTCGTTGAATTCGGCGGCCCGGAAAGCTGTATCGTCGGGTTTTGATGCACTTTATTCTGTGTCATCTCATCTATGACTACCCGTATCACGTTTTGCACACAATTTTGATGTATGATGACGAGAAGAACGCTTCCAAGGTGAAAGGCTTCTTGAAAACGATATTTGACGCGCGAGCTGACCAACGGGATTCGTCGAAGCTTAAGGAGATTGTGATTACTATTCGTGAGGCTCACCAGGCTTATAGAGAAATTGCAATGCTTGACGTGAGAGGAAACGTTCGGATACAACGTGTTGAAATAAACGGAAAAACGATGTACCGATGGCCACATGATTTGAAGATTTTCAAGTGCAAATTGCGTCAATTGCCGATTCCCACCATTTCGCAAAAGATTGGTTGCCCGGGTGACTACTCGACGACTGACCTAATCACGTGGAAACGATGGAAAGATGTATTCACAATTGCCGACGGAATTTCCACTCCAAAAATCTGGGAAATTGAGGGTAGCGATGGAAAATGGTATAAAACTGTGTGGAAGAAGGACGACGTACGACAGGATGTGCTTGTTGAGCAAATGTTTGATGTTACGAATAATATGTTGGAGAAGGCGATGCTTCGGACGTATAATGTTGTCCCGTTAGACACTGAATGTGGAGTTATAGAGTTTTGTGGTGGAACTGTTAGCTTAAAAGAAGTTATGTGTGGTGTGACACGAGAAGGCGGTCTCCACCGGGAGTTCAATTCAGAAGAAGTTTCGGCGAGTAAAGTGTCGTCAATGATGAGACAAGTACAGACAGAGTCCACAGAGACACGACGACAAGTTTTTGTGGAGATTTGTCAGCAGTATTCTCCAGTTTTTAGACACTTCTTCTACACCAACTTTTCGACGGCGCAGATTTGGCGGCAAAAAATCATAAATTACAGGCAGAGCTTGGCCACGTGGAGTATTGTGTGCTACATCGTTGGCCTCGGCGATCGACACGCGTCGAATATTCTATTCGATCAGAAATTGTGCACATTTGTGCATATCGATTTGGGAATGATTTTGGAGTATAGTAAACGAACGTTGCCAGTTCCCGAACAAGTTCCATTCCGTATAACTCGAGATGTGCTCGACCCGATTCTGATTGAAGGAATCGAGAATGGACAGTTGGCTGAGGAATGTACGCAGATTATGGAGAAATTGAAGGAAAATGGAAAGGTAATCCTCGGTGTCGCCTCTGCTCTACTCCGCGAAACGATGACAAATTTCCGAGAAGCCGAACAGGCTGCCGGCCGCCCGTCCTACATTTCTGAAATGGCCATCGGACGGCTTCGAGAAAAGCTACGGGGCACCGATGACGGTGTGACGGCCCAATCGTCGAATCTTCAAATTCGGCGACTTTTACGAGAAGCTACGAGTGCTGACAATTTATCGCGAATGTTCTGCGGATGGATGCCGTTTTTGTAG C.2 atm-1.b ATGAGCGTTACTCAGCTAAAAAATCTGAAACACGCGATAGCTCAGCTTCTGGAGTGGGATGGAACGAAAACGGCGAGAAAGAAAATCGTCGACGAGGTGGTTCTTCTCTATCACGCTCTGGGAGCGGAGGCATTAAGTGAGGATAACCAGGAGATATATGATTTGTATGATTTATCTGCACGTATATTCAACTTAGCTAAGAAAGAAATCGAAGAAGCCAACCAACAATTCGAAAAAGAACGAAAAAAGGGCACAAGACGAAGCGAAAAACCAGTCCCAACTCCACTTTTCGAGCTATCAATACAACATTTAAAACGTTGCTGTCAGCAGGGAATCGATCACAATCAGGTGCCATGGATAGCATATTGTCTGAAACTACTGGAATTTCCAATCACAATCACCGAAAAATCGATCGAAAACGAGATTTCCAACGTGCTCCTATTGAGCTCCAACGCCTCACAGCTCCATTGGGCCGAGCATGCTCATTTGAGCAGCTTATGGAAATGGATTTGGAGCCGTGTCGAGACCGCCGATATTGGGGCACTCGCCATGAGAAATTATATGGAATTGGCGGCGAACTTGCTGGAAAACGTGGATTACGTGGTTTTCGAGAAGTCGCCTATTGACCTGATGGCGAAAGTGATGGGAACACTGAAGAAAAGTGTGGAAATGGGAAATCCCAAAGAATATTGGTGTCATTACCGAAAATTCTCGGTCGTCCACATTCTCTCATATATCGTACATCGTTGGGGTCTCGAAGCGAGAGATTTCATTCTTCGTGAGATTTTCGAGCTCACTGGTAGTTTGTCCAACTTGATATCAGTGGCTCATAAGGATGGAGAACAGTCCAAGAAATGCATAATGCGTTTGATTGATGATCTCGTGAAGCTCGCCATGATCGAAACCGTACACGGCCACCGTACCATGAACGAAGTGACACGTGGAAATATTCAAAAACTCGTGAAAACCGGAATCCAAGAGTCTCTGAAATCGGCGCACCGAAATTTCTCAAGGAGTTCGACATTTTCGATTTCCGAAGAATGCGTGAGATATCTGACGAGATGGTTGTTGGCCGAACGAAGACTTGAACAGCCGTCTGCGGCTATGAAT 74 GAATCGTTTGAATTGACCGGTGATAGCAGTAGTAAGAAGAAGGACGATGCCACGTTTGATAGCCTTATCGATCTCTCCTTTGGCTCGACGATTTCCGGAAAACATAAATTGAACGCGTGGAATGGTGTAATGCAAATCCTGAATGAGCTCCTAAAAAGTCGACGACTCGAACTTCAAGTCACTGAAAAAATCGTGACAATCCTCTGGGAAAAGCGAAAATCCTACACAACGGAGCCACTCCGTACTGTGTTCTGCTCCATTCTCTCCACAGTCGTCTGCCAGGCCGATGTTCGATTCGGTCATCGGAAAGTGCCGACAATCGACTCGATTCTCAAATATTCGCTGTCTCTAATGCCAAATGTCGCATCTCTTCCCAGTGCCGCTGCGTTGACCGAAACGATTGTCAGATTCAGGACAGTATCACGAGAGGGTCTCCGTAACACGTGGGATACCGTATCCCGAACTAGCTCCGGCTCATTTGAAGTTGTTCGGCTGATTTCGGCGTTGATCTCCGTTACGGAATTTGATGAGAATTCGAGATTCGCCAACGATGAGAGAGTTCGGAGTTGGAGTTTTCGAAAAGACATAATCGAATGGGTATTGCTGGATCCGAATGCACATAGTCACAAATTACTCTATCAGTTGTGCCAGTATCATCCAACGTATTGTTATGAATCAGAAGCTTCTTCTAGTGACGACTCCCTGCTTCAAACTCTGAAACTATGTAAATTAGCTTGCTCTCCAGCTCCCCCATCGGCTCCAAAAGCCCTCCGACCACTCGAAGCTTCAATTGAAGAGATTGTGAGATATGTGCATGATAAGCTCAAGAGCATTCTCGCGACTGAAATCACTCTGCCTGCATTTGTGCTCTGCCACGAATTTGCTCTGAAGTATCCGGATAGATCTTATGAGTTTAATAAAATGTACAAAAAGCTCTACCAAATCATGGAAGATCAAGAAGAAGACGAGTTTCTCCAATCAGCTCGCCATTTCTCAAAATGGCCTCAAAATCTGACACTACCAATACAAAAACAGACAATAAATTGCATGGCTGTCTTTTTCGAAGCGAATCTTGACAATCAGCTCGTCGATCTCTGTCAGTGGAGTGACCGACGAAAAGTGCTTGTCGAGATGCTCGCCGAGCTGGCCGCCACAAGATCTGAAATTCGAGATAAACTTCAAAAATCGATGCCGTTCAACAAATTCGTCAAGGAGTGTATAATGGAGAATCGCGGTGATTTGTATGAAATGACAAAGAGATTTGAGAAATATTCGTTTTTGCTCTCGATTCGGAATTTAATTGTTACTAGGATGATTATAACAAACGAAGCCGCCCGACTCCTAGGAGATGGTGAAACAATCAGTGAAACCGATATCTTCATAATCGAAAAGCGTACTCTTTCCACGTGTATTCGTAATGTGTCCGAAGGAAAAGAGTTGAGCGGCTACACACTGGACCCCTATACGGTAGCTGCCAACGTGCACAATGTGCATTTCGATCACATAAACGTCGAAATCTATCTGGAATTGCTGAAAAAGTCGCCATTTTTCGCTCAGAACATTGTACGCCACTTGTTACGGCAGAATGGAAAAGAAGCAGAAGAAGAGACGTGGCACTTGCATGCCACTGTGCTGAAAATTGTGATGAAAGATGAAAAGTTGCTGGCGGTATGCGTGGCCACAATTCCAAATATGGTTCGATATCTCAAAGTCTATCAGATTCATTTCAGTCCGAAATCCAACGCGGCGAAGTTCTTACACCTCGACATGGAATCGATTTCCCACTGCCAATCATATTTGCGAAAACCAACAAAATCATCCAATCTGATCACGGCCGCCAACTTTTTGACACTTTTCGGATGTGAAAAGCGCACGTGGAAGCGCCCGATTCTCAGATTTTGGAGCATTTTCAAGCAGCAACCGGCTATGTGTTGCGAGAAATTGCTCATTTTTGCTGAAGAATGTGTCGAACTTGGCCTGAACCACCGAATCGCTTGTCTTTTACGCGCACTGACAACCAGTGAATTCTGCCGAAAAGCTCTATGTGATGAATATCTGAAAATCGCGTTTCAGCTGACTTATCGATCGATTTTTCTGATTTTAAGCAAAAATGAGTGCAGACCAGAAATTGTGGAGCTCTGCGATGACATGAATCTTCGGTACGATCTTCTCCAGCACCAGATCAAACATGTCGCGGCTCACCATTTGGAGCACTTTGAGCGTTTCGAAACGAAAATCGCATTTTCTGTTGAGAAATTTCTGAAATCTGGAATTGATGGAATCGATTTTGAAGATTTAGGATTAGTCGAGTTTTATAAGCAGTTGAACGAAAATTTGACAGAAGACGCGATCAGGAGCAATGAGGCGAGAAACATCTACATCGTCGACATTCTCTCAACAATTTGGCTCCAGCTCCCCTCAATTCGTCCTCAAATTCTACCGATTCTCGCCCGCTTCAAGCACATTTCCCCAGCGTGGACTAATTTCCCACAGCCGCCTCATATTTCGACAAACGAGAAATCATTTCTTCAACATCTACGATTTCATCTTTATCTAAAAATGATGAATATCTCGAAATCCATGACGCAAGGCGAGTATGCCACGTGTATTATGATGCTCCTGACGAGCTACGACTCGAGCCATTTCGTTGCGGATTTGATAGAGAAAAAGCAGCTGGGAAAGCTGAAATTGCAGCAGAGAAGGAATGTTTTGTGCATTTTGAGCCGACTTTTGAAAGATCAAGCTGTGATGGGTGATGAAGATGAGACGATTATCGATCCGATTCTTTTTAAGGCAATCACCAAAGCTTCCG 75 CCGTTTTTGAAGACACCGCCGCGTGCATCGTACCATTTTTGTTCAAAATTTGTGTGGATTTCAAGGGAAAATACGATAAATGTGTGATAAATTTGCTGGGATGTCTTAAGGGAGTTAATGCAGAAGACGAAATAGTAGTCCGCTGCCTTGCCGAATGCGTCGACTCCATTGGACTCAACGTGATTGCTCGTTACGAACGCCTGAACATTGAGACTCATTCGGAATTCGGTGTGAAATGGTTCTTCAAATTATCCCGTCTCTTTCTGAAACATGGATTTACTACGCATTCCTTCGCGATTGCGAATATATTATTTGACCGACTTTCGGCGAGAAAACGAAATACAATGATGATAGATCGTACGAGTTTAGACCGAATTGACAGATCACAGGAACTTATCAATCTTTTGGTGGAGATCTATGTGGCCGAGGGTAACTCGGTAGCGTTGTCCTCTCTGCCACCTGCGGTACAAAATAGACCGGATGTTCGGCAGGTTATGAATAAGAGTTCAAAGGAATGGTTGAAATTGCTGTCTTCGAATCAAATGGACTCGTGGGAGTTGACTATTGTGCAGTGGATGTGTGGCATCCAATTCAATGCAATCACCGGCGATAAATACCTGAATTCAATTCTTCGTTGCAATTTCAACGAGTACACCAAAAAAATCGATTCCCCATTAAAATTCGTCTATTTTCAACTATTCCATCTTTCGACGAGCACTCTGGAAATCGAAGAAGCCATTTCTAGCATGCCGTTGGCTCCAACAATCGATCAAATGCGGCTTATGATTATAGCCAACGCGACGGCAAGCTTCGAGCCACAATCCGTTGAAGAGCACGTTGTTCGAGCTGTTCGAGAGCTCCGAGAGACGTCAAATCGGCGAAAATCCGGTGGAAATGTGAAGGGAATTAATGAAAAAACAACGAGAATGGTGAAACTGGCCGAGATGCTCACCGAGAATAAAGCATACGATGCGGCAATAAACCTGCTCGACACGTGGGAGCACGAGTGTCTCCAATGGACATCTGTCGCTGCCGAATCAATCGATATCGATTTAATTCGAATCTGCAAACAACACGTGACGTGTCGGTCGGGAGATCCAAGAATGGCGGACATAAATCTACGAACAATGCATCCACGTGTCCCGGTGATGAGTGACCTGGCGATTGCCGAATGGTCACTCGCGTTGAGCAAGATTACAATTGAATATCGGAATGATATGGAAGAGGGTATTCGGATTTTGGAATTTGGATGCAAACATTTACAGAATAAGGATTCTGTAGAGACGAGGTTAAAGGTTCTCCTAAAACTCCATTCCGTCTGTATTGGCCAACTGTCGAAACTCGAAGAATATCGCGAAACGCGTACCTACCGTATGAAGCAGCAGGCGGTCACCGCATTCGAACAACAAATTCAAAATTCGTGTCGAACCAGTCTGGCACGTGGCAATTCGGGTGACGAATGGACGAAAAAAACGGTGCAACGGATTCTGTGTCATCTCATCTATGACTACCCGTATCACGTTTTGCACACAATTTTGATGTATGATGACGAGAAGAACGCTTCCAAGGTGAAAGGCTTCTTGAAAACGATATTTGACGCGCGAGCTGACCAACGGGATTCGTCGAAGCTTAAGGAGATTGTGATTACTATTCGTGAGGCTCACCAGGCTTATAGAGAAATTGCAATGCTTGACGTGAGAGGAAACGTTCGGATACAACGTGTTGAAATAAACGGAAAAACGATGTACCGATGGCCACATGATTTGAAGATTTTCAAGTGCAAATTGCGTCAATTGCCGATTCCCACCATTTCGCAAAAGATTGGTTGCCCGGGTGACTACTCGACGACTGACCTAATCACGTGGAAACGATGGAAAGATGTATTCACAATTGCCGACGGAATTTCCACTCCAAAAATCTGGGAAATTGAGGGTAGCGATGGAAAATGGTATAAAACTGTGTGGAAGAAGGACGACGTACGACAGGATGTGCTTGTTGAGCAAATGTTTGATGTTACGAATAATATGTTGGAGAAGGCGATGCTTCGGACGTATAATGTTGTCCCGTTAGACACTGAATGTGGAGTTATAGAGTTTTGTGGTGGAACTGTTAGCTTAAAAGAAGTTATGTGTGGTGTGACACGAGAAGGCGGTCTCCACCGGGAGTTCAATTCAGAAGAAGTTTCGGCGAGTAAAGTGTCGTCAATGATGAGACAAGTACAGACAGAGTCCACAGAGACACGACGACAAGTTTTTGTGGAGATTTGTCAGCAGTATTCTCCAGTTTTTAGACACTTCTTCTACACCAACTTTTCGACGGCGCAGATTTGGCGGCAAAAAATCATAAATTACAGGCAGAGCTTGGCCACGTGGAGTATTGTGTGCTACATCGTTGGCCTCGGCGATCGACACGCGTCGAATATTCTATTCGATCAGAAATTGTGCACATTTGTGCATATCGATTTGGGAATGATTTTGGAGTATAGTAAACGAACGTTGCCAGTTCCCGAACAAGTTCCATTCCGTATAACTCGAGATGTGCTCGACCCGATTCTGATTGAAGGAATCGAGAATGGACAGTTGGCTGAGGAATGTACGCAGATTATGGAGAAATTGAAGGAAAATGGAAAGGTAATCCTCGGTGTCGCCTCTGCTCTACTCCGCGAAACGATGACAAATTTCCGAGAAGCCGAACAGGCTGCCGGCCGCCCGTCCTACATTTCTGAAATGGCCATCGGA 76 CGGCTTCGAGAAAAGCTACGGGGCACCGATGACGGTGTGACGGCCCAATCGTCGAATCTTCAAATTCGGCGACTTTTACGAGAAGCTACGAGTGCTGACAATTTATCGCGAATGTTCTGCGGATGGATGCCGTTTTTGTAG    77 Appendix D  : atm-1 protein translations D.1 atm-1 MSVTQLKNLKHAIAQLLEWDGTKTARKKIVDEVVLLYHALGAEALSEDNQEIYDLYDLSARIFNLAKKEIEEANQQFEKERKKGTRRSEKPVPTPLFELSIQHLKRCCQQGIDHNQVPWIAYCLKLLEFPITITEKSIENEISNVLLLSSNASQLHWAEHAHLSSLWKWIWSRVETADIGALAMRNYMELAANLLENVDYVVFEKSPIDLMAKVMGTLKKSVEMGNPKEYWCHYRKFSVVHILSYIVHRWGLEARDFILREIFELTGSLSNLISVAHKDGEQSKKCIMRLIDDLVKLAMIETVHGHRTMNEVTRGNIQKLVKTGIQESLKSAHRNFSRSSTFSISEECVRYLTRWLLAERRLEQPSAAMNESFELTGDSSSKKKDDATFDSLIDLSFGSTISGKHKLNAWNGVMQILNELLKSRRLELQVTEKIVTILWEKRKSYTTEPLRTVFCSILSTVVCQADVRFGHRKVPTIDSILKYSLSLMPNVASLPSAAALTETIVRFRTVSREGLRNTWDTVSRTSSGSFEVVRLISALISVTEFDENSRFANDERVRSWSFRKDIIEWVLLDPNAHSHKLLYQLCQYHPTYCYESEASSSDDSLLQTLKLCKLACSPAPPSAPKALRPLEASIEEIVRYVHDKLKSILATEITLPAFVLCHEFALKYPDRSYEFNKMYKKLYQIMEDQEEDEFLQSARHFSKWPQNLTLPIQKQTINCMAVFFEANLDNQLVDLCQWSDRRKVLVEMLAELAATRSEIRDKLQKSMPFNKFVKECIMENRGDLYEMTKRFEKYSFLLSIRNLIVTRMIITNEAARLLGDGETISETDIFIIEKRTLSTCIRNVSEGKELSGYTLDPYTVAANVHNVHFDHINVEIYLELLKKSPFFAQNIVRHLLRQNGKEAEEETWHLHATVLKIVMKDEKLLAVCVATIPNMVRYLKVYQIHFSPKSNAAKFLHLDMESISHCQSYLRKPTKSSNLITAANFLTLFGCEKRTWKRPILRFWSIFKQQPAMCCEKLLIFAEECVELGLNHRIACLLRALTTSEFCRKALCDEYLKIAFQLTYRSIFLILSKNECRPEIVELCDDMNLRYDLLQHQIKHVAAHHLEHFERFETKIAFSVEKFLKSGIDGIDFEDLGLVEFYKQLNENLTEDAIRSNEARNIYIVDILSTIWLQLPSIRPQILPILARFKHISPAWTNFPQPPHISTNEKSFLQHLRFHLYLKMMNISKSMTQGEYATCIMMLLTSYDSSHFVADLIEKKQLGKLKLQQRRNVLCILSRLLKDQAVMGDEDETIIDPILFKAITKASAVFEDTAACIVPFLFKICVDFKGKYDKCVINLLGCLKGVNAEDEIVVRCLAECVDSIGLNVIARYERLNIETHSEFGVKWFFKLSRLFLKHGFTTHSFAIANILFDRLSARKRNTMMIDRTSLDRIDRSQELINLLVEIYVAEGNSVALSSLPPAVQNRPDVRQVMNKSSKEWLKLLSSNQMDSWELTIVQWMCGIQFNAITGDKYLNSILRCNFNEYTKKIDSPLKFVYFQLFHLSTSTLEIEEAISSMPLAPTIDQMRLMIIANATASFEPQSVEEHVVRAVRELRETSNRRKSGGNVKGINEKTTRMVKLAEMLTENKAYDAAINLLDTWEHECLQWTSVAAESIDIDLIRICKQHVTCRSGDPRMADINLRTMHPRVPVMSDLAIAEWSLALSKITIEYRNDMEEGIRILEFGCKHLQNKDSVETRLKVLLKLHSVCIGQLSKLEEYRETRTYRMKQQAVTAFEQQIQNSCRTSLARGNSGDEWTKKTVQRVRKEHQFEKNDLEKVDNSLNSAARKAVSSGFDALLCISQLEDDDEAIRASSLIIFPLIDVIYKYETDVGVIALLKEHTKSKLPSKLWISATSHIASKCFSIEKSQITRHLSQILCHLIYDYPYHVLHTILMYDDEKNASKVKGFLKTIFDARADQRDSSKLKEIVITIREAHQAYREIAMLDVRGNVRIQRVEINGKTMYRWPHDLKIFKCKLRQLPIPTISQKIGCPGDYSTTDLITWKRWKDVFTIADGISTPKIWEIEGSDGKWYKTVWKKDDVRQDVLVEQMFDVTNNMLEKAMLRTYNVVPLDTECGVIEFCGGTVSLKEVMCGVTREGGLHREFNSEEVSASKVSSMMRQVQTESTETRRQVFVEICQQYSPVFRHFFYTNFSTAQIWRQKIINYRQSLATWSIVCYIVGLGDRHASNILFDQKLCTFVHIDLGMILEYSKRTLPVPEQVPFRITRDVLDPILIEGIENGQLAEECTQIMEKLKENGKVILGVASALLRETMTNFREAEQAAGRPSYISEMAIGRLREKLRGTDDGVTAQSSNLQIRRLLREATSADNLSRMFCGWMPFL    78 D.2 atm-1.a * Indicates a PTC MSVTQLKNLKHAIAQLLEWDGTKTARKKIVDEVVLLYHALGAEALSEDNQEIYDLYDLSARIFNLAKKEIEEANQQFEKERKKGTRRSEKPVPTPLFELSIQHLKRCCQQGIDHNQVPWIAYCLKLLEFPITITEKSIENEISNVLLLSSNASQLHWAEHAHLSSLWKWIWSRVETADIGALAMRNYMELAANLLENVDYVVFEKSPIDLMAKVMGTLKKSVEMGNPKEYWCHYRKFSVVHILSYIVHRWGLEARDFILREIFELTGSLSNLISVAHKDGEQSKKCIMRLIDDLVKLAMIETVHGHRTMNEVTRGNIQKLVKTGIQESLKSAHRNFSRSSTFSISEECVRYLTRWLLAERRLEQPSAAMNESFELTGDSSSKKKDDATFDSLIDLSFGSTISGKHKLNAWNGVMQILNELLKSRRLELQVTEKIVTILWEKRKSYTTEPLRTVFCSILSTVVCQADVRFGHRKVPTIDSILKYSLSLMPNVASLPSAAALTETIVRFRTVSREGLRNTWDTVSRTSSGSFEVVRLISALISVTEFDENSRFANDERVRSWSFRKDIIEWVLLDPNAHSHKLLYQLCQYHPTYCYESEASSSDDSLLQTLKLCKLACSPAPPSAPKALRPLEASIEEIVRYVHDKLKSILATEITLPAFVLCHEFALKYPDRSYEFNKMYKKLYQIMEDQEEDEFLQSARHFSKWPQNLTLPIQKQTINCMAVFFEANLDNQLVDLCQWSDRRKVLVEMLAELAATRSEIRDKLQKSMPFNKFVKECIMENRGDLYEMTKRFEKYSFLLSIRNLIVTRMIITNEAARLLGDGETISETDIFIIEKRTLSTCIRNVSEGKELSGYTLDPYTVAANVHNVHFDHINVEIYLELLKKSPFFAQNIVRHLLRQNGKEAEEETWHLHATVLKIVMKDEKLLAVCVATIPNMVRYLKVYQIHFSPKSNAAKFLHLDMESISHCQSYLRKPTKSSNLITAANFLTLFGCEKRTWKRPILRFWSIFKQQPAMCCEKLLIFAEECVELGLNHRIACLLRALTTSEFCRKALCDEYLKIAFQLTYRSIFLILSKNECRPEIVELCDDMNLRYDLLQHQIKHVAAHHLEHFERFETKIAFSVEKFLKSGIDGIDFEDLGLVEFYKQLNENLTEDAIRSNEARNIYIVDILSTIWLQLPSIRPQILPILARFKHISPAWTNFPQPPHISTNEKSFLQHLRFHLYLKMMNISKSMTQGEYATCIMMLLTSYDSSHFVADLIEKKQLGKLKLQQRRNVLCILSRLLKDQAVMGDEDETIIDPILFKAITKASAVFEDTAACIVPFLFKICVDFKGKYDKCVINLLGCLKGVNAEDEIVVRCLAECVDSIGLNVIARYERLNIETHSEFGVKWFFKLSRLFLKHGFTTHSFAIANILFDRLSARKRNTMMIDRTSLDRIDRSQELINLLVEIYVAEGNSVALSSLPPAVQNRPDVRQVMNKSSKEWLKLLSSNQMDSWELTIVQWMCGIQFNAITGDKYLNSILRCNFNEYTKKIDSPLKFVYFQLFHLSTSTLEIEEAISSMPLAPTIDQMRLMIIANATASFEPQSVEEHVVRAVRELRETSNRRKSGGNVKGINEKTTRMVKLAEMLTENKAYDAAINLLDTWEHECLQWTSVAAESIDIDLIRICKQHVTCRSGDPRMADINLRTMHPRVPVMSDLAIAEWSLALSKITIEYRNDMEEGIRILEFGCKHLQNKDSVETRLKVLLKLHSVCIGQLSKLEEYRETRTYRMKQQAVTAFEQQIQNSCRTSLARGNSGDEWTKKTVQRVRKEHQFEKNDLEKVDNSLNSAARKAVSSGFDALYSVSSHL*LPVSRFAHNFDV**REERFQGERLLENDI*RAS*PTGFVEA*GDCDYYS*GSPGL*RNCNA*RERKRSDTTC*NKRKNDVPMAT*FEDFQVQIASIADSHHFAKDWLPG*LLDD*PNHVETMERCIHNCRRNFHSKNLGN*G*RWKMV*NCVEEGRRTTGCAC*ANV*CYE*YVGEGDASDV*CCPVRH*MWSYRVLWWNC*LKRSYVWCDTRRRSPPGVQFRRSFGE*SVVNDETSTDRVHRDTTTSFCGDLSAVFSSF*TLLLHQLFDGADLAAKNHKLQAELGHVEYCVLHRWPRRSTRVEYSIRSEIVHICAYRFGNDFGV**TNVASSRTSSIPYNSRCARPDSD*RNREWTVG*GMYADYGEIEGKWKGNPRCRLCSTPRNDDKFPRSRTGCRPPVLHF*NGHRTASRKATGHR*RCDGPIVESSNSATFTRSYEC*QFIANVLRMDAVFV    79 D.3 atm-1.b MSVTQLKNLKHAIAQLLEWDGTKTARKKIVDEVVLLYHALGAEALSEDNQEIYDLYDLSARIFNLAKKEIEEANQQFEKERKKGTRRSEKPVPTPLFELSIQHLKRCCQQGIDHNQVPWIAYCLKLLEFPITITEKSIENEISNVLLLSSNASQLHWAEHAHLSSLWKWIWSRVETADIGALAMRNYMELAANLLENVDYVVFEKSPIDLMAKVMGTLKKSVEMGNPKEYWCHYRKFSVVHILSYIVHRWGLEARDFILREIFELTGSLSNLISVAHKDGEQSKKCIMRLIDDLVKLAMIETVHGHRTMNEVTRGNIQKLVKTGIQESLKSAHRNFSRSSTFSISEECVRYLTRWLLAERRLEQPSAAMNESFELTGDSSSKKKDDATFDSLIDLSFGSTISGKHKLNAWNGVMQILNELLKSRRLELQVTEKIVTILWEKRKSYTTEPLRTVFCSILSTVVCQADVRFGHRKVPTIDSILKYSLSLMPNVASLPSAAALTETIVRFRTVSREGLRNTWDTVSRTSSGSFEVVRLISALISVTEFDENSRFANDERVRSWSFRKDIIEWVLLDPNAHSHKLLYQLCQYHPTYCYESEASSSDDSLLQTLKLCKLACSPAPPSAPKALRPLEASIEEIVRYVHDKLKSILATEITLPAFVLCHEFALKYPDRSYEFNKMYKKLYQIMEDQEEDEFLQSARHFSKWPQNLTLPIQKQTINCMAVFFEANLDNQLVDLCQWSDRRKVLVEMLAELAATRSEIRDKLQKSMPFNKFVKECIMENRGDLYEMTKRFEKYSFLLSIRNLIVTRMIITNEAARLLGDGETISETDIFIIEKRTLSTCIRNVSEGKELSGYTLDPYTVAANVHNVHFDHINVEIYLELLKKSPFFAQNIVRHLLRQNGKEAEEETWHLHATVLKIVMKDEKLLAVCVATIPNMVRYLKVYQIHFSPKSNAAKFLHLDMESISHCQSYLRKPTKSSNLITAANFLTLFGCEKRTWKRPILRFWSIFKQQPAMCCEKLLIFAEECVELGLNHRIACLLRALTTSEFCRKALCDEYLKIAFQLTYRSIFLILSKNECRPEIVELCDDMNLRYDLLQHQIKHVAAHHLEHFERFETKIAFSVEKFLKSGIDGIDFEDLGLVEFYKQLNENLTEDAIRSNEARNIYIVDILSTIWLQLPSIRPQILPILARFKHISPAWTNFPQPPHISTNEKSFLQHLRFHLYLKMMNISKSMTQGEYATCIMMLLTSYDSSHFVADLIEKKQLGKLKLQQRRNVLCILSRLLKDQAVMGDEDETIIDPILFKAITKASAVFEDTAACIVPFLFKICVDFKGKYDKCVINLLGCLKGVNAEDEIVVRCLAECVDSIGLNVIARYERLNIETHSEFGVKWFFKLSRLFLKHGFTTHSFAIANILFDRLSARKRNTMMIDRTSLDRIDRSQELINLLVEIYVAEGNSVALSSLPPAVQNRPDVRQVMNKSSKEWLKLLSSNQMDSWELTIVQWMCGIQFNAITGDKYLNSILRCNFNEYTKKIDSPLKFVYFQLFHLSTSTLEIEEAISSMPLAPTIDQMRLMIIANATASFEPQSVEEHVVRAVRELRETSNRRKSGGNVKGINEKTTRMVKLAEMLTENKAYDAAINLLDTWEHECLQWTSVAAESIDIDLIRICKQHVTCRSGDPRMADINLRTMHPRVPVMSDLAIAEWSLALSKITIEYRNDMEEGIRILEFGCKHLQNKDSVETRLKVLLKLHSVCIGQLSKLEEYRETRTYRMKQQAVTAFEQQIQNSCRTSLARGNSGDEWTKKTVQRILCHLIYDYPYHVLHTILMYDDEKNASKVKGFLKTIFDARADQRDSSKLKEIVITIREAHQAYREIAMLDVRGNVRIQRVEINGKTMYRWPHDLKIFKCKLRQLPIPTISQKIGCPGDYSTTDLITWKRWKDVFTIADGISTPKIWEIEGSDGKWYKTVWKKDDVRQDVLVEQMFDVTNNMLEKAMLRTYNVVPLDTECGVIEFCGGTVSLKEVMCGVTREGGLHREFNSEEVSASKVSSMMRQVQTESTETRRQVFVEICQQYSPVFRHFFYTNFSTAQIWRQKIINYRQSLATWSIVCYIVGLGDRHASNILFDQKLCTFVHIDLGMILEYSKRTLPVPEQVPFRITRDVLDPILIEGIENGQLAEECTQIMEKLKENGKVILGVASALLRETMTNFREAEQAAGRPSYISEMAIGRLREKLRGTDDGVTAQSSNLQIRRLLREATSADNLSRMFCGWMPFL 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