Adv Physiology and Molecular Biology (OPTO 5344) Midterm 1

Genes

organized information encoded in the DNA

genetic units in the DNA that code for the RNAs necessary for cell function and structure

Gene products

RNA, proteins

transcriptome

collection of all RNA transcripts in a cell

proteome

collection of all proteins in a cell or tissue

result of transcriptome

genome

each cell nucleus contains an identical complement of chromosomes in two copies; copy is a genome

identical in all cells

chromosomes

way genome is organized

number varies between species

humans contain how many chromosomes?

46

23 from each parent; 22 autosomes and a sex chromosome

RPE65

enzyme required in retinoid recycling

processes retinyl esters during retinoid (visual) cycle

chromophore (11-cis-retinal)

when bound to opsin, absorbs light and starts signal transduction in photoreceptor

light isomerizes 11-cis-retinal to?

all-trans-retinal

Leber congenital amaurosis can be caused by?

mutations in RPE65

what occurs when a RPE65 mutation leads to a depletion in 11-cis-retinal

lack of light recognition

rhodopsin does not function

blindness

gene therapy strategies

replace missing gene product

stimulate normal &/or pathological processes

eliminate/inactivate toxic proteins

replace missing gene product strategies

add back biosynthetic component (e.g. dietary supplement)

introduce new correct and functional copy of gene into affected cells

how to rescue mutation and eliminate disease in biosynthetic pathway

block pathway entirely

replace intermediates (nutritional supplements)

replace missing or non-functional enzyme

replace mutant/missing gene strategy

sub-retinal injection with adeno-associated virus

contains normal gene for RPE 65 and infects RPE cells

delivery of gene in somatic cell gene therapy

identify mutation in patient

clone correct/normal gene (RPE 65)

package DNA for normal gene in viral vector (AAV)

virus infects cells and sends DNA to nucleus

virus DNA cargo expresses long-term and makes the correct gene product

stimulate normal &/or block pathological processes strategies

block proliferation of target cells

block excess blood vessel formation and growth

preventing cell death via survival factors or genes that block apoptosis

excess proliferation of cells solutions

laser ablation destroys neovascular tufts

block of vessel growth signaling pathways

chemotherapy to kill abnormal cells

surgery to remove tumor

antibiotics to block proliferation

excess cell death solutions

eliminate/inactivate toxic proteins

add survival factors or genes to block apoptosis

neovascular retinal diseases

wet amd

corneal neovascularization

retinopathy of prematurity

diabetic vasculopathy

neovascular retinal disease characteristics

excessive blood vessel growth

leakage and edema

vision loss

retinal detachment

therapeutic strategies for neovascular retinal disease

laser ablation/photocoagulation

photodynamic therapy

anti-VEGF therapy

laser ablation

"hot" laser to destroy abnormal vessels

results in blind spot

photodynamic therapy

systemic injection of drug verteporfin

laser activates drug and forms blood clots to stop leakage

molecular blocking of blood vessel growth

inhibit, block, and/or destroy VEGFA

requires repeated intraocular injections and can block normal functions of VEGF

excess production of VEGF stimulates?

pathological neovascularization

damage caused by ____ leads to increased VEGF production

hypoxia

complement activation

cellular damage/death

disease

high levels of VEGF-A causes

vasoproliferation

vasomigration

vasopermeability

vasopermeability leads to

choroidal neovascularization

treatment of pathological neovascularization

block VEGF function

first anti-VEGF therapy

aptamers

short RNA oligonucleotides that bind to and inhibit VEGF

screen/isolate compounds with high binding affinity

result of aptamers in anti-VEGF therapy

VEGF bound by aptamers cannot bind receptor

blocks VEGF function in endothelial cells

antibodies role in anti-VEGF therapies

bind to VEGFA and inactivate it

VEGF bound by antibodies cannot bind receptor

two common medications used to treat AMD

ranibizumab and bevacizumab

Aflibercept (EYELA)

medication to treat wet AMD

uses soluble VEGF receptor proteins to bind excess VEGF

"decoy"

reduces amount of free VEGF and prevents/reduces activation of receptors on endothelial cells

goals for gene-based therapies

stop pathological processes

eliminate/fix malfunctioning proteins

replace missing metabolites/proteins/genes

add genes/cells that will make corrected or therapeutic proteins

problems for gene-based therapies

targeting affected cells

reducing unwanted side-effects

eliminate need for frequent doses

minimize need for repeated surgical interventions

soluble "decoys"

proteins that mimic a binding receptor of a target protein

bind target protein and prevent normal binding, blocking signaling pathway

central dogma of gene expression and protein synthesis

DNA is replicated, undergoes transcription into RNA, which is translated into protein

how do we know genes are in DNA (1928)

Griffith experiment with mice and Streptococcus pneumoniae

Characteristics of dead bacteria could be passed to other bacteria by something that remains in the cell extracts

how do we know DNA contains the virulence factor genetic material

Avery, MacLeod, and McCarty (1944)

only the destruction of DNA allowed for the mouse to live

no live S strain recovered; gene was in the DNA

chemical composition of DNA

deoxyribose sugar + phosphate + nitrogenous base

nucleotide composition

deoxyribose sugar + nitrogenous base + phosphate

nucleoside composition

deoxyribose sugar + nitrogenous base

deoxyribose sugar composition

ribose = 5 carbon sugar

#1 carbon attachment site for base

#2 carbon contains an -H instead of a hydroxyl (-OH)

#3 carbon must contain -OH for DNA polymerization

#5 carbon attachment site for phosphate

which carbons on deoxyribose are where nucleotides are joined during DNA synthesis

5' and 3'

nitrogenous bases classified as Purines

adenine and guanine

nitrogenous bases classified as pyrimidines

cytosine and thymine

purines contain how many rings in their structure

2 rings

pyrimidines contain how many rings in their structure

1 ring only

Chargaff's rule for DNA

%A = %T and %C = %G

%(A+T) + %(G+C) = 100%

does Chargaff's Rule apply to RNA

does not apply to RNA

single-stranded

what kind of bonds stabilize bases in DNA

hydrogen bonds

how many hydrogen bonds between A-T? C-G?

A-T = 2 hydrogen bonds

C-G = 3 hydrogen bonds

which nitrogenous base pair is more stable between A-T and C-G

C-G

contains more hydrogen bonds

structure of DNA

double-stranded alpha helix w/ sugar phosphate backbone

one full turn of helix contains 10 base pairs (3.4 nm)

minor and major groove

DNA base pairs are more exposed where

major groove

where do regulatory proteins preferentially bind on DNA

major groove

what is the bond type between deoxyribose sugars in DNA and where on the sugars

phosphodiester bonds

5' and 3' carbons

orientation of two strands in DNA

anti-parallel

what does anti-parallel orientation allow for in DNA

carrying of information

what kind of DNA contains a higher order structure of chromatin

Eukaryotic DNA

octamer

mature histone with 8 subunits

nucleosomes

the basic unit of chromatin

consists of DNA wrapped around eight histone proteins

what makes up chromatin?

DNA + histones

open chromatin consists of?

11 nm fibers

beads on a string-like

what is chromatin organized onto

protein scaffolds

packing density increases in what kind of chromosomes

mitotic chromosomes

eukaryotic chromatin organization

organized into chromosomes even when not condensed for mitosis

semiconservative DNA replication

each parent strand serves as the template for the synthesis of a new daughter strand

daughter cell receives chromosomes that each consist of one original and one new strand of DNA

T/F a newly synthesized strand remains paired with a parent strand

TRUE

chain elongation of DNA polymerase

the addition of a new nucleotide to the end of a growing nucleic acid polymer requires a 3'-OH at the 3' end

phosphate group at a 5' C of the correct new nucleoside triphosphate is joined to the 3' end of the elongated chain (phosphodiester)

what occurs if there is no 3'-OH for chain elongation

new nucleotide cannot be added and DNA synthesis stops

DNA synthesis by DNA polymerase occurs where

the replication fork

function of DNA polymerases

enzymes that synthesize DNA

can only extend with a primer

DNA replication starting point

origin of replication

DNA replication movement

moves in both directions at the replication fork

leading strand to lagging strand

okazaki fragment

short segment of DNA synthesized discontinuously in small segments in the 3' to 5' direction by DNA polymerase

located on lagging strand

lagging strand purpose

initiates replication from RNA primer

dissociates once okazaki fragment forms

leading strand of prokaryotes

DNA polymerase III

prokaryotic leading and lagging strand synthesis

primase synthesizes short RNA oligonucleotides copied from DNA

DNA polymerase III elongates RNA primers with new DNA

joinging okazaki fragments steps

DNA polymerase I removes RNA at 5' end of neighboring fragment and fills gap

DNA ligase connects adjacent fragments

Eukaryotic leading and lagging strand synthesis

DNA polymerase A synthesizes short RNA oligonucleotides copied from DNA

DNA polymerase D elongates RNA primers with new DNA

which DNA polymerase does most of the elongating in eukaroytes

DNA polymerase D

Eukaryotic joining okazaki fragments

RNAse H removes RNA at 5' end of neighboring fragment and polymerase D fills in gaps

DNA ligase connects adjacent fragments

DNA replication of circular DNA

begins from single origin and goes both ways

DNA helicase separates DNA strands

DNA gyrase removes supercoiled twists and rejoins the DNA strands

the separation of DNA strands by DNA helicase leads to

super-coiled (overwound) areas

Replication of linear chromosomes

DNA synthesis goes in both directions from multiple origins

topoisomerase enzyme removes supercoiled twists that form when DNA strands are separated

what happens to histones during DNA replication

removed

what's required to reassemble histones after DNA replication

Chromatin assembly factor 1 (CAF-1)

Proliferating cell nuclear antigen (PCNA)

CAF-1 purpose

scaffold for histones

assembles histone octamers

proliferating cell nuclear antigen (PCNA) purpose

wraps the DNA around octamers to form nucleosomes

marker for cell division

major problems after DNA replication

gap remains at end of chromosome

if gap isnt filled, the cell removes the unpaired end and chromosomes will be shortened after replication

telomeres

specialized sequences at the ends of chromosomes that contain telomerase

without telomerase, telomeres continue to shrink after each replication and cell division

telomerase function

enzyme that lengthens telomeres after DNA replication

has RNA that acts as a template, allowing DNA to continue replication without shortening

what occurs if chromosomes lose their telomeres

cell stops dividing

location of transcription in prokaryotes

cytoplasm

co-transcriptional translation

Prokaryotic process where mRNA is translated while still being transcribed; impossible in eukaryotes, because transcription and translation happen in separate locations

location of transcription in eukaryotes

nucleus