Species within the family Cimicidae (bed bugs) are hematophagous ectoparasites of mammals and birds. Many cimicids are of socio-economic importance. Despite the global resurgence of these pests, there is currently a paucity of information regarding the mitochondrial (mt) DNA sequences of cimicids. Therefore, I used a PCR-based primer walking strategy to amplify and sequence the near complete mitogenome of the common bed bug (Cimex lectularius), and several mitochondrial gene regions of the Eastern bat bug (Cimex adjunctus) and swallow bug (Oeciacus vicarius). I compared the mitochondrial genetic variability between C. lectularius from two populations to look for molecular markers useful for population genetic studies. Furthermore, the mt DNA sequences of these species of medical and veterinary importance were compared to those of other heteropterans to infer the evolutionary relationships of species in the family Cimicidae.

Programmable site-specific endonucleases are useful tools for genome editing and may lead to novel therapeutics to treat genetic diseases. TALENs can be designed to cleave chosen DNA sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. We engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome engineering agents with improved DNA specificities.

No description available.

In 2005 an equine rhinitis A virus (ERAV) isolate was recovered from a febrile horse during a respiratory outbreak in Ontario. This isolate (ERAV/ON/05) was propagated in cell culture and used to study its genomic characteristics and to investigate the clinical features in experimentally infected ponies. The fulllength genome of this isolate was sequenced and compared with other ERAV available in GenBank. The isolate genome is 7839 nucleotides (nts) in length with a variable 5’UTR and a more conserved 3’UTR. When the isolate was compared to other reported ERAV, an insertion of 13 nts in the 5’UTR was identified. Phylogenetic analysis demonstrated that ERAV/ON/05 was closely related to the ERAV/PERV isolate, which was recovered in 1962 in the United Kingdom. An experimental model was developed to study the clinical infection in naïve healthy ponies (ERAV/ON/05 n=4 and placebo n=4). ERAV/ON/05 induced clinical respiratory disease compared to placebo. The clinical signs consisted of pyrexia, nasal discharge, increased and abnormal lung sounds, increased size of submandibular lymph nodes and persistent mucopus in the trachea (up to 21 days post-infection). The virus was isolated from the lower and upper airways up to day 7 post-infection, corresponding with the detection of neutralizing ERAV antibodies. Assessment of the cytokine profile from bronchoalveolar lavage (BAL) cells demonstrated that this infection induced down-regulation of the mRNA expression of IL-4. One year later, four previously infected ponies with neutralizing antibodies to ERAV were assigned to a reinfection trial. None of the re-infected ponies developed clinical disease, and only one animal had a four-fold increase in antibody titres to ERAV. Attempts to recover the virus from the re-infected ponies using cell culture were negative; however, a down-regulation of the mRNA expression of IL-4 and IFN-β was identified in BAL cells. In conclusion, this study shows that the genome of ERAV has not significantly changed in the last 50 years and more importantly the virus induces clinical respiratory disease similar to other common equine respiratory viruses.

Necrotic enteritis (NE) is an economically important, but poorly understood, disease of poultry, typically caused by Clostridium perfringens Type A strains that carry the NetB toxin gene. The objective of the current research was to identify additional genes associated with NE-causing C. perfringens strains, and thus putatively involved in virulence. To identify novel NE-associated genes, the draft genome sequences of seven C. perfringens NE isolates and one isolate from a healthy chicken were compared against nine non-poultry genomes, and three highly-conserved NE-associated loci (NELoc-1 – 3) were identified. The largest locus (NELoc-1) encoded 37 putative proteins, including NetB, an internalin-like protein, a ricin-domain protein, two leukocidins, several cell-surface proteins and a cyclic-di-guanidine monophosphate (c-di-GMP) signaling system. NELoc-1 and -3 were both localized to separate plasmids that are both predicted to undergo conjugative transfer. These findings suggest that NE pathogenesis involves multiple virulence factors that are encoded on discrete pathogenicity loci, some of which are plasmid-borne. To further elucidate the genetic basis of NE pathogenicity, a microarray was developed based on two of the sequenced NE bird isolates, and used to assess the gene content of 54 isolates from chickens with and without NE. Variable genomic regions associated with netB-positive isolates were identified, including several chromosomal fitness-related loci, such as a carbohydrate ABC transporter, ferric-iron siderophore uptake system, and adhesion locus. Additional loci were related to plasmid maintenance. This study suggests that chromosomal background confers a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism and plasmid maintenance Finally, the relationship between netB presence, NetB production and host NE status was examined to assess the hypothesis that netB-positive isolates from healthy birds frequently do not express NetB toxin. The expression of NetB toxin was determined in 57 poultry isolates, demonstrating that NetB expression is closely correlated with the presence of netB, and independent of host disease status. In conclusion, these studies have identified a number of C. perfringens genes predicted to play a role in NE pathogenesis, and suggest that NE is a complex, multifactorial disease involving both host and plasmid-encoded virulence factors.

A species threshold was used to assign unidentified Hymenoptera into DNA barcode Operational Taxa (DbOT) for both an assessment of species richness in rose gall communities and as part of a broad scale survey of genome size diversity. The species threshold of 2.2% was calculated from minimum interspecific divergence of DNA barcode (COI, mtDNA) and internal transcribed spacer region 1 (ITS1, rDNA) sequences from both identified and unidentified Hymenoptera associated with rose galls induced by Diplolepis (Cynipidae). Analysis of both DNA barcodes and ITS1 sequences suggested that several described species of Diplolepis (Cynipidae), Periclistus (Cynipidae), and Torymus (Torymidae) require re-examination to define species boundaries. It was also determined that the total number of DbOTs is higher than previous estimates of species richness of Hymenoptera associated with rose galls induced by Diplolepis. Additionally, genome size estimations were determined for 51 DbOTs from all eight families of Hymenoptera associated with rose galls induced by Diplolepis, five of which did not have any previous genome size estimates. A subsequent large-scale survey of Hymenoptera enabled by the use of the DbOT approach produced genome size estimations for 309 DbOTs from 36 families in 13 superfamilies. It was shown that Hymenoptera do not have smaller genome sizes than other holometabolous orders, and that a parasitoid lifestyle does not appear to constrain genome size. The suggested positive relationship between genome size and development time was investigated by comparing mean genome size of taxa with known or apparent differences in development rate. It was concluded that statistical comparisons between taxa that are grouped in broad categories would be unlikely to detect significant differences in mean genome size because the range of biological features within such categories is highly variable. However, comparisons between interacting groups with narrowly defined development strategies determined that mean genome size was statistically smaller in taxa that obtained resources within a narrow window of opportunity. This result suggests that rapid development in relation to competitors may be important in species of Hymenoptera with higher mortality risk.

No description available.

Telomeres are the ends of linear chromosomes tasked with preventing their recognition by the DNA damage machinery and providing a mechanism to solve the end replication problem. The telomeric DNA is mostly double-stranded, but it terminates in a 3’ protrusion termed the G-overhang. Telomeres utilize telomerase, a reverse transcriptase, to elongate the telomere, and thus, solve the end replication problem. Both the double strand region and the G-overhang are bound by specific proteins to facilitate the objectives of the telomere. First discovered in budding yeast, the CST (Cdc13(CTC1)/Stn1/Ten1) complex binds to the G-overhang and is important for both chromosome end protection and telomere replication. Work reported in this dissertation provided the first evidence that CST was present outside of yeast, which led to its subsequent identification in a number of vertebrates. Here I present the identification and characterization of the three components of CST in Arabidopsis thaliana. Similar to yeast, Arabidopsis CST is required for telomere length maintenance, for preventing telomere recombination and chromosome end-to-end fusions. Mutations in the CST complex result in severe genomic instability and stem cells defects. My research also shows that CST and telomerase act synergistically to maintain telomere length. Together these data provide evidence for an essential role for CST in maintaining telomere integrity. Unexpectedly, I discovered that the TEN1 component of CST may have a more complex role than other members of the heterotrimer. The majority of telomere-related functions we can assay using molecular and cytological approaches are shared by CTC1, STN1 and TEN1, though TEN1 has additional roles in maintaining genome stability, modulating telomerase activity and possibly non-telomeric functions in the chloroplast. I also present genetic evidence that TEN1 and STN1 act in the same pathway for the maintenance of telomere length and chromosome end protection. Interestingly, however, disrupting the STN1/TEN1 interaction reveals a separation of STN1 function for chromosome end protection versus telomere length maintenance. Finally, I describe the design and creation of a library of STN1 and TEN1 mutants that will be used to further characterize their functions and their interaction partners. By disrupting such interactions, it will be possible to elucidate the functional significance of these interactions, and thus, provide new insight into how CST functions in Arabidopsis.

The gene encoding chicken growth hormone (GH) was isolated from a chicken genomic library. The size of the gene was 4 kb. It was digested with PstI and subcloned into pUC18. Three of the PstI fragments were used for restriction fragment length polymorphisms (RFLPs) analysis at the GH locus in two chicken strains (fat and lean line). Four polymorphic sites were detected using a PstI fragment (PII) as a probe. One polymorphism was located at a SacI restriction site (PS1), and three at MspI sites (PM1, PM2 and PM3). A method based on polymerase chain reaction (PCR) was developed for detecting polymorphisms at PM3 site. A fragment of 823 base pairs which contained the PM3 polymorphic site was amplified. Three genotypes (+/+,$-$/$-$ and +/$-$) were distinguished by examining the MspI digested PCR products in either agarose or polyacrylamide gel. / Ten anonymous cDNA clones were also isolated from a chicken liver cDNA library and used for RFLPs analysis. Three of these clones were found to be able to detected RFLPs at MspI sites in chicken strains (strain 7, 8, 9, 8R, S and K) indicating that a high frequency of genes are polymorphic and can be used as markers in mapping experiments. One of the three clones was present on a haploid genetic element. Segregation analysis showed that the inheritance of this haploid gene was determined by the genotype of the female parent.

We report here the use of RAPD-PCR (Random Amplified Polymorphic DNA - Polymerase Chain Reaction) to identify segregating loci in the haploid progeny of an Armillaria ostoyae basidiocarp and the construction of the first genetic linkage map of this fungus, one of the causal species of Armillaria Root Disease. Upon screening 75 RAPD primers, 18 were found to identify a total of 43 loci segregating with a 1 : 1 Mendelian ratio. These loci were analysed for linkage among 58 monospore progeny. The map constructed with Mapmaker (LOD = 3.0, r = 0.38) was confirmed by GMendel (LOD = 1.5, r = 0.38). This map arranged 30 loci into 6 linkage groups and 4 linkage pairs. Thirteen markers remained unlinked. Using the Kosambi mapping function the linked loci accounted for approximately 450 cM and the genome was estimated to be 1600 cM. This preliminary map covers approximately 28% of the A. ostoyae genome.