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Title: Molecular cloning of chicken transforming growth factor β1 and isolation of microsatellites
Author: Long, Qiaoming
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Transforming growth factor-Bs (TGFBs) are candidate genes in the control of chicken development and growth. It is very important to study the expression, regulation and functions of these genes. Microsatellites are ideal DNA markers for genetic linkage studies and genome mapping. An increasing numbers of studies have demonstrated that they may also be involved in DNA homologous recombination, gene regulation and genome rearrangement in vivo. The [CAG/CTG]n triplets have an important role in transcriptional factors. We have attempted to clone the 5' region of the chicken TGFB1 gene to facilitate a more detailed analysis of the expression and developmental role of TGFBs. We have developed a method for enriching microsatellites in chicken DNA libraries to facilitate the generation of polymorphic markers and the identification of potential transcriptional factors. We have shown that chicken TGFB1 exists as an single copy gene with an upper size limit of approximately 15 to 22 kb. However, screening of a lambda phage chicken genomic library using both the TGFB1 cDNA and oligo probes failed to obtain chicken TGFB1 clones. Procedures were developed for the construction of specific microsatellite-enriched DNA libraries. [CA/TG] n-enriched genomic DNA libraries were constructed using "genetic marker selection" and DNA affinity hybridisation procedures; whereas [CA/TG] n- and [CAG/CTG] n-enriched liver cDNA libraries were constructed by a DNA affinity hybridisation procedure. The frequency of positive clones in the DNA libraries constructed ranged from 0.5% to 5% depending on the type of microsatellite repeat. An enrichment of 50 fold over the classical small-insert DNA libraries has been achieved. Microsatellite-positive clones from both the genomic DNA libraries and the cDNA libraries were identified and characterised by sequencing. Microsatellite polymorphisms were studied by polymerase chain reaction and are being mapped using the EAST LANSING and COMPTON reference mapping crosses. A search of the non-redundant databases using the available information of expressed sequences revealed chicken homologues of human transcriptional factor, myocyte enhancer factor 2D (MEF2D) and human Fragile X syndrome (FMR1) as well as a substantial number of new other genes. The differential pattern of expression of the chicken MEF2D gene was examined in different chicken tissues and at various ages. A ubiquitous distribution of chicken MEF2D transcripts was revealed. Chromosome mapping and a study of the pattern of expression of these genes is underway. Comparative mapping of closely related avian species using microsatellite markers from chicken cDNA library is discussed. The isolation of these microsatellites will facilitate the mapping of economically important quantitative traits in chicken. The mapping of expressed sequences will help to define candidate genes of these and other traits.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available