Exam 2 Lecture Notes Based

What is the advantage of using intact tissue for experiments

Provides the most realistic source of material, and some proteins can be obtained without breaking the tissue

Why are cell cultures often used instead of tissue

They provide a more homogeneous population, are convenient, and allow extraction of material for lab purposes

What does in vitro mean

“in glass” experiments on culture cells that are no longer alive, performed in a test tube

what does in vivo mean

“in the living organism” experiments are conducted on intact organisms where reactions occur within cells

What is a callus in plant cell cultures

A mass of relatively undifferentiated cells

What is replicative cell senescence

When a cell stops dividing in culture after a finite number of divisions

what is an immortalized cell line

A cell line coaxed to proliferate indefinitely by introducing a gene encoding the catalytic subunit of telomerase to maintain telomeres

What is culture shock in cell lines

Cells stop dividing after limited divisions due to excessive stimulation of replication. It can be overcome by introducing oncogenes

What are transformed cell lines

Cell lines made from cancer cells. They grow without needing attachments, grow at higher density, and cause tumors if injected into organisms

What are primary cultures

Cell cultures prepared directly from tissues. They can be made with or without an initial fractionation step and can be separated into a second culture

What are hybridoma cell lines used for

They are used to produce an unlimited monoclonal antibodies by propagating a clone from a single B lymphocyte

What is a hybridoma

A hybrid cell line created by fusing a tumor cell with an antibody-secreting B cell

What is a monoclonal antibody

An antibody from a hybridoma that is identical and recognizes a single antigenic site

What are some uses of monoclonal antibodies

Localizing proteins in cells/tissues, tracking protein movement, purifying proteins, and treating diseases

What is subcellular fractionation

A method to reduce material complexity before protein purification

How are cells separated into components

By breaking membranes to make a homogenate, then centrifuging at specific speeds to pellet organelles based on size and density

What is velocity sedimentation

Separates components by sedimentation speed using a salt solution

What is equilibrium sedimentation

separates by buoyant density using sucrose and centrifugation

What is column chromatogrpahy

Separation of substances in solution through a porous matrix based on interactions like charge or size

What is affinity chromatogrpahy

Separation based on a specific macromolecular binding interaction. It is highly selective

What is HPLC

High-performance liquid chromatography. It uses tiny beads under high pressure to efficiently separate solutes

What is immunoprecipitation

Affinity purification using antibodies attached to beads to capture specific proteins from extracts

How are genetically engineered tags used in protein purification

Proteins are expressed with a recognition tag to allow specific purification

What is SDS-PAGE

Separates proteins by size. SDS and B-mercaptoethonal unfolds proteins and masks charge, so migration depends on size

What is 2D gel electrophoresis?

Combines isoelectric focusing (charge) and SDS-PAGE (size) to separate proteins

What is Western blotting

Also called immunoblotting. Proteins are electrophoresed, immobilized on a membrane, and detected with labeled antibodies

How are hydrodynamic measurements used

Centrifugation and gel-filtration chromatography reveal size, shape, and mass of protein complexes

How does mass spectrometry work

Separates ions by mass-to-charge ratio using an ion source, mass analyzer, and detector toe generate a mass spectrum

What is co-immunoprecipitation

Identifies protein complexes by pulling down a target protein with associated partners, analyzed by mass spectrometry

How can protein interactions be studied optically

Using equilibrium/ kinetic binding experiments and fluorescent anisotropy to measure association/ dissociation rates

How is protein structure determined

X-ray crystallography and NMR

What is X-ray crystallography

using a crystal of proteins and shooting X-rays at them to form a pattern on a detector, and studying the patterns to determine shape and thus function

How can protein function be predicted

By comparing amino acid sequences and structures to known proteins. Similar sequences usually indicate similar functions

What is recombinant DNA technology?

Combining DNA segments from different sources to make new DNA, used in gene cloning, modification, and protein production

What are restriction nucleases

Enzymes that cleave DNA at specific sequences, producing fragments of defined sizes

How does gel electrophoresis separate DNA

DNA moves toward the positive electrode. Fragments are separated by size, and then stained with ethidium bromide to visualize

How can DNA be labeled in vitro

Using DNA polymerase to copy DNA with radioactive or chemical markers

How are genes cloned using bacteria

DNA fragments are inserted into plasmid vectors or BACs. Recombinant DNA is replicated in the bacteria

What is a DNA library

Collection of cloned DNA molecules representing an entire genome from a genomic library, or cDNA from mRNA (cDNA library)

What is hybridization and what is it used for

Complimentary nucleic acid strands form base-paired duplexes. It is used for detecting specific sequences with DNA probes

How does PCR work

Polymerase Chain Reaction (PCR) is a revolutionary technique used to amplify small segments of DNA, enabling the production of millions of copies from a minimal initial sample.

What are some applications of PCR

DNA cloning, diagnostics, pathogen detection, forensics, and verification of food authenticity

What is Gibson Assembly

A method of joining DNA fragments and plasmids using exonuclease, DNA polymerase, and ligase to produce recombinant DNA

How can proteins be produced in large amounts

DNA cloning with expression vectors, producing stable mRNA, translating into proteins, followed by purification

What is dideoxy (Sanger) sequencing

Uses DNA polymerase and ddNTPs to synthesize fragments ending at different positions to determine sequence

What is the difference between genotype and phenotype

Genotypes are genetic constitution and phenotypes are observable characteristics

How are mutations used to study gene function

Mutations causing loss or gain of protein function can be identified through DNA analysis. Genomics accelerates discovery this way

Where do transcription and splicing occur in eukaryotic cells

In the nucleus

Where does translation occur

In the cytoplasm

Are transcription and translation coupled in eukaryotes

No, they are separate processes with different machinery and mechanics

What happens to mRNA abundance as DNA transcription increases

The more DNA is transcribed, the more abundant mRNA becomes

Where does DNA replication occur and when does it stop

Occurs in the nucleus, stops during mitosis/meiosis

What are introns and exons

Segments of DNA within genes. Introns are noncoding and exons are coding

How can gene expression vary

Efficiency, timing, and location

What is temporal regulation

Control of when a gene is expressed (Ex: at specific ages in life)

What is spatial regulation

Control of where a process happens (in specific parts of the body)

What are the key differences between RNA and DNA

RNA is single stranded and has ribose instead of deoxyribose, and uracil instead of thymine

How does RNA fold into 3D structures

Hydrogen bonding between purines and pyrimidines

What are purines

Adenine and Guanine

What are pyrimidines

Cytosine and Uracil and Thymine

What enzymes carry out transcription

RNA polymerase

What categories of RNA molecules exist

Coding RNAs and noncoding RNAs

What are the coding RNAs

mRNA

What are the noncoding RNAs

tRNA, rRNA, snRNA, miRNA, siRNA, piRNA….

What is a promoter

A nucleotide sequence indicating the start site for RNA synthesis, usually upstream at the 5’ end of a gene

Can either DNA strand serve as a template

Yes, depending on promoter location, but only one strand is used per gene

What is required for transcription initiation in eukaryotes

RNA polymerase, transcription factors, and general transcription machinery

What do enhancers and distant regulatory sequences do

They bind activator proteins to regulate transcription from a distance

Which RNA polymerases transcribe which genes

RNA poly 1: Transcribes three rRNA gene

RNA poly 2: Transcribes all protein coding genes, and some other coding RNA genes

RNA poly 3: Transcribes for tRNA and other small RNAs

What is the TATA box

Promoter sequence where general transcrption factors bind to recruit RNA poly 2

What occurs during transcription elongation

RNA synthesis is tightly coupled to RNA processing

What is 3’ polyadenylation

Addition of adenines to the 3’ end of RNA to protect it from degradation

What is RNA splicing

Removal of introns from pre-mRNA to produce mature mRNA

How much of a gene is typically spliced out? 

80%, all the introns

What is alternative splicing

Producing different mRNAs from the same gene (in 10% of cases)

What enzymatic machinery performs splicing

The spliceosome, composed of snRNAs and associated proteins (snRNPs)

What energy is required for splicing

ATP to synthesize phosphodiester bonds

Where are mature mRNAs exported

Through nuclear pores to the cytoplasm

Where are noncoding RNAs synthesized

In the nucleus

What is the nucleus

A ribosome producing factory

How are mRNAs decoded

In sets of three nucleotides (codons)

What molecules match amino acids to codons

tRNA molecules

Where are amino acids added during translation

To the C-terminal end of the growing polypeptide

What is a ribosome

A ribozyme that decodes mRNA and coordinates protein folding and modification

What signals start and stop of protein synthesis

Start codons (AUG) and stop codons (UAA, UAG, UGA)

What are polyribosomes

Multiple ribosomes simultaneously translating the same mRNA, forming a spiral structure

What happens to misfolded proteins

They get marked for destruction via polyubiquitylation

What is polybiquitylation

The addition of multiple ubiquitin molecules marking proteins for proteasome degradation

What is the proteosome

Cytoplasmic complex of proteases that degrades marked proteins. It has sequestered active sites

What is the role of ubiquitin hydrolase

Gatekeeper protein allowing only target proteins into the proteasome

What determines the final amout of protein in a eukaryotic cell

Efficiency of each step and stability versus degradation

List the steps affecting protein production

1. Initiation of transcription

2. Capping, elongation, splicing

3. Cleavage, polyadenylation, termination

4. Export to cytoplasm

5. mRNA degradation

6. Initiation of translation

7. Completion of translation and folding

8. Protein degradation if it misfolds, this is an optional step ofc

What can single-stranded RNA molecules do

Fold into complex structures, store information, transcribe genomes for translation, and catalyze reactions (ribozymes)

Evolutionarily, what came first, DNA, RNA, or proteins

RNA

Where do transcription and splicing occur in eukaryotes

The nucleus

Where does translation occur

The cytoplasm

Are transcription and translation coupled in eukaryotes

No, the are separate processes with different machinery and mechanics

How does DNA transcription affect mRNA abundance

more transcription means more mRNA

When does DNA replication have to stop

During mitosis and meiosis,

What enzyme performs transcription

RNA polymerase