Cell Replication
Inquiry question: How important is it for genetic material to be replicated exactly?
Students model the processes involved in cell replication, including but not limited to DNA replication, using the Watson and Crick DNA model, including nucleotide composition, pairing and bonding.
Introduction and the historical context
Thus far, we have learned that living things reproduce. All reproduction occurs at the cellular level. Before cells divide, the genome must be replicated. Why is it important? It ensures that the daughter cells produced through binary fission or mitosis contain the same amount of DNA as the parent cells. Although meiosis halves the amount of DNA in gametes, fertilisation restores the DNA content of the offspring.
The search for the genetic material
Although Mendel had worked out the principles of heredity (i.e. the principles that apply to the inheritance of biological traits), he needed to learn how information about the traits was passed from one generation to the next. He called the units of inheritance ‘factors’. Charles Darwin also speculated on the nature of inheritance. He proposed that all cells in an organism are capable of shedding minute particles, called gemmules, which circulate throughout the body and finally congregate in the gonads”. Darwin’s idea, called Pangenesis, is now discredited.
Theodore Boveri's work on sea urchin development
Theodore Boveri was a German biologist who studied reproduction in various animal species. He noticed that in the worm, Ascaris megalocephala, the number of chromosomes in mature egg cells were half that in the other cells of the animal. He also studied cellular and chromosomal events during cell division and described features such as the centrosome, centriole and spindle fibres in dividing cells (Fig X1).
Figure X1. Cell division in Ascaris megalocephala. The black bodies in the middle of the cell are chromosomes, which are attached to the centrosomes via spindle fibres.
Boveri then moved to Naples, where he experimented with sea urchins. Marine invertebrates, including sea urchins, produce large numbers of eggs and sperm that are released into the external environment. This makes them useful organisms to study fertilisation and embryological development. Indeed, work with gametes from marine organisms has demonstrated that during fertilisation, the sperm contributes only to its nucleus. Thus, the goal of fertilisation is to bring together the male and female gamete nuclei, which fuse to form the zygote. Both the gamete nuclei are haploid, while the zygote is diploid.
Boveri then conducted a series of experiments to produce hybrid sea urchin embryos. He worked with two species of sea urchins, Sphaerechinus granularis and Psammechinus microtuberculatus: these species were sufficiently closely related (same number of chromosomes) so that they could be interbred. When Boveri fertilised S. granularis eggs with P. microtuberculatus sperm, the offspring contained features of both species, suggesting that the chromosomes of the gametes carried the genetic information of the animals. He also identified offspring that contained only paternal features. He inferred from those observations that the paternal features in the offspring were contained in sperm chromosomes. The correct number of chromosomes from both fertilising gametes was important to ensure normal embryonic development.
Walter Sutton's discovery of meiosis in grasshoppers
The same year that Boveri published his work on sea urchins (1902), the American biologist, Walter Sutton, published his research findings that suggested that chromosomes were the physical basis for Mendel's observations on the inheritance of traits in pea plants.
“I may finally call attention to the probability that the association of paternal and maternal chromosomes in pairs and their subsequent separation during the reducing division . . . may constitute the physical basis of the Mendelian law of heredity."
Sutton (1903)
Sutton's drawings of Brachystola magna chromosomes during nuclear division, show the three smallest chromosome pairs (i, j, k) and the accessory chromosome (x) (Sutton, 1902, figs. 1, 2, 3).
Chromosomes of Brachystola magna during prophase I. The panel on the left shows chromosomes in an earlier stage of prophase I than those in the right panel. Sutton named the chromosomes a to k, in order of decreasing size. The unequal chromosome pair was called the accessory chromosome, labelled X. Image credit: McKusick (1960).
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Sutton's findings |
Modern interpretation |
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The somatic chromosome groups are made up of two equivalent chromosome groups, one of maternal derivation and one of paternal. |
This is the description of the diploid state - two sets of chromosomes, with each set derived from one parent. |
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Synapsis is the pairing of corresponding or homologous maternal and paternal chromosomes1. |
The crossing-over of homologous during prophase I of meiosis. |
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Chromosomes retain their morphologic and functional individuality throughout the life cycle. |
Although chromosomes change their shape (e.g. condensation) and move to different parts of the dividing cell, every chromosome remains an intact and discrete unit. Each chromosome retains its full complement of genes throughout meiosis. |
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The synaptic mates contain the physical units that correspond to the Mendelian allelomorphs; that is, the chromosomes contain the genes. |
The alleles of a gene are located on homologous
chromosomes. |
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The position of the bivalent chromosomes in the equatorial plate of the reducing division is purely a matter of chance - that is, any chromosome pair may lie with maternal or paternal chromatid indifferently toward either pole irrespective of the positions of other pairs-and hence a large number of different combinations of maternal and paternal chromosomes are possible in the mature germ-products of an individual. The number of possible combinations in the germ-products of a single individual of any species is represented by the simple formula 2n in which n represents the number of chromosomes in the reduced series. |
The maternal and paternal chromosomes of a homologous
pair arrange themselves randomly along the equatorial plate
during metaphase. Since each member of a homologous
chromosome pair moves to a different daughter cell, the
total number of genetic variants possible is described by
2n, where n is the haploid number of the organism. |
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Some chromosomes at least are related to a number of different allelomorphs. All the allelomorphs represented by any one chromosome must be inherited together. The same chromosome may contain allelomorphs that may be dominant or recessive independently.2 |
This is the basis for gene linkage - genes that are
located closely together on a chromosome will be inherited
together, but will behave independently (i.e. the
dominance/recessiveness of an allele will not affect the
behaviour of other linked genes). |
Table XX. This table summarises Sutton’s conclusions about the behaviour of chromosomes during meiosis and how the model provides a physical basis for Mendel's Laws of Inheritance. Table adapted from McKusick (1960).
1 Sutton did not discover the crossing over of homologous chromosomes. Frans Alfons Janssens first described it, and later, other biologists, such as Thomas Hunt Morgan, developed it.
2 Here, Sutton predicted gene linkage. However, he did not have any evidence for such behaviours of genes.
Other works, such as Theodore Boveri’s and Walter Sutton’s research, highlighted the importance of chromosomes in inheritance. Boveri’s work on sea urchin development showed that normal development required a complete set of chromosomes. Sutton, who investigated grasshopper reproduction, showed that the behaviour of chromosomes during meiosis could explain the segregation and assortment of characters that Mendel had described many years before. The chromosome theory was eventually accepted when Thomas Hunt Morgan published his work on the inheritance of eye colour in the fruit fly, Drosophila melanogaster. The inheritance of eye colour in this organism (red and white) demonstrates a dominant (red)-recessive (white) pattern. This follows Mendel’s Laws of inheritance. However, Morgan also noticed that only male flies showed the white eye colour trait. Thus, the inheritance of this trait appears to be associated with the sex of the flies.
Fred Griffith
Oswald Avery, Colin MacLeod, and Maclyn McCarty
Alfred Hershey and Martha Chase.
1. Griffith’s experiment: the transforming principle
Fred Griffith was a bacteriologist who researched and developed a vaccine against Streptococcus pneumoniae, which causes pneumonia. Different variants of S. pneumoniae exist, and Griffith experimented with Types II and III. These types exist in two different forms (phenotypes): R (Rough) and S (Smooth).
The S form is the virulent form of the bacterium and is lethal to hosts when injected into animals. However, only living S-form Streptococcus bacteria are deadly to the host animals - if they are killed by heating before injection into animals, the bacteria will not harm the hosts. Griffith injected different S. pneumoniae bacteria into mice, as Figure XX shows. As expected, the mice injected with live S-form Streptococcus bacteria died, while those that received the heat-killed Type S survived the challenge. Those animals injected with R-form Streptococcus also survived the challenge.
For one group of mice, Griffith injected a mixed cocktail of R and S Streptococcus bacteria - live R and dead S. He had hypothesised that since the Type S bacteria were heat-killed and that the Type R bacteria were non-virulent (non-lethal), the mice should survive. However, to his surprise, those mice died. The autopsy revealed that the mice contained live S-form bacteria. G Griffith concluded that something from the heat-liked Type S bacteria had transferred to the R-fom bacteria and transformed them into the S-form bacteria. As he did not know the identity of this substance, he called it the Transforming Principle.
A pictorial representation of Griffith’s experiment on bacterial transformation. Note that this investigation uses two forms (R, S) and two types (II, III) of pneumococcus bacteria. Griffith concluded that, based on the mixed inoculation data, the Type IIR pneumococcus bacteria became transformed into the Type IIIS bacteria. He further postulated that some molecular factor, which he called a transforming factor, was responsible for this transformation. Image credit: NSDU.
Before Fred Friffith’s work was published, bacteriologists believed that the R and S forms of Streptococcus pneumoniae were distinct strains and were not interchangeable. However, Griffith’s research indicated that the R form could be transformed into the S form. The transformation of the R form to the S was a genetic change (a non-virulent form of the bacteria changed into a virulent form). However, what substance, in the transforming principle caused this change?
2. Avery, Macleod and McCarty’s experiment: DNA is the transforming principle
DNA, RNA or protein are three leading candidates for Griffith’s transforming principle. Avery and his team of researchers conducted the following experiment to distinguish between these possibilities. Following Griffith’s experiment, R-form Streptococcus cells were mixed with the transforming principle from heat-killed S-type Streptococcus bacteria. The transforming principle was treated with proteases, RNase or DNAse in the experimental groups before mixing with the R-form cells. Proteases destroy proteins in the transforming principle sample, while RNAse and DNAse destroy RNA and DNA, respectively. Figure XX shows no transformation occurred when the DNase-treated transforming principle was mixed with the R-type bacteria. This study, published 16 years after Griffith’s publication, confirmed that DNA, not RNA or protein, was the carrier of genetic information in living things.
This figure shows the design and outcomes of the investigation conducted by Avery, Macleod and McCarty to identify Griffith’s transforming factor. Each enzymatic treatment destroys the target molecules, thus eliminating them from consideration as the candidate for the transforming factor. As shown in the figure, DNase treatment prevented the transformation of Type IIR cells into Type IIIS. This confirmed that DNA was the transforming factor. Image credit: NSDU.
3. The Hershey-Chase experiment
Alfred Hershey and Martha Chase researched bacteriophages. Bacteriophages (also called phages) are viruses that infect bacterial cells. Pages are composed of an inner core of nucleic acid (DNA or RNA) surrounded by a protein coat. To reproduce, phages attach to the surfaces of bacteria and inject their genetic material into the cells. Hershey and Chase realised this could be a useful experimental system to determine if DNA or protein is the carrier of genetic information.
Atomic structural model of bacteriophage T4. Dr. Victor Padilla-Sanchez, PhD. CC 4.0
Hershey and Chase worked with the T2 phage, which uses DNA as its genetic material. But how could they distinguish between DNA and protein in cells? Chemical analyses revealed elemental differences between them: phosphorus is found in DNA but not in protein. Similarly, sulfur occurs in protein but not DNA. Both phosphorus and sulfur exist as isotopes, and some of those isotopes are radioactive. Hershey and Chase discovered that radioactivity would be incorporated into the phages’ DNA or protein if the phages were cultured in media containing the radioisotopes. The researchers monitored the radioactivity in the samples using a Geiger counter.
An overview of the Hershey-Chase experiment. CNX OpenStax. CC 4.0.
Hershey and Chase discovered that only the DNA entered the bacterial cells during virus replication. The viral proteins remained extracellular. In their paper, Hershey and Chase describe this as the “physical separation of the phage T2 into genetic and non-genetic parts” After replication, the progeny viruses contained radioactive DNA, not radioactive protein.
Perspective
These three classic experiments, together with many other studies, describe the complex investigations that led to our understanding that DNA carries the genetic information passed to the next generation. By the 1950s, scientists accepted that DNA was the molecule of heredity.
The structure of DNA
What is the nature of genetic information? How is it used in living organisms? How is it transmitted to the next generation? Scientists realised that the answers to these questions must lie in the structure of the DNA molecule.
Historical perspectives
In 1869, the Swiss physician and biologist, Friedrich Miescher, discovered a substance in the nuclei of white blood cells, which he called nuclein. This name was later changed to nucleic acid and then to deoxyribonucleic acid. The Russian biochemist, Phoebus Levene, published his work on the composition of DNA. He indicated that DNA was composed of nucleotides, which, in turn, consisted of three components of nucleotides - sugar, phosphate and nitrogenous bases.
The chemical structure of a nucleotide. A single nucleotide comprises three components: a nitrogen-containing base, a five-carbon sugar, and a phosphate group. The nitrogenous base is either a purine or a pyrimidine. The five-carbon sugar is either a ribose (in RNA) or a deoxyribose (in DNA) molecule. Image credit: Nature Education.
Erwin Chargaff, an Austro-Hungarian biochemist, analysed the chemical composition of DNA from different sources. As shown in the following table, he observed a curious property of nucleotides in DNA: the ratio of purine nucleotides (adenine and guanine) to pyrimidine nucleotides (thymine and cytosine) was almost always equal to one. Furthermore, he discovered that the molar concentration of adenine is approximately equal to thymine's, while cytosine's concentration is roughly equal to guanine. These ratios of nucleotides in DNA came to be known as Chargaff’s rules. The following tables show the nucleotide composition of DNA from different organisms. The first table shows the detailed nucleotide composition of DNA from different sources, while the second table provides an organism-level summary of the same data.
|
Tissue or organism |
A |
G |
C |
T |
A+G |
C+T |
[A+G]/[C+T] |
A/T |
C/G |
|
Ox thymus |
0.26 |
0.21 |
0.16 |
0.25 |
0.47 |
0.41 |
1.1 |
1.0 |
0.8 |
|
Ox thymus |
0.25 |
0.24 |
0.18 |
0.24 |
0.49 |
0.42 |
1.2 |
1.0 |
0.8 |
|
Ox thymus |
0.30 |
0.22 |
0.17 |
0.25 |
0.52 |
0.42 |
1.2 |
1.2 |
0.8 |
|
Ox Spleen |
0.25 |
0.20 |
0.15 |
0.24 |
0.45 |
0.39 |
1.2 |
1.0 |
0.8 |
|
Ox Spleen |
0.26 |
0.21 |
0.17 |
0.24 |
0.47 |
0.41 |
1.1 |
1.1 |
0.8 |
|
Human sperm |
0.29 |
0.18 |
0.18 |
0.31 |
0.47 |
0.49 |
1.0 |
0.9 |
1.0 |
|
Human sperm |
0.27 |
0.17 |
0.18 |
0.30 |
0.44 |
0.48 |
0.9 |
0.9 |
1.1 |
|
Human thymus |
0.28 |
0.19 |
0.16 |
0.28 |
0.47 |
0.44 |
1.1 |
1.0 |
0.8 |
|
Avian Tubercle bacillus |
0.12 |
0.28 |
0.26 |
0.11 |
0.4 |
0.37 |
1.1 |
1.1 |
0.9 |
|
Yeast |
0.24 |
0.14 |
0.13 |
0.15 |
0.38 |
0.28 |
1.4 |
1.6 |
0.9 |
|
Yeast |
0.30 |
0.18 |
0.15 |
0.29 |
0.48 |
0.44 |
1.1 |
1.0 |
0.8 |
Table showing the proportions of nucleotides in the DNAs from different sources. The two conclusions that can be inferred from this data are that, in any DNA, (1) the amount of purine nucleotides (A+G) is approximately the same as the amount of pyrimidine nucleotides (C+T), and (2) the amount of adenine is the same as thymine, and the amount of cytosine is the same as guanine. This table is adapted from Chargaff E. Chemical specificity of nucleic acids and mechanism of their enzymatic degradation. Experientia. 1950 Jun;6:201-9.
|
Organism |
Species |
A/G |
T/C |
Pu/Py |
|
Human |
Homo sapiens |
1.56 |
1.75 |
1.0 |
|
Wheatgerm |
Triticum vulgare |
1.56 |
1.75 |
1.0 |
|
Yeast |
Saccharomyces cerevisiae |
1.22 |
1.18 |
1.0 |
|
Bacterium |
Escherichia coli, K-12 |
1.05 |
0.95 |
1.0 |
Molar proportions of purines and pyrimidines in DNA from different species and organisms. Adapted from Manchester (2008).
Erwin Chargaff’s paper chromatography analysis of nucleotide extracts from DNAs from different organisms. A = Adenine, C = Cytosine, T = thymine and G = Guanine. Also included are nucleotides from RNA: H = Hypoxanthine, X = Xanthine, and U = Uracil. Image credit: Chargaff E. Chemical specificity of nucleic acids and mechanism of their enzymatic degradation. Experientia. 1950 Jun;6:201-9.
What is Chargaff’s rule? All DNA follows Chargaff’s Rule, which states that the total number of purines in a DNA molecule equals the total number of pyrimidines. Image credit: Nature Education.
James Watson and Francis Crick, working in Cambridge, England, assembled a model of DNA. One critical piece of evidence for their model was an X-ray crystallographic DNA image produced by Rosalind Franklin.
Photo51, an X-ray crystallographic image of DNA. Photo 51. (2023, April 28). In Wikipedia.
Photo51 suggested that DNA had a regular (fixed diameter) helical structure. Using this information (including the distances between the atoms in the DNA crystal) and Chargaff’s law, Watson and Crick constructed a structural model of the DNA molecule. According to this model, DNA is composed of two intertwining polynucleotide chains. Each polynucleotide chain is a linear arrangement of nucleotides. The nucleotides in the polynucleotide chain are arranged so that the deoxyribose sugar and the phosphate molecules form a “backbone” The nitrogenous bases project away from the backbone.
The double-helical structure of DNA. This is a model of a right-hand DNA helix (the molecule twists to the right). Each DNA molecule has a fixed width of 2 nm. One turn of the helix consists of 10 base pairs and spans a length of 3.4 nm. Image credit: Nature Education.
The nitrogenous bases on the polynucleotide chains of DNA interact with each other. The intermolecular distance formed by a G-C base pair is identical to that created by an A-T base pair. This was one of the crucial insights that Watson and Crick had about the structure of DNA. It explains the fixed width predicted by photo51, the equimolar amounts of A and T, and G and C that Chargaff discovered. Another feature of the molecule explained by the Watson-Crick model is the attractive force between the polynucleotide chains in DNA. When a G on one strand is located next to a C on the other strand, hydrogen bonds can form between certain atoms of the nitrogenous bases of the two nucleotides. The following figure shows three hydrogen bonds that can form between G-C and two between A-T. Thus, in a large DNA molecule, thousands of hydrogen bonds form between the polynucleotide strands, thus forming a stable double helix. The G-C and A-T interactions in DNA are called complementary base pairing.
Base pairing in DNA. Two hydrogen bonds connect T to A; three join G to C. The sugar-phosphate backbones (grey) run antiparallel to each other to align the 3’ and 5’ ends of the two strands. Image credit: Nature Education.
Thus the Watson-Crick model of DNA explains many chemical and physical features that other scientists discovered. Two critical features of the model that we will explore in other sections of this resource are:
The hydrogen bonds between the two strands of DNA explain how the molecule can be ‘opened up’ for processes such as DNA transcription and replication and enable biotechnologies such as DNA sequencing and PCR amplification.
The complementary base pairing means that each strand can act as a template to produce the other. This is the basis for DNA replication.
DNA Replication
If a DNA molecule, which carries genetic information, can make copies of itself, it answers a profound question in biology: how is genetic information passed from parents to offspring? As each DNA molecule acts as a template, new DNA molecules can be produced. How does that happen?
Proposed models of DNA replication
Inspired by the double-helical structure of DNA, scientists proposed three possible models of DNA replication: conservative, semi-conservative and dispersive. These are shown in the following figure. These models differ in the arrangement of parental and newly-synthesised DNA.
Conservative: After DNA replication, one daughter DNA molecule is new, while the other is the parental DNA molecule.
Semi-conservative: After DNA replication, every new DNA double helix would be a hybrid that consisted of one strand of old DNA bound to one strand of newly synthesized DNA.
Dispersive: After DNA replication, DNA double helices are part original DNA and part new DNA.
Three models of DNA replication were proposed by scientists in the 1950s: conservative, semi-conservative and dispersive. In this diagram, the grey boxes represent DNA from the parental DNA sequences, while the blue boxes are newly synthesised DNA. Image credit: CNX OpenStax. CC by 4.0
The Messelson and Stahl experiment
Scientific models possess two critical features. They are;
Explanatory: scientific models explain current observations and data. For example, the double-helical model of DNA structure explains the nucleotide composition data and the X-ray diffraction patterns.
Predictive: scientific models predict outcomes that were not evident previously. For example, the double-helical model of DNA predicted that the structure of DNA allows for the replication of the molecule.
Despite knowledge of DNA’s structure, the mechanism of DNA replication was not evident to scientists in the 1940s and early 1950s. Thus, the conservation, semi-conservative and dispersive models of DNA replication were proposed. Those models predicted different outcomes when daughter DNA molecules were compared to their parents.
To determine which of these models correctly represents how DNA replicates, Matthew Meselson and Franklin Stahl used radioisotopes to investigate the products of DNA replication. They used two isotopes of nitrogen (14N ('light' nitrogen) and 15N ('heavy nitrogen')) in their experiments. They added these nitrogen isotopes as ammonium chloride salts in bacterial growth media. Once the salts enter bacterial cells, they will be used to synthesise DNA nucleotides. During DNA replication, those nucleotides will be incorporated into the daughter DNA molecules.
In addition to using nitrogen isotopes, Meselson and Stahl used a technique known as cesium chloride density gradient centrifugation. This technique separates DNA, based on the size (density) in a cesium chloride gradient. This is described in the following figure.
Cesium chloride density gradient centrifugation of DNA. The centrifuge tube contains a solution of cesium chloride. The solution is densest at the bottom of the tube and least dense at the top. DNA travels from the top of the tube towards its bottom in this solution. The DNA stops moving when it reaches a region of cesium chloride with the same density. This figure shows 3 DNA bands. DNA containing only the light nitrogen isotope is the least dense and occupies a position towards the top of the tube. Similarly, DNA containing only the heavy nitrogen is densest and is located towards the bottom of the tube. DNA that contains both the light and heavy nitrogen isotopes has an intermediate density and is located between those 2 bands. The author created this figure using Biorender (https://BioRender.com).
Meselson and Stahl’s approach to resolving the DNA replication model involved the following (note this is a simplified account for students - experimental details have been omitted).
E. coli bacteria were grown for several generations in media containing heavy nitrogen. As a result, the DNA of those bacteria contained only heavy nitrogen.
The bacteria were then transferred to fresh media containing light nitrogen.
After the DNA was allowed to replicate, the DNA was extracted and analysed using cesium chloride density gradient centrifugation.
Their result, summarised in the following figure, provides conclusive evidence in support of the semi-conservative model of DNA replication.
Figure showing the result of the cesium chloride density centrifugation experiment. The reference tube shows the relative locations of the DNA bands based on the amounts of heavy and light nitrogen in them. Tube A shows the DNA from bacteria grown in heavy nitrogen. After DNA replication in the presence of light nitrogen, the DNA is a hybrid of heavy and light nitrogen isotopes.
Reference
HERSHEY AD, CHASE M. Independent functions of viral protein and nucleic acid in growth of bacteriophage. J Gen Physiol. 1952 May;36(1):39-56. doi: 10.1085/jgp.36.1.39. PMID: 12981234; PMCID: PMC2147348.
Griffith F. The significance of pneumococcal types. Epidemiology & Infection. 1928 Jan;27(2):113-59.
AVERY, 0. T., C. M. MACLEOD and M. MCCARTY, 1944 Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exp. Med. 79: 137-158.
Hernandez, Victoria, “The Hershey-Chase Experiments (1952), by Alfred Hershey and Martha Chase”. Embryo Project Encyclopedia (2019-06-23). ISSN: 1940-5030 http://embryo.asu.edu/handle/10776/13109.
McKusick VA. Walter S. Sutton and the physical basis of Mendelism. Bulletin of the History of Medicine. 1960;34:487.
Manchester KL. Historical Opinion: Erwin Chargaff and his ‘rules’ for the base composition of DNA: why did he fail to see the possibility of complementarity? Trends in biochemical sciences. 2008 Feb 1;33(2):65-70.
Sutton, W. S., 1902 On the morphology of the chromosome group in Brachystola magna. Biol. Bull. 4:24–39.
Sutton, W. S., 1903 231–251. The chromosomes in heredity. Biol. Bull. 4:231-251