Dissection of gene function: mutational analysis. Using Model organisms Defining the genes Forward Genetics Reverse Genetics. A. Using Model organisms. Defining a Model – Organisms suitable for genetic experimentation
Yeast – genes easily manipulated due to their haploid/diploid lifecycle. Can easily detect recessives in haploid cells, and diploid makes complementation tests possible.
Balancer chromosome – prevents the recovery of crossover products, so together w/ it’s homologous normal chromosome – they act like a haploid, no recombinants passed on to progeny.
Progeny carrying chromosomes that are the products of recombination between balancer and normal chromosomes are not viable.
P element = 2907-bp sequence features a perfect 31-bp terminal inverted repeat and an 11-bp subterminal inverted repeat for efficient transposition, along with other repeat units of unknown function plus a transposase gene. Can be used to assist in cloning (insertion mutations).
Model for human diseases
Determine if the mutant alleles are alleles of one gene or of different genes
Cross two mutant strains and analyze the F1 generation:\
There are two alternate outcomes -
case 1 – all offspring are normal
case 2 – all offspring are mutants
The two mutations are in separate genes and are not alleles of one another. Each F1 is heterozygous at both loci
The two recessive mutations affect the same gene and are alleles of one another
If two recessive mutations are alleles of the same gene, then the phenotype of an organism that contains one copy of each mutation is mutant;
if they are alleles of different genes, then the phenotype of an organism that contains one copy of each mutation is wildtype.
Gene then mapped using techniques described, molecular markers linked to the gene can be used to isolate clones from an existing library
Only the desired phenotypes evade the filter.
Oligonucleotide synthesized & then hybridized to SS M13, complementary region anneals, however the non-complementary region doesn’t. DNA pol extends the 3’ end of the oligo to create a DS M13 DNA molecule, ligase seals the deal. The DS molecule is then transformed into E. coli.
C1-THF synthase in 9.5 day mouse embryo. Tissues that stain specifically are neural tube, vasculature of heart, limb bud, first brachial arch, cranialfacial region, umbilicus, inner ear.
Epithelial cells undergoing apoptosis in mammary gland. Immunofluorescence staining of caspase 3 activity in shed, apoptotic cells (green). Nuclei of surrounding luminal cells are stained in red.