Detailed Notes for the 10/4/05 Lecture
Solves the problem of the relative position of genes.
Textbook approach has flaws (see handout)
An alternative approach that uses only the existing strains ab/ab and c/c for which the distance between a and b is known (p).
Mate the ab/ab and c/c animals
Mate the resulting A or B progeny by c/c animals
Score the resulting progeny for the C phenotype
Theoretical considerations derived by drawing chromosomes and considering what happens during recombination
Predict parental progeny
Look at progeny resulting from one parental chromosome and one recombinant chromosome (if more than one recombination occurs, it will be an extremely rare event, so it can be ignored.).
Only the A and B recombinant progeny are important by these criteria.
These animals will have one parental ab chromosome and one recombinant chromosome that has a or b and may also have c
The second cross will determine whether the c mutation is present.
If c is between a and b, the proportion of recombinants in each interval gives the relative map distance of c from a and b.
If not, then c is at either a or b or to the left or right, depending on the results.
In C. elegans this is even easier, since the A and B progeny can simply be selfed to see whether they have a c-containing chromosome.
Only have to look at the recombinants.
Can map in very small intervals
Uses previously known information
Disadvantages or problems
If c is very far away from a and b
A and B animals may have c from a second recombination (i.e., it will not be rare) and this will give the spurious result that c is between a and b.
Can be corrected by mating with another known pair.
Doesn't give information about interference (but probably want to use a large interval for such studies anyway).
These and other mapping procedures generate a series of maps for genes that are said to be on the same linkage group
Chromosome is the cytological manifestation
Linkage group is the genetically-derived operation definition
Bridges' experiment tied them together
Translocations and “Pseudolinkage”
Region of one chromosome is attached to another
Many times these are reciprocal
Different segregation pattern according to independent assortment
Adjacent -1 T1 and N2 separate from T2 and N1
Alternate T1 and T2 separate from N1 and N2
Adjacent-2 T1 and N1 separate from T2 and N2
Adjacent-1 and Alternate patterns are most common
Think of them resulting from different twists in the molecule
Adjacent-2 is rare because it is difficult for homologous centromere to go to the same pole.
Because Adjacent-1 products delete and duplicate large regions of the chromosomes, they often result in lethality
Because of interactions of two sets of chromosomes, mutations on different chromosomes can appear to be linked, this is referred to a pseudolinkage (actually the linkage is real).
The majority of viable products are from the alternate segregation pattern.
One cell gets N1 and N2 (i.e., both mutations) and the other gets T1 and T2 (i.e., both wild-type alleles).
This results because the translocation really links the two genes.
Think over what would happen if recombination occurred.
Combining translocations with slight differences can produce organisms that are effectively deleted for various regions of the genome.
Segmental aneuploids in Drosophila
Find out how these are made
Used to make deletions and duplications
Robertsonian translocations in mammals
Translocations at centromeres
Whole chromosomes attached to each other in mice because mouse chromosomes are acrocentric
Making a trisomy 16 mouse
17:16/17:16 9/9 X 16:9/16:9 17:17
17/17:16/16:9/9 X WT
Possible germ cells (given that two chromosomes must go to a germ cell)
17; 17:16 -> lethal
17; 16:9 -> WT
17; 9 -> lethal
17:16; 16:9 -> trisomy 16
17:16; 9 -> WT
16:9; 9 -> lethal
So 2 die as early embryos; 1/3 of remaining trisomics
Inversions will affect pairing
Lowers recombination frequency
Difficulty in pairing
In some species the recombined products are not fertilized
A useful feature is that inversions can be used to prevent recombination
Involvement of centromere affects segregation
Involves the region around the centromere
The products of meiosis all are monocentric, but can contain duplications or deletions
Involves a region outside the centromere
The products of meiosis can include dicentric chromosomes (which will break) and acentric fragments that will be lost.
These losses reduce the number of recombinations and condenses the map (genes appear to be closely linked).
Chromosomes that prevent the appearance of recombinant products
Inversions (usually multiple)
Marker mutations (dominant)
Recessive lethal mutations
Speciation also involves inversions and translocations
Look up how synteny changes with more divergent species
Problems with mapping human genes
Data from V.A. McKusick Mendelian Inheritance in Man
X-linked (1/25 of genome)
Proportionally more X-linked and dominant mutations
X-linked because the effect of single mutations can be seen in males.
Dominant because, again, effect of single mutations can be seen.
Mapping is important because of getting at genes, but recombination mapping has problems
Takes considerable time
Not all that many markers (even for worms and flies)
For humans - special considerations
Is it a genetic defect or caused by an external agent
Familial, e.g. Alzheimer's dementia
Obviously can't test the crosses
Families with traits are rare: the problem of two rare events
Inborn errors of metabolism
How can genetic diseases be studied
One needs markers - Enzymes can be such markers.
Advantages and Problems of Using Mice
Complex behaviors and activities
Many genes identified by mutation -
Many years of activity
Large breeding colonies
Lots of cells and tissues (complexity creates a problem)
Takes a lot of time - e.g. mapping a gene
Until recently not many markers
Solution: Recombination using sequence differences
Sequence as a phenotype - suddenly there are many markers
RFLP (Restriction Fragment Length Polymorphisms)
SNP (Single nucleotide polymorphisms)
Snip-SNP (SNP that can be cut with a restriction enzyme)
Duplications and repeats (e.g., microsatellite DNA and CAn repeats)
Many of these are silent mutations
Example: Linkage analysis in C. elegans
Two strains of wild-type C. elegans
N2 has 30 copies of a transposon
Hawaiian has >500 copies
Any single polymorphic site can be identified by PCR
Choose on primer for the common end of the transposon
Choose the second primer for a site outside the transposon
Length of PCR product identifies the product
Pick one polymorphism for each chromosome
Pick primers so each product is a different length
Mate mutant N2 strain with the Hawaiian strain
Let the F1 animals self and pick homozygous mutants
Amplify all the polymorphism from each animals and display on an agarose gel
If linked, one should not see a band
If unlinked, three quarters of the animals should have the band
Mapping on a chromosome
Similar to linkage, but choose sites along a single chromosome
Repeat steps 2 – 5 as above
The closer the polymorphism site is to the mutation, the fewer animals will have the band