We are searching data for your request:
Upon completion, a link will appear to access the found materials.
I am learning about the genetic code, replication, and transcription, and I have a question about whether or not both strands of DNA are really "necessary".
In replication, at a high level, we are taking 2 strands of DNA and creating 4 of them. Part of my question is: suppose hypothetically that the genome only consisted of a single strand (instead of the double helix), couldn't replication just as well occur by turning 1 strand into 2 (instead of 2 into 4)? I suppose this could be done by taking advantage of complementary base pairing as usual, and then splitting the two strands?
In transcription, I believe only one strand of a gene is read by RNA polymerase, so I don't think think the second strand is "necessary" here.
Can someone shed some light on this for me? I realize this is pretty speculative, and DNA is what it is, and does what it does. but I think this sort of question will allow me to see deeper into tits finer workings.
From a strictly information theoretical perspective, no, a second strand does not provide any additional information that cannot be inferred from the complementary strand. But as many comments have pointed out, there are many practical reasons for DNA's double strandedness.
- DNA is biochemically more stable in a double stranded state.
- The redundancy of two strands allows for maintenance and repair mechanisms to ensure the integrity of the genome.
- Proteins that bind to DNA can only recognize the sequence of that strand, so even though the complementary strand can always be reconstructed from the primary strand, DNA binding proteins cannot target that complementary strand unless it physically exists. In some cases, a DNA binding site encompasses both strands simultaneously.
- Proteins (such as polymerases) traverse DNA in the 5' to 3' direction, so if information from the complementary strand was required it would have to be read "backwards".
Although the title refers only to an “information perspective”, the question itself brings in the processes of replication and transcription. It is not entirely clear whether the poster envisages 'piggybacking' on existing DNA- and RNA polymerases which operate on double-stranded DNA (dsDNA) or whether he is envisaging completely different enzymes. I shall consider how the existence of single-stranded DNA and RNA viruses make it possible to argue that, both scenarios are possible in theory.
My answer to the question, therefore, like that of @DanielStandage, is no.
Single-stranded genomes relying on a double-stranded replicative form
Many viruses exist in which the genome packaged in the virion is single-stranded DNA (ssDNA). Replication occurs using either virally-encoded or host-coded DNA polymerases, often going through a double-stranded replicative intermediate (but see next section). Transcription normally occurs using the nuclear host DNA-dependent RNA polymerase, acting on the dsDNA.
In order to have all the information for transcription and translation on a single strand in a non-cryptic form, the individual genes would all have to have the same orientation so that the codons of all the transcribed single-stranded mRNA made sense. There seems no reason why this should not be so, although in practice most DNA genomes of which I am aware have genes in both orientations. However the banana buchy top virus has a genome composed of six individual ssDNAs, each apparently with a single gene.
Single-stranded genomes without a double-stranded replicative form
The rolling circle mechanism of DNA replication involves generation of a single stranded form without a double-stranded replicative intermediate, and, although the most well characterized examples are dsDNA viruses, the ssDNA banana buchy top virus replicates by the same mechanism. If we think of what might be possible in linear ssDNA genomes by extrapolating from what exists in linear ssRNA viruses, it is not hard to envisage replication mechanisms involving the production of many copies of a ssDNA of opposite sense to a single copy of a ssDNA template. Replication is therefore not a problem with ssDNA-dependent ssDNA polymerases analogous to extant viral RNA-dependent RNA polymerases. For transcription it would seem more logical to make a further step of a ssDNA-dependent ssRNA polymerase, rather than 'mutating' extant dsDNA-dependent RNA polymerases.
As things don't operate this way it may seem to be a waste of time discussing them. However:
- The poster is reassured that his reasoning is - in principle - correct.
- The poster and reader is made aware of the varieties of viral genomes and replication strategies of which he might previously have been ignorant.
- The answer provides food for thought in relation to the idea that a world in which cellular organisms had RNA genomes preceded the current one in which they have DNA genomes.
A final word about information
The way in which the question is posed relates to information as a linear sequences of nucleotides in DNA, specifically codons. This can be extended to other signals such as transcriptional start sites, splicing sites, polyadenylation/cleavage sites etc. However it is also possible to conceive of three-dimensional information. Although three-dimensional structures are possible with ssDNA, some. e.g. cruciform DNA do require dsDNA.
Closer look offers clues on how DNA strands are separated during replication
A side view of double hexameric ring complex stacked at the N-terminal end in a twisted and tilted manner. Credit: Division of Life Science, HKUST
(Phys.org)—A team of researchers working in China has used newly developed cryo-electron technology to get a closer look at the mechanism involved in separating DNA strands necessary for natural replication of DNA. In their paper published in the journal Nature, the team describes their study, what they were able to observe, and their theory on how the separation actually takes place. Mathew Bochman with Indiana University and Anthony Schwacha with the University of Pittsburgh offer a News & Views piece on the work done by the team in the same journal issue.
In order for DNA to be replicated naturally, the two strands that make up its double helix must be separated—how that process occurs has been the subject of much debate. In this new effort, the researchers were able to get the closest look ever at the components involved allowing them to offer a theory on the process.
Under the new microscope, the researchers were able to see the structure of the helicase enzyme, Mcm2–7, which prior research had suggested was a major player in separating DNA strands (by somehow binding to one strand or another). The structures were doughnut shaped hexamers, two of which came together to form a single unit offset and tilted at a 14° degree angle to one another. The DNA strands would pass through both holes, via a central channel, the team noted, providing the basis, they believe for separating the two strands. Because the researchers were not able to watch as the helix was split, they were left to make an educated guess as to how the hexamers did their work. They believe that melting occurs rather than unwinding and that it occurs due to the angle between the hexamers—the narrowness of the channel combined with the angle causes, they believe, a kink to occur in the DNA strands, which they believe initiates melting of the bonds between the strands, allowing them to separate.A helix 2 insertion loop (H2I) and Beta turns contributed by each subunit form constriction points that could immobilize DNA Credit: Division of Life Science, HKUST
As for how the melting itself works, Bochman and Schwacha note that prior work has shown that an SV40 antigen has been seen to serve as a pump—they believe the hexamers might work in a similar way, noting also that the unique alignment of the hexamers allows for two exit channels—one for each of the separated strands.Axially staggered turns provide tight fitting for helical DNA. Credit: Divison of Life Science, HKUST
DNA replication in eukaryotes is strictly regulated by several mechanisms. A central step in this replication is the assembly of the heterohexameric minichromosome maintenance (MCM2–7) helicase complex at replication origins during G1 phase as an inactive double hexamer. Here, using cryo-electron microscopy, we report a near-atomic structure of the MCM2–7 double hexamer purified from yeast G1 chromatin. Our structure shows that two single hexamers, arranged in a tilted and twisted fashion through interdigitated amino-terminal domain interactions, form a kinked central channel. Four constricted rings consisting of conserved interior β-hairpins from the two single hexamers create a narrow passageway that tightly fits duplex DNA. This narrow passageway, reinforced by the offset of the two single hexamers at the double hexamer interface, is flanked by two pairs of gate-forming subunits, MCM2 and MCM5. These unusual features of the twisted and tilted single hexamers suggest a concerted mechanism for the melting of origin DNA that requires structural deformation of the intervening DNA.
Structure of Nucleic Acids
Nucleic acids are key macromolecules in the continuity of life. They carry the genetic blueprint of a cell and carry instructions for the functioning of the cell.
Figure 2. A nucleotide is made up of three components: a nitrogenous base, a pentose sugar, and a phosphate group.
The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the genetic material found in all living organisms, ranging from single-celled bacteria to multicellular mammals.
The other type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus, but instead use an RNA intermediary to communicate with the rest of the cell. Other types of RNA are also involved in protein synthesis and its regulation.
DNA and RNA are made up of monomers known as nucleotides. The nucleotides combine with each other to form a polynucleotide, DNA or RNA. Each nucleotide is made up of three components: a nitrogenous base, a pentose (five-carbon) sugar, and a phosphate group (Figure 2). Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to a phosphate group. The nucleotides link together by phosphodiester bonds to form the polynucleotide.
DNA Double-Helical Structure
Figure 3. The double-helix model shows DNA as two parallel strands of intertwining molecules. (credit: Jerome Walker, Dennis Myts)
DNA has a double-helical structure (Figure 3). It is composed of two strands, or polymers, of nucleotides. The strands are formed with covalent bonds between phosphate and sugar groups of adjacent nucleotides.
The two strands are bonded to each other at their bases with hydrogen bonds, and the strands coil about each other along their length, hence the “double helix” description, which means a double spiral.
The alternating sugar and phosphate groups lie on the outside of each strand, forming the backbone of the DNA. The nitrogenous bases are stacked in the interior, like the steps of a staircase, and these bases pair the pairs are bound to each other by hydrogen bonds. The bases pair in such a way that the distance between the backbones of the two strands is the same all along the molecule.
Meselson and Stahl
Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a &ldquoheavy&rdquo isotope of nitrogen ( 15 N) that is incorporated into nitrogenous bases and, eventually, into the DNA. The E. coliculture was then shifted into medium containing the common &ldquolight&rdquo isotope of nitrogen ( 14 N) and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge in a tube in which a cesium chloride density gradient had been established. Some cells were allowed to grow for one more life cycle in 14 N and spun again.
Figure: Meselson and Stahl: Meselson and Stahl experimented with E. coli grown first in heavy nitrogen ( 15 N) then in ligher nitrogen ( 14 N.) DNA grown in 15 N (red band) is heavier than DNA grown in 14 N (orange band) and sediments to a lower level in the cesium chloride density gradient in an ultracentrifuge. When DNA grown in 15 N is switched to media containing 14 N, after one round of cell division the DNA sediments halfway between the 15 N and 14 N levels, indicating that it now contains fifty percent 14 N and fifty percent 15 N.. In subsequent cell divisions, an increasing amount of DNA contains 14 N only. These data support the semi-conservative replication model.
During the density gradient ultracentrifugation, the DNA was loaded into a gradient (Meselson and Stahl used a gradient of cesium chloride salt, although other materials such as sucrose can also be used to create a gradient) and spun at high speeds of 50,000 to 60,000 rpm. In the ultracentrifuge tube, the cesium chloride salt created a density gradient, with the cesium chloride solution being more dense the farther down the tube you went. Under these circumstances, during the spin the DNA was pulled down the ultracentrifuge tube by centrifugal force until it arrived at the spot in the salt gradient where the DNA molecules&rsquo density matched that of the surrounding salt solution. At the point, the molecules stopped sedimenting and formed a stable band. By looking at the relative positions of bands of molecules run in the same gradients, you can determine the relative densities of different molecules. The molecules that form the lowest bands have the highest densities.
DNA from cells grown exclusively in 15 N produced a lower band than DNA from cells grown exclusively in 14 N. So DNA grown in 15 N had a higher density, as would be expected of a molecule with a heavier isotope of nitrogen incorporated into its nitrogenous bases. Meselson and Stahl noted that after one generation of growth in 14 N (after cells had been shifted from 15 N), the DNA molecules produced only single band intermediate in position in between DNA of cells grown exclusively in 15 N and DNA of cells grown exclusively in 14 N. This suggested either a semi-conservative or dispersive mode of replication. Conservative replication would have resulted in two bands one representing the parental DNA still with exclusively 15 N in its nitrogenous bases and the other representing the daughter DNA with exclusively 14 N in its nitrogenous bases. The single band actually seen indicated that all the DNA molecules contained equal amounts of both 15 N and 14 N.
The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N and the other corresponded to the band of exclusively 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Dispersive replication would have resulted in exclusively a single band in each new generation, with the band slowly moving up closer to the height of the 14 N DNA band. Therefore, dispersive replication could also be ruled out.
Meselson and Stahl&rsquos results established that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are synthesized. The new strand will be complementary to the parental or &ldquoold&rdquo strand and the new strand will remain basepaired to the old strand. So each &ldquodaughter&rdquo DNA actually consists of one &ldquoold&rdquo DNA strand and one newly-synthesized strand. When two daughter DNA copies are formed, they have the identical sequences to one another and identical sequences to the original parental DNA, and the two daughter DNAs are divided equally into the two daughter cells, producing daughter cells that are genetically identical to one another and genetically identical to the parent cell.
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD, USA
Xuemin Chen, Huaibin Wang, Wei Yang & Martin Gellert
California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA
Yanxiang Cui & Z. Hong Zhou
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, MD, USA
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
X.C. carried out all experiments and structure determination. Y.C. collected cryo-EM micrographs on the Krios microscope at UCLA and helped with structure determination and refinement. H.W. helped with cryo-EM data collection on the TF20 and Krios at NIH. R.B.B. carried out molecular dynamics simulations. Z.H.Z., W.Y. and M.G. supervised the research project. X.C., R.B.B., W.Y. and M.G. prepared the manuscript.
Anti-parallel configuration of DNA strands
The final revelation that allowed Watson and Crick to complete their model came in a moment described as "a stroke of inspiration" when Watson realized that the nucleotides would fit together if one was "upside down" relative to the other. (According to Watson, he saw this possibility as he sat across a small table from Crick, both of them working with small models of nucleotides.) This upside down orientation would occur if the two strands that wrap around each other are not pointed in the same direction, but in opposite directions. Thus, these two strands are said to be anti-parallel, like the traffic on a two-lane highway (Figure 8).
Figure 8: Antiparallel nature of the DNA double helix. Notice how the sugar-phosphate backbone is on the outside of the "ladder" while the bases point inward. Notice also how the orientation of the two strands is "antiparallel" and thus look upside down compared to each other. This is most easily seen by looking at the pentose sugars (orange). image © Visionlearning, Inc.
Suddenly, everything made sense! With the two strands wrapping around each other in an anti-parallel configuration, Watson and Crick were able to fit the strands very close together, as Franklin's picture shows them to be, and the structure is regular and symmetrical. Most importantly, the nitrogen bases fit perfectly together through a type of chemical attraction called a hydrogen bond. Hydrogen bonds hold the two strands together stably, but not permanently. Specifically, an adenine–thymine "base pair" has two hydrogen bonds and a cytosine–guanine base pair has three hydrogen bonds. (See Figure 8 above.)
Given this anti-parallel structure, to distinguish the two strands of DNA, scientists say that one strand is oriented "5' to 3' " and the other strand is "3' to 5'." This is in reference to the 5'-3' connections in the phosphate-sugar backbone. The machinery of the cell also uses this orientation to select which direction to read the genetic information contained in the nucleotide sequence. Imagine trying to read an English sentence going from right to left. This would make no sense because the proper direction of reading English is left to right. Similarly, the DNA code must be read in the correct direction, which is 5' to 3'.
The beauty of the double-stranded anti-parallel configuration is found in the complementary base pairing according to Chargaff's law. If we know the sequence of nucleotides on one strand, we can accurately predict the nucleotides on the other. An adenine on one side of the DNA molecule would be paired with a thymine on the other side, and so on. Thus, if the two strands are separated, we could look at either strand and know exactly what was on the complementary strand. In fact, this is precisely what happens during DNA replication: The DNA double helix is pried apart or "unzipped" and both of the single strands then serve as copy templates for synthesizing a new strand. The result is two new DNA double helixes, both of which are identical to each other and to the original strand (Figure 9). Figure 9: Schematic of DNA replication method proposed by Watson and Crick. In this model, the two strands of the original DNA molecule are first pried apart. Then, complimentary nucleotides (A with T, G with C, etc.) are added opposite of both of the original strands. The result is two DNA molecules, both identical to the original strand (and thus to each other), and both with one old strand and one new strand. image © Visionlearning, Inc.
Once Watson and Crick had built the correct model, all could see that the anti-parallel configuration and the hydrogen bond base-pairing allowed this simple and effective means of DNA self-replication. In fact, the final sentence of their 1953 research article announcing the structure of DNA was, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Watson and Crick published their model of DNA in the journal Nature in 1953, a model which earned them the Nobel Prize in 1962.
There has been much debate about whether Rosalind Franklin, as a rare female scientist in the 1950s, received enough credit for her crucial contributions to this important discovery. Unfortunately, she died from ovarian cancer just five years after the model was built and Nobel Prizes are not given posthumously. In the 1950s, scientists were not aware of the cancer risks involved with repeated X-ray exposure and did not properly protect themselves from the radiation given off by these instruments. Thus, it is conceivable that Franklin's premature death was a direct result of her dedication to scientific research and her pursuit of the structure of the DNA molecule.
From the sequence of nucleotides on one DNA strand, we can predict
27.4.4. DNA Replication Requires Highly Processive Polymerases
Enzyme activities must be highly coordinated to replicate entire genomes precisely and rapidly. A prime example is provided by DNA polymerase III holoenzyme, the enzyme responsible for DNA replication in E. coli. The hallmarks of this multisubunit assembly are its very high catalytic potency, fidelity, and processivity. Processivity refers to the ability of an enzyme to catalyze many consecutive reactions without releasing its substrate. The holoenzyme catalyzes the formation of many thousands of phosphodiester bonds before releasing its template, compared with only 20 for DNA polymerase I. DNA polymerase III holoenzyme has evolved to grasp its template and not let go until the template has been completely replicated. A second distinctive feature of the holoenzyme is its catalytic prowess: 1000 nucleotides are added per second compared with only 10 per second for DNA polymerase I. This acceleration is accomplished with no loss of accuracy. The greater catalytic prowess of polymerase III is largely due to its processivity no time is lost in repeatedly stepping on and off the template.
From the Latin procedere, “to go forward.”
An enzyme that catalyzes multiple rounds of elongation or digestion of a polymer while the polymer stays bound. A distributive enzyme, in contrast, releases its polymeric substrate between successive catalytic steps.
These striking features of DNA polymerase III do not come cheaply. The holoenzyme consists of 10 kinds of polypeptide chains and has a mass of
900 kd, nearly an order of magnitude as large as that of a single-chain DNA polymerase, such as DNA polymerase I. This replication complex is an asymmetric dimer (Figure 27.30). The holoenzyme is structured as a dimer to enable it to replicate both strands of parental DNA in the same place at the same time. It is asymmetric because the leading and lagging strands are synthesized differently. A τ2 subunit is associated with one branch of the holoenzyme γ2 and (δδ′χψ)2 are associated with the other. The core of each branch is the same, an αϵθ complex. The α subunit is the polymerase, and the ϵ subunit is the proofreading 3′ → 5′ exonuclease. Each core is catalytically active but not processive. Processivity is conferred by β2 and τ2.
Proposed Architecture of DNA Polymerase III Holoenzyme. [After A. Kornberg and T. Baker, DNA Replication, 2d ed. (W. H. Freeman and Company, 1992).]
The source of the processivity was revealed by the determination of the three-dimensional structure of the β2 subunit (Figure 27.31). This unit has the form of a star-shaped ring. A 35-Å-diameter hole in its center can readily accommodate a duplex DNA molecule, yet leaves enough space between the DNA and the protein to allow rapid sliding and turning during replication. A catalytic rate of 1000 nucleotides polymerized per second requires the sliding of 100 turns of duplex DNA (a length of 3400 Å, or 0.34 μm) through the central hole of β2 per second. Thus, β2 plays a key role in replication by serving as a sliding DNA clamp.
Structure of the Sliding Clamp. The dimeric 㬢 subunit of DNA polymerase III forms a ring that surrounds the DNA duplex. It allows the polymerase enzyme to move without falling off the DNA substrate.
How is DNA anti-parallel?
DNA is double stranded, and the strands are antiparallel because they run in opposite directions.
Each DNA molecule has two strands of nucleotides. Each strand has sugar phosphate backbone, but the orientation of the sugar molecule is opposite in the two strands.
Both of the strands of DNA double helix can grow in 5' to 3' direction, but they grow in opposite directions due to opposite orientation of the sugar molecule in them.
The antiparallel orientation allows for the base pairs to compliment one another. Antiparallel DNA is also more structurally stable than parallel DNA.
The antiparallel orientation of DNA has important implications for DNA replication, as at the replication fork one strand allows steady replication, thereby known as leading strand while the other becomes lagging strand.
Francis Crick (1916-2004)
Francis Harry Compton Crick was born in a small town near Northampton, England. As a child, Crick was very inquisitive and he read all of the books of Children's Encyclopedia that his parents bought him. He found the sections that dealt with science most interesting. This interest led to "kitchen" experiments and eventually serious study and a second-class Honours degree in physics at University College, London.
The physics Crick learned in class was already out of date, so he taught himself the rudiments of quantum mechanics while doing graduate research on the viscosity of water. World War II interrupted his graduate studies. During the war, Crick worked for the Admiralty doing mostly research and design on magnetic and acoustic mines.
When the war ended, Crick continued to work at the Admiralty but he knew he did not want to design weapons for the rest of his life. The problem was that he was unsure what he did want to do. In the end, he decided to enter the life sciences. He liked reading, thinking, and talking about the new discoveries being made in the life sciences. Crick found that "what you are really interested in is what you gossip about." To pursue his interests, Crick visited several labs and scientists. He finally settled in for a two year stint at Strangeways Laboratory where he did work on the effects of magnetism on chick fibroblast cells.
In 1947, armed with this biology experience, Crick joined Max Perutz at the Cavendish Laboratory in Cambridge. Sir Lawrence Bragg was directing a new unit of the Laboratory where they were using X-ray crystallography to study protein structure. Max Perutz was working on the structure of hemoglobin and Crick's thesis project was on X-ray diffraction of proteins.
In 1951, Francis Crick met James Watson who was visiting Cambridge. Although Crick was twelve years older, he and Watson "hit it off immediately." Watson ended up staying at Cavendish, and using available X-ray data and model building, the two solved the structure of DNA. The classic paper was published in Nature in April 1953. A flip of the coin decided the order of the names on the paper. Francis Crick, James Watson and Maurice Wilkins shared the 1962 Nobel Prize for Physiology or Medicine for solving the structure of DNA. Maurice Wilkins and Rosalind Franklin provided some of the X-ray crystallographic data.
After the "double helix" model, there were still questions about how DNA directed the synthesis of proteins. Crick and some of his fellow scientists, including James Watson, were members of the informal "RNA tie club," whose purpose was "to solve the riddle of RNA structure, and to understand the way it builds proteins." The club focused on the "Central Dogma" where DNA was the storehouse of genetic information and RNA was the bridge that transferred this information from the nucleus to the cytoplasm where proteins were made. The theory of RNA coding was debated and discussed, and in 1961, Francis Crick and Sydney Brenner provided genetic proof that a triplet code was used in reading genetic material.
For most of his career, Crick was at Cambridge working for the Medical Research Council. In 1976, Crick moved to the Salk Institute in La Jolla where he focused his research on developmental neurobiology. In 1988, he wrote about his experiences in What Mad Pursuit: A Personal View of Scientific Discovery. Crick has been described as having a keen intellect and a dry, British sense of humor.
DNA was first crystallized in the late 70's &mdash remember, the 1953 X-ray data were from DNA fibers. So, the real "proof" for the Watson-Crick model of DNA came in 1982 after the B-form of DNA was crystallized and the X-ray pattern was solved.
If the DNA of one human cell is stretched out, it would be almost 6 feet long and contain over three billion base pairs. How does all this fit into the nucleus of one cell?
Meselson and Stahl Experiment Steps
Meselson and Stahl performed a series of an experiment, which includes the following steps:
- Growth of E.coli: First, the E.coli were grown in the medium containing 15 NH4Cl for several generations. NH4 provides the nitrogen as well as a protein source for the growth of the E.coli. Here, the 15 N is the heavy isotope of nitrogen.
- Incorporation of 15 N: After several generations of E.coli, Meselson and Stahl observed that the 15 N heavy isotope has incorporated between the DNA nucleotides in E.coli.
- Transfer of E.coli cells: The DNA of E.coli labelled with 15 N isotope were transferred to the medium containing 14 NH4Cl. Here, the 14 N is the light isotope of nitrogen. The E.coli cells were again allowed to multiply for several generations. The E.coli cells will multiply every 20 minutes for several generations.
- Processing of DNA: For the processing or separation of DNA, the E.coli cells were transferred to the Eppendorf tubes. After that, caesium chloride is added, having a density of 1.71 g/cm 3 (the same of DNA). Finally, the tubes were subjected to high-speed centrifugation 140,000 X g for 20 hours.
After centrifugation, the DNA separates based on mass or density. Different DNA bands like heavy, intermediate and light DNA forms as a result of the concentration gradient created by CsCl.
The light DNA will consist of a pure 14 N isotope. An intermediate DNA band will indicate the combination or mixture of both 15 N and 14 N isotopes. The occurrence of heavy DNA bands will consist of a pure 15 N isotope.
The result, after two generations of E.coli, the following results were obtained:
In the F-1 generation: According to the actual observations, two DNA strands (with a mixture of both 15 N and 14 N isotopes) will produce in F-1 gen. The above diagram shows that the semiconservative and dispersive model obeys the pattern of growth explained by Meselson and Stahl.
Thus, it is clear that the DNA does not replicate via “Conservative mode”. According to the conservative model, the DNA replicates to produce one newly synthesized DNA and one parental DNA. Therefore, the conservative model was disapproved, as it does not produce hybrid DNA in the F-1 generation.
In the F-2 generation: According to the actual observation, four DNA strands (two with hybrid and the remaining two with light DNA) will produce in the F-2 generation. The hybrid DNA includes a mixture of 15 N and 14 N. The light DNA strands contain a pure 14 N. The diagram shows that only semi-conservative type of replication gave similar results conducted by Meselson and Stahl. Thus, both the conservative and dispersive modes of replication were disapproved.
Therefore, we can conclude that the type of replication in DNA is “Semi conservative”. The offsprings have a hybrid DNA containing a mixture of both template and newly synthesized DNA in the semi-conservative model. After each multiplication, the number of offspring will double, and half of the parental DNA will be conserved for the next generation.
Epigenetics: DNA Isn&rsquot Everything
Research into epigenetics has shown that environmental factors affect characteristics of organisms. These changes are sometimes passed on to the offspring. ETH professor Renato Paro does not believe that this opposes Darwin&rsquos theory of evolution.
A certain laboratory strain of the fruit fly Drosophila melanogaster has white eyes. If the surrounding temperature of the embryos, which are normally nurtured at 25 degrees Celsius, is briefly raised to 37 degrees Celsius, the flies later hatch with red eyes. If these flies are again crossed, the following generations are partly red-eyed &ndash without further temperature treatment &ndash even though only white-eyed flies are expected according to the rules of genetics.
Environment affects inheritance
Researchers in a group led by Renato Paro, professor for Biosystems at the Department of Biosystems Science and Engineering (D-BSSE), crossed the flies for six generations. In this experiment, they were able to prove that the temperature treatment changes the eye colour of this specific strain of fly, and that the treated individual flies pass on the change to their offspring over several generations. However, the DNA sequence for the gene responsible for eye colour was proven to remain the same for white-eyed parents and red-eyed offspring.
The concept of epigenetics offers an explanation for this result. Epigenetics examines the inheritance of characteristics that are not set out in the DNA sequence. For Paro, epigenetic mechanisms form an additional, paramount level of information to the genetic information of DNA.
Such phenomena could only be examined in a descriptive manner in the past. Today, it has been scientifically proven, which molecular structures are involved: important factors are the histones, a kind of packaging material for the DNA, in order to store DNA in an ordered and space-saving way. It is now clear that these proteins have additional roles to play. Depending on the chemical group they carry, if they are acetylated or methylated, they permanently activate or deactivate genes. New methods now allow researchers to sometimes directly show which genes have been activated or deactivated by the histones.
Cells have a memory
Epigenetic marks, such as the modifications of the histones, are also important for the specialisation of the body&rsquos cells. They are preserved during cell division and are passed on to the daughter cells. If skin cells divide, more skin cells are created liver cells form liver cells. In both cell types, all genes are deactivated except the ones needed by a skin or liver cell to be a skin or liver cell, and to function appropriately. The genetic information of the DNA is passed on along with the relevant epigenetic information for the respective cell type.
Paro&rsquos group is researching this cell memory. It is still unclear how the epigenetic markers are passed on to the daughter cells. During cell division, the DNA is doubled, which requires the histones &ndash as the current picture suggests &ndash to break apart. The question is therefore how cellular memory encoded by epigenetic mechanisms survives cell division.
Emerging area of research
A similar question remains for the inheritance of the epigenetic characteristics from parents to offspring. They now know that when the gametes are formed, certain epigenetic markers remain and are passed on to the offspring. The questions, which are currently being researched, are how much and which part of the epigenetic information is preserved and subsequently inherited.
The research is also looking at the influence of various substances from the environment on the epigenetic constitution of organisms, including humans. Diet and epigenetics appear to be closely linked. The most well known example is that of the Agouti mice: they are yellow, fat and are prone to diabetes and cancer. If Agouti females are fed with a cocktail of vitamin B12, folic acid and cholin, directly prior to and during pregnancy, they give birth to mainly brown, slim and healthy offspring. They in turn mainly have offspring similar to themselves.
Contradiction to Darwin?
Environmental factors, which change the characteristics of an individual and are then passed on to its offspring, do not really fit into Darwin&rsquos theory of evolution. According to his theory, evolution is the result of the population and not the single individual. &ldquoPassing on the gained characteristics fits more to Lamarck&rsquos theory of evolution&rdquo, says Paro.
However, he still does not believe Darwin&rsquos theory of evolution is put into question by the evidence of epigenetics research. &ldquoDarwin was 100 percent right&rdquo, Paro emphasises. For him, epigenetics complement Darwin&rsquos theory. In his view, new characteristics are generated and passed on via epigenetics, subject to the same mechanisms of evolution as those with a purely genetic origin.
Materials provided by ETH Zurich. Note: Content may be edited for style and length.