Module 5

what is molecular cloning- cloning or replicating a chosen segment of DNA resulting in GMOs

what are the two methods for molecular cloning? recombinant DNA technology or PCR, polymerase chain reaction

what are the main differences between recombinant DNA and PCR- PCR can be done in a test tube and doesn’t require a living cell

recombinant DNA technology- bacterial plasmids are cut with restriction endonuclease (EcoRI) and specific genes are added in.

what enzyme cuts the plasmid at specific sites and how- restriction endonucleases and they cut it in 4, 6, or 8 palindromic sequences with sticky ends

What sticks together the DNA fragment through ligation and the sticky ends of the plasmid? DNA ligase through covalent phosphodiester bonds

what’s the final step after the DNA fragment is attached to the plasmid?- the recombinant plasmid is the vector and will replicate in host cells.

recombinant plasmid containing whatever gene you’re wanting to study

how to get the inserted gene sequence? polymerase chain reaction (PCR)

polymerase chain reaction (PCR)- design DNA primers that are complementary to sequence (forward binding to 5’ and other one binding to 3’).

what does temp 94 to 96 degrees celsius in PCR do? denatures DNA strands.

why is temp then lowered to 68 degrees in PCR? primers are able to attatch to sequence.

finally, what does raising the temp slightly to 72 degrees celsius do? allows for DNA polymerase to create a full sequence (the process of elongation) (bc of DNA polymerase being used that lives in hot springs)

why is bacteria a good way to study a specific protein in more detail? bc easy to insert a gene and produce multiple copies due to the fast reproduction of bacteria

why is cloning eukaryotic genes difficult/impossible? contains numerous, large introns which need to be removed and PCR + plasmid vectors have a 10 kilo base-pair-limit

why is the mRNA used in cloning eukaryotic genes? bc it lacks introns (unlike template DNA) so it’s a compact version of a eukaryotic gene that retains all of the protein coding information.

what is done to mRNA to synthesize a cDNA? The enzyme Reverse Transcriptase is used, along with an oligo-dT primer that is complementary to the polyA tail, to synthesize a complementary DNA

after reverse transcriptase is used on mRNA and creates a cDNA what’s it used for? the complementary DNA can be inserted into a plasmid and cloned or amplified by PCR

genetically modified organism (GMO)- any organism that has been manipulated to carry new genetic material, from either a different species or synthesized in the laboratory.

what do Plasmid vectors for cloning and expression in bacteria to make genetically modified bacteria need? origin of replication, restriction endonuclease sites (polylinker), a selectable marker gene so cells that do not contain the plasmid can be eliminated, gene to distinguish between recombinant and non-recombinant plasmid, a promoter to drive transcription (and translation) of the inserted foreign gene

why is a promoter to drive transcription (and translation) of the inserted foreign gene so important? because without it the plasmid would just re-ligate without the foreign gene so resulting in an empty plasmid

GMOs genetically modified organisms: engineered crops

have no effect on health

transgenesis adds a new gene

genome editing

stem cells- those cells that will continue to divide and replenish the body’s cells for the rest of the patient’s life

CRISPR gene editing

CRISPR- stands for __C__lustered __R__egularly __I__nterspersed __S__hort __P__alindromic __R__epeats and functions as a library of foreign DNA samples that represent viruses or pathogens that could harm a bacterial species

crispr-cas9 in modern medicine

what is Cas9? it’s a protein enzyme that binds short RNAs made from the CRISPR gene library

sickle cell disorder (SCD)

why is cas9 binding to a short guide RNA segment important? when it binds to the RNA it cuts the DNA at a programmable site

what happens after Cas9 cuts the DNA? creates a double-strand DNA break where non-homologous end-joing occurs

non-homologous end-joining: the cell’s DNA repair system will trim the broken ends and ligate them together which often creates a small deletion as a result of the trimming.

if it’s a homologous end-join then what occurs: homology-dependent repair system kicks in when the cell’s DNA repair system can use the matching DNA as a template to repair the break in the DNA

single nucleotide substitution that leads to a glutamic acid to valine substitution

hydrophilic to hydrophobic amino acid change: causes a lot of problems

hemoglobic clumps together leading to sickle-shaped red blood cells

LEARNING CATALYTICS

4 ^6 comes from that it can be any of the four bases

mutation in intron does nothing to protein product

Q8 can be B or C

stem cells- self-renewing cells that can divide and go thru mitosis and divide into multiple different types of cells. can continue to divide and both replace themselves and produce progeny cells that differentiate into new blood and immune system cells, or skin cells, or cells that line the gut and airways, or muscle cells.

differentiated cells- cells that have a specific function within a multicellular body

what is somatic cell nuclear transfer (SCNT)- nuclei (DNA) from a person’s skin cells that are in cell cycle arrest due to starvation are transferred into egg cells with no nucleus (DNA) to create stem cells to make new therapies like creating liver, muscle, or nerve cells. it’s limited to vertebrate animals

what are the difficulties or obstacles for animal cloning- low success rate, high complications, possible premature aging

pluripotent or totipotent cells- early human embryo cells that can become any cell type in the body (except the amniotic sac) and it’s part of of the inner cell mass

multipotent- stem cells that can become a specific branch of cells (ex: nerve stem cell can create all diff cells in neuro system)

therapeutic cloning- uses enucleated human eggs and somatic cell nuclear transfer technology to create a human embryo that is a genetic clone of the patient. is done in a lab and forms new tissues/organs that won’t be rejected by the patient’s immune system

hematopoietic stem/progenitor cell lineage includes…

red blood cells, erythrocytes, Which immune cells, multipotent cell

how to obtain embryonic stem cells- induced pluripotent stem cells (iPSCs) which transforms adult differentiated (multipotent) cells into pluripotent cells by using 4-6 different transcription factors. but cancer or deformed cells are an issue

ethical concerns for sourcing of human eggs

• remove DNA from a donor egg
• process is inefficient and it destroys the embryo
• implementation into a human’s uterus has high risk of fatality

different between reproductive and therapeutic: doing the same process but instead of implementing the nucleus into the surrogate mother it’s put into tissue culture to potentially grow into specific tissue that can be utilized for person (ex: skin graft)

explain dolly: ex of mammilian reproductive cloning, took egg from scottish black sheep and took nucleus from finn-dorset egg and dolly was born and dolly looked like from finn-dorset nucleus donor not egg donor. 1 success case out of 277

what is alternatives to embryonic stem cells (avoid ethical issues): induced pluripotent stem cells (IPSC)

1 purpose of IPSC- results in induced pluripotent cells that can be used for cellular differentiated and then observe genetic abnormality with diff drugs/treatments

who has innate immunity- everyone from bacteria to trees to worms to humans, and can include physical barriers to anti-microbial chemicals defense

who has a more complex adaptive immune system- only jawed vertebrates featuring antibodies and cytotoxic “killer” cells that recognize billions of different molecules with high specificity.

antigen receptors- a T-cell receptor (TCR) or an antibody molecule (immunoglobulin, Ig).

what are T cells and B cells? they are lymphocytes, types of white blood cells, that are able to recognize foreign (non-self) antigens.

what type of receptors do T cells have? T-cell receptors on their cell surface;

what type of receptors do B cells have? cell-surface antibodies.

what do helper T-cells do? APCs phagocytose pathogens or foreign cells and digest them into peptide fragments and then send the fragments bound to Class II MHC to cell surface for T cells to inspect. Once the T cell recognizes peptide-MHC II complex via its T-cell receptor it activates and divides

what do B cells do? they recognize and bind antigen via their cell surface Ig. They endocytose, process and present the peptide as fragments on their cell surface MHC II proteins. responsible for humoral (noncellular; mediated by soluble proteins) branch.

what do cytotoxic T cells (CTLs) do? they look for cells that are presenting non-self or altered peptides on Class I MHC molecules. their T-cell receptors kill the virus or cancer infected cell or cells that express a diff MHC I allele than the host thru signaling apoptosis and then if needed secretes granzymes (destructive enzymes) and perforins,

perforins- proteins that assemble in the target cell membrane to create holes and destroy the target cell.

antigen-presenting cells (APCs) that have phagocytic activity and express Class II Major Histocompatibility (MHC) proteins.

First antigen has to be presented to the rest of the cells in the immune system

activates helper T cells, includes B cell, helper T cell, or cytotoxic T cell

cancerous cell, or virus infecting own cell then cytotoxic T cell is activated

memory helper T cells are clones or mugshots of antigens exposed in the past

“effector” cells- activate and execute the humoral and cellular immune responses.

is MHC Class I bound to internal or external cell? internal peptides

does MHC Class I present to CTL or helper T cells? CTL

does MHC Class II present to CTL or helper T cells? helper T cells

MHC class I (all cells) activate cytotoxic T cells to destroy cells

is MHC Class II bound to internal or external cells? extracellular peptide antigens

when antibodies are making receptors to fit antigens they are making as many variations as possible.

if the heavy chain has the V domains and D segments FIGURE OUT MATH!!!!!!!! ON FINAL

heavy chains is VDJ

light chains is VJ

if antibody uses both heavy chain and light chain to make a single antigen binding pocket

quaternary structure with light and heavy chains

vaccines contain antigens for specific viruses

MVP is helper T cells some pathogens directly target helper T cells like HIVS. virus attatces to cell surface and insert its own RNA to reverse transcribe to DNA and stick the DNA into the genome of the helper T cell

HIV Drugs reduce viral load aka trying to stop the process of making new viruses

Class II is associated with helper T cell immune response

helper T cells dont eliminate but cytotoxic does eliminate

100,000-200,000 years ago humans evolved from shared common ancestors of Neanderthals and Denisovan. Eurasian has mixing with 1-4% of neanderthal DNA in nuclear DNA not mitochondrial.

originated from Africa (has greatest genetic diversity in continent, founder effect as other countries have less genetic diversity so that’s where other people are moving into)

Haplotype- set of DNA markers that are inherited together

Single Nucleotide Polymorphisms- differentiate different lineages through different nuclutoides

where is mitochondria DNA from? (maternal lineage)

where is Nuclear DNA from? (inherited from all female and male ancestors)

why is the Y chromosome or mitochondrial DNA used for lineage tracking? because it doesn’t undergo recombination or shuffling during meiosis

where is Y chromosome from? (inherited from father)

where was Y chromosome point of origin? 275,000 years ago

how did humans evolve and adapt to malaria? there are high number of sickle cell disease and thalassemias due to balancing selection

Learning objective 2

1000 genome project- sequence 1000 healthy genomes from around the world. were able to identify when different traits evolved through time

blue eye color

• mutation in OCA2 gene so lack of melanin
• common ancestor had this mutation 6000-10,000 years ago and then passed it down
• neutral mutation bc no benefit or drawback

CCR5-delta32

• \
• makes T cell less effective BUT it protects against HIV because the antigens can attach to T cell properly
• more prominent in Europe, South America

UV Radiation & Human Skin color

• UV is good for vitamin D production but bad for breakdown of folate and causes cancer
• melanin (dark pigment) which acts like a natural sunscreen which balances vitamin D and folate levels
• Equator with a lot of sunlight can break down folate → favors dark skin
• at poles w/o sunlight can cause vitamin d deficiency→ favors light skin

current human evolution

• gene flow: mixing of alleles between populations or preference when mating

walking upright

• separates us from ancestor and other primates
• we have a shortening o pelvis and lengthening of femus
• 6 million years ago Sahelanthropus is oldest evidence

cost of walking upright

females have a greater angle from leg to pelvis and ischial spine is more spread apart. to help with childbirth and help fit baby through birth cannal. Babies have least developed brains because human babies are born at earlier development stage to fit through pelvis OR brains require a lot of energy to make then babies are born earlier to stop sapping a lot of nutrients from mother

humans have a lot higher brain size (relative to body mass) than chimpanzees.

selection on baby head size (babies need to be big enough to survive, but not so large they can’t pass through the hips) stabilizing selection.

baby sizes have been increasing due to better nutrients and higher weights of mothers so science helps with c-sections.

having more C-sections causes relaxed selection so less selection towards smaller babies. More mothers are needing surgery and also more elective surgery is increasing. Artificially changing traits