Biology Primer

1
Biology Primer

• Basic principles
• Organisms/Cells as Basic Units
• Biochemical Components of Cells
• Genetic processes
• Fundamental Molecular Genetics

BIO520 Bioinformatics Jim Lund
2
Phylogeny
3
Prokaryotes
4
Gram-negative Prokaryotes
5
Eukaryotes
What cell compartments are present? What cell
processes are different between prokaryotes and
eukaryotes? What are protein families are unique
to eukaryotes? How is gene annotation
different?
6
Central Dogma of Molecular Biology
DNA
RNA
Protein
7
(No Transcript)
8
DNA

• G,A,T,C
• 5 to 3
• antiparallel double helix
• GC, AT

5-GATTC-3
3-CTAAG-5
9
RNA

• Single-stranded polymer
• G,A,C,U
• rRNA (90)
• tRNA (5-10)
• mRNA (1-5)
• informational
• many kinds
• snRNA, siRNA, miRNA, snoRNA

10
Other Molecules

• Lipids
• Carbohydrates
• Small molecules
• Precursors, metabolites

Proteins
11
Polypeptides

• Polymers of 20 amino acids
• amide linkages
• 100-1000 residues typical
• amino (N)-----carboxyl (C) end
• encoded by mRNAs
• modified (Pi, CH3)

Protein 1 or more polypeptide
12
Protein structure

• Primary
• AA sequence
• Secondary
• Alpha helix, Beta sheet
• Tertiary
• 3D structure
• Quaternary
• 3D structure of 2 or more protein chains bound
  together.

13
JOE AVERAGE Polypeptide

• 350 amino acids
• Rare vs common aas
• Amino acids
• MW110 Da

Know your aa's
14
Protein lengths
Zhang et al., 2000
15
Protein lengths
Zhang et al., 2000
16
Proteins

• Enzymes
• Receptors
• Transporters
• Structural components
• Regulatory factors

17
Translation Reading Frames
AUGGUACCACC...
Frame 1
MetValProPro..
AUGGUACCACC...
Frame 2
TrpTyrHis....
AUGGUACCACC...
Frame 3
GlyThrThr...
18
Genetic Code
STOP
19
Long ORFs are RARE
P(ORF)(61/64)n
P(20)(61/64)20.38
P(100)0.008
P(200)10-4
20
Prokaryotic Gene Expression
Promoter
Cistron1
Cistron2
CistronN
Terminator
Transcription
RNA Polymerase
mRNA 5
3
1
2
N
N
N
C
N
C
C
1
2
3
Polypeptides
21
Eukaryotic Gene Expression
Transcribed Region
Promoter
Terminator
Enhancer
Transcription
RNA Polymerase II
Primary transcript 5
3
Intron1
Exon1
Exon2, etc
Cap Splice Cleave/Polyadenylate
Translation
7mG
An
N
C
Transport
7mG
An
Polypeptide
22
Joe Average Gene

• Encodes 350 amino acid product
• Euk
• Exons and introns, typically 5-10
• Enhancer/promoter region
• Prok 1 cistron (part of operon)

10 kbp
1.5 kbp
23
Genome elements

• Genes
• Special regions
• Origins of replication
• Telomeres
• Intergenic Regions
• Enhancers
• Chromsomal attachment sites
• Origins of replication
• Junk??

24
Genome Structure
25
E. coli genome
--------------- 10 kb
26
H. sapiens genome
----------- 50 kb
27
Genome Size and Gene Number in Prokaryotes
LINEAR
28
Genome Size and Gene Number in Eukaryotes
29
Joe Average Genome

• Human
• 3,000,000 kb
• 20,000 genes (as many as the worm C. elegans)
• 10 genes

• E. coli
• 5,000 kb
• 4,200 genes
• 90 genes!

30
Commonly used molecular biology techniques
31
DNA Cloning
32
The cloning process

• Source of Nucleic Acid
• Cloning Vector
• Host
• Selection/Screen

33
Polymerase Chain Reaction (PCR)

• Template (DNA to be amplified)
• Primer Sequences
• 18-27 base pairs (bps)
• Conditions
• Annealing temperature for primers (55-65C)
• Extension time (1-3 minutes)
• Cycles (20-35).

34
PCR
35
Sanger DNA Sequencing
36
DNA-DNA hybridization
Southern blotting
37
DNA-DNA hybridization
Dot Blot/Gene Chip
38
Transcript Analyses

• Northern Blot-1977

• Genomic transcript profiling

DeRisi, Iyer, Brown Science, 1998
Northern blot DNA microarray
39
Questions addressed by protein analysis techniques

• Identity/Amount
• modifications
• Structure
• 3D
• Detailed information about binding to ligands and
  other proteins
• Location
• Function
• Protein-protein interaction
• Enzymatic activity

40
Protein Expression
Western blot 2D PAGE
Charge
Size
41
Protein structure
42
Microscopy(location of a protein in a cell)
43
Investigating gene function

• Biochemistry
• Variation
• Sequence
• Expression
• Where, when, expression level?
• Knockout
• Overexpression
• Homology to well-studied proteins.

44
DNA mutation
Promoter
Transcribed Region
Terminator
Enhancer
Amount Nature
3
Intron1
Exon1
Exon2
nucleus
X
7mG
An
N
C
cytoplasm
7mG
An
45
Variation-Population

• Most variation unimportant to function
• SNPs (Single Nucleotide Polymorphism)
• Some variation has functional importance
• Eg. HBA1 mutants
• Thalassemia
• Sickle Cell Anemia

46
Phylogenetic Variation
Consensus 1 MLEARLVQASLLKKLLEAIKDLVNDANFDCSESGISLQAMDSSHVSLVSLLLRSEGFEHY 60 query 1 MFEARLVQGSILKKVLEALKDLINEACWDISSSGVNLQSMDSSHVSLVQLTLRSEGFDTY 60 1PLR 1 MLEAKFEEASLFKRIIDGFKDCVQLVNFQCKEDGIIAQAVDDSRVLLVSLEIGVEAFQEY 60 gi 400740 1 MLEAKLNNASILKKLFECIKDLVNDANVDADESGLKLQALDGNHVSLVSLHLLDSGFSHY 60 gi 129696 1 MFEAEFKTGAVLKRLVETFKDLLPHATFDCDNRGVSMQVMDTSHVALVSLQLHAEGFKKY 60

• Evolutionary changes in DNA/protein sequence.
• Variation depends on distance
• Orthologs, paralogs
• Variants function in context.

47
Induced/Directed MutationExperimental

• Often GROSS mutations
• May not reflect in any way the population or
  phylogenetic variation
• Critical to functional study