Genetics Exam 2

What is a Virus

Genetic material protected by a capsid protein shell that can infect host cells and replicate within them.

How do Viruses Enter a Cell

Viral proteins interact with host cell membrane proteins to facilitate fusion or endocytosis, allowing the viral genome to enter.

What are Types of Viral Enzymes Used to Replicate Viral RNA

RNA Dependent RNA Polymerase (RdRp) - directly replicates viral RNA in viruses like covid/flu

Reverse Transcriptase (RT) - makes a DNA “provirus” that is then transcribed by host cell into viral RNA

What do (+) and (-) Mean for ssRNA Viruses

(+) strands can be used directly as mRNA and translated into protein

(-) strands are base-pair compliments of mRNA, so an RNA replication step is required to make mRNA for translation

Describe the Difference between Coronavirus and Influenza Replication

Coronavirus is a (+) genomic RNA strand, so translation of RdRp enzyme is the first step

Influenza is a (-) genomic RNA strand, so RdRp is carried in the viral particle so RNA can immediately begin replication

What are the steps of Retrovirus Replication (HIV)

1. RT makes ssDNA from viral ssRNA

2. ssDNA undergoes replication to give dsDNA

3. Viral dsDNA is inserted via integrase into host genome - “provirus”

4. Provirus is transcribed by RNA polymerase to make new viral RNA genome

5. Viral RNA is translated to build viral proteins

6. Virus assembles and takes part of cell membrane as envelope

What is cDNA

Complimentary DNA made from RNA by Reverse Transcriptase

Can be used in diagnostics to measure RNA levels, which allows us to identify viral RNA or measure gene expression

Clones EXON-ONLY copies of genes

Can be made with poly-T primers (match poly-A tail)

What is Unique about HIV’s proteins

Some are translated as one long string of amino acids, so HIV has its own protease to cut them apart

Define Zoonosis

Transmission of disease from animals to humans

How do Antiviral Drugs Work

These drugs target specific viral proteins - RdRp or other viral enzymes

• Tamiflu blocks enzyme that cuts carb from influenza molecule that make them bind to cells

• Protease inhibitors stop HIV and CoV-2 from slicing non-functional protein chains

What do Antiretrovirals target in HIV

• Reverse Transcriptase, so ssDNA strand is not synthesized.

• Integrase Enzyme, so viral DNA will not be spliced into host DNA

• Protease, so nonfunctional protein chains will not be divided

Examples of proteins coded for in a viral genome.

• Structural proteins that form viral particle (capsid, envelope glycoproteins)

• Enzymatic proteins that perform genome replication (RdRp, RT, protease)

• Regulatory/Accessory proteins that interfere with immune defense

Describe Fluorecsent Microscopy

Nucleic acid based technique for identifying target genes

Fluorophores can have affinity for target or attach to molecules with affinity for target

They can be excited by a fluorescent microscope, and absorb/emit colored light

Describe Fluorescent In Situ Hybridization (FISH)

Nucleic acid based technique for identifying complementary nucleic acids in a cell

Fluorescent tagged nucleic acids are viewed through fluorescent microscope

Tells if sequence is present, where it is, and how many

Describe RT-PCR (Reverse Transcriptase)

Nucleic acid based technique to create cDNA from a mRNA sample

RT makes cDNA that LACKS INTRONS

How is cDNA used to measure RNA levels?

• qPCR measures amount of few specific mRNA

• Microarrays can compare mRNAs found in two different cells for known genes

• RNA sequencing gives complete view of mRNAs, High through-put and gives SPLICE VARIANTS

Describe qPCR

Nucleic acid based technique

More mRNA means more cDNA, so faster production of PCR product

Low through-put

Describe Microarray Analysis

Nucleic acid based technique w/ high through-put

cDNA is fluorescent tagged and washed over microarray tray, with pieces binding to probes and indicating gene presence in each sample.

Describe Reporter Genes

Nucleic acid based technique using easily measurable reporter protein (fluorescently-labeled)

Reporter gene is inserted into a genome near a regulatory sequence to be studied

What are antibodies (immunoglobulins)?

Proteins of the immune system designed to attach to a specific 3D area (epitope) of antigen

Can be derived from lab animals or genetically modified to contain peptide epitope tags recognized by other antibodies

Describe Western (Immuno) Blotting

Protein based technique that determines where a protein is found

Mixture of proteins is separated by gel electrophoresis and stained with antibody that identifies protein location

Uses LOADING CONTROL / positive control

Tells whether protein is present and what abundance (more qualitative)

Describe Enzyme Linked Immunosorbent Assay (ELISA)

Protein based technique that identifies presence of protein in solution using antibodies

If present, protein binds to capture antibodies and detection antibodies “sandwich” it

Identifies if protein is present quickly, and can give yes/no and quantitative amnt

Describe Fluorescent Microscopy w/ Conjugated Antibodies

Protein based technique that identifies in what cells and where in cells a protein is expressed

Fluorescent proteins are genetically attached to target protein and can be traced

DOES NOT destroy the sample in process

Describe Chromatin Immunoprecipitation (ChIP)

Protein based technique that identifies DNA sequence a protein binds to & where

Covalent bond forms between protein and chromosome before DNA is broken into pieces

Antibody is added to immunoprecpitate the protein/chromatin combination

Define contact, paracine, and endocrine signaling

Contact - cells touching to exchange signals

Paracrine - Most common, local chemicals

Endocrine - can be chemical (endocrine) or electrical (nervous) signals throughout the body; hormones travel through bloodstream, action potentials and neurotransmitters through nervous system

What are the steps of cell signaling

1. Signal generation

2. Signal reception

3. Changes in signaling components (direct effect or transduction)

4. Response (protein activity - nuclear, or abundance - cytoplasmic)

What type of signaling molecules can pass the cell membrane?

Hydrophobic (O2, CO2, N2)

Nuclear receptors - estrogen receptors are both receptors and TFs so signal doesn’t need transduction

What are kinases?

Protein enzymes that transduct cellular signals

Kinases ADD a phosphate group

Phosphatases REMOVE a phosphate group

How does the ubiquitin proteosome chain work?

Ubiquitin labels targeted proteins for destruction with help from E3 ubiquitin ligase

Proteosome breaks polypeptides into small pieces

Ubiquitin hydrolase removes chain for reuse

What is NFkB and what does it do?

Transcription factor for immune response

Has low nuclear concentrations until TNF (fluorescent microscopy) or LPS (immuno blot)

Describe the NFkB pathway

TNF binds to surface receptor and activates the IKK complex that phosphorylates IkB

Phosphorylated IkB is ubiquitylated and degraded by proteosome

NFkB is liberated and translocated into nucleus to activate transcription of NFkB target genes

How is NFkB a negative feedback loop?

NFkB activated the transcription of IkB to inhibit itself. Short and long signals therefore have different effects.

What are the phases of the cell cycle and what happens in each?

G1 - Cell grows and copies organelles (growth checkpoint - external)

S - Replicated genome and centromeres
G2 - Finishes growth (DNA checkpoint - internal)

Prophase - Chromosomes condense, spindle forms

Prometaphase - Nuclear envelope dissolves, microtubules attach to chromosomes

Metaphase - Chromosomes line up (quality checkpoint - internal - before APC activates)

Anaphase - Sister chromatids separate, cell elongates

Telophase/Cytokinesis - Daughter nuclei form, chromosomes de-condense

Chromatid vs. Chromosome

Chromatid is one DNA double helix

Chromosome is EITHER one chromatid or two identical joined at centromere

#chromosomes = #centromeres

Kinetochore vs. Non-kinetochores microtubules

Kinetochore connects proteins at centromere and pulls chromosomes apart

Polar microtubules overlap and push MTs apart to elongate the cell

How does Flow Cytometry measure cell division

Droplets w/ single cells pass through laser to determine DNA content

Cells sorted by stage, longest phase generally G1 (G2 and Mitosis expect 2x DNA content, S 1-2x)

What is BrdU staining?

A unique nucleotide is incorporated DURING S PHASE to be identified with antibody staining

Internal vs external checkpoints

Internal - surveillance for DNA damage and proper alignment

External - growth inhibition, nutrients, growth factor presence

What are cyclins and what do they do?

Proteins that regulate steps of cell cycle

Levels cycle - they are transcribed/translated when needed and ubiquitylated when unneeded

What are CDKs

Kinases that control cell cycle progression (activate mitosis promoting factor MPF)

CONSTANT LEVEL throughout cycle, where activity depends on cyclins

Name a CDK inhibitor and what it does

P21 is an inhibitor of the G1 CDK

Activated by cell stress and damage when P53 transcription factor (tumor suppressor) is activated

What is the Anaphase Promoting Complex

When APC is activated by cdc20 after the M checkpoint, securin is ubiquitylated, which released separase

Separase breaks the cohesin proteins that hold chromosomes medially during metaphase

*also degrades cyclins and innactivates M-CDKs

What are Mitogens

Signaling molecules that stimulate cell division

Describe the mitogen signaling pathway (from mitogen to DNA synthesis)

Mitogen is received by surface receptor tyrosine kinases (RTKs)

GEF phosphorylates GDP to GTP, which activates Ras protein

RAS activates Mitogen Activated Protein (MAP) Kinases and leads to cell division

MAP activates transcription of Myc in nucleus that leads to cyclin synthesis

G1 CDK transmits signal and phosphorylates Rb to activate E2F protein

S phase gene transcribed and S-CDK activates DNA synthesis

What is anchorage and density dependence? How are these solved?

Anchorage dependence - Cells need something to grow onto

Density dependence - cells stop dividing when lacking space

To solve: Membrane proteins link cytoskeleton to ECM (E-cadherin, integrin), regulated by YAP/TAZ TFs

How does Myc affect p21 protein?

Myc attaches to MdM2 (ubiquitin ligase for p53 / oncogene) before it binds to p53 (tumor suppressor)

If binding to p53, then p 53 unable to be the TF factor for p21

No inhibition of g1 CDK and no cell cycle arrest when necessary

High levels of Myc does what?

Triggers binding to MdM2, which allows for p53 to act at TF for p21

Cells experience fail safes and apoptosis

What is E2F and Rb proteins?

Rb - tumor suppressor protein that inhibits function of E2F protein until active G1CDK is present

E2F - family of TFs that turn on genes needed for DNA synthesis

What is spontaneous hydrolysis?

Endogenous DNA damage

Depurination - G is spontaneously lost (deletion or frameshift)

Deamination - amino acid lost and carbonyl added, turning C into U (substitution of A instead of G)

What are DNA Replication Errors?

Endogenous DNA damage

One incorrect nucleotide in every million (mismatch occurs)

What are transposable elements?

Endogenous DNA damage

Transposons code for proteins that move DNA segment(CUT & PASTE) Uses transposase

Retrotransposons code for proteins that copy transposon and move it (COPY & PASTE) Uses reverse transcriptase

Describe the effects of UV radiation.

Exogenous DNA damage

Adjacent pyrimidine bases for covalent bonds (dimers)

Causes stall in replication, alternative splicing, gives future permanent mutations

Describe the effects of chemical mutagens.

Exogenous DNA damage

Molecules may attach to DNA, creating insertion or deletions

Describe the effects of ionizing radiation.

Backbone breaks on both strands (DSB)

Most severe, can lead to translocation and signals telling cells to always divide

Describe base excision repair.

Uracil-DNA glycosylase removed the incorrect uracil from a deamination reaction

Endonuclease removed sugar backbone

DNA polymerase and ligase repair

Describe nucleotide excision repair.

Excision nuclease and helicase remove a strand segment

DNA polymerase and ligase repair the portion

Describe non-homologous repair.

Nucleotides are removed off ends and strands rejoin

Does NOT return accurate sequence, ERROR PRONE

Describe Homologous Recombination

Sister chromatids are used as template after nuclease processes 5’ ends

Returns accurate sequence, ERROR FREE

How are embryonic and adult stem cells different?

Embryonic stem cells are used to make an entire fetus, - totipotent and pluripotent

Adult stem cells are founds in certain locations to make specific sets of cells - unipotent or multipotent

Types of Stem Cells

Totipotent: Can produce all cell types, including tissues and organs needed to form an entire organism.

Pluripotent: Can produce nearly all body cell types but cannot independently form an entire organism.

Multipotent: Can produce several related cell types, such as the different types of blood cells produced by blood stem cells.

What are iPSC

Induced pluripotent stem cells

Adding certain TFs can reprogram cells to be pluripotent

Used to determine drug efficacy or reverse T1D

What does MyoD do?

Master regulatory gene that triggers TFs to transcribe cell-specific genes (differentiation)