Unit 2: From Genomes to Genes - Mechanisms of Inheritance & Cell Division

Human Genome Project

Began in 1990, completed in 2022.
Goal: Sequence the entire human genome.

The Human Genome Project found that the human genome is…

Majority of our genome is non-coding proteins. 

Alleles

Part of the Gene.
Dominant or Recessive
Mutated or Normal

Humans have the same genome, then why different phenotypes? In other words, what are some ways we get genetic variations? 

Mutations
Homologous Recombination (Crossing-Over)

Independent Assortment 
Random Fertilization
Transposable Elements

What is a mutation? 

A mutation is a permanent change in our DNA sequence. 

Methods of Gene Alterations

1. Direct mutations to the DNA Sequence (within a gene — coding region).

2. Mutation in regulatory DNA (promotor, enhancer, silencer).

3. Gene Duplication & Divergence

4. Exon Shuffling.

5. Transposons (Mobile genetic elements).

6. Horizontal Gene Transfer (Commonly seen in prokaryotes)

7. Insertion/Deletion

Direct mutations in our genes occurs when

a base pair is modified/removed/added, affecting the stability, transcription, splicing, protein structure, and/or function of that gene. 

Mutation in regulatory DNA (promoter, enhancers, silencers) occurs when

changes in our DNA lead to changes in transcription and thus, translation.

Gene Duplication & Divergence occurs when

a mutated gene reproduces, producing new sets of genes called gene family. These mutated genes assume new function and gene expression, which is also passed on to generations.

How are gene duplication and divergence predicted to be generated by? 

Homologous recombination, which is the process of mending a broken DNA fragment, can cause crossovers or misalignments, creating mutations in our genes that are passed down to other somatic cells and germline cells. 

What is an example of a homologous recombination creating a new set of genes and thus proteins?

The globin genes were mutated and created new alpha and beta globin genes, which combined created hemoglobin.

Mutations during exon shuffling aka RNA splicing can…

shuffle our exons randomly which creates new proteins and therefore new functions.

What are Mobile Genetic Elements (Transposons)?

Non-coding regions that jump into other locations within a gene. They can jump into splicing sites, regulatory DNA sites, or coding-regions, affecting the gene expression. 

What types of genes did MGE or transposons create?

Novel genes which are a new set of genes that were never found in ancestors.

What can transposons be useful for? 

Domesticating plants/crops by giving them new traits that can be lucrative such as fruit size, etc.

How did lactose tolerance develop?

As a young mammal, we had lactase in our small intestines, but when we became adults, this transcription was stopped.

When a mutation occurred, the lactase transcription wasn’t stopped, giving some people lactose tolerance even in adulthood. 

Horizontal Gene Transfer is…

when prokaryotes can randomly swap their genes through direct contact (conjugation).

Zygote

Fertilized egg (sperm + egg)

Zygotes create…

Germline cells + Somatic cells 

What are germline cells?

These cells create gametes for future generations. They go through meiosis.

What are somatic cells?

They are the body’s regular cells like muscles, neurons, etc. Support daily activities and the germline cells by keeping the organism alive. 

Point Mutations are…

Mutations that affect only 1 base pair. 

Types of point mutations…

Silent (same amino acid is produced.)
Nonsense (a stop amino acid is produced.)
Missense (a new amino acid is produced.)

Conservative missense mutation is a…

A type of point mutation where the new protein produced has similar chemical properties to the original.

Non-conservative missense mutation is a…

A type of point mutation where the new protein produced has different chemical properties to the original.

Repressor Protein binds to which DNA regulator?

Silencer DNA 

Activator Protein binds to which DNA regulator?

Enhancer DNA 

The transcription factors (form protein complexes to help polymerase find the promoter site) binds to which DNA Regulator?

Promotor

Phylogenetic Tree is a tree that…

shows the relationships of species based on their genes. 

Homologous genes are…

DNA sequences that are similar to each other b/c of common ancestry.

Highly-conserved genes are…

genes that remained perfectly recognizable by all organisms.

Conserved synteny is a…

phenomena where between 2 species the genes are found in the same order because those regions are intolerable to mutations (purifying selection). 

A purifying selection is… 

DNA regions that are intolerable to mutations 

Retroviruses such as HIV have a…

reverse transcriptase, so they use RNA to make DNA double strands. Then, integrase will integrate the virus’ DNA into the host’s DNA, where the provirus (integrated viral genome) will replicate and spread.

Reverse Transcriptase converts…

RNA to DNA

Integrase integrates…

the virus’ DNA into the host’s DNA. 

What is a provirus? 

Integrated viral genome

After the host’s DNA becomes a provirus…

The host’s RNA polymerase transcribes the provirus and produced virus coated proteins. 

½ of our genome are…

transposons, which shows us the evolution.

Around 1-2% fo our genomes are…

actual protein-coding exons. 

Most of our DNA is made up of

non-protein coding DNA.

Single Nucleotide Polymorphisms (SNPs) are…

mutations that occur in more than 1% of the population.

What is the difference between a SNP and mutation?

The amount of ppl.

SNPs occur in more than 1% of the population, but a mutation typically changes 1 individual’s DNA.

Polymerase Chain Reaction Goal

Amplifies desired DNA with high specificity. 

Polymerase Chain Reaction Uses

DNA Testing, Cloning, Forensics, Evolution

Polymerase Chain Reaction Steps

1. Denature = DNA strands are separated by heat

2. Annealing = Primers are added to the DNA strands.

3. Extension = DNA polymerase is added which polymerizes the DNA.

Gel Electrophoresis Goal 

separate DNA based on size. 

Gel Electrophoresis steps…

1. Add a restriction enzyme to cut the DNA at specific sites to create fragments.

2. Insert the DNA fragments into a gel + expose it to an electric field. 

3. Wait for the DNA to separate and then watch under UV light. 

Where do Restriction Enzymes come from and why? 

Prokaryotes because they need to protect their DNA from outsiders. R.E cut the invader’s gene and ligase ligated it using phosphodiester bonds.

Recombinant DNA Technology Goal

Inserts desired DNA into a host, which then gets replicated.

Recombinant DNA Technology Steps

1. Cut a cloning vector (Bacterial Plasmid) and DNA with the same restriction enzyme. 

2. Insert the DNA into the plasmid. 

3. Ligase ligates the DNA. 

4. Introduce to a host. 

5. Cloning 

   1. Host’s polymerase are used to replicate the recomb. DNA.

Recombinant DNA + Cloning Uses

Creating recombinant proteins like insulin

Aiding in understanding gene function

Creates transgenic (inserted DNA from other orgs) organisms. 

Plasmid Map…

Origin of Replication (DNA poly will bind — from host) 

Survival Gene 

Reporter Gene/Selectable Marker

Promoter (RNA poly will bind) 

Restriction Sites (where you cut the plasmid)

Insertion/Multiple Cloning site (where you insert/all the possible locations for DNA insertion)

What is a splice site? 

Sites in the pre-mRNA that guide the removal of introns by Spliceosomes.

Real life examples of Recombinant DNA technology 

Insulin is an example. The human Insulin gene is inserted into a plasmid and then into a bacteria. After that, the DNA is cloned to create multiple recombinant proteins like insulin. 

Crispr Cas 9 Goal

Cut DNA + modify it for a new one. 

Crispr Cas 9 Mechanisms

Cas-9 enzyme cuts out a specific DNA sequence with the help of a gRNA.

The gRNA guides the Cas-9 to the target sequence.

You need the gRNA and the enzyme together.

Crispr Cas 9 Steps

1. gRNA will be attached to the Cas 9 enzyme creating the Cas-9 Complex

2. Introduce to target cells.

3. Cas-9 scans for the PAM site (where it will cut). Once it find that…

4. Double checks to see if gRNA matches

5. Forms a phosphodiester bond with the complementary DNA strand.

   1. Using hydrolysis, it cuts the DNA sequence.

DNA Repair processes (2 types)…

1. Non-Homology End Joining = prone to mistakes because cell tries ligating the broken DNA.

2. Homology-Directed Pathways = using a template DNA, the DNA repairs itself.

DNA Sequencing Goals

determines the exact nucleotide sequence of a genome. Used to find unknown DNA sequences (for human genome project)

SANGER

1. Add a primer to the Unknown DNA.

2. DNA polymerase begans polymerizing by adding the ddNTPs to the DNA.

3. When it stops polymerizing, the gel undergoes PCR and gel electrophoresis to determine which base was added.

ddNTP vs dNTP

dNTPs are nucleotides and ddNTPs are the modified nucleotides. ddNTPs have a fluorescence dye and are without a hydroxide group (at the 3`), preventing the further extension of the DNA.

NGS (Next-Generation Sanger)

Removes the hydroxide to allow extension, but uses the same ddNTPs and needs primer, etc. Just doesn’t need gel-electrophoresis. 

Blotting Goal

The transfer of macromolecules such as DNA, RNA, and proteins from a gel into a membrane.

Blotting Steps

1. Extract the macromolecules from a cell.

2. Run a gel

3. Transfer the gel to a membrane.

Benefits of Blotting

Can help determine protein concentration, DNA + RNA expression/presence. 

Visualization for Proteins is called…

Immunolocalization

Visualization for DNA/RNA is called…

In-situ hybrid within cells/tissues.

Importance of Visualization techniques

Critical for studying gene expression, cell differentiation, and tissue-specific localization. 

Cytoskeleton is

the foundation of a cell. Allows for cellular movement, stability, shape, intracellular transport.

Cytoskeleton is made up of filaments. What are filaments?

Filaments are a polymer of monomers such as proteins —> dimers —> tetramer —> filament. The only covalent bond is the peptide bond within proteins.

What are the 3 filaments found in cytoskeleton?

1. Actin

2. Intermediate filaments

3. Microtubules 

Intermediate filaments (the springs of a mattress) are…

Structure: rope-like…made up of lamin (found in the nuclear envelope)

Function: they provide the cell stability and mechanical strength.

Microtubules (The highway for cellular transport) are…

Structure: hollow tubes with lumen in middle + alpha/beta tubulin wrapped around. 

Function: Support intracellular transport, mitotic spindles, cilia + flagella movement, and organelle positioning. 

Actins are…

Structure: made up of actin…globular structure

Function: muscle contraction, cell shape, crawling/movement.

Filaments need what to do their tasks?

Motor proteins which are proteins that help intracellular movement.

Which motor proteins help which filament?

Myosin — Actins
Dynesin & Kinesin — Microtubules. 

Function of Kinesin

Organelle & Vesicle transport.
Direction: Moves from in —> out, so from the - end to + end.

Dynein function 

retrograde (out—> in) transportation. moves from + to - end. 

Myosin function

aid muscle contraction.

There are 4 types of intermediate filaments which are… and found….

1. Cytoplasmic

   1. Keratin

   2. Vimentin

   3. Neurofilaments

2. Nuclear

   1. Nuclear lamins

Diseases found by issues in the intermediate filaments…

Emery-Dreifuss Muscular Dystrophy (EDMD) & Epidermolysis Bullosa Simplex (EBS) 

Which tubulin monomer holds the GTP and which hydrolyzes it?

Alpha - Attract (hold) the GTP

Beta - Break (hydrolyze) the GTP

There are 2 types of microtubules…

Cilia = hair-like, which act like the Custodians of our respiratory and fallopian tubes. 

Flagella = found in the sperm-tail. 

Centrosome is…

the organizing factory for the microtubules in eukaryotes.

Cytoskeleton is very advanced and complex found in eukaryotes. What about in Prokaryotes?

They do not have microtubules and centrosomes, so they do not undergo spindle formations, but they do have special proteins that help them with similar functions. 

Centrosome is the site for…

Nucleation which is the foundation-setting process of assembling microtubules.

Which tubulin helps during nucleation and how?

the y (gamma)-tubulins help forms the structure of microtubule assembly by mimicking the minus end, providing a general foundation for the alpha and beta tubulins to form.

How are the microtubules unstable or stable? 

When they are extending, the stable ones are stuck to a microtubule capping protein, while the unstable ones are not.

The stable microtubules help…

serve as tracks for intracellular transport.

The unstable microtubules form…

the mitotic spindle fibers and respond to signals.

Kinesin and Dynesin help microtubulins. What are the directions they move in? 

Kinesin (in —> out) towards the plus end. 

Dynesin (out —> in) towards the minus end (centrosome). 

Disease related to malfunction in micro tubulins…

Kartagener’s Syndrome (specifically dynesin)

Actins like microtubules also have…

minus and plus end, but intermediate filaments don’t.

Diseases correlated with Actin

Usher syndrome which occurs in usher-related myosin, disrupting the maintenance of structure and function of sensory cells —> deafness & blindness.

How do actins move?

They undergo a process called crawling…

1. where the actin is polymerized in the (+) end.

2. myosin contracts & slides in the cell forward (protrusion)

3. more actin is polymerized

4. cycle repeats.

The cell cycle consists of which phases…

Interphase (G1, G0, S, G2)
M Phase (Mitosis, Cytokinesis)

What does the cell do in interphase?

Prepares for cellular division. 

In G1 phase the cell…

grows and does its daily activities, while preparing for DNA replication.

The cell enters G0 phase after…because…so it (function)

G1 phase…the cell doesn’t think it is environmentally favorable…so the cell just continues its daily activities, but doesn’t duplicate.