Mendelian_Genetics Flashcards
complementation test
used to distinguish whether mutations are allelic variants of a single gene, or of different genes.
what types of genes must you use for complementation tests? why?
recessive, since in practice you will need to overcome (compensate for) the mutant allele with the wild type
what happens during a complementation test if both mutants are in the same gene?
they are both recessive, so no complementation, and the organism has mutant phenotype
what happens during a complementation test if mutants are in different genes?
Gene complement each other (heterozygotes in trans), and you get wild type phenotype
how do you tell how many complementation groups are in your set of mutants?
make series of punnett squares, mark a + for complement, and - for noncomplement, then advance out until you’ve assigned groups
what are two broad ways you can become an exception to a complementation test?
intragenic complementation, and non-allelic non-complementation (second site)
what are three ways you can mechanistically achieve intragenic complementation?
different domains in a multi-domain protein could interact, different alleles could have reduced dosage affects, and stabilization of products created by multiple complementary changes in the protein structure (suppression)
what is non-allelic non-complementation?
also called haploinsufficiency, happens if one wild-type copy of a gene is not sufficient to rescue a system to WT phenotype
what are two mechanistic ways you can get haploinsuffiency?
if your protein has a dosage threshold and needs to make a certain amount to function at all (vesicles fusing at synaptic cleft) or mutant alleles poison the system
give me some yeast life cycle/mating facts! (5)
- can be haploid or diploid
- haploid state = 16N, and are called mating type alpha
- Haploids can make other haploids or mater with another alpha to produce a diploid
- Diploid can stay diploid or undergo meiosis
- Both haploids and diploids do cell cycle in 90 min
what is reverse genetics?
start with a gene mutation, examine resulting phenotype
how is a wild type gene denoted?
italicized, all upper case
how is a mutant gene denoted?
italicized, all lower case
how is a protein denoted?
no italics, first letter capitalized and the rest lower case
how is a mutant protein denoted?
no italics, all lower case
what are two types of selectable markers?
auxotrophic, antibiotic
how does an auxotrophic marker work?
it provides an essential gene that a particular organism cannot live without. commonly used are biosynthetic pathways (Ura3 or HIS3). so these experiments have to be carried out in organisms with these essential genes deleted.
how are selectable markers incorporated?
you position them right beside the gene of interest’s ORF (close enough to link), target mutant allele to the locus using 50-500bp of endogenous locus (homology arms, everything introduced to cell by transformation), then homology arms find the locus and take advantage of recombination machinery
how often does recombination happen in mammalian vs yeast cells?
1/20,000 in mammalian, 1/100 in yeast
advantages to working with yeast?
haploid or dipoid, quick cell cycle, highly recombinagenic, can see cell cycle under light microscope
what is sporulation?
production of haploid spores post meiosis of diploid yeast
amorph
no phenotype
T/F. Null allele behaves the same way as a deletion
True
hypomorph (loss of function)
recessive allele with less function than a WT, but more gene function than a null
Classic way to distinguish allelic classes (phenotypic analysis)
mutant/deletion, and mutant/2X WT overexpression, then see if phenotype gets “better” or “worse”
what is a neomorph?
gain of function allele - protein misexpression or novel ligand binding
what if a mutation is lethal?
can use conditional alleles
what are some triggers for a conditional mutation?
heat, cold, mechanical stimulation, addition of a specific molecule
reverse genetics examples?
delete a specific gene to look at its null phenotype, make a particular mutation to see how it affects protein function
common tools for reverse genetics?
crispr, RNAi, TALENs, selectable markers
forward genetics examples?
ID mutants with interesting phenotype, random mutations
common tools for forward genetics?
Drosophila P-elements, chemical mutagens, Tn-seq
why does percent cell survival tank with increasing concentration of a mutagen?
likelihood of hitting an essential gene increases
caveats to mutagenesis
can have multiple mutations in one cell, you must back-cross to wildtype, you will miss some genes (essential, short, redundant)
T/F. All phenotypes are selectable
False, this is a drawback to selection experiments
T/F. Screens are more biased than selection
False
