THE UNIVERSITY OF CALGARY

FACULTY OF SCIENCE

MIDTERM EXAMINATION I

CMMB 403

Answers in bold-face

October 9, 1996 TIME: 120 minutes

TOTAL POINTS: 20

1. Fill in the blanks (0.5 points each).

The issue of how the Xenopus egg develops dorsal-ventral polarity during the cleavage stages has recently become clarified. Antisense oligo-knockout experiments have shown that elimination of ß-catenin is sufficient to eliminate the organizer (credit also given for Nieuwkoop center or dorsal mesoderm or dorsal-ventral axis). Likewise, elevated ß-catenin on the ventral side of a Xenopus embryo is sufficient to duplicate the embryonic axis. During normal development, ß-catenin is found in greater abundance on the dorsal side than on the ventral side. One way by which the abundance of ß-catenin can be regulated is by degradation, which is regulated by the process of phosphorylation by Xgsk-3. According to this hypothesis, the dorsal-ventral differences in ß-catenin abundance result in the formation of the region called the Nieuwkoop center on the dorsal side of the embryo.

ß-catenin is homologous with armadillo in Drosophila, whereas the protein that directly regulates it is homologous with shaggy. These proteins are in the Wnt (or wingless) signal transduction cascade. ß-catenin presumably functions by interacting with Xtcf-3 and regulating transcription of target genes in the nucleus (either directly or indirectly).

The organizer is formed as a result of signals that emanate from the Nieuwkoop center.

The original dorsal-ventral asymmetry of the Xenopus embryo is caused by cortical rotation, which is triggered by entry of the sperm.

2. Describe how the synthesis and accumulation of 5S RNA are thought to be controlled during Xenopus oogenesis (2.5 points).

Transcription is regulated by the interaction of the transcription factor TFIIIA with the internal promoter. After it is synthesized, the 5S RNA binds to free TFIIIA, thus titrating it, resulting in its eventual depletion and cessation of transcription.

3. Fill in the blanks (0.5 points each):

Both doral-ventral and anterior-posterior patterning of the Drosophila oocyte are dependent upon a signal that is encoded by the gurken gene. Precise localization of this signal depends upon redistribution of the gurken mRNA, which is a consequence of the function of microtubules. Once the anterior-posterior axis has been established,

bicoid mRNA becomes localized to the anterior end of the oocyte, whereas oskar mRNA becomes localized to the posterior end. The latter messenger is localized as the result of the function of microtubules.

Cite your evidence for the last statement (1.5 points):

oskar mRNA localization is abolished by treatment with colchicine.

What portion of this mRNA interacts with this entity (0.5 points)?

3' UTR

4. Provide clear definitions of the following terms (1.5 points each):

A. the zona reaction

The zona reaction is analogous to the cortical reaction in sea urchins. Hydrolytic enzymes released from the cortical granules into the perivitelline space harden the zona pellucida, making it refractory to sperm penetration and inactivating sperm receptors. The latter may involve a chemical modification to ZP-3, whereby sugar groups are hydrolyzed by glycosidases released from the cortical granules. The zona hardening mechanism is unknown, but it does not involve formation of glycoprotein cross-linkages.

b. MPF

MPF stands for Maturation Promotion Factor. MPF was first demonstrated by Yoshio Masui, who demonstrated that oocytes could be caused to undergo maturation by injecting them with cytoplasm from progesterone-treated eggs. Serial transfers of cytoplasm from maturing recipients will continue to induce maturation in recipients even though only the original donor was exposed to progesterone. MPF activity has also been found in cells of cleaving Xenopus embryos, oscillating with the period of the cell cycle, appearing in late G2, peaking in metaphase and disappearing by interphase. MPF is now known to be present with the same periodicity in a variety of cells and is thus referred to as M-phase Promotion Factor.

MPF was shown by Lohka and Maller to be comprised of two subunits: p34 (homologous to cdc2) and p45 (cyclin). Both of these constituents are kinase enzymes. MPF pre-exists in the oocyte in an inactive form. Progesterone triggers maturation by causing a reduction in the levels of cyclic AMP, which initiates translation of c-mos mRNA. c-mos activates the cyclin component of MPF by phosphorylation. The cyclin, in turn, activates p34 by phosphorylating it. p34 phosphorylates a number of proteins, including lamins and histone H1. Phosphorylation of lamins causes dissociation of the nuclear envelope, and phosphorylation of histone H1 facilitates chromosome condensation. c-mos also functions as cytostatic factor (CSF), which stabilizes cyclin and retaining MPF activity until fertilization. In somatic cells, cyclin is degraded during each cell cycle, which allows for progression through the cell cycles.

c. phospholipase C

An enzyme in the inositol triphosphate (IP3) pathway that catalyzes the conversion of PIP2 into IP3 and diacylglycerol (DAG). Its activity is activated by G protein in response to binding of sperm to a sperm receptor. The IP3 is released into the cytoplasm, where it releases Ca++ from the endoplasmic reticulum. The liberation of Ca++ triggers the cortical reaction that produces the slow block to polyspermy, whereas DAG remains in the membrane and activates the Na+/H+ pump that raises internal pH and triggers egg activation, including activation of protein synthesis. Phospholipase C is bound to the inner surface of the plasma membrane. Students note: A drawing of the IP3 pathway was helpful to demonstrate that you understood the relationships among the various components:

d. Pole cells of Drosophila (define and describe their origin and formation).

Pole cells are the presumptive germ cells in Drosophila. The Drosophila egg is centrolecithal and undergoes superficial cleavage to form a syncytial blastoderm. However, at the posterior pole, a group of energids enter the pole plasm and complete cleavage furroews segregate the nuclei into distinct cells that will later form the germ cells.

**************************