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DNA the GENE

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					DNA the GENE
History of DNA’s
Discovery
1.Griffith Experiment
 demonstrates the
 Transformation of
 bacteria : DNA is later
 found to be the
 transforming principle
2. Hershey-Chase demonstrate
DNA is hereditary material not
 proteins by using radioactive
isotopes
3. Meselson-Stahl
demonstrate the
Semiconservative
Replication
of DNA using radioactive
nitrogen
   Discovery of DNA
1. 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
      Discovery of DNA
2. Avery, McCarty and MacLeod
  –   What was the genetic material in Griffith’s
      experiment?
  –   Purified the heat–killed S-bacteria
      •   Into DNA, RNA, and Protein
  –   Mixed each with the R cells to see which one
      transformed
      Discovery of DNA
3. 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
P*
P*
Discovery of Structure
    The Structure of DNA:
       a double helix?




• Rosalind Franklin and Maurice Wilkins use
  X-Ray diffraction to view structure
Watson and Crick propose a
      double helix.
From Chromosomes to Genes
     DNA Basic Composition
• DNA is made up of nucleotides
• Nucleotides are made of
   …………...Deoxyribose sugar
   ……………Phosphate
   ……………Base
 bases are guanine,cytosine, thymine and
  adenine
      The Structure of DNA:
         a double helix?
•    Chargaff’s Nucleic Acid Ratios
    1. Measured the base compositions of several
       species
    2. Percentage of each base present
       •   Human DNA
           1. A = 30% and T = 29%
           2. G = 20% and C = 19%
DNA: The Deoxyribose Sugar
DNA: The Phosphate
  DNA: The Nitrogenous Bases
• Purines
     • Adenine and Guanine
     • Double Ring Structure


• Pyrimidines
     • Thymine and Cytosine
     • Single Ring Structure
Single Stranded DNA

Nucleotides can only
be added to the 3’ end
of the nucleotide and
therefore addition of
new nucleotides is
always 5’-----> 3’

DNA is
anti-parallel!!
        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
                  Purines
• Adenine
• Guanine
• All double ring structures
DNA BASE PAIRS
                Pyrimidines
• Cytosine
• Thymine
• single ring
     How does it know to pair up?
• ADENINE ALWAYS
  PAIRS WITH
  THYMINE
    • Two hydrogen
      bonds
• GUANINE ALWAYS
  PAIRS WITH
  CYTOSINE
    • Three hydrogen
      bonds
DNA STRUCTURE
            One last look




Why does
it twist?
DNA Replication
 Why must DNA Replicate?

• Species Survival
 – DNA must replicate BEFORE cell
   division
    • Synthesis during Interphase
• All genes must be present in
  the daughter cells
   DNA Replication
   Number the following events 1-5
Enzymes unwind and unzip
Hydrogen bonds break, forming bubbles
Free nucleotides in the nucleus start
process of complementary base pairing
Nucleotides are fused together by
  DNAPolymerase only 5’ to 3’

Results in two identical double helixes
     How does DNA
       Replicate?
• Enzymes unwind and unzip
• Hydrogen bonds break, forming
  bubbles
• 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
How does DNA Replicate?
How does DNA Replicate?
DNA Structure
Single stranded
Nucleotides can only be added to the 3’ end
of the nucleotide and therefore addition of
new nucleotides is always 5’-----> 3’
replication
Leading strand is continious
 Lagging strand is done in
segments as each primer is
 added only after a coding
   segment is exposed.
          Replication Steps
• DNA polymerase and helicase enzymes
  start the following :
• DNA uncoils and unzips exposing the DNA
  template and free nucleotides
• bases pair A-T and G-C as new strand is
  added in a 5’ to 3’ direction
• Two identical strands of DNA are made
replication3
DNA and RNA functions

 Replication, Transcription, and
           Translation
function of
   DNA
           RNA Nucleotides
• Made of the following:

• ………Ribose sugar
• ………Phosphate
• one of four bases ( uracil replaces thymine)
             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
        Transcription Steps
• DNA uncoils and exposes template
• RNA nucleotides base pair with DNA
  template A-U, G-C via RNA polymerase
• This new mRNA is then processed and
  leaves the nucleus to be translated.
Transcription
Promoter Regions direct
    Transcription
     Processing Genetic Material
• After transcription mRNA is PROCESSED

•   INTRONS ARE DELETED
•   A CAP AND TAIL IS ADDED(G-p-p-p)5’
•   poly a tail AAA-AAA
•   start and stop codon
DNA Processing
             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)
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
Aminoacyl-tRNA syntase
 matches each amino acid to
 the correct tRNA
      Steps in Translation
• Initiation
• Elongation-elongation
  factors enable addition of
  tRNAs to A site.
  codon recognition
  peptide bonds form
• translocation -tRNA
  move from A site to P site
• Termination-UAA, UAG
  UGA stop the process
   Polyribosomes-
clusters of ribosomes
translating the same
       mRNA
    At the ribosome
                                        Growing
                                        polypeptide




tRNA molecule exits        P A
                                        Next amino
                                        acid to be
                                        added


             tRNA molecules with
             amino acids
 P site--- peptidyl-tRNA binding site

 A site- aminoacyl-tRNA binding site
               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
             Transposons
• Stretches of DNA that can move from one
  location to another ( Jumping genes)


                                    DNA




                     transposase
Immunoglobulin genes undergo permanent
rearrangement during antibody production.
     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
 PCR-polymerase chain reaction
• Makes several copies of DNA
• adjust temperature and enzyme
• addition of nucleotides with DNA
  polymerase
Prepare single strand
complimentary DNA
Mix hybrid (Known fragment as
           Hybrid )
Hybrid 3
Blot on Nylon Film
Use probe to identify position of
    gene on a chromosome
PROBES
       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
          Sequencing Methods
•   Chain termination- Sanger Method
•   Restriction enzymes specific
•   Restriction fragments vary in size
•   gel electrophoresis resolves the fragments
 Chain Termination Method-uses
dedeoxynucleotides that terminate
 the synthesis of DNA strands at
          specific bases
Electrophoresis
Restriction Analysis
RFLP restriction fragment length
          polymorph
                  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
DNA Fingerprints
VNTRvariable number tandem
         repeats
Restriction Analysis
  Other Technologies
• Recombinant DNA - gene
  splicing
• Transgenic organism- an
  organism that contains another
  organism’s DNA
           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
Transgenic Organism
Genetic Therapy

				
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