Molecular Biotechnology

1
Molecular Biotechnology

• Don Seto
• Department of Bioinformatics and Computational
  Biology
• College of Science
• George Mason University _at_Manassas, VA USA
• dseto_at_gmu.edu [phone]
• http//binf.gmu.edu/dseto/molecbiotec
• Course Text Molecular Biotechnology Principles
  and Applications of Recombinant DNA.
• BR Glick and JJ Pasternak. ASM., 5th edition
  (2003)
• Format
• 12 x2.5hrs lecture 1.5hr, discussion 0.5hr,
  student oral pres 0.5hr
• gtthree exams _at_100pts ea
• gttwo student oral presentations _at_12.5pts ea
• gtthree homework _at_25pts ea, question format from
  exam
• gttotal 400pts
• Grading structure 85A, 70B, 55C

2
Escaping superstition

• Flammarion Woodcut, 18th cent.? (1888 reference)
• A medieval missionary....found the point where
  heaven and Earth meet
• heliocentrism
• flat earth
• gravity
• creationism

CPurrington (Swarthmore)
3
CPurrington (Swarthmore)
4

Who should be interested in biology (science) and
why

• Feb 14, 2006 Alan Leschner. CEO, AAAS
• on Earth Sky Radio program (Deborah Byrd and
  Joel Block)
• A new tension between science and people?
• Last 4-5 yrs, the relationship between science
  and society has seemingly deteriorated.
• Encroachment or abutment of scientific findings
  onto issues of either core human moral values or
  issues of economic values.
• For the scientific community, theres a feeling
  of greater disengagement between science and
  society.
• Rising tide of fundamentalism that is an overlay
  to their (the publics) views of science.
• Scientists believe that scientific truths have
  value.
• If the purpose of science is to tell us about
  the natural world and the way it actually is, we
  shouldnt be saying, tell us about the nature of
  the world as long as we agree with it and so long
  as it doesnt make us uncomfortable.

earthsky.org sciencedaily.com
5

Policies and attitudes affect you more than you
know ex., Cervical cancer can be caused by a
common virus

• http//tell-someone.hpv.com/ (Merck 2006)
• Human papillomavirus (HPV)
• One of most common STI gt100 different types
• CDC estimates 20 million Americans have HPV 06
• NIH 5.5 million new cases reported every year
• Cervical cancer- kills 3,500 US women every year
• Genital warts
• Cancer of the vagina, vulva, penis and anus
• ACS (06) estimates 2,420 new cases of vaginal
  cancer in US 3,740 vulvar cancer 4,660 anal
  cancer
• Recurrent respiratory papillomatosis (RRP)
• (noncancerous tumors in throat)
• Abnormal Pap tests

Complex to simpler
6

....not required to follow the recommendations of
its panel of outside experts, but...

• Merck
• Developed HPV vaccine
• Gardasil against four HPVs
• Two responsible for cervical cancer
• Two responsible for 90 genital warts
• Under FDA review
• Conservative groups opposed on the grounds that
  it would encourage promiscuity.
• FDA panel of outside experts met 5/18/06 to
  recommend to FDA recommended approval, but
• FDA is not required to follow the recommendation,
  but usually does.

• SC Harrison, EMBO J 02
• Structure and Function of Macromolecular
• Assemblies
• Hybrid technique of EM and X-ray crys data
• Generates atomic model
• Spatial relationships of coat proteins affect
• host immunological response
• Vaccine development

7

Life.......is AMAZING! (for the beauty of
knowledge)

• Chemistry/Biology
• Cell Biology/Biochemistry/Genetics
• Molecular Biology
• Genomics
• Bioinformatics/ Computational Biology
• Systems Biology
• nothing is really new.....
• There is grandeur in this view of life.....
• most beautiful and most wonderful have been, and
  are being, evolved.

8
Biology Science in Transition
Genetic Engineering DNA Sequencing
Human Genome Project
Genetic Fingerprinting
H. Influenzae C. elegans
Human Genome
PCR
DNA Chips

• Biology is changing rapidly from a data-poor to a
  data-rich science
• New Fields
• Bioinformatics
• Computational Biology

JGref04
Growth of Genome Databases
9
Lecture 1

• Intro to life
• .cells
• chemistry, biochemistry
• molecular biotechnology

10

• Cells come in all shapes, sizes, functions

11
(No Transcript)
12
(No Transcript)
13
(No Transcript)
14
(No Transcript)
15
Prokaryotes vs eukaryotes
16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20

• But, single cellular does not mean simple.

21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24

• Cells and the world are divided into two types
• ..exception!

25
(No Transcript)
26
Exception- What is a virus?

• Mimi virus
• LaScola, B......Raoult, D. Science 299 2033
  (03).
• 1992 influenzae outbreak
• 1.2Mb
• 1260 genes
• 50 not seen in viruses previously

27

• To make sense of it, need
• reductionism
• tools
• imagination

28
What is life? how is life molecules of life
29
metabolism centered around ATP
30
What is life? sorta...
31
Simple structures
32
Complex structures
33
Complicated processes
34
Mutations Phenotypes and the molecular basis
of....
Good, bad, indifferent sickle cell anemia gene
Hb-? high-altitude variant Hb-? Germ-line vs
somatic
35
The first science technology drives research
drives technology dri...
36
Cell biology Size, shape, location of parts
37
Biochemistry chemistry of living systems
Genetics heritableness of living systems
38
Genomics Structure and consequences of genomes
39
Developmental biology......
40
Model organisms Cellular biology,
biochemistry... molecular biology
41
(No Transcript)
42
Jaenisch, R.Nat. Genet. 2001 27 327-331
43
Rett syndrome

• X-linked, primarily in females.
• Neuropsychiatric condition
• developmental disorder healthy until 6-18
  months
• autistic-like symptoms
• seizures/ mental retardation
• scoliosis
• muscle wasting
• Mecp2 (methyl-CpG-binding protein)
• X-linked nuclear protein, binding to methylated
  DNA
• general txn repressor, by assoc with
  chromatin-remodeling complexes
• high level expression in postnatal brain
• Mecp2-null mice
• normal till 5 weeks
• develop disease
• death at 6-12 weeks
• rescueable

44
Genomics and bioinformatics Dog genome

• Lindblad-Toh, K.......Lander, ES
• Domesticated ca. 15,000 years ago from gray
  wolves
• 2.41 x 109 bases _at_ 19,300 genes vs 22,000 human
• 5 coding 0.2 of genome comprises highly
  conserved -not coding
• half of this in neighborhood of 200 genes, some
  in embryonic dev and nerve growth
• (eg regulation)
• Certain subspecies or breeds at higher risk for
  certain ailments
• Samoyeds- diabetes
• Rottweilers- osteosarcoma
• Springer spaniels- epilepsy
• Doberman- narcolepsy

• Morey, D. J Archaeological Sci 06
• Dog burial ca 14,000 years ago

45
Genomics/bioinformatics and Developmental biology
Convergence of fly genetics and developmental
biology, ca. 1900s with molecular biology, ca.
1977 and with genomics and bioinformatics, ca.
1990s

• Developmental patterns and homeostasis genes

46
Fly mutation eyeless
The fly and you are not much different.
47
Counterpart gene in humans, Pax6
Summary of the universality of the central dogma
of molecular biology What was true for E. coli
would be true for the elephant -Jacques Monod
60s
48
Evolution Back to the future
Galapagos finches
Cambridge University Library
Tree of Life
49
The beauty of knowledge and appreciation of
nature True then, true now, questions-
Charles Darwin, The Origin of Species There is
grandeur in this view of life, with its several
powers, having been originally breathed into a
few forms or into one and that, whilst this
planet has gone cycling on according to the
fixed law of gravity, from so simple a beginning
endless forms most beautiful and most wonderful
have been, and are being, evolved. -1859
50

• Chemical basis of life
• biochemistry

51
Recent tools development Biotechnology is driven
by tools!
52
Chemistry of life key concepts- bonds
overview
53
(No Transcript)
54
(No Transcript)
55
(No Transcript)
56
(No Transcript)
57
Carbon chirality optical isomers
stereoisomers
58
(No Transcript)
59
Polar covalent bonds, resonance
60
(No Transcript)
61
Breaking and making bonds, in life
62
Ionic interactions cation/anion
63
water solubility H-bonding potential
64
Noncovalent bonds H bonds
between nonpolars
65
van der Waals interactions not bonds
66
Hydrophobic interactions
like dissolves like
67
Molecular complementarity
lock and key
68
Chemical building blocks of cells
AND....
69
(No Transcript)
70
Amino acids differ in side chains R
71
Amino acids specials
72
Amino acids common structure
73
(No Transcript)
74
(No Transcript)
75
(No Transcript)
76
Nucleotides common structure
77
(No Transcript)
78
(No Transcript)
79
(No Transcript)
80
Monosaccharides CH2O
81
(No Transcript)
82
(No Transcript)
83
Fatty acids precursors for lipids
84
(No Transcript)
85
(No Transcript)
86
(No Transcript)
87
(No Transcript)
88
amphipathic
89
(No Transcript)
90
Chemical equilibrium
91
Equilibrium constants
A B--gt Z Keq Z/AB
PD PD Kd PD/PD
92
pH
H2Oltgt H OH-
pure water at 25C pH -logH log1/e-7 7
93
Biochemistry of pH
zwitterion
94
Buffers maintain intracellular and extracelluar pH
95
(No Transcript)
96
-bio- chemistry of pH
97
energetics
98
-bio- energetics
99
Coupling reactions- allows unfavorables to go
A lt-gt B X ?G 5 kcal/mole X lt-gt Y
Z ?G -10 kcal/mole A lt-gt B Y Z
?G -5 kcal/mole
100
Where X is usually ATP
101
Another lower energy molecular
102
ATP is generated by photosynthesis and respiration
C6H12O6 6O2-gt 6CO2 6H2O
103
Biological Redox reactions
-Transferred to FAD, reducing to FADH2
104
Redox carriers
105
Redox carriers
106
The big picture Glycolysis and fates
107
Glycolysis carriers
108
Metabolic pathways reductionism
109
Metabolic pathways expanded
110
Systems biology Reactions to pathways to cells
to tissues to critter