Question

1. What are the official names of the genes that code for the C.bo two-component proteins that control toxin expression?

2. What does a protease do? A nuclease? A kinase? A DNA polymerase? An RNA polymerase?

3. What is the function of the wHTH effector protein? Of the BotR protein? Of the HA70 (hemagglutinin) protein (or any hemagglutinin protein)? Of the botA protein?

4. You have a 5kb linear fragment of DNA and you cut it with a Hind III restriction enzyme that has a recognition site at 500 bp from the left end and 1500 bp from the right end. Then you run the digested DNA on an agarose gel. Drae a picture placing the expected sizes of DNA on the gel.

5. You have the same DNA fragment as above but it is circular and not linear. You cut it with the same restriction enzyme (Hind III) and the restriction recognition sites are in the same places. What would this look like on an agarose gel?

6. You have a 4.5 kb linear fragment of DNA and you cut it with Pst I restriction enzyme that has a recognition site at 1500bp from the left end and 1500bp from the right end. What would this look like on an agarose gel?

7. You did a restriction digest on a linear DNA fragment 3000bp long. You ran it on the gel and discovered that the DNA was not cut. What reasons could yield this result?

8. If you want to cut a fragment of DNA with a restriction enzyme and you know that the DNA sequence has an EcoRI restriction site in it. (You use a computer to identify the EcoRI restriction site.) You look in your freezer for restriction enzymes and find that you only have Hind III and have no EcoRI. Could you substitute HInd III and have it cut the DNA at the same place as EcoRI?

Answer 1

1. The names of genes enoded by Clostridium botulinum for toxin expression are - CBO0787 - a sensor histidine kinase and CBO0786- a Response Regulator protein. These two together form a signaling pathway called 2 component histidine kinase pathway.

2. Protease - enzymes which degrades protein or peptide molecules. Eg pepsin, trypsin

Nuclease - enzymes that degrade nuclic acid these can be both exonuclease (cleaving from ends) or endogenous ( cleaving at sites within the sequence). Eg. ECORI, HINDIII.

Kinase - are enzymes that add phosphate to Hygroxyl group of certain amino acids like histidine, serine and theories. They are post translational modifications on the protein. Eg histidine kinase, protein kinase C.

DNA polymerase - required to make multiple copies of DNA. An enzyme that synthesizes DNA from deoxyribonucleotide using a template DNA.

RNA POLYMERASE- this is an enzyme that synthesizes RNA from a DNA template using ribonucleosides.

Question no. 4 is in the image.