In contrast to water-soluble vitamins, which must be a part of our daily diet, fat-
soluble vitamins can be stored in the body in amounts sufficient for many
months. Suggest an explanation for this difference, based on solubilities.
Fat-soluble vitamins are found in high-fat food sources like yolks, liver, red meat like
beef, and some dairy products. Unlike water-soluble vitamins, any excess of fat-soluble
vitamins don't immediately leave the body. Instead, they're stored in the liver or those
fatty tissues for later use.
Cellular membranes are self-sealing—if they are punctured or disrupted mechanically, they
quickly and automatically reseal. What properties of membranes are responsible for this
important feature?
The driving force for these membrane formation is the Hydrophobic Interactions
The gram-negative bacterium Vibrio cholerae produces a protein, cholera toxin
(Mr 90,000), that is responsible for the characteristic symptoms of cholera:
extensive loss of body water and Na+ through continuous, debilitating diarrhea. If
body fluids and Na+ are not replaced, severe dehydration results; untreated, the
disease is often fatal. When the cholera toxin gains access to the human
intestinal tract it binds tightly to specific sites in the plasma membrane of the
epithelial cells lining the small intestine, causing adenylyl cyclase to undergo
prolonged activation (hours or days).
(a) What is the effect of cholera toxin on [cAMP] in the intestinal cells?
(b) Based on the information above, suggest how cAMP normally functions in intestinal epithelial cells.
(c) Suggest a possible treatment for cholera.
a) cAMP is cyclic AMP-dependent chloride channel known also as the cystic fibrosis
transmembrane conductance regulator or CFTR. When cholera toxin by the bacteria, it
binds to the intestinal cells known as enterocytes through the interaction of the
pentameric B subunit of the toxin with the GM1 ganglioside receptor on the enterocyte,
triggering endocytosis of the toxin. As a next step, the A/B cholera toxin undergoes
cleavage of the A1 domain from the A2 domain in order for A1 to become an active
enzyme. Once inside the enterocyte, the enzymatic A1 fragment of the toxin A subunit
enters the cytosol, where it activates the G protein Gs- alpha through an ADP-
ribosylation reaction that acts to lock the G protein in its GTP-bound form, thereby
continually stimulating adenylate cyclase to produce cAMP. The high cAMP levels
activate the cystic fibrosis transmembrane conductance regulator (CFTR), causing a
dramatic efflux of ions and water from infected enterocytes, leading to watery diarrhea.
b) cAMP is present as an ion channel in the apical or luminal membrane of crypt epithelial
cells. cAMP is responsible for secretion of water by the following steps: Chloride ions
enter the crypt epithelial cell by co-transport with sodium and potassium; sodium is
pumped back out via sodium pumps, and potassium is exported via a number of
channels. Activation of adenylyl cyclase by secretagogues leads to generation of cyclic
AMP. Elevated intracellular concentrations of cAMP in crypt cells activate the CFTR,
resulting in secretion of chloride ions into the lumen. Accumulation of negatively charged
chloride anions in the crypt creates an electric potential that attracts sodium, pulling it
into the lumen, apparently across tight junctions - and as a result sodium chloride is
secreted. Secretion of sodium chloride into the crypt creates an osmotic gradient across
the tight junction and water is drawn into the lumen.
c) Cholera is treated with the use of antibiotics. Tetracycline are more effective compared
to furazolidone. chloramphenicol and sulfa guanidine in reducing cholera morbidity.
Treatment with a single 300mg dose of doxycycline has shown to be equivalent to
tetracycline treatment. Stimulation of enkephalins, which regulate intestinal secretion
acts directly on enterocytes is an effective treatment. Enkephalins bind to the opioid
receptors on enterocytes, which act through G proteins to inhibit the stimulation of cAMP
synthesis induced by cholera toxin, thereby directly controlling ion transport and
diarrhea.
The base composition of phage M13 DNA is A, 23%; T, 36%; G, 21%; C, 20%.
What does this tell you about the DNA of phage M13?
The number of A residues doesn't equal the number of T residues, nor does the number
of G equal the numbers of C. Therefore, the DNA is not a base-paired double helix, the
M12 DNA is single stranded.
The E. coli chromosome contains 4,639,221 bp.
(a) How many turns of the double helix must be unwound during replication of the E. coli
chromosome?
Unwinding of the complementary strands completely allow the synthesis of new
strand on each template. With a given of 10.5 bp/turn of B-DNA and
approximation of 4.64x10^6 bp. 4.64x10^6/10.5 = 4.42x10^5 turns
(b) how long would it take to replicate the E. coli chromosome at 37 °C if two replication
forks proceeded from the origin? Assume replication occurs at a rate of 1,000 bp/s.
Under some conditions E. coli cells can divide every 20 min. How might this be
possible?
Chromosomal DNA replication in E coli starts at a fixed origin and proceeds bi-
directionally. Each replication for travels (4.64 x 10 bp)/2 = 2.32 x 10 bp during
replication. If we assume a replication rate of 1,000 bp/s, the time required for the
completion of DNA synthesis in each replication fork is (2.32 x 10 bp)/(1000 bp/s)
(60 s/min)) = 40 min
E. coli chromosome starts from two origins, and each proceeding bi-directionally
to yield four replication forks. It would take 20 min to complete the replication at
this point. However, we know there is only one replication origin in the E coli
chromosome.
(c) ) In the replication of the E. coli chromosome, about how many Okazaki fragments
would be formed? What factors guarantee that the numerous Okazaki fragments are
assembled in the correct order in the new DNA?
The Okazaki fragments in E. coli are 1,000 to 2,000 nucleotides long, so (4.64 x
10 nucleotides)/(2000 nucleotides) to (4.64 x 10 nucleotides)/(1000 nucleotides)
= 2,000 to 5,000 Okazaki fragments are formed. The fragments are firmly bound
to the template strand by base pairing, and each fragment is quickly joined to the
lagging strand by the successive action of DNA polymerase I and DNA ligase,
thus preserving the correct order of the fragments. A mixed pool of Okazaki
fragments, detached from their template, does not form during normal replication.
In a nutrient medium that lacks histidine, a thin layer of agar containing ~109
Salmonella typhimurium histidine auxotrophs (mutant cells that require
histidine to survive) produces ~13 colonies over a two-day incubation period at
37 °C. How do these colonies arise in the absence of histidine? The experiment
is repeated in the presence of 0.4 μg of 2-aminoanthracene. The number of
colonies produced over two days exceeds 10,000.What does this indicate about
2-aminoanthracene? What can you surmise about its carcinogenicity?
With the help of Ames test these results describes evaluation of 2-
aminothracene carcinogenicity. Ames test is widely used, relatively
inexpensive, rapid and accurate screening test. For the Ames test, an
auxotrophic, histidine-requiring strain of Salmonella enterica serotype
Typhimurium is used. . If inoculated onto a plate of nutrient medium
lacking histidine, no colonies will appear because in this auxotrophic strain
the gene inducing histidine synthesis is mutated and hence not active. The
presence of 13 colonies from 109 inoculated cells can be explained by
spontaneous mutations. The same experiment with the presence of 0.4
micrograms of 2-aminoanthracene resulted in 10,000 colonies. The agent
2-aminoanthracene mutated the bacterial (his-) gene back to the wild type
(his+), so visible colonies appeared on the medium. Because the agent 2-
aminoanthracene is a mutagen, it is therefore a possible carcinogen in
humans
