MBB 222 - M2 material

origin of replication

Location on chromosome where DNA replication begins in eukaryotic and prokaryotic cells.

• Replication-initiator protein recognizes (binds specifically) to particular sequence in the origin site

Chlamydomonas reinhardtii

unicellular algae with phototaxis

• cell swims towards light that is

  sensed by its eyespot organelle

• eyespot has specialized protein “channelopsin”

• (green algae)

channelopsin

protein that absorbs light + responds by transporting Na+ ions

into cell to create an electrical signal

• found in green algae

** human eye needs multi[;e proteins to respond to light

Optogenetics

biological technique that uses light to control neurons within living tissue, often through genetically encoded light-sensitive proteins.

• Light-sensing electrical

  signaling capability can be introduced to any cell!

coding sequence

DNA sequence that encodes amino acids in a protein

non-coding sequence

• other regions of the chromosome

that do not encode proteins, often involved in gene regulation and expression.

• “binding sites for proteins that preform functions”

Electrophoresis/ miracle of polymerization

method used to separate charged particles, such as DNA or proteins, based on their size and charge under an electric field.

takeaway:

DNA + NTPs had RNAP added next to it for comparison and we saw that a RNA was polymerized

DNA template is catalytic (not altered, not annealed to the RNA product and is not used up)

all RNA product molecules have the same length (+ non-random sequence) showing how RNAP starts and stops at unique locations to make specific products intentionally.

Transcription factors

(transcription)

Proteins that bind to specific DNA sequences to regulate transcription of genes, influencing the recruitment of RNAP and the transcription process.

• Expression of a gene relies on these proteins

• recognize other specific DNA sites than RNAP (it’s before)

often clustered in a "promoter region" just upstream of the promoter ... but might be after the promoter, or even inside the coding sequence!

Upstream

Upstream in DNA orientation refers to the direction toward the 5' (five-prime) end of the coding (sense) strand, opposite to the direction of transcription.

non-coding (non-template)

Downstream

Downstream in DNA orientation refers to the direction toward the 3' (three-prime) end of the coding (sense) strand, in the same direction as transcription.

coding strand (non-template)

RNAP

(transcription)

RNA polymerase, the enzyme synthesizing RNA from a DNA template during the transcription process.

• binds specifically to promoter sequence to initiate transcription, aided by

gene regulation proteins called TFs

Other sequences in mRNA are termination sequences - signaling RNAP to end transcription

ribosome

(translation)

a non-membrane-bound cellular structure made of ribosomal RNA (rRNA) and proteins that synthesizes proteins by translating genetic code from messenger RNA (mRNA)

binds mRNA at a specific regulatory sequence ("ribosome binding site", RBS) ... translation starts at the nearest downstream 5' aug 3' sequence, which is the initiation (start) codon.

• therefore, Translation does not start at the end of the mRNA strand!

*start codon encodes Met, so Met is the first (N-terminal) a.a. in every polypeptide ... translation subsequently continues in the same reading frame until the termination (stop) codon is reached.

(ORF) open reading frame

a consecutive sequence of codons in mRNA that begins with a start codon and ends with a stop codon, capable of being translated into a protein.

operons

cluster of bacterial genes controlled by 1 promoter and transcribed together into 1 mRNA molecule.

(genes in 1 operon are transcribed from 1 promoter)

Each coding sequence has its own ribosome binding site, start codon & stop codon

polycistronic

1 mRNA that encodes multiple proteins, typically seen in operons of prokaryotic cells. (bacteria)

• possible adaptive benefit…

less non-coding DNA = rapidity of DNA rep.

proteins that need to work tgt. are expressed at same time, in same amount

monocistronic

1 mRNA that encodes 1 protein, typical in eukaryotic cells and some viruses. (us fr).

RNAP vs. DNAP (7 thing table)

Feature	DNA Polymerase (DNAP)	RNA Polymerase (RNAP)
Primary Function	DNA replication (copies DNA to DNA)	Transcription (copies DNA to RNA)
Product	Double-stranded DNA (daughter strand)	Single-stranded RNA (mRNA, tRNA, rRNA)
Nucleotides Used	Deoxyribonucleotides (dNTPs: A, T, C, G)	Ribonucleotides (rNTPs: A, U, C, G)
Primer Requirement	Requires a primer with a free 3'-OH group (cannot start de novo)	Does not require a primer (starts de novo) polymerize single NTPs w/o primer
Helicase Needed	Yes (cannot unwind DNA strands on its own)	No (opens duplex to expose template)
Proofreading	Yes (3'→5' exonuclease activity; very high accuracy)	Minimal/different (lower fidelity, higher error rate)
Speed	Very fast (~1000 nucleotides/sec)	Slower (~20 to 80 nucleotides/sec)

• RNAP has no proofreading according to slides; fidelity is less important than speed

• RNAP can’t start from a single DNA strand or a single DNA strand w/ a primer; needs duplex.

• RNAP acts processively without any external sliding clamp

bacterial RNAP

Bacteria have only 1 RNAP responsible for nearly** all transcription (mRNA, tRNA, etc.)

primase

(DNA replication)

A specialized RNAP that synthesizes a short RNA primer to initiate DNA replication, providing a starting point for DNA polymerase.

(template-hybrid strand)

NTPs

Nucleotide triphosphates, the substrates of RNA synthesis: new phosphodiester linkage forms, w/ release of diphosphate.

RNA(n) + NTP →←RNA(n+1) + PPi

what direction does DNA duplex get renatured/denatured in transcription

happen simultaneously at opposite ends of a moving transcription bubble as the RNA polymerase enzyme moves along the DNA template from the 3' to 5' direction.

renatured in rear, denatured in front.

topoisomerase I

Topoisomerase I relieves supercoil strain (Wr.) in DNA ahead of RNAP

RNAP core

The core enzyme of RNAP: binds DNA non-specifically

• requires sigma protein

  for recognizing promoter

holoenzyme

core + sigma

(transcription)

“+1” position

position on DNA coding strand matching 1st base of mRNA

-35 and -10 box

Regions in the promoter of bacterial DNA that are crucial for the binding of RNAP;

the -35 box is typically recognized by the sigma factor, while the -10 box is known as the Pribnow box.

What is a consensus sequence, and how can differences affect gene expression?

A consensus sequence is the idealized DNA sequence recognized by a protein.

Real sequences may differ from it, potentially reducing protein binding and gene expression.

imagine a bacterial promoter has this consensus sequence:

TATAAT

But an actual promoter has this sequence:

TATGAT

One base is different from the ideal sequence.

• Perfect match: RNAP may recognize the promoter more effectively, leading to more transcription.

• Imperfect match: RNAP may bind less effectively, leading to less transcription.

sigma factors

proteins that facilitate RNAP binding to promoters in bacterial transcription

• 1 bacterial species has multiple sigma factors,

  each targeting a different promoter sequence,

  for use in different circumstances

Sigma 70 “housekeeping”

Sigma 32 “heat shock/expression of proteases and chaperone”

steps in the transcription initiation process

closed (pre-initiation) complex formation, opening of the DNA helix (open complex), and promoter escape by RNA polymerase (promoter clearance). Then elongation complex.

closed (pre-initiation complex)

A formation where RNA polymerase and the sigma factor bind to the promoter region of DNA without unwinding the helix.

1. RNA polymerase binds nonspecifically to DNA

2. RNAP binds w/ high affinity to promoter sequences in the -35 and -10 region

3. DNA is partially unwound at initiation site of transcription

open complex

intermediate state during transcription initiationthe double-stranded DNA is unwound so that RNAP can access the template strand

• Binding of sigma to promoter triggers conformational change in RNAP that denatures DNA ~10 bp

1. RNA synthesis is initiated in a primer-independent RX’N

2. sigma factor is released and promoter clearance occurs

promoter clearance

when RNAP moves away from the promoter after synthesizing RNA, allowing elongation to proceed. (transitional phase btwn open complex and elongation complex)

• sigma factor leaves

elongation complex

a dynamic molecular assembly formed when an RNAP transitions from the initiation phase to synthesize a nascent RNA strand along a DNA template.

• occurs in bacterial cytoplasm, so ribosomes can start translation on the mRNA while transcription is ongoing!

nascent strand

a newly formed, growing strand of DNA or RNA that is actively being synthesized by an enzyme like DNA polymerase or RNA polymerase

polynucleotide template

a single-stranded or double-stranded nucleic acid molecule that serves as a pattern for the synthesis of a complementary strand during processes like transcription or replication or translation.

rely on specific sequences in the template for initiation of their function, such as:

• replication origin (for DNA rep)

• promoter (for transcription)

• ribosome binding site + start codon (for translation)

IN BACTERIA

initiation of

(RNAP) action is guided by regulatory sequences, recognized by a sigma factor that later leaves during promoter clearance

chromosomes

structure composed of DNA and proteins that carries genetic information, (replication + division)

in humans, compacted by supercoiling in nucleosomes, with higher coiling levels forming heterochromatin that suppresses transcription.

much DNA is non-coding, with structural or regulatory purposes

• introns that interrupt coding sequences (exons) are

removed by splicing the RNA that is initially transcribed

nucleosomes

the basic repeating structural unit of chromatin in eukaryotic cells that packs and organizes DNA inside the nucleus]

Histone proteins + DNA

• H2A,H2B,H3,H4 (packaged inside octamer)

• H1 histone binds OUTSIDE octamer

prokaryotes DONT have histones or nucleosomes

Duplex is supercoiled by nucleosomes

Beads on a string

a model describing the structure of nucleosomes where DNA wraps around histone proteins, resembling beads (nucleosomes) strung on a string (the linker DNA). This configuration helps organize and compact the DNA within the nucleus.

arrangement leaves space for enzymes to access individual nucleosomes ... for replication and expression

Histone core octamer

2 copies each of subunits of 4 histones = 8 core histone subunits alltogether

• H2A,H2B,H3,H4

• packages and organizes eukaryotic DNA into repeating units (nucleosomes)

• H1 histone binds OUTSIDE octamer

Histone proteins

Proteins that package and order DNA into structural units called nucleosomes.

Histone proteins associate with the backbone of any DNA duplex ... thus not specific for a sequence**

• subtle differences in how histones bind to G:C-rich vs. A:T-rich sequences

Draw histone octamer and DNA duplex coiled up; differences?

heterochromatin

a tightly packed form of DNA that is transcriptionally inactive, often found at the centromeres and telomeres.

Centromere & telomere are used for physical structure

- not for coding - sequence is repetitive

how centromere and telomere work together on a chromosome

NOTE: both sequences are repetitive

Centromere: combines with

proteins in a kinetochore

- will be grabbed for pulling

sister chromatids apart

(during mitosis) AKA anaphase

Telomere: combines

with proteins to

protect strand ends

PROKARYOTES DONT HAVE THESE

Chromatin

DNA in a cell is always packaged with protein to form a complex called chromatin, which regulates gene expression and DNA replication.

30-nm fibre

A higher-order structure of chromatin formed by the coiling of the nucleosome fibers, facilitating DNA packaging in the nucleus.

typical in interphase

(between cell divisions)

- 30-nm fibre is

supercoiled & again

supercoiled etc.

Euchromatin

varying degree of access for transcription

and is typically associated with actively expressed genes, found at the gene-rich regions of the genome.

• 10nm- before 700 ish nm

heterochromatin

visibly denser in

light microscope

- no access for

transcription

• 750ish nm to 1,400 nm

DNA is replicated to make sister chromatids here

• Prokaryotes DONT have this

Hierarchical chromatin structure in the chromosome

introns

non-coding sections of a gene that are transcribed into precursor mRNA (pre-mRNA) but are removed during RNA splicing before translation.

PROKARYOTES DONT HAVE INTRONS OR SPLICING

exons

coding sections of a gene that remain in the mature mRNA after splicing and are translated into protein.

Exon shuffling

The process by which exons from different genes are mixed and matched during recombination or gene duplication, potentially leading to the creation of new proteins with novel functions.

can occur in chromosome ... exons rearranged into different order, or wholly lost!

eukaryote

Protein is made in cytoplasm, but DNA resides in nucleus

Replication & transcription both happen in nucleus, but RNA is exported to cytoplasm for translation

• numerous other internal compartments ...some of which evolved from

bacteria that invaded the cell!

prokaryote

bacterial cell

DNA & RNA & proteins are

all made in the cytoplasm

Eukaryotic mRNA processing

In eukaryotes, extensive maturation modifications are made to the premature mRNA (pre-mRNA) as it is being synthesized by RNAP II ...

1. transcription initiation

2. 5’ capping

3. splicing

4. 3’ processing

5. transcription termination

introns that interrupt coding sequences (exons) are

removed by splicing, and a 5' cap and 3' poly-A tail

are covalently attached

transcription in nucleus

3 RNAP in nucleus:

RNAP II: dedicated to mRNA

- the focus in MBB 222

RNAP I: for rRNA

RNAP III: for tRNA plus some rRNA,

other non-coding RNAs; ALL must be exported out of nucleus since aminoacyl-tRNA synthesis and translation (by the ribosome) ALL happen in the cytosol (of cytoplasm)

NOTE: mitochondria + chloroplasts have separate RNAPs

General; mRNA maturation?

RNA polymerase II, an enzyme responsible for synthesizing messenger RNA (mRNA) from DNA during transcription in eukaryotic cells.

• RNAP II transcription complex

  changes its structure during elongation:

  covalent modifications and even protein subunits

  are added or subtracted

1. 5’capping

2. splicing

3. 5’poly A-tail addition

(Prokaryotic mRNA doesn't have this maturation)

If a mature mRNA transcript loses a 5' cap or 3' tail, it is excluded from translation and degraded by exonucleases

exonuclease

An enzyme that removes nucleotide residues from the ends of DNA or RNA molecules.

what’s 5’ cap? specifically state what happens to it during transcription

The 5' cap is a modified guanine nucleotide added to the 5' end of eukaryotic mRNA during transcription.

• 5' triphosphate at end of pre-mRNA (not a “sequence”): recognized by

  capping enzymes which bind to RNAP II

  soon after transcription initiation.

• 5' phosphate gets linked to 5' end of a

  methylated G for 5' - 5' triphosphate link

  - no free 5' phosphate remains !

splicing

The process of removing introns from pre-mRNA and connecting exons to produce mature mRNA, which can then be translated into protein.

• regulated - some exons may or may not

  be excluded (alternative splicing of mRNA)

One gene's DNA can thus produce

multiple variant mRNAs,

hence variant proteins

by the splicesome complex

- composed of protein and RNA, where RNA is the

catalytic component, similar to the ribosome

5’poly A-tail addition

at termination of transcription,

polyA polymerase (not RNAP II) adds A nucleotides

(up to 250!) to 3' end of mRNA, not using any template

polyA polymerase

An enzyme responsible for adding a long chain of adenine nucleotides to the 3' end of eukaryotic mRNA during transcription termination.

addition of up to 250 adenines, forming the poly A tail that stabilizes the mRNA and aids in its export from the nucleus.

What does Mature mRNA do?

assembles into a circular complex where both 5' cap and polyA tail are

recognized by specific proteins

- names of proteins are not in Objectives

Circular complex associates with small ribosomal subunit:

ATP hydrolysis is used to pull mRNA past the ribosomal

subunit until the RBS (ribosome binding site) is found

- the RBS in eukaryotes typically overlaps the start codon

extrachromosomal genetic elements

DNA outside a cell’s chromosomes

can be replicated and / or expressed using host cell resources, and inherited through host cell divisions.

Plasmids

circular DNA duplex (extrachromosomal circular DNA duplex)

that:

- is found inside a host cell ...

(usually a bacterium)

- and is copied & inherited through

host cell divisions ...

- but is not part of the host cell's chromosome(s) ...

(i.e. it is not found in every cell of the host species!)

• entirely made of DNA, and typically encode genes giving adaptive benefit to the host

has a replication origin sequence

for DNAP to start replication,

mimicking the host chromosome!

Thus the plasmid propagates along with the host cell population

Replication: (with replication origin) can't prevent

random loss of the plasmid over many host generations

(e.g. mutation or replication failure)

Expression of genes conferring fitness advantage

ensures that the plasmid persists over generations

= SELFISH ITS ADAPTED TO ENSURE ITS REPLICATION

Viruses

(parasitic, infectious particle)

parasitic and encode coat proteins and other enzymes that enable exiting the host cell and infecting other cells (as an extracellular virion particle)..

• some viruses have an RNA genome that encodes an RNA-dependent RNAP for replication

polynucleotide with genes, but protected by a protein

coat (capsid), sometimes also with lipid envelope

- not a cell itself: no metabolism outside of a host cell*

Plasmids & viruses are adapted (by natural selection)

to replicate efficiently inside a host cell

capsid

The protein shell that encases and protects the viral genome, allowing the virus to survive outside the host cell and facilitating infection of new host cells.

Viral genome

The complete set of genetic material, either DNA or RNA, found within a virus.

contains a replication origin (like plasmids)

plus genes: for coat proteins &

for enzymes to do assembly of new

particles (virions) ready to exit cell

(don’t benefit host)

viruses carry genes for proteins that:

- maximize replication efficiency to out-compete the host

using host resources (parasitism)

- enable exiting the host & invading a new host (infection)

Eukaryotes don't naturally take up plasmids!

But they certainly can be infected by viruses

HPV cycle

The HPV cycle refers to the lifecycle of the human papillomavirus, which includes attachment to host cells, entry, uncoating, viral gene expression, replication of the viral genome, assembly of new virions, and eventual exit from the host cell to infect new cells. This process often involves integration of viral DNA into the host genome, leading to persistent infection.

RNA viruses ( SARS-CoV2)

encode an RNA-dependent RNAP (RdRp)

• for rep the genome strand is used as a template for RdRp to make a complementary (non-genome) strand → used as template for RdRp to make a new genome strand.

transformation

when a cell takes up foreign DNA from its surroundings, incorporating it into its own genome.

Bacteria can acquire new DNA!

- importing plasmids or linear DNA

directly from outside

conjugation

receiving a plasmid from another bacterium via a connecting pilus

recombination

(breaking & re-joining DNA strands)

Inside the cell, new DNA fragments (e.g. resistance genes)

can get incorporated into a plasmid through

spinocerebellar ataxia

neurological disease traced to mutation in a gene ATXN1 residing on chromosome 6, encoding a transcription factor called ataxin-1.

ATXN1 gene (pre-mRNA) covers 462,349 bases (excluding polyA)

Mature mRNA is only 10,557 bases from 9 exons

Coding sequence is only 2,448 bases (start codon to stop codon)

using only exons 8 & 9 ... for 815 a.a. polypeptide

There are numerous 5' AUG 3' triplets in the long 5' UTR which

are somehow not recognized by the ribosome as start codons

... thus the RBS is poorly understood for this particular gene

7

Eukaryotes have a lot of non-coding DNA

Human genome is >95% non-coding DNA!

(3.1 billion bp total, of which 60 million bp are coding)

Contrast prokaryotes & viruses: < 15% non-coding DNA

e.g. E. coli: 4.6 million bp total, of which 4 million bp are coding

HPV: 7.9 kbp total, of which 7 kbp are coding

Pros of a high percentage of non-coding DNA

- more possibilities for regulatory sequences

e.g. larger number of transcription factor binding sites

- some DNA is used for structural purposes

e.g. centromere for moving chromosomes around the cell

Cons of a high percentage of non-coding DNA

- genome replication takes longer

- human genome has about 50,000 replication origin sites!

- proteins recognizing specific sequences

have more DNA to search through

- chromosomes occupy more space in cell

Polymerase Chain Reaction (PCR)

uses DNAP in a test tube (in vitro) to exponentially replicate (amplify) a targeted region of DNA duplex.

Target specificity is achieved with single-stranded primers and cycles of annealing & melting.

Combining PCR with reverse transcriptase enables diagnostic detection of RNA

e.g. from RNA viruses

helps get a targeted gene fragment for analysis (pedigree)

• think of dominant allele disease, we can use PCR to amplify a test segment of exon 8 in ATXN1 gene …

• design PCR primers that flank the test segment

reverse transcriptase

an enzyme that synthesizes complementary DNA (cDNA) from an RNA template; converting RNA into DNA for studies such as PCR.

DNAP

DNA polymerase, an enzyme responsible for synthesizing new DNA strands by adding nucleotides to a pre-existing chain during DNA replication.

• cannot recognize an

  isolated single-strand DNA template ...

  and cannot link two single nucleotides to start!

  Rather, DNAP can only extend a pre-existing strand

  (called a primer) that is already paired to the template

• Without primer-template annealing, replication

  won't happen: this specificity is exploited in PCR

  etc.

  
  

How does PCR start A-C

A) synthesize 2 primers, one per duplex strand

B) heat & cool to anneal (shape w/ heat) primers

C) DNAP RXN w/ dNTPs: Any non-target DNA in the original sample is not replicated

Steps B. & C. make one PCR cycle (= simple doubling)

For "chain reaction": repeat the PCR cycle many times!

taq DNAP

A thermostable DNA polymerase derived from the bacterium Thermus aquaticus, used widely in PCR for its ability to withstand high temperatures during DNA denaturation.

processive

with a built-in clamp ...

AND it is heat-stable so it

withstands extreme heating

processivity

The ability of a polymerase enzyme to continuously synthesize DNA or RNA by adding nucleotides without dissociating from the template.

Exponential amplification:

a process where the amount of a target (such as DNA, RNA, or an electrical signal) doubles or multiplies at a rapid, compounding rate with each cycle or reaction step

After 1st cycle: TWO duplexes

- each has original template with new strand

After 2nd cycle: FOUR duplexes (two with original template)

After n cycles: 2^n duplexes (most are new DNA strands)

Electrophoresis detects extra DNA in one ATXN1 allele

A laboratory technique used to separate DNA fragments based on their size and charge. It helps visualize and identify extra DNA segments within a specific allele, such as ATXN1.

Here, PCR has a

preparative purpose

(obtaining a desired material)

and electrophoresis has an

analytical purpose

(measuring a property)

DNA sequencing results from previous flashcard

DNA sequencing (= determining sequence of a DNA sample)

reveals that this region of ATXN-1 has multiple repeats

of 5'cag3' codon (for amino acid Glu, Q)

• Disease-associated allele has

  many extra 5'cag3' repeats - but

  no disruption of the reading frame!

How does: SARS-CoV2 testing detects RNA using PCR

SARS-CoV-2 testing uses RT-PCR to convert viral RNA into complementary DNA (cDNA) and make many copies of it, allowing the virus to be detected even when only small amounts are present.

• single stranded RNA, not DNA

• Special enzyme that reverse transcriptase (RT) (derived from different virus)

Reverse transcriptase (RT)

An RNA-dependent DNA polymerase that synthesizes complementary DNA (cDNA) using a single-stranded RNA template.

• Mechanism:

  • Synthesizes DNA in the $5' \rightarrow 3'$ direction, requiring a primer annealed to the RNA template to initiate extension.

  • Often exhibits RNase H activity to degrade the RNA template in RNA:DNA hybrid intermediates, facilitating double-stranded cDNA synthesis.

• Biological Origin:

  • Derived from retroviruses to transcribe their viral RNA genome into double-stranded DNA for host genome integration.

• Key Applications:

  • RT-PCR: Enables detection and amplification of RNA viruses (e.g., SARS-CoV-2) by converting unstable viral RNA into stable cDNA before PCR cycles.

  • Transcriptomics: Converts total cellular mRNA into cDNA libraries for high-throughput sequencing and gene expression

Recombinant DNA

made by combining DNA fragments from different sources into one DNA molecule.

• Restriction endonucleases: Cut DNA at specific sequences.

• DNA ligase: Joins DNA fragments together.

• Plasmid vector: Carries the recombinant DNA into host bacteria, where it can be copied.

Example: A gene of interest is inserted into a bacterial plasmid to create recombinant DNA, which can then be copied inside bacteria.

amplification

the process of multiplying cells that contain the desired recombinant DNA to produce large amounts of the desired cells or product.

Steps:

1. Insert the desired gene into a plasmid to create recombinant DNA.

2. Introduce the plasmid into host cells, such as bacteria.

3. Select the cells with the desired genetic trait.

4. Multiply these cells to produce large amounts of the desired cells or product.

Remember: Amplification = making many copies or increasing the amount of something desired.

controlling amplification

To amplify a DNA with a sequence of interest we need..

• Host cells (growth we can control like E. coli bacteria)

• Vector (plasmid that can replicate in the host containing a selectable marker gene like antibiotic resistance)

vector carry our DNA of interest inside the host

ampillicin antibiotic (amp^R) vector

used to select for bacteria containing plasmids with antibiotic resistance.

• added to nutrient agar before inoculation with bacteria

• provides selection for the marker gene amp^R

• bacteria can’t grow UNLESS they carry the vector with amp^R

Growth Hormone (GH)

Growth Hormone (GH) Production Using Bacteria

• GH: A protein made by the pituitary gland into blood; helps with childhood growth and tissue repair.

• Problem: Donated pituitary glands provide very little GH.

• Solution (genetic engineering):

  1. Insert the human GH gene into a plasmid (small circular DNA).

  2. Introduce the recombinant plasmid into bacteria (transformation).

  3. Grow the bacteria in culture so they multiply.

  4. The bacteria produce GH, which is then harvested for medical use.

Key idea: Use genetically engineered bacteria to produce large amounts of human GH for treating GH deficiency.

replacement therapy

A medical treatment that involves replacing a missing or deficient substance in the body, often using hormones or enzymes, to restore normal function.

GH deficiency (dwarfism) can be treated by injections of GH

PCR helps get starting material for recombinant DNA

Our DNA of interest is a tiny proportion of a chromosome:

We can amplify our DNA of interest using PCR

Primer 1: sequence matches strand A, annealing to strand B

Primer 2: sequence matches strand B, annealing to strand A

DNA then prepped for recombination

DNA Cloning (Recombinant DNA)

the host cells in the isolated population are clonal,

genetically identical to the first transformed cell

selected for the desired trait (carrying the plasmid)

The desired gene DNA inside the plasmid has been

replicated exactly as part of the host cell growth

- so the desired gene DNA exists as a population

of "molecular clones"

Key idea: One bacterium multiplies into a colony of genetically identical cells, each carrying copies of the desired gene.

DNA cloning

Making many identical copies of a desired DNA sequence.

Clonal cells

• Genetically identical cells that come from one original transformed cell containing the desired plasmid.

Molecular cells

• Copies of the desired gene made as bacteria multiply and replicate their DNA

Nutrient agar plate

• Gel containing nutrients that allows bacteria to grow.

Bacterial colony

A visible group of bacteria that grows from a single bacterium.

Each of these colonies contains many cells ...

but each colony is a clonal population that grew

from a single bacterium placed on the plate

What’s the work flow for DNA cloning?

1. Recombine DNA of interest (“insert”) w/ a plasmid vector, to make a new recombinant plasmid (only tiny amount of this recombinant plasmid DNA is needed)

2. Transform host cells with recombinant plasmid

   (Only a tiny amount of transformed cells are needed)

3. Amplify (grow) lots of host cell culture

   - make sure we select cells carrying the plasmid

   (e.g. treat with antibiotic to select for resistance)

4. A) Isolate lots of clone DNA of recombinant plasmid

   and / or

   B) Isolate lots of protein from expression of insert

what are some common types of enzymes for recombination?

endonucleases:

(cleave strands)

-ligases:

("ligate" or join strands)

-recombinases

(all-in-one enzymes: cleave 2 duplexes & re-ligate their strand ends in a new arrangement)

Typically, endonucleases & recombinases have a specific recognition sequence in DNA where they bind and act

expression vector

a plasmid "engineered" for high expression of a protein of interest

in a host cell, allowing for the production and purification of large quantities of that protein.