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The human genome of is found where in the human body?
A. NucleusB. RibosomeC. Smooth ERD. Cell membrane
The cellular structure where proteins are made is called the
A. NucleusB. Smooth ERC. RibosomeD. Cell membrane
DNA and Biotechnology
Announcements
• Ch 21- today• Ch 21, 17- Wednesday• Bone & muscle labs- Due Wednesday• Ch 21, 17 online quizzes- Due Friday• Quiz 10- Friday (chs 21, 17)• Lab today- pGLO (DNA transformation)
Lecture Outline
• DNA- Structure, function, and importance• How DNA works
– The central dogma– Transcription and Translation– The DNA code– DNA replication
The importance of DNA
The DNA double helix is the code of life
• The blueprint for all structures in your body which are made of protein
• DNA is comprised of nucleotides
Nulceotides are the monomers of nucleic acid polymers
• Consist of a sugar, a phosphate, and a nitrogen-containing base
• Sugar can be deoxygenated
• Bases contain the genetic information
There are 4 kinds of DNA bases
Adenine always matches with
Thymine, Cytosine always
matches with Guanine-
Hydrogen bonds hold bases together
Living things are extremely complex• Cellular machinery is
sophisticated and required for life
• Cellular machinery is made largely of proteins
• Blueprints for all cellular machinery are contained in genes
• Genes are inherited from parents
• Humans have ~30,000 genes
Proteins give living things the variety of their structures
Protein variety is generated by 1o structure- the sequence of amino acids
which make the protein
Figure 2.12
Amino Acids
• Proteins consist of subunits called amino acids
How DNA works
• Replication• Transcription• Translation
The sequence of DNA bases is the code for the primary structure of
proteins
All cells require a copy of the genome
• Genome- all the genes of the cell • Human genome is made of DNA• DNA is similar in all cells• Gene- 1 DNA Molecule (+
proteins the genetic information to produce a single product (protein)
• DNA replication copies all cellular DNA
Replication of DNA
Figure 21.2
In vivo, enzymes such as DNA polymerase make DNA replication happen
The DNA code
Computers use binary digital code
• 01100001 = A• 01100010 =B• 01000011 =c• 00100111 = apostrophe• Etc.
• http://www.geek-notes.com/tools/17/text-to-binary-translator/
01000011 01101000 01100101 01100101 01110011 01100101 01100010 01110101 01110010 01100111 01100101 01110010 00100000 01000100 01100101 01101100 01110101 01111000 01100101 = cheeseburger deluxe
How does the DNA code work?
• atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttc=GFP
The DNA code is (nearly)
universalIt uses groups of 3 bases (codon)
3 bases = 1 codon = 1 amino acid
And what are these U’s for?
RNA is ribonucleic acid
• Ribose sugar is not deoxygenated
• RNA is single-stranded
• RNA has Uracil, not Thymine
• There are many kinds: mRNA, rRNA, tRNA, siRNA, etc.
RNA can fold back on itself
• Single strand offers greater flexibility
Kinds of RNA
mRNA tRNA
The Central Dogma of Molecular Biology
• DNA RNA Protein • DNARNA :
Transcription• RNA Protein:
Translation
When one DNA molecule is copied to make two DNA molecules, the new DNA contains
1. A) 25% of the parent DNA. 2. B) 50% of the parent DNA. 3. C) 75% of the parent DNA. 4. D) 100% of the parent DNA. 5. E) none of the parent DNA.
DNA RNA Protein Trait
The Universality of the DNA code makes
this possible
Firefly gene (Luciferase) in a tobacco plant
Transcription and Translation
Transcription: DNA RNA
DNA Codes for RNA, Which Codes for Protein
Figure 21.3
DNA information is transcribed into mRNA
Note in DNA: sense strand vs. antisense strand
Translation: RNA Protein
tRNA’s carry an amino acid at one end, and have an anticodon at the other
Figure 21.6
Amino acid(phenylalanine)
mRNA
Anticodon
Amino acidattachment site:Binds to a specific amino acid.
Anticodon:Binds to codon on mRNA, following complementary base-pairing rules.
The ribosome matches tRNA’s to the mRNA, thereby linking amino acids in
sequence
tRNA’s add amino acids one by one according to mRNA instructions until the protein is complete
We would expect that a 15-nucleotide mRNA sequence will direct the production of a polypeptide that consists of
1. A) 2 amino acids. 2. B) 3 amino acids. 3. C) 5 amino acids. 4. D) 7 amino acids. 5. E) 15 amino acids.
Viruses exploit the universality of the DNA code to take control of cells
Basic life cycle of Viruses
• Viruses are obligate intracellular parasites
• They inject their genetic material into their host
• Host machinery is commandeered to mass-produce virus
• Viruses burst host cell to infect other cells
Virus Structure
• Many viruses contain only:– Protein capsid– Genome (DNA or RNA)
• Some viruses have a phosopholipid bilayer envelope
Bacteria are infected by viruses, too
Bacteriophages attacking bacterial cell The lytic bacterial life cycle
Bacteriophages
• Infect bacteria• Cause formation of
plaques on a lawn of agar in bacteria
Bacteria use restriction enzymes to defend against viral DNA
Restriction enzymes cut very specific sequences of DNA
• Hundreds of different restriction enzymes have been found
• Named after bacteria in which they are found
Viruses such as T4 fight back with DNA ligase
• Ligase glues DNA back together
• Viral countermeasures against bacterial cell defenses
Scientists commandeer these enzymes to perform DNA manipulations
• Ligase and restriction enzymes allow any sequence of DNA to be cut and pasted at will
• Plasmids, small loops of bacterial DNA, can be modified with any DNA
• Because the genetic code is universal, DNA will be read in the same way
Plasmid DNA
manipula-tion is at the heart
of biotech-nology
Bacterium
Bacterialchromosome
Plasmid
Gene inserted intoplasmid
Cell containing geneof interest
Gene ofinterest DNA of
chromosome
RecombinantDNA (plasmid)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Protein expressedby gene of interest
Protein harvested
Gene ofinterest
Copies of gene
Basicresearchon gene
Basicresearchon protein
Basic research andvarious applications
Gene for pestresistance insertedinto plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growth hor-mone treats stuntedgrowth
The pGLO plasmid has
• ori- origin of replication1. GFP- green fluorescent
protein2. bla- beta-lactamase
(confers ampicillin resistance)
3. araC- Arabinose regulator protein (regulates GFP expression)
1. GFP: Green Fluorescent Protein• discovered in 1960s by Dr. Frank
Johnson and colleagues
• closely related to jellyfish aequorin
• absorption max = 470nm
• emission max = 508nm
• 238 amino acids, 27kDa
• “beta can” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore
• amino acid substitutions result in several variants, including YFP, BFP, and CFP
40 Å
30 Å
2008 Nobel Prize- GFP
• GFP mice
Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
GFP can be fused to cellular proteins
GFP fusions are useful in Biology and medicine
Nervous system of C. elegans worm illuminated by GFP
GFP fused to Huntingtin protein in monkey to study Huntington’s disease
2. bla: β-lactamase enzyme can destroy penicillin and other β-lactam antibiotics
Antibiotic resistance genes are found commonly on plasmids and can be shared between
bacteria by conjugation
Bacterial conjugation is sex without reproduction
Genes for making a sex pilus also are often found encoded on plasmids
3. araC: araC regulates pGLO expression through the presence of arabinose
Arabinose is a 5-carbon sugar, different from ribose
Gene Regulation
Q: What is “regulation”?*
"When I was warning about the danger ahead on Wall Street months ago because of the lack of oversight, Senator McCain was telling the Wall Street Journal -- and I quote -- 'I'm always for less regulation.' " – Sen. Barack Obama
“Senator Obama was silent on the regulation of Fannie Mae and Freddie Mac, and his Democratic allies in Congress opposed every effort to rein them in…last year he said that subprime loans had been, quote, “a good idea.””- Sen. John McCain
* Slide created, September 2008
Regulation means controlProkaryotic cells require efficiency
Eukaryotic cells must differentiate
Eukaryotic genes can be turned on and off
• Females only use a single X chromosome per cell• Genes from the other chromosome are not used in that
cell
Random inactivation of one X chromosome creates a tortoiseshell pattern in cat fur
Genes are regulated in eukaryotes in more complex ways
• Each step in the process of gene expression is a possible point of control
• The cell capitalizes on each one
The job of master control genes is to turn many genes on or off
The gene eyeless turns on many genes involved in formation of eyes
When eyeless is mutated, eyes do not form
Master control genes control formation of entire organs
-Drosophila eyeless gene can be artificially turned on in non-eye cells
Prokaryotes must also regulate genes
Example: β- Galactosidase can hydrolyze lactose
11
lactose
galactose
glucose
H2O
- galactosidase(aka lactase in humans)
-galactosidase
10
When lactose is present, transcription is activated
In the absence of lactose, the lac operon is repressed by the lac repressor protein
04/19/23 73
araC allows expression of arabinose-digesting genes in the presence of arabinose
In pGLO, arabinose-digesting genes are removed, and araC is fused to GFP
• How do you think this fusion was made?
• What are the structural sequences? The regulatory sequences?
• What happens when we add arabinose sugar to these bacteria?
• What do you think is meant by “reporter gene”?
Gene Regulation
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
On pGLO, the regulatory regions of the Arabinose operon have been glued to the structural sequences for GFP
What will happen on the Ara (+) plates?What will happen on the Ara (-) plates?
04/19/23 76
Grow? Glow?
Follow protocol On which plates will
colonies grow? Which colonies will
glow?
Which colonies will glow?
Plasmids with novel structural and regulatory genes are now easily manipulated by undergraduates
iGEM is an annual undergrad bioengineering contest Engineering bacteria to smell better
Which strand carries the DNA's instructions for synthesizing a particular protein from the nucleus to
the cytoplasm?
• A) Transfer RNA • B) Messenger RNA • C) RNA transcriptase • D) Ribosomal RNA
Why are the complementary base pairing rules so important when a cell needs to copy its DNA?
• A) The cells need both strands to be accurate because each strand codes for one half of the gene.
• B) As long as there is one strand, a copy can be made by following the pairing rules.
• C) Unless the bases pair up correctly, the DNA strand can break apart.
• D) Transcription cannot proceed unless the pairs are in the proper sequence.