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Browsing by Author "McFadyen, David A."

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    Developing microsatellite markers for Cypripedium passerinum (Sparrow’s egg lady’s slipper)
    (2022) Lim, Lina; McFadyen, David A.
    Natural and anthropogenic disturbances contribute to increased habitat loss and fragmentation and subsequently, species loss. Integrated conservation approaches combine both in-situ and ex-situ approaches whereby natural habitats of endangered species are conserved, and the genetic diversity of the threatened population is retained outside of their natural habitat. Therefore, an essential component of an effective conservation strategy is to assess genetic variation to ensure that the conservation approach employed is effective in preserving the diversity of the whole population. Microsatellites, highly polymorphic repetitive DNA sequences in the genome of all organisms, have proven to be a valuable tool in the assessment of genetic diversity. This project aimed to isolate microsatellite markers from Cypripedium passerinum, a native North American terrestrial orchid at risk of extinction. Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) was employed to generate a genomic DNA library enriched for AT, AC, and AAG microsatellites. Clones were selected from the libraries and bidirectionally sequenced to identify those which contain microsatellites. A total of 158 microsatellite loci were identified, of which 83% were perfect microsatellites. PCR primers were developed using the unique sequences flanking the identified microsatellites and were evaluated for their utility. Primers amplifying polymorphic loci can be used to assess the genetic diversity of C. passerinum populations both within the Wagner Natural Area, Alberta, Canada and elsewhere in its range of distribution. The project findings will contribute to the integrated conservation efforts to protect species found in Wagner Natural Area and contribute to our understanding of C. passerinum.
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    DNA barcoding of Masdevallia orchids using the matK locus
    (2018) Drever, Josh; McFadyen, David A.
    Maintaining global biodiversity is becoming more of a focus as the quality of this biodiversity declines. Conservation efforts need to be targeted at areas where this loss of biodiversity is most critical. Orchids are a family of plant facing significant survival pressures. Masdevallia is a genus of neotropical orchids which is poorly represented in orchid studies. When not flowering, individuals of this genus are morphologically indistinguishable from each other. DNA barcoding will assist in targeting conservation efforts by genetically identifying unknown species in threatened ecosystems. Many different loci in the orchid genome have been examined for use as a barcode, and the matK locus has had the best results. The objective of this study is to use the matK locus to create a DNA barcoding system which distinguishes between individuals of the Masdevallia genus. DNA has been isolated from samples and PCR has been done to amplify the matK locus. PCR products were sequenced using ABI sequencing, and the resulting sequences were aligned to create a phylogenetic tree. This tree contains unedited sequencing data, so while not conclusive, it indicates that this DNA barcoding system is sufficient to distinguish between samples at the species level. This will contribute to a DNA library, so unknown orchid individuals may be better identified in threatened ecosystems.
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    Genomic organization of the rat alpha(2u)-globulin gene cluster
    (1999) McFadyen, David A.; Addison, William; Locke, John
    The α2u-globulins are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many α2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 α2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the α2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the α2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing α2u-globulin genes indicated that the α2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the α2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes.
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    High-resolution FISH mapping of the rat alpha(2u)-globulin multigene family
    (2000) McFadyen, David A.; Locke, John
    The rat α2u-globulins are a group of similar proteins that are encoded by a family of approximately 20 genes located a single locus of ≤880 kbp on Chromosome (Chr) 5q. Individual members of this gene family demonstrate complex tissue, hormonal, and developmental expression patterns despite a high degree of sequence similarity among the members and consequently provide an interesting system for studying the evolution of differential gene expression. Hybridization analysis indicated that gene classes, similar to those identified at the homologous MUP locus in the mouse, do not exist within the rat α2u-globulin locus. Furthermore, cross-hybridization analysis revealed the presence of conserved sequences in the 5′ and 3′ regions flanking the α2u-globulin genes, some of which were present in an inverted orientation. We have used high-resolution fiber FISH to examine the structural organization of the α2u-globulin locus, and found the genes to be arranged as an array of both direct and inverted repeats. The organization of the rat α2u-globulin genes differs from the MUP genes and suggests different evolutionary events have reorganized these homologous sets of genes.
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    Three subsets of genes whose tissue specific expression is sex and age-dependent can be identified within the rat alpha(2u)-globulin family
    (1997) Wang, Kathy S.; McFadyen, David A.; Locke, John; Hodgetts, Ross B.
    The rat α2u‐globulins are encoded by a multigene family whose 20–25 members are subjected to multihormonal regulation that is dependent upon the sex of the animal, the developmental stage and the tissue being examined. Using RT‐PCR and diagnostic restriction analysis of the products, we have examined the specificity of the expression of different members of the gene family. All family members can be classified into three subsets, depending on how the amplified cDNA responds to digestion with ApaLI, SstI and VspI. Subset A contains the restriction sites for both ApaLI and SstI but not VspI and typifies the genes expressed in the salivary glands of both mature and juvenile animals of both sexes, where it is the only subset expressed. This subset of genes also accounts for all the transcripts observed in the kidneys and mammary glands of juvenile males. Although subset A was represented in the transcript populations of all the other tissues examined, its proportion relative to the total varied greatly. The two other subsets were subset V, which contains only the restriction site for VspI, and subset N, which lacks all three restriction sites. In all the other tissues examined, two or all three of the subsets were expressed, usually in a manner that was unique to the sex and age of the tissue in question. The proportion of each of the three α2u‐globulin subsets in the α2u‐globulin gene family was determined by quantitation of the restriction products of amplified genomic DNA. Interestingly, the most prevalent subset in the genome (N) has the most limited tissue expression pattern, but is found in liver and preputial glands, the tissues expressing the most substantial quantities of α2u‐globulin. These results indicate the complexity of the regulation of the α2u‐globulins and point to the necessity for gene specific analyses if the expression of the family is to be understood in molecular terms.