Bio Lab Quiz #2

Question 1

Advantages as a model organism

Answer 1

short life cycle (~ 10 days) –> takes 1 day for egg to fertilize, pupa before egg hatch
small
easy to care for
genetic tools

Question 2

Distinguishing the sex of fruit flies

Answer 2

Female slightly bigger with larger strips along stomach and males have a black but

Question 3

Oogenesis - germline to mature cell steps

Answer 3

Egg has polarity (A/P, D/V)
- anterior head
- posterior but
- d/v is dorsal and ventral

Set up by mother (maternal genes)

Micropyle (anterior dorsal)

Dorsal appendages (anterior dorsal)

Once fertilized by sperm called embryo

Question 4

The Drosophila Reproductive Tract

Answer 4

2 ovaries, oviduct, uterus (takes up most of abdomen)

About 16 ovarioles/ovary

Ovariole-assembly line of egg production

Question 5

The basic unit of the Drosophila ovary
oogenesis starts
egg chamber

Answer 5

Oogenesis starts in the germarium

The germarium contains germline stem cells

Egg chambers bud off from germarium

The egg chamber has three cell types:
1 oocyte (germline), 15 nurse cells, lots of follicle cells

Question 6

Cyst formation in the Drosophila ovary steps

Answer 6

Germ line stem cells divide to form daughter stem cell and cystoblast

Cystoblast divides 4x (mitosis) but cytokinesis is incomplete

Cells remain connected by intercellular bridges (ring canals)

One cell becomes the oocyte, the rest nurse cells

Nurse cells provide mRNAs, proteins and organelles to the oocyte and eventually die give their life for oocyte

Question 7

Early embryogenesis

Answer 7

fertilized egg is called a zygote or one cell embryo

Nucleus divides several times w/o cell division

Nuclei migrate out
(syncytial blastoderm)

Nuclei become enclosed by membranes
(cellular blastoderm)

Maternal products in egg turn on zygotic genes that set up embryo polarity (A/P)

Question 8

Segmentation

Answer 8

Embryo arranged into segments
(3 head, 3 thoracic, 8 abdominal)

Question 9

Zygotic segmentation genes are embryonic lethal and controlled by a regulatory hierarchy of genes

Three genes which are

How Id?

Answer 9

Three classes of genes:
Gap genes
Pair rule genes
Segment polarity genes

ID mutants by looking at the larval cuticle (epidermis), denticle belts on ventral side in each segment

Question 10

Gap genes

Answer 10

Mutants lack: large region
Gene activated by: maternal genes
Normal expression pattern: broad region

Question 11

Pair rule genes

Answer 11

Mutants lack: every other segment
Gene activated by: gap genes
Normal expression pattern: seven stripes

Question 12

Segment polarity genes

Answer 12

Mutants lack: part of each segment
Gene activated by: pair rule genes
Normal expression pattern: 14 stripes (every segment)

Question 13

Direct Immunostaining

Answer 13

fluorophore attached directly to antibody of interest

Question 14

Indirect Immunostaining

Answer 14

primary antibody of interest is not attached to fluorophore

secondary antibody recognized primary antibody

Question 15

Immunostaining details

Answer 15

Fix: formaldehyde (preserves tissue, proteins

Washes: Triton (detergent), BSA (bovine serum albumin) both help to eliminate nonspecific staining

Propidium iodide: labels nucleic acids (DNA/RNA)

RNase: get rid of RNA (so only DNA—to visualize nucleus)

Vectashield- anti-fade

Anti-Orb (labels oocyte)

Anti-phosphotyrosine (labels intercellular bridges/ring canals)

Question 16

Orb protein is expressed in the

Answer 16

oocyte

Propidium iodide (nucleus) helps show protein/DNA

Question 17

Phosphotyrosine antibody labels

Answer 17

ring canals of developing egg chambers

Question 18

Larval Structures

Answer 18

imaginal disks
- Cells set aside during embryogenesis
- Packaged into disks
- Form adult structures (ex:wing)
- Larval body parts histolyzed (degraded)

polytene chromosomes
- Found in some tissues of insect larvae (including salivary glands)
- Chromosomes replicate but don’t separate
- Distinct pattern for each chromosome (can locate genes)
- Drosophila has 4 chromosomes (x, 2, 3, 4)

Why? Make multiple copies of gene so can quickly make a lot of gene product (protein)

Question 19

Polytene Chromosomes

Answer 19

“Puffs” in polytene chromosomes indicate transcription activation

Occurs in response to ecdysone (hormone important for molting and metamorphosis) or environmental factors such as heat shock

Heat shock induces expression of new genes (9 heat shock genes)

Called the heat shock response (can observe puffs at the 9 heat shock genes)

Heat shock proteins (hsps) are made

Hsps bind to other proteins to protect them during heat shock (called molecular chaperones). Prevent aggregation and help proper folding.

Question 20

Dark field blocks out direct light (increases contrast)

Answer 20

Used to look at Drosophila larvae

Question 21

Fluorescent Microscopy

Answer 21

Light = mercury bulb

Sample is labeled with a fluorophore (fluorescent molecule)

Absorbs light at one wavelength (excitation wavelength)

Emits light (photons) at another wavelength (emission wavelength)

Microscope detects photons

Question 22

Confocal Microscopy

Answer 22

Confocal Laser Scanning Microscopy (LSM)

Thick specimens appear blurry because light outside the focal plane increases background and lowers contrast.

Confocal microscopy eliminates the light outside the focal plane.

Illuminate sample with Laser light. Use pinhole to eliminate out of focus light.

Optical sectioning- obtain a stack of sections at different focal planes and make a 3D image.

Question 23

What cell type does orb label?

Answer 23

Anti-Orb (labels oocyte)
Propidium iodide (nucleus) helps show protein/DNA

Question 24

What are the three cell types that make up a Drosophila egg chamber?

Answer 24

1 oocyte (germline), 15 nurse cells, lots of follicle cells

Question 25

What is a fluorophore?

Answer 25

(fluorescent molecule)

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.

Question 26

What is the phenotype of a segment polarity mutant in Drosophila?

Answer 26

mutant lack part of each segment

Question 27

Maternal effect genes

Answer 27

female sterile mutants, depends on mothers genotype

Question 28

Zygotic genes

Answer 28

embryonic lethal mutants, depends on embryos genotype