Molecular Biology and Cell Membrane Concepts

Molecular biology

Group of processes to manipulate, analyze, expand, and express nucleotide sequences

Gene transfection

Inserting intact functioning genes

regulated GFP reporter

two types transient and stable

Genomics

Studying an organism's genome

Gene editing

Adding or removing a few bases or full genes

ex. CRISPR or organ xenotransplantation

CRISPR

A method used in gene editing. CRISPR-Cas9 can be used to edit genes in cells and tissues, potentially correcting the genetic basis of inherited diseases like sickle cell anemia, cystic fibrosis, and hemophilia.

Organ xenotransplantation

Transplanting organs from one species to another.

Controlling gene expression

Manipulating the expression of genes.

Gene knock in/knock out

Techniques used to add or remove genes from an organism.

embryonic cells are injected to mice

siRNA (small interferring)

DSRNA (double stranded) that will silence genes complementary to its sequencing via RNAi (RNA interference).

used for transient trasnfection

used to treat huntingtons disease (break down nerve endings) (less likely to have cancer)

shRNA

Same idea as siRNA but is in a hairpin shape.

used for both transfections

Oxford nanopore technology

1.Enzyme will pull DNA through a pore in a naturally occurring membrane protein.

2. The enzyme will unwind the DNA and the nucleotide

sequencing can be read as the DNA is brought through the

pore

3. Ion current can be used to read the nucleotide sequence

Transcriptomes

Umbrella term for RNA.

a). mRNA

b). tRNA

Proteomics

Study of all proteins.

Epigenetics

Change in DNA expression that can be reversed.

methylation

histone phorphorylation

Methylation

A process that can affect gene expression without changing the DNA sequence.

Histone phosphorylation

Modification of histone proteins that can influence gene expression.

Mutation

Damage to DNA bases.

many causes:

uv light

photo will hit the bases and destroy them

when bases are repaired they might not be correctly fixed

-chemicals

Metabolome

Small molecules found in a biological sample.

Secretome

Anything secreted by a biological sample.

ex. protein, cfDNA, exosomes

Cytokine storms

Overactivation of inflammatory immune cells.

can cause ARDS and MOF

STEM cell exosomes decrease cytokin storms

***Downgrades CD4 T cells, CD8 T cells, regulatory T cells

***Upregulates hyperactivated T cells

Temperature sensitive mutants

Used to study in vivo (living) and in vitro (laboratory) protein function at higher temperatures.

genes that exhibit permissive (normal function) temp at 23 degrees celsius and non permissive temp (non functional) at 36 degrees celsius

cant grow at non permissive anymore because growth related proteins are denatured

ex. yeast

Complementation analysis

Used to see if two mutations are on the same gene.

if they were on the same gene then there would be no growth because the mutation makes them temperature sensitive so the dominant gene couldnt grow even at 36 degrees Celsius

Restriction nucleases/enzymes

Used to manage DNA and enable gene transcription (RNA copy)

discovered in 1963 in bacteria so they can ward off viruses

600+ commercially available

cleaves DNA or RNA at restriction sits

Hybridization

Used for techniques such as FISH, antisense DNA/RNA, Northern and southern blots, cDNA microarrays, and molecular beacons.

DNA gel electrophoresis

A technique that separates DNA fragments based on size.

evolved from SDS gel electrophoresis

-uses restriction enzymes

-uses polycrimaline sieve for smaller dNA fragments

-uses agarose sieve for larger DNA fragments

Band/gel shift experiment

An experiment to determine if a protein of interest (POI) binds to a gene of interest (GOI) by comparing gel bands.

if ban is higher up, poi has bound to GOI and hold it up from falling

apopotosis necrosis

apoptosis will show up as a ladder

necrosis= a continuous streak

electrophoresis

SDS gel electrophoresis

A method that DNA gel electrophoresis evolved from.

Restriction enzymes

Enzymes used to cut DNA at specific sequences.

Polyacrylamide sieve

A medium used for separating smaller DNA fragments.

Agarose sieve

A medium used for separating larger DNA fragments.

Apoptosis

A form of programmed cell death that induces non-random DNA cleavage.

Necrosis

A form of cell death that induces random DNA cleavage.

Southern blots

(1) DNA gel electrophoresis bands placed onto blotting paper (nitrocellulose membrane) (a) Nitrocellulose membrane placed on top of gel inside alkaline solution (b) Bands transfer from the gel to the membrane

Nitrocellulose membrane

A type of blotting paper used in Southern blots.

Complementary DNA/RNA probe

A probe used to detect specific DNA/RNA sequences, often radioactive.

Variable Number Tandem Repeats (VNTRs)

Regions of the genome where short strands of nucleotides are repeated adjacent to each other

used in forensic medicine

Single nucleotide polymorphisms (SNPs)

Genetic variations that looks at sigle nucleotide differences primarily in noncoding genes

98-99% of genes are non coding so 1 base can make a big difference in the coding region

used for personalized medicine

Northern blots

A technique similar to Southern blots but used for tracking RNA instead of DNA.

-used to measure gene expression (mRNA)

-can only analyze 1 mRNA transcript at a time

-if we look at multiple mRNA transcripts we will get mixed up on what is used to code what

Snow=drop

Southern= DNA

Northern=RNA

Western= proteins

Gene expression

The process by which information from a gene is used to synthesize functional gene products, typically proteins.

Molecular beacons

(1) Takes RNA or ssDNA complementary probe that has fluorophore and quencher dye on either end (2) The probe region is circular so the fluorophore and quencher dye are next to each other (3) Complementary probe bound to the target (a) Fluorophore and quencher dye are farther away and the fluorophore will fluoresce (b) Tells us the same thing as FISH but is more efficient

cDNA (complementary DNA) microarrays

used to examine up to 8600 genes expressed

harvest mRNA cells and reverse transciptast them to form sscDNA

transfection delivery methods

Transient transfection (i) cDNA placed onto a vector (ii) (iii) Electroporation used to allow vector into cells Used for short term protein synthesis (b) Stable transfection (i) Same as transient transfection except this introduces the GOI permanently in the cell (ii) Will cause permanent protein synthesis c) Both types of cloning require cutting out pieces of bacterial plasmid vectors to place the DNA or cDNA i

Reverse transcriptase

An enzyme used to convert mRNA into complementary DNA (cDNA).

Fluorescent dyes

Dyes used to tag cDNA in microarray experiments for visualization.

Oligonucleotides

Short DNA or RNA sequences used in microarrays to capture cDNA.

Single cell RNA sequencing (scRNA-seq)

Sequences RNA to determine gene expression of a cell.

-used in tissue engineering

-RNA signature

(1) Part of mRNA that is reflective based off the state of the cell (a) Healthy

(b) Diseased

(c) Old

(d) Etc

RNA signature

Part of mRNA that is reflective based off the state of the cell.

Single cell genome sequencing

Taking a cells genome and comparing it to sequencing library for analysis.

-can tell you cell type

-cells from a tissue sample MUST be sorted before sequenced

Genetic cloning

The process of creating copies of DNA fragments.

Genomic cloning

Take all the DNA of one organism, place it into vector, place vector into bacteria (E.coli).

(a) When bacteria undergo mitosis, the gene will be cloned along with the entire genome that we inserted into the vector (2) GOI will always be in the DNA but it is harder to search for it specifically

cDNA cloning

Reverse transcript mRNA to form cDNA of GOI that we place into vector that we place into bacteria (E.coli).

GOI will only be clones if it is used by the E.coli

takes longer than genomic cloning

can make humulin from E.coli by taking out lac operon, place G-SCF cDNA instead

GOI

Gene of Interest.

Humulin

Insulin produced from E.coli, commercially available since 1982.

it works with e.coli bc or pur luck

Transient transfection

cDNA placed onto a vector and electroporation used to allow vector into cells for short term protein synthesis.

Stable transfection

Introduces the GOI permanently in the cell, causing permanent protein synthesis.

PCR

Polymerase chain reaction, used to expand a DNA sample.

c) Uses taq polymerase (1) Heat resistant DNA polymerase

Taq polymerase

Heat resistant DNA polymerase used in PCR.

Transgenic mice

a) Take foreign DNA that you want to study

b) Inject it into fertilized egg cell nucleus

c) Place egg into mother rat

(1) 10-30% chance offspring will have foreign DNA

d) Mice expressing foreign dna are rebred so we can have 100% if

them having foreign DNA

Antisense DNA/RNA

Used to knock in or knock out genes by blocking mRNA coding for a specific protein.

-less effective than si/shRNA

siRNA

Simpler to make than shRNA, used for transient transfection.

short term protein synthesis

-hard to transfect

-used to treat huntingtins disease

Huntington's disease (HD)

Causes breakdown of nerve cells, associated with high levels of huntingtin mRNA.

shRNA

Harder to make than siRNA, used for transient and stable transfection.

gene stays in the genome

Schleiden and Schwann

Suggested there is a 'barrier' between cells in 1830s.

Overton

Used lipid soluble dyes to penetrate cells and determined 'barrier' was made of lipids in the 1890s.

Langmuir trough experiment

Used water surface and placed phospholipids on the water to measure lateral pressure.

when too much pressure the phospholipids formed a double layer

membrane was either mono or bi layered

Phospholipids

Molecules that form the bilayer structure of cell membranes.

Electroporation

A technique used to introduce DNA into cells by applying an electrical field.

Bacterial plasmid vectors

Circular DNA molecules used to transfer genetic material into bacteria.

Gorter and Grended

Extracted phospholipids from RBC to see if membrane is mono or bi layered.

membrane was bi layered

Mudd's Experiment

Placed RBC and WBC in water and mixed them; RBC stayed on top and WBC on bottom.

Membrane Composition

Determined that membrane is made up of phospholipids AND proteins.

Davson-Danielli Model

Depicted membrane as phospholipid bilayer sandwiched between 2 protein layers.

Robertson's TEM Observation

Used TEM to observe cell membrane and used OsO4 for staining.

proved the davson-danielli model

Freeze Fracture Technique

Originally developed for quick fixation like cryosectioning; did not work for this.

E Face Freeze Fracture

Shows inner membrane of cell.

P Face Freeze Fracture

Shows cytoplasm; more bumps than E face.

mosaic freeze fracture

shows most bumps

Cell Fusion Experiment

Fused human and mice cells; integral membranes can be fluid

Cell Patching

We add anti-X Abs; protein X will start to patch up in certain parts.

Capping

Visualizing the concentration of the protein in the part of the cell it clusters in.

Con A FRAP

Uses concanavalin A. binds to carbohydrates and glycosylated proteins.

-most proteins on membrane are glycosylated

used for FRAP

Fluidity of Membrane Proteins

Showed that half of proteins are fluid but not all; stationary proteins stay in place due to cytoskeleton and extracellular matrix.

Mosaic Model

Accurate description of how the cell membrane actually looks.

Advantages of Using RBCs

-Easily purified

-simple centrifugation can separate RBC from WBC.

-RBC has no toher membranes or organelles

-has few cell proteins

-RBC ghosts

RBC Ghosts

RBC undergoes hypotonic lysis; disturbed and resealed to see inner and outer membrane proteins.

RBC Half Life

RBC has half life of ~ 120 days; when RBC gets old, it undergoes shape changes and the body will replace the old ones.

Sickle Cell Anemia

RBC has shorter half life because it is oddly shaped when first produced.

Phosphoglycerides

part of inner membrane that moves to outer membrane in apoptosis.

Sphingolipids

Sphingomyelin is important for nerves and myelin; associated with demyelinating diseases like multiple sclerosis and dementia.

Cholesterol (steroids)

50% of all cholesterol is found in cell membrane; precursor to hormones, Vit D, steroids.

Statins

Gets rid of LDL by interfering with pathway we don't need; LDL causes several health problems.

Flippase

Also known as ABCB4; is an ATPase; can take phospholipids from inner membrane and flip them to outer membrane.

Phospholipid synthesis

Phospholipids are synthesized in the smooth ER inside the cell.

Transport of phospholipids and cholesterol

Not really known; 3 possibilities: vesicles transport them, proteins pinch membrane inwards, or proteins in cytosol transport them.

Integral cell membrane proteins

Typically transmembrane proteins; protein is sticking out of membrane inside and outside the cell.

shows up one only one layer

Peripheral membrane proteins

Lipid anchored proteins; can be single pass or triple pass transmembrane proteins.

Glycophorin

Single pass dimer in RBC; is glycosylated;

is why RBC has a hydrophilic coat

all hydrophobic AAs

Porin

Barrel shaped membrane protein; serves as a passive diffusion channel for small molecules, water, and ions.