Transcript
MCDB 4650 DEVELOPMENTAL BIOLOGY Spring 2006
Name_______________________________ Lab Section______
Quiz 3 (Answers) (Classes 7-10) 2/21/06
You will have 25 minutes to complete this quiz. Note that 1
point is equivalent to 1 minute, and budget your time Page 1 (12) ______
accordingly. Be sure to put your name on both pages. Page 2 (13) ______
Quiz is worth 25 total points. Continue answers
on reverse of the same page if you need more space. Total (25) _______
1. (6) In a genetic screen for additional mutations affecting vulval development, you have isolated a mutation that causes a Muv phenotype and does not map near any of the known vulval genes. You conclude the mutation defines a new gene, which you name lin-101.
a) (3) What is the most likely normal function of the lin-101 gene, and what experiment would you do to provide more evidence for your answer?
Probably lin-101 turns off vulval development (in non-VPC cells), but this is only true if it is an lf mutation. As a first test, you could determine whether it is recessive, which generally indicates an lf mutation.
b) (3) What experiment would you do to find out if lin-101 acts upstream or downstream of the let-60(ras) gene in the ras pathway controlling vulval development, and how would you interpret your answer?
Do an epistasis test by making the double mutant, let-60(lf);lin-101(lf) (best to use actual mutants; RNAi is an alternative). Your answer had to also include interpretation: if the phenotype is Muv, lin-101 is probably downstream; if Vul, it is probably upstream.
2. (6) The LIN-3 protein is an essential component of the RTK/ras pathway that controls vulval formation in C. elegans. Animals homozygous for a lin-3(lf) mutation form no vulva (Vul phenotype). Remember that all cells in the somatic gonad are descended from P1, while all the vulval precursor cells (VPCs) in the ventral hypodermis are descended from AB. In experiments on LIN-3 function, you start with a strain that is homozygous for a lin-3(lf) mutation, but that carries a small free duplication including a wild-type lin-3 gene, so that most of the animals have a wild-type phenotype. You isolate several animals developed from embryos that lost the free duplication in the AB cell at the 2-cell stage, but maintained the duplication in all cells derived from P1. Vulval formation is normal in these mosaic animals.
a) (2) Does the LIN-3 gene product act cell-autonomously or non-autonomously in vulval development? Explain your choice briefly.
Non-autonomously. The genotype of the VPCs (lin-3-) does not reflect their phenotype.
b) (2) Explain why these mosaic animals display no vulval phenotype even though none of the VPCs from which the vulva forms have a functional lin-3 gene. The lin-3(+) gene is expressed in the AC, which is located in the somatic gonad. The LIN-3 ligand transmits an inducing signal to the VPCs, which do not require the ligand for the subsequent function of the ras pathway in causing vulval development; therefore, it makes no difference to the VPCs that they lack functional lin-3, since they do not normally express it anyway. They still have the receptor for lin-3, thus they receive the signal just fine.
c) (2) What do you expect would be the phenotype (Vul, Muv, or normal vulval formation) in animals developed from embryos that lost the duplication in the P1 lineage and retained it in all AB-derived cells? Explain briefly. It would be Vul. The AC would not produce the inducing signal; therefore the VPCs, although lin-3(+), would not be induced to form the vulva.
3. (3) What special features of the early Drosophila embryo allows patterning of its anterior and posterior axis to be determined by overlapping gradients of transcription factors? Explain.
Its syncytial nature allows the spread of transcription factors that normally would remain inside individual cells. Most of these factors are maternally transcribed and deposited into the embryo. The proteins can diffuse along the length of the embryo through the common cytoplasm. The identity of individual cells is determined by relative levels of different tx factors.
(answer should mention syncytium, maternally transcribed factors, and that there is a gradient or that they act as morphogens)
4. (5) You have cloned a gene you think is involved in posterior development in the Drosophila embryo. Curious about how important this gene is during early development, you wish to overexpress this transcript in the anterior of the cellularized embryo. How do you do it? Explain how you generate the flies, what the constructs look like, and how the system works to actually target the production of your gene product only where you want it.
Pick a gene you know is transcribed in the anterior of the cellularized embryo (for example, hunchback). You don’t actually have to remember the name of the gene. Fuse the promoter region for this gene to the GAL4 sequence. Make a transgenic fly carrying that construct. (2 pts) Make a second transgenic fly carrying the UAS sequence fused to the cDNA for your posterior gene. (2 pt) Mate the two flies together and look at their progeny. In the progeny, GAL4 will be expressed in the anterior cells only. All cells in the fly embryo have the UAS-posterior gene construct, but the posterior gene will be activated only when GAL4 is around to bind to the UAS. (1 pt for description demonstrating understanding that the GAL4 is expressed only in anterior)
Another good answer would be to make a transgenic fly where a promoter for an anterior gene was hooked to the gene you’ve cloned.
If you suggested simply injecting posterior cytoplasm, you weren’t testing the specific gene you had just cloned.
If you suggested taking your cloned gene and just injecting it into the anterior cytoplasm, that might work as long as it is incorporated and transcribed. The best method, however, is the one described.
5. (5) You are studying a human disease known as Familial Hypertrophic Cardiomyopathy (FHC), which causes enlargement of the heart and is responsible for sudden deaths in some young athletes. The disease has a dominant pattern of inheritance, and is caused by mutations in the cardiac myosin II gene.
a) (3) What would be the simplest and quickest approach to making a mouse model of FHC in which a mutant version of the gene was expressed only in the heart muscle? (You should be able to think of an answer that does NOT use the specialized techniques described in lecture today). Include all information about how you generate the mouse.
Make a mutant version of cardiac myosin II, fused to a heart-specific promoter (so it will only be expressed in the heart), then inject this DNA into the pronucleus of a fertilized mouse egg. (Three components to this answer: promoter, mutant version of gene, inject into pronucleus). If you said generate a cre-lox mouse with homologous recombination, and target removal only to the heart, and you actually described it fully, that of course also receives full credit (even though technically, didn’t need to know that yet, since the quiz didn’t cover today’s lecture).
b) (2) The strains you generate in (a) display a wide range of phenotypes. What is a reasonable explanation for why each strain is slightly different? Incorporation of the transgene is random—it could go into a region of DNA that is not highly transcribed, not transcribed at all, or highly transcribed.
