Molecular Genetics

Molecular Genetics

BACKGROUND:• 1860 - Gregor Mendel determined

patterns of inheritance

• 1868 Friedrich Miescher discovered material inside the cell nucleus (chromosomes) is half protein and half something else

• - other half later discovered to be DNA (deoxyribonucleic acid)

• 1902 - Walter Sutton= genetic material is found on chromosomes

Conclusion:• 1. Chromosomes are made up of DNA & Protein

• 2. Which one makes up the GENES?

Experiments to Determine DNA or Protein

• 1. Frederick Griffith (1928): was attempting to develop a vaccine against pneumonia. He never succeeded but did make some important discoveries concerning DNA .

Griffith’s Experiment

Took 2 strains of bacteria Streptococcus pnuemoniae; inject them into mice in 4 different experiments:

• #1) Bacteria Enclosed in a smooth mucous coat (smooth = S strain) = kill mice

• #2) Bacteria with Coat absent (rough = R strain)= mice live

• #3) Heated strain S bacteria = made harmless, mice lived

• #4) Mixed heated S strain with R strain = MICE DIED!!

Conclusion:

• Transformation had taken place. Transformation = process by which bacterial cells incorporate DNA from dead bacterial cells (transfer of genetic information). The question remains: Is DNA or protein portion of the chromosome responsible for transformation?

Avery, McCarty, & Macleod

• 2. Oswald Avery, Colin Macleod, Maclyn McCarty (1944):

• Strong evidence for DNA as the transforming principle.

• Used Enzymes: (repeated Griffith experiment)

• Use a Protein destroying enzyme = transformation still occurs

• Use a DNA “ “ = NO transformation!

Hershey & Chase

• 3. Martha Chase & Alfred Hershey (1952) : Proved DNA is the hereditary material

• Used a Bacteriophage = a virus that infects a bacteria cell; made of a DNA core & protein coat; attached radioactive labels (32P to DNA; 35S to Protein) in two different batches.

Viruses given time to attach to bacteria and inject their genetic material

Separated the mixture using a high speed centrifuge, this removes any viral material remaining on the outside

35S radioactivity found only in liquid 32P radioactivity found only in bacteria

ALL NEW VIRUSES produced in future generations

contained only radioactive 32P

• CONCLUSION: DNA and NOT protein must be the genetic material!

The Structure of a DNA Molecule

• Nucleotides = subunits of DNA; made up of 3 components:

• 1. 5 - Carbon Sugar molecule (deoxyribose)

• 2. Phosphate group

• 3. Nitrogen Base (4)

Nitrogenous Bases

• Purines

• - Adenine & Guanine• - Double ring structure

• Pyrimidines

- Cytosine & Thymine- Single ring structure

Hydrogen bonds hold the nitrogen bases together

Determining the Structure of a DNA Molecule

• Erwin Chargaff (1950) : discovered in cells that equal amounts of A & T and G & C always exist.

• Chargaff’s Rule: A=T ; C=G (Purine always bonded to a pyrimidine)


• Rosalind Franklin (1954) : used X-ray diffraction to determine that DNA is a long, thin molecule. She interpreted the shape of a DNA molecule to be in the shape of a helix (single coil)


• James Watson & Francis Crick (1962) : determined the structure of a DNA molecule to be in the shape of a Double Helix (twisted ladder)

DNA STRUCTURE

DNA molecule is made of COMPLEMENTARY strands:

• one strand : A T T G C A T

• Complement : T A A C G T A

DNA STRUCTURE

Twisted ladder structure:

• Sugar - Phosphate backbone = outside rails of the ladder, held together by strong covalent bonds

• Nitrogen Base Pairs = make up the inside rungs (steps) held together by weak hydrogen bonds

DNA Replication:How does DNA make a copy of itself before Mitosis?• Replication: process by which genetic

information gets copied such as during Interphase of the cell cycle

Involves separating “unzipping” the DNA molecule into 2 strands

Each strand serves as a template for making a new complementary strand

The process is SEMI CONSERVATIVE = each new molecule consists of one new and one old strand of DNA

the sequence of bases gets preserved

Steps in the process of Replication

• 1. Enzyme Helicase unwinds the DNA helix (1A)

• 2. A Y-shaped Replication Fork results (1B)

• 3. Single stranded DNA binding proteins prevent the strands from recombining (1C)

Steps in the Process of Replication (cont.)

• 4. Topoisomerase removes any twists or knots that form (1D)

• 5. RNA Primase initiates DNA replication at special nucleotide sequences called origins of replication using RNA Primers


• 6. DNA Polymerase attaches to the RNA primers and begins elongation = adding DNA nucleotides to the complement strand DNA polymerase moves in the 3’ 5’ direction along each template (3)


• 7. The Leading Complementary Strand ( 5’ 3’ ) is assembled continuously (4)

• 8. The Lagging Complementary Strand ( 3’ 5’ ) is assembled in short Okazaki fragments which are joined by DNA Ligase (5A, 5B)


• 9. RNA primers get replaced by DNA nucleotides

Mutations: any sequence of nucleotides that does not match the original DNA molecule from

which it was made

• Mutagen = anything that causes a mutation to occur (UV light, radiation, drugs, chemicals etc.)

DNA can “proof read” itself DNA polymerase often does this • Excision repair enzymes can fix mistakes

Types of Mutations

• Original DNA MESSAGE:• THE DOG RAN AND THE FOX DID TOO

• Dna is read by the cell 3 base letters (CODON) at a time, this is called a Reading Frame

Point (substitution) = an incorrect nucleotide

• THE HOG RAN AND THE FOX DID TOO

Deletion = missing nucleotide

• THE DOG RAN AND THE FOX DID TO

Insertion = additional nucleotide is added

• THE DOG RAA NAN DTH EFO XDI DTO O

• Frameshift mutation = reading frame is every 3 bases (Codon)

Duplication = section of nucleotides gets repeated

• THE DOG THE DOG THE DOG RAN AND THE FOX DID TOO

Inversion = sequence of nucleotides gets turned

around

• THE GOD RAN AND THE FOX DID TOO

Translocation = sequence of nucleotides gets moved to

another chromosome

• THE DOG RAN AND THE CAT HAS FUN ALL DAY

Protein Synthesis DNA in chromosomes contains genetic

instructions Those instructions regulate development, growth,

and metabolic activities. They also determine cell type and characteristics DNA controls the cell by using codes of

Polypeptides (Proteins)

• Polypeptides (Proteins) = enzymes that regulate chemical reactions or structural components

• GENE (genotype) = genetic information for a particular trait

• From a molecular viewpoint = traits are the end product of metabolic processes regulated by enzymes!

• The GENE is the DNA segment that codes for a particular polypeptide (protein) = One-gene-one-polypeptide hypothesis

Protein Synthesis = process by which enzymes and other proteins are manufactured

from the information contained in DNA

• Consists of three steps:– 1. Transcription = transfer of information

from a strand of DNA to a strand of RNA– 2. RNA Processing = modifies the RNA

molecule with deletions and additions– 3. Translation = processed RNA used to

assemble amino acids into a polypeptide

3 types of RNA are involved in the process:

• 1. Messenger RNA (mRNA) = carries protein building instructions out of the nucleus

• 2. Transfer RNA (tRNA) = carries amino acids to ribosomes

• 3. Ribosomal RNA (rRNA) = building blocks of ribosomes which coordinate the activities of mRNA and tRNA

How is RNA Different from DNA?

• RNA Is single stranded Bases = A, G, C and U (Uracil) replaces T Sugar = Ribose

CODON vs. ANTICODON

• Codon = a triplet group of 3 adjacent nucleotides in mRNA; codes for one specific amino acid

• Anticodon = a triplet group of 3 adjacent nucleotides in tRNA; complementary to mRNA

PROTEIN SYNTHESIS• Transcription:

• .Initiation = RNA polymerase attaches to promoter regions on DNA and begins to unzip the DNA into 2 strands. Promoter region contains the sequence T-A-T-A (called the TATA box)

• .Elongation = RNA nucleotides are assembled using one side of the DNA molecule as a template (5’ 3’)

• Termination = RNA polymerase reaches a special sequence of nucleotides that serve as a stop point; Usually AAAAAAA

PROTEIN SYNTHESIS

• Alterations take place before the mRNA leaves the nucleus:

A 5’ Cap is added to the 5’ end of the molecule5’ Cap = GTP (guanosine triphosphate)This provides stability to the mRNAProvides a point of attachment for the ribosome

(small unit)

Poly-A Tail added to the 3’ endA sequence of 150 to 200 adenine nucleotidesThe tail provides stabilityControls the movement of the mRNA across

the nuclear membraneSome mRNA segments get removedExons = sequences that express a code for a

proteinIntrons = intervening sequences that are

noncodingSnRNPs (small nuclear ribonucleoproteins) =

delete out the introns and splice the exons

PROTEIN SYNTHESIS

• Translation: • 1. Initiation = small ribosomal subunit attaches

to a special region near the 5’ end of the mRNA• 2. A tRNA with the anticodon UAC attaches to

the mRNA start codon AUG• 3. Large ribosomal subunit now attaches to the

mRNA

• 4. Elongation = tRNA’s deliver their amino acids to the growing polypeptide

• 5. Ribosome moves over to the next codon and repeats the process

• 6. Polypeptide chain elongates one amino acid at a time• 7. Termination = occurs when ribosome encounters a stop

codon

• The completed protein can now be used by the cell as a structural unit or as an enzyme!!

DNA Organization:

DNA packaged with proteins forms a matrix called Chromatin

During cell division DNA = compact Chromosomes

Transposons = segments of DNA able to move to new locations on the same chromosome or to a different chromosome altogether

Transposons have the effect of a mutation

Control of Gene Expression

Every cell in a human contains the exact same sequences of DNA

Cells obviously have different functions however

Gene expression is regulated by the activation then of only certain genes

• Example: gene regulation in E. coli (well understood)

• OPERONS = sequence of DNA that direct particular biosynthetic pathways.

4 Major Parts of an Operon

• 1. A regulatory gene produces a repressor protein that prevents gene expression by blocking the action of RNA polymerase

• 2. Promoter region of DNA attaches to RNA polymerase to begin transcription

• 3. Operator region blocks the action of RNA polymerase

• 4. Structural Genes contain DNA that codes for several related enzymes that direct the production of a product

Lac Operon = in E. coli controls the breakdown of

Lactose Lactose is required to turn on the operon that

codes for the enzymes that break down lactose.

If lactose is not present the enzymes are not made.

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Molecular Genetics