U2: Virology & Gene Regulation

DIfferent methods pf gene sequncing and regulation & virus basics

What is a virus? 

piece of genetic material protected by a protein shell called a capsid that invades and takes over a cell in order to replicate itself.

What are the two shapes of viruses?

Capsid (gene inside) and some have membrane envelopes with membrane capsid & protein spikes on the outside.

Life cycle virus

Virus use host cells enzymes and monomers to replicate their genome and make protein. Some virus have dormant phase inside host cell.

Viral Entry 

viral proteins interact with host cell membrane proteins to initiate entry. 

Viral Genomes

Code for viral proteins needed in entry replication and viral particle creation. Can be RNA/DNA & SS/DS

Central Dogma for viruses

RNA template is used to create DNA then protein, then transcribe to RNA to protein 

RdRP

RNA dependent RNA polymerase that can replicate RNA direactly from viral RNA

Retrovirus

Rare viruses (HIV) use reverse transcriptase to make DNA “provirus” that can be transcribed by host cell to viral RNA

Positive RNA

strands that the genome can be used directly as a mRNA & translate (RdRP)

Negative RNA 

genome is the base-pair compliment of mRNA, so an RNA replication step is needed to make the proper mRNA for translation

In which strand is RdRP needed to be carried? 

Negative 

Why are Neg and Pos strands significant?

Both are needed for RNA replication in viral replication

Where does RNA replication occur?

Nucleus of the host cell

Retrovirus replication

RT RNA—> ssDNA, replication dsDNA—> Insertion Provirus —> transcribed in host DNA —> translated

RT-PCR

reverse transcriptase is used to make DNA from RNA into cDNA for research and diagnostic purposes 

cDNA

Measures RNA levels'; identify viral RNA, measuring gene expression

What makes cDNA unique?

No introns only clones exons; gene sequences only

Fluorescent

Microscopy visualizes content and location of biomolecules. 

Fluorophores

absorb light of one color and then emit light of a different color.

Affinity of Fluorophores

Fluoro has inherent affinity for target cells

Conjugating Fluorophores

Fluoro will attach to molecules with affinity for target

FISH

Fluoro In Situ Hybridization uses labeled nucleic acid to identify complementary nucleic acids present in cells 

Purpose of FISH?

Highlights cell of a particular high concentration of RNA/mRNA; which cells are making the gene/mRNA? 

Measurement techniques cDNA 

q-PCR, Microarrays, RNA sequencing

Q-PCR

Measures amount of a few specific mRNA ( few specific genes); more precious

Microarrays

Compare mRNA found in two different cells for known genes; is specific gene present? Compare multiple gene at a time 

Benefits of qPCR

more mRNA (starting material) = more cDNA rapid PCR production. Less cycles are highest

Reporter Gene

Help identify when and where certain promoters are actively expressed. More activity = higher intensity light

Protein Identify

Anitbodies

Antibodies

immunoglobulins. proteins attach to molecules with specific 3D shape called epitopes. Antigen finds the antibody. 

Western (immuno) Blotting 

separate mixture of proteins by gel electro, transfer to membrane and then stain with antibody. When and where protein found? 

ELISA

Enzyme linked immunosorbent assay; identify presence of protein or other molecule in solution using antibodies 

Fluorescent microscopy

Antibodies conjugated with fluoro; spital information of protein in cells

Fluorescent proteins

proteins that naturally fluoresce based on sequence; genetically attached to target protein to make fusion gene

ChIP

Identifies DNA sequences that specific proteins bind to

What cis-regulator does it bind to? Under what conditions?

ChIP (Chromatin immunoprecipitation)