The effect of the nonsense mutations would be revealed under intermediate trp levels where the attenuator normally modulates the transcription termination efficiency. Normally at intermediate trp concentrations some transcripts terminate and some proceed through the operon, depending on how fast the trp codons in the leader are translated. Changing them to stop codons would cause the ribosomes to stall there, effectively mimicking extreme trp starvation leading to maximum expression of the operon.
The trp operon would be expressed at maximum levels in the absence of tryptophan, since the trp repressor would be unbound and the mutant leader region would allow full expression.
b)
A lacI-lacZ fusion gene has been identified that produces a chimeric protein with both repressor and ß-gal function. What Lac phenotype(s) would you expect for a strain carrying this fusion gene.The mutant should express lacZ activity constitutively but at very low levels since expression of the fusion protein would be driven from the lacI promoter.
This would be phenotypically Lac- since the low-level expression would not allow growth on Lactose. Remember there is no way to induce the synthesis of the fusion protein or the lacY gene because the lac promoter-operator region is deleted.
c)
When an Hfr strain carrying a lambda prophage is mated to a non-lysogenic recipient the prophage is induced to grow lytically shortly after transfer to the recipient. What does this suggest about the mechanism by which the lambda prophage is maintained in the bacterial chromosome?This is exactly analogous to the PaJaMo experiment and indicates that lysogeny is controlled by repression.
d)
When a male from a P strain of Drosophila is crossed to a M type female the progeny are usually sterile. If an M or P type male is crossed to a P type female the progeny are fertile.i)
What does this imply about the mechanism by which P element transposition is controlled?This is exactly analogous to the case with lambda above and to the PaJaMo expt. You bring P elements into a M cytotype and get transposition. The other way around or P into P nothing happens. Control is negative.
ii)
Why are of the progeny of the P � X ™ M cross viable but sterile?Because transposition is limited to the germ-line. The mobilization of the P elements occurs only in the gonads of the progeny. Thus the progeny are normal somatically but because of the massive mobilization of P elements in their gonads they are sterile.
Q)
A pre-med teaching assistant devised an experiment to demonstrate the concept of cis-dominant mutations to a laboratory class. The TA had the class create two recombinant clones in the multicopy plasmid vector pBR322. The first plasmid carried the wild-type lac promoter-operator region and mutant Z- and Y- structural genes. The second plasmid carried the wild-type promoter-operator region and the Z+ and Y+ genes. The plasmids were introduced into E. coli strains of genotypes I+ oc Z+ Y+ and I+ oc Z- Y- and assayed for lacZ activity by plating on X-gal plates with the following results:Colony phenotype on X-gal plates
- IPTG
+ IPTG
I
+ o+ Z+ Y+ control strainwhite
blue
I
+ oc Z+ Y+ control strainblue
blue
pBR322 o
+ Z- Y- / I+ oc Z+ Y+blue
blue
pBR322 o
+ Z+ Y+ / I+ oc Z- Y-blue
blue
a)
Why were the results surprising to the TA?The TA expected the strain in the bottom row of the table to be inducible and not constitutive. The implication of the results in the table is that the o
c mutation is trans-dominant and not cis-dominant.b)
What went wrong? Provide an explanation for the seemingly anomalous results.The problem is due to the multicopy plasmid. The TA wanted to mimic the diploid state but instead of using a single-copy F', used the multicopy pBR322. This means that the operator sequence was present in multicopy and titrated the limited amount of repressor (~10 molecules) present in the wild-type strain. Thus, most of the plasmids expressed the lacZ and Y genes in the bottom row giving the appearance that o
c acts in trans.c)
Suggest an experiment to test your answer to part b).If true, one should be able to mimic the situation by cloning the wild-type operator only (no Z
+ or Y+ genes) on the plasmid and introducing this into a wild-type strain. One would expect constitutive lac expression since the plasmid operator will titrate the repressor.A second test would be to include the repressor gene on the multicopy plasmid. In this case sufficient repressor should be made to repress all o
+ operators and one would see the cis-dominant effect of the oc mutation.A third test would be to use a single-copy plasmid. There the situation would be like the case with an F' and the cis-acting character of the o
c allele would be apparent.Q)
The following three graphs chart the growth of E. coli and the production of ß-galactosidase (lacZ product) when grown in a medium with both glucose and lactose as carbon sources.Q)
The following three graphs chart the growth of E. coli and the production of ß-galactosidase (lacZ product) when grown in a medium with both glucose and lactose as carbon sources.(a)
Explain the significance of left graph.The graph illustrates the process of diauxic growth. The cells use the better carbon source (glucose) first and only use lactose when the glucose is exhausted. The lac genes remain repressed while glucose is present even though the inducer (lactose) is present, and are only synthesized when glucose is used up.
(b)
Are the results shown in the middle and right graphs consistent with the idea that glucose-lactose diauxie (catabolite repression) is regulated directly by cellular cAMP concentration? Explain why or why not.No the data are inconsistent with the idea. The central graph indicates that B-gal is synthesized at a high rate in the presence of glucose if there is no repressor protein. This indicates that the CRP-cAMP complex must be bound to the lac operon promoter even in the presence of glucose. According to the cAMP concentration model, cAMP levels should be low in glucose and thus the lac operon should be essentially off even in the absence of repressor. The data in the right hand graph suggests essentially the same thing. When the inducer IPTG is added in the presence of glucose the lac operon is immediately induced. This also indicates that CRP-cAMP must be bound to the lac promoter in the presence of glucose.
6
) HIV infects human cells through a process that involves physical interaction between the viral envelope glycoprotein gp120 and the CD4 protein present on the surface of many cells of the immune system. In order for HIV to enter the cells it is thought that gp120 must also interact with the ccr-5 gene product (aka ckr-5). An alternative possibility is that the viral capsid protein interacts with CCR-5 after the cell membrane and viral envelopes have fused, and that this interaction allows the viral capsid to enter the cytoplasm.a) Propose a genetic test that will distinguish whether it is gp120 or the capsid protein that physically contacts CCR-5. Outline the experiments, including any controls you think would be useful, and state the results expected if the first hypothesis is true.
Use the two-hybrid system. Fuse CCR-5 and CD4 to the lexA DNA binding domain and gp120 and capsid protein to the GAL4 transcription activation domain. Construct a reporter construct that places lacZ under the control of a yeast basal promoter and lexA operator sites. Test the various fusion proteins by looking at lacZ expression. High levels of ß-gal activity indicate a positive interaction between the protein fused to the DNA binding domain and that fused to the activation domain.
LexA DNA binding domain fusion
Gal4 Activation Domain fusion
ß-gal activity
lexA-
Gal4 A.D.-
none--negative control
lexA-CCR-5
Gal4 A.D.-
none--negative control
lexA-CCR-5
Gal4 A.D.-gp120
High
lexA-CCR-5
Gal4 A.D.-capsid protein
none
lexA-CD4
Gal4 A.D.
none--negative control
lexA-CD4
Gal4 A.D.-gp120
High
--Positive controllexA-CD4
Gal4 A.D.-capsid protein
none
lexA-
Gal4 A.D.-capsid protein
none--negative control
lexA-
Gal4 A.D.-gp120
none--negative control
b) Several years after HIV infection, coincident with the onset of clinical AIDS, an HIV variant often appears that no longer "interacts" with CCR-5 protein. The modified virus, called T-tropic, instead infects immune cells that express both CD4 and another protein called CXCR-4. If the first model presented above is correct, in which viral gene would you expect to find the key sequence differences between the early and late forms of the virus? (Give the gene not the protein.)