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UNIT C CHAP 18 CELL DIVISION, GENETICS, AND MOLECULAR GENETICS

Miescher
- Used the term nucleic acid – DNA was found to be slightly acidic, contained phosphorus
Levene
- Isolated nucleic acid into RNA and DNA
Griffith
- Studied 2 strains of bacterium on mice
- R bacteria had been transformed into pathogenic S bacteria by an unknown heritable
substance from dead S cells
- Transforming principle
Avery, MacLeod, and McCarty
- Identified that DNA was the agent responsible for transformation
Hershey and Chase
- Used viruses called bacteriophages which infected bacteria
- Radioactive labelling:
o Protein = sulfur
o DNA = phosphorus
o Centrifuge and sulfur on top, phosphorus on bottom
Chargaff
- Provided support for Levene that nucleotides were present in equal and characteristic
proportions
Rosalind Franklin
- X-ray scans of DNA
- Helped with double helix
Watson and Crick
- Double helix
Structure of DNA
- Four building blocks
o Nucleotides
o 5-carbon sugar (deoxyribose for DNA and ribose for RNA)
o Phosphate group (nucleic acid)
o Nitrogenous bases (AGCT)
 Purines (double ringed) = AG
 Pyrimidines (single ringed) = TC
 Uracil replaces thymine in RNA

DNA
- Generic DNA nucleotide:
o Phosphate on 5’ carbon
o Base on 1’ carbon
o Hydroxyl on 3’ carbon
- RNA nucleotide would have a hydroxyl off the 2’ carbon
- The difference between RNA and DNA is that DNA nucleotide has no oxygen
- A chain of nucleotides forms a single strand of DNA
- Nucleotide + nucleotide = phosphodiester bonds between phosphate and sugar groups:
which creates the “backbone” of DNA strand
- Bond between 5’ end with phosphate group and 3’ end with the -OH group
- Hydrogen bonds for between nitrogen bases
o 3 between GC
o 2 between AT
o Rungs of the ladder

Matching up nucleotides
- Purine always match up with pyrimidines
- Antiparallel
o Run 5’ – 3’ on one strand and 3’ – 5’ on complementary strand
o Very important for DNA replication and protein synthesis

RNA
- 3 main differences:
- Ribose instead of deoxyribose
- Uracil instead of thymine
- Single strande

Genes and the genome


- Gene – functional subunit of DNA, directs production of one or more polypeptides
- Genome – all the DNA carried in each cell of organism
- Chromosomes have different numbers of genes
- Human genome = 3 billion base pairs approx. 25000 genes

DNA replication
- S phase (replication of DNA) -> semi conservative replication (each new molecule of
DNA contains one original strand and one newly made strand)

Initiation, elongation, termination

Initiation:
- Replication origin
- Helicases: enzyme that bind to replication origin, break H bonds, creates replication
bubble and replication forks
- Single strand binding proteins stabilize DNA strands
- Single strands = templates for complementary base pairing to create molecules of DNA
- Enzymes that build DNA CANNOT start the process
- RNA strand (primer): synthesized by the enzyme primase and starts the process, about
5-10 nucleotides long
o Starts at 3’ end of the RNA primer – always goes in 5’-3’ direction
Elongation and termination
- DNA polymerase adds DNA nucleotides to primer
- Creates a complementary strand: A-T and G-C
- Elongation only takes place in the 5’ – 3’ direction!!! (adds nucleotides to the 3’ – OH of
previous nucleotide)

Leading and lagging strands:


- Antiparallel nature
- Both strands are built in the 5’ – 3’ direction
- Leading = continuously
- Lagging = replicated in Okazaki fragments
o Primase creates many primers
o DNA polymerase adds to primers and detaches when gets to next primer
o Primase jumps back into opening fork
o DNA ligase forms bonds between fragments

DNA polymerase
- Adds nucleotides in 5’ – 3’ direction
- Removes RNA primer fragments
- Proofreads (recognizes error and replaces incorrect nucleotides) -> only during
replication

Termination
- Replication machine = collection of polypeptides (enzymes and other proteins) and DNA
interacting at replication fork
- Replication machine comes apart
o Two strands of DNA rewind back into double helix structure

Telomeres
- Ends of chromosomes
- Specific nucleotide sequences that repeat
- Do not contain genes
- Protects genes from degradation
- Plays role in aging and preventing cancer
- Telomere shortening: the end Okazaki fragment cannot produce a new primer

Protein structure
- Frederick Sanger
o Proteins consist of amino acids
o Specific sequences of amino acids determine chemical properies of protein
o Proteins determine structure, function, and development of cells

Protein structure
- Primary protein (sequences of amino acids)
- Secondary (local folding of polypeptide)
- Tertiary (three dimensional looking pattern due to side chain interactions)
- Quaternary (protein consisting of more than one amino acid chain)

Proteins
- Combinations of 20 amino acids
- DNA determines the sequences of amino acids and controls the proteins the cells make
- One gene = one protein

Gene types
- Regulatory
o Regulates other genes, activates/stops other genes
o Creates proteins that regulate other genes
- Structural
o Produce proteins that are part of physical structures such as enzymes
- Oncogenes
o Normally directs cell growth
o P 53

Central dogma of gene expression:


- DNA -> transcription -> mRNA -> translation -> protein

The genetic code (see formula sheet)

- 3 bases = codon = 1 amino acid


- Genetic code is in mRNA NOT DNA
- Some codons do not code for amino acids, but rather intiating and terminating codons

Why 3 letter codons?


- 1 base only 4 combinations (ACGT)
- 2 bases only 16 combinations
- 3 bases = 64 different codes

Characteristics of the genetic code


1. Redundant (more than one codon can code for same amino acid)
2. Continuous (genetic code reads as a series of 3 letter codons without spaces or overlap,
knowing where to start and stop is essential)
3. It is nearly universal (almost true for all organisms)

RNA
- Ribose
- Uracil
- Single strand
Transcription

5’ – 3’ direction

Sense and anti-sense strands


- Sense strand = coding strand (segment of DNA running 5’ – 3’ , same sequences as
mRNA to be transcribed)
- Anti-sense strand = template strand (the one that runs 3’ – 5’)

Transcription
- mRNA builds itself in 5’ – 3’ direction off of the template/ anti-sense strand
- template strand is being “read” in the 3’ – 5’ direction
- RNA polymerase does the job of helicase, as well as the jobs of other enzymes
o Binds to promoter, unwinds DNA, initiates RNA synthesis

Translation – mRNA to polypeptide


- mRNA moves from nucleus to cytoplasm
- finds ribosomal subunits
- tRNA carries the amino acid
o synthesized from DNA
o single stranded RNA nucleotides
o one end of tRNA attaches to an amino acid while the other end has the anti-
codon to mRNA, complementary)

Ribosome
- rRNA + protein
- made in nucleoli
- Holds the mRNAs, tRNAs, and amino acids to assemble amino acids into new protein
-
- p site (binding site for the growing polypeptide chain)
- a site (amino acid binding site)
- e site (exit site for empty tRNAs)

Translation
- DNA instructions -> mRNA -> tRNA + amino acids -> proteins
- Order of codons on mRNA dictates order of tRNA and anticodons and amino acids
- Stops when a stop codon is present
- Start in P site, add in A site, and exits in E site
- Uses GTP (energy) for anticodon to bind to codon

Mutations
- Gene mutation
o A change in base sequences of organism’s DNA
o Chromosomal: large alterations that can affect structure and/or number of
chromosomes
o Gene or point: alteration of base sequence of individual genes
- Usually DNA repair enzymes detect and fix, in case of failure is a permanent mutation

Passing on mutations
- All mutations are inheritable
o Copied during DNA replication and passed on to daughter cells
- Not all mutations are passed on to future generations
o Mutation must be in germ cell of gamete to affect offspring
- Body cell mutations (somatic)
o Cancer
- Reproductive cell mutations -> germ line mutations
o Mutation passed to offspring

Types of mutations
- Point: affects one of a few nucleotides, can be substitution, deletion, or insertion
- Frame shift: insertion or deletion of one of more nucleotides (and then every single set
or a codon is different and codons all shifted are now different)
- Silent: no effect on cell’s proteins or metabolism
- Missense: altered protein (difference sequence of amino acids) (e.g. sickle cell anemia)
- Nonsense: premature stop codon

Other mutations
- Chromosomal: translocation (relocation of groups of bases from one part of genome to
another)
- Inversions
o Section of chromosome gets reversed, gene may be disrupted

Causes of mutations
- Spontaneous mutations
o Occurs naturally in cells
o Incorrect base pairing by DNA polymerase, and other errors during replication or
protein synthesis
- Induced
o Caused by agent outside of cell
o Mutagens are substances

Physical mutagens
- Cause physical changes in structure of DNA
- Tear through DNA and cause point mutations or loss of large portions of DNA
o X-rays, UV rays

Chemical Mutagens
- Molecule that enters nucleus of cell and induce mutations by reacting chemically with
DNA
o May insert itself in DNA causing frameshift or substitution
o May have base pairing properties similar to another base
 E.g. carcinogens

DNA repair
- Double strand provides mechanism for repair
- DNA polymerase
- Nucleases

Genetic variation
- Mutations ca accumulate over time
- Use these to track evolution of different species
- Endosymbiont theory: the thought that organelles used to be their own organisms that
were engulfed by a larger one

Genetic variation and evolution


- Same mutations in mtDNA -> likely have recent maternal ancestor
- 2 theories:
o Multiregional: homo sapiens evolved around the world
o Monogenesis: modern ethnic groups are descendants of second migration of
homo sapiens, traced back to Africa (holds true)

Genetic recombination and engineering


- Genetic recombination – new arrangement of genes
- Naturally occur between homologous chromosomes
- Artificial recombination
o DNA from different sources -> recombinant DNA
o Transgenics - gene from one species spliced into another
- GMO

Restriction endonucleases – cutting out DNA


- Enzymes that catalyze cleavage of DNA at specific nucleotide sequences (restriction
enzyme)
- Cut out DNA molecule in middle
- Recognize short sequences of nucleotides – target sequence
- Restriction site – point in target sequence that is cut
- Glue together with ligase
o Mass produce bacterial DNA, e.g. insulin, hGH

Plasmid insertion
- Produces artificial recombinant DNA
- E.g. insulin production in bacteria
Gel electrophoresis
- Separates nucleic acids or proteins according to mass and charge
- Smaller fragments move further

Gel electrophoresis – DNA finger prints

Polymerase chain reaction PCR – mass producing DNA


- Uses Taq polymerase from bacterium Thermos aquaticus
- Functions at high temperatures
- PCR requirements:
o Taq polymerase
o DNA to copy
o Large amounts of 4 deoxyribonucleotides
o Short DNA primers

Genetic engineering applications


- To get mass production of proteins
- Gene therapy
- Improving plant and animal stock

Biotechnology products
- Medicinal bacteria
- Cloned and transgenic animals
- Transgenic plants
o Golden rice

Diagnosis and treatment of genetic disorders