Discovery Talk Questions and Answers: The Semi-Conservative Replication of DNA

Matthew Meselson
Assessments created by Dr. Kirk Ehmsen

Questions

In his talk, Dr. Meselson states that his density gradient experiment with Frank Stahl had a profound ‘psychological effect’ on those studying molecular biology – it validated not only the semi-conservative DNA replication model proposed by Watson and Crick in 1953, but moreover it ‘made the DNA seem real’.
In the DNA schematic below, which region of this very real molecule was in fact labeled by heavy isotope in the Meselson and Stahl experiment? (Choose one letter corresponding to the indicated DNA feature)

(a) (b) (c) (d) (e)

Illustration by Kirk Ehmsen for iBiology.
Dr. Meselson states in his talk that his mentor, Max Delbrück, was ‘pessimistic about semi-conservative replication’. Which of the following statements best accounts for why Dr. Delbrück did not favor this model for DNA replication?
In the semi-conservative model for DNA replication . . .
1. Phosphodiester bonds in the backbone would need to be broken and rejoined.
2. The two strands of the helix would have to be unwound from one another.
3. The structure of DNA remained only hypothetical, given that it had been deduced by model-building.
4. All of the above statements explain why Dr. Delbrück believed that semi-conservative replication was too ‘inelegant’ to be the likely mode by which DNA is copied.
5. None of the above
Dr. Meselson refers in his talk to three models for DNA replication discussed in the 1950s. These models differed from one another ‘in the predictions they make concerning the distribution among progeny molecules of atoms derived from parental molecules’ (p. 671).

Consider the schematics for the three scenarios below, showing DNA molecules from up to two E. coli generations following a switch from N15- to N14-containing growth media. Each block represents one strand of the DNA double helix; black regions are labeled with heavy nitrogen (N15) and gray regions are labeled with light nitrogen (N14):

Illustration by Kirk Ehmsen for iBiology.

Which of the centrifuge schematics below represents the daughter molecule banding patterns that would be seen for these three different modes at Generation 1?

Illustration by Kirk Ehmsen for iBiology.

In their paper, Meselson and Stahl note that, in the density gradient experiments, it is difficult to distinguish between semi-conservative and dispersive mechanisms for DNA replication. What evidence do they provide to rule out the dispersive replication hypothesis? Choose all that apply.
1. They show that across four generations of E. coli DNA replication, only three discrete DNA species are observed: completely heavy, ½-heavy / ½-light, and completely light.
2. They show that across four generations of E. coli DNA replication, DNA molecules progressively become 50% lighter in every generation following a switch to N14-containing media.
3. They heat hybrid-density 1st-generation daughter DNA molecules to 100 degrees C, and show that both strands of each molecule are partially light and partially heavy.
4. They heat hybrid-density 1st-generation daughter DNA molecules to 100 degrees C, and show that each molecule had one completely light strand and one completely heavy strand.
5. None of the above
Tracking the density of DNA molecules over multiple generations (rounds of DNA replication) allowed Meselson and Stahl to infer the manner in which DNA is copied.

Of all the DNA molecules present in the density gradients below, what % of molecules contain some heavy nitrogen at each of the four generations?

Illustration by Kirk Ehmsen for iBiology.

Consider the UV absorption photographs of the DNA bands following density-gradient centrifugation of bacterial lysates at ten different generation times in Figure 4 (p. 675). If you were asked to select only 3 or 4 of these photographs to display together, which ones would you consider sufficient to demonstrate that DNA is replicated semi-conservatively?
Choose 3 or 4 of the images, indicate the generation time that each image depicts, and explain how together the images you chose demonstrate that DNA replication is semi-conservative.
Watson and Crick proposed in 1953 that DNA consists of two strands, held together by hydrogen bonds between complementary base pairs. In his talk, Dr. Meselson explains that the DNA structure proposed by Watson and Crick was itself ‘a proposal for how DNA would replicate.’
Briefly explain how DNA structure relates to the semi-conservative model for its replication.
In Figure 4 of their paper, Meselson and Stahl present the data that they interpreted to support semi-conservative DNA replication. In Figures 1, 2 and 3, they present data they believe is important to interpreting the results in Figure 4. Briefly describe the results shown in each of the first three figures (Fig. 1, 2 and 3) and how it is relevant to the data shown in Figure 4.
Dr. Meselson explains in his talk that when he and Dr. Stahl were writing their paper, they debated with their mentor Dr. Delbrück whether to start with the premise that they had specifically tested the Watson-Crick hypothesis for DNA replication (the semi-conservative model), or whether to start by describing their results and then to explain whether the results agreed with any of the contending hypotheses.
Which approach did Drs. Meselson and Stahl choose for their 1958 paper, and what explanation does Dr. Meselson give for their decision?
In his talk, Dr. Meselson suggests that DNA was unique among biomolecules in that “this molecule essentially gave orders to a whole period in molecular biology” – “the DNA molecule [was] essentially telling you what the problems were, what you had to go out and solve.” (11:36)
What major questions in molecular biology does Dr. Meselson suggest were ‘problems’ posed by DNA? Briefly explain what he means by these ‘problems.’

Answers

a; the nitrogenous bases are the only nitrogen-containing subunits of DNA, and therefore it is the adenine, guanine, thymine and cytosine subunits of DNA that are labeled when E. coli is cultured in medium containing heavy nitrogen (N15). Both the talk and the paper refer to N15 repeatedly.p. 672: “By use of this method, we have observed the distribution of the heavy nitrogen isotope N15 among molecules of DNA following the transfer of a uniformly N15-labeled, exponentially growing bacterial population to a growth medium containing the ordinary nitrogen isotope N14.”
p. 677: “The nitrogen base (adenine, guanine, thymine, or cytosine) at each level on one chain is hydrogen-bonded to the base at the same level on the other chain.”Video (7:00): “So to make a long story short, we grew bacteria in heavy nitrogen medium and then after many generation of growth, so that everything would be labeled with heavy nitrogen, we switched the bacteria, centrifuging them and resuspending them in a light medium and took samples at various times.”

Note: Assessment goal: This question asks students to relate the isotopically heavy atom (nitrogen) to its physical component in the DNA molecule (nitrogenous bases), evaluating a pre-requisite understanding of DNA molecular structure. Regardless of prior background, sufficient information is provided in the talk and the paper to conclude that it is the subunits between the backbones that contain nitrogen: Dr. Meselson describes the structure of DNA in his talk, and the paper describes the Watson-Crick model (naming the nitrogen bases between the chains) on p. 677.
b; Dr. Meselson explains that Max Delbrück was very concerned that in the semi-conservative model for DNA replication, the two strands of the double helix ‘had to unwind’. He found it difficult to envision how two new double helices could be disentangled from one another without breaking and rejoining pieces of each double helix; therefore, he favored a dispersive model for DNA replication.(a) is incorrect because it refers to the dispersive model (see below). (c) is not mentioned as a reason why Max Delbruck was skeptical about this model.
Video (01:42) “At Caltech, Max Delbruck who knew Jim Watson and was in correspondence with him was pessimistic about this replication scheme, pessimistic about semi-conservative replication. The chains were wound around each other, so to get them apart unless you break them, you would have to unwind the basic double helix to get the two arms separate. Max thought this would be impossible, hydronamically impossible. And so he proposed that maybe the two chains separated by a system of breaking every several nucleotides along the chain. And he and Gunther Stent published a paper which proposed three different methods for DNA replication. Semi-conservative, as Watson and Crick had predicted, conservative, in which at least conceptually maybe there was a way that a double helix could guide the formation of another double helix just like it nearby and so you would never have to separate the chains at all. You’d have a brand new double helix and a completely old double helix, and that would be the act of replication. And then dispersive, the third way, break the single chains, separate the pieces, and then put everything back together again.”
Note: Assessment goal: this question asks students to identify predictions of the semi-conservative model for DNA replication, and to distinguish a testable prediction from a personal judgment (Delbrück’s belief that unwinding DNA duplexes was ‘too inelegant to be efficient’ [PNAS 40:783-788, 1954]).
e; Based on Fig. 5 in the paper (p. 677) and the corresponding schematics provided for alternative modes of replication, at Generation 1 there would be:
- Two bands if DNA replicated conservatively (one completely heavy, one completely light),
- One band if DNA replicated semi-conservatively (intermediate density: 50% light, 50% heavy),
- One band if DNA replicated dispersively (intermediate density: 50% light, 50% heavy).
Video (01:42) “At Caltech, Max Delbruck who knew Jim Watson and was in correspondence with him was pessimistic about this replication scheme, pessimistic about semi-conservative replication. The chains were wound around each other, so to get them apart unless you break them, you would have to unwind the basic double helix to get the two arms separate. Max thought this would be impossible, hydronamically impossible. And so he proposed that maybe the two chains separated by a system of breaking every several nucleotides along the chain. And he and Gunther Stent published a paper which proposed three different methods for DNA replication. Semi-conservative, as Watson and Crick had predicted, conservative, in which at least conceptually maybe there was a way that a double helix could guide the formation of another double helix just like it nearby and so you would never have to separate the chains at all. You’d have a brand new double helix and a completely old double helix, and that would be the act of replication. And then dispersive, the third way, break the single chains, separate the pieces, and then put everything back together again.”

Note: Assessment goal: this question asks students to identify predictions of all three proposals for DNA replication, and to link the schematic provided by Meselson and Stahl in their paper (p. 677) with a schematic of the results as they would have been seen in a density gradient. One of the key goals is to assess whether students can recognize that daughter molecules can be either completely light, completely heavy, or intermediate in density depending on the mode of DNA replication. Finally, this question also assesses students’ recognition that they can first make predictions based on each schematic (each hypothesis) and then find the corresponding result among the choices. It is not necessary to examine each possibility (a-e) a priori.
a & d;
(a)-Meselson & Stahl show in their paper (Fig. 4) that when DNA is sampled from up to four generations of replicating E. coli, only three discrete species of DNA appear: the fully-heavy DNA band seen at the starting point (Generation 0) is converted entirely to a DNA band that is intermediate in density between fully-heavy and fully-light DNA bands (Generation 1), and over the course of Generations 2 – 4, the intensity of this intermediate-density band is successively depleted as fully-light DNA molecules appear. Had the dispersive model been true, the intermediate-density band would also have depleted in Generations 2 – 4, but instead of a fully-light band accumulating, a series of bands transitional in density between the ‘intermediate’ and fully light bands would have been observed;(d)-Meselson & Stahl also show in their paper that heat-denaturation of 1st-generation hybrid DNA molecules results in loss of the original intermediate density band, and the appearance of two new density species (Fig. 9B, p. 680). The two new density species correspond to bands seen when fully light and fully heavy DNA are denatured, mixed, and separated on a density gradient (Fig. 9C).Note: Assessment goal: This question asks students to evaluate the evidence provided in the paper that distinguishes between two competing models for DNA replication – one supported by Watson and Crick (semi-conservative) and one supported by Max Delbrück (dispersive). It requires that students (1) predict the outcome (banding patterns) of dispersive replication over the same generation time examined by Meselson and Stahl in their paper, and (2) to evaluate the purpose of the conventionally little-noted heat denaturation experiment in the final figures of the 1958 paper.
Sample answer:
Generation 1: 100% of the molecules contain N15 (all are intermediate-density)
Generation 2: 50% of the DNA molecules contain N15
Generation 3: 25% of the DNA molecules contain N15
Generation 4: 12.5% of the DNA molecules contain N15Note: Assessment goal: This question asks students to relate the qualitative banding patterns typically used to describe the results of the Meselson-Stahl experiment to a quantitative description of how much DNA (relative to the total DNA from E. coli lysates) is present in each band.
Sample answer: Key to the Meselson-Stahl experiment is that molecules of DNA that are fully light, fully heavy, and ‘hybrid’ were identified as separate entities by density gradient centrifugation. Matching these three distinct entities to the generation times that correspond to the doubling of an E. coli cell population is what allowed Meselson and Stahl to conclude that in each generation (each DNA or cell ‘doubling time’), the DNA molecules are hybrids of an ‘old’ (pre-existing) strand and a ‘new’ (freshly made) strand – the essence of the semi-conservative model. A number of possible image choices could work here, but the minimal set sufficient to demonstrate that freshly replicated DNA molecules are hybrids of old and new consists of those images that allow comparison between at least three discrete generations: 0, 1.0/1.1, 1.9/3.0/4.1. The image where 0 and 1.9 are mixed would be an excellent choice to include, as well, because it shows that the intermediate-density molecules indeed migrate at 50% the density of fully N15-labeled molecules, and at 50% greater density than fully N14-labeled molecules.Note: Assessment goal: This question asks students to work directly with data as presented in the paper: (1) to evaluate a collection of data (the multiple images), (2) to make a judgment regarding the minimal data points required to support the semi-conservative hypothesis, and (3) to interpret in their own words how these images demonstrate semi-conservative replication.
Sample answer: The semi-conservative model for DNA replication predicts that each strand of the DNA double helix serves as a template for the synthesis of a new second strand – thus the concept of each new DNA molecule being a hybrid between a ‘completely old’ strand and a ‘completely new’ strand. Complementary base-pairing between adenine and thymine, guanine and cytosine explains how one strand can serve as a template for the assembly of a completely new strand.Video (0:39): “It is hard to know where to start any particular story but let me start with the publication in 1953 of the paper by Watson and Crick. There were actually two papers in Nature. The first was about the structure which was based on model building and a little bit of information from X-ray diffraction. The X-ray diffraction certainly did not tell you the structure. It told you something about the repeat distance along the helix. It told you that it was a helix and very little more. The model building was model building, so the structure certainly wasn’t proven. This was a proposal and many people didn’t believe it or maybe didn’t even pay any attention to it. The second paper proposed how the molecule might replicate. The two chains would separate, each would guide on its surface the formation of a new chain, so you would end up with two double helices, each one has one of the old chains and one brand new chain. That’s called semi-conservative replication.”
Note: Assessment goal: This question asks students to apply an understanding of DNA structure to a prediction for how one molecule could specify the construction of two copies.
Sample answer: Figure 1- Here Meselson and Stahl demonstrate that they can concentrate a sample of DNA into a single, discrete band by centrifugation in CsCl under sufficient force for sufficient time (here, 31,410 rpm for up to >40 hrs). They can visualize this band under ultraviolet light, as DNA fluoresces under UV light. This figure is relevant to Figure 4 because it demonstrates that density gradient centrifugation can yield a discrete band of DNA.
Figure 2- Here Meselson and Stahl demonstrate that when E. coli are grown in media with heavy vs. light nitrogen sources, the resulting DNA molecules have different densities that can readily be separated by the CsCl density gradient centrifugation they 1st showed in Fig. 1. Here they have used a higher centrifugation speed (44,770 rpm) and apparently this is why they achieved discrete banding patterns in a shorter time than in Figure 1 (24 hrs. vs. >40 hrs). This figure is relevant to Figure 4 because it demonstrates that lysates from E. coli grown in heavy vs. light media yield DNA molecules that are distinguishable in density by CsCl density gradient centrifugation.

Figure 3- Here Meselson and Stahl demonstrate that they can readily keep track of the E. coli doubling time in both heavy and light media, by counting the number of cells in a sample under a microscope or by colony counting (spreading a sample on a plate and counting the visible colonies that grow). This figure is relevant to Figure 4 because it is the basis from which Meselson and Stahl determine ‘Generations’ – and therefore the basis from which they are able to describe the type of DNA molecules present (heavy, intermediate, light) at each DNA doubling time (1st generation, 2nd generation, etc.)

Note: Assessment goal: This question asks students to identify the conclusions that can be drawn from data presented in three primary figures, and then to interpret why these conclusions were important to a subsequent experiment (Figure 4) that was critical to the demonstration of semi-conservative replication.
Sample response: Drs. Meselson and Stahl chose to focus on a presentation of their result, and then to describe whether it agreed with any of the hypotheses that had been put forth (p. 671-Introduction & p. 677,678-Figs. 5, 6 ). Dr. Meselson explains that he and Dr. Stahl were not convinced that they were testing any one particular hypothesis, and that the structure of DNA was still insufficiently clear that their results could in fact suggest something entirely different from the three prevailing possibilities suggested from the Watson-Crick model for DNA structure. For these reasons, they began with ‘first principles’ rather than specifically setting out to test hypotheses based on the Watson-Crick model for DNA structure.Video (08:29): “But there was a question how to write this up. There were two ways that we discussed. One way is to start with a hypothesis, the Watson-Crick hypothesis, and say: ‘here’s a test, we do this experiment,’ and see if it works out the way they said it should. And that is certainly one way to do an experiment, and Richard Feynman, who was very close to students in those days (he would come over to our parties and so on), he thought that we should write it up that way. The other way would be to write up exactly what your experiment said, no more, no less without reference to any hypotheses at all, and then at the very end say whether it may agree with some hypothesis. We chose the last way, partly because we thought, well, if you are trying to test a hypothesis, the only way to really be sure that you are going to be right is to know all possible hypotheses. And since nobody can know all possible hypotheses just because your experiment might agree with one of them doesn’t prove that that is the right hypothesis. There could be another one that your experiment agrees with So it seemed to us more elegant to write our paper up in terms of subunits, and only at the end say, “ah, these subunits could be the single chains of the Watson-Crick model,” which of course they were. So that is what we did.”
Assessment goal: This question asks students to evaluate two different possible contexts for the presentation of data in the 1958 paper, which would probably not even come to mind without Dr. Meselson’s reference to the two alternative approaches in his talk. Students are asked to provide evidence from the paper and/or the talk that explains why one approach was chosen over another. Furthermore, this question reminds students that although testing specific hypotheses is often the goal of many scientific experiments, some experiments are performed primarily from the perspective of collecting observations about a phenomenon – observations from which hypotheses can then be formed. It was in this latter context that Drs. Meselson and Stahl preferred to view their historic experiment.
Sample answer: Dr. Meselson notes the following ‘problems’ or questions (Q) posed by DNA (iBio video, 11:43 min):
1. Q: I have two chains – how do I replicate?
  Dr. Meselson refers here to the challenge of determining the mechanisms of DNA replication
2. Q: I have 4 bases – how are these converted to protein?
  Dr. Meselson refers here to the challenge of determining how information in DNA is converted to information in protein sequence (later described by the ‘central dogma of molecular biology’ and the genetic code).
3. Q: In eukaryotes, I reside in the nucleus – how do my instructions get out to the cytoplasm where proteins are made?
  As in Q2, Dr. Meselson refers here to the challenge of determining how information in DNA is transferred to a useable unit in the cytoplasm (later described by the ‘central dogma of molecular biology’).
4. My sequence can change over time – how do genetic alterations occur?
  Dr. Meselson refers here to the challenge of understanding how mutations take place and other ways that DNA sequence can change over time or exchange between molecules (as in recombination).
Assessment goal: This question asks students to summarize a set of conceptual challenges articulated by Dr. Meselson, and to interpret his ‘short-hand’ description of these challenges by defining what he meant in each case.