DNA the GENE

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DNA the GENE
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History of DNAs Discovery

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Discovery of DNA

• Frederick Griffith
• Was studying Streptococcus Pneumonia
• Smooth vs. Rough Strains
• Smooth had a mucous coat and were pathogenic
  (caused pneumonia)
• Rough were non-pathogenic
• Conducted an experiment with mice
• Found out that the Rough bacteria became
  transgenic with the Smooth and killed the mouse

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Discovery of DNA

• Avery, McCarty and MacLeod
• What was the genetic material in Griffiths
  experiment?
• Purified the heatkilled S-bacteria
• Into DNA, RNA, and Protein
• Mixed each with the R cells to see which one
  transformed

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3. Meselson-Stahl demonstrate the
Semiconservative Replication of DNA using
radioactive nitrogen
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Discovery of DNA

• Hershey-Chase Experiment
• Studied viruses that infect bacterial cells
  called Bacteriophages
• Viruses use Bacteria to multiply
• Protein or DNA responsible for multiplying within
  the bacteria
• Tagged the Protein with radioactive S
• Why?
• Tagged the DNA with radioactive P
• Why?
• Checked the Virus Progeny for Radioactive Elements

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• 1. Griffith Experiment demonstrates the
  Transformation of bacteria DNA is later found
  to be the transforming principle

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2. Hershey-Chase demonstrate DNA is hereditary
material not proteins by using radioactive
isotopes
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3. Meselson-Stahl demonstrate the
Semiconservative Replication of DNA using
radioactive nitrogen
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From Chromosomes to Genes
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From Chromosomes to Genes
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The Structure of DNAa double helix?

• Chargaffs Nucleic Acid Ratios
• Measured the base compositions of several species
• Percentage of each base present
• Human DNA
• A 30 and T 29
• G 20 and C 19

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The Structure of DNAa double helix?

• Rosalind Franklin and Maurice Wilkins use X-Ray
  diffraction to view structure
• Watson and Crick propose a double helix using
  their X-Ray pictures

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DNA Helix
1.0nm
3.4nm
.34nm
.34nm
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DNA Structure

• Watson and Crick propose a double helix and
  construct a model of DNA based on an accumulation
  of various researchers.

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James and Francis
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DNA Basic Composition

• DNA is made up of nucleotides
• Nucleotides are made of
• ...Deoxyribose sugar
• Phosphate
• Base
• bases are guanine,cytosine, thymine and adenine

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DNA The Deoxyribose Sugar
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DNA The Phosphate
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DNA The Nitrogenous Bases

• Purines
• Adenine and Guanine
• Double Ring Structure
• Pyrimidines
• Thymine and Cytosine
• Single Ring Structure

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Single Stranded DNA Nucleotides can only be
added to the 3 end of the nucleotide and
therefore addition of new nucleotides is always
5-----gt 3 DNA is anti-parallel!!
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Why do they pair up?

• Double helix had a uniform diameter
• Purine Purine
• too wide
• Pyrimidine Pyrimidine
• too narrow
• Purine Pyrimidine
• fits the x-ray data

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DNA Double Helix
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How does it know to pair up?

• ADENINE ALWAYS PAIRS WITH THYMINE
• Two hydrogen bonds
• GUANINE ALWAYS PAIRS WITH CYTOSINE
• Three hydrogen bonds

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Purines

• Adenine
• Guanine
• All double ring structures

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DNA BASE PAIRS
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Pyrimidines

• Cytosine
• Thymine
• single ring

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DNA Structure
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Single stranded
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Nucleotides can only be added to the 3 end of
the nucleotide and therefore addition of new
nucleotides is always 5-----gt 3
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DNA STRUCTURE
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One last look
Why does it twist?
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DNA Replication
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Why must DNA Replicate?

• Species Survival
• DNA must replicate BEFORE cell division
• Synthesis during Interphase
• All genes must be present in the daughter cells

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How does DNA Replicate?

• Hydrogen bonds break, forming bubbles
• Enzymes unwind and unzip
• Free nucleotides in the nucleus start process of
  complementary base pairing
• Nucleotides are fused together by DNA Polymerase
  only 5 to 3
• Results in two identical double helixes

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How does DNA Replicate?
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How does DNA Replicate?
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DNA and RNA functions

• Replication, Transcription, and Translation

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replication
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Replication Steps Leading Strand

• DNA helicase uncoils and unzips exposing the DNA
  .
• Single stranded binding proteins hold them apart.
• Topoisomerase helps relieve strain on open strand
• Primase adds RNA primer
• DNA polymerase III adds free nucleotides
• bases pair A-T and G-C as new strand is added in
  a 5 to 3 direction

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Lagging strand is done in segments as each primer
is added only after a coding segment is exposed.

• That means the end of it can not be replicated.
  Last primer only has a 5 end .
• Telomeresends of chromosomes have repeating
  nonsense sequences. TTAGGG
• As cells age the ends shorten until it can no
  longer replicate the DNA

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Proteins are critical

• Helicase-unwinds parental DNA
• Single-strand binding protein-stabilize DNA
  strands
• Primase-makes single RNA primers at 5 end of the
  leading strand
• DNA polymerases
• I. Removes primer and replaces with DNA
  
• III. Continuously synthesizes the leading strand
  and replaces it with DNA adding to 3 end only
• elongates each Okazaki fragment

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• Telomerase replaces DNA nucleotides where RNA
  primer was located on the leading strand.

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Lagging Strand

• Lagging strands begin with addition of Primase
  and then RNA primer
• DNA polymerase I adds nucleotides in segments
  known as Okazaki Fragments
• Ligase glues fragments together of lagging strand

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Proofreading and Repairing DNA

• Mismatch repair enzymes remove incorrectly
  paired nucleotides
• Nuclease enzymes cut out damage
• mutagens and carcinogens can cause these
  mismatches( uv light , x rays, reactive
  chemicals)
• Nucleotide excision repair

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replication3
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function of DNA
Processing
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RNA Nucleotides

• Made of the following
• Ribose sugar
• Phosphate
• one of four bases ( uracil replaces thymine)

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Types of RNA

• M RNA- messenger RNA carries the DNA
  instructions(gene) out of nucleus to ribosome
• tRNA-transfer RNA carries amino acids to their
  appropriate location during protein synthesis (
  gene expression )
• r RNA - ribosomal RNA makes up much of the
  ribosome and is essential to translation

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Transcription in 3 steps

• 1. Initiation
• RNA polymerase binds to promoter region
• DNA unwinds and
• 2. Elongation polymerase initiates RNA synthesis
  one nucleiotide at a time 5-3
• 3.Termination- transcript released at terminator
  sequence

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Eukaryotic Transcription Steps

• RNA polymerase II-binds to promoter regions
  mediated by proteins called transcription factors
  keys on upstreamTATA box with help of protein
  transcription factors-transcription initiation
  complex
• DNA uncoils and exposes template-RNA nucleotides
  base pair with DNA template A-U, G-C via RNA
  polymerase
• Elongation-nucleotides added to 3 end of
  transcribed single strand
• The sequence called polyadenylationAAUAAA-signals
  proteins to cut mRNA free

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Prokaryotic Transcription

• RNA Polymerase binds to promoter region
• Signal ends at Terminator sequence
• RNA polymerase detaches several nucleotides down
  stream

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Promoter Regions direct Transcription
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Transcription
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• Termination AAATAAA- release mRNA

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DNA Processing
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Processing Genetic Material

• After transcription mRNA is PROCESSED
• INTRONS ARE DELETED
• A CAP 5 end is capped with a modified guanine
  (G-p-p-p)
• AND TAIL IS ADDED 50-250 nucleotides added to 3
  end( poly A tail AAA-AAA)
• Introns are non coding units
• Exons are expressed regions of RNA

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RNA splicing cut and paste

• Small sequences at the end of each intron contain
  a signal for splicing.
• snRNPs small nuclear ribonucleoproteins join
  together to form a SPLICEOSOME ---these release
  the introns and join the exons
• alternative RNA splicing
• Ribozymes- rna acts as an enzyme

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Translation Steps

• Messenger RNA is at the ribosome and the tRNA
  nucleotides will base pair A-U, G-C
• The tRNA has the amino acid attached to it and
  when it finds the right codon the RNA anticodon
  places the amino acids in their proper sequence
  for protein synthesis
• The bond that forms between two amino caids is
  called a peptide bond.
• Base ( Uracil replaces Thymine)

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45 different anticodons exist AUG is initiation
codon GTP supplies energy intiator tRNA carries
methionine small ribosomal unit
attaches intiiation factors-proteins bring all
parts together
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• Aminoacyl-tRNA syntase matches each amino acid to
  the correct tRNA

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Steps in Translation

• 1. Initiation
• 2. Elongation-elongation factors enable addition
  of tRNAs to A site.
  codon recognition
  peptide bonds form

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• 3. translocation -tRNA move from A site to P site
• 4. Termination-UAA, UAG UGA stop the process

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Polyribosomes- clusters of ribosomes translating
the same mRNA
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At the ribosome
Growing polypeptide
P
A
E
tRNA molecule exits
Next amino acid to be added
tRNA molecules with amino acids
P site--- peptidyl-tRNA binding site
A site- aminoacyl-tRNA binding site, E site- exit
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Gene Expression

• Various cells express different genes
• Organization of chromatin controls expression
• Regulation of expressed genes occurs at each step
• Control of transcription is most important
  regulatory mechanism ( binding factors and
  enhancers)
• some binding factors are sensitive to hormones

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Prokaryotic

• gene control or feedback---- a metabolite or
  substrate causes gene to be expressed by acting
  as a transcription factor.
• - neg. feedback the metabolite or substrate
  causes the removal of a blocking regulator

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Key to lac operon inducible enzymeNegative
Feedback

• a. regulatory gene b. promoter c. operator d.
  structural genes e. operon f. RNA polymerase g.
  active repressor h. inducer metabolite lactose
  i. mRNA for variousenzymes to degrade lactose

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Eukaryotic Gene Expression

• Heterochromatin to euchromatin-uncoiling and loss
  of DNA compaction
• Attachment of acetyl groups to histone proteins
• Metylation of DNA makes it unable to be expressed

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2. Transcriptional control

• Promoter region where a transcription initiator
  complex( including RNA polymerase II ) must bind.
• General Transcription factors bind to RNA
  polymerase, this may be facilitated by binding
  specific transcription factors
• Proximal Control elements
• Distal Control elements-enhancersbend in DNA

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3. Post-transcriptional Control

• Processing- alternative exon splicing
• can produce a variety of genes
• mRNA degradation-nuclease hydrolysis
• Micro RNA complimentary binds and blocks m RNA
  transcript
• Binding proteins can attach to mRNA and prevent
  translation

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Protein Processing

• Polypeptides can be cleaved or groups attached,
  and quaternary structure will result in
  additional folding
• Proteins can be degraded
• Selective Transport

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Transposons

• Stretches of DNA that can move from one location
  to another ( Jumping genes)

DNA
transposase
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Immunoglobulin genes undergo permanent rearrangeme
nt during antibody production.
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DNA TECHNOLOGIES

• SEQUENCING-determine order of bases
• PCR (polymerase chain reaction)-makes repeated
  copies of desired DNA
• RFLPS(restriction fragment length polymorphs)
  -unique gene fragments used as a fingerprint
• Gel Electrophoresis- separate DNA by size on a
  gel bed
• Probes- Radioactive tags label DNA

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PCR-polymerase chain reaction

• Makes several copies of DNA
• adjust temperature and enzyme
• addition of nucleotides with DNA polymerase

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Prepare single strand complimentary DNA
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Mix hybrid (Known fragment as Hybrid )
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Hybrid 3
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Blot on Nylon Film
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Use probe to identify position of gene on a
chromosome
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PROBES
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Reverse Transcriptase

• Viral enzyme
• transcribes DNA from RNA
• if you know the protein you can dtermine the mRNA
  which makes the protein
• revers the transcription process to make DNA

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Sequencing Methods

• Chain termination- Sanger Method
• Restriction enzymes specific
• Restriction fragments vary in size
• gel electrophoresis resolves the fragments

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Chain Termination Method-uses dedeoxynucleotides
that terminate the synthesis of DNA strands at
specific bases
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Electrophoresis
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Restriction Analysis
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RFLP restriction fragment length polymorph
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RFLPS

• Restriction fragments are created by cutting DNA
  with enzymes that cut at specific locations and
  create fragments of various size. These
  fragments can then be amplified and separated by
  gel electrophoresis

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DNA Fingerprints
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VNTRvariable number tandem repeats
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Restriction Analysis
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Other Technologies

• Recombinant DNA - gene splicing
• Transgenic organism- an organism that contains
  another organisms DNA

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Recombinant DNA

• Plasmid DNA
• Ligase enzyme Bacterial Cell
• Restriction Enzyme Bacterial cell wall
• Host cell Sticky ends
• Vector
• DNA fragment desired gene to be cloned

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Transgenic Organism
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