Human Genetics - Final Exam

Indels

Insertion and Deletion

Mutational Hot Spots

repetitive sequences and palindromes

strand slippage

DNA polymerase slips can create a hairpin

transition mutation

purine → purine

pyrimidine → pyrimidine

transversion mutation

purine → pyrimidine

Depurination or depyrimidination

loss of base from nucleotide

creates an Apurinic Site

if not repaired before DNA replication, an A typically gets incorporated

Deamination

loss of amino (NH2) group from a nucleotide base 

deaminated cytosine usually repaired

deaminated 5-methylcytosine more complicated

Types of DNA damage caused by mutagens

mutagens: radiation, chemicals, infectious agents

base modifications, single-strand break, bulky lesion, cross link, double-strand break

Ames Test

determines mutagenicity of a substance

dosage matters

more colonies mean there is more mutagenicity

extragenic regions 

the change has no effect 

coding sequence mutations

affect the protein created

amino acid encoding and splice sites

non-coding mutations

affect level of expression

regulatory sequence

introns

UTRs

silent mutations 

synonymous 

third base wobble

creates the same amino acid

nonsense mutations

creates a stop codon

shortens the amino acid chain

missense

codes for a difference amino acid

Frameshift mutations

insertion/deletion of one or more bases into coding sequence

messes up the rest of the sequence

Base Excision Repair

fixes damaged bases

small addings

reverses deamination

Nucleotide Excision Repair

distortion to DNA helix (only one strand has damage)

nuclease cuts backbone up and downstream of damage site

Mismatched Repair

fixes newly replicated DNA

recognizes parent strand because of the methyl group

Synthesis Dependent Strand Annealing

repairs double strand breaks

uses sister chromatid as template to fix damage

only during S and G2 phase after replication

Nonhomologous End Joining

damage commonly from ionizing radiation or chemicals

no template used to repair so mutations often introduced

Core Promotor CRE

promotor region close to the gene that binds general transcription factors 

minimum amount of promotor needed for basal expression

Enhancer and Silent CRE

DNA sequences far away from the gene but still influence gene expression

Activator TFs

proteins that activate gene expression

Repressor TFs

proteins that repress gene expression

Coactivators 

does not bond to the DNA but helps to recruit to activate gene expression

Corepressors

does not bond to DNA but helps to recruit to repress gene expression

Locus Control Regions (LCRs)

group of CRE that regulates a cluster of genes

Insulator Sequence

restrict range of CRE influence

block regulation of genes even though activator/repressor is within the range

bound by insulator-binding proteins

prevent CRE from regulating the wrong genes

nucleosome

147 base pairs wrap 1.7 times around histone protein (8 proteins)

open promotors

express genes

TFs are able to bind to the area and express the gene

closed promotor

silenced genes

wrapped within a nucleosome

activator protein cannot bind

housekeeping genes

tend to be open

inducible genes 

can change from open to closed based on cell type or stimuli 

Nucleosome positioning code

repeating “dinucleotides” every 10 base pairs

controls nucleotide positions

1) DNA sequence

2) compitition from other DNA binding proteins

3) ATP-dependent chromatin remodelers

4) histone and DNA modification

Nucleosome and DNA-binding proteins compete to bind DNA

levels of TFs matter to overcome histone proteins to bind to DNA sequence

pioneer transcription factors 

first TFs to lead to moving the nucleosome 

Nucleosomes moved by ATP-dependent enzymes

chromatin remodelers: enzymes that couple ATP hydrolysis to move nucleosomes

Histone Modifications

influence nucleosome positions

histone tails: 20 amino acids at end of each histone protein

tails are post-translational modified

Modified tails regulate binding of other proteins to chromatin

Acetylation (Ac)

Methylation (Me)

Phosphorylation (P/Ph)

Nomenclature for histone modifications 

1) histone

2) amino acid and number 

3) modification

ex. H3k9Ac

Histone modifications

are added, removed, or read by proteins

histone code means that the modifications have meaning

constitutive heterochromatin

permanently condensed and silent

facultative heterochromatin

can switch between condensed and uncondensed based on stimuli 

Cytosine Nucleotide Methylation

only occurs to Cs immediately followed by Gs 

modifications occur in major groves

does not affect GC base pairing 

alters protein interactions and influences which ones can bind to it

DNA methylation in promoter region

indicates gene silencing

active genes are not DNA methylated

silenced genes are DNA methylated 

Epigenetics 

heritable changes in gene expression without changing DNA sequence 

comes from histone modifications or DNA methylation

Waddington’s Epigenetic Landscape 

metaphor for gene regulation influencing development 

DNA Methyltransferase I

reads the parent strand and methylates the new daughter strand

during S-phase

X-Inactivation

monoallelic expression of most X chromosome genes

stable through mitosis but not meiosis

½ of cells are silenced for one x chromosome and other ½ of cells are silenced for the other x chromosome

bar body: silenced x gene in ½ cells

X-Inactivation Center (XIC)

x-inactivation starts here

XIC encodes an RNA (XIST) that is expressed only by the inactive X chromosome

the inactive X chromosome is coated with XIST RNA

the RNA recruits transmission factors to add heterochromatin “marks”

Epigenetics are erased

occurs during sperm and egg development

164 places in the genome stay put

imprinted loci

Methylation not erased during embryogenesis

sperm and egg have specific methylation

164 methylation sites kept and passed down

Genomic imprinting

describes gene expression regulation depending on parent

imprinting = silenced

gamete-specific expression

different syndrome based on if maternal or paternal alleles are expressed

Alternative Splicing

A single gene gives rise to multiple proteins

regulated by RNA sequencing and RNA binding proteins

isoforms

different proteins encoded by the same gene

alternate promoters and terminators

splicing regulatory elements

RNA sequence bound by splicing factors

splicing factors

RNA-binding proteins that recruit or block the spliceosome

RNA Interference

block gene expression using double-stranded RNA

siRNA

Micro RNA

non-coding

initially single strand, forms hairpins

Drosha processes into pre-micro RNA

DICER cuts into 21-22 double stranded RNA

RISC selects guide and they bind to complementary target mRNAs

perfect base pair

degraded mRNA

decreased gene expression

imperfect base pair

blocked translation

blocks translation of the target gene

Post-translation expression

Reversible covalent modifications regulate protein activity

alter binding interactions with other proteins

activate/inhibit activity

alter cell localization

molecular cloning

set of experimental methods used to assemble and replicate recombinant DNA molecules within host organisms

recombinant DNA (rDNA)

DNA from 2 or more organism combined into a single molecule

plasmid/vector

small circular DNA double helix that stably replicates in host cell

structure:

replication of origin

marker genes for selection

multiple cloning site (gene of interest insertion

optional promotor

transgenic cell

holds something foreign inside

gene addition - knock in

can either

deliver vector in without integrating into the genome

deliver vector into cell and integrates into genome

random loci more efficient

target loci more precise 

gene deletion - knock out

targeted insertion into genome to replace endogenous gene with selectable markers

creates loss-of-function alleles interested into genome 

gene expression regulation - knock down

blocks expression but doesn’t alter gene sequence

can either be transient or long term knockdown

not always full loss of function

gene editing

CRISPR

CRISPR

precisely edits genome

clustered regularly interspaced short palindromic repeats

based on bacterial immune system 

Cas9 

cuts the targeted DNA

endonuclease: in the middle 

Guide RNA

directs Cas9 to the specific loci through complementary base pairing

crRNA

20 bp complementary to target

tracrRNA

binds Cas9 and crRNA

PAM sequence 

protospacer adjacent motif

present in target DNA only 

determines self vs viral 

Damage fixed by either

random repair: non-homologous end joining

precise repair: requires donor DNA, only for dividing cells

CRISP limitations

delivery into target cells

vector can cause immune response

guide RNA cause of target mutations

ethical uses

gene therapy in somatic cells vs germline cells

less controversial

changes are heritable, more controversial

in vivo vs. ex vivo gene therapy

therapy delivered into the body

cells removed, altered, then delivered back

gene therapy today

safer delivery vectors, precise targeting, and expression

comparative genomics

systematic comparison of genomic sequences

sequence homology

similar sequence, shared ancestry, conserved sequences

BLAST

program used to align sequences

Syntenic Sequences

conservation of a gene order between two organisms

synteny between human and mouse genomes

negative selection

sequence cannot change, otherwise the organism would not survive

evolutionary constrained

mutations that decrease fitness are selectively eliminated

Comparing protein sequences

protein sequences typically show greater sequence homology than DNA sequences

occurs because of synonymous sequences

Positive Selection

new mutations that benefit the fitness of the organism

rare within the population

neutral mutations

have no effect on the fitness of the organism

Genes are identified by

open reading frames: miss genes with introns

cDNA library: collect RNA from cell and convert to cDNA with NGS. Cell type specific because not all genes are expressed

comparative genomics

paralogs

different genes in one species, products have related functions

orthologs

same gene in different species, products have equivalent functions

errors of gene duplications

gives rise to gene families and gene super families

pseudogene

features of a gene but not expresses

subfunctionalization

have both copies. one copy codes for one thing the second codes for a different thing

neofunctionalization

one copy gains mutation that does something new

phylogenomic

evolutionary tree of life incorporates analysis of multiple genomics/proteomic/physical features

divergent evolution

process by which groups from the same common ancestor evolve and accumulate differences, leading to the formation of new species