Bio 1a 2

Principles of chromosomes replication

Chromosomes are copied by a semi-conservative mechanism where the parental strands are copied to form a hybrid duplex, rather than used to make a fully new duplex with conservation of the parental duplex.

Explanation:
Semi-conservative means each new DNA molecule has one old parental strand and one newly made strand. The original DNA strands separate, and each one is used as a template to make a matching new strand.

Replication proceeds bidirectionally on circular bacterial and linear eukaryotic chromosomes.

Explanation:
Replication starts at an origin and moves in two directions at the same time. This creates two replication forks moving away from the origin. Bacteria usually have circular chromosomes, while eukaryotes have linear chromosomes.

Lagging strands polymerize away from the replication fork to form short “Okazaki” fragments that are stitched together by the action of a repair DNA polymerase and DNA ligase.

Explanation:
DNA polymerase can only build DNA in the 5′ to 3′ direction. Because of this, the lagging strand cannot be made continuously. It is made in short pieces called Okazaki fragments. These fragments are later connected together. DNA polymerase replaces the RNA primer with DNA, and DNA ligase seals the remaining gaps.

A special RNA “primase” makes a short oligonucleotide on the leading and lagging template strand to initiate replication by a separate chromosome replication DNA polymerase.

Explanation:
DNA polymerase cannot start making DNA by itself. Primase first makes a short RNA primer. That primer gives DNA polymerase a place to start adding DNA nucleotides. The leading strand usually needs one primer, while the lagging strand needs many primers because every Okazaki fragment needs its own starting point.

The meson stahl experiment

• Bacteria were first grown in ¹⁵N (heavy nitrogen) → DNA became heavy.

• Bacteria were transferred to ¹⁴N (light nitrogen) → new DNA strands used light nitrogen.

• After the 1st replication, DNA showed one intermediate-density band.

• After the 2nd replication, DNA showed two bands: one light and one intermediate.

• DNA was separated by centrifugation based on density.

• These results were later used to determine how DNA replicates.

It showed that DNA replicates semi-conservatively.

That means each new DNA molecule contains:

• 1 original parental strand

• 1 newly made strand

• this means the band has this

Why the experiment showed this:

• After the 1st replication, all DNA had intermediate density → each molecule had one heavy old strand + one light new strand.

• After the 2nd replication, there were intermediate and light bands → some DNA was still hybrid, while some was completely light.

Bacterial chromosome replication vs eukaryotic chromosome replication

Bacterial chromosome replication

• DNA is circular

• Usually has one origin of replication

• Replication moves in both directions from the origin

• Creates one replication bubble

• The bubble has two replication forks

• Ends with two circular daughter DNA molecules

Eukaryotic chromosome replication

• DNA is linear

• Has multiple origins of replication

• Each origin forms its own replication bubble

• Each bubble has two replication forks

• Bubbles grow and eventually merge together

• Ends with two linear daughter DNA molecules

Easy distinction:
Bacteria = circular + one origin
Eukaryotes = linear + many origins

Leading vs Lagging strand

Helicase: Unwinds DNA, creating the replication fork.

• Single-strand binding proteins (SSBs): Keep the DNA strands separated.

• Topoisomerase: Relieves supercoiling ahead of the replication fork.

• Primase: Synthesizes RNA primers that provide a starting point for DNA polymerase III.

• DNA polymerase III: Synthesizes new DNA from 5′ → 3′.

  • Leading strand: Synthesized continuously toward the replication fork.

  • Lagging strand: Synthesized discontinuously away from the replication fork, creating Okazaki fragments.

• DNA polymerase I: Removes RNA primers and replaces them with DNA nucleotides.

• DNA ligase: Seals the remaining gaps (nicks) between DNA fragments by forming phosphodiester bonds.

Nucleotide addition / polymerization

• the precursor molecules for DNA and RNA are nucleoside triphosphates.

  • DNA uses dNTPs

  • RNA uses NTPs

• Nucleic acids are polymerized by forming phosphodiester bonds in the sugar-phosphate backbone.

• During polymerization, the 3′ OH of the growing strand attacks the phosphate of the incoming nucleotide.

• The incoming nucleotide loses pyrophosphate (PPi), which helps drive the reaction forward.

• New nucleotides are added only to the 3′ end, so nucleic acids are synthesized 5′ → 3′.

DNA vs RNA starting point

• DNA polymerase cannot start de novo

  • it needs a pre-existing 3′ OH

  • usually provided by a primer

  • A primer is a short piece of nucleic acid that gives DNA polymerase a place to start.

• RNA polymerase can start de novo

  • it does not need a primer

DNA helices and single strand binding protein

1. DNA helicase

• Unwinds the DNA double helix

• Separates the two parental DNA strands by breaking the hydrogen bonds between bases

• Uses ATP for energy

• As helicase moves forward, it opens more DNA and creates the replication fork

Think: Helicase = unzipper

2. Single-strand binding proteins (SSBs)

• Bind to the DNA strands after helicase separates them

• Keep the separated strands from pairing back together

• Prevent the single strands from folding into structures like hairpins

• Keep the DNA stretched out so DNA polymerase can copy it

Think: SSB = holds the unzipped DNA open

But helices cause torsional stress to build up and causes the fork to form supercoils so its like knots think about when you pull a water hose it coils up

Helicase

• unwinds the double helix

• separates the two DNA strands

• as it keeps opening DNA, the still-double-stranded DNA ahead of it gets overwound

• this creates supercoils / torsional stress

If that stress kept building up, the DNA would get too tightly twisted and replication would have trouble continuing.

That’s where topoisomerase comes in:

• makes a temporary nick/cut in the DNA

• lets the DNA rotate and relax

• removes the supercoiling

• then reseals the DNA

So for your flashcard:

Helicase unwinds DNA → causes supercoiling ahead of the fork → topoisomerase relieves the twisting stress by temporarily cutting and resealing DNA.

DNA Repair + Telomeres

DNA repair / sequence fidelity

• Cells use multiple repair mechanisms to keep DNA replication highly accurate

• DNA polymerases can detect and correct some mismatched bases

• Other repair enzymes scan DNA for mismatches and distortions

• If DNA repair fails, mutations can accumulate

• Repair defects are associated with diseases, including some cancers

Telomeres

• Telomeres = repetitive DNA sequences at the ends of linear eukaryotic chromosomes

• Their main job is to protect chromosome ends

• They prevent chromosome ends from being mistaken for damaged/broken DNA

Main distinction:
DNA repair = protects the DNA sequence
Telomeres = protect the ends of chromosomes

Ways to correct tanslation

DNA polymerase proofreading its own work

• DNA polymerase adds a nucleotide.

• If the new base is mismatched, DNA polymerase detects that the 3′ end is wrong.

• The polymerase shifts the DNA to its editing site.

• Its 3′ → 5′ exonuclease activity removes the incorrect nucleotide.

• Then DNA polymerase goes back to the polymerizing site and continues adding the correct nucleotides.

So the key idea is:

DNA polymerase builds DNA 5′ → 3′, but proofreads backward 3′ → 5′ to remove mistakes.

1. Mismatch repair

This fixes a base-pairing mistake that escaped DNA polymerase proofreading.

• Repair proteins recognize the mismatch

• The cell identifies the newly made strand

• A section containing the mistake is removed

• DNA polymerase fills the gap with the correct nucleotides

• DNA ligase seals the backbone

MutS and MutL in mismatch repair

• MutS recognizes and binds the mismatched base pair

• MutL binds with MutS and helps coordinate the repair process

• The repair system identifies the newly synthesized strand

• The incorrect section is removed

• DNA polymerase fills in the correct DNA

• DNA ligase seals the strand

Easy way to remember:

MutS = spots the mismatch
MutL = helps organize the repair

So this is basically:

detect mismatch → remove wrong section → DNA polymerase replaces it → ligase seals it

2. Thymine dimer repair

The middle/right side is showing damage called a thymine dimer, where two neighboring thymines become abnormally linked.

• Nuclease cuts out the damaged DNA section

• DNA polymerase fills in the missing DNA

• DNA ligase seals the final gap

The big difference from the previous slide is:

Proofread = DNA polymerase proofreads while DNA is being replicated
Repair = repair systems fix mistakes or damage after they are detected

What happens to the last RNA primer?

• On the lagging strand, DNA is made in Okazaki fragments

• Each fragment starts with an RNA primer

• Normally, the RNA primer is removed and replaced with DNA

The problem is at the very end of a linear chromosome:

• The last RNA primer gets removed

• But there is no 3′ OH upstream for DNA polymerase to extend from

• So DNA polymerase cannot fill that final gap

• This would make the chromosome shorter after each round of replication

How telomerase fixes it

• Telomerase binds to the 3′ end

• It uses its own RNA template

  • Telomerase contains a short RNA oligonucleotide (short chain of nucleotides) sequence

  • That RNA acts as a template

  • Telomerase uses it to add many repetitive, noncoding DNA sequences to the chromosome end

  • Those repeats form the telomere

  So the key correction is: the RNA itself does not become the long sequence. It is the template used to build the long repetitive DNA sequence.

• It adds repetitive DNA sequences to extend the chromosome end

• That gives the cell extra template so the lagging strand can be completed

gene expression

the process of using DNA information to make RNA and usually a protein

Genetic complementation test

This slide is showing how a genetic complementation test tells you whether two mutations are in the same gene or different genes.

A — Mutations are in different genes → complementation

Here:

• m1 is a mutation in Gene 1

• m2 is a mutation in Gene 2

When you put the two mutant copies together:

• one chromosome has a working Gene 1

• the other chromosome has a working Gene 2

So together, the cell can still make:

• Protein 1

• Protein 2

✅ Normal function is restored → the mutations complement each other → they are in different genes.

B — Mutations are in the same gene → no complementation

Here:

• m1 is in Gene 1

• m2 is also in Gene 1

So neither chromosome has a functional copy of Gene 1.

Result:

• ❌ No Protein 1

• ✅ Protein 2 is still made

Therefore the mutant phenotype remains.

❌ They do not complement → the mutations are in the same gene.

The shortcut to memorize is:

Complementation = different genes
No complementation = same gene

Transcription in prokaryotes and eukaryotes

1. Gene expression can be controlled at transcription

   • Transcription = making RNA from DNA.

   • If a cell makes more mRNA from a gene, it can usually make more of that gene’s protein.

   • So controlling transcription is a major way cells control gene expression.

2. Only one DNA strand is used as the template for a given transcript

   • DNA has two strands.

   • For a particular gene, RNA polymerase normally reads one strand as the template strand.

   • Different genes can use different DNA strands.

3. Prokaryotic transcription starts at a promoter

   • RNA polymerase + regulatory proteins bind to a promoter.

   • The DNA locally unwinds.

   • RNA polymerase reads the DNA template 3′ → 5′.

   • It builds RNA 5′ → 3′.

A really important rule to memorize:

DNA template read: 3′ → 5′
RNA made: 5′ → 3′

4. Elongation and termination

   • RNA polymerase moves along the DNA.

   • DNA opens briefly in front of RNA polymerase and then re-anneals/re-zips behind it.

   • The newly made RNA separates from the DNA as it gets longer.

   • When RNA polymerase reaches a termination signal, transcription ends and the RNA is released.

5. Eukaryotic transcription is more complicated
   Eukaryotes have more regulatory proteins and their initial RNA must usually be processed before becoming mature mRNA.

The big 3 processing steps are:

• 5′ cap added

• Introns removed by splicing

• 3′ poly-A tail added

So a simple way to remember the difference is:

Prokaryotes: DNA → mRNA, relatively direct
Eukaryotes: DNA → pre-mRNA → processing → mature mRNA

And then that mature mRNA can be used for translation into protein.

Gene expression is often controlled at the level of transcription

Gene expression can be controlled at transcription

• Transcription = making RNA from DNA.

• If a cell makes more mRNA from a gene, it can usually make more of that gene’s protein.

• So controlling transcription is a major way cells control gene expression.

Gene A is transcribed a lot, so it makes many RNA molecules, which then get translated into many A proteins.

Gene B is transcribed much less, so it makes less RNA, which leads to less B protein.

So the key idea is:

More transcription → more mRNA → more protein
Less transcription → less mRNA → less protein

Transcripts may use either DNA strand as a template, but usually not both for the same gene

Only one DNA strand is used as the template for a given transcript

• DNA has two strands.

• For a particular gene, RNA polymerase normally reads one strand as the template strand.

• Different genes can use different DNA strands.

• we can see in the photo that there is nearly no overlap

sequence of events in the trasncirption of prokaryotic mRNA

• Initiation

  • RNA polymerase binds to the promoter.

  • The promoter tells RNA polymerase where transcription should begin.

  • The DNA unwinds near the start point.

  • One strand becomes the template strand.

  • The other strand is the nontemplate strand.

  • RNA synthesis begins.

• Elongation

  • RNA polymerase moves along the template DNA strand.

  • It reads the template DNA 3′ → 5′.

  • It builds the RNA 5′ → 3′.

  • The DNA opens in front of RNA polymerase and then rewinds behind it.

  • The growing RNA transcript comes out of the polymerase.

• Termination

  • RNA polymerase reaches a termination signal.

  • The completed RNA transcript is released.

  • RNA polymerase comes off the DNA.

eukaryotic transcription is more complex because the RNA has to be processed after transcription

Eukaryotic transcription is more complicated
Eukaryotes have more regulatory proteins and their initial RNA must usually be processed before becoming mature mRNA.

The big 3 processing steps are:

• 5′ cap added

• Introns removed by splicing

• 3′ poly-A tail added

So a simple way to remember the difference is:

Prokaryotes: DNA → mRNA, relatively direct
Eukaryotes: DNA → pre-mRNA → processing → mature mRNA

And then that mature mRNA can be used for translation into protein.

• remember mcn strcture with exons and introns

Eukaryotic coding sequences-exons- often correspond to independent domains of the protein

• Exon 1 contributes to Domain 1

• Exon 2 contributes to Domain 2

• Exon 3 contributes to Domain 3

A protein domain is a region of a protein that can have its own structure or function.

So the flow is:

DNA with exons + introns → transcription → RNA processing → exons joined → translation → protein with different domains

In eukaryotes, introns are removed and exons are spliced together; exons often encode separate functional domains of a protein.

remember how to read and interpret this dont memorize

You can see that this single mRNA has several separate regions, each with:

• its own ribosome-binding site

• its own AUG start codon

• its own coding region

That means the same mRNA can be translated into:

• protein α

• protein β

• protein γ

• each mRNA can translate different start signals with the start signal in between

redundancy of the genetic code

Redundancy means that more than one codon can code for the same amino acid.

For example, leucine can be coded by several different codons:

• UUA

• UUG

• CUU

• CUC

• CUA

• CUG

So even if one nucleotide changes, the amino acid sometimes does not change.

That matters because it can make the genetic code more tolerant of mutations. Some mutations are silent mutations, meaning the DNA or mRNA codon changes, but the same amino acid is still added to the protein.

Single nucleotide mutations in the 3rd codon position are usualyl silenced

Still gly this is because of redundancy

missense mutation

mutations in the 1st or 2nd nucleotide position of a codon are more likely to change the amino acid, because those positions are usually more important for determining which amino acid is coded.

stop chain mutation

Remmeber stop chains are UAA UAG and UGA

Single nucleotide deletion (or insertion) shifts the reading frame downstream of the insertion

very substatial because think about a story if you take a part out of a story or add a part the story would not make sense

more substantial depending on how early it arrives

ribosome during translation

The ribosome has 2 subunits:

• Small ribosomal subunit: binds the mRNA

• Large ribosomal subunit: helps form the protein by making peptide bonds

Inside the ribosome are 3 important tRNA-binding sites:

• A site = Aminoacyl site/ Acceptor site

  • The new tRNA enters here

  • It carries the next amino acid

• P site = Peptidyl site

  • Holds the tRNA carrying the growing polypeptide chain

  • This is where the protein chain is being built

• E site = Exit site

  • The empty tRNA moves here before leaving the ribosome

The easiest way to memorize the order is:

A → P → E

Arrive → Protein → Exit

So during elongation:

1. A charged tRNA enters the A site

2. Its amino acid gets added to the growing chain

3. The tRNA shifts to the P site

4. The old empty tRNA shifts to the E site and leaves

translating the code to the placement of amino acids in a polypeptide

1. Transfer RNA – tRNA – folds into a tertiary structure recognized by enzymes that

attach specific amino acids to the 3’OH terminus. A folded loop of the tRNA presents

the triplet information to base pair to a codon.

2. Amino acids are attached to the 3’ terminus of a tRNA in a two-step process

requiring ATP to form a high energy phosphoanhydride bond between the α phosphate

of ATP and the aa COO- which is then transferred to the 3’OH of the tRNA with the

release of AMP and PPi.

3. The full ribosome has two large multisubunit RNA-protein complexes which contain

three sites engaged in polypeptide assembly: A (the acceptor site), P (the peptide-

tRNA site and E (the exit site free tRNA). mRNA is positioned along the surface of the

smaller ribosome subcomplex. A growing polypeptide chain threads through a

channel in the large subcomplex.

4. Each step in the cycle of polypeptide growth is organized by GTPases involved in

initiation, elongation and chain termination. Peptide bonds are formed by a catalytic

site in the large subunit with RNA and protein involved in catalysis.

tRNA

tRNA, or transfer RNA, is basically the molecule that brings the correct amino acid to the ribosome during translation.

Think of it as an adapter between the mRNA code and amino acids.

It has two especially important parts:

• The 3′ end at the top is the amino acid attachment site. A specific amino acid gets attached there.

• The anticodon at the bottom is a 3-base sequence that pairs with a complementary codon on the mRNA.

For example, if the mRNA codon is:

5′-AUG-3′

the tRNA has a complementary anticodon that pairs with it, and that tRNA carries methionine.

The left side of the figure is the simplified 2D cloverleaf shape, while the right side shows the actual folded 3D shape of tRNA.

how a tRNA gets “charged” with the correct amino acid

• On the left, the enzyme aminoacyl-tRNA synthetase recognizes:

  • the correct amino acid

  • the correct tRNA

In this example, the amino acid is tryptophan and the tRNA is tRNAᵀʳᵖ.

• The enzyme uses ATP to help attach tryptophan to the 3′ end of the tRNA.

• ATP is converted to AMP + PPi.

• This forms a high-energy covalent bond between the amino acid and the tRNA.

Now the tRNA is called a charged tRNA or aminoacyl-tRNA.

Then on the right:

• The anticodon of the tRNA base-pairs with the matching mRNA codon

• Here, the mRNA codon is UGG

• UGG codes for tryptophan

how a peptide bond forms during translation

It connects directly to the ribosome A and P sites

On the left, the growing polypeptide chain is attached to the tRNA in the P site. A new amino acid arrives attached to another tRNA in the A site.

Then the key reaction happens:

• the amino group of the new amino acid attacks the bond holding the growing peptide to the old tRNA

• a new peptide bond forms

• the entire growing polypeptide chain gets transferred onto the tRNA in the A site

• the old tRNA is released from the peptide

So before:

P-site tRNA = holds growing chain
A-site tRNA = holds new amino acid

After the peptide bond forms:

A-site tRNA = now holds the longer growing chain
P-site tRNA = empty

Then the ribosome moves forward and the empty tRNA can eventually leave through the E site.

The protein grows from the N-terminus toward the C-terminus, so each new amino acid is added to the C-terminal end.

Prokaryotes transcription and translation

In prokaryotes, transcription and translation can happen at the same time.

Here’s what is happening:

• RNA polymerase is moving along the DNA and making mRNA.

• As soon as part of the mRNA comes out, ribosomes can bind to it immediately.

• Those ribosomes start translating the mRNA into protein before transcription is even finished.

That is what “coupled” means here.

The long mRNA has many ribosomes attached to it at once. That group is called a:

polyribosome / polysome

So one mRNA can be translated by many ribosomes at the same time, producing many copies of protein quickly.

The reason prokaryotes can do this is that they do not have a nucleus, so there is no nuclear membrane separating DNA transcription from ribosome translation.

So the main takeaway is:

Prokaryotes: transcription and translation are coupled

• RNA polymerase makes mRNA

• ribosomes begin translating that mRNA immediately

• multiple ribosomes can translate the same mRNA at once

This is different from eukaryotes, where transcription happens in the nucleus and translation happens in the cytoplasm, so the two processes are separated.

What is genetics?

Genetics is the scientific study of heredity and variation

• Heredity is the transmission of traits from one generation to

the next

• Variation is demonstrated by the differences in appearance

that offspring show from parents and siblings

Inheritance of genes

• Genes are hereditary units made of DNA that parental cells transmit to their progeny cells

• Most DNA is packaged into chromosomes, each of which is one very long double-stranded

• DNA molecule During asexual reproduction, somatic cells divide to give rise to two genetically identically progeny cells via mitosis and cytokinesis

• During sexual reproduction, genes are recombined and passed to the next generation via reproductive cells called gametes (sperm and eggs)

Chromosomes in a human somatic cell

Humans have 46 chromosomes in the nuclei of their somatic cells (all cells of the body except gametes): 22 pairs of autosomes and 1 pair of sex chromosomes (2n=46).

We are diploid (2n), except for our gametes, which are haploid (n).

Sister chromatids are held together by cohesin proteins, especially around the centromere.

mitosis

cell cycle

The cell cycle has 2 major parts: Interphase and M phase.

• Interphase = about 90% of the cell cycle

  • G₁: cell grows and carries out normal metabolic activity.

  • S phase: DNA is replicated. This is the only phase where chromosomes are duplicated.

  • G₂: cell keeps growing and prepares for division.

The important chromosome idea is:

Before S phase: each chromosome has 1 chromatid.
After S phase: each chromosome has been copied, so it has 2 sister chromatids joined at the centromere.

Then comes M phase:

• Mitosis: separates the duplicated chromosomes so each new nucleus gets the same chromosomes.

• Cytokinesis: divides the cytoplasm, physically making 2 daughter cells.

So the flow is:

G₁ → S → G₂ → Mitosis → Cytokinesis → back to G₁

Checkpoints

G₁ → safe to replicate?
G₂ → replication finished correctly?
M → chromosomes properly attached to spindle?

Human life Cycle

haploid

having a single set of unpaired chromosome

diploid

containing 2 complete sets of homologous chromosomes one from each parent

meiosis

Law of Segregation

The maternal and paternal copies of a given chromosome (or gene) separate during meiosis and end up in different gametes at random (with equal probability).

• meiosis 1 is haploid

• When an organism makes gametes, each gamete receives

  just one copy of each chromosome, which occurs randomly.

• Probability of receiving the maternal or paternal

  version of a chromosome is completely random

Law of Independent Assortment

The maternal and paternal chromosomes of any homologous pair segregate into gametes independently of all other homologous pairs of chromosomes

• The pairs of homologous chromosomes get segregated

  into gametes independently of one another

• The version of chromosome 1 selected has no effect

  on which version (maternal or paternal) of the

  other chromosomes will be selected.

  
  

  
  

Crossing Over

SPO11 creates double-strand breaks in the DNA of homologous chromosomes during prophase I of meiosis. Those breaks start the recombination process.

Then:

• the broken DNA is processed

• one broken strand invades the homologous chromosome

• homologous DNA is used as a repair template

• this can produce a crossover, where corresponding DNA segments are exchanged

• The synaptonemal complex keeps the homologous chromosomes tightly paired and aligned, so when SPO11 makes a double-strand break, the broken DNA can find the matching sequence on the homologous chromosome and use it during repair.

• 1-3 crossovers per pair of homologous chromosomes

• Chiasmata- crossover+cohesin

Genetic variation related to meiosis and fertilization

• The number of combinations possible when chromosomes

assort independently into gametes is 2n, where n is the

haploid number

• For humans (n = 23), there are ~8.4 million (223) possible

combinations of chromosomes

• Crossing over adds even more variation!

• Random fertilization adds to genetic variation because

any sperm can fuse with any unfertilized egg

• The fusion of two gametes (each with 8.4 million possible

chromosome combinations from independent assortment)

produces a zygote with any of about 70 trillion diploid

combinations

• You really are genetically unique!

helicases

unwinds parental double helix at replication forks

Single strand binding protein

Binds to and stabilizes single stranded DNA until it is used as a template

Topoisomerase

Relieves overwinding strain ahead of replication forks by breaking, swiveling, and rejoining DNA strands

Primase

Synthesizes an RNA primer at 5’ end of leading strand and at 5’ end of each Okazaki fragment of lagging strand

DNA polymerase 3

Using parental DNA as a template, synthesizes new DNA strand by adding nucleotides to an RNA primer or a pre-existing DNA strand

DNA polymerase 1

Removes RNA nucleotides of primer from 5’ end and replaces then with nucleotides added to 3’ end of adjacent fragment

DNA ligase

joins Okazaki fragments of lagging strand; on leading strand, joins 3’ end of DNA that replaces primer to rest of leading strand DNA

What are the 2 Competing hypothesis for heredibility

• Blending inheritance = traits from the parents mix together, like paint. A tall father + short mother would tend to produce offspring with an intermediate height, and the original traits would become “blended.”

• Particulate inheritance = traits are passed as separate units that do not permanently blend. Those units are what we now call genes/alleles.

Mendel’s pea experiments supported particulate inheritance. For example, when he crossed purple and white flowers, the white trait could disappear in one generation and then reappear later. If traits truly blended, you wouldn’t expect the original white trait to come back unchanged

Why peas?

• There are many true-breeding varieties of pea plants with different characters (flower color, seed color, morphology, etc.)

• Each variant for a character is called a trait (for example,purple vs. white)

• Peas have a short generation time

• Peas produce a lot of offspring (progeny)

• Mating can be controlled; pea plants can self-pollinate or can be cross-pollinated

Mendals Pea plant experiment

1. P generation = true-breeding parents

He started with plants that were true-breeding, meaning they consistently produced offspring with the same trait when self-fertilized.

For flower color, you can represent them as:

• Purple true-breeding = PP

• White true-breeding = pp

So Mendel crossed:

PP × pp

“True-breeding” basically means homozygous for the trait.

---

2. F₁ generation

Every offspring receives:

• P from the purple parent

• p from the white parent

So all the F₁ plants are:

Pp

And because purple is dominant:

100% purple flowers

This was important because if inheritance worked by blending, Mendel might have expected some intermediate color. Instead, the white trait seemed to disappear.

---

3. He crossed/self-fertilized the F₁ plants

Now:

Pp × Pp

The offspring possibilities are:

PP, Pp, Pp, pp

So the F₂ generation has:

• 1/4 PP

• 1/2 Pp

• 1/4 pp

Phenotypically:

• 3/4 purple

• 1/4 white

That's why his actual data, 705 purple and 224 white, is close to a 3:1 ratio.

What does “monohybrid cross” mean?

Mono = one

A monohybrid cross follows inheritance of one trait at a time.

Here, Mendel is only examining:

flower vs. white

So this whole experiment is a monohybrid cross.

If he were studying two traits simultaneously, like flower color and seed shape, that would be a dihybrid cross.

What did medals model show us

1. There are alternative versions of heritable factors (genes) that we call alleles

2. Two copies (alleles) of a gene are inherited by an individual, one from each parent (in the flowers P, p are alleles)

3. If the alleles differ, then the dominant allele determines the appearance of the individual; the other allele is recessive

4. The law of segregation: the two alleles separate from each other during gamete formation and end up in gametes

• an egg or sperm gets only one of the two alleles present in the parent

• this is explained by the behavior of chromosomes in meiosis!

Meiosis in terms of allele P is the wild type and p is the one with a mutation

What do you do to distinguish homozygous dominant and heterozygous.

Breed them with a known recessive the homozygous would 100 percent show the dominant trait while the heterozygous would show the dominant trait ½ of the time and show the recessive trait the other half.

Dihybrid cross

Hypothesis of dependent assortment: the alleles stay paired the way they came from the parents. Since the original parents contributed YR and yr, the F₁ would only make:

YR or yr gametes

So Y stays with R, and y stays with r. They are “dependent” on each other.

Hypothesis of independent assortment: the Y/y gene and R/r gene separate independently when gametes are made. So the F₁ can make:

YR, Yr, yR, yr

each about 1/4 of the time.

That allows new combinations like yellow wrinkled and green round, which were not present in the original parents.

So the easiest way to remember it is:

Dependent = genes stay together
Independent = genes mix and match independently

Incomplete dominance

neither allele is completely dominant

Codominance

Two dominant alleles affect the phenotype in separate, distinguishable ways. Ina heterozygote, both alleles are fully expressed at the same time

Epistasis

The phenotypic expression of one gene affects that of another

Polygenic inheritance

additive effect of two or more genes on a single phenotypic character

Chromosome theory of inheritance

The chromosome theory of inheritance says that genes are located on chromosomes, and the way chromosomes behave during meiosis explains Mendel’s inheritance patterns.

Symbols for Drosophila genetics

The wild type allele denoted with a plus:w+

A recessive mutant allele is show in lowercase: w

The chromosomal basis of sex-linked inheritance

The chromosomal basis of sex-linked inheritance means that some genes are located on the sex chromosomes, especially the X chromosome, so their inheritance depends on which sex chromosome a person receives.

For an X-linked gene:

• XX individuals have two copies of the gene.

• XY individuals usually have only one copy on their single X chromosome.

That is why an X-linked recessive allele is more likely to show up in an XY individual: there usually is no second X allele to mask it.

A gene that is located on either sex chromosome is called a

sex-linked gene

• Some disorders are caused by recessive alleles on the X chromosome in humans:

• Color blindness (mostly X-linked)

• Duchenne muscular dystrophy

• Hemophilia

For a recessive X-linked trait to be expressed,

• a female needs two copies of the allele (homozygous)

• a male needs only one copy of the allele (h)

Morgans discovery

Why fruit flies (Drosophila melanogaster)?

• Fruit flies have a short generation time (2 weeks)

• Fruit flies produce a lot of offspring (progeny)

• Mating can be controlled, but unlike pea plants, fruit flies must

be crossed

• Fruit flies have only four pairs of chromosomes, which can be

seen with a microscope

• In the early 1900’s, Morgan isolated mutants that could be

easily distinguished from the wild type

X inactivation in female mammals/dosage compensation

• In female mammals, one of the two X chromosomes in each cell is randomly inactivated during embryonic development

• The inactive X condenses into a Barr body super condensed no expression anymore

• If a female is heterozygous for a particular gene located on the X chromosome, she will be a mosaic for that character

• Tortoiseshell cats are a common example of genetic mosaicism in female mammals

What happens when genes are on the same chromosome?

• Genes that are near each other on the same chromosome deviate from Mendel’s law of independent assortment

• Such genes tend to be inherited together and are called linked genes

• Morgan observed linkage of genes in his experiments with fruit flies

-testcross- You cross the unknown organism with a homozygous recessive organism.

Recombination frequency

Those are called recombinant chromosomes.

So the main idea is:

same chromosome = linked genes → usually inherited together

but

crossing over can separate linked genes → recombinant offspring

And the farther apart two genes are on the same chromosome, the more likely crossing over will happen between them.

Using recombination datât to map the distance between genes

• The recombination frequency depends on the distance between genes on a chromosome

• The farther apart two genes are, the higher the probability that a crossover will occur between them

• Distances on a linkage map are expressed as map units, where 1 map unit = 1% recombination frequency

• Very close together on the same chromosome → low recombination frequency, maybe 1%, 5%, 10%

• Farther apart on the same chromosome → higher recombination frequency

• So far apart that crossing over happens very often → can approach 50%

• Also recombination frequency can never be more than 50 percent

Alterations of chromosome number

• Large-scale chromosomal alterations in humans and other mammals often lead to miscarriages or cause a variety of

developmental disorders

• Aneuploidy: when there is an abnormal number of a

particular chromosome

• Monosomy (2n - 1)

• Trisomy (2n + 1): for example, trisomy of chromosome

21 causes Down syndrome

• Polyploidy: having multiple complete sets of chromosomes

(e.g. 3n, 4n, 6n, or even 8n)

• Plants tolerate such genetic changes better than animals do.

In fact, many plants are polyploid, and polyploidization is an

important factor in plant diversification and speciation.

Non disjunction

Nondisjunction causes alterations of chromosome number

• In nondisjunction, pairs of homologous chromosomes do not

separate normally during meiosis

• As a result, one gamete receives two of the same type of

chromosome, and another gamete receives no copy

Alterations of chromosome structure

Gene expression is often controlled at the level of transcription

The more transcription the more RNA that will be produced meaning more translation would happen but this could also be just the amount of a gene the body needs.

E coli and Tryptophan biosynthesis in E coli

It is a single-celled, prokaryotic organism, Scientists commonly study E. coli to understand genetics, metabolism, and gene regulation.

Biosynthesis of Tryptophan

Tryptophan- one of the amino acids acids that the human body can’t produce by itself but e coli does

How does E coli control how much tryptophan it makes

(A) Feedback inhibition — Regulation of enzyme activity

• E. coli uses a series of enzymes (1, 2, and 3) to convert a precursor into tryptophan.

• When enough tryptophan is produced, tryptophan goes back and inhibits enzyme 1.

• This slows down the entire pathway, preventing more tryptophan from being produced.

• The enzymes are still present, but their activity is inhibited.

Key idea: Feedback inhibition regulates enzymes that already exist.

(B) Regulation of gene expression — Regulation of enzyme production

• E. coli has genes (trpE, trpD, trpC, trpB, and trpA) that encode the enzymes needed to produce tryptophan.

• When tryptophan levels are high, E. coli reduces the expression of these genes.

• This means fewer enzymes are produced, which decreases tryptophan synthesis.

Genes are organized in operons in bacteria and archaea

Operon- An operon is a group of genes controlled by the same promoter and transcribed together into a single mRNA molecule.

Instead of controlling each gene individually, bacteria can regulate multiple genes at once.

In this example, the trp operon contains five genes:

• trpE

• trpD

• trpC

• trpB

• trpA

Each DNA has their own coding region with a start and a stop sequence

A. Regulatory gene (trpR)

• Located outside the trp operon.

• Contains instructions for making the trp repressor protein.

• The repressor helps regulate whether the operon is transcribed.

Important: trpR is a separate gene and is NOT part of the trp operon.

B. Promoter

• A DNA sequence where RNA polymerase binds.

• RNA polymerase begins transcription from the appropriate start site downstream.

Think of the promoter as the place where transcription machinery is recruited.

C. Operator

• A DNA sequence where the repressor protein can bind.

• When the active repressor binds the operator, it prevents transcription of the operon.

Think of the operator as a control switch.

D. Structural genes (trpE, trpD, trpC, trpB, trpA)

• These five genes contain the instructions for making proteins involved in tryptophan synthesis.

• All five are transcribed together into one long mRNA molecule.

Allosteric regulation

a molecule binds to a protein at a specific site, causing the protein to change shape and altering its activity

Repressible operon

A repressible operon is one that is usually on; binding of a repressor protein to the operate shuts off transcription. TRP operon is a repressible operon

Inducible operon

An inducible operon is one that usually off; a molecule called an inducer inactivates the repressor and turns on transcription. The lac operon is an inducible operon and contains genes that code for enzymes used in hydrolysis and metabolism of lactose (a sugar that cells can use as a carbon and energy source)

Lac Operon

• A small proportion of lactose is converted to an isomer called

allolactose (natural inducer-molecule that naturally occurs in a cell or its environment) , which induces production of b-galactosidase

• For our discussion, we consider lactose, allolactose, and IPTG

to be “inducers” of the lac operon

Jacob and Monod testing the hypothesis of how: a repressor blocks transcription of the lac operon when the inducer is absent

1. Their hypothesis

They proposed that a repressor protein acts like an OFF switch for the lac operon.

When the inducer is absent:

• The repressor binds to the operator.

• This blocks transcription of the lac operon.

• The enzymes needed for lactose metabolism are not produced in significant amounts.

When the inducer is present:

• The inducer (naturally, allolactose) binds to the repressor.

• This causes an allosteric shape change.

• The repressor releases from the operator.

• Transcription of the lac operon can occur.

2. What were they trying to prove?

Jacob and Monod wanted to determine whether gene expression was controlled by a negative regulator (repressor) rather than simply being activated directly by lactose.

3.The experiment

A. X-gal — Detects enzyme activity

• X-gal is a colorless substance.

• When the enzyme β-galactosidase breaks down X-gal, a blue pigment forms.

• Therefore, blue bacterial colonies indicate β-galactosidase activity.

Remember: X-gal is used to detect whether the enzyme is active.

B. IPTG — Inducer

• IPTG is an artificial inducer that acts similarly to allolactose, the natural inducer.

• IPTG binds to the lac repressor and causes an allosteric shape change.

• This prevents the repressor from binding the operator.

• RNA polymerase can then transcribe the lac operon, allowing β-galactosidase to be produced.

Remember: IPTG helps turn ON the lac operon.

C. Glycerol — Energy source

• E. coli needs energy to grow and reproduce.

• Glycerol provides a carbon and energy source.

• It is present in both experimental conditions so the bacteria can grow regardless of whether the lac operon is ON or OFF.

4.Results

1. Scientists introduce random mutations

They started with a population of E. coli bacteria and introduced random mutations into their DNA.

Their goal was to find bacteria with mutations that prevented the lac operon from being regulated normally.

2. They grow the bacteria on a plate

The plate contains three important conditions:

• X-gal: Turns blue when broken down by β-galactosidase.

• Glycerol: Provides energy for bacterial growth.

• No IPTG: There is no inducer to turn on the lac operon.

Normally, without IPTG, the repressor should prevent transcription.

Therefore, they expected most colonies to be white.

3.Most were white but a colony with a mutation in the repressor gene transcribes the B-galactosidase gene when there is no inducer turned blue

This demonstrated that the lac operon uses a negative regulatory system, meaning a repressor normally blocks transcription.

They identified two types of mutations:

1. Regulatory gene mutation (lacI): The gene encoding the repressor is mutated, causing a defective repressor that cannot properly block transcription.

2. Operator mutation: The DNA sequence where the repressor normally binds is mutated, preventing the repressor from binding even if the protein is functional.

Key takeaway: Both mutations can cause the lac operon to remain ON regardless of whether an inducer is present. This is called constitutive gene expression.

Negative and positive regulators

• Loss of function mutations in positive regulators cause non inducible active (can’t be turned off)

• Loss of function mutations in negative regulators cause constitutive activity (on all the time)

How does lac Operon sense the presence of glucose

Cells prefer glucose

cAMP

• small signaling molecule

• cAMP is used as a measure of glucose in the cell

  • Glucose high- cAMP low

  • Glucose low- cAMP high

• cAMP binds to CRP and activates its binding to the lac promotor

CRP

• (cAMP Receptor Protein) is a transcription factor that helps activate the lac operon when glucose levels are low.

• it is an activator not a repressor

• If there is glucose CRP is inactive and the lac operon is not expressed

eukaryotic cells regulate gene expression at different stages

Gene expression is the process of using information in DNA to produce a functional product, usually a protein.

The important idea is that cells can control how much protein is produced at multiple steps, not just during transcription.

Regulation inside the nucleus

1. Chromatin modification: Chromatin is DNA wrapped around histone proteins. When chromatin becomes less tightly packed, genes become more accessible for transcription.

2. Transcription: DNA is used as a template to produce RNA. Cells can regulate whether a gene is transcribed and how much RNA is made.

3. RNA processing: The primary RNA transcript is modified. Introns are removed, exons are joined, and a 5′ cap and poly-A tail are added.

4. Transport to cytoplasm: Mature mRNA exits the nucleus through nuclear pores.

Regulation inside the cytoplasm

5. mRNA degradation: mRNA can be broken down. Generally, the faster mRNA is degraded, the less protein can be produced from it.

6. Translation: Ribosomes read mRNA and assemble amino acids into a polypeptide.

7. Protein processing: The polypeptide folds and may undergo additional modifications to become a functional protein.

8. Protein transport: Proteins are delivered to their appropriate cellular locations.

9. Protein degradation: Proteins can be broken down when they are damaged or no longer needed.

Regulation can happen at every stage.

For example, a cell can decrease protein production by:

• Packing DNA more tightly, reducing transcription.

• Degrading mRNA before it can be translated.

• Inhibiting translation.

• Degrading the protein after it is produced.

Heterochromatin vs euchromatin

Chromatin: DNA wrapped around histone proteins.

Heterochromatin: Tightly packed DNA; genes generally inactive (less transcription).

Euchromatin: Loosely packed DNA; genes accessible for transcription.

Histones: Proteins that DNA wraps around to help package and regulate DNA. Histone can open up heterochromatin and make it into euchromatin.

the 3 types of RNA polymerase

RNA Polymerase: Enzyme that uses DNA as a template to synthesize RNA during transcription.

RNA Polymerase I: Transcribes most rRNA genes (except 5S rRNA).

RNA Polymerase II: Transcribes mRNA genes and some noncoding RNA genes.

RNA Polymerase III: Transcribes tRNA, 5S rRNA, and some snRNA genes.

RNA Polymerase II Structure: Complex containing more than 10 protein subunits.

RNA Polymerase II Initiation: Cannot initiate transcription alone; requires general transcription factors.

Initiation of Transcription in Eukaryotes

Core Promoter: DNA region where general transcription factors and RNA polymerase II assemble to initiate transcription.

TATA Box: DNA sequence within some core promoters recognized by TATA-binding protein (TBP).

General Transcription Factors (GTFs): Proteins that bind the core promoter and help recruit RNA polymerase II to initiate transcription.

TFIID: General transcription factor that recognizes the core promoter and helps recruit RNA polymerase II. Contains TBP and approximately 13 TBP-associated factors (TAFs).

Transcription Initiation Complex: RNA polymerase II + general transcription factors assembled at the promoter to begin RNA synthesis.

Steps of Transcription Initiation:

1. TFIID recognizes and binds the core promoter (often at a TATA box).

2. Other general transcription factors and RNA polymerase II assemble.

3. Transcription initiation complex forms and RNA synthesis begins.

Specific Transcription Factors and Control Elements

Control Elements (Cis-Regulatory Elements): DNA sequences that bind specific transcription factors to regulate gene expression.

Proximal Control Elements: Regulatory DNA sequences located near the core promoter.

Distal Control Elements: Regulatory DNA sequences located farther from the promoter, sometimes thousands of base pairs away.

Enhancer: DNA region containing control elements that bind activators to increase transcription.

Specific Transcription Factors: Proteins that bind specific DNA control sequences and act as activators or repressors.

Activators: Proteins that bind positive control elements and increase transcription.

Repressors: Proteins that bind negative control elements and decrease transcription.

Activator Protein Structure: Contains a DNA-binding domain that recognizes specific sequences and activation domains that help stimulate transcription.

Bacterial Operators vs. Eukaryotic Control Elements: Both are regulatory DNA sequences that bind proteins controlling transcription.

DNA Bending and Mediator Proteins

DNA Bending: Allows distant enhancer-bound activators to interact with proteins at the core promoter.

Mediator Complex: Group of proteins that connects activators with RNA polymerase II and general transcription factors to regulate transcription.

How Enhancers Activate Transcription:

1. Activators bind enhancer sequences.

2. DNA bends, bringing activators near the promoter.

3. Mediator proteins connect activators to the transcription machinery.

4. RNA polymerase II initiates transcription.

Combinatorial Control of Gene Expression

Combinatorial Control: Different combinations of transcription factors determine which genes are expressed in different cell types.

Reporter gene

Reporter Gene: A gene that produces an easily measurable product (e.g., GFP fluorescence) to study gene expression.

Negative control - removed will increase the level of reporter mRNA

positive control- removed will decrease the level of reporter mRNA

GAL4 Regulation of Gene Expression in Yeast

GAL Genes (GAL1, GAL7, GAL10): Encode enzymes that convert galactose into glucose-1-phosphate, which can enter glycolysis.

GAL4: A specific transcription factor that acts as a positive regulator, activating GAL1, GAL7, and GAL10 when galactose is available.

GAL4 Structure: Contains two important domains:

• Zinc finger DNA-binding domain: Binds specific enhancer sequences.

• Acidic activation domain: Helps recruit transcription machinery.

How GAL4 Activates Transcription:

1. GAL4 binds enhancers located upstream of GAL genes (more than 250 base pairs from the TATA box in the example).

2. DNA bends, bringing GAL4 near the core promoter.

3. GAL4 interacts with general transcription factors (TFIID, TFIIB), with help from mediator proteins.

4. RNA polymerase II initiates transcription of GAL genes.

GAL4 Mutation: Without functional GAL4, adding galactose does not induce GAL1, GAL7, or GAL10 expression.

Coordinated Gene Expression: GAL1, GAL7, and GAL10 are not in an operon. Each has its own promoter and GAL4-binding regulatory sequences, allowing GAL4 to activate all three genes.

Yeast is a good model of why eukaryotes don’t operate in operons

why yeast

• Unicellular, haploid, but one can do crosses and test phenotypes in the diploid phase

• small genome

• a collection of knockout mutations in every non essential gene is available this allows us to pinpoint what we want to focus on

In Eukaryotes (Yeast): GAL1, GAL7, and GAL10 have separate promoters but are all activated by the same transcription factor, GAL4.

Exam Takeaway: Eukaryotes generally coordinate gene expression through shared transcription factors rather than operons.

Gene expression in archae

Gene expression in archae has features of both bacteria and eukaryotes

• Genes in archaeology are often organized in operons like bacteria

• Specific transcription factors in archaeology resemble bacterial transcription factors and generally bind close to the promoter like bacteria

• The general transcription machinery ( TOTA box and binding protein) in archaeology is a simplified version of the eukaryotic machinery

• Some archaeologists have histones and simplified chromatin

Alternative RNA splicing

• A primary transcript is made from a eukaryotic gene

• This RNA can be processed by alternative splicing to make multiple mRNAS that are translated to make multiple proteins from one gene

• Alternative splicing is very common mammalian cells

In Situ Hybridization

Technique used to detect the location and relative amount of a specific mRNA in cells or tissues.

• DNA in situ hybridization: Identifies where a specific DNA sequence is located on a chromosome.

• RNA in situ hybridization: Shows where a particular gene is being expressed by detecting its mRNA.

• FISH (fluorescence in situ hybridization): Uses fluorescent probes to visualize DNA or RNA.

Pathways of Eukaryotic mRNA Degradation

mRNA Degradation: Breakdown of mRNA to regulate how much protein can be produced.

5′ Cap: Protects the 5′ end of mRNA from degradation and helps initiate translation.

Poly-A Tail: Protects the 3′ end of mRNA from degradation and increases mRNA stability.

Deadenylation: Shortening/removal of the poly-A tail, making mRNA more susceptible to degradation.

Decapping: Removal of the 5′ cap, allowing mRNA to be degraded from the 5′ end.

Exonuclease: Enzyme that degrades RNA one nucleotide at a time from an end.

• 5′ → 3′ degradation: Begins at the 5′ end.

• 3′ → 5′ degradation: Begins at the 3′ end.

Endonuclease: Enzyme that cuts RNA internally, creating fragments that can be degraded.

Steady-State mRNA Level: Determined by the balance between mRNA synthesis and degradation.

Three degradation pathways

1. Deadenylation → Decapping → 5′ to 3′ degradation.

2. Deadenylation → 3′ to 5′ degradation.

3. Endonuclease cleavage → RNA fragments degraded.

MicroRNAs (miRNAs) and Gene Regulation

MicroRNA (miRNA): Small noncoding RNA (~22 nucleotides) that regulates mRNA degradation or translation.

RNA-Induced Silencing Complex (RISC): Complex containing miRNA that base-pairs with specific target mRNAs to regulate gene expression.

How miRNA Is Produced:

1. RNA Polymerase II: Transcribes miRNA genes into primary miRNA (pri-miRNA).

2. Drosha: Cleaves pri-miRNA into precursor miRNA (pre-miRNA).

3. Exportin-5 + Ran-GTP: Transport pre-miRNA from the nucleus to the cytoplasm.

4. Dicer: Cleaves pre-miRNA into a ~22-nucleotide miRNA duplex.

5. RISC: Mature miRNA is assembled into RISC to target specific mRNAs.

How RISC Regulates Gene Expression:

1. mRNA Cleavage: RISC cuts target mRNA, leading to degradation.

2. Translational Repression: RISC blocks ribosomes from translating mRNA.

3. mRNA Deadenylation: RISC promotes poly-A tail shortening.

Amplification of DNA using the polymerase chain reaction and analysis of DNA fragments by gel electrophoresis

The polymerase chain reaction (PCR) can produce many copies of a specific target segment of DNA

Three steps of PCR

1. Denaturation (95°C): Heat separates double-stranded DNA into two single strands by breaking hydrogen bonds.

2. Annealing (50°C): Forward and reverse primers bind to complementary sequences on opposite DNA strands.

3. Extension (72°C): DNA polymerase extends the primers, synthesizing new DNA in the 5′ → 3′ direction.

Repeat 25–35 cycles: DNA approximately doubles each cycle, resulting in exponential amplification ((2^n)) under ideal conditions.

Fun fact- there are no lagging strand in a PCR reaction

DNA Cloning Using Recombinant Plasmids

DNA cloning: Produces multiple identical copies of a specific DNA segment (gene).

Plasmids: Small, circular DNA molecules that replicate independently of bacterial chromosomes.

Vectors: Plasmids can act as vectors that carry foreign DNA into bacterial cells.

Recombinant DNA: A DNA molecule formed by combining DNA from two different sources.

• Restriction enzymes: Cut the plasmid and target DNA at specific recognition sequences, often creating complementary sticky ends.

• DNA ligase: Joins the target DNA to the plasmid, forming a recombinant plasmid.

• Transformation: Bacteria take up the recombinant plasmid.

• Selection: An antibiotic-resistance marker helps identify bacteria containing a plasmid.

• Cloning: As bacteria divide, they replicate the plasmid, producing many copies of the inserted DNA.

Transforming the recombinant plasmid into E. coli

• To make lots of copies of the recombinant plasmid, we let cells do the work of replicating it

• The plasmid is introduced into bacterial cells by transformation (Introduces the recombinant plasmid into E. coli bacterial cells.)

• Because the plasmid contains a selectable marker gene (A gene, usually for antibiotic resistance, that allows researchers to identify cells containing the plasmid.) only the bacterial cells that get the plasmid can survive and grow on the antibiotic

• PSY is the gene we want to replicate

• If we reuse ECORI and cut the PSY it would separate making 2 strands of DNA

RT-PCR(reverse transcription pcr)- analyzed RNA expression of one or a few genes at a time

Step 1: cDNA Synthesis

1. Isolate mRNA from cells at different times or from different tissues.

2. Oligo-dT primer: Binds to the poly-A tail at the 3′ end of mRNA.

3. Reverse transcriptase: An RNA-dependent DNA polymerase that uses mRNA as a template to synthesize complementary DNA (cDNA).

4. DNA polymerase: Synthesizes the second DNA strand using a gene-specific primer, producing double-stranded cDNA.

Step 2: PCR Amplification

• Forward and reverse primers bind to sequences surrounding the specific gene of interest.

• PCR amplifies the corresponding cDNA sequence.

• More starting mRNA generally produces more amplified DNA under comparable PCR conditions.

Step 3: Gel Electrophoresis

Separates the amplified DNA fragments based on size and allows researchers to compare gene expression.