The document provides an overview of microbial genetics and DNA manipulation techniques. It discusses [1] the central dogma of biology where DNA is transcribed into RNA and then translated into protein, [2] DNA structure including replication, transcription and translation, and [3] common genetic engineering techniques like using restriction enzymes to cut DNA and recombining DNA fragments into plasmids.

1. Microbial genetics
Bios 10115
February 18, 2010
Additional reading from your book is suggested-Ch 4 pp 61-80

2. “Central Dogma” (DNARNAProtein)
 Genetic information of all
cells is in DNA
 DNA is duplicated during
the process of DNA
Replication
 Transfer of genetic
information to RNA occurs
during Transcription
 RNA is converted to protein
during translation

3. DNA
 4 nucleotides
 GATC
 Nucleotides consist of
pentose sugar (5’ and 3’
positions), phosphate and
backbone

4.  Double stranded-held together
by hydrogen bonding-base
pairs
 A-T (2 H bonds)
 G-C (3 H bonds)
 Chromosome
 Threadlike Double helix of DNA
 Prokaryotes-Circular (most)
 Eukaryotes- linear
 Virus-circular or linear

5. Double Helix – ________ strands

6. DNA higher order structure
E. coli has 1.5mm of DNA

7. Figure 10.1a: An electron micrograph of an E. coli cell immediately after
disruption. The tangled mass is the organism’s DNA.
© H. Potter-D. Dressler/Visuals Unlimited.

8. Eukaryotic DNA higher order structure
“beads on a string”

9. Information transfer
 Replication
 DNA makes new (is the template for) DNA
 Transcription
 DNA is the template for RNA
 Translation
 RNA is the template for proteins
A template is a pattern that is faithfully copied

10. DNA replication
 New strand is made as
complement of template
strand
 Each daughter double
helix has one parental
strand and one newly
synthesized strand
 DNA replication always
occurs 5’-3’
 5’ Phosphate attaches
to 3’ hydroxyl using
DNA Polymerase

11. The DNA replication fork (E. coli)
Leading and lagging strands
DNA polymerases can only move in one direction

12. http://www.dnai.org/a/index.html
(animations of replication,
E. Coli replicates 1000 bases per second

13. Replication of circular DNA
Also see fig 10.2 of your book

14. RNA
 Ribose instead of deoxyribose
 Uracil instead of Thymine
 Single Stranded (except some viruses)
 Messenger (mRNA)
 Transfer (tRNA)
All transcribed from DNA
 Ribosomal (rRNA)

15. Transcription
 An RNA copy of DNA must be made first before
proteins can be synthesized
 Transcription requires the enzyme RNA Polymerase

16. Translation
 Translation is the
process of
synthesizing
protein from RNA
 Three types of
RNA involved

17. Figure 4.8: The translation process

18. The Genetic Code
 Codon-set of 3 nucleotides
that encodes an amino
acid
 Amino acid-building block
of protein
 Start Codon - AUG-
methionine
 Stop codon- terminator-
UAA, UAG, UGA

19. Figure 4.9: A summary view of protein synthesis

20. Open reading frames

21. Mutations- changes in DNA
Genotype -genetic info
 Always changed by a mutation
Phenotype -outward appearance
 May not change with mutation
Point mutations
 A single nucleotide has been
changed
 A single codon is changed
 May or may not change amino
acid sequence (silent mutation)

23. Mutations (cont.)
Frameshift mutations
 A deletion or insertion of
one or more bases
 Changes ORF
 Usually change
phenotype

25. Mutations (cont.)
 Spontaneous mutations
 Occur in the absence of any known agent
 Arise during DNA replication
 Bacterial genes have a relatively high
spontaneous generation rate
 Induced mutations
 Caused by mutagens

26. Mutagens
 Chemical
 Many different effects
at molecular level
 Base analogs
 Alkylating agents
 Deaminating agents
 Acridine derivatives
 Radiation
 Thymine dimers

27. Plasmids
Small, circular extra-chromosomal
DNA
Do not carry essential genes
May carry helpful genes
Replicate independently of
chromosome
Over 300 naturally occurring
plasmids have been isolated
from E. coli

29. Gene transfer
 Vertical gene transfer-parents to offspring
 Lateral (or horizontal) gene transfer-passing
of genes within a generation
 Transformation
 Transduction (virus mediated)
 Conjugation
http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/
Mondomedia/4hirez.mov

30. Also see figs 10.5-10.9 in your book

31. Figure 10.9a: Bacterial transformation
Free DNA taken up by cell
© National Library of Medicine

32. Genetic exchange-Transformation
Streptococcus pneumoniae (pneumococcus) +/- capsule
1928-pneumococcus expt (Griffith)
1944-transforming compound is DNA
1953-structure of DNA
Also fig 4.2 of your book

33. Transduction
 Transfer of DNA by a _____________

34. Conjugation: _______ transfer of plasmids
 Ability is encoded by
_________
 Donor cell produces ____
 New population can gain
plasmid very quickly

35. Manipulation of DNA and
Genetic engineering

36. Tools to manipulate DNA
 ___________________
Sequences usually palindromic
(enzymes)
 Derived from _______
 Protect bacteria from
_______________
 Recognize specific small
sequences of DNA and ___
_________ leaving
“sticky” (staggered) or
“blunt” (straight) ends
 Bacteria’s own DNA
_________ by methylation

37. Figure 10.10a: A restriction enzyme cuts through two strands of a DNA
molecule to produce two fragments.

38. Figure 10.10b: The recognition sites of several restriction enzymes

39. Recombinant DNA technology
 Recombinant DNA
 DNA from _________
 Transgenic or recombinant
organism
 Organism derived from
recombined DNA
 _______
 Self replicating DNA
(plasmid)

40. Figure 10.11A-B: Construction of a recombinant DNA molecule

41. Figure 10.11C-E: Construction of a recombinant DNA molecule

42. Figure 10.12A-C: Developing new products using genetic engineering

43. Figure 10.12D-G: Developing new products using genetic engineering