Lecture 7 - Genome Tools

(Chapt 9: Molecular genetic techniques) 

Preview: Genomics/Bioinformatics to Systems 

Genetics 

Molecular genetic techniques 

Molecular biology 

Recombinant DNA technology

Fly genetics to fly molecular biology 

•WT 

•Dominant mutation (ey) 

•Suppressor mutation 

•Nature of… genetics v mol biol v biochem

Worm cell biology to molecular biology 

•RNAi can silence genes 

•Transgenic worm (right) with GFP reporter in head neurons 

•Expresses dsRNA to muscle gene unc-15 

•Degrades mRNA 

•Causes complete paralysis 


Function, structure and location of gene products 


Bacterial genetics: original expts in bacteria and bacteriophages

Bacterial genetics: original expts in bacteria and bacteriophages

into minimal medium---> 

PREVIEW: Molecular biology and applications. 

Genome and manipulation of genome define organism 

(simple case) 

--> growth 

--> no growth

Molecular basis- metabolic pathways 

(simple case) 

•Example: genetics and biochemistry 

to molecular biology to Systems 

•Metabolic pathways 

•Lac operon 

•cis/trans


(simple case or complex case???) 

-albinism, 1:17,000 

-metabolic pathways 

-phenotype/genotype 

-affects entire organism 

--> simple or complex?

Genetic and molecular basis- metabolic pathways 

“simple case” 

•One gene, one mutation 

•DNA-> protein sequence 

•Two copies vs one copy 

•[Different from skin color]

Chemical basis- metabolic pathways 

“simple case”

Genetic and molecular basis- metabolic pathways 

“fish to human” 

•(What is the difference between a fish and a human?) 

•RLLamason…KCCheng. Sci310: 1782 (05) 

•SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans 

•Zebrafish pigmentation gene and human counterpart 

•One variant - natural selection for lighter skin in Europeans 

•But, 93% Africans and East Asians share same allele 

•“East Asians are usually light skinned too”- so, other genes? 

•“golden” zebrafish plus human SLC4A5 -> darker stripes (rescue)

•Epigenetic 

•Environmental 

•External 

•Internal 

•Expression and pools 



But, more data leads to more questions…. complexity 

-Systems biology

•Very different heart structures

Molecular basis- developmental pathways 


But, more data leads to more questions…. complexity TO SIMPLICITY 

•http://sandwalk.blogspot.com/2006/11/sea-urchin-genome-sequenced.html 

•http://scienceblogs.com/pharyngula/2006/06/modules_and_the_promise_of_the.php 

•0.5B years apart 

•Seemingly complex [they are!]

Molecular basis- developmental pathways 


But, more data leads to more questions…. complexity TO SIMPLICITY 



•0.5B years apart 

•Seemingly simpler [they are!]

Genomics/bioinformatics…. complexity TO SIMPLICITY 



•ESodergren…RWright. Science, Nov 06 

•and The Sea Urching Genome Sequencing Consortium 

•Strongylocentrotus purpuratus 

•814Mb genome 

•A model for developmental and systems biology

From 


(complex case)

Leads to....... 

(‘The good, the bad and the ugly [?])’....

Leads to.......


(how) 

•Gurkin is sim to EGF; receptor is RTK •PIPE protein- sulfation of glycosaminoglycans 

•[attached to proteins- proteoglycans]- cell fates

•Activation of Toll by Spatzel leads to 

•Gradient of Dorsal: 

ventrally high and dorsally low. 

•[Dorsal] not high enough to bind to 

•low affin twist/snail; binds to 

•High affin rhomboid with activator bHLH. 

•Sharp boundary of Rhomboiddistinct 

cell types. 

•And.... 

Spatzel binds Toll....

[visually] Flower development fn of spatially regulated txn factors

Complex, simple- same basis 

-It’s in the genome!

‘Baby’ genetics 

•terminology •Diploid/haploid 

•Dominant/recessive, alleles 

•What is a gene?

‘Baby’ cell biology: asexual reproduction

‘Baby’ cell biology: sexual reproduction

•Br/bl x bl/bl 

•F1: Br allele lost 

•random event 

Forces that cause populations and 

species to evolve 

CPurrington (Swarthmore)

Forces that cause populations and 

species to evolve 

•mutations can be 

•good- improves species fitness and/or reproductive success 

•bad- decreases “ 

•neutral- no effect 

•[ugly- totally subjective... ‘only a mother...’] 

•basis for evolution via natural selection, which operates when there 

is variation caused by genetic differences 

CPurrington (Swarthmore)

What is a mutation? Molecular level

Segregation of alleles in yeast

Identification of gene, gene product (gp), pathways…. 

-similar to bacteria… and viruses

Tools for genetics: conditional mutations

•cdc28 arrest pre-budding 

•cdc7 arrest at budding

Complementation: temperature sensitive mutants

Interacting or redundant proteins, compensating mutants 

•Nature of ‘rescue’ vs suppression 

•Temperature sensitive mutants 

•ACT1 actin 

•SAC6 actin-binding protein 

•WT: asymmetric distribution 

•Each mutant: random distribution 

•Dbl mutant: “WT-like” 

•(restores function, “suppresses”)

Recombinant DNA and 

molecular biology techniques: 

Paradigm shift. 

(and bioinformatics)

Molecular biology: recombinant DNA technology. Key technique 

•summer ‘71 SV40 DNA to E. coli- postponed 

•Feb ‘75 Asilomar Conference: most recombinant DNA work should continue, with safeguards 

•Sanctions could be severe

Host restriction (and modification): organism view

At the molecular level

Host modification

Palindromes: 

1) 55, 212, 1331, 45654 

2) pop, level, racecar 

3) Madam, I’m Adam 

Was it a rat I saw? 

A man, a plan, a canal, 

panama 

4) GGATCC 

CCTAGG 

Restriction enzymes and recognition sites

•Lambda repressor 

•EcoRV 

•http://en.wikipedia.org/wiki/DNA 

Restriction enzymes and recognition sites: DNA binding

Restriction enzymes and restriction sites: use 1

Construction of ‘ideal’ vectors

Construction of ‘ideal’ vectors: pUC series

RE, use 2: molecular cloning

Cloning into plasmids

Variations: cloning into eukaryotic hosts

Once you have a recombinant “genetic material,” 

Then what? 

Moving genomic elements into bacteria

Plasmid 

(Recombinant 

DNA) 

Phage 

Conjugation 

Moving DNA, mechanisms

Vector and insert size 

Vector type Insert Capacity Concerns Host 

M13 virus 1.5kb Bias of sequences E coli 

Plasmids 0.1-10kb Size instability bacteria, yeast 

Lambda phage 10-20kb Size limitation •Î/E.coli 

Cosmids 35-45kb Size limitation E coli 

P1 80-100kb E coli 

BAC 50-300kb E coli 

P1-BAC 100-300kb E coli 

YAC 0.1-2Mb Up to 60% chimera Yeast

Need for bigger inserts: Lambda phage vector

Larger inserts, eg, from euk genes: cDNA by reverse transcription 

•cDNA libraries reflect gene expression states 

•[using protein chemistry methods]

cDNA libraries in plasmids 

•cDNA libraries reflect gene expression states

Characterization of inserts: Nucleic acids hybridization 

[what do you have? how do you know?] 

•Discovery limted by: 

•Labels 

•Types and conditions 

•Probes 

•Specificity 

•Non-specificity

Hybridization to inserts within lambda genome

Characterization of inserts: Nucleic acids hybridization 

Edwin Southern 1975, “Southern blot”

Variations on Southern blotting 

•Combine with restriction enzyme digestion and gel electrophoresis 

•Northern blots 

•Western blots 

•Southwestern blots

RE digest, gel electrophoresis resolution, blot transfer, and 

Southern hybridization 

•PAGE, polyacrylamide 

•Vertical gels

Gel electrophoresis, blot and probe- in more detail 

•Agarose gels 

•Horizontal gels

Hybridization results: Comparison of signals, alternative labels 

•Southern: single copy INO2 gene 

•5 ug yeast genomic DNA with biotinylated cRNA probe 

•H3, R1, Sal1 digests, with one minute exposure 

•Biotinylation/streptavidin (nucleic aicds) 

•Digoxygenin/antidigoxygenin Ab (nucleic acids) 

•Color (alkaline phosphatase), ferritin, gold, chemiluminescent dye, fluorescent dye 

•Northern: TCM1 expression: 5, 2.5, 0.625, 0.313 and 0.156 ug yeast genomic DNA with 

•A/B: DIG-label; C/D: biotinylated; E: 32 P 

•Exposure times: A: 5’; B: 25’; C: 5’; D 25’; E: 72hrs

End-labeling- using biochemistry of cell 

•www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/Dig12-18.pdf 

•Can be difficult to make- 

•Clone, amplify, purify, label, clean, use 

•Kinase end-labeling 

•Fill-in end-labeling

•http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.464 

Labeling by in vitro synthesis: multiple ways 

•Nick translation labeling 

•Random primed labeling

•http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.467 

Alternative labeling protocols

Zoo blots: Comparative genomics, pre-genomes 

•Defined highly conserved sequence, 250 bp CSB probe ( 32 P) 

•Low stringency: 2xSSC/50C/15min 

•Higher stringency: 1xSSC/50C (-chicken and -xenopus) 

•Higher stringency: only primates left 

•ca. late 1980s

Zoo blots: Comparative genomics, pre-genomes 

•http://opbs.okstate.edu/~melcher/MG/MGW4/MG412.html 

•vs “BLAST”

Second generation nucleic acids probes 

through oligonucleotide synthesis 

•Inorganic chem 

•Automation

•Synthesis of poliovirus from oligos 

•“Driven by “strong curiosity” 

•Not using a “natural” template 

•Assembled DNA 

•Transcribed into RNA ss genome 

•Self-replicates 

•5,700 bp 

•[social and political ramifications] 

•[no cure for poliomyelitis] 

•JCello et al. Sci 297:1016 (2002) 

Synthetic genomics 

•JCVenter: “irresponsible”, “inflammatory without scientific justification” 

•Ditto: 

•HOSmith (70s) and CAHutchinson (60s) - all-ighters to creat genome of phi-X174 (5386 bp) 

•…Venter. PNAS 100:15440. 2003 

•“Minimal microbial genome needs just 271 genes” 

•KKobayashi, et al. PNAS 100:4678 (2003) 

•http://www.genomenewsnetwork.org/articles/07_02/polio_create.shtml

Biochemistry techniques into molecular biology techniques: 

Bridging protein and DNA elements 

Characterizing genes and recombinant elements, 

and gene products (gps)

Analysis of DNA-binding protein 

Identical three SDS PAGE lanes 

•1- Silver stained yHSTF 

•2- Western: anti-yHSTF MAb 

•3- Southwestern: HSE 

->Three different purposes of assays

Northern blot analysis: increased expression of β-globin mRNA 

in differentiated erythroleukemia cells 

•UN= uninduced 

•Induced to stop growing, 

to differentiate 

•1000x increase at 96 hr

Over-expression in E. coli 

Western blot assay 

•A1: uninduced in E. coli 

•A2: induced in E.coli 

•A3: positive control 

DNase I footprinting assay 

•B1, 10: no protein 

•B2, 3: pure HSTF 

•B4, 5, 6: uninduced crude 

•B7, 8, 9: induced crude 

•Very high resolution of binding

Deletion analysis of binding domain 

Localization of binding domain: 

Deletion analysis and SW assay 

•A1- 3926 native 

•A other lanes- 3’ endpoints of deletions 

DNase I footprinting assay 

•B1, 18: no protein 

•B2, 3: pure HSTF 

•B4: uninduced native 

•B5, 6, 7, 8: induced native 

•B9: uninduced deletion 1 

•B10, 11: induced deletion 1, two levels 


•B13, 14: induced deletion 2, two levels 


•B16, 17: induced deletion 3, two levels

Over-expression in S. cerevisiae 

Galactose-induced overexpression GAL-HSF1 fusion 

•GAL1/GAL10 promoter+HSF1 

•cotransformed with UAS HSE replacing UAS CYC -lacZ 

A- galactose 

B- glucose 

‘blue= lacZ gene on’

Genomics 

and the ‘new’ Big biology 

“discovery science” 

Recombinant DNA technology 

genomes, esp human 

human disease genes 

“next” lecture [8]: Tools and tools development 

Applications and ‘new’ applications

Complementation: metabolic intermediate mutants

Examples of pathway mutations and pathway dissections: 

Genetics to biochemistry 

•In same pathway 

•Intermediate accumulate preceding mutation step

Use of double mutants to order steps in biosyn path 

•In same pathway 

•Intermediate accumulate preceding mutation step

Use of double mutants to order steps in signal path 

•Analysis works if the two mutations have opposite effects 

•Define constitutive vs basal levels

Lecture 7 - Genome Tools

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