Lake Michigan College

Principles of Biology I (111)

[DNA Replication] [Transcription & Translation] [Viral genetics] [Bacterial genetics] [DNA Technology ]

Human Genome Project educational site
Talking Glossary of Genetic Terms - National Human Genome Research Institute

MOLECULAR GENETICS (DNA & RNA)

DNA Structure and Function

Primer on Molecular Genetics Department of Energy
Milestones in DNA History
Evidence for DNA as the genetic structure
- Transformation of non virulent Rough strain of Streptococcus to virulent Smooth strain. [F. Griffith, 1928]
- Transformation due to transfer of DNA [O. Avery, 1944]
- Phage DNA entered the host cell [ Hershey & Chase Experiment, 1952]
Discovery of structure of DNA
- Chargaff's Base pairing rule
- Rosalind Franklin's research led to discovery of DNA double helix structure
- DNA [Watson & Crick Model, 1953] is double helix
- The 2 DNA strands are antiparallel [5'--->3' pairs to 3'<---5']
See interactive DNA structure also DNA Gif Animation]
DNA replication
- Semiconservative model [ Meselson & Stahl Experiment]
- Antiparallel DNA strands have 2 different modes of replication
  1. Continuous replication [leading strand]
  2. Discontinuous replication in fragments [lagging strand]
- DNA replication enzymes include
  - Helicase - unzips double helix between base pairs
  - DNA polymerases - extends polymer from 3' end
  - Primase - starts replication with short RNA primer
  - Ligase - joins discontinuous fragments with sugar phosphate bonds
- Replication fork [one end of double helix unwinds]
  - Leading strand (template 3' --> 5') replication continuously
  - Lagging strand (template 5' --> 3') replication in fragments
- See animated interactive DNA replication
- Telomers protect the tips of chromosomes during replication
  - See Telomers animated
  - primer at the 5' end of leading strand cannot be replaced by DNA
  - last primer on end of lagging strand cannot be replaced by DNA
  - After each replication of linear DNA, the daughter strands have a reduced number of base pairs at their ends.
  - Telomerase using RNA template can extend the telomer [another animation]

Central Dogma of Biology DNA --> RNA --> Protein

Genes (DNA) to protein synthesis requires two processes [transcription & translation]

Comparing steps in prokaryotic and eukaryotic cells.

Transcription of mRNA occurs in the nucleus on DNA template.
RNA polymerase binds to promoter on single strand (sense strand) of DNA
Code is transfered from DNA template to RNA by base pairing rule
See Transcription animation
Posttranscriptional mechanism: eukaryotic RNA is modified before leaving nucleus
- RNA splicing - introns are excised and exons joined
- Modified Guanine added as 5' cap
- Poly A tail added to 3' end
- Edited mRNA tansported from nucleus to cytoplasm
Genetic code
- each 3 base sequence (codon) codes for an amino acid
- 61 codons for 20 amino acids
- AUG is start codon + 3 "stop" codons
- reading the mRNA codons
Translation of mRNA on ribosome produces protein
See Translation animation Excellent!
- Correct amino acid attached to correct tRNA by tRNA synthetase
- Each tRNA has anticodon that has complementary bases to mRNA codon
- Transfer RNA (tRNA) transports amino acids to the ribosome to form polypeptides
- Three stages to translation [animation] [interactive animation]
  1. Initiation [ribosome comes together on start codon and first 2 tRNAs align their anticodon on mRNA codon and thereby position their amino acids]
  2. Elongation [as ribosome moves to each codon, another tRNA enters to pair its anticodon on mRNA codon, and an amino acid is added to growing polypeptide.]
  3. Termination [when ribosome moves to stop code, ribosome releases polypeptide]
This program transcribes and translates any DNA sequence you enter.

Mutations are base changes. See how a mutation effects amino acid sequence

MICROBIAL GENETICS

Virus ultrastructure - See examples of viruses
- Genome is entire set of genes (DNA or RNA)
- Capsid is protein coat that excloses viral genome.
- Envelope is host membrane that enclose capsid of some viruses.
Bacteriophage attach, penetrate and reproduce in bacterium host
Phages exhibit two reproductive cycles
1. Lytic [virulent phage lyses host cell] see animation
2. Lysogenic [temperate phage DNA is incorporated into host genome as prophage]
  - When host bacterium replicates, prophage is replicated
  - Prophage can produce active phage which lyses host
  - See animation
Reproductive cycles of animal viruses
- Viruses can only reproduce within host cell
- DNA viruses
  - Herpes viruses
- RNA viruses
  - RNA serves as mRNA [Picornavirus]
  - RNA is template for mRNA [influenza viruses]
  - See life cycle of HIV and interactive animation
  - RNA serves as template for DNA [Retrovirus has reverse transcriptase]
Viroids and Prions are smaller and simpler than viruses
- Viroids are small circular RNAs and are pathogenic in plants
- Prions are infectious proteins that cause brain disease
  - mad cow disease
  - scrapie in sheep
  - Creutzfeldt-Jakob in humans

Prokaryotic chromosome is single loop of DNA

Chomosome replication is bidirectional
Some prokayotes in addition to chromosome also possess plasmid (small circle of DNA).
Prokaryotic genetic recombination (3 methods of genetic transfer)
1. Transformation - Competent bacteria can take in external DNA fragments
  - See animation
2. Transduction - DNA fragment transferred between bacteria hosts by bacteriophage
  - Generalized -any fragment of host DNA incorporated in phage genome
  - Specialized - requires infection by temperate phage - Host DNA adjacent to prophage is incorporated into phage genome
3. Conjugation - DNA transferred from one bacteria to another through sex pili
  - F+ transfer copy of plasmid to F-
  - In Hfr plasmid incoporated in chromosome and only first part of chromosome is transferred to F-.
  - See conjugation F+and Hfr
  - Genetic mapping of Hfr strains by interrupting conjugation
Transposons are genes capable of moving from one site to another
- Mobile DNA sequence that includes a gene for enzyme transposase and insertion sequences on ends.
- Genetic transposition (transposons) discovered in corn by Barbara McClintock
- Isertion sequences (IS) carry one gene
  - Mobile genetic elements with gene for transposase
  - Transposition of IS
- Transposons reduce the expression of eye pigment in fruit flies
Control of gene expression Operon model
- Real Player animation
- Animation of The LAC Operon [Inducible System]
- The LAC Operon the movie
- Animation of The TRP Operon [normally on] [repressible system]
- Animation of The TRP Operon [off]
Problems on Molecular genetics of prokaryotes

DNA TECHNOLOGY

Recombinant DNA Technology - isolate, cut and recombine DNA from different species

Restriction enzymes are used to cut DNA at unique restriction sites
- See examples of restriction enzymes and target sequences cut
- Enzymes recognize restrictions sites or target DNA sequences (palindromes)
- Cut DNA fragments have "sticky ends" (extension of single strands)
- "Sticky ends" created by same restriction enzyme will base pair
- DNA ligase joins sugar to phosphate in DNA fragments whose sticky ends have base paired
Cloning DNA
- Cloning Vectors (plasmids or viruses), are vehicles that transport genes into host cells for cloning.
- Inserting DNA into a plasmid [joining with ligase]
- Plasmid mixed with host bacteria and enters cell by transformation
- Screen bacteria to find cells with correct gene for cloning
- Genes cloned as host cell divides.
- Cloning with bacteria and plasmid
Sources of DNA for cloning
- Reverse transcriptase to make cDNA
- Genomic Libraries - bacteria or viruses that contain cloned DNA fragments of an organism's genome.
  - Constructing a genomic library of a yeast
  - Construction of a Human Genomic Library
Transgenic organisms have foreign genes inserted into their genome.
- The application of transgenic plants and animals in agriculture
- Transgenic plants animation demo
- Transgenic mice
DNA Fingerprinting
- Paternity case: Can you determine the father?
- Crime scene: DNA Forensics
- DNA fingerprints of twins
- Gel electrophoresis separates DNA fragments
- Transfer DNA fragments to nylon sheet with Southern Blot.
- Denature DNA and hybidize with labeled probe
Polymerase Chain Reaction PCR replicates large quantities of a DNA fragment.
- Robotic cycler repeats cycles of heating and cooling of DNA samples
- Denature with heat and cool to anneal primer and extend new strand
- PCR animation
- Kary Mullis wins Nobel in chemistry for invention of PCR
DNA sequencing [determing exact order of bases]
- Dideoxy method or Sanger Method - use four samples
  - Unknown single stranded DNA + labeled primer to each sample
  - Add all four normal deoxynuleotides
  - To each sample add one of the four dideoxynucleotides (ddNTPs).
  - When a dideoxynucleotide (ddNTP) is incorporated in polymer, the chain is terminated
  - Resulting DNA chain length products in each sample indicate the occurence of specific bases in the sequence
  - Separate the four samples by gel electrophoresis [one base differences can be separated]
  - Read the base sequence in the gels
- Human Genome project [sequencing 30,000 genes and areas controlling them]
Visit the DNA Learning Center at Cold Springs Harbor Laboratory
- An excellent Polymerase Chain Reaction Animation
- Other Biotechnology Animations from the DNA Learning Center

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Talking Glossary of Genetic Terms

Online Biology Book

Online Biology Glossary