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This homework assignment turned out to be more difficult than I had intended, particularly because I did not have as much time in lecture to review these concepts as I had hoped. To compensate for this unanticipated difficulty, I awarded one point of extra credit to each student who completed the assignment. Overall, I was very impressed with the quality of your answers.

a. They may appear as if they were homozygous for the wild-type allele

b. They may appear mutant

c. They breed true

d. They may have altered enzyme activity

2. (1 pt) Causes of heritable mutation include all of the following except

a. radiation

b. chemicals

c. errors in DNA replication

d. errors in copying DNA into mRNA

3. (1 pt) Crossing over (recombination) occurs during (circle all that apply)

a. metaphase of meiosis I

b. metaphase of mitosis

c. prophase of meiosis II

d. metaphase of meiosis II

The best answer to this question is "none of the above," since crossing over between homologs occurs during prophase of meiosis I. I accepted any answer except b, since reasonable arguments can be made for a, c, and d. SOME crossing over may occur during meiosis II, even in the absence of synapsis (and, in fact, in the absence of homologous chromosomes). Since the sister chromatids are not identical at this point (because of crossing over between homologs in meiosis I), recombination between them CAN shuffle the genetic material.

It is even true that some recombination can occur during phases of the cell cycle other than meiosis or mitosis, although at much lower frequencies than during meiosis I.

This was not a well-written question--my apologies.

4. (3 pts) You are studying a bacterium that has the remarkable property of being able to survive exposure to high levels of radiation. Although the general phenomenon has been characterized and described, the genetics underlying this ability have not yet been elucidated. Describe how you would design and carry out a genetic screen to do just that. You may make the following assumptions:

• This bacterium has a small genome (roughly 4,000 kb), which is compact. Like other bacteria, this organism lacks introns.
• Existing transformation technologies (calcium chloride, electroporation) and plasmids will work for your organism.
• Other than radiation, standard methods of mutagenesis will work for your organism.
• This bacterium is haploid and grows clonally. It can be maintained on standard agar plates.

You should describe how you would isolate and characterize mutants, determine the order of the genetic loci (i.e. which gene product acts first, which acts next, and so on), and isolate individual genes.

[student answer, slightly edited, with some extra comments from me]

First, I would mutagenize the strain in order to find out what kind of mutations caused death at high radiation levels. I would allow the mutagenized strain to grow on an agar plate and make a replica of the original plate so that I had two agar plates, each with duplicate bacterial colonies [this technique is called "replica plating." I'll discuss it briefly on Wed morning]. Then I would expose one plate to high radiation and observe which of the colonies were still able to survive. By identifying dead mutants and matching them up to their live counterparts on the replica plate I know which bacteria have muations in genes that are essential for survival at high radiation. I would characeterize the dead mutants based on variations in phenotype after radiation. [You might think that "dead is dead," but there may be observable differences in these cells. For example, if you add a stain that fluorescently labels DNA and inspect the cells under a microscope, you may discover that some mutant strains have fragmented chromosomes, while others do not. With other stains, you may observe alterations in cell structure or membrane integrity. If you can find such phenotypic differences between your different mutant isolates, you can use them to determine the functional order of the genetic loci, as described below.]

To determine the order of gene loci involved in radiation resistance I would first look for different phenotypes in the dead mutants. Having isolated two mutants with different phenotypes I would create a haploid bacterial strain that would be a double mutant. Depending on which of the two phenotypes appear in this double mutant, you can determine which comes first in the pathway that allows resistance. [Yes, this assumes that the gene products act in a linear and ordered pathway--that one event must occur in order for the next to occur. This is not always true, but sometimes it is the case.]

In order to isolate the individual genes responsible I would first creat a genomic library of wild-type DNA using plasmid vectors. I'd transform this library into the mutant bacteria and look for restoration of the wild-type phenotype (radiation resistance) in the transformants. Mutants that are now able to survive high radiation would have to have been transformed by a plasmid carrying either the wild-type version of the gene that was responsible for the original loss of function, or a gene that was able to suppress the mutant phenotype. To isolate the actual gene, I would grow cultures of these bacteria, isolate the plasmids and sequence them, and then sequence the same gene from the mutant strain and compare. If this same gene in the mutant is in fact mutated then it's pretty safe to assume that I've identified my gene. If the gene in the mutant is not mutated then the plasmid gene is merely suppressing the mutant phenotype and is not the same gene as the one causing loss of function in the mutant.

5. (4 pts) You are studying a human neurological disorder which strikes children between the ages of 6 months and 3 years. Once it strikes, it is rapidly degenerative and invariably fatal. In studying inheritance patterns, you have learned that this illness is single-gene recessive, non-sex-linked, and strictly genetic. In other words, children who inherit two disease-bearing alleles at this locus inevitably get the disease, while heterozygotes are phenotypically normal. You have also discovered a small religious community in New York among whom this disease is extraordinarily prevalent. The members of this community are willing to collaborate with you in providing blood samples for testing and research.

Describe how you could use RFLP and/or PCR fingerprinting analysis to determine the chromosomal region associated with this illness. Then describe how you could identify the actual gene involved. You may assume that the human genome project has been completed, but that the gene for this illness has not been identified as such or functionally characterized.

[You may be interested to know that this is a real illness by the name of Tay-Sachs disease. The gene responsible has been isolated and characterized, although at this point there is no effective treatment and little understanding of the mechanism of disease.]

[Student answer, slightly edited]

When analyzing an autosomal recessive gene to determine the location, you must first look at the linkage with other known markers to give you a general idea of location. Unfortunately, In humans there is not an excess of markers to isolate the general area of the gene as with other organisms. To circumvent this issue, RFLPs are used as markers. Restriction fragment length polymorphisms result due to various mutations that give rise to or eliminate unique restriction sites within the DNA. By isolating these predetermined RFLPs (via PCR followed by restriction digests, or via Southern Blots), it can be determined which RFLPs an afflicted individual carries. Genetically related individuals will carry similar RFLPs. By analyzing the linking between the RFLPs and the disease, an approximation of gene location can be determined. The more people analyzed the more accurate the location becomes. Again, RFLPs range from family to family so if a founder effect of the disease is possible as in this problem, the RFLPs will be highly similar and therefore accurate in gene location.

Once a general location is isolated (approximately within 100 genes) a process of elimination begins. The first step is to create probes for the possible genes. Then tissue type gene expression should be checked with the probes. For example, in the above problem, the tissue afflicted is the neuronal tissue. Neuronal cells could be isolated and expressed mRNA could be tested against the probes. This allows a narrowing down of the original 100 genes. You would not want to test diseased tissue first. Many diseases are recessive and may not be expressing the mRNA, therefore unknowingly eliminating your gene during the mRNA screening. The remaining genes could be reduced by a number of methods:

1) Literature searches of previous research on the genes of interest: do any of them function in a manner that could be related to your phenotype?

2) Genes could be checked across phyla or species to determine if the gene is highly conserved. Obviously the defected gene is an important one. Usually, genes essential for species natural selection are highly conserved. The wanted gene is probably highly conserved.

3) Now there are programs that predict protein structure based on gene sequence (although not very accurately). Form follows function; the predicted structure could be compared with analogous proteins for possible functions. [This last approach would probably only be useful if you had a hypothesis about the specific cellular function of your gene product, which you often will not.]

[When you've narrowed your search to one or a few genes, sequence them from DNA samples of people who you know to carry the disease allele. Compare these sequences with the human genome database, looking for differences that correlate with the presence of the disease allele (according to your pedigree analysis.) When you find a strong correlation, you have probably found your gene.]

With this knowledge, the mutated site can be analyzed and compared to any restriction enzymes. If you're lucky, a restriction site will be created or destroyed by the new mutation. This would give a direct test to determine whether individuals carried the mutation. If this is not an option, the RFLP associated with the disease could be used as a restriction marker for the disease.

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