Genetic Exam 2

half as much gene product is NOT enough
Aa and aa = mutant phenotype
haploinsufficient
AA is the only wt genotype
mutation CAN change the phenotype in a heterozygote

half as much gene product IS enough
only aa = mutant
haplosufficient
mutation CANNOT change the phenotype in a heterozygote

look at the phenotype of a heterozygote
cross true breeding wt x mut, observe F1

produces a mutant gene product that changes the wt gene parent

intermediate phenotype (blend)
e.g. red x white = pink

both phenotypes
e.g. blood type, A x B = AB

heterozygote looks like only one of the parents

allele interactions

independent assortment

more than 4 phenotypes, "doses"

recessive epistasis
two recessive alleles mask the dominant allele of the other trait
bb masks A

dominant epistasis
one dominant allele masks two recessive alleles of the other trait
A_bb and A_B_ are wt
A is epistatic to B

2 genes interacting in the same pathway
one gene makes the transcription factor, the other makes the product
A_B_ is wt, all others are mutant

being homozygous recessive for one gene (aa) can mask the defect caused by being homozygous recessive (bb) for the second gene

suppressor
A_B_, aabb, aaB_ = wt
A_bb = mut
aa suppressor is wt alone: does not cause mutant phenotype

suppressor is like mutant
A_B_ or aabb = wt
aaB_ or A_bb = mut
aa suppressor is mutant alone: caused mutant phenotype

synthetic lethality
aabb = DEATH

what genes or gene products interact with my gene or gene product of interest
start with mutant #1, perform a mutagenesis, look for death/lethality with a second mutation(conditional phenotype)

adding a chemical to make mutations

phenotype only present under certain conditions

two different mutations in the heterozygous condition are exhibited as the wild-type phenotype; indicates that the mutations are at different loci.
two wrongs make a right

mutations in the same gene

one gene, one protein/polypeptide/enzyme
for any enzyme in any biological pathway, there is a gene coding for it
1. is there a genetic control of chemistry?
2. irradiated Neurospora(fungus) and isolated mutants
3. tested mutants for biological function - classified auxotrophs on minimal media

catalyzes a reaction
turns reactants into products
RNA or protein encoded by genes

the same genotype yields different phenotypes
complicated gene analysis
% of individuals with an allele that exhibit the corresponding phenotype

the degree/severity of a phenotype varies with the same genotype

holds together two nucleotides
dehydration synthesis
1. lose H2O
2. lose pyrophosphate (pp)

2 rings
adenine and guanine

1 ring
cytosine, thymine, uracil

two hydrogen bonds

three hydrogen bonds

an enzyme that untwists the double helix at the replication forks, separating the two parental strands and making them available as template strands

relieves tension in DNA molecule by cutting and reannealinh DNA nuclease (cuts) and ligase (glues) for untangling
endonuclease
e.g. gyrase

an enzyme that joins RNA nucleotides to make the primer using the parental DNA strand as a template
DNAP needs a 3' OH to add nucleotides to

synthesizes new DNA only in the 5' to 3' direction
exonuclease
polymerize and proofread

removes RNA nucleotides of primer from 5' end and replaces them with DNA nucleotides
exonuclease

molecular scissors
splits nucleotides

pac man (if mistake)

Chromatin Assembly Factor 1, targets histones to the replication fork where they can be assembled with newly synthesized DNA to form chromatin after replication in eukaryotes

protein molecules around which DNA is tightly coiled in chromatin

repeating subunit of chromatin fibers, consisting of DNA coiled around histones
8 histones
repositioning affects gene expression

an enzyme that catalyzes the lengthening of telomeres
the enzyme includes a molecule of RNA that serves as a template for new telomere segments
still results in a 3' overhang, but lengthens DNA so that genes are not lost
made of protein and RNA
reverse transcriptase

synthesis 5' to 3' (add to the 3' end)
strands are complimentary and antiparallel
DNAP needs a 3' OH to add to, cannot start from scratch, DNAP III starts at primer

phosphate group
deoxyribose sugar
nitrogenous base

an enzyme that connects two fragments of DNA to make a single fragment
phosphodiester bonds

keep the strands of DNA separate during replication

a protein that encircles the DNA like a donut, keeping the enzyme DNA pol III attached to the DNA molecule at the replication fork

1. Is there a heritable molecule?
2. What is the heritable molecule?
3. What is the structure of the heritable molecule?
4. How does the heritable molecule replicate?

Griffiths 1928
used pathogenic bacteria (S), non pathogenic bacteria (R), and mice
injected mice with S, R, and boiled S
S = dead, R = live, boiled S = live (killed pathogenic bacteria)
boiled S + R = dead? HGT, R became S
conclusion: YES!

Avery 1944
took boiled pathogenic bacteria and performed an elimination experiment
removed one type of biological molecule in non pathogenic bacteria... do mice live?
when DNA removed, mouse lived
conclusion: DNA!

Hershey and Chase 1953
label: radioactivity
DNA virus T2
S35 = labeled sulfur, protein
P32, labeled phosphate, nucleic acid
S35 label stayed outside the cell after infection
P32 label went into the cell
conclusion: DNA!

1. Chargaff
analyzed % of AGCT in different organisms
%A = %T and %G = %C
2. Franklin
took a picture (x-ray crystallography) of a double helix
3. Watson and Crick
built the model by trying to put pieces together
conclusion: DOUBLE HELIX!

Messelson Stahl 1958
semi-conservative, conservative, or dispersive?
N14 = normal (light) nitrogen
N15 = isotope (heavy) nitrogen
used centrifugation: heavy on bottom light on top
1. start with E.coli in N15
2. put E.coli in N14 for one round of replication
saw strand in middle of tube, ruled out conservative
3. put E.coli in N14 for another round of replication
saw 2 strands in the tube, ruled out dispersive
conclusion: SEMI-CONSERVATIVE!

a molecule or molecular complex consisting of two identical molecules linked together

does not last long
very flexible
ribose sugar
ACGU
intramolecular bonds = different foldings, not just double helix

using a gene, making RNA

1. initiation
promoter sequence (not transcribed), where basal TFs (eukaryotes) are for RNAP binding
2. elongate
5' to 3' synthesis
3. termination
termination sequence

typicalsual/same sequence for some gene feature

bind enhancers to promote initiation
variation in cell type

where the gene is, new RNA will match exactly but with U instead of T

1. 5' cap
2. splice introns out of RNA
3. poly A tail

the end of a protein that has a free amino group

the end of a protein that has a free carboxyl group
phosphorylation RNAP's domain changes structure which changes function (for 5' cap, slicing, and poly A tail)

modified nucleotides
7-methoguanosine
5' to 5' linkage
RNAP needs enzymes for this because it only recognized 3' OH and adds, but this is a 5' to 5' linkage

the way a spliced intron looks

introns are always removed, but sometimes exons are also removed
one gene can produce multiple RNAs/proteins

more exons
splicing factors regulate where splicing happens on the RNA

expression data
1. extract all RNA
2. turn RNA into cDNA (DNA copy of RNA) by reverse transcriptase
3. sequence cDNA
4. map/align cDNA to the genome

modified by phosphorylation
sequentially recruits capping enzymes, spliceosome, and poly A machinery

enhancer: can go anywhere on the same gene, needs specialized TF
promoter: needs basal TF to bind
termination sequence: transcription ends, poly A tail
gene
exons
introns
start and stop codon
splice donors and acceptors

want to make light purple petunia dark purple
added purple making enzyme
ended up with white petunia?
conclusion: the purple enzyme added must have been double stranded, double stranded RNA is the trigger for RNAi, blocks and destroys double stranded molecules and others that look like it

what triggers gene silencing?
knew: adding antisense RNA complementary to a protein transcript (mRNA) blocks protein production
control: adding more sense RNA
surprise! adding more sense RNA also blocked protein production
1) sense RNA = wt
2) antisense RNA = wt
3) doble stranded RNA = twitcher (unc-22)
1st Nobel Prize 1998

discovered miRNAs
our cells make microRNAs (dsRNAs) that pick on other genes
about 2,000 in our genome
regulate genes
2nd Nobel Prize 2025

RNA induced silencing complex
gets rid of dsRNA and RNA with the same sequence as the dsRNA (targets)
+ 1/2 of the dsRNA:
-binds to the 3' UTR of the target
-degrades then blocks translation (interferes) with the target DNA

between amino acids (monomer) to make a protein (polymer)
formed by RNA

sequence of amino acids

alpha helix or beta pleated sheet

3D protein

multiple peptides coming together

3 nucleotides
codes for one amino acid

DNA to RNA

RNA to protein
mRNA to amino acid
changes languages

brings anticodon amino acid complimentary to the codon on the mRNA into the ribosome

ribosomal RNA; type of RNA that makes up part of the ribosome
catalysis
makes peptide bond

Crick and Brenner
we know there are 20 amino acids:
1 nt = 4^1 = 4 total a.a.
2 nt = 4^2 = 16 total a.a.
*3 nt = 4^3 = 64 total a.a.
INDELS (insertions and deletions)
vII mutations in T4 phage (mutants)
(+) + (-) = wt
(+) + (-) = mut
A suppressor mutation in the same gene gets phenotype back to wild type

provides energy for translation processes.

no!
if the code overlapped, 3 codon changes = 3 a.a. changes
however, we see that 1 nucleotide change = 1 a.a. change

a "fake tRNA" protein called the release factor

Nirenberg and Matthaei
synthetic -UUUUUU- = phe - phe... 3 mean something!
(UA)n = ile - tyr - ile- tyr... which is which?
(AUA)n = ile - asn - ile - asn... AUA present in both codes, must be ile!
conclusion:
UAU = tyr
AUA = ile
AAU = asn
ribosome can jump on sequence anywhere

certain a.a. are changed to STOP
specifically codons for 6 a.a. become STOP because they are already close

glu, lys, g.a., tyr, trp, and ser are the most likely to be changed to STOP because only 1 nucleotide has to change

how we know the codon table, 1 START, and 3 STOP

joines tRNA and amino acid together

in the A site, first 2 nucleotide positions are "locked"
3rd does not really matter, tRNA is planted enough to peptide bond its amino acid to the a.a. chain

AaBb x AaBb = non-mendelian
13:3 or 10:6
double recessive either causes mut or wt alone but suppresses other mutation when aabb

INDEL leading to +++ or ---
(+ for ++) (- for --) (+ for -)
restores reading frame after first INDEL

suppresses a premature STOP codon from a missense mutation in a tRNA
tRNA recognizes UAG (STOP) and puts AUC (tyr)

translation initiation relies on... ribosome needs to use the right AUG to start

consensus sequence in prokaryotes
light 70s ribosome

consensus sequence in eukaryotes
heavy 80 ribosome
cap binding

they target bacterial ribosomes, so it kills bacteria but not us

mitochondria is very similar to prokaryote so antibiotic can target it
antibiotics also kill good bacteria in our bodies

lead to antibiotic resistance

an organism's complete set of proteins

direct alteration of one or more nucleotides in an mRNA that has already been synthesized

add or subtract certain chemical groups by phosphorylation for activity
ubiquitinationbiquitylation

ubiquitin added (76 a.a. peptide), tags proteins for recycling via the proteosome (recycling bin of cell), tags with ub to change structure + function

a lot faster
can respond quickly to the environment

proteins encode for localization
outside the cell --> membrane --> nucleus
protein domains that interact with transport machinery

regulation of gene expression (transcription of RNA)

in prokaryotes
bacteria responding to lactose need an enzyme to break it down
make enzyme when lactose present, don't make is glucose present

breaks down lactose into glucose and galactose in prokaryotes
glucose is prokaryotes preferred food

DNA region encoding multiple genes (multigenic, polycistonic) and includes an adjacent regulatory sequence

codes for the repressor of the lac operon

a gene system whose operator gene and three structural genes control lactose metabolism in prokaryotes
if lactose present, repressor is taken off
effector = lactose
lactose binds to the repressor so that B-gal can be made

encodes B-galactosidase

codes for permease, a protein which transports lactose into the cell

codes for transacetylase

if bound to operator (seq), stops protein production

binds to a repressor so it no longer associates to the DNA
changes transcriptional regulators (allosteric proteins)

binds to an activation site (seq)

can change in response to an inducer/effector

only affects the same piece of DNA

genes that are expressed all the time

a protein that regulates the process of RNA splicing
determines which GU is a splice donor and which AG is a splice acceptor
protein acting in trans, binding to sequence to dictate which way splicing happens

middle of intron that starts to move

branch point moves to splice donor (GU), creates a loop
splices at splice donor
exon 1 connects to exon 2
splices at splice acceptor

lasso
what a spliced intron resembles

a group of proteins that are structurally and functionally related

made up of snRNPs (small nuclear RNAs and proteins)
splices introns out of RNA
localized in the nucleus

binds to 3' UTR
carries an RNA complementary to the 3' UTR
binds to target RNA strand

target degraded
blocks translation

dsRNA triggers
unwinds the dsRNA, holds on to a strand
degradeslocks translation of other RNA with a complementary sequence

acting on the same DNA molecule
promoters, enhancers, operators

acting on different DNA molecules
repressors, transcription factors, splicing factors

gene that RISC carries the complimentary RNA to in order to silence it