THE UNIVERSITY OF CALGARY

FACULTY OF SCIENCE

MIDTERM EXAMINATION I

CMMB 403

Answers in bold-face

October 1, 1997 TIME: 50 minutes

TOTAL POINTS: 20

1. Answer either question A or B (5 points).

A. Discuss an experiment that demonstrates the role of a 3' UTR in regulation of translation of a specific transcript. Be as complete as possible. Be sure to interpret the results.

The 3' untranslated region (UTRs) of mouse protamine transcripts appears to determine when they are translated. The role of the 3' UTR was demonstrated by fusing the 3' UTR of mouse protamine 1 mRNA to the human growth hormone gene. Mice made transgenic for this transgene transcribed it before histone replacement but did not translate it until late in spermiogenesis when the endogenous protamine messengers were translated. Conversely, replacement of the protamine 3' UTR in the protamine 1 gene with that of human growth hormone allowed translation to commence immediately after transcription.

The translation of oskar transcripts is repressed outside the posterior domain. This repression is dependent upon the binding of a protein called Bruno to elements in the 3' UTR of oskar mRNA. In the absence of a functional bruno gene, oskar is translated prematurely. The Oskar protein that is synthesized accumulates throughout the oocyte, causing posteriorization of the embryo.

B. Discuss the complete sequence of events that lead to fusion of the egg plasma membrane and the sperm plasma membrane in mammals. Cite experimental evidence in support of your answer.

The egg is surrounded by the zona pellucida. One of the components of the zona, ZP-3, functions as a sperm receptor. The sperm-binding activity of ZP3 is mediated by the oligosaccharide side chains of ZP3. The role of the oligosaccharides is demonstrated by experiments in which either removal or modification of the sugars causes loss of sperm-binding activity.

A lectin in the plasma membrane overlying the acrosome of mouse sperm called sp56 binds to the oligosaccharide moieties of ZP3. Purified sp56 binds to the zona of unfertilized eggs, but not to that of zygotes. This observation suggests that the oligosaccharides on ZP3 trap incoming sperm at the zona surface of unfertilized eggs and that this activity is lost after fertilization.

There is a correlation between the presence of sp56 and species specificity of sperm-egg recognition. Mouse and hamster sperm contain sp56 and bind to the mouse egg. Guinea pig and human sperm, however, lack sp56 and do not bind. Different lectins may be involved in human and rabbit sperm-egg binding.

Binding of the sperm to the zona triggers the acrosome reaction, which allows the sperm to penetrate the zona. The sperm form a slit in the zona that is apparently formed through a combination of force and digestion by the enzymes released by the acrosome reaction.

After penetrating the zona, incoming sperm enter the perivitelline space surrounding the egg and land on the egg plasma membrane, where the equatorial segment of the sperm head initiates sperm-egg adhesion. A sperm protein called fertilin is thought to be involved in mediating adhesion in mammals. The disintegrin domain of fertilin is thought to interact with integrins on the egg surface, which serve as the sperm receptor. Various observations support this hypothesis, including: peptide analogs of the guinea pig fertilin disintegrin domain inhibited fertilization in vitro; integrins have been detected on mammalian egg surfaces.

The final step in fertilization is sperm/egg fusion. The initial site of fusion on the sperm is the equatorial segment. The antigenic properties of the equatorial segment change after the acrosome reaction. Furthermore, neutralizing antibodies against the equatorial segment of acrosome-reacted sperm block sperm/egg fusion. These results suggest that alterations to the equatorial segment facilitate sperm/egg fusion.

2. Answer either question A or B (5 points).

A. Describe how the synthesis and accumulation of 5S RNA are thought to be controlled during Xenopus oogenesis.

The promotor site for 5S RNA is located within the coding sequence. Initiation of transcription is initiated by an interaction between the promoter and three oocyte transcription factors. One of these factors is TFIIIA, which is a zinc finger protein. TFIIIA also binds to the 5S RNA after its synthesis. Thus, as 5S RNA begins to accumulate, it titrates out the available TFIIIA, thus attenuating further 5S RNA synthesis. After oocyte maturation, no additional 5S RNA is detectable due to the attrition of TFIIIA.

B. What effects does alpha-amanitin have on progesterone-induced oocyte maturation? Why?

Alpha-amanitin has no effect on maturation. Alpha-amanitin is an inhibitor of transcription. All events of maturation are mediated by cytoplasmic constituents.

3. Answer either question A or B (5 points)

A. What are the consequences for the organization of the oocyte microtubule cytoskeleton and localization of grk mRNA in top/DER mutant egg chambers? What are the developmental consequences?

The oocyte microtubule cytoskeleton is not properly polarized in egg chambers of these mutants, and they lack both anterior-posterior and dorsal-ventral patterning. For example, the GV fails to migrate to the anterior dorsal region of the oocytes and grk mRNA is not localized.

B. What are the developmental implications of the localization of oskar mRNA?

The localization of oskar mRNA has important implications for formation of the pole plasm. The pole plasm contains elements that are necessary for translation of nanos mRNA. This establishes a gradient in Nanos protein that is necessary for patterning of the posterior of the embryo. The pole plasm also contains determinants of the germ line. Oskar protein induces pole plasm assembly.

4. Provide clear definitions for five of the following terms (1 point each).

A. Totipotency

Totipotency is the capacity of a nucleus to promote the development of all cell types that make up an organism.

B. Homeotic transformation

Homeotic transformations convert a body part into another body part, as a result of mutation. An example is the conversion of the Drosophila antenna into a leg by the Antennapedia mutation.

C. Embryonic stem cells

Embryonic stem cells are undifferentiated cells that are derived by culturing an early mammalian embryo. Stem cells have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene knock-outs.

D. Germ plasm

The germ plasm is a cytoplasmic constituent that promotes development of the germ cell line. In electron micrographs it contains granulofibrillar components and mitochondria. In the amphibian oocyte, components with these characteristics are first seen in the mitochondrial cloud and later at the vegetal pole. In Drosophila, the germ plasm is found in the pole plasm at the prospective posterior end of the embryo. It is incorporated into the pole cells, which are the first cells to be formed in the otherwise syncytial embryo.

E. Leydig (interstitial) cells

Leydig cells are found in the mammalian testis in the connective tissue between seminiferous tubules. They secrete testosterone and other adrogens.

F. BMP8b

BMP8b is a growth factor in the bone morphogenetic protein family that is produced by developing germ cells in the male mouse and is essential for spermatogenesis. It is required for the resumption of male germ-cell proliferation at puberty and the maintenance of the germ cells in the adult.

G. Inhibin

Inhibin is a growth factor in the TGF-ß family. It is related to activin. It is secreted by the Sertoli cells in the male and by the corpus luteum in the female. It functions to suppress secretion of Follicle Stimulating Hormone (FSH) from the pituitary.

H. Nurse cells

Nurse cells are found in the ovaries of organisms with meroistic oogenesis. Nurse cells are derived from the germ cell line. In Drosophila, they are highly polyploid. They produce messenger RNA and organelles that are transported into the oocyte.

I. What is Vera, and what does it do?

Vera is a protein that binds Vg1 mRNA to vesicles of the endoplasmic reticulum and is proposed to assist in its transport to the vegetal cortex during oogenesis in Xenopus. Vera binds to a sequence element in the 3' UTR of the Vg1 mRNA

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