Video #2 DNA: The Blueprint of LifeName the technology used in the movie Jurassic Park.Where did Meissner extract the “nuclein” material that later was identified as

DNA?How did Hershey & Chase separate the virus from its bacterial host? How did they

trace (track) the DNA and protein?What did x-ray crystallography reveal about DNA?What purpose do enzymes serve in the replication process?Segment #2:Name the disorder that Andrew and his sister inherited. What were the major

symptoms of this disorder?How can this genetic defect be treated? Name the gene that is defective.How can a gene be transported and carried to a cell?What is a vector? Give an example.What purpose do restriction enzymes serve? What about ligase?What does PCR stand for?Segment #3:What is the first step of gene therapy? How long would all of the DNA contained in all of the chromosomes in a human

cell be if they were connected end to end? Which chromosome consists of 5% of all the genes in the human genome?

Protein Synthesis: Chapter 17Bridging the gap between Genotype & Phenotypes

(proteins are thought to be that link)Trace the Flow of Information from Gene to Protein

Key Topics:• Garrod• Beadle & Tatum• Transcription (nucleus)• Processing mRNA• Translation (cytoplasm)• Completed polypeptide (protein)

Introductory Questions #31) Name the substance that accumulates in a person’s urine causing

alkaptonuria. 2) Why did Beadle and Tatum use breadmold spores to determine

that one gene forms one polypeptide allowing for the first metabolic pathway to be defined?

3) Transcribe & Translate the following sequence of DNA by determining the nucleotide sequence for mRNA, the anticodon for tRNA, and the overall amino acid sequence:

TACTCAGGACCTGCAACGATTmRNA: ???????????????????????????????Amino acids Sequence: ???????????????????????????????Anticodon: ???????????????????????????????

4) How does the DNA and amino acid sequences differ from a person with sickle cell anemia and a person with normal hemoglobin in their RBC’s? (pg. 328)

5) When mRNA is “processed” what is taken out (spliced)?

Key Discoveries• Miescher (isolated “nuclein” from soiled bandages) 1869• Garrod (Proteins & inborn errors) 1902• Sutton (Chromosome structure) 1903• Morgan (Gene mapping) 1913• Sumner (Purified Urease, showed it to be an enzyme) 1926• Griffith’s Experiment (Transforming Principle) 1928• Avery, McCarty, and Macleod 1944• Chargaff (Base pairing & species specific) 1947• Hershey and Chase 1952• Pauling, Wilkins, and Franklin 1950’s• Watson and Crick 1953• Meselson & Stahl 1956

Archibald Garrod (1902-1908)• First to suggest that genes dictate phenotypes through

enzymes and their metabolic, catalytic properties.• Studied a rare genetic disorder: Alkaptonuria• Thought to be a recessive disorder• Tyrosine is not broken down properly into carbon dioxide

and water.• An Intermediate substance: “Homogentisic acid”

accumulates in the urine turning it BLACK when exposed to air.

• An enzyme was thought to be lacking• A genetic mutation was thought to be the cause “An Inborn Error of Metabolism”

Metabolic Pathway for the breakdown of Tyrosine

Tyrosine↓

Hydroxyphenylpyruvate↓

Homogentisic acid

Alkaptonuria Maleyacetoacetate Inactive (lacking) enzyme (active ↓ enzyme)

CO2 & H2O

Garrod’s Conclusion

• A mutation in a specific gene is associated with the absence of a specific enzyme.

• Led to the idea of:“One gene, One Enzyme”

• Not validated until Beadle & Tatum’s work in the 1940’s with Neurospora (breadmold)

James Sumner (1926)

• Isolated the enzyme “Urease”

• First to identify an enzyme as a protein

• First to crystallize an enzyme

• Awarded the Nobel prize in 1946 in chemistry for his crystallization of an enzyme.

• Studies of inherited metabolic disorders first suggested that phenotype is expressed through proteins

• Studies of the bread mold Neurospora crassa led to the one gene-one polypeptide hypothesis (Beadle & Tatum)

Figure 10.6B

George Beadle & Edward Tatum

• Discovered the “One Gene, One Enzyme” Principle• Analyzed mutations that interfered with a known metabolic

pathway• Organism they chose to work with: Neurospora

(breadmold)

-Grows easily

-Grows as a haploid: (no homologs)

-Mutants are easily identified: Dominant allele won’t be expressed

• Neurospora (wild type) can grow easily in only: salt, sugar, & Biotin (vitamin) = “Minimal Medium”

George Beadle & EdwardTatum cont’d

• Breadmold spores were bombarded with x-rays & UV• Separated out the survivors: Mutants• Haploid spores were crossed, grown in a variety of media to

determine what kind of mutation was occurring• Mutants (had diff. nutritional needs vs. the wild types) –were

unable to make certain organic molecules: amino acids, lipids, etc. when grown on the minimal media.

• Mutants cold grow on complete growth medium• These mutants were grown in minimal media with one added

nutrient to determine the metabolic defect.• Ex. Arginine was one nutrient that supported growth of the

mutants. • Conclusion: These mutants had defective biochemical pathways

that allowed for the synthesis of arginine.

• **They examined the effect of the mutation instead of identifying the enzyme.

Beadle & Tatum Experiment w/Neurospora

Beadle & Tatum’s Conclusion

“One Gene affects One Enzyme”Later Revised

“One Gene affects One Protein”

Later Revised

“One Gene affects One Polypeptide Chain”

• The information constituting an organism’s genotype is carried in its sequence of bases

THE FLOW OF GENETIC

INFORMATION DNA → RNA → PROTEIN

• The “words” of the DNA “language” are triplets of bases called codons– The codons in a gene specify the amino acid

sequence of a polypeptide

Genetic Information Written in Codons is Translated into Amino acid Sequences

• Virtually all organisms share the same genetic code

• 1st codon determined was “UUU” by Marshal Nirenberg in 1961.

• All of the codons were determined by the mid 1960’s

The Genetic Code Dictionary

Figure 10.8A

Figure 10.7

DNA molecule

Gene 1

Gene 2

Gene 3

DNA strand

TRANSCRIPTION

RNA

Polypeptide

TRANSLATIONCodon

Amino acid

Introductory Questions #31) Name the substance that accumulates in a person’s urine causing

alkaptonuria. 2) Why did Beadle and Tatum use bread mold spores to determine

that one gene forms one polypeptide allowing for the first metabolic pathway to be defined?

3) Transcribe & Translate the following sequence of DNA by determining the nucleotide sequence for mRNA, the anticodon for tRNA, and the overall amino acid sequence:

TACTCAGGACCTGCAACGATTmRNA: ???????????????????????????????Amino acids Sequence: ???????????????????????????????Anticodon: ???????????????????????????????

4) How does the DNA and amino acid sequences differ from a person with sickle cell anemia and a person with normal hemoglobin in their RBC’s? (pg. 328)

5) When mRNA is “processed” what is taken out (spliced)?

Figure 10.7

DNA molecule

Gene 1

Gene 2

Gene 3

DNA strand

TRANSCRIPTION

RNA

Polypeptide

TRANSLATIONCodon

Amino acid

Protein Synthesis: overview

• One gene-one enzyme hypothesis (Beadle and Tatum)

• One gene-one polypeptide (protein) hypothesis

• Transcription: synthesis of RNA under the direction of DNA (mRNA)

• Translation: actual synthesis of a polypeptide under the direction of mRNA

4) How many sites are present in the ribosome? Name the enzyme that is used to attach an amino acid to the tRNA molecule.

• An exercise in translating the genetic code

Figure 10.8B

Startcodon

RNA

Transcribed strand

StopcodonTranslation

Transcription

DNA

Polypeptide

• In transcription, the DNA helix unzips

– RNA nucleotides line up along one strand of the DNA following the base-pairing rules

– The single-stranded messenger RNA peels away and the DNA strands rejoin

RNA polymerase

DNA of gene

PromoterDNA Terminator

DNAInitiation

Elongation

Termination

Area shownin Figure 10.9A

GrowingRNA

RNApolymerase

Completed RNA

Figure 10.9B

Transcription

• Occurs in the nucleus

• RNA Polymerase II is needed

-Adds nucleotides to the 3’ end only

-Eukaryotes have three types vs. Bacteria

with only one type

• Elongation occurs from 5’ 3’ direction

• TATA Box : initiation site for the attachment of RNA polymerase

• 3 Steps: Initiation Elongation Termination

Transcription: Initiation

• RNA Polymerase II binds to the “Promoter” region on the DNA RNA Polymerase recognizes this region because of the “TATA” box

• TATA Box is (upstream about 25 nucleotides from starting pt)

• Other proteins also are needed: – Transcription factors

• These proteins must bind to the DNA first before RNA Polymerase can bind and begin Transcription.

• No Primer is needed, mRNA can made from scratch

Transcription produces genetic messages in the form of RNA

Figure 10.9A

RNApolymerase

RNA nucleotide

Direction oftranscription

Newly made RNA

Templatestrand of DNA

Transcription: Elongation

• DNA is untwisted (hydrogen bonds are broken)

• About 10 base pairs are exposed

• Nucleotides are are added to the 3’ end of the growing mRNA molecule

• Proceeds at a rate of: 60 nucleotides/sec

Transcription: Termination

• Termination site is reached by RNA Polyermase

• In Eukaryotes “AATAAA” is the signal

• In Bacteria Translation can occur as it is released from the first transcription event

• Final mRNA molecule is made consisting of “Coded” and “Non-coded” regions

Transcription- The three Phases

• Noncoding segments called introns are spliced out

• A cap and a tail are added to the ends

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

Eukaryotic RNA is processed before leaving the nucleus

Figure 10.10

DNA

RNAtranscriptwith capand tail

mRNA

Exon Intron IntronExon Exon

TranscriptionAddition of cap and tail

Introns removed

Exons spliced together

Coding sequence

NUCLEUS

CYTOPLASM

Tail

Cap

mRNA Structure• 1) 5’ cap: modified guanine; protection; recognition site

for ribosomes• 2) 3’ tail: poly(A) tail (adenine); protection;

recognition; transport• 3) RNA splicing: involves introns & Exons• Exons (expressed sequences) retained• Introns (intervening sequences)

-These are spliced out / spliceosome

Key Regions on Newly Transcribed mRNA

Various Types of RNA Molecules

Splicesomes w/ SNRP’s(Small nuclear Ribonuclearprotein)

Animated View of Transcription

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

Translation

• Occurs in the Cytoplasm

• Key molecules and structures include:– mRNA– tRNA– Ribosome (30s and 40s subunits)– Free floating amino acids– Endoplasmic reticulum

Translation: Overview

• In the cytoplasm, a ribosome attaches to the mRNA and translates its message into a polypeptide

• The process is aided by transfer RNAs

Transfer RNA molecules serve as interpreters during translation

Figure 10.11A

Hydrogen bond

Amino acid attachment site

RNA polynucleotide chain

Anticodon

A Typical tRNA Molecule

Translation: Transfer RNA (tRNA)-Pg. 273

mRNA from nucleus is ‘read’ along its codons by tRNA’s anticodons at the ribosome

tRNA – has the anticodon and amino acid attached

• Each tRNA molecule has a triplet anticodon on one end and an amino acid attachment site on the other

Figure 10.11B, C

Anticodon

Amino acidattachment site

Attachment of an Amino Acid (Pg 321)

Translation- the Ribosome

rRNA site of mRNA codon & tRNA anticodon coupling

P site holds the tRNA carrying

the growing polypeptide chain

A site holds the tRNA carrying

the next amino acid to be added to the chain

E site discharged tRNA’s

Animated View of Transcription

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

Video #4:Proteins-Building Blocks of Life

• Name the structures identified by Dr. James Lake that has helped him to trace the hereditary path of life back to the first cell.

• What type of therapy is suggested by Dr. Richard Firtel that may provide long term help for patients suffering from sickle cell anemia?

• In the third segment what type of organism is profiled? How do prokaryotic cells switch protein production on and off?

Be sure to write the title for all three segments and list five key statements for each segment.

Translation

• Initiation~ union of mRNA, tRNA, small ribosomal subunit; followed by large subunit

• Elongation~ •codon recognition •peptide bond formaton •translocation

• Termination~ ‘stop’ codon reaches ‘A’ site

• Polyribosomes: translation of mRNA by many ribosomes (many copies of a polypeptide very quickly)

Transcription & translation in Prokaryotes

Mutations & Errors During Transcription & Translation

DNA Repair• Mismatch repair:

DNA polymerase

• Excision repair:Nuclease

• Telomere ends:telomerase

• Mutations are changes in the DNA base sequence– These are caused by errors in DNA replication or by

mutagens– The change of a single DNA nucleotide causes

sickle-cell disease

Mutations can change the meaning of genes

DNA Mutations & Modifications• Point mutations

-changes in 1 or a few base pairs in a single gene

-most common

• Base-pair substitutions: •silent mutations no effect on protein

•missense ∆ to a different amino acid (different protein)

•nonsense ∆ to a stop codon and a nonfunctional protein

• Base-pair insertions or deletions:

-additions or losses of nucleotide pairs in a gene; alters the ‘reading frame’ of triplets

-frameshift mutation

• Mutagens: physical and chemical agents that change DNA

• Types of mutations

Figure 10.16B

mRNA

NORMAL GENE

BASE SUBSTITUTION

BASE DELETION

Protein Met Lys Phe Gly Ala

Met Lys Phe Ser Ala

Met Lys Leu Ala His

Missing

Figure 10.16A

Normal hemoglobin DNA

mRNA

Normal hemoglobin

Glu

Mutant hemoglobin DNA

mRNA

Sickle-cell hemoglobin

Val

Mutations: genetic material changes in a cell

Chapters 18 & 19

Bacteria

Viruses & Operon Systems

Key Topics and Text Pgs to ReviewTopic Pgs.

Bacteria: Genetic recombination 346-350Plasmids & ConjugationTransformation (Lab #8)

Transposons: 351-352Lac Operon System 353-356Regulating Gene ExpressionViruses: DNA, RNA (retroviruses) 338-342Lytic & Lysogenic Cycle 337-339

Relative size Differences between of Viruses, Prokaryotes, and Eukaryotes

Bacterial Reproduction of DNA

The Transfer of a Plasmid

Detecting Genetic Recombination in Bacteria

Regulation of a Metabolic Pathway

Introductory Questions #41) Name the two scientists that discovered the Lac

operon system.2) How are repressible operons different from inducible

operons? Give an example of each.3) What is the difference between an operator and a

promoter?4) Why are transposons called “jumping genes”? What

purpose do the insertion sequences play?5) Name three example of a virus that has DNA as its

genetic material and three examples of Viruses with RNA as its genetic material.

6) Briefly explain what a vaccine is and what it does.

Repressible Operons (trp operon)• Usually “ON” - to turn OFF:

– Co-repressor needs to bind to an inactive repressor and activate it

– RNA Polymerase then cannot bind and transcribe mRNA

Ex. trp operon is a repressible operon: -trancription is usually on-inhibited only by tryptophan

(corepressor)

Trp Operon when Tryptophan is Absent

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html#

INDUCIBLE Operons (ex. lac operon)• Usually “OFF” - to turn ON:

– INDUCER needs to bind to an active repressor and inactivate it

– RNA Polymerase can then bind and transcribe mRNA

Ex. Lac operon is an inducible operon

Lac Operon• Lactose ONLY used when glucose is not present

in large quantities• When glucose is present, cAMP levels are low,

cAMP cannot bind to CAP and initiate enzyme production

Inactive Repressor-Lactose Presenthttp://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html

Lac Operon• In absence of glucose, cAMP levels are

HIGH, binding to CAP can occur

• Beta-Galactosidase is made

Lac Operon

• RNA polymerase only binds efficiently when cAMP-CAP complex is in place

• Lac Operon = an INDUCIBLE Operon• Lactose = an INDUCER

– Binds to repressor and inactivates it