LS1B: PIE 1

PIE 1: Lectures 2 - 6

C-value

"constant" value of DNA content in a cell

C-value paradox

lack of correlation between genome size and the biological complexity of an organism

What did the Humane Genome Project reveal?

• 2% of genome: encodes proteins

• 9% of genome: conserved by natural selection (similar between species), implying function importance

• 89% of genome: "junk" DNA of unknown clear function

natural selection only acts against _____, which affect protein production, and doesn't act on ______

coding mutations; mutations in the "junk" region

What % of the human genome is functionally important?

11% (2% codes for proteins, 9% is conserved by natural selection)

Central dogma

DNA -> RNA -> Protein

gene

functional unit of heredity/DNA sequence that can be transcribed to produce a functional product (RNA or mRNA)

information storage molecule

DNA, because it's very stable and maintained over time

information transfer molecule

RNA, because it acts as intermediary between DNA & proteins

gene expression can be regulated at the levels of

transcription, mRNA processing, translation, protein activity, and chromosomal activity

transcribed DNA sequence

what is transcribed into mRNA

regulatory DNA sequence

what controls gene expression by forming DNA-protein complex w/ DNA binding protein

RNA coding region

specific portion of DNA that's transcribed from the transcription start site to the transcription termination site

transcription start/end sites

location in DNA corresponding to the first/last RNA nucleotide incorporated into the transcribed RNA

terminator

DNA sequence that disrupts activity of the RNA polymerase & induces it to stop transcription

promoter

sequence at which proteins use DNA as template to make RNA bind & initiate synthesis

upstream

sequences before the transcription start site (usually promoter & regulatory elements)

downstream

sequences after transcription start site including coding sequence of gene & transcriptional termination site

non-template/coding strand

has same sequence as the transcribed RNA (except w T rather than U)

template strand

RNA polymerase base pairs with this strand to make complementary RNA

RNA polymerase

enzyme that copies DNA to RNA

negative regulation of transcription in bacteria (using repressor protein)

• E.coli regulates expression of sugar-digesting genes

• bacteria prefer glucose, but will also take lactose if available

• repressor protein inhibits expression when lactose is absent by binding to specific repressor binding site near transcription start site, stopping RNA polymerase

• lactose is inducer of gene expression b/c its presence activates expression

• lactose binds to and deactivates the repressor to allow for transcription

positive regulation of transcription in bacteria

• activator proteins bind to DNA sequences near RNA Polymerase binding site to recruit RNA polymerase

• activates gene transcription

What makes eukaryotic regulatory sequences different from prokaryotes?

core/minimal promoter (contains TATA box and the protein complex TFIID)

TATA box

A promoter DNA sequence crucial in forming the transcription initiation complex; bound by TATA binding protein (TBP) and core transcriptional machinery such as protein complex TFIID

deletion/disruption of the TATA box will

prevent transcription entirely, because the general transcription factors & RNA polymerase won't be able to bind & initiate RNA synthesis

eukaryotic core transcriptional machinery transcribes genes at a ____ level, requiring regulatory transcription factors to either ____ the transcription level or ____ it entirely

very low; raise; repress

Where do eukaryotic regulatory transcription factors bind?

to cis regulatory elements (DNA sequences)

regulatory elements adjacent to the core promoter are part of the _____, which is where activators and repressors bind to _____

regulatory promoter; increase/decrease binding of general transcription factors to the core promoter

regulatory elements far from the core promoter are called ____, which bind to regulatory transcription factors & inhibit/activate transcription by influencing binding of the ________

enhancers; core transcriptional machinery (TFIID & RNA polymerase, etc.)

Eukaryotic post-transcriptional RNA processing (occurs in the nucleus)

• adding 5'-cap to mRNA

• poly-A tail to mRNA

• remove introns and splice together exons

• form of gene expression

alternative splicing

different exons differentially included/excluded in mature mRNA to increase # of proteins that are made by a single gene

gene expression regulation in translation in the cytoplasm: RNAi (RNA interference) --> mRNA-mediated regulation

• RNA in nucleus via nuclear pore

• small double-stranded RNA molecules around 21 bp long interfere w/ translation

• siRNAs and miRNAs

siRNAs and translational interference

• siRNAs are picked up in cytoplasm by enzyme complex Dicer

• dices them into 21bp sequences that join with proteins to produce the RISC, which acts to silence the target gene

• RISC eliminates one of the two strands of the double-stranded RNA

• RISC now has 21 nucleotide RNA called the guide RNA (gRNA) that's complementary to the target sequence on the mRNA of the gene that's being regulated

• gRNA is perfectly complementary to the target sequence (all 21 bp), making siRNA binding highly specific

• results in cleaving of the mRNA at the site of the siRNA attachment, degrading the snapped mRNA

miRNAs and translational interference

• miRNAs are picked up in cytoplasm by enzyme complex Dicer

• dices them into 21bp sequences that join with proteins to produce the RISC, which acts to silence the target gene by interfering w/ translation

• RISC eliminates one of the two strands of the double-stranded RNA

• RISC now has 21 nucleotide RNA (gRNA) that's complementary to the target sequence on the mRNA of the gene that's being regulated

• guide RNA is only complementary to a short segment of the target sequence (not all 21 bp), making miRNA binding less specific (will bind to multiple sequences)

• miRNAs inhibit translation by physically interfering with ribosome assembly

spontaneous genetic mutations

mutations that arise de novo in the current generation (no family history of the problem)

every human is born with around ____ de novo mutations

70-80

simple genetic traits

traits determined by a single gene

polygenic genetic trait

traits that have their roots in variation at many loci

heterozygous advantage in sickle cell disease

being heterozygous for sickle cell disease protects from malaria w/o severe sickling effects

gene therapy challenges

1. getting genetic construct into the cell (some viruses have immune response)

2. getting genetic construct into the right cell

ex vivo gene therapy

cells taken out of the body, genetic manipulation performed in the lab

in vivo gene therapy

most effective when target cells are easily accessed, the gene is delivered in a viral vector via injection into the cells

embryonic manipulation gene therapy

• early embryonic manipulation affects germline

• changes to zygote will be present in the germline cells and the next generation

• inherited by every cell in the body

How to target a sequence in gene therapy

• ZFN: use nucleases (DNA-cutting enzymes) that recognize longer DNA sequences via engineered nucleases (DNA binding domain binds 3 nucleotides at a time)

• TALENs: protein-DNA binding domains recognize one nucleotide at a time, but makes proteins too bulky

• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

What makes CRISPR-Cas9 better than TALENs and ZFNs?

• easier to design and deliver in vivo

• more specific

• less toxic

• naturally part of the bacterial immune system

How does bacterial DNA lend itself to CRISPR?

• repeated bacterial DNA has spaces in between each stretch that matches viral DNA, with each repeat forming palindromes (read same forward and back)

• bacterial genome has operons encoding Cas nucleases (DNA cutting enzymes)

adaptive immunity and CRISPR

mechanism of remembering past infections & creating an immune response on reinfection (as opposed to immunity to first-time infection)

3 stages of CRISPR

1. Spacer Acquisition (executed by Cas9 & Cas 1/2 enzymes):

   • bacteria first infected by virus, captures snippet (protospacer) of the virus & stores in memory system

   • the protospacer fills space between palondromic repeats & starts close to the PAM

2. crRNA processing:

   • CRISPR locus w/ various protospacers is transcribed into long cRNA precursor that's processed by Cas enzymes and RNaseIII to make short cRNAs, each with one protospacer sequence

   • tracrRNA (naturally occurring in CRISPR locus) anneal to cRNA to make a hairpin structure

   • cRNA will anneal to target DNA sequence (reinfecting viral DNA), Cas is employed to cut the target DNA if it contains a PAM

3. Destruction of New Virus by Cleavage:

   • if virus returns, bacteria's tracrRNA-cRNA complex will anneal to it

   • if PAM site is present in the viral sequence, Cas will digest the viral DNA & eliminate the infection by cleaving the double stranded DNA of the invading virus

   • sometimes, the virus can repair itself, but this is prone to error & usually leads to loss of function anyways

importance of the PAM in CRISPR

• short sequence that varies between bacterial species

• must be present in intruding viral DNA for Cas to cut

• w/o PAM, the Cas-9 would attack the bacteria's own CRISPR genome

• w/ PAM, the bacteria isn't attacked because PAM isn't in the palindromic sequences between the protospacers in the CRISPR array, so it won't be attacked

• pretty common, expected around every 16 bp or so

• usually is "NGG", which is very common in genomes

What makes CRISPR unique and good for genetic engineering?

it can use the same nuclease complex to cleave any sequence of a genome

sGRNA (single guide RNA) in CRISPR

• artificial mix of tracrRNA and cRNA

• simpler than having to separately add tracrRNA and cRNA to

Steps:

• sGRNA & Cas9 protein form effector complex

• complex associates with PAM, unwinds DNA, and pairs with the complementary sequence on the DNA

• Cas cleaves the DNA

CRISPR to introduce mutation or sequence: Homology-Directed Repair (works well!)

• natural DNA mechanism that uses template homologous DNA sequence to generate missing nucleotides at a double stranded break point

• scientists introduce desired sequence into the cell at the cut

• this repair mechanism is also not controllable

What's the other type of DNA repair other than Homology-Directed Repair that doesn't allow for introduction of desired DNA material?

Nonhomologous end joining, which introduces duplications or deletions & frameshifts

GMO (genetically modified organisms)/ transgenic organisms

organism whose genome has been altered by insertion of a gene or genes from other species or breeds

How do you identify genes of interest to clone before doing expression cloning? (ex: GMOs, etc.)

Steps:

1. Reverse transcription

   • DNA copies of RNA made using enzyme reverse transcriptase

   • oligo (dT) primers bind to RNA of interest (poly-A tail) to generate RNA-DNA hybrid

   • the RT (reverse transcriptase) also adds a few bases to the 3' end of the new DNA to act as a primer for a second DNA strand using the first

2. Making double stranded DNA

   • RNA degraded via NaOH or RNAase enzyme

   • DNA polymerase added to synthesize 2nd DNA strand using the overhang

   • S1 nuclease added to remove the hairpin between the DNA strands

   • Now you're left with complementary DNAs, or cDNAs that can be purified for later use

OVERALL GOAL:

• obtain cDNAS corresponding to all the genes expressed in the cells you test

• can help you find the genes encoding specific functions you are interested in (ex: GFP expression)

Why is it easy to reverse-transcribe eukaryotic mRNAs to DNA?

• they all share a common sequence: the poly-A tail

• This means that an oligo (dT) primer of only thymine residues can be used to make a complementary DNA strand

Expression cloning general idea

• each cDNA is inserted in different bacterium to screen for activity of gene of interest

• we are looking for cells that exhibit a specific characteristic after taking up a particular cDNA

Expression cloning steps after cDNAs of expressed genes are obtained

1. insert cDNA copy of the gene into a plasmid

2. plasmid must have

   • A) antibiotic resistance gene (to determine which cells took up a plasmid)

   • B) origin of replication (initiates replication of plasmid)

   • MCS/multiple cloning site (DNA sequence with restriction enzymes to be inserted in the plasmid)

Generating transgene constructs:

3. cut plasmid w restriction enzyme that leaves blunt ends (easier to insert cDNA)

4. mix cut plasmid w/ cDNA library, add DNA ligase to connect each plasmid DNA to cDNA, making many transgene constructs w/ a different cDNA insert

Transforming transgene constructs into bacteria:

5. use electroporation to shock bacteria w/ an electric current, allowing the bacteria to take up a plasmid

6. plate bacteria onto antibiotic-containing media to select bacteria w/ plasmids and grow/clone them

PCR-based cloning vs expression cloning

• DIRECTIONAL CLONING (can adjust orientation accordingly)

• much more specific, much easier, more efficient

• use two primers to amplify the DNA sequence you want to clone

• add sequence of interest into this sequence and use different restriction sites at the end of each primer to control the orientation of insertion

PCR cloning uses restriction enzymes that cut and leave _____, while expression cloning single restriction enzymes leave ____

sticky ends/single-stranded overhangs; blunt ends

Why are sticky end overhangs in PCR cloning beneficial?

DNA can be inserted more efficiently because an insert & plasmid cut by the same enzyme will have complementary "sticky ends" and will find/stick to each other more easily

the plasmid used in expression & PCR cloning is called

expression vector, which has antibiotic resistant gene, origin of replication, MCS, AND all sequences you need to express an inserted gene

vector

anything you can use to move transgenes around

expression vectors to drive expression of gene insert in high level in bacteria

vector would require strong bacterial promoter & transcription termination sequences

expression vectors to drive expression of gene insert in high level in eukaryotes

vector would require eukaryotic promoter & transcription termination sequences

when are transgenes randomly incorporated into the genome?

when they're introduced into eukaryotic cells; when added to cells giving rise to egg/sperm

reporter gene assay

substitutes reporter gene (one that reports where it's active), such as GFP, for the actual gene --> to hook the gene's promoter to a GFP gene to see GFP expression

What causes Huntington's disease

repeated CAG codons that lead to too many glutamine residues

PCR general purpose

to isolate & amplify specific DNA sequence from a sample

PCR main components

1. template DNA

2. primers

3. Taq DNA polymerase (can withstand high temps without being denatured)

4. dNTPs (deoxynucleotides)

Typical PCR cycle

1. Heating DNA at 94C to denature it into single strands

2. Lowering temp to allow for DNA to anneal to primers

3. Increase temp again to have Taq polymerase extend the primers & make a new DNA strand

Each cycle doubles # of target DNA sequences

gel electrophoresis

• agarose gel

• smaller fragments move faster (further)

• larger fragments move slower (closer)

Using PCR to determine if one's genome contains a given sequence

if primers bind & produce a PCR product, the sequence is present

PCR detects only ______, while gel electrophoresis detects _____

whether or not the genome contains a specific sequence; mutations that result in a change in the length of a gene/sequence

How do you look for smaller scale genetic changes, since PCR and gel electrophoresis only look at large-scale changes?

Using RFLP Analysis Requires (Restriction Fragment Length Polymorphism)

1. position/s that can be recognized by a restriction enzyme (where one variant is cut, and the other is not)

2. PCR amplification of the site recognized by the restriction enzyme

3. Gel electrophoresis to view the DNA and determine size fragments

ex: if the mutation disrupts a restriction enzyme recognition site, this prevents the enzyme from cutting DNA

Sanger sequencing

a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication

next-generation sequencing

entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences.

whole genome sequencing

• process that determines the DNA sequence of an entire genome; has greater "read depth" (# of times a base is sequenced in a given sequence)

• read depth helps distinguish between genuine mutations vs sequencing errors

genotype vs phenotype

• phenotype: appearance/way in which people manifest a characteristic

• genotype: genetic makeup

alleles

two different versions of a gene, one from mom and one from dad

heterozygous for a gene

two different alleles of a gene

homozygous for a gene

two copies of the same allele

dominant vs recessive trait

dominant traits requires only a single copy of the allele for the phenotype to be expressed, recessive requires two

many recessive traits are due to ____ mutations because having a single copy of the normal allele is ____ for function, but can cause heterozygous individuals to exhibit the mutant phenotype

loss-of-function/null; sufficient

haplosufficiency

where one working copy will do

many dominant traits are due to ____ mutations because having a single copy of the normal allele is ____ function, which affects both homozygous and heterozygous individuals, altering the ____ of the genes

gain-of-function; insufficient to ensure normal; behavior

what does null/knockout/loss of function mutation do?

completely eliminates gene function (ex: deleting a gene)

what does gain of function/neomorphic mutation do?

causes gene to do something new like binding to a new partner or catalyzing a new reaction

regulatory mutation

mutation in DNA sequence involved in regulating gene expression (enhancer, promoter, splice site, terminator)

coding mutation

mutation in the body of the gene that codes for the protein

molecular DNA changes

• base-pair substitution: one base pair in DNA duplex replaced with another

• insertion/deletion: one or more extra/missing nucleotides

Effects of base-pair subs/insertions/deletions

1. synonymous (silent) mutation: no change in amino acid sequence

2. non-synonymous (missense) mutation: changes original amino acid to different one

3. nonsense/termination mutation: amino-acid codon changes to stop codon (truncated protein)

4. frameshift mutation: insertion/deletion of non-divisible by 3 bases, leads to "out of phase" shift & completely different amino acid sequence

sporadic disease

• novel appearance of a disease in offspring that's absent from either side of the family

• often caused by de novo mutations

single cell genomics

isolating single cell, preparing DNA from that cell, amplifying the DNA from that cell, and sequencing the product of that cell

Crick & Brenner discovered

that DNA code is read in triplet codons, and adding/removing one or two base pairs causes a frameshift mutation where all downstream info is out of phase

in eukaryotes, mRNA is sent from the nucleus via nuclear pores to the ____ to be translated in a ____

cytoplasm; ribosome

Key mechanical players in translation

1. ribosome- where translation takes place

2. tRNA- pairs to mRNA with anticodon 3. aminoacyl tRNA synthetase- enzyme connecting specific amino acids to tRNAs via covalent bond

uncharged vs charged tRNA

• uncharged: not bound to amino acid

• charged: bound to amino acid

Initiation phase of translation (eukaryotes)

• initiator tRNA charged w/ Met joins w small ribosome at 5'-mRNA cap to make initiation complex

• complex scans mRNA for AUG codon so tRNA can base pair, setting the start site

• after pairing, the large ribosome joins