Browsing by Subject "sequencing"
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Item ALLELE CHARACTERIZATION OF FIFTEEN SHORT TANDEM REPEAT LOCI OF NORTH AMERICAN GOLDEN (AQUILA CHYSAETOS) AND BALD (HALIAEETUS LEUCOCEPHALUS) EAGLES USING NEXT-GENERATION SEQUENCING(2014-03) Howard, Taylor E.; Kreutzer, McKensie; Curtis, Mary; Allen, Michael; Planz, JohnPurpose (a): The purpose of this study was to characterize by deep sequencing fifteen short tandem repeat (STR) loci in North American bald and golden eagles currently utilized by the US Fish and Wildlife Service. These STR loci are used for eagle identification in North America, however they were all developed from related species of European eagles. We hypothesized that using next-generation sequencing techniques we would be able to not only show concordance between allele calls using capillary electrophoresis (CE) methods and our new sequencing method, but also have an increased discrimination power of individuals by using this technique. Methods (b): Five samples of both bald and golden eagles were provided by the US Fish and Wildlife Service and deoxyribonucleic acid (DNA) was extracted from each using organic extraction. The current method of analysis through polymerase chain reaction (PCR) using fluorescent CE size based detection was adapted to create a protocol with increased specificity for the 15 sets of primers. Once the amplicon pool was developed, the amplicons were combined into a library using Library Prep Set for Ion Torrent™ kit for sequencing on the Ion Torrent™ Personal Genome Machine (PGM™) Sequencer® (Life Technologies™, Carlsbad, CA) with individual samples identified through barcodes embedded in the sequencing adaptors of the kit. After sequencing, the alleles were analyzed to identify the allele classification and determine the repeat structure of the STR motifs. The sequence data was used to determine if the sequences of North American population differed from existing reference sequences in GenBank®. Data was evaluated with the NextGENe® software using a virtual allelic ladder developed for each locus and contigs were assembled by anchoring the reads with the primer sequence. Results (c): Variants of alleles and allele distributions were recorded. The data revealed that the presence of sequence motif variation can increase the power of discrimination when using sequence analysis as compared to CE. Concordance was observed between the allele calls made using CE and with the sequence determined alleles. Conclusions (d): The method developed can be used to identify individual eagles with a higher discrimination power, which will be helpful in determining identity of individuals when needed for law enforcement investigations of harmed protected species such as bald and golden eagles.Item ALLELE CHARACTERIZATION OF TEN SHORT TANDEM REPEAT LOCI OF NORTH AMERICAN BEARS (URSIDS) USING NEXT-GENERATION SEQUENCING(2014-03) Kreutzer, McKensie; Howard, Taylor; Curtis, Mary; Allen, Michael; Planz, JohnPurpose (a): In the areas of conservation genetics and wildlife forensics, it is important to be able to accurately identify an individual and relate that individual back to the population from whence it came. With the low levels of genetic diversity possessed by some isolated North American bear populations, an identification method is needed that can provide higher resolution in short tandem repeat (STR) regions than current capillary electrophoresis assays. High resolution sequencing methods, such as those provided by next-generation sequencing (NGS) technologies, allow sequence motif changes within STR regions to be detected, whereas capillary electrophoresis only detects STR size. In this study, I hypothesized that when compared to capillary electrophoresis, NGS of STR loci would attain better genetic resolution among bear populations and thereby improve the accuracy of assigning an individual to its true population. Methods (b): Polymerase chain reaction (PCR) conditions were optimized for ten Ursid STR loci. An amplicon pool was generated and used to develop a library for NGS on the Ion Torrent™ Personal Genome Machine™ (PGM™) Sequencer (Life Technologies™, Carlsbad, CA). Deoxyribonucleic acid (DNA) barcode adaptors were ligated to the amplicons, thus allowing multiple samples to be sequenced in one run. Sequencing reads were aligned to a virtual ladder, first by sample via barcode, then by locus via primer. Sequence analysis was then performed using NextGENe® (SoftGenetics®, State College, PA) software. Results (c): Successful sequencing of ten loci for seven black bear (Ursus americanus) samples was carried out in one run. Allele call concordance was shown between capillary electrophoresis and NGS technologies. Variants within alleles (base changes) were evaluated and showed that NGS provided higher genetic resolution. Conclusions (d): In conclusion, NGS provided better genetic resolution than current capillary electrophoresis assays. Higher resolution has implications for increasing the accuracy of assigning an individual to its true population. Better assignment could improve genetic monitoring for conservation officers, as well as help wildlife forensic analysts improve evidentiary weight in wildlife crime cases that go to court.Item Molecular Cloning and Regulation of Expression of an NK Cell Receptor(2001-07-01) Medina, Miguel Angel; Porunelloor Mathew; Rafael Alvarez-Gonzales; Neeraj AgarwalNatural killer (NK) cells are large granular lymphocytes derived from bone marrow. They form the first line of defense against virally infected and tumor cells. Unlike B and T cells, they are not MHC restricted therefore do not require prior antigen stimulation (1-4). NK cell functions include producing various cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and granular-macrophage colony stimulating factor (GM-CSF) and cytotoxicity (5,6). A number of cell surface molecules have been identified, cloned and characterized that modulate NK cell recognition and activation by target cells (1). Most of these molecules are also expressed on other leukocytes. NK cell function is regulated by the balance of the positive and negative signaling through these receptors (3, 7-10). In the past attention has primarily focused on major histocompatibility complex (MHC) recognizing receptors that are mostly inhibitory (11). It is through these inhibitory receptors that levels of MHC molecules and associated peptides are monitored. Cells that have lost the expression of MHC class I molecules or have altered peptides-class I complexes are not able to transmit an inhibitory signal to NK cells and are consequently killed. Members of the CD2 subset of receptors play a major role in lymphocyte functions and do not recognize MHC molecules. The signaling lymphocyte activation molecule, SLAM (CD150), a member of the CD2 subset, is expressed on T cells and B cells. SLAM regulates T cell activation and production of immunoglobulins by B cells (12,13). 2B4 is a member of the CD2 subset and is expressed on NK cells as well as other leukocytes (14, 15). 2 B4 is a surface molecule implicated in the activation of NK cell-medicated cytotoxicity (15-17). Human 2B4 is a 60-70 kDA glycoprotein surface molecule found on all NK cells and a small subset of T cells that exhibit NK-like activity. CD48 has been identified as the high affinity ligand for 2B4 and implicates a broader role for 2B4 in immune regulation (18, 19). Recent reports have demonstrated the importance 2B4 and the functional role 2B4 plays in immune regulation. In X-linked lymporoliferative (XLP) disease NK cells can not be activated through surface 2B4 (20-23). The molecular adaptor protein, SLAM-associated protein or SH2 domain containing adaptor molecule (SAP/SH2D1A) is associates with cytoplasmic tail of 2B4 or SLAM (24, 25). Defective signaling via 2B4 and SLAM may contribute to the pathogenesis of X-linked lymphoproliferative disease due to mutations in SAP. The cytoplasmic domain of 2B4 contains four novel tyrosine motifs (TxYxxV/I) (14, 15). SLAM, a close relative of 2B4, also contains these novel tyrosine motifs. The signaling mechanism for 2B4 remains unclear. Along with other members of the CD2 subset 2B4 also localizes to chromosome 1. The genes that encode the CD2 family of receptors are locatedon human chromosome at 1q21-24 (24, 26-30). The murine genes for 2B4, CD48, Ly49, Ly108, and CD84 are located on the syntenic region of the long arm of the chromosome 1 (30-33). The exon arrangement for 2B4 is consistent with other CD2 subset members and consists of an exon per domain for the leader sequence, V-like domain, C2-like domain, and the transmembrane domains (27, 34-37). Differential exon usage leads to splice variants of the receptors, which complicates understanding the functional relevance between the cytoplasmic domains between receptors. Both murine 2B4 and SLAM demonstrates splice variants that alter the number of novel tyrosine motifs within the cytoplasmic domains (14, 34, 38). The murine 2B4 gene consists of 9 exons with one exon dedicated to each leader sequence, V-like, C2-like, and transmembrane domains. The total gene size is approximately 27 kilobases with the first intron consisting of 16 kilobases. Variable exon usage gives rise to two isoforms of 2B4, 2B4-L and 2B4-S, in the mouse (38). Four exons encode the 2B4-L cytoplasmic domain, giving rise to four tyrosine motifs. 2B4-S is identical to the 5’end of 2B4-L, differing only at the 3’ end in a portion of the cytoplasmic domain and the 3’untranslated sequence. 2B4-S is the product of the same first five exons in 2B4-L with the usage of a novel exon at the C-terminal. Although splice variants exists there Is no direct biochemical evidence to support their expression. In vitro analysis of the m2B4 variants suggest potential signaling differences. Murine 2B4 variants and mutants were transfected into a rat NK cell line, RNK-16. Interestingly, the two forms of 2B4 had opposing functions (39). Murine 2B4 is expressed on all NK cells, a subset of T cells, dendritic epidermal T cells, and monocytes (40). Expression levels of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 with anti-2B4 monoclonal antibody (mAb) causes secretion of interferon-γ, increased 2B4 expression, and elevated cytotoxicity (41). Characterization of how 2B4 and its related receptors are expressed is critical to the understanding not only the receptors’ biology but also NK cell biology. My first project will focus on mastering the techniques involved in the isolation and characterization of genes. Previously two genomic clones were isolated from 129 Sv/J mouse liver, 531 and 532. The first clone, 531, has been fully characterized and revealed to be 2B4. 532 has been partially characterized and revealed to the related form of mouse 2B4. In order to determine the function of 532 on mouse NK cells, 532 cDNA has to be isolated. I attempted to isolate 532 cDNA through PCR using previously isolated clones from the BALB/c cDNA library. My next aim was to isolate 532 genomic DNA for automated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated response molecule. The final portion of my thesis will focus on the isolation of the 2B4 activated response molecule (2ARM). Human peripheral blood NK cells were isolated incubated with interleukin-2 or C1.7. C1.7 is a monoclonal antibody that specifically recognizes human 2B4. RNA was extracted from these NK cells at various time points and used for RT-PCR to monitor the expression levels of 2B4. Aside from the expression of human 2B4, the expression of a 160 base pair transcript was also detected. Sequencing analysis revealed this transcript to be novel. I screened a human NK cDNA library constructed by Dr. J. Houchins (R & D System, Minneapolis, MN) using this 160 base pair transcript as a probe. Upon isolation of 2ARM cDNA, functional analysis can be performed to determine its role on human NK cells.