Detailed Notes for the 10/4/05 Lecture

  1. Three-factor crosses

    1. Solves the problem of the relative position of genes.

    2. Textbook approach has flaws (see handout)

    3. 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).

      1. Crosses

        1. Mate the ab/ab and c/c animals

        2. Mate the resulting A or B progeny by c/c animals

        3. Score the resulting progeny for the C phenotype

      2. Theoretical considerations derived by drawing chromosomes and considering what happens during recombination

        1. Predict parental progeny

        2. 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.).

        3. Only the A and B recombinant progeny are important by these criteria.

          1. These animals will have one parental ab chromosome and one recombinant chromosome that has a or b and may also have c

          2. The second cross will determine whether the c mutation is present.

        4. 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.

        5. If not, then c is at either a or b or to the left or right, depending on the results.

        6. 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.

      3. Advantages

        1. Only have to look at the recombinants.

        2. Can map in very small intervals

        3. Uses previously known information

      4. Disadvantages or problems

        1. If c is very far away from a and b

          1. 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.

          2. Can be corrected by mating with another known pair.

        2. Doesn't give information about interference (but probably want to use a large interval for such studies anyway).

  2. These and other mapping procedures generate a series of maps for genes that are said to be on the same linkage group

    1. Chromosome is the cytological manifestation

    2. Linkage group is the genetically-derived operation definition

    3. Bridges' experiment tied them together

  3. Translocations and “Pseudolinkage”

    1. Region of one chromosome is attached to another

    2. Many times these are reciprocal

    3. Different segregation pattern according to independent assortment

      1. Adjacent -1 T1 and N2 separate from T2 and N1

      2. Alternate T1 and T2 separate from N1 and N2

      3. Adjacent-2 T1 and N1 separate from T2 and N2

    4. Adjacent-1 and Alternate patterns are most common

      1. Think of them resulting from different twists in the molecule

      2. Adjacent-2 is rare because it is difficult for homologous centromere to go to the same pole.

    5. Because Adjacent-1 products delete and duplicate large regions of the chromosomes, they often result in lethality

    6. 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).

      1. The majority of viable products are from the alternate segregation pattern.

      2. One cell gets N1 and N2 (i.e., both mutations) and the other gets T1 and T2 (i.e., both wild-type alleles).

      3. This results because the translocation really links the two genes.

    7. Think over what would happen if recombination occurred.

    8. Combining translocations with slight differences can produce organisms that are effectively deleted for various regions of the genome.

      1. Segmental aneuploids in Drosophila

        1. Find out how these are made

        2. Used to make deletions and duplications

      2. Robertsonian translocations in mammals

        1. Translocations at centromeres

        2. Whole chromosomes attached to each other in mice because mouse chromosomes are acrocentric

      3. Making a trisomy 16 mouse

        1. 17:16/17:16 9/9 X 16:9/16:9 17:17

        2. 17/17:16/16:9/9 X WT

        3. Possible germ cells (given that two chromosomes must go to a germ cell)

          1. 17; 17:16 -> lethal

          2. 17; 16:9 -> WT

          3. 17; 9 -> lethal

          4. 17:16; 16:9 -> trisomy 16

          5. 17:16; 9 -> WT

          6. 16:9; 9 -> lethal

        4. So 2 die as early embryos; 1/3 of remaining trisomics

  4. Inversions

    1. Inversions will affect pairing

      1. Lowers recombination frequency

        1. Difficulty in pairing

        2. In some species the recombined products are not fertilized

      2. A useful feature is that inversions can be used to prevent recombination

    2. Involvement of centromere affects segregation

      1. Pericentric inversions

        1. Involves the region around the centromere

        2. The products of meiosis all are monocentric, but can contain duplications or deletions

      2. Paracentric inversions

        1. Involves a region outside the centromere

        2. The products of meiosis can include dicentric chromosomes (which will break) and acentric fragments that will be lost.

      3. These losses reduce the number of recombinations and condenses the map (genes appear to be closely linked).

    3. Balancer chromosomes

      1. Chromosomes that prevent the appearance of recombinant products

      2. Components

        1. Inversions (usually multiple)

        2. Marker mutations (dominant)

        3. Recessive lethal mutations

  5. Speciation also involves inversions and translocations

    1. Define synteny

    2. Look up how synteny changes with more divergent species

  6. Problems with mapping human genes

    1. Data from V.A. McKusick Mendelian Inheritance in Man





      auto. dom.





      auto. rec.




      X-linked (1/25 of genome)










    2. Unusual distribution

      1. Proportionally more X-linked and dominant mutations

      2. X-linked because the effect of single mutations can be seen in males.

      3. Dominant because, again, effect of single mutations can be seen.

  7. Polymorphism mapping

    1. Mapping is important because of getting at genes, but recombination mapping has problems

      1. Takes considerable time

      2. Not all that many markers (even for worms and flies)

      3. For humans - special considerations

        1. Fundamental question

          1. Is it a genetic defect or caused by an external agent

          2. Familial, e.g. Alzheimer's dementia

        2. Obviously can't test the crosses

        3. Families with traits are rare: the problem of two rare events

        4. Inborn errors of metabolism

        5. How can genetic diseases be studied

          1. Map

          2. Diagnosis

          3. Possible cure

        6. One needs markers - Enzymes can be such markers.

    2. Advantages and Problems of Using Mice

      1. Advantages

        1. Mammal

          1. Development known

          2. Biology known

          3. Synteny

          4. Complex behaviors and activities

        2. Many genes identified by mutation -

        3. Many years of activity

        4. Large breeding colonies

      2. Disadvantages

        1. Lots of cells and tissues (complexity creates a problem)

        2. Large genome

        3. Takes a lot of time - e.g. mapping a gene

        4. Until recently not many markers

        5. Expensive

    3. Solution: Recombination using sequence differences

      1. General considerations

        1. Sequence as a phenotype - suddenly there are many markers

        2. Types

          1. RFLP (Restriction Fragment Length Polymorphisms)

          2. SNP (Single nucleotide polymorphisms)

          3. Snip-SNP (SNP that can be cut with a restriction enzyme)

          4. Deletion

          5. Duplications and repeats (e.g., microsatellite DNA and CAn repeats)

          6. Transposon insertion

        3. Detection

          1. Restriction enzymes

          2. PCR

          3. Sequencing

        4. Many of these are silent mutations

      2. Example: Linkage analysis in C. elegans

        1. Two strains of wild-type C. elegans

          1. N2 has 30 copies of a transposon

          2. Hawaiian has >500 copies

        2. Any single polymorphic site can be identified by PCR

          1. Choose on primer for the common end of the transposon

          2. Choose the second primer for a site outside the transposon

          3. Length of PCR product identifies the product

        3. Linkage

          1. Pick one polymorphism for each chromosome

          2. Pick primers so each product is a different length

          3. Mate mutant N2 strain with the Hawaiian strain

          4. Let the F1 animals self and pick homozygous mutants

          5. Amplify all the polymorphism from each animals and display on an agarose gel

          6. If linked, one should not see a band

          7. If unlinked, three quarters of the animals should have the band

        4. Mapping on a chromosome

          1. Similar to linkage, but choose sites along a single chromosome

          2. Repeat steps 2 – 5 as above

          3. The closer the polymorphism site is to the mutation, the fewer animals will have the band