MBB 141 Question 19 and 20

Sara is an undergraduate student who is doing an internship in the research laboratory described above. Just before Sara started working in the lab, the restriction map above was made of the 47-kb NotI restriction fragment containing the prion-protein gene (distances between restriction sites are in kb).
Since smaller DNA fragments cloned into plasmids are more easily analyzed than large DNA fragments cloned into BACs, Sara has been asked to “subclone” the 6.1-, 10.5-, 4.1-, and 8.2-kb BamHI DNA fragments containing the prion-protein gene into the pUC19 plasmid vector. Her mentor gives her some intact pUC19 plasmid DNA, some of the purified 47-kb NotI fragment, and shows her where the lab’s stocks of DNA ligase and BamHI are stored. Describe the steps Sara should take to complete her task. In your answer, address how she will identify plasmids that contain genomic DNA inserts, and how she will verify that she has identified clones containing each of the desired genomic BamHI fragments.

20. Imagine that you have been able to clone the structural gene for an enzyme in a catecholamine biosynthesis pathway from the adrenal gland of rats. How could you use this cloned DNA as a probe to determine whether this same gene functions in the rat brain?

MBB 141 Questions 18-

18. The investigators in Question 17 were successful in purifying a BAC-DNA clone containing the gene for the mouse prion protein. They narrow down which region of the BAC DNA contains the prion-protein gene, they purified the BAC DNA, digested it with the restriction enzyme NotI, and separated the products of the enzymatic digestion by size using gel electrophoresis. Then, they purified each of the relatively large NotI DNA fragments from the gel, digested each individually with the restriction enzyme BamHI, and separated the products of each enzymatic digestion by size using gel electrophoresis. Finally, they transferred the size-separated DNA fragments from the agarose gel onto a membrane filter using the Southern blot technique, and allowed the DNA fragments on the filter to hybridize with a labeled cDNA probe. The figure above shows the results that were obtained: The pattern of DNA bands seen after the BAC DNA fragment is digested with NotI is shown in panel A; the pattern of DNA bands seen after each NotI fragment is digested with BamHI is shown in panel B; and the pattern of hybridizing DNA fragments visible after probing the Southern blot is shown in panel C.

MBB 141 Questions 1-17

1. A piece of DNA 900 bp long is cloned and then cut out of the vector for analysis. Digestion of this linear piece of DNA with three different restriction enzymes singly and in all possible pairs of enzymes gave the following restriction fragment size data

Enzymes	Restriction fragment sizes (bp)
EcoRI	200, 700
HindIII	300, 600
BamHI	50, 350, 500
EcoRI + HindIII	100, 200, 600
EcoRI + BamHI	50, 150, 200, 500
HindIII + BamHI	50, 100, 250, 500

Construct a restriction map from these data

2. The ability of complementary nucleotides to base pair using hydrogen bonding, and the ability to selectively disrupt or retain accurate base pairing by treatment with chemicals (e.g., alkaline conditions) and/or heat is critical to many methods used to produce and analyze recombinant DNA. Give three examples of methods that rely on complementary base pairing, and explain what role complementary base pairing plays in each of these methods.
3. A new restriction endonuclease is isolated from a bacterium. This enzyme cuts DNA into fragments that average 4,096 base pairs long. Like many other known restriction enzymes, the new one recognizes a sequence in DNA that has twofold rotational symmetry. From the information given, how many base pairs of DNA constitute the recognition sequence for the new enzyme?
4. E. coli, like all bacterial cells, has its own restriction endonucleases that could interfere with the propagation of foreign DNA in plasmid vectors. For example, wild-type E. coli has a gene, hsdR, that encodes a restriction endonuclease that cleaves DNA that is not methylated at certain A residues. Why is it important to inactivate this enzyme by mutating the hsdR gene in strains of E. coli that will be used to propagate plasmids containing recombinant DNA?
5. Suppose you have cloned a eukaryotic cDNA and want to express the protein it encodes in E. coli. What type of vector would you use, and what features must this vector have? How would this vector need to be modified to express the protein in a mammalian tissue culture cell?
6. Suppose you wanted to produce human insulin (a peptide hormone) by cloning. Assume that you could do this by inserting the human insulin gene into a bacterial host where, give the appropriate conditions, the human gene would be transcribed and then translated into human insulin. Which would be better to use as your source of the gene: human genomic insulin DNA or a cDNA copy of this gene? Explain your choice.
7. You have inserted human insulin cDNA in the cloning vector pUC19 and transformed the clone into E. coli, but insulin was not expressed. Propose several hypotheses to explain why not.
8. Genomic libraries are important resources for isolating genes and for studying the functional organization of chromosomes. List the steps you would use to make a genomic library of yeast in a plasmid vector. In what fundamental way would you modify this procedure if you were making the library in a BAC vector?
9. The human genome contains about 3 x 10^9 bp of DNA. How many 20 kb fragment would you have to clone in a BAC library to have a 90% probability of including a particular sequence?
10. Suppose a researcher wants to clone the genomic sequences that include the human gene for which a cDNA has already been obtained. She has available a variety of genomic libraries that can be screened with a probe made from the cDNA.
1. Assuming that each library has an equally good representation of the 3 x 10^9 base pairs in a haploid human genome, about how many clones should be screened if the researcher wants to be 95% sure of obtaining at least one hybridizing clone and

i. The library is a plasmid library with inserts that are, on average, 7 kb?

ii. The library is a YAC library with inserts that are, on average, 1 Mb?

2. What advantages and disadvantages are there to screening these different libraries?
3. What kinds of information might be gathered from the analysis of genomic DNA clones that could not be gathered from the analysis of cDNA clones?
10. A researcher interested in the control of the cell cycle identifies three different yeast mutants whose rate of cell division is temperature-sensitive. At low, permissive temperatures, the mutant strains grow normally and produce yeast colonies having a normal size. However, at elevated, restrictive temperatures, the mutant strains are unable to divide and produce no colonies. She has a yeast genomic library made in a plasmid shuttle vector, and wants to clone the genes affected by the mutants. What steps should she take to accomplish this objective?
11. It’s 3 am. Your best friend has awakened you with yet another grandiose scheme. He has spent the last 2 years purifying a tiny amount of a potent modulator of the immune response. He believes that this protein, by stimulating the immune system, could be the ultimate cure for the common cold. Tonight, he has finally been able to obtain the sequence of the first seven amino acids at the N-terminus of the protein: Met-Phe-Tyr-Trp-Met-Ile-Gly-Tyr. He wants your help in cloning a cDNA for the gene so that he can express large amounts of the protein and undertake further testing of its properties. After you drag yourself out of bed and ponder the sequences for a while, what steps do you propose to take to obtain a cDNA for this gene?
12. A piece of DNA 5,000 bp long is digested with restriction enzymes A and B, singly and together. The DNA fragments produced are separated by DNA electrophoresis and their sizes are calculated, with the following results:

Digested with

A	B	A+B
2,100 bp	2,500	1,900
1,400	1,300	1,000
1,000	1,200	800
500		600
		500
		200

Each A fragment is extracted from the gel and digested with enzyme B, and each B fragment is extracted from the gel and digested with enzyme A. The sizes of the resulting DNA fragments are determined by gel electrophoresis, with the following results

A fragment	Fragments produced by digestion with B	B fragment	Fragment produced by digestion with A
2,100 bp	1,900; 200	2,500	1,900; 600
1,400	800; 600	1,300	800, 500
1,000	1,000;	1,200	1,000; 200
500	500

Construct a restriction map of the 5,000 bp DNA fragment.

14. A 10-kb genomic DNA EcoRI fragment from a newly discovered insect is ligated into the EcoRI site of the pUC19 plasmid vector and transformed into E. coli. Plasmid DNA and genomic DNA from the insect are prepared and each DNA sample is digested completely with the restriction enzyme EcoRI. The two digests are loaded into separate wells of an agarose gel, and electrophoresis is used to separate the products by size.
1. What will be seen in the lanes of the gel after it is stained to visualize the size-separated DNAs?
2. What will be seen if the gel is transferred to a membrane to make a Southern blot, and the blot is probed with the 10-kb EcoRI fragment? (Assume that the fragment does not contain any repetitive DNA sequence.)
15. During Southern blot analysis, DNA is separated by size using gel electrophoresis, and then transferred to a membrane filter. Before it is transferred, the gel is soaked in an alkaline solution to denature the double-stranded DNA, and then neutralized. Why is it important to denature the double-stranded DNA? (Hint: Consider how the membrane will be probed.)
16. A researcher digests genomic DNA with the restriction enzyme EcoRI, separates it by size on an agarose gel, and transfers the DNA fragments in the gel to a membrane filter using the Southern blot procedure. What result would she expect to see if the source of the DNA and the probe for the blot is as described as follows?
1. The genomic DNA is from a normal human. The probe is a 2.0-kb DNA fragment obtained by excision with the enzyme EcoRI from a plasmid containing single-copy genomic DNA.
2. The genomic DNA is from a normal human. The probe is a 5.0-kb DNA fragment that is a copy of a LINE (long interspersed elements) sequence with an internal EcoRI site.
3. The genomic DNA is from a normal human. The probe is a 5.0-kb DNA fragment that is a copy of a LINE sequence that lacks an internal EcoRI site.
4. The genomic DNA is from a human heterozygous for a translocation (exchange of chromosome parts) between chromosomes 14 and 21. The probe is a 3.0-kb DNA fragment that is obtained by excision with the enzyme EcoRI from a plasmid containing single-copy genomic DNA from a normal chromosome 14. The translocation breakpoint on chromosome 14 lies within the 3.0-kb genomic DNA fragment.
5. The genomic DNA is from a normal female. The probe is a 5.0-kb DNA fragment containing part of the testis determining factor gene, a gene located on the Y chromosome.
17. A molecular genetics research laboratory is working to develop a mouse model for bovine spongiform encephalopathy (BSE, “mad cow”) disease, which is caused by misfolding of the prion protein. As part of their investigation, they want to investigate the structure of the gene for the prion protein in mice. They have a mouse genomic DNA library made in a BAC vector and a 2.1-kb long cDNA for the gene. List the steps they should take to screen the BAC library with the cDNA probe.

BSIT Practical Exams 8 Oct 2008, 1:00 pm

1. The order of exam takers will be group 6, 2, 4, 1, 3, 5. Please arrive on time. If you are not around when your group takes the exam, you will be given one more test that does not appear in the pointers. This extra question will have something to do with the algae experiment.

2. Each person will answer 1 out of the 7 test items given in the pointers. Persons will be randomly assigned to items before the exam. You have 15 minutes to answer the item assigned to you. Answers will be submitted on the exam sheets, except for those assigned computer problems; in their case, they will show their solutions on the computer.

3. Grading will be as follows. Each item is worth 100 points, distributed as follows: 0, 20, 40, 60, 80, 100, with no in-betweens. Your exam grade will be the average between your performance in the item and the group performance average. Thus, if you get a performance grade of 0, and your group as a whole got a performance grade of 60, your personal grade for the exam will be 30. If in the same group you got 100 for performance, your exam grade will be 80.

4. Bring your notebooks. I will check them during the exam.

5. You have had two weeks to prepare. I wish I could say "good luck", but 2 weeks was more than enough to bring you above luck. See you on Wednesday!

BSIT pointers for final exams bio lab 081008

1. Computer work with given data (lettuce lengths)
a. generate the histogram
b. find the average
c. find the standard deviation
d. get the 95% confidence interval (2pts)

2. Computer work with given data (algal growth)
a. calculate theoretical population numbers from a sigmoid curve or carrying capacity model
b. calculate chi-squares between observed and theoretical models
c. determine the best-fitted rmax using chi-squares (3pts)

3. Draw correctly
a. insect versus arachnid: highlight the differences (2pts)
b. annelid versus millipede: highlight the differences (2pts)
c. hydra

4. Computer work with given data (lettuce and hydra growth)
a. Calculate the IC50 from lettuce data
b. Calculate the EC50 from hydra data (2pts)
c. Calculate the LD50 from hydra data (2pts)

5. Draw correctly and label parts
a. dissected cockroach gastrointestinal tract with malpighian tubules
b. paramecium
c. vorticella with its stalk
d. euglena
e. tardigrade

6. Various
a. diagram how to prepare a serial two-fold dilution
b. draw the lettuce complete set-up: 8 petri dishes and their contents (including volumes and concentrations)
c. draw the hydra complete set-up: two 12-well culture plates with their contents (including volumes and concentrations)
d. draw the two forms of abnormal hydra and a normal hydra
e. describe how to prepare a solution of a given ppm

7. Draw and label
a. Warburg apparatus and ant set-up
b. rotifer
c. planaria
d. coleoptera
e. lepidoptera

BioIT S1 08-09: Assignment for 30 July 2008

The following groups will demonstrate the following projects on Aug 6, 2008.

All of them involve research, checking out Internet sites and biology books.

They also involve some trial and error, practice, and planning. You will be making mistakes. I therefore suggest you start immediately.

I am available the whole week to provide assistance.

I. Group 6
4 Gatorade bottles containing the following:
1. Fruitflies, all female.
2. Fruitflies, all male.
3. Fruitfly larvae only
4. Fruitfly pupae only

Discuss how you went about your project.
Describe the life cycle and anatomy of the fruitfly.
Briefly present a simple research article on fruitflies that demonstrates their usefulness to man.
Note: Your main problem is that it takes up to 10 days to get larvae and pupae; so start immediately.

II. Group 2
Construct an ant farm.
Describe how you went about your project.
Describe the life cycle, anatomy, and social structure and behavior of ants.
Briefly present a simple research article on ants that demonstrates their usefulness to man.
Note: the farm itself is easy to construct. The trick is finding the ants.

III. Group 3
Construct a Warburg apparatus and demonstrate that it works for insects.
Describe how you went about your project, from construction to calculation.
Briefly present a simple research article that used a Warburg apparatus or something similar.
Note: Any insect will do, but larger ones give better readings. The difficult part is the construction of the apparatus itself. Though most components are simple materials, the capillary tubes are very fragile, and it is a challenge to seal the connections without breaking them. The apparatus is also very sensitive--even the temperature from your fingers will cause you problems. You'll get the hang of it, though.

IV. Group 1
Determine the wavelengths of light to which Euglena are most phototaxically attracted.
Describe how you went about your project.
Describe Euglena and phototaxis in Euglena.
Briefly present a simple research article that involves Euglena and/or phototaxis.
Note: The challenge here is to construct two mini dark rooms that allows you to split light and expose parts of the culture to only one color of light. The first dark room will use multi-colored cellophane; a second darkroom will use a prism. Differences in results are expected.

V. Group 4
(Warning: Some amoeba are potentially deadly, and their cysts or eggs can remain undetected in the water sample. Thus, the project entails some risk, particulary when isolating pure amoeba. Do not touch any part of your face when working with amoeba. Wear gloves and always wash your hands before and after working.)

Isolate amoeba using agar plates and pond water.
Describe amoeba and their anatomy.
Briefly present a simple research article on amoeba, demonstrating their importance to man.
Note: Although amoeba are very common in the wild, it is not guaranteed that you will be able to isolate them. Knowing where to look and how to collect ups your chances. And since they are transparent, they will not be easy to detect. That's what the agar is for: we will detect them because they eat the agar and will form visible canals.

VI. Group 5
Produce zebrafish embryos, and present a slide show of photographs you took in the course of observing their development.
Describe how you went about the project.
Describe zebrafish, their anatomy and life cycle, and their embryonic development.
Briefly present a simple research article on zebrafish, demonstrating their importance to man.
Note: The big challenge here is how to separate the females from the males. Getting the embryos is easy after that.

BioIT Sem 1 08-09: Protists

Microscopic animals and their parts

1. Paramecium
a. Cytoplasmic streaming: moving cytoplasm around the edges of the animal
b. Contractile vacuole: located centrally; circular; tends to shrink and expand; there may be two of them
c. Nucleus: dark-colored central body; usually circular
d. Peristome: median location; the funnel-shaped opening that leads to the esophagus
e. Esophagus: constriction that leads from the peristome to the food vacuole
f. Food vacuole: end of the esophagus; dark-colored if it contains food

2. Rotifer (multicellular)
a. Corona: the anterior ring lined with ciliated cells
b. Eyespot: small dark-colored spot located immediately posterior to the corona
c. Stomach: large, central, dark-colored chamber (if it contains food)
d. Vitellarium: the outer transparent skin
e. Foot: the long posterior organ that serves for attachment
f. Toe: the end of the foot

3. Planaria (multicellular)
a. Ocelli: the two eyespots
b. Mouth: the opening of the gastrointestinal tract, located ventral and medial
c. Pharynx: passage from the mouth to the two lateral branches of the gastrointestinal tract

4. Hydra (multicellular)
a. Mouth: located at the base of the tentacles
b. Tentacles: arms
c. Ectoderm: the outer layer of the skin
d. Gastroderm: the lining of the digestive cavity
e. Digestive cavity: hollow cavity where digestion takes place
f. Mesoglea: layer of cells between the ectoderm and the gastroderm

5. Euglena
a. Flagellum: whip-like motor organelle located posteriorly
b. Nucleus: large, round, central, dark-colored body; distinguished from the smaller eyespot
c. Eyespot: small, red-colored photoreceptor located anteriorly
d. Chloroplasts: green-colored organelles generally distributed throughout the body

6. Vorticella
a. Three rows of cilia: motile organelles located anteriorly forming three rows
b. Buccal funnel: or mouth; at the base of the rows of cilia
c. Superficial pellicle: the outer covering of the animal
d. Vacuole: contractile organelle located inside the animal
e. Macronucleus: central, dark body containing DNA; in the form of a horse-shoe
f. Peduncle: coiled spring-like appendage that attaches the animal to the substrate

REPORT ON

1. Taxonomy, anatomy, habitat, and food requirements.

2. Significance to humans. Summarize a recent research article to illustrate. For example: Upadhyaya A et al. Power limited contraction dynamics of Vorticella convallaria: an ultrafast biological spring. Biophys J. 2008, 94(1):265-272. “Vorticella convallaria is one of the fastest and most powerful cellular machines. The cell body is attached to a substrate by a slender stalk containing a polymeric structure---the spasmoneme. Helical coiling of the stalk results from rapid contraction of the spasmoneme, an event mediated by calcium binding to a negatively charged polymeric backbone. We use high-speed imaging to measure the contraction velocity as a function of the viscosity of the external environment and find that the maximum velocity scales inversely with the square root of the velocity. This can be explained if the rate of contraction is ultimately limited by the power delivered by the actively contracting spasmoneme…etc.”. Explain this to us in layman’s terms, especially the significance to humans. You may use illustrations and other strategies to make the presentation good. Suggested sources: PUBMED (a search engine that will give mostly summaries); Scientific American.