Bio 314 - Exam 3

Process of early cloning experiment

1. Isolate nucleus from frog skin cells

2. Inject nucleus into unfertilized egg that’s been altered with UV light

3. Egg grows into normal embryo →tadpole clone of parent

What did early cloning experiments prove?

Different cell types contain the same DNA, with only the expression of differential genes altering their function

housekeeping genes

Universally expressed in every cell type like actin for the cytoskeleton, DNA and RNA polymerase, and DNA repair enzymes

selective expression of genes

Requires signals from the surrounding environment to influence expression (ex. hemoglobin only expressed in red blood cells)

Examples of gene control beyond expression

Molecular switches (GDP/GTP), phosphorylation, cellular location

Transcriptional regulators

Proteins like activators and repressors that recognize cis-regulatory sequences in DNA. Have a-helix or b-sheet structural motif to read/form dimers with DNA

non-cooperative binding

The binding of transcriptional regulators to DNA does not influence the subsequent binding of more regulators. Steady increase in proteins, dimers stable

cooperative binding

Transcriptional regulator binding makes it easier for subsequent regulators to bind. Most common, dimers less stable

Repressible operon (bacteria)

Always on. TRP = biosynthesis of tryptophan, contains trp promoter with RNA pol and operator, and trp genes. TrpR encodes trp repressor that binds with tryptophan, and interact with trp operator to turn expression off

Inducible operon (bacteria)

Always off. Lac operon normally repressed, LacI gene makes repressor in the absence of lactose. When lactose binds repressor is no longer expressed and lac operon is transcribed

inducer

inactivates repressor (like LacI)

anabolic pathway

Builds proteins, can be repressed

catabolic pathway

Breaks down proteins, can be activated

Transcription initiation complex (TIC)

Contains promoter (TATA box and start codon), transcription factors that bind to control elements like the TATA box, and RNA pol II (makes mRNA)

distal control elements

Non-coding DNA sequences made of enhancers that interact with activators/repressors and the transcription initiator complex when bent

mediator proteins

Bind DNA strand and connect enhancers to transcription initiator complex

insulator elements

sit between genes to prevent regions of different genes from interacting

introns

Non-coding nucleotide sequences that must be spliced to translate mRNA into proteins

How does the cell get positional information?

Gap genes (hunchback, Kruppel, knirps, giant) respond to transcription regulators that are unevenly disturbed along the length of the embryo to give the cell positional information early on in development

Ways to control transcriptional regulators and post-translationally

synthesize when needed, activate through ligand binding or covalent modifications, formation of complex to bind DNA, unmask an activation domain, removal of inhibitory proteins, release regulator from a membrane

pluripotent cell

original undifferentiated embryonic cell with maternal RNA (womb env’t drives function), each round of mitosis/meiosis divides transcriptional regulators between daughter cells

How can transcriptional regulators impact a cell?

example → fly with eyeless gene artificially expressed in leg precursor cells can form an eye on its leg

induced pluripotent stem cells (iPS)

de-differentiated cells that look and behave like embryonic stem cells

master transcriptional regulators

transcriptional regulators whose expression can reprogram cells into iPS (ex. skin cells can be re-differentiated into muscle cells, neurons, etc)

How do cells have “memory”?

Things like DNA methylation, x-inactivation, CG islands, genomic imprinting, and master cell-type transcription factors (activate the transcription of their own genes) influence transcription in cells and their descendants. A differentiated cell maintains its identity and passes it on

How does positive feedback relate to cell memory?

A master cell-type transcription regulator activates transcription of its own gene, or a transient signal turns on transcription of a gene and after the signal is gone the gene continues to be transcribed

What questions does a cell have to consider?

What genes do I express, how much, and when?

alternate mRNA splicing (post-transcriptional control)

Production of different RNAs from the same gene since segments can be removed by spliceosome

poly-A shortening (post-transcriptional control)

Acts as “timer” for how long mRNA can stay in the cytosol before degradation

RNA interference (RNAi) (post-transcriptional control)

Short ssRNA serve as a guide to target specific RNAs in a cell (miRNA and siRNA). Inhibit translation of RNA or destroy entirely

microRNA (miRNA)

Synthesized by nucleus and is a guide to destroy target mRNA

1. miRNA synthesized, forms double strand structure

2. Gets “cropped” at cleavage sites and exported to nucleus

3. Gets “diced” by Dicer, single strand siRNA is complementary to target

4. ss siRNA incorporated into RISC complex

5. Proteins in RISC slice exact match target, if not exact match then translation is inhibited

small interfering RNA (siRNA)

Used in cellular defense to degrade foreign dsRNA molecules

RNAi use in research

siRNA in cell cultures or whole organisms allow for the inactivation of any gene by evoking RNAi response, is used in medical applications where human disorder results from misexpression of genes

CRISPR-Cas9

Endonuclease, Cas9 protein complex uses guide RNA to cut complementary DNA strand. Based on bacterial immune system genes where cut ends are ligated w/random nucleotide b/t them or repair enzymes correct mutation or insert transgene

cell signaling

cell is stimulated or inhibited by extracellular signals, usually chemical signals (can be light, temp, touch)

Three components of cell signaling

1. Reception- ligand binds to receptor inside or outside cell on PM, organelles, or nuclei

2. Transduction- Each step can amplify the ligand response to create large change, allows multiple levels of control through intracellular signaling proteins

3. Response- nuclear response, gene expression, cytoplasmic change, protein function

short range (local) signaling

paracrine, cells secrete ligand into ECF and it can only influence the cell it binds to (ex. coordinates tissue growth, prokaryote communication)

long range signaling

endocrine, secrete signaling molecules into the bloodstream, slower (ex. hormones)

synaptic, neuronal network for rapid communication where neurotransmitter is the messenger

example of ligands

proteins, peptides, AAs, nucleotides, fatty acids, steroids, gas

What are the ways signaling molecules (ligands) can be released?

1. exocytosis (transferring things out of the cell using a secretory vesicle, for neurotransmitters and hormones)

2. diffusion (for small hydrophobic molecules like gas and steroids)

What does the speed of cellular response depend on?

Existing proteins present in the cell

Fast = pre-existing proteins only need to be modified (milliseconds-seconds)

Slow = Gene expression must be altered to build something (minutes to days)

How does the concentration of signaling molecules alter cell response?

The concentration needed to trigger a response is variable (hyperbolic, discontinuous, sigmoidal)

hyperbolic

smooth graded response over a range of concentrations, response is