Modes of replication. Not everyone was immediately convinced that the Meselson-S

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Modes of replication. Not everyone was immediately convinced that the Meselson-Stahl experiment had ruled out all modes of replication besides the semiconservative mode. One investigator proposed that the observed data was consistent with a conservative mode of replication. In this case, it was proposed that DNA was actually a paired double helical molecules [two associated double helices—with a total of 4 strands, 2 per each], and that each double helix served as a template to make another identical double helix. At this time, the X-ray data could not have ruled out this possibility.

Assume that experiments were conducted exactly as those described in the Meselson-Stahl paper (15N-labeled cells shifted to 14N culture media at time = 0).

Assuming the specific conservative mode of replication described above was correct, what specific experimental predictions would be made for the density of observed DNA molecules at generation 1 and at generation 2. Note that this is a different type of conservative replication than that described in the M-S paper or your textbook.

Is this prediction the same or different from the prediction made for a single double helix undergoing semi-conservative mode of replication? Explain.

Describe a specific experiment that could potentially rule out a conservative mode of replication as described above. Be sure you explain your logic.

2. Side-by-side DNA and topoisomerases. Even in the 1970s, alternative models for the structure of DNA were still be proposed. One controversial proposal (that did gain some attention) was the so-called side-by-side model of DNA. In this model, the strands of DNA do twist around each other, but there was no net twist. The basic idea is that over 10 base pairs, the first 5 base pairs twisted one way, while the next twisted the opposite way. These opposite directed twists would cancel each other out. Overall it would be as if the two strands were lying side-by-side and the strands could be pulled apart without causing the building of stress in adjacent DNA segments. [It would be similar to the structure shown in simple ladder diagrams, with bases as the rungs in DNA.]

This hypothesis was proposed to avoid the problems associated with stresses generated by the separation of DNA strands in the double helix described by Watson and Crick. (1) What specific problems arise with strand separation in the Watson and Crick model? (2) How are they avoided by the side-by-side model? (3) How do topoiosmerases solve this problem for a DNA double helix as described by Watson and Crick.

The gel at right shows the migration of plasmid DNA in an agarose gel (stained with ethidium bromide). Lane 1 shows a plasmid preparation, with mostly supercoiled DNA and a small amount of relaxed (nicked) DNA.   If the side-by-side model of DNA were true, what banding pattern would you expect to observe?

An experiment was designed to rule out the side-by-side model. The two complementary strands of a circular double-stranded DNA molecule from a bacteriophage were isolated as closed circular single-stranded DNA molecules. The two complementary circular DNA molecules were mixed together to attempt to reform a double-stranded DNA molecule. The original double-stranded DNA molecule and this experimentally created double-stranded DNA molecule were separated by gel electrophoresis. Even though they had exactly the same sequence on both strands, they migrated very differently in gels. Would this result be consistent with the Watson-Crick model or the side-by-side model for DNA structure? Explain your reasoning.

3. DNA sequencing. The Sanger method of sequencing DNA molecules was ultimately based on Kornberg’s demonstration that DNA could be replicated in the test tube using defined chemical components.

What is the minimal set of required components for carrying out DNA replication in the test tube?

How does this differ from what is required for DNA replication in a living cell? Be specific.

In the Sanger method, four reactions are set up. In each reaction, there is a mixture of the 4 normal deoxyribonucleotides, but there is also a smaller amount of a dideoxy version of one of the 4 nucleotides. For example, reaction 1 would include the 4 dNTPs but also a smaller amount of dideoxy-ATP. Dideoxy nucleotides lack a free 3’-OH group. Explain why this reaction will produce a nested set of DNA fragments, each one ending in dideoxy-ATP, which will produce a ladder of bands when separated by gel electrophoresis. [Individual DNA bands can be visualized by either radioactive or fluorescent labels.

Suppose that a mistake was made in preparing a mixture of the four deoxyribonucleotides, so that dCTP was left out. If this dNTP mixture was used to carry out reaction 1 (part c above) , what would you predict that the outcome would be? Be specific and explain your logic.

4. X-chromosome inactivation and Barr bodies. In order to provide the same dosage of X-linked genes in males and females, one of the two X-chromosomes in females is inactivated, forming a structure called a Barr body. The Barr body is a special form of chromatin.

(a) Barr bodies are a different form of highly condensed chromatin that that seen in spermatozoa. How are these two forms of chromatin different in make up?

(b) Inactivated X-chromosomes contain nucleosomes with a different mixture of histone proteins. What is different about the histone composition of an inactivated X-chromosome that sets it apart from the active form of the X chromosome?

(c) Would a Barr body best be described as euchromatin, constitutive heterochromatin, or facultative heterochromatin? Explain.

Explanation / Answer

1.Conservative mode of DNA replication involves the synthesis of duplicate DNA strand in such a way that their is a newly synthesized DNA duplex template to the parental one is called as Conservative mode of DNA replication.After one round of replication one strand remain as two strand synthesized.DNA are separated on cesium chloride density gradient solution.

2(1) DNA are supercoiled in a double helix acc. to Watson model ,it is hard to separate them as base pair are hydrogen bonded with each other.

(2) side by side model involves simple ,linear arrangement of base pair and it is easy to separate before replication.

(3)DNA topoisomerase or gyrase remove tension from supercoiled DNA by introducing negative supercooling or relieving positive supercoil.

3.Sanger method of DNA sequencing is based on the synthesis of DNA strand rather than DNA denaturation.Radiolabelled nucleotides are added in the test tube.DNA sequence are introduced in M13 phage to replicate in E.coli. Dideoxynucleotides are introduced in each test tube ,they have no 2'OH on sugar and hence bring out chain termination.After gel electrophoresis ,DNA are analysed and sequence is determined.

4(a)Barr bodies are the inactive X-chromosome found in female somatic cell, it consist of heterochromatin type of chromatin.

(b)Histone present on inactive X-chromosome is highly methylated and it is found in highly condensed form,active chromosome Histone is highly acetylated for full expression.

(c)Barr body is facultative heterochromatin as it can become active under certain conditions.

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