genetics exam 4

cell theory

• living organisms are composed of one or more cells

• cell is a basic unit of life of the structural organization of an organism

• cell arrive from pre-existing cells and are NOT spontaneously generated

protobiont

• a precursor to living cells

• favor the idea that rna was the first macromolecule found in protobionts 

• consisted of an aggregate of molecules and macromolecules that acquired a boundary

• maintained an internal chemical environment distinct from that of its surroundings

rna world hypothesis

• rna can perform both info storage and enzymatic activity

• rna is involved in each of the major steps of gene expression

• maybe rna predates both dna and proteins

noncoding rna

• genes that don’t encode polypeptides

• binds to different types of molecules

• form different structures via intermolecular base pairing

• rna molecules can form stem-loop structures which may bind to pockets on the surface of proteins

long non coding rna

• longer than 200 nucleotides

• mis regulation of lcRNAs are is involved in many diseases

small regulatory rna (short ncRNA)

• shorter than 200 nucleotides 

• microRNA

ribozyme

• ncRNA molecules with catalytic function

• RNA enzyme/catalytic RNA

scaffold

• ncRNA binds a group of proteins at multiple binding sites

guide

• ncRNA binds to a protein and guides it to a specific site in the cell

• use base-pairing to direct proteins to specific locations (CRISPR)

decoy

• ncRNA recognizes another ncRNA and sequesters it 

• provides an alternate binding site for an inhibitory miRNA 

• miRNA normally binds to mRNA inhibiting translation

• decoy binds to the miRNA preventing binding to the mRNA 

blocker

• ncRNA physically prevents or blocks a cellular process from happening

• translation is repressed by ncRNA called micF, which does not code for a protein and is complementary to the to-be-translated gene

rRNA large subunit 

• ribozyme catalyzes peptide bond formation

RNase P

• a ribozyme endonuclease that cuts the 5’ end of precursor tRNAs to the correct position

small nucleolar RNAs (snoRNAs) 

• found in high amounts in the nucleolus 

• synthesis of rRNAs and the assembly of ribosomal subunits occurs in nucleolus 

• guide enzymes to covalently modify rRNAs in important locations 

• methylation of ribose on the 2’ hydroxyl group

• conversion of uracil to pseudouracil

snoRNAs act as scaffolds for modification proteins

• C/D box snoRNA methylation of ribose

• H/ACA box snoRNA converts uracil to pseudouracil

• scaffold function of snoRNAs to create snoRNPs

snoRNAs act as guides

• use base pairing to bring the modifying enzymes to the correct location on the rRNA

sense vs antisense rna 

• antisense rna: complementary to the mrna 

• sense rna: the mrna 

DsrA

• trans-acting ncRNA that can positively and negatively regulate translation in bacteria

• inhibits hns (histone-like nucleoid structuring protein) by blocking the RBS (ribosome biding site/shine-dalgarno) and part of DsrA is antisense

• activates rpoS (alternative sigma factor) by binding to the part of the rpoS mRNA that is complementary to the RBS (thus freeing it from stem-loop)

DsrA is complementary to multiple mRNAs

• different parts of DsrA have complementarity base-pairs to the hns or the rpoS mRNAs

• DsrA also has complementarity to other mRNAs

• the same sRNA can affect expression of multiple genes

HOTAIR (Hox transcript antisense intergenic RNA)

• recently discovered ncRNA alter chromatin structure

• HoxC genes act as a scaffold that guides two histone-modifying complexes to their target genes

mechanism of HOTAIR transcriptional repression

• Scaffold function binds:

  • PRC2 (Polycomb Repressive Complex 2)

    • Repressive – Adds trimethylation to histone H3K27

  •  LSD1 (Lysine Specific Demethylase 1)

    • Repressive – Removes methyl groups from H3K4 (Histone 3, lysine 4. Makes Histone 3 more positively charged)

• Guide function:

  • Base-pairing with GA-rich regions on the chromosome brings the scaffold to the appropriate location

  • Represses/Silences transcription

HOTAIR and cancer

• HOTAIR overexpression is implicated in many cancers 

discovery of RNAi

• hypothesis: inject antisense RNA into organisms to inhibit mRNA translation by complementary base-pairing

• this worked! and the effects of antisense rna persisted for a long time

• used a technique called FISH

• make sense and antisense mex3 RNA by in vitro transcription

• mixing in vitro synthesized sense and antisense RNA before injection allowed them to base-pair and form double stranded RNA (dsRNA)

FISH: Fluorescent in situ Hybridization

• use a probe DNA (or RNA) that is labelled fluorescently

• add probe to cells and allow it to hybridize to complementary sequences via base-pairing

• detect fluorescence by microscopy

miRNA (microrna)

• endogenous encoded by genes in eukaryotic organisms

• encode in the genome

• miRNA genes do not encode a protein

• give rise to small RNA molecules, typically 21 to 23 nucleotides

• not usually a perfect match to mRNAs

• act as guide ncRNA

siRNAs (short-interfering RNAs)

• exogenous encoded by foreign/invading genes (virus)

• foreign rna

• usually a perfect match or close to a perfect match to specific mRNAs

• act as guide ncRNA

Drosha 

• RNase located in the nucleus 

• cleaves pri-miRNAs into pre-miRNAs

dicer

• multisubunit complex RNase

• cleaves pre-miRNAs and pre-siRNAs into 20-25 bp miRNAs and siRNAs

RISC (rna inducing silencing complex)

• RNase

• gets rid of one RNA strand

• argonaute - RNase component of RISC

mechanism of RNA interference (siRNA)

• dicer 

• RISC/argonaute 

• perfect base pairing 

• target rna cleavage and protects against viral dna 

mechanism of RNA interference (miRNA)

• drosha

• dicer

• RISC/argonaute

• imperfect base-pairing

• translational inhibition or RNA degradation or p body localization

functions and benefits of RNA interference

• miRNA: important form of gene regulation; production of miRNAs silences the expression of specific mRNAs

• siRNA: provide a defense against viruses 

PIWI-interacting RNA

• found in animals

• ncRNA interacts with PIWI proteins and inhibits the movement of transposable elements

CRISPR-Cas

• defense against bacteriophages, plasmids and transposons

• ncRNAs play a key role

ncRNAs Called piRNAs Interact with PIWI Proteins

• transposable elements: segments of DNA that can become integrated into chromosomes 

• if TE is inserted into a genes, the event is likely to inactivate the gene

different transposition mechanisms of transposable elements

• simple transposition: cut and paste info, preserves or increases the number of transposons

• retrotransposition: goes thru an RNA intermediate, duplicates number of transposons

transposable elements influences on mutation and evolution

• TEs exist because they simply can!

  • They survive as long as they do not harm the host “selfish DNA”

• TEs exist because they offer some advantage

  • Bacterial TEs carry antibiotic-resistance genes

  • TEs may cause greater genetic variability through recombination

  • TEs may cause the insertion of exons into the coding sequences of structural genes

  • This phenomenon, called exon shuffling, may lead to the evolution of genes with more diverse functions

Purpose of PIWI RNAs & Proteins:

• prevent transposition-induced mutations from being passed on to the next generation

CRISPR

• Clustered Regularly Interspaced Short Palindromic Repeats

• adaptive immune defense system found in bacteria and archaea

CRISPR array

• a locus for memory storage of previous infections

• memories are short sequences derived from the infectious source that are incorporated into the genome as spacers between repeated sequences

cas genes 

• CRISPR associated proteins

• machinery driving immunity 

CRISPR-Cas system

bacterial cell must first be exposed to an agent to elicit a response

occurs in 3 phases:

• adaptation

• expression

• interference

adaptation phase

• also called spacer acquisition

• cas1 and cas2 protein complex recognize phage DNA fragments (protospacers) and add them into CRISPR array in the bacterial genome

• leader sequence is an A/T-rich sequence downstream of the CRISPR promoter adjacent but preceding to the CRISPR array

• spacers from newer infections are closer to leader sequence

expression phase 

• exposure results in the expression of CRISPR and cas genes 

• pre-crRNA: noncoding RNA that the repeats and spacers are transcribed as  

• tracrRNA: noncoding RNA in class II CRISPR systems 

interference phase

• resembles rna interference

• each spacer in a crRNA is complementary to one strand of the bacteriophage dna

• crRNA acts as a scaffold for the Cas protein and as a guide that brings the Cas complex to the invading dna

• cas endonuclease functions make double-strand breaks in bacteriophage dna

• cleavage of the phage dna stops the infection

gRNA

• guide RNA

• Uses spacer sequence to form base pairs with the foreign/infecting genome (guide function)

• Recognizes the Protospacer in the foreign genome

• Associates with Cas proteins (scaffold function, contains the endonuclease enzyme)

protospacer 

• sequence in the infectious genome recognized by the bacterial spacer

PAM

• protospacer adjacent motif

• sequence in the infectious genome next to the protospacer

• recognized by Cas proteins

• distinguishes self from non-self

CRISPR: phage response

• initial discovery that phage-encoded Anti-CRISPR (Acr) protein inhibit endonuclease activity

• the protein AcrIIA4 looks like DNA and inhibits Cas9 binding to DNA

• Acr proteins have since been found that inhibit CRIPSR in different ways

recombinant dna: cloning 

• gene cloning or genetic engineering

• joining of 2 or more dna molecules usually from different biological sources that are not found together in nature 

• dna fragments are obtained by treating dna samples with restriction enzymes

• these fragments are put into plasmid vector

• dna ligase joins the fragments and vector

• recombinant dna molecules are formed into bacteria 

plasmid vector

• isolate a gene or DNA sequence you’re interested in

• replicate this dna

• make copies of the cloned gene

• amp^R: used to select for cells with a plasmid

• lacZ: used to screen for with a plasmid that have acquired a piece of DNA

• unique restriction site: place to insert a piece of DNA

• origin of replication: allows plasmid to replicate in the cell

restriction enzymes

• binds to DNA at a specific recognition sequence and cleaves the dna to produce restriction fragments

• biological: produced by bacteria as a defense against infection by phages

• biotech: used by researchers to cut dna at specific places

transform recombinant plasmids into bacteria 

• need to put plasmids into cells

• transformation: mix ligated DNAs with E.coli

• plate on selective agar media 

• get bacteria with the gene cloned into the plasmid 

blue/white screening

• lacZ gene used to detect recombinant plasmids

• uses a substrate analog called x-gal

• restriction enzyme site is located within the lacZ gene but doesn’t disrupt beta-galactosidase function

• cells with this plasmid will appear blue with x-gal

• inserting dna into the restriction site disrupts lacZ gene eliminating beta-galactosidase function

• cells with this plasmid will appear white on x-gal

DNA Sequencing: Sanger Method

• takes advantage of dna replication

• dna synthesis with small amounts of fluorescently labeled nucleotides that contain the sugar dideoxyribose (ddNTP) instead of deoxyribose

• once a ddNTP is incorporated DNA polymerization stops

dna sequencing 

• dna replication

• requires dna polymerase and primer

• modified sugars cause chain termination bc no 3’ -OH

• products of DNA synthesis are separated by electrophoresis 

• each ddNTP has different fluorescence 

cloning eukaryotic protein coding genes

• contain introns which interrupt the coding sequence

• mRNA is used to get uninterrupted coding sequence

• complementary dna is made using reverse transcriptase which is a dna polymerase that uses rna as a template to synthesize dna

making cDNA

requires

• mRNA

• oligo-dT primer: complmentary to polya tails

• reverse transcriptase: synthesizes dna using rna template

• RNaseH: endonuclease that cuts rna in rna/dna hybrid

• DNA polymerase I: synthesizes second strand and removes rna primers

• Ligase

genomic library 

• clone all dna sequences in a genome

• contains at least one copy of all the sequences int eh genome of interest

• want to clone many different pieces of dna

• into separate plasmids

• all at the same time

cDNA library

• clone all mRNA sequences as cDNA

• represents the genes being expressed in a particular cell type at a given time

• only has genes that rae expressed and with a poly-a tail

• will have different sets of genes depending on cell type

genomic library construction

• cut genomic dna and vector dna with the same restriction enzyme

• ligate the fragments into vectors

• transform the ligation products into e.coli

• plate on selective media

cDNA library: construction

• genes are treated with reverse transcriptase and dna polymerase to produce cDNA copies of mRNA 

• number of cDNA fragments is proportional to gene expression 

pcr

• copies a specific dna sequence

• uses dna polymerase and designed primers to amplify the amount of dna from specific region of a genome

*primers in pcr*

• primers are complementary to genomic DNA

• need a primer for EACH strand

• the 3’-OH of both primers point towards each other!

• provide the 3’-OH for DNA Polymerase to extend

• dna between the primers gets copied

*pcr: how it works*

• uses dna polymerase and designed primers to amplify the amount of dna from specific region of a genome 

• no replication fork

• dna strands separate due to heating 

• dna synthesis of each strand is independent of the other 

3 steps of pcr

• denaturation of template dna: heat to separate strands (>= 95)

• primer annealing to template dna: cool to anneal primers (50-60)

• extension of primers by dna polymerase: dna synthesis (70-75) 

measuring RNA levels with RT-PCR & cDNA

• uses cDNA as the initial template

• PCR only works on DNA

• RNA must be converted to cDNA

• all other aspects of PCR are the same as any other PCR

• can be used as a template for qPCR

• indirect measurement of RNA amount

• used to test for COVID-19 and other viruses with RNA genomes and gene expression levels

Electrophoretic Mobility Shift Assay

• binding of a protein to a fragment of DNA or RNA slows its rate of movement through a gel

• EMSA assays must be preformed under non-denaturing conditions

• buffer and gel should not cause the unfolding of the proteins nor the separation of the DNA double helix not SDS PAGE

• can also be done with RNA

dna footprinting 

• segment of dna that is bound by a protein will be protected from digestion by the enzyme DNase I 

• one dna strand is isolated with radioactivity on one end 

• bind protein to DNA 

• cut with DNase I randomly cuts many places 

• protein prevents DNase I from cutting where it is bound 

• electrophoresis separates the pieces of dna 

• compare differences with and without protein 

• get a “footprint” where the protein binds at single nucleotide level

southern blotting

• digest dna into small fragments and separate by electrophoresis

nothern blotting

• isolate total rna and separate by electrophoresis

western blotting

• isolate proteins and separate by electrophoresis (SDS-PAGE)

hybridization 

• allow you to see where a piece of dna or an rna is located after gel electrophoresis 

Population genetics

Study of how allele and genotype frequencies behave and change within populations.

Population

A group of interbreeding individuals sharing a gene pool in the same geographic area.

Gene pool

All genetic information (alleles) in all individuals in a population.

Phenotype frequency

Proportion of individuals expressing a specific phenotype.

Genotype frequency

Proportion of individuals with a specific genotype.

Allele frequency

Proportion of a specific allele among all alleles for a gene in a population.

Allele frequency formula

Number of copies of allele divided by total number of alleles (2N).

Genotype frequency formula

Number of individuals with a genotype divided by total individuals.

Sum of allele frequencies

p + q = 1 for two alleles.

Sum of genotype frequencies

p² + 2pq + q² = 1.

p

Frequency of the dominant allele.

q

Frequency of the recessive allele.

p²

Frequency of the homozygous dominant genotype.

2pq

Frequency of the heterozygous genotype.

q²

Frequency of the homozygous recessive genotype.

Hardy-Weinberg equilibrium

State where allele frequencies remain constant across generations in ideal populations.

Hardy-Weinberg assumptions

No mutation

Hardy-Weinberg prediction 1

Allele frequencies remain constant over generations.

Hardy-Weinberg prediction 2

Genotype frequencies become p²

Finding q from phenotype

q = square root of the recessive phenotype frequency (q²).

CCR5 Δ32 allele

Mutation deleting 32 bp in CCR5

Estimating allele frequencies

Use p = [2(AA)+Aa]/2N and q = [2(aa)+Aa]/2N.

PTC tasting inheritance

Dominant allele controls tasting ability; non-tasters are tt.

X-linked allele frequency

Male phenotype frequency equals allele frequency because males have one X chromosome.

Color blindness example

q equals the fraction of males who are colorblind.

Darwinian fitness

A genotype’s reproductive success relative to others.

Natural selection

Differential survival or reproduction based on phenotype.