THE UNIVERSITY OF CALGARY

FACULTY OF SCIENCE

FINAL LECTURE EXAMINATION

CMMB 403

Answers in bold-face

December 14, 1996

TIME: 120 minutes


TOTAL POINTS: (35)

1.
a) Two genes, A and B, required early in Drosophila development are expressed in a spatially distinct pattern in the embryo. Explain the type of genetic experiment that would be used to determine whether these genes function in a hierarchy.

Take flies that are mutant for gene A and look at the expression of gene B. Then do the reverse experiment, looking at gene A expression in a B mutant. Expression is studied by in situ hybridization or immunochemistry. If the expression of one gene is altered from the wild-type patterns in a mutant of the other, then they interact. If there is no change in expression from the wild-type patterns in both crosses, then they don't interact according to this genetic test.

Assuming that they do, diagram the outcome of such an experiment using two known Drosophila spatial patterns of gene expression (2 points total for a).

Full marks were awarded only if the outcomes of both parts of the experiment were diagrammed.

b) How could germ-line transformation (P-element mediated gene transfer) be used to understand this interaction in more detail? (2 points).

There is no single right answer to this question. Answers must include a suggestion that something is feasible; e.g., insert a gene back into a mutant fly to see if the other gene's expression is returned to normal or overexpress one gene (i.e., using multiple copies) to see the effect on the expression of the other gene or use a heat shock promoter to express the gene ectopically and determine whether there is any change in the other gene's expression. Some people even mentioned putting a promoter region for the gene in front of a reporter gene and checking to see whether the expression of the reporter was normal for that gene. No mark was given for explaining P-element-mediated gene transfer. If the experiment describing Ubx ectopic expression in flies was described, it was credited with 1/2 mark unless it was extended to describe how these types of experiments could be used to study the interaction of the two genes mentioned in part 1(a). Full marks were given for two feasible suggestions.

c) Homologues of these Drosophila genes have been identified in the sea urchin and you want to know which parts of these genes are important for their spatial patterns of expression. Explain how fusion genes and mobility shift (gel retardation) assays could be used to study this. (3 points)

Put control regions (5'-promoter regions/enhancer elements) in front of a reporter gene (or replace the coding region with a reporter gene). Check for normal temporal and spatial expression patterns of the reporter gene to show that all the required elements are present. Trim away/dissect these control elements to determine what is minimally required for normal expression. Mobility shift assays are conducted by incubating labeled DNA fragments with nuclear extract ("proteins" accepted along with an explanation of where the proteins came from). Run on a gel along side the labeled DNA fragment alone. If the DNA fragment binds a protein(s) from the nuclear extract, it will run more slowly than the labeled DNA fragment alone. Check for specificity with cold competitor DNA.

2. Describe some of the characteristics of the control (promoter or enhancer) regions of spatially-regulated genes. (2 points)

1/2 mark was given for any of the following to 2 maximum:

3. You have just isolated a new embryonic lethal mutation that is a little peculiar because it displays three distinct phenotypes:

 

Phenotype 1 is a viable, minor anterior deformity.

Phenotype 2 is more severe: an anterior defect that is embryonic lethal.

Phenotype 3 is most severe: an embryonic lethal with a major anterior defect.


They appear in the following crosses:


+/- x +/- : 1/4 phenotype #1; 3/4 normal

+/- x -/- : 1/2 phenotype #2; 1/2 phenotype #3

-/- x +/- : 1/2 phenotype #1; 1/2 normal

-/- x -/- : all display phenotype #3

a) Please define and distinguish between maternal effect and zygotic mutations. (1 point)

Maternal effect mutations are mutations in maternally-encoded gene products. Phenotypes arise in the embryo when the mother is homozygous mutant for the gene, even if the embryo is heterozygous. Zygotic mutations occur in genes that are expressed in the embryo following fertilization. In this case the phenotype that arises in embryo is the result of the embryo's genotype and not the mother's.

b) How do maternal and zygotic genes differ in their RNA and protein expression? (1 point)

Maternal genes are transcribed and stored as maternal RNA during oogenesis. Depending on the organism, these maternal mRNAs are translated after oocyte maturation or fertilization. Zygotic genes are transcribed after fertilization (in some cases as late as the midblastula transition). Translation of zygotic mRNAs is coordinated with transcription as well as the degradation of maternal mRNAs.

c) Explain the difference between phenotype #1 and phenotype #2. Which phenotype represents the maternal effect phase, the zygotic phase and why? (3 points)

Phenotype 1 arises in a Mendalian fashion (based on cross 1) and represents the zygotic phase. Phenotype 1 cannot represent the maternal effect phase as it does not occur when the mother is homozygous mutant (cross 2). Only phenotype 2 and 3 arise when the mother is homozygous mutant. Therefore as the question is written phenotype 2 must represent the maternal effect phase (Note that when the reciprocal cross (cross 3) is performed the same phenotypes do not arise. The difference in this case is that the father is the homozygote and not the mother).

d) Why does this gene have both a maternal and a zygotic phase? How does phenotype #3 arise? (3 points)

This gene has both a maternal and a zygotic phase because both zygotic and maternal effect phenotypes arise. Therefore this gene is expressed and is important both maternally and zygotically. Phenotype 3 arises from the combined absence of maternal and zygotic product. It only occurs when the mother is homozygous mutant and the resulting embryo is homozygous mutant (crosses 2 and 4). This situation arises in all of the progeny resulting from cross 4 (because both parents are homozygous mutant) and 50% of the progeny in cross two because the father is heterozygous.

4. Define and state the significance of the following terms:

a) Zone of polarizing activity (3 points)

The zone of polarizing activity is a region at the posterior junction of the chick limb bud with the body wall that determines the anterior-posterior axis ofthe limb. The significance of the ZPA is demonstrated by transplanting it to ectopic positions in the limb bud. For example, if it is transplanted to the anterior margin, it causes a duplication of digits in mirror image symmetry. The ZPA is thought to emit a diffusible signal that determines anterior-posterior polarity depending upon its concentration. The highest concentration, which is at the posterior margin, specifies digit 4, whereas the lowest concentration, which is at the anterior margin, specifies digit 2. Insertion of a barrier at the apex of the limb bud to prevent diffusion of the putative morphogen into the anterior half prevents digit formation in front of the barrier. This suggests that the putative morphogen originates on the posterior side of the limb bud. Earlier work indicated that the morphogen might be retinoic acid, but recent work has failed to confirm that hypothesis.

b) Apical ectodermal ridge (3 points)

The AER is a ridge of thickened ectoderm that covers the distal tip of the limb bud of higher vertebrates. The AER is a transient structure that is necessary for the outgrowth of the limb. A region of undifferentiated mesenchyme (the progress zone) below the AER is necessary for its maintenance. In turn, the progress zone is dependent upon signals emanating from the AER. The essential role of the AER is demonstrated by experiments in which it is removed, which causes cell division in the progress zone to cease. If the AER is removed at progressively later stages of development, outgrowth of the limb ceases, resulting in distal truncations in elements of the limb.

The AER is sufficient to promote limb development from competent mesoderm. This is demonstrated by grafting a supernumerary AER to the side of a young limb, which causes additional limb elements to be formed. If an AER is combined with limb mesoderm in vitro, an outgrowth is produced. The eudiplodia mutation in the chick also provides evidence for the role of the AER. A dorsal extension if the AER in these mutants causes a second limb axis to form.

Extra credit: There is evidence that members of the FGF family of signaling molecules are produced by the AER and are responsible for its function.

c) Apoptosis (3 points)

Apoptosis is defined as the orderly and characteristic sequence of structural changes resulting in programmed cell death. Apoptosis is characterized by maintenance of intact cell membranes during the suicide process so as to allow adjacent cells to engulf the dying cell so that it does not release its contents and trigger a local inflammatory reaction. Cells undergoing apoptosis usually exhibit a characteristic morphology, which include: nuclear and cytoplasmic condensation, endolytic cleavage of the DNA into small oligonucleosomal fragments and eventual fragmentation of the dying cell into a cluster of membrane-bound segments (apoptotic bodies) that often contain morphologically intact organelles. Apoptosis during development is used to eliminate transitory tissues or to remodel tissues.

5. You have isolated two related cDNAs from a fibroblast cell line, U-DIE, library which was undergoing programmed cell death in response to serum starvation. You have determined that the RNAs corresponding to these cDNAs are alternatively spliced products of the YUCK gene and produces a 900 nucleotide (nt) long mRNA (YUCK-1) and a 700 nt long mRNA (YUCK-2). Since alternative splicing occurred in the open reading frame, these mRNAs encode two different-sized proteins identical in all aspects EXCEPT for a 30 amino acid N-terminal extension in the YUCK-1 protein.

a) You first confirmed that the RNA and corresponding proteins for YUCK-1 and YUCK-2 are specifically expressed in the serum-starved U-DIE cells. How did you go about doing this? (2 points)

Probes specific for the alternative RNA species and proteins are necessary to confirm expression. Expression of the two RNA species could be confirmed by Northern blotting. RNA would be isolated, separated on a gel, blotted and the blot probed with probes for the two RNA species. The probe for YUCK-1 will detect a 900 nt transcript, whereas the probe for YUCK-2 will detect both it and a 900 nt transcript. Nuclease protection assay would not allow for unambiguous detection of YUCK-2, although it would allow for detection of YUCK-1.

The proteins could be detected by Western blotting, using antibodies against both the unique component of YUCK-1 and the common elements. Probing blots with the YUCK-1-specific antibody would detect a single band, whereas the antibody against the common elements would detect two proteins.

A potential target for the YUCK proteins is the potent killer gene, CROAK. To test this possibility, you perform mobility shift assays on the two known CROAK promoters (A and B) using pure preparations of each protein. You obtain the following results:

 

b) What do the above results tell you about YUCK-1 and YUCK-2's ability to potentially regulate CROAK gene expression? (3 points)

Both proteins bind to promoter B specifically, as indicated by the ability of cold competitor B to abolish the binding of these proteins to labeled promoter B. There is no specific binding of either protein to promoter A.

6. Discuss the properties of the basic helix-loop-helix (bHLH) group of proteins, focussing on the prototypical bHLH protein, MyoD. Include in your answer how MyoD was discovered, its biochemical and functional properties, and its putative role in myogenesis, both in tissue culture cells and in the embryo. (4 points)

Fibroblasts treated with 5-azacytidine differentiated into myoblasts that fused to form myotubes. The gene responsible for this transformation was designated MyoD. 5-Azacytidine is thought to demethylate CpG islands upstream of the MyoD gene and divert fibroblasts onto the myogenic pathway. Transfection of MyoD into fibroblasts converted them into myogenis cells. Likewise, infecting a variety of cells with an RNA virus carrying MyoD caused the infected cells to express muscle-specific genes and to assume a myoblast morphology.

MyoD encodes a basic helix-loop-helix protein that functions as a transcriptional activator. The HLH domain facilitates dimerization, whereas the basic region (which contains positively-charged amino acids) mediates binding to DNA. Since the discovery of MyoD, a family af myogenic regulatory factors (MRFs) has been discovered, including Myf-5, myogenin and MRF4. These factors are expressed in a hierarchical fashion during myogenesis. Myf-5 and MyoD are expressed in myoblasts, myogenin is expressed after myoblast fusion and MRF4 is expressed after muscle differentiation.

The MRFs dimerize with members of the E-box family to form functional heterodimers. Although any E protein can pair with the MRFs, the most prevalent partner is E12. The MRF heterodimers bind to a sequence in the promoters of target called called the E box (CANNTG). Genes encoding MRFs, themselves, also have E boxes, by which they can regulate their own and one-another's transcription.

The MRFs are expressed sequentially in the somites during development. Myf-5 is expressed initially and is first seen in the medial somite cells. Next is myogenin, followed by MRF4. MyoD expression is first localized to lateral portions of the somites. Initially, Myf-5 and MyoD expression is mutually exclusive, but their expression later overlaps.

Mice that were null for MyoD overexpressed Myf5. Myf5 expression was also prolonged. This suggests that MyoD and Myf 5 are redundant and that Myf5 expression can compensate for the absence of MyoD expression. Knocking out both genes eliminated skeletal muscle development, indicating that their expression is essential to initiate myogenesis. Myogenenin knockouts developed myoblasts that were deficient in muscle differentiation. These mice express MyoD and Myf-5 but fail to express MRF4 and functional muscle proteins.

In summary, MyoD and Myf-5 initiate myogenesis in myoblasts. Myogenin controls myotube differentiation. MRF4 may be responsible for maintaining the differentiated stae.