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1. modern genetics (2010) 1. modern genetics (2010) Presentation Transcript

    • Modern Genetics
    • Modern Genetics:
    • Study of DNA and how it serves as the molecular basis of heredity began in the 1920s. It became clear that chromosomes contained genes for genetic traits. A number of experiments were important in establishing that DNA was indeed the genetic material of living organisms.
      • Griffith’s experiments (1928)
      • Hershey and Chase experiments (1954)
    • Frederick Griffith’s Experiments (1928)
    • Griffith was studying two strains of the bacteria Streptococcus pneumoniae, which causes pneumonia and other infections.
      • Smooth strain (S): Produced a polysaccharide capsule that gave its colonies a smooth appearance.
      • Because of the capsule, the bacteria can evade the immune system and cause disease .
      • Rough strain (R): Does not produce a capsule, rough appearance.
      • Not pathogenic, doesn’t cause disease .
    • Frederick Griffith’s Experiments (1928)
    • Griffith treated his mice with the following bacterial preparations:
    • Treatment Outcome
    • 1. Live rough Mouse lives
    • 2. Live smooth Mouse dies
    • 3. Heat killed smooth Mouse lives
    • 4. Heat killed smooth + live rough Mouse dies
    • To his surprise, he did not get the expected results in the last treatment (#4). The mice got sick and died. Additionally, he recovered smooth bacteria from these mice.
  • Griffith’s Experiments: Transformation of Harmless Bacteria into Deadly Bacteria
    • Frederick Griffith’s Experiments (1928)
    • Why were smooth bacteria present in the last group of mice (live rough + dead smooth)?
      • Griffith concluded that the genetic instructions had been transferred to the rough bacteria, from the dead smooth bacteria.
      • He called this transformation .
      • However, he was unable to identify what type of molecule was responsible for transformation .
    • Hershey and Chase Experiments (1952):
    • Proofed that DNA rather than Protein carries the hereditary
    • information of life.
    • E. Coli a bacteria. Bacteriophages is a virus that infects
    • bacteria. It contains only a protein coat ( capsid ) and DNA.
    • They wanted to find out whether the protein or DNA carried the genetic instructions to make more viruses.
    • Their experiment
    • They labeled either the viral proteins or DNA:
      • Protein capsid: Labeled with radioactive sulfur ( 35 S)
      • DNA: Labeled with radioactive phosphorus ( 32 P)
    • Radioactive labeled viruses were used to infect cells.
  • Bacteriophages Are Viruses that Infect Bacteria
  • Either Bacteriophage DNA or Proteins Can be Labeled with Radioactive Elements
  • Hershey Chase Experiment: DNA is Genetic Material
    • Hershey and Chase Experiments (1952):
    • Bacterial cells that were infected with the two types of bacteriophage, and examined.
    • Results :
      • Labeled viral proteins did not enter infected bacteria
      • Labeled viral DNA did enter bacteria during viral infection.
    • Conclusion :
      • DNA is the molecule that carries the genetic information to make new viruses!!!!
    • DNA Nucleotide
    • A DNA nucleotide is composed of three parts:
    • 1. A phosphate group
    • 2. A deoxyribose (5-carbon sugar) molecule
    • 3. A nitrogenous base of either adenine, thymine, guanine, or cytosine
  • Nucleotide as part of DNA
    • Rosalind Franklin, James Watson, and Francis Crick 1953 Nature )
      • Rosalind Franklin : X-ray crystallography of DNA, trying to determine the structure of the molecule. Franklin’s work laid the foundation for Watson and Crick. Died of cancer in 1958.
      • James Watson and Francis Crick : Determined the exact three dimensional structure of DNA as a double helix held together by H bonds . Won 1962 Nobel Prize.
      • DNA is an antiparallel double helix: 5’ end of one strand is paired to 3’ end of other strand.
        • A & T and G & C are paired up by hydrogen bonds
        • Two strands are complementary to each other.
        • If you know sequence of one strand, can determine sequence of the other one.
  • DNA Structure: Double Helix Held Together by H Bonds
  • Chargaff’s Rule
    • DNA from any cell should have a 1:1 ratio of pyrimidine and purine bases.
    • The amount of guanine is equal to cytosine and the amount of adenine is equal to thymine.
  • Hydrogen Bonding Between Complementary Nucleotides Explains Chargaff’s Rule
    • How exactly does DNA replicate?
    • Findings :
      • Replication is carried out by DNA polymerase.
      • DNA strands unzip and each one acts as a template for the formation of a new strand.
      • Nucleotides line up along template strand in accordance with base pairing rules .
      • Enzymes link the nucleotides together to form new DNA strands.
  • DNA Replication
  • DNA Replication: Leading Strand is Made Continuously; Lagging Strand is Made in Fragments
    • DNA Genotype is Expressed as Protein
      • Gene
      • Segment of DNA that codes for a protein or RNA product.
      • Fundamental unit of heredity.
      • DNA sequences specify order of amino acids in
      • protein; but do not produce protein directly.
      • Proteins are crucial to cell activity
            • Cell movement
            • Oxygen and carbon dioxide transport
            • Active transport across membranes
            • Cell division
            • Enzymatic reactions
  • RNA (Ribonucleic acid)
    • RNA is the molecule responsible for copying DNA.
    • RNA is a nucleic acid, like DNA, composed of nucleotide building blocks.
    • There are three major differences between the structure of DNA and RNA:
    • 1. In RNA, ribose is substituted for deoxyribose.
    • 2. uracil (U) is substituted for thymine (T)
    • 3. RNA consists of only a single strand of
    • nucleotides.
    • Flow of Genetic Information in the Cell
      • DNA does not produce protein directly. DNA the genetic blueprint do not leave nucleus. It requires two steps
        • Transcription : RNA copies of DNA (a gene), the RNA copy is called messenger RNA or mRNA .
        • Translation : Process in which a protein is made from instructions contained in messenger RNA .
      • *Translation is carried out by ribosomes in the cytoplasm or rough E.R.
    • Flow of Genetic Information in the Cell
      • Transcription Translation
      • DNA m RNA Protein
  • Transcription and Translation Occur in Different Parts of the Cells
    • RNA is made by Transcription
      • Occurs in the cell’s nucleus.
      • Transcription is carried out by RNA polymerase .
      • A single strand of DNA (“ coding strand ”) serves as the template for RNA synthesis.
      • RNA nucleotides are matched to complementary DNA nucleotides in 5’ to 3’ direction.
      • RNA contains uracil (U) instead of thymine (T).
      • mRNA transcript leaves nucleus through nuclear pores and goes to cytoplasm.
  • Transcription of a Gene
    • Proteins are made by Translation
      • Occurs on ribosomes in the cell’s cytoplasm or rough ER.
      • Translation is a carried out by several types of RNA molecules:
        • 1. Messenger RNA (mRNA)
        • 2. Transfer RNA (tRNA)
        • 3. Ribosomal RNA (rRNA)
    • Necessary Components for Translation:
        • 1. Messenger RNA (mRNA): Encodes for a specific protein sequence.
        • Variable length (depending on protein size)
        • Information is read in triplets ( codons )
        • 64 possible codons (4 x 4 x 4 = 64 = 4 3 )
          • 61 codons specify amino acids
          • 3 codons are termination signals .
  • mRNA is complementary to DNA and read in triplets (codons)
    • Necessary Components for Translation:
        • 2. Transfer RNA (tRNA): Brings one amino acid at a time to the growing polypeptide chain .
        • Small molecule (70 to 90 nucleotides)
        • Forms a cloverl eaf structure
        • Anticodon : Base pairs to mRNA codon during translation.
        • Amino acid binding site : At 3’ end of molecule.
  • Transfer RNA (tRNA) Carries Amino Acids to the Growing Polypeptide Chain
  • The anticodon of tRNA matches the codon of the mRNA.
  • Over View of Protein Synthesis
    • Necessary Components for Translation:
        • 3. Ribosomal RNA (rRNA)
        • Ribosome is site of protein synthesis.
        • Facilitates coupling of mRNA to tRNA
  • Ribosome is the Site of Translation
    • STEPS OF TRANSLATION: ( Also referred to as Protein Synthesis)
    • In the cytoplasm, the mRNA becomes associated with a ribosome.
    • Amino acids in the cytoplasm are “picked-up” by molecules of transfer RNA (tRNA).
    • Each codon on the mRNA bonds with a corresponding anticodon on a tRNA, which carries a specific amino acid.
    • These amino acids are joined together by peptide bonds.
    • The resulting chain of amino acids is a polypeptide
  • Translation: Initiation at Start Codon
  • Translation: During Elongation one Amino Acid is Added at a Time
  • Elongation: Ribosome Travels Down mRNA, Adding One Amino Acid at a Time
  • Termination: Once Stop Codon is Reached, Complex Disassembles
    • Genetic Code
      • Twenty amino acids are found in proteins
      • Four nucleotides are found in RNA and DNA
      • How can nucleic acids with only 4 bases encode proteins with 20 amino acids?
      • Each amino acid is encoded by more than one nucleotide
        • If 1 base = 1 amino acid, Can only determine 4 amino acids
        • If 2 bases = 1 amino acid, Can only determine 16 amino acids
        • If 3 bases (Codon)= 1 amino acid, Can determine 64 amino acids
      • Genetic code is redundant , more than one codon per several amino acids.
      • Genetic code is universal
  • Universal Genetic Code
    • Mutations are permanent changes in DNA
      • DNA replication is never 100% accurate
      • Bases may be inserted, deleted, or mismatched during replication.
      • Mutations: Any mistakes that cause changes in the nucleotide sequence of DNA.
      • Mutations may be either harmful, beneficial, or have no effect on a cell or individual.
  • Biotechnology
  • Karyotypes
    • Pictures of chromosomes, cut out and placed in order of size and location of centromere. Placed in homologous pairs.
    Normal male karyotype
  • Can show chromosomal problems:
    • Down Syndrome: Trisomy 21 ; mental retardation, many physical differences
  • Turner Syndrome – female, small stature, infertile
  • Klinefelter Syndrome – male, tall stature, infertile
  • These disorders result from NONDISJUNCTION
    • Failure of chromosomes to separate normally during meiosis –
      • Eggs or sperm get one too many chromosomes or one too few.
      • Ex – Down syndrome has one extra #21
      • Klinefelter has one extra X chromosome
      • Turner has one too few – only one X
  •  
  • Other Human Disorders
    • Sickle Cell Anemia –
      • autosomal recessive
      • Found most often among people of African ancestry
      • Blood cells sickle (change shape) when oxygen-deprived (exertion, increase in altitude)
      • Causes sickle cell event – pain and immobility
      • and death of tissue ( dangerous if in organ)
      • Treatment – hospitalization and oxygen
      • Carriers are resistant to malaria
  •  
  • Tay Sachs Disease
    • Autosomal recessive
    • Found most often among Jews of Mediterranean ancestry
    • Child born appearing normal, but fat builds up in brain and child dies by age 5
    • No treatment, no cure
  • Huntington Disease
    • Autosomal dominant
      • Symptoms do not appear until age 30-40.
      • Death takes about 5-10 years
      • No treatment, no cure – but there is a test to see if you have it before symptoms begin
      • Results in mental impairment and uncontrollable spastic movements
  • Phenylketonuria - PKU
    • Person can’t breakdown phenylalanine (one of the 20 amino acids)
    • OK when born, but if phenylalanine not restricted by diet, mental retardation will result, getting worse the longer phenylalanine is in the diet.
    • Diet prevents PKU
  • Deletions
    • Chromosome fragment breaks off and is lost
    • Cri du chat syndrome – mental retardation and many physical problems
  • Prenatal Tests to detect chromosomal problems:
    • Amniocentesis – removes a little amniotic fluid from around baby – fluid is then tested for abnormal proteins and the cell in it can be karyotyped.
    • Risk of miscarriage
  •  
  • Bioethical Dilemma
    • Once a prenatal diagnosis of a genetic disorder is made, what are the parents to do?
      • Do nothing and give birth to child with disorder
      • Abort embryo/fetus
    • Who should make the decision?
    • What should enter into making the decision?
  • Genetic Counseling
    • Genetic counselor:
      • educates the parents about the disorder,
      • tells them of their options without influencing their decision,
      • and tells them of the consequences of each option
  • Genetic Engineering
    • We can manipulate DNA and genes to alter organisms or make them produce a product we need.
            • Two ways:
            • Gene cloning
            • Organism cloning
    • Toolkit for DNA technology includes:
            • 1. Restriction enzymes
            • 2. Vectors
            • 3. Host organisms
  •  
  • 1. Restriction Enzymes
    • 1 st discovered in the 1960’s
    • They naturally occur in bacteria
    • Most recognize a short, specific nucleotide sequence (restriction site).
    • These enzymes cut DNA up into fragments at restriction sites.
    • When a restriction enzyme cuts DNA, there are ends that are exposed with no complement.
    • These overhangs are called sticky ends .
    • This creates a “hole” where you can insert a new piece of DNA from other sources.
    1. Restriction Enzymes
  •  
  • 1. Restriction Enzymes
    • The new piece of DNA with the insert is called a recombinant .
    • What carries this new DNA into the site?
    • Vectors.
  • 2. Vectors
    • Usually a virus or a bacterial plasmid.
    • A plasmid is a small, circular piece of DNA.
    • The virus will insert the gene on its own.
    • A plasmid will be taken up in bacteria through transformation.
  • Bacteria (Host)
    • Very diverse
    • No nucleus
    • Exhibit 3 different shapes
    Rod shape Spherical Helical
  • Recombinant DNA – DNA from two different sources joined together.
    • Cut the DNA and the plasmid using the same restriction enzyme (these enzymes recognize the same base sequences.
    • Insert the foreign DNA into the plasmid.
    • Place the plasmid into the bacterium
    • Allow the bacterium to reproduce – all future generations have the new DNA
    • Collect the product – it might be insulin or growth hormone, or some other molecule.
  • Plasmids
    • Are small DNA fragments, are known from almost all bacterial cells.
    • Plasmids carry between 2 and 30 genes. Some seem to have the ability to move in and out of the bacterial chromosome
  •  
    • Cloning and Biotechnology
      • Definition of Cloning —To make an exact genetic copy of; can be a gene, a cell, or an entire organism. In 1997, researcher Ian Wilmut at PPL Therapeutics cloned Dolly the sheep.
      • How…
  • a) A cell was taken from udder of adult sheep and grown in culture in a laboratory to create many daughter cells.
  • b) An egg was taken from another sheep, and its nucleus was removed.
    • c) The udder cell and the denucleated egg were fused by electricity, stimulating the egg to develop as if it had been fertilized using the diploid udder cell nucleus instead of the sperm and egg nuclei.
  •  
          • d) The embryo that developed was implanted into a surrogate mother sheep, and was born as Dolly, with the exact DNA from the original udder cell.
  •  
  •  
      • Benefits of cloning
        • 1. Creating livestock that contain human genes needed to treat genetic disorders like hemophilia.
        • 2. Creating livestock to serve as organ donors, or blood donors.
    • Visualizing DNA Sequences (DNA finger printing)
    • Gel Electrophoresis
    • So many bases, it is best to visualize them all in some organized fashion.
        • 1. Restriction enzymes can be used to cut the chromosomes from many cells into manageable pieces.
        • There will be a collection of fragmenting.
        • 3. The pieces can be ordered according to size using gel electrophoresis (moving the fragments in an electric field through a gel matrix). Smaller pieces are more easily in the gel than larger pieces.
        • 4. Dyes that bind DNA can then be used to visualize the fragments as bands that can be compared to reference DNA fragments of known size.
  •  
  •  
  • O.J. Simpson capital murder case,1/95-9/95
    • Odds of blood in Ford Bronco not being R. Goldman’s:
    • 6.5 billion to 1
    • Odds of blood on socks in bedroom not being N. Brown-Simpson’s:
    • 8.5 billion to 1
    • Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:
    • 21.5 billion to 1
    • Number of people on planet earth:
    • 6.1 billion
    • Odds of being struck by lightning in the U.S.:
    • 2.8 million to 1
    • Odds of winning the Illinois Big Game lottery:
    • 76 million to 1
    • Odds of getting killed driving to the gas station to buy a lottery ticket
    • 4.5 million to 1
    • Odds of seeing 3 albino deer at the same time:
    • 85 million to 1
    • Odds of having quintuplets:
    • 85 million to 1
    • Odds of being struck by a meteorite:
    • 10 trillion to 1
    • The Human Genome Project
      • A. Massive undertaking to locate and catalogue every bit of genetic information in the human genome.
      • B. Budget of $300 million in 1998
      • C. Limitations:
        • 1. Knowing all the sequence is not the same as knowing what all the genes do,
        • 2. Just a good reference point to start.
    • Uses of Biotechnology
      • A. Biopharmaceuticals: drugs produced from recombinant DNA technology
      • B. Human Gene Transfer—gene therapy, replacing defective gene with a working one to treat genetic conditions
        • 1. Insert gene into vectors which will allow it to be added to human cells.
        • 2. Best cells to infect are stem cells.
        • 3. Problems—specificity, triggering immune response, keeping cells producing the protein over generations.
      • C. Biotechnology and food:
        • 1. Boost milk production 25 percent in cows with bovine growth hormone made in bacteria.
        • 2. Fast-growing salmon:
      • C. Biotechnology and food:
        • 3. Genetically altered crops—First came to market in 1994, by 1998 there were 45 million acres in production. Two categories of genetic alterations:
          • a) Added genes for herbicide resistance
          • b) Added genes for killing pests: Figure 15.11 ( Bacillus thuringensis Bt toxin). Concerns about insects building resistance, plants passing genes to wild relatives, or inadvertently killing off beneficial insects like butterflies. (Interactive Activity 5)
    • Ethical Questions in Biotechnology
      • Five percent of the Human Genome Project is devoted to ethical ramifications
        • 1. Ethical to modify humans, how far should we go? Chickens without legs or eyes?
        • 2. Will biotech produce new harmful organisms?
        • 3. Are biotech diagnoses running far ahead of treatments?
        • 4. Genetic discrimination? Who can have access to this information?
  • Menu Appetizers Spiced Potatoes with Waxmoth genes Juice of Tomatoes with Flounder genes Entrée Blackened Catfish with Trout gene Scalloped Potatoes with Chicken gene Cornbread with Firefly gene Dessert Rice Pudding with Pea gene Beverage Milk from Bovine Growth Hormone (BGH) Supplemented Cows