1. UNIT VII – MOLECULAR GENETICS
Big Campbell – Ch 18, 19, 20
Baby Campbell – Ch 10, 11, 12

2. UNIT OVERVIEW Microbial Genetics Gene Expression in Prokaryotes
Viruses
Bacteria
Gene Expression in Prokaryotes
Regulation of Gene Expression
DNA Technology
DNA Testing Techniques
PCR
Recombinant DNA
Extensions

3. I. MICROBIAL GENETICS – VIRUSES
Discovery of Viruses
First isolated by Ivanowsky in 1890s from infected tobacco leaves
Crystallized by Stanley in 1935 – proved viruses were not cells
Not organisms; correctly referred to as particles
Not capable of carrying out life processes without a host cell
Parasites

4. I. VIRUSES, cont

5. I. VIRUSES, cont Characteristics
Viral genome may be either single-stranded or double-stranded DNA or RNA.
Protein coat surrounding virus is known as a capsid made up of protein subunits called capsomeres.
Some viruses are also surrounded by a viral envelope
Typically derived from host cell membrane
Exception is Herpes virus, synthesized from nuclear envelope of host cell
Aid in attachment. Envelope glycoproteins bind to receptor molecules on host cell
Most viruses that infect animals have envelope

6. I. VIRUSES, cont
Bacteriophages
Defense Mechanisms

7. I. VIRUSES, cont - Reproduction

8. I. VIRUSES, cont - Reproduction
Viral Entrance into Host Cell

9. I. VIRUSES, cont - Reproduction
LYTIC CYCLE
1. Lytic Cycle – Results in death of host cell.

10. I. VIRUSES, cont - Reproduction
LYSOGENIC CYCLE

11. I. VIRUSES, cont - Retroviruses

12. I. VIRUSES, cont – Animal Viruses
Coronavirus
Filovirus
Herpesvirus
Herpes simplex I and II
Epstein-Barr virus
Varicella zoster

13. I. VIRUSES, cont – Animal Viruses
Influenza Virus
Papillomavirus
Rhinovirus

14. II. OTHER INFECTIOUS AGENTS
Viroids
Prions

15. III. MICROBIAL GENETICS - BACTERIA
Genetic Make-up of Bacteria
Prokaryotic
Single chromosome wrapped in much less protein than found in euks
DNA concentrated in region known as nucleoid
Reproduction
Reproduce asexually
Advantages of asexual reproduction
Disadvantages of asexual reproduction
Have developed mechanisms for genetic variability . . .

16. III. BACTERIA, cont Transformation
Ability to take up DNA from surrounding environment
Streptococcus pneumoniae
Used to introduce human genes into bacterial cells to produce human protein
Transduction
Use of viruses to carry bacterial genes from one host cell to another
Conjugation
“Bacterial sex” 
One-way transfer of a self-replication piece of DNA known as a plasmid
Donor (male) extends pilus
Pulls cells together
Cytoplasmic bridge forms
Plasmid is transferred

17. III. BACTERIA, cont A Closer Look at Plasmids
Bacterium’s ability to produce plasmids and form pili due to specific piece of DNA known as the F factor
F factor may be integrated into chromosome, or separate as a piece of plasmid
Contains an origin of replication
Copy of F factor may be transferred to recipient cell; allow recipient cell to become “male”
R Plasmids – Carry genes for enzymes that destroy antibiotics

18. III. BACTERIA, cont A Quick Look Back at Protein Synthesis in Euks

19. III. BACTERIA, cont Protein Synthesis Similar to euks
Different in that transcription & translation occur virtually simultaneously

20. IV. REGULATION OF GENE EXPRESSION IN PROKARYOTES
Important adaptation for bacteria
Two basic mechanisms for metabolic control
Regulation of Enzyme Activity
Feedback Inhibition
Regulation of Gene Expression
Operons

21. IV. PROKARYOTE GENE EXPRESSION, cont
Operon Model
Operon = Promoter + Operator + all genes required for a given metabolic pathway
Operon acts as a single transcription unit
Promoter → Binding site for RNA polymerase
Operator → “On-off” switch located either close to or within the promoter
Operator controls whether or not RNA polymerase can bind to the promoter region
Therefore operator determines whether operon genes are transcribed & translated

22. IV. PROKARYOTE GENE EXPRESSION, cont
Operon Control
Operon can be turned off by a protein known as a repressor
Repressor binds to operator and prevents attachment of RNA polymerase to promoter
Repressor is a protein controlled by a gene known as a regulatory gene in a different location on chromosome; not part of operon
Expressed continuously
Always a small supply of repressor protein present

23. IV. PROKARYOTE GENE EXPRESSION, cont
Types of Operons
Inducible Operons
Operons that are usually off; that is, not usually transcribed
Can be stimulated when a specific molecule interacts with regulatory protein
Example is the lac Operon
Repressible Operons
Transcription normally occurs
Can be inhibited when a specific molecule binds allosterically to regulatory protein
Example is the trp Operon

24. IV. PROKARYOTE GENE EXPRESSION, cont Inducible Operons
lac Operon
Regulates transcription of genes required for breakdown of lactose
Inducible – Typically off
Bacterium is metabolizing glucose, other carbs; lactose is not present

25. IV. PROKARYOTE GENE EXPRESSION, cont Inducible Operons
lac Operon, cont
When lactose is available, lactose itself binds with repressor; inactivates it by changing its shape
Repressor cannot bind to regulator
Therefore, RNA polymerase is able to bind to promoter; operon is “on”
3 enzymes required to metabolize lactose are synthesized

26. IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons
Tryptophan (trp) Operon
Operon controls production of 5 enzymes required to synthesize amino acid, tryptophan when it is not available to bacterium in surrounding
Operon normally on; repressor inactive

27. IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons
When tryptophan is present, it binds to the repressor of the trp operon, activating the repressor, and turning off enzyme production.
Tryptophan acts as a
co-repressor, a molecule that works with a repressor protein to switch an operon off.

28. IV. PROKARYOTE GENE EXPRESSION, cont Repressible Operons

29. IV. PROKARYOTE GENE EXPRESSION, cont
Positive Gene Regulation
In addition to repressors, some operons are also under the control of proteins known as activators
Essentially the opposite of repressors
They “turn up” an operon by making it easier for RNA polymerase to bind to DNA, therefore facilitating transcription of operon genes
In the lac operon . . .
If both glucose and lactose are available, bacterium utilizes glucose until its supplies are depleted
As glucose ↓, concentration of cyclic AMP (cAMP) ↑
Increase in cAMP triggers release of activator protein known as CAP; CAP binds to promoter, facilitates binding of RNA polymerase to promoter of operon to enhance synthesis of enzymes of lac operon
When glucose concentration is high, decrease in cAMP results in decrease in CAP → RNA polymerase has very low affinity for lac operon promoter so lactose metabolism does not occur

30. IV. PROKARYOTE GENE EXPRESSION, cont

31. DNA TECHNOLOGY & GENOMICS

32. V. TECHNIQUES IN DNA TECHNOLOGY
Restriction Enzymes
Used by bacteria to “chop up” viral DNA
Bacterial DNA protected by _________
Very specific
Each enzyme recognizes a particular nucleotide sequence
Called a restriction sequence or restriction site
Palindromic
Cuts made at specific points
May create “sticky ends”
Used in gel electrophoresis
Also used to form recombinant DNA
Fragments may be pasted together with DNA ligase to form recombinant DNA

33. V. TECHNIQUES, cont Polymerase Chain Reaction (PCR)
In vitro method of amplifying small amounts of DNA
DNA is heated to separate the double helix.
Mixture is allowed to cool, DNA primers attach to target
Heat-stable polymerase is used to extend the primers in the 5’–3’ direction.

34. V. TECHNIQUES, cont Gel Electrophoresis
Separates DNA fragments based on size
Restriction fragment analysis
DNA treated with restriction enzymes
Resulting fragments migrate based on size
Produce a pattern characteristic of original DNA and restriction enzyme used

35. V. TECHNIQUES, cont Southern Blotting Northern Blotting
Designed by Dr. Southern
Detects particular DNA sequences
Northern Blotting
Detects particular mRNA sequences
Western Blotting
Used to detect proteins

36. VI. EXTENSIONS IN DNA TECHNOLOGY
Recombinant DNA
DNA containing nucleotides from other sources
Process utilizes restriction enzymes that make jagged cuts in DNA; creates sticky ends
When DNA from different sources treated with same restriction enzyme, sticky ends “mix & match”
Often use reporter genes to determine success; for example, ampicillin resistance

37. VI. EXTENSIONS, cont cDNA - complementary DNA
Procedure for “cloning DNA” that uses mRNA, reverse transcriptase
STRs – short tandem repeats
Short segments of DNA that are highly repetitive, polymorphic
Repeat patterns are inherited
Useful for identifying individuals
SNPs – single nucleotide polymorphisms
Single base-pair that shows variation in a significant % of population
SNPs that alter the fragment length following exposure to restriction enzymes called RFLPs (restriction fragment length polymorphisms)
Genetic markers

38. VI. EXTENSIONS, cont DNA Microarray Assays AKA DNA Chips
Test used to determine gene function, gene interactions
May be used to determine agressiveness of cancers, method of treatment, etc

39. VI. EXTENSIONS, cont Gene Cloning
Process of preparing multiple copies of a particular segment of DNA
Requires host and vector
Hosts
Initially done using bacterial cells
Now eukaryotic hosts are used
Yeast
Plants
Vector
Should have 4 characteristics
Ability to replicate independently of host cell DNA
Recognition sequence
Reporter gene
Small size
Possible vectors include
Plasmids
Viruses
YAC = Yeast Artificial Chromosome

40. VI. EXTENSIONS, cont Gene Cloning Use of plasmid as vector
Plasmid isolated from bacterial cell
Foreign DNA inserted into plasmid
Plasmid returned to bacterial cell; described as recombinant bacterium
Foreign gene is cloned as bacteria reproduce
Common bacterium used for plants is Agrobacterium tumefactiens

41. VI. EXTENSIONS, cont
A CLOSER LOOK AT GENE
CLONING

42. VI. EXTENSIONS, cont Reproductive Cloning Nuclear Transplantation
Process of using unfertilized egg cell & replacing nucleus with DNA
In 1997, scientists were able to produce first reproductive clone, “Dolly”, by culturing somatic cells in a nutrient-poor medium to de-differentiate them and force them back to totipotency.
Reproductive cloning in animals has enjoyed limited success.

43. VI. EXTENSIONS, cont Gene Silencing Knockout Genes
Use of genetic recombination to create an inactive , “knocked out” gene
Based on principal of homologous recombination
Mutated allele introduced into embryonic stem cells
Forms chimeras
Often used in mice to study gene expression
RNAi
Based on principal of microRNA
Small-interfering RNA (siRNA) synthesized complementary to mRNA
Base-pairing occurs
Translation is blocked
Has been used to block production of growth factors in certain cancers

44. VII. GENOMICS Human Genome Project
International government effort begun in 1990
Goals
identify all the approximately 20,000-25,000 genes in human DNA,
determine the sequences of the 3 billion chemical base pairs that make up human DNA,
store this information in databases,
improve tools for data analysis,
transfer related technologies to the private sector, and
address the ethical, legal, and social issues (ELSI) that may arise from the project.
Celera Genomics
Shotgun sequencing
Completed early and under-budget in 2003
Genomics has given rise to proteonomics