Biology Primer

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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
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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?
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Central Dogma of Molecular Biology
DNA
RNA
Protein
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(No Transcript)
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DNA

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

5-GATTC-3
3-CTAAG-5
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RNA

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

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Other Molecules

• Lipids
• Carbohydrates
• Small molecules
• Precursors, metabolites

Proteins
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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
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Protein structure

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

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JOE AVERAGE Polypeptide

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

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

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

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Translation Reading Frames
AUGGUACCACC...
Frame 1
MetValProPro..
AUGGUACCACC...
Frame 2
TrpTyrHis....
AUGGUACCACC...
Frame 3
GlyThrThr...
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Genetic Code
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Long ORFs are RARE
P(ORF)(61/64)n
P(20)(61/64)20.38
P(100)0.008
P(200)10-4
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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
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Eukaryotic Gene Expression
Promoter
Transcribed Region
Terminator
Enhancer
Transcription
RNA Polymerase II
Primary transcript 5
3
Intron1
Exon1
Exon2
Cap Splice Cleave/Polyadenylate
Translation
7mG
An
N
C
Transport
7mG
An
Polypeptide
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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
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Genome elements

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

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Genome Structure
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E. coli genome
--------------- 10 kb
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H. sapiens genome
----------- 50 kb
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Genome Size and Gene Number in Prokaryotes
LINEAR
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Genome Size and Gene Number in Eukaryotes
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Joe Average Genome

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

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

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Commonly used molecular biology techniques
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DNA Cloning
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The cloning process

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

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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).

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PCR
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DNA Sequencing
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DNA-DNA hybridization
Southern blotting
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DNA-DNA hybridization
Dot Blot/Gene Chip
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Transcript Analyses

• Northern Blot-1977

• Genomic transcript profiling

DeRisi, Iyer, Brown Science, 1998
Northern blot DNA microarray
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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

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Protein Expression
Western blot 2D PAGE
Charge
Size
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Protein structure
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Location of a protein in a cell
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Investigating gene function

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

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

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

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Phylogenetic Variation

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

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Induced/Directed MutationExperimental

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