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Introduction to Genetic Analysis (Introduction to Genetic Analysis (Griffiths))

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Chapter 12: The Genetic Control of Development I) Organizers—regions that are able to organize the development of the surrounding tissues A) Produce morphogens (molecules that induce responses in surrounding tissue in a concentration dependent manner) II) Four questions about number, identity, and function of genes taking part in development A) Which genes are important in development? B) Where in the developing animal and at what times are these genes active? C) How is the expression of developmental genes regulated: D) Through what molecular mechanisms do gene products affect development? III) Genetic toolkit for Drosophila (a small fraction of all genes) A) Housekeeping genes—encode proteins that function in essential processes in all cells of the body B) Toolkit—sets of genes that are concerned with building of organs and tissues and the specification of cell types. In order to understand gene function, need to know: 1) Mutant phenotype 2) Pattern of gene expression 3) Nature of gene product C) Many genes classified according to their function D) Homeotic mutants cause one body part to be transformed to resemble another body part 1) Single gene mutation can alter developmental pathway dramatically 2) Transform the identity of serially reiterated structures (body parts that are members of repeated series like limbs, digits, segments, etc.) 3) Mutation may cause loss of homeotic gene function where gene normally acts or may cause a gain of homeotic function where it doesn’t normally act 4) Homeotic genes (which controls the fate of segments along the anterior- posterior axis) are required throughout most of fly’s development E) Hox genes—eight loci that affect the identity of segments and associated appendages 1) Complete loss of Hox-gene function is lethal in early development 2) Located on the third chromosome 3) Bithorax and Antennapedia complexes contain the Hox genes 4) Order of genes in complexes and on chromosome corresponds to the order of body regions 5) Determined where each gene is expressed through molecular cloning of sequences 6) Visualization of gene expression in embryos and other tissues: a) Expression of RNA transcripts visualized by in situ hybridization i) Transcribe cDNA to contain single-stranded RNA probe w/modified nucleotides complementary to mRNA sequence. ii) Incubate embryos with RNA probe iii) Wash away unbound probe
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iv) Add enzyme-conjugated antibody to specific modified nucleotide v) Wash away unbound antibody vi) Add enzyme substrate vii) Visualize mRNA expression in light microscope b) Expression of Hox proteins visualized by immunological methods i) Express cDNA protein in bacteria ii) Inject protein into vertebrate host iii) Extract antibodies to protein iv) Incubate embryos with antibody which binds to protein v) Wash away unbound antibody vi) Add fluorochrome-conjugated antibody vii) Wash away unbound antibody viii)Visualize protein expression in fluorescence microscope 7) In absence of function of all Hox genes, segments form but they have the same identity (other genes control formation of segments, limbs, etc.)
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Chapter 12 - Chapter 12 The Genetic Control of Development...

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