Introduction

Question 1

Does a gene include coding AND regulatory regions?

Answer 1

Yes

Question 2

What is gene expression?

Answer 2

process of converting genes to “observable product” usually a protein

Question 3

mutation

Answer 3

• transmissible permanent change in nucleotide sequence of chromosome, often in gene
• may change function

Question 4

mutant

Answer 4

organism having a mutant gene that expresses itself in phenotype (silent mutations do not count)

Question 5

organism with silent mutations still counts as mutant

Answer 5

false!

Question 6

exon vs intron

Answer 6

exon - coding
intron - non-coding

Question 7

prokaryotes have introns AND exons

Answer 7

false, they have only exons

Question 8

CDS vs ORF

Answer 8

CDS = coding sequence
ORF = open reading frame
SAME thing!

Question 9

on a DNA strand, is +x up or downstream?

Answer 9

DOWNstream

Question 10

tR = ?

Answer 10

terminator - stops trnscription

Question 11

UTR =?

Answer 11

untranslated regions

Question 12

where are promoters?

Answer 12

upstream of CDS

Question 13

splicing

Answer 13

to remove non-coding regions (introns, Euk) in mRNA, AKA intragenic, and joining exons

Question 14

what are some protein functions?

Answer 14

structural proteins, enzymes, regulators, hormones, receptors, neurotransmitters, transporters, antibodies

Question 15

What are the (2) ways to study gene expression and function of specific proteins?

Answer 15

1) biochemical approach
2) genetic approach

Question 16

biochemical approach

Answer 16

• 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

Question 17

how is cDNA made?

Answer 17

reverse transcription from RNA
(so no introns)

Question 18

genetic approach

Answer 18

• 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

Question 19

gene suppression determines?

Answer 19

interactions between diff genes(proteins)

Question 20

complementation analysis determines?

Answer 20

whether mutations are in the same or different genes

Question 21

what is WGS? (incl steps)

Answer 21

whole genome shotgun sequencing
- very efficient
- “shotgun” - untargeted

DNA extraction -> DNA shearing -> DNA library prep -> DNA library sequencing -> DNA seq anal

Question 22

model organisms: pros/cons? (general)

Answer 22

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)

Question 23

Escherichia coli bp#?

Answer 23

4 million bp

Question 24

Caenorhabditis elegans?

Answer 24

developmental studies, nematode

Question 25

Saccharomyces cerevisiae bp# and pupose?

Answer 25

14 million bp unicellular eukaryote

Question 26

Drosophila?

Answer 26

insect/invertebrate model developmental studies

Question 27

Arabidopsis thaliana

Answer 27

smallest plant model

Question 28

Xenopus laevis bp # and purpose

Answer 28

3 billion bp (similar to humans) vertebrate development

Question 29

mice/rats bp # and purpose?

Answer 29

3 billion bp developmental/disease/protein studies

Question 30

humans bp #

Answer 30

3 billion bp

Question 31

transgenic mice used to analyze ___

Answer 31

promoters

Question 32

promoter can be attached to ___ gene (e.g., GFR, fluoresce), _____

Answer 32

reporter gene "transcription fusion"

Question 33

transgenic or knockout organisms

Answer 33

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!

Question 34

solution to model organism cons: (1) - ethics - economics - reproducibility of results

Answer 34

solution = Cell Culture - ethical - economical - high reproducibility

Question 35

in vitro, ex vivo, in vivo, in silico

Answer 35

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

Question 36

do all models have limitations? how does extrapolation affect that? how can we have greater understanding?

Answer 36

YES more limitations, can lead to misinfo study of both in vitro & in vivo -> greater understanding

Question 37

cell culture (in vitro) usage cons:

Answer 37

- sometimes difficult to extrapolate some results to whole organism (e.g., developmental processes, immune system, etc) - consider in vivo data vs in vitro data