Introduction Flashcards
Does a gene include coding AND regulatory regions?
Yes
What is gene expression?
process of converting genes to “observable product” usually a protein
mutation
- transmissible permanent change in nucleotide sequence of chromosome, often in gene
- may change function
mutant
organism having a mutant gene that expresses itself in phenotype (silent mutations do not count)
organism with silent mutations still counts as mutant
false!
exon vs intron
exon - coding
intron - non-coding
prokaryotes have introns AND exons
false, they have only exons
CDS vs ORF
CDS = coding sequence
ORF = open reading frame
SAME thing!
on a DNA strand, is +x up or downstream?
DOWNstream
tR = ?
terminator - stops trnscription
UTR =?
untranslated regions
where are promoters?
upstream of CDS
splicing
to remove non-coding regions (introns, Euk) in mRNA, AKA intragenic, and joining exons
what are some protein functions?
structural proteins, enzymes, regulators, hormones, receptors, neurotransmitters, transporters, antibodies
What are the (2) ways to study gene expression and function of specific proteins?
1) biochemical approach
2) genetic approach
biochemical approach
- start from PROTEIN, go towards DNA
isolate protein,
determine AA,
synthesize oligonucleotides that correspond to AAs (predict sequence),
use oligonucleotides as probes to select cDNA or library genomic clone protein,
sequence isolated gene
GOAL: deduce nucleotide sequence of gene
how is cDNA made?
reverse transcription from RNA
(so no introns)
genetic approach
- start from GENE (wildtype vs mutated) -> go towards protein
isolate genomic clone corresponding to altered trait in mutants,
use genomic DNA to isolate cDNA for mRNA (reverse transcription),
sequence cDNA to deduce AA,
compare AA with known proteins (to gain info abt function),
use expression vector (plasmid) to produce said protein in vivo
gene suppression determines?
interactions between diff genes(proteins)
complementation analysis determines?
whether mutations are in the same or different genes
what is WGS? (incl steps)
whole genome shotgun sequencing
- very efficient
- “shotgun” - untargeted
DNA extraction -> DNA shearing -> DNA library prep -> DNA library sequencing -> DNA seq anal
model organisms: pros/cons? (general)
pros: simple, no introns in prok, small/cheap, easy maintenance, short reproductive period, avail data, availability
cons: SOMETIMES results can be extrapolated to more complex organisms, but sometimes it won’t work! ethics, economics, reproducibility of results (due to differences between organisms)
Escherichia coli bp#?
4 million bp
Caenorhabditis elegans?
developmental studies, nematode
Saccharomyces cerevisiae bp# and pupose?
14 million bp
unicellular eukaryote
Drosophila?
insect/invertebrate model
developmental studies
Arabidopsis thaliana
smallest plant model
Xenopus laevis bp # and purpose
3 billion bp (similar to humans)
vertebrate development
mice/rats bp # and purpose?
3 billion bp
developmental/disease/protein studies
humans bp #
3 billion bp
transgenic mice used to analyze ___
promoters
promoter can be attached to ___ gene (e.g., GFR, fluoresce), _____
reporter gene
“transcription fusion”
transgenic or knockout organisms
transgenic - an organism with foreign DNA introduced artificially (one or more cells)
- DNA can be integrated in random fashion by injecting into pronuclei of fertilized ovum
controlled experiments (consequences/ethics?)
knockout - no protein (phenotype) for knocked out gene; DNA is introduced first into embryonic stem cells - targeted insertion -> knockout!
solution to model organism cons: (1)
- ethics
- economics
- reproducibility of results
solution = Cell Culture
- ethical
- economical
- high reproducibility
in vitro, ex vivo, in vivo, in silico
in vitro = test performed OUTSIDE of normal physiological env (living organism) but certain env conditions are maintained
ex vivo = test performed OUTSIDE of normal physiological env (living organism) BUT with a direct link to living organism, which provides a continuous supply of material
- e.g., connected to circulatory (blood) supply
- e.g., work done on/in tissue taken from organism, put back into organism
in vivo = test performed WITHIN normal physiological env
in silico = modelling in computers
do all models have limitations? how does extrapolation affect that? how can we have greater understanding?
YES
more limitations, can lead to misinfo
study of both in vitro & in vivo -> greater understanding
cell culture (in vitro) usage cons:
- sometimes difficult to extrapolate some results to whole organism (e.g., developmental processes, immune system, etc)
- consider in vivo data vs in vitro data
