Advanced Genetics

Advanced Genetics

Why Study Genetics?

Why Study Genetics? 1. Historical and aesthetic appreciation

Why Study Genetics? 1.  Historical and aesthetic appreciation

2.  Practical applications - Recognize genetic experiments

Why Study Genetics? 1.  Historical and Aesthetic appreciation

2.  Practical applications -  Recognize genetic experiments -  Interpret genetic experiments

Why Study Genetics? 1.  Historical and Aesthetic appreciation

2.  Practical applications -  Recognize genetic experiments -  Interpret genetic experiments -  Conduct genetic experiments

What is a genetic experiment?

Classification of Experiments 1. Observational 2. Interventional

Classification of Experiments 1.  Observational

-  Visual (microscopy)

-  Physiology (blood pressure)

-  Chemical analysis (serum glucose)

-  Biochemical analysis (sequence genome, proteome, metabalome, etc.)

Classification of Experiments

Interventional 1. Environmental - pharmacology - nutrition - temperature

Classification of Experiments

Interventional 1. Environmental - pharmacology - nutrition - temperature

2. Mechanical - biochemistry - physiology - surgical (transplantation)

Classification of Experiments

Interventional 1. Environmental 2. Mechanical

3. Genetic - manipulate genes

Interventional experiments are powerful because cause/effect relationships can be inferred

Classification of Experiments

Interventional Genetic Experiments 1. Manipulate genes 2. Observe phenotype

3. Conclusion

Interventional experiments are powerful because cause/effect relationships can be inferred

Cause How does an experiment establish cause? Why is it important to establish cause?

Cause Observation 1 Observation 2

Cause Observation 1 Observation 2

Correlation

Cause Observation 1 Observation 2

Correlation

Cause Experiment

Intervention 1 Observation 1

Control No Intervention Observation 1 Positive Conclusion: Intervention causes change Negative Conclusion: Intervention does not cause change

Cause Experiment

Intervention 1 Observation 1 Observation 2

Control No Intervention Observation 1 Observation 2 Positive Conclusion: Intervention causes change of 1 and 2 What conclusion about relationship of 1 and 2?

Cause Experiment

Intervention 1 Observation 1 Observation 2

Control No Intervention Observation 1 Observation 2 Positive Conclusion: Intervention causes change of 1 and 2 What conclusion about relationship of 1 and 2?

CORRELATION

Cause

Intervention 1 JUSTIFIED

Observation 1 Observation 2

Intervention 1

Observation 1 NOT JUSTIFIED

Observation 2

Categories of genetic experiments 1.  Method

- Classical (mutagens and breeding) - Molecular (Molecular biology)

2. Logic

- Forward (phenotype to gene) - Reverse (gene to phenotype)

Example: Mendel’s peas Found mutant pea strains – wrinkled and smooth. Bred to generate heterozygotes. Observed 3:1 segregation ratio. Conclusion regarding dominant and recessive alleles.

Example: Transcription factor binding Perform ChIP-seq with histone mark. Perform ChIP-seq with transcription factor. Identify transcription factor binding sites that have the histone mark. Draw conclusion regarding how histone mark affect transcription factor binding.

Example: Protein crystallography Express wild-type protein from plasmid in bacteria, purify, crystallize and solve 3-D structure. Perform site directed mutagenesis on plasmid. Express mutant protein from plasmid in bacteria, purify, crystallize and solve 3-D structure. Draw conclusion regarding residue and structure.

Example: Dominant negative protein Design mutant ERK kinase protein that interferes with function of wild-type protein. Express in cells from plasmid, measure signal transduction. Draw conclusion regarding role of ERK in signaling.

Example: Dominant negative protein Design mutant ERK kinase protein that interferes with function of wild-type protein. Synthesize protein with a machine. Inject protein into cells, measure signal transduction. Draw conclusion regarding role of ERK in signaling.

Proposition: The essence of genetics is the analysis of the relationship between GENOTYPE and PHENOTYPE Geneticists primarily: 1.  Manipulate genotype (Intervention)

2. Analyze phenotype (Observation)

3. Draw conclusions

Genotype 1. Definition: All the genetic information contained in an organism; the genetic constitution of an organism with respect to one or a few genes under consideration. 2. Distinctions: A.  One gene vs. Entire genome B.  Genetic vs. Molecular

Alleles known by Alleles known by DNA phenotype they cause sequence

Allele Phenotype DNA unc-1(+) wild type wild type unc-1(e1) mild uncoordinated missense codon unc-1(e2) severe uncoordinated deletion C. Wild type is by definition D. Complete description

- genetic: wild type or mutant at every locus - molecular: sequence of the genome

Phenotype

1.  Definition: The observable characteristics of an individual, which are a result of genotype and environment.

2.  Common ways to determine phenotype:

- visual inspection, including microscopy, staining, etc. - biochemical assay - chemical assay - behavioral assay

Caenorhabditis elegans

Brenner,  Sidney  (1974).  The  gene5cs    of  Caenorhabdi+s  elegans.  Gene5cs  77:  71-­‐94    (9,310  cita5ons,  2002  Nobel  prize)  

What makes an organism suitable for genetic analysis?

1.  Ease  of  culture.  

2.  Reproduc5ve  system  (self  fer5le  hermaphrodites  and  males).  

3.  Small  genome  and  limited  number  of  chromosomes.  

Introduction/Rationale 1.  “How  genes  might  specify  the  complex  structures  

found  in  higher  organisms  is  a  major  unsolved  problem  in  biology”  

2.  Necessary  to  find  mutants  (genotype)  and  analyze  the  structure  of  the  nervous  system  (phenotype).  

3.  “Some  eight  years  ago,  when  I  embarked  on  this  problem,  I  decided  that  what  was  needed  was  an  experimental  organism  which  was  suitable  for  gene5cal  study  and  in  which  one  could  determine  the  complete  structure  of  the  nervous  system.  Drosophila,  with  about  105  neurons,  is  much  too  large,  and,  looking  for  a  simpler  organism,  my  choice  eventually  seWled  on  the  small  nematode,  Caenorhabdi+s  elegans.”  

Properties of C. elegans    -­‐  Self-­‐fer5lizing  hermaphrodite:    XX      Male:                      XO  

 -­‐  1  mm  length      -­‐  3.5  day  life  cycle  

   -­‐  Small,  possibly  fixed        number  of  cells  

Results  1.    Isolate  muta5ons    

 -­‐  Mutagenize  with  chemical  mutagen    

 -­‐  Screen  for  mutant  animals  

Results  2.    Posi5on  in  the  genome  (mapping)    

 A.    Autosomal  vs.  sex  linked    

 B.  Two-­‐factor  mapping  (Distance)  Measure  recombina5on  frequency  between  two  muta5ons  

       a  b          +  +  

     

Results  2.    Posi5on  in  the  genome  (mapping)    

 A.    Autosomal  vs.  sex  linked    

 B.  Two-­‐factor  mapping  (Distance)  Measure  recombina5on  frequency  between  two  muta5ons  

       a  b          +  +  

   C.  Three-­‐factor  mapping  (Order)  

   +  a  b          a  +  b    c  +  +          +  c  +  

Results  3.    Complementa5on    

   -­‐  Purpose,  determine  if  two  muta5ons  affect  the        same  gene  

                 m1                  m2    

Results  Table  4    1.  Linkage  to  par5cular  autosome  or  X  chromosome  2.  Complementa5on  

Results  Figure  3  and  maps    1.  2-­‐factor  mapping  2.  3-­‐factor  mapping  

Discussion/Conclusions  Brenner  developed  methods  for  manipula5ng  the  genotype  of  C.  elegans,  making  gene5c  analysis  possible.    1.  Isola5on  of  muta5ons  2.  Mapping  

 -­‐    Linkage  to  chromosome    -­‐  Posi5on  on  a  chromosome        -­‐  2-­‐factor  

 -­‐  3-­‐factor  3.  Complementa5on  tests    These  techniques  made  it  possible  to  address  biological  ques5ons.