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investigated by searching A. mexicanum limb regeneration ESTs against UNIGENE zebrafish fin regeneration ESTs (Figure 2.3). This search identified 1357 significant BLAST hits (TBLASTX; E 400,000 zebrafish ESTs that were sampled from non-regenerating tissues was then performed to assess whether any of these potential regeneration homologues were represented uniquely in limb and fin regeneration databases (and not in databases derived from other zebrafish tissues). These comparisons revealed 43 that were unique to the zebrafish regeneration database (Table 2.10). Conceivably, these 43 ESTs may represent transcripts important to appendage regeneration. For example, this search identified several genes (e.g. hspc128, pre-B-cell colony enhancing factor 1, galectin 4, galectin 8) that may be expressed in progenitor cells that proliferate and differentiate during appendage regeneration. Overall, these results suggest that regeneration is achieved largely through the reiterative expression of genes having additional functions in other developmental contexts, however a small number of genes may be expressed uniquely during appendage regeneration. DNA sequence polymorphisms within and between A. mexicanum and A. t. tigrinum The identification of single nucleotide polymorphisms (SNPs) within and between orthologous sequences of A. mexicanum and A. t. tigrinum is needed to develop DNA markers for genome mapping (Parichy et al, 1999), quantitative genetic analysis (Voss and Shaffer et al, 1997), and population genetics (Fitzpatrick and Shaffer, 2004). The frequency of within species polymorphism was estimated for both species by calculating the frequency of SNPs among ESTs within the 20 largest contigs (Tables 2.6 and 2.7). These analyses considered a total of 30,638 base positions for A. mexicanum and 18,765 base positions for A. t. tigrinum. Two classes of polymorphism were considered in this analysis: those occurring at moderate (identified in 10–30% of the EST sequences) and high frequencies (identified in at least 30% of the EST sequences). Within the A. mexicanum contigs, 0.49% and 0.06% of positions were polymorphic at moderate and high frequency, while higher levels of polymorphism were observed for A. t. tigrinum (1.41% and 0.20%). Higher levels of polymorphism are expected for A. t. tigrinum because they exist in larger, out-bred populations in nature. 19
To identify SNPs between species, it was necessary to first identify presumptive, interspecific orthologs. This was achieved by first performing BLASTN searches between the A. mexicanum and A. t. tigrinum assemblies, then filtering the resulting alignments were to retain only those alignments between sequences that were one another's reciprocal best BLAST hit. As expected, the number of reciprocal 'best hits' varied depending upon the E value threshold, although increasing the E threshold by several orders of magnitude had a disproportionately small effect on the overall total length of BLAST alignments. A threshold of E
Page 1 and 2: ABSTRACT OF DISSERTATION Jeramiah J
Page 3 and 4: ABSTRACT OF DISSERTATION AMBYSTOMA:
Page 5 and 6: RULES FOR THE USE OF DISSERTATIONS
Page 7 and 8: AMBYSTOMA: PERSPECTIVES ON ADAPTATI
Page 9 and 10: Materials and Methods..............
Page 11 and 12: LIST OF FIGURES Figure 2.1, Results
Page 13 and 14: LIST OF FILES File Name Size Supple
Page 15 and 16: under which the female phenotype is
Page 17 and 18: CHAPTER 2: From Biomedicine to Natu
Page 19 and 20: Materials and Methods cDNA library
Page 21 and 22: minimum 100 bp match length, and 85
Page 23 and 24: indeed, there were approximately tw
Page 25: Informatic searches for regeneratio
Page 29 and 30: ESTs and to assess likelihood that
Page 31 and 32: Table 2.1: Tissues selected to make
Page 33 and 34: 26 Table 2.2 (continued) Locus ID F
Page 39 and 40: 32 Marker ID Primers a Table 2.3 (c
Page 41 and 42: 34 Table 2.6: Top 20 contigs with t
Page 43 and 44: Table 2.8: Functional annotation of
Page 45 and 46: 38 Table 2.9: Ambystoma contigs tha
Page 47 and 48: 40 TABLE 2.9 (continued) Ambystoma
Page 49 and 50: 42 Table 2.9 (continued) Ambystoma
Page 51 and 52: Table 2.10 (continued) A. mexicanum
Page 53 and 54: Figure 2.3: Results of BLASTN and T
Page 55 and 56: CHAPTER 3: A comprehensive EST Link
Page 57 and 58: Materials and Methods Study species
Page 59 and 60: additionally constrained to flank a
Page 61 and 62: linkage groups that consisted of on
Page 63 and 64: combined map length of LG1 and LG2
Page 65 and 66: needed to elucidate fully the effec
Page 67 and 68: and BLAST alignments can also be ob
Page 69 and 70: Table 3.2: Distribution of markers
Page 71 and 72: Figure 3.1: Statistical tests for M
Page 73 and 74: Figure 3.2 (continued) 66
Page 75 and 76: CHAPTER 4: Sal-Site: Integrating Ne
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elated databases. To create the AES
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as hyperlinks to separate marker fi
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Figure 4.1 - Schematic showing the
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CHAPTER 5: Evolution of Salamander
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metamorphic timing. These results s
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coding sequence for this EST was ob
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modes in WILD2, all individuals wer
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Genetic basis of discrete variation
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met alleles that increase or decrea
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Figure 5.1: Larval and adult phases
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CHAPTER 6: Gene Order Data from a M
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sequence alignments between transla
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Results Identification of putative
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N=309 for Ambystoma-human), and 2)
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identifiable between Ambystoma and
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and more variable rates of genome r
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Fissions derived within the mammali
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Table 6.2: Distribution of human/Am
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Figure 6.2: Frequency distributions
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Figure 6.5: Oxford plot of the posi
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Figure 6.10: Oxford plot of the pos
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evolution) that has evolved since t
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Sequence Alignment Similarity searc
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Software Requirements: MapToGenome
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substantial proportion of zebrafish
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segments within which linear order
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shows much stronger correspondence
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Figure 7.2: Oxford plots of alignme
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Figure 7.4: Plot of the λ values f
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Figure 7.6: Oxford plots of vertebr
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CHAPTER 8: Bird and Mammal Sex Chro
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(XCR) (Graves, 1995; Ross et al, 20
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uild 2.1 (http://www.ncbi.nih.gov/m
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identified between amniote sex chro
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evidence for ancestral linkage of X
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Figure 8.1: An abridged phylogeny o
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etween sex chromosomes, or by gene
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Rearing conditions At approximately
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AGGGCCTTCACATATTTTTCTGCAAAATAT). Th
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were females (respectively, Z A. me
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Localization of the major sex-deter
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and possibly other members of the t
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Table 9.1: Segregation of sex among
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Figure 9.3: Diagram of the crossing
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Other amphibians As genetic maps be
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REFERENCES Adrian, E. K. Jr. and B.
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Collins, J. P., J. L. Brunner, J. K
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Gardiner, D. M., M. A. Torok, L. M.
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Humphrey, R.R., 1957 Male homogamet
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Licht, L. E., L. A. Lowcock, 1991 G
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Ohno, S., 1967 Sex chromosomes and
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Sakata, N, Y. Tamori, M. Wakahara,
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Straus, N. A., 1971 Comparative DNA
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(superorder Afrotheria) reveals the
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Page, R. B., J. R. Monaghan, A. K.
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