HSC – Biology – Maintaining a Balance



HSC Biology – Blueprint of Life

4. The structure of DNA can be changed and such changes may be reflected in the phenotype of the affected organism.

The phenotype of an organism is its total appearance determined during development by an interaction between its genetic make-up (genotype) and the environment.

A genome is all of the genetic material (DNA) within a cell and is specific to each organism. Genomes influence nearly all the traits or phenotypes. The phenotypic appearance is therefore directly affected by gene expression. The extent of phenotypic differences depends on how different the DNA sequences are in individuals, but may also be influenced by the environment.

• Outline evidence that led to Beadle and Tatum’s ‘one gene-one protein’ hypothesis and explain why this was altered to the ‘one gene – one polypeptide’ hypothesis

Beadle and Tatum carried out experiments with red bread mould. The normal variety of mould can manufacture certain substances that it needs for living, including vitamin B1, B2, B4 and B12. The normal moult possesses specific enzymes that catalyse the different reactions that produce these vitamins.

Beadle and Tatum produced several varieties of the bread mould, each of which had a change in one of its genes. They tested these varieties and found that some had lost their ability to make vitamin B2 while others could no longer make vitamin B4 and so on.

The results obtained by Beadle and Tatum showed that a change in various genes of the bread mould resulted in the loss of different enzymes and the failure of specific products to appear. These results indicated that one gene controlled vitamin B2 production and a different gene controlled vitamin B4 production.

CHANGED GENE -> NO ENZYME -> NO REACTION -> NO VITAMIN

Beadle and Tatum concluded that different genes are involved in making different enzymes that catalyse different reactions in a cell. They stated that there was a ‘one to one relationship between a gene and specific enzymic reaction.

ONE SPECIFIC GENE -> ONE PROTEIN (ENZYME) THAT CATALYSES REACTION

Many proteins consist of a single polypeptide that is built of amino-acid subunits. However some proteins consist of several polypeptides that become associated to form an active protein. For example, haemoglobin consists of four polypeptides. Recognition that proteins may consist of one or more polypeptides means that Beadle and Tatum’s conclusion is now expressed more accurately as:

One particular gene -> one polypeptide chain => one cellular activity

Extra:

The proposal that a gene is responsible for the production of a specific protein was first put forward in 1909. This idea, together with work on the inheritance of eye colour in Drosophila, led biologists to investigate the importance of genes in enzyme production. George Beadle and Edward Tatum worked on mutants of the fungus Neurospora crassa (a mould), leading to their groundbreaking discovery that genes provide the instructions for making proteins.

They put forward the hypothesis that one gene controls the production of one enzyme. This was based on observations made in Beadle’s earlier experiments on fruit flies—he found that if fruit flies with normal eye colour were exposed to X-rays, their offspring would show a change in eye colour. He hypothesized that this occurred as a result of a defective enzyme for the eye pigment. Since evidence was needed to support this hypothesis, Beadle and Tatum designed and carried out an experiment to attempt to mutate genes of the mould Neurospora crassa, the enzyme functioning of which they could test fairly simply. Experimental evidence led them to propose a hypothesis that genes affect enzyme production—the details of this were later modified as the understanding of the relationship between genes and proteins advanced.

Beadle and Tatum’s experiment

1. They irradiated the bread mould Neurospora crassa with X-rays to induce mutations. The resulting mould forms they called mutants.

2. Further experimentation showed that some of the mutants could no longer produce an essential amino acid (implying that a particular enzyme was no longer functioning).

3. To test that whether this loss of function had a genetic basis, they then crossed these mutant moulds with the normal moulds and found that some of their offspring shared the mutant phenotype, proving that the inability to produce the amino acid could be inherited—that is, it was due to a genetic defect or mutation.

4. With further analysis it was found that different enzymes in different mutants had been altered or were missing, proving that a gene determines the structure of a specific enzyme. This led them to propose their fi rst hypothesis: the ‘one gene—one enzyme’ hypothesis.

A changed hypothesis

Beadle and Tatum’s ‘one gene—one enzyme’ hypothesis changed to the ‘one gene—one protein’ hypothesis, once it was demonstrated that there are other proteins besides enzymes that are encoded by genes. After the discovery by biologists that one gene is not necessarily responsible for the structure of an entire protein, but for each polypeptide chain making up that protein, the current one gene—one polypeptide hypothesis was adopted. This is the currently accepted theory and has stood up to rigorous testing, holding true for all predictions so far.

• Describe the process of DNA replication and explain its significance

The active functioning of DNA in the everyday activities of cells takes two forms:

1. DNA replication: In its hereditary role, DNA must be able to make an exact copy of itself so that when a cell divides, the resulting daughter cells each have a full complement of DNA.

2. Protein synthesis: Genes are expressed in terms of the protein products they produce. Many of these proteins are enzymes, which control the chemical functioning of cells. Other proteins produced nay form a structural part of the cell (e.g. the protein in cell membranes, pigment in skin and eyes, and silk in insect cocoons) and some proteins form essential chemicals such as hormones (e.g. insulin).

Process of DNA Replication

The process of DNA replication is termed semi-conservative, as the two strands of the original DNA molecule separate and each gives rise to a new complementary strand. This mechanism ensures that the genetic material is copied exactly.

DNA replication begins when a region of double-stranded DNA unwinds to form two short lengths of single-stranded DNA. An enzyme called helicase causes the DNA helix to progressively unwind.

Each of the single strands acts as a template for building a new complementary strand. Nucleotide building blocks are the raw material for DNA replication. The nucleotides come into place – where G is the template strand, a C-containing molecule is brought into place. Where there is a T in the template strand, an A-containing molecule is brought into place. Where there is a T in the template strand, an A-containing molecule is brought into place, and so on. In this way, the base sequence of a double-stranded molecule of DNA controls the order of the nucleotides in two new single strands of DNA. This process is catalysed by the enzyme, DNA polymerase. Each nucleotide joins to its neighbour in the chain by a strong sugar phosphate bond. The direction in which the nucleotide insertion occurs is antiparallel on the opposite strands – on one strand it begins at the replication fork and goes towards the end of the strand whereas on the other, it begins at the end single strand and goes towards the replication fork. The DNA polymerase is essential for editing any incorrect additions to ensure accuracy. Incorrect base pairing will result in a change in the DNA base sequence, known as a mutation.

DNA replication results in the formation of two double-stranded molecules of DNA, each of which is identical to the original double stranded DNA molecule. The two DNA molecules that are produced contain one old strand from the original molecule and one new synthesised strand and the genetic instructions they carry are precisely copied.

Where does DNA replication occur?

DNA replication must occur before mitosis takes place in tissues such as the germinal layer of the skin.

DNA replication is part of a process of cell reproduction (mitosis) that is responsible for growth of organisms and for replacement of cells and tissues.

Growth involves many cycles of cell reproduction and during each cycle, the DNA of the chromosomes is replicated. Likewise, tissue replacement involves cell reproduction such the replacement of skin tissue and blood cells. The precision of DNA self-replication means that the new body cells produced by an organism during growth or replacement have exact copies of the double set of genetic instructions present in the fertilised egg from which that organism developed.

The Significance of DNA Replication

DNA has two main functions:

1. Heredity – this relies on DNA replication.

2. Gene Expression – this relies on protein synthesis

Heredity and the need for replication

The genetic material of a cell must be transmitted from:

- One cell to another during mitosis, allowing for growth, repair and maintenance of an organism.

- One generation to another during meiosis.

In each of these situations, it is necessary for the DNA to exactly replicate. This replication ensured that the genetic code of a cell is passed onto each new daughter cell that arises from it. An exact replica must be produced so that the new cells have the same, distinctive message that the original cell had. If DNA replication goes wrong, this has a direct effect on the phenotype of the individual.

• Explain the relationship between proteins and polypeptides

Proteins are large, complex macromolecules made up one or more long chains called polypeptides. Each polypeptide chain consists of a linear sequence of many amino acids joined by a peptide bond. One or more polypeptides can be twisted together into a particular shape, resulting in the overall structure of a protein. The sequence and arrangement of amino acids determines the configuration of the protein. Any change in the amino acid sequence that results in a change in the shape of the protein molecule could affect the ability of the protein to carry out its function in the cell.

• Outline, using a simple model, the process by which DNA controls the production of polypeptides

Multicellular organisms are made up of a variety of different cells; e.g. humans have over 200 different types of cells including skin cells, muscle cells, blood cells and many others. Despite differing in structure, every cell that has a nucleus has a full copy of the same coded genetic information in its DNA. This encoded information directs the production of cell products such as polypeptides which form proteins, the key to cell specialisation and differentiation. In specialised cells, coded instructions for the production of a particular protein are ‘switched on’. This ensures that the cell develops a particular structure, in keeping with the type of tissue to which it belongs.

The process of protein synthesis

DNA never leaves the nucleus. In order for a cell to make proteins, only the relevant instructions for those proteins are accessed in the DNA nucleotide sequence. Since the DNA instructions must remain in the nucleus, an intermediate molecule – messenger RNA (mRNA) – is created; this carries a transcribed copy of the relevant instructions from the nucleus to the ribosomes in the cytoplasm. The ribosomes can be considered as the ‘machinery’ that translated the message carried by the mRNA into a cell product such as protein.

The sequence of information transfer necessary for DNA to direct the production of proteins is summarised below, in a framework known in genetics as the central dogma:

DNA => RNA => Protein

The chemicals involved in protein synthesis

DNA:

DNA consists of long chain of nucleotides wound into a double helix. The sequence of nucleotide bases determines the meaning of the message – because it codes for the sequence of RNA nucleotides and ultimately the sequence of amino acids that form the polypeptide chain.

RNA:

Like DNA, RNA is a nucleic acid made from a chain of nucleotides, but it differs from DNA in the following ways:

- Most RNA is single-stranded

- The sugar in RNA is ribose sugar

- RNA has a nitrogenous base uracil (U) instead of thymine.

There are three types of RNA:

- messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA):

- mRNA is single-stranded and is not twisted into a helix. mRNA molecules are a few thousand bases long, mush shorter than DNA. They are found in both the nucleus and cytoplasm. They function as an intermediate molecule, carrying information from DNA in the nucleus to the ribosomes in the cytoplasm.

- tRNA molecules occur in the cytoplasm. Each one is 75 nucleotides long and twisted into the shape of a clover leaf. On one end of the tRNA there are three unpaired bases called the anticodon, which attach the tRNA to its complementary bases on the mRNA strand. The other end of the tRNA is able to bind with an amino-acid temporarily. Each tRNA molecule will only attach to one particular amino acid. The specific sequence of these three bases at the anticodon end determines which amino acid will be carried by the tRNA.

- rRNA forms a structural part of the ribosomes

The Steps involved in Protein Synthesis

1) An enzyme, RNA polymerase, binds to a part of the DNA called the promoter and the DNA ‘unzips’ – that is, the DNA unspirals, hydrogen bonds break between the two strands and the strands separate over a short length, just in that part of the DNA that holds the gene to be used. Only one strand of DNA contains the genetic information to make a protein; rather confusingly, it is called the non-coding strand or sense strand. The other strand is called the coding strand, or anti-sense strand.

2) Transcription of the gene occurs, controlled by the enzyme RNA polymerase: the sense strand of the DNA acts as a template and the RNA nucleotides are assembled forming a complementary single stranded mRNA molecule. The sequence of nucleotide bases on the mRNA molecule is the same as the DNA coding strand, except that is has U instead of T.

3) The mRNA moves out of the nucleus and into the cytoplasm where it encounters a ribosome. In eucaryotes, ‘editing’ of the mRNA may take place at this point.

4) Translation: The ribosomes move along the mRNA molecule and as they do so, they attach tRNA molecules by temporarily pairing the bases of the tRNA anticodons with their complementary triplets of bases (codons) on the mRNA.

5) The amino acids are linked together by another enzyme to form a polypeptide chain. The amino acids are then spliced off their tRNA carries.

6) The tRNA s move away from the mRNA leaving a growing chain of amino acids and move back into the cytoplasm where they can pick up another amino acid and be reused.

7) The polypeptide chain may be joined by one or more other polypeptides; they are further processed and folded into their correct shape, forming a protein.

8) The mRNA is broken down into its individual nucleotides which can be reused.

Defining a Gene

A gene was at first defined as a sequence of nucleotides that codes for one protein. Advances in understanding of the biochemical functioning of cells led to the definition changing to a sequence of nucleotides that code for one polypeptide chain. More recent research has shown that some genes code mRNA and tRNA which are not proteins at all, and that in other instances one gene may code for more than one polypeptide sequence (due to splicing and rearrangement of blocks of mRNA before translation).

Therefore the definition of a gene may need to change to a more functional concept: a sequence of nucleotides that codes for any molecular cell product.

Structural and Regulator Genes

Genes vary in the functions that they carry out in the cells of an organism. Some genes produce proteins that become part of the structure and functioning of the organism. These genes are termed structural genes.

Some genes, however, produce proteins that control the action of other genes. These genes are termed regulator genes and their actions determine whether other genes are active or not and, if active, the rate at which their products are made. These genes are important in the embryonic development.

• What are Mutations?

A mutation is a change in the genetic material of a cell – that is, a change in the sequence of nucleotides of DNA. All mutations do not arise in the same manner – some mutations arise spontaneously whereas other are induced. Mutations differ in their effect – some mutations may produce no phenotypic change, whereas others produce little phenotypic effect or significant effect. Mutations that are inheritable are a direct source of new alleles.

Mutation is a collective term for change in the DNA, but the different types of mutation can be distinguished according to three things:

1: The amount of genetic material changed: While most mutations affect only a single gene (gene mutations), there are some mutations that move whole blocks of genes to other parts of a chromosome or to another chromosome entirely. These are called chromosome mutations.

2: The effect of mutation on phenotype: mutations that do not change the phenotype of an individual may be harmful, beneficial or neutral in their effect on the individual and its survival. The phenotypic difference that a mutation produces may be present in the individual only (somatic), or may be transmitted to future generations (gametic). Changes in DNA sequences that may occur in somatic body cells are called somatic mutations and can only be passed onto daughter cells within an individual when the cells carrying the mutation divide by mitosis. Eg: mutations leading to skin cancer in a person. Gametic mutations are those where the mutation occurs in the sexual reproductive cells which give rise to gametes and these mutations are inheritable – that is, they can be transmitted to future generations.

3. The origin of the mutation: Spontaneous mutations arise randomly as a result of an error in the natural process such as DNA replication in cells, whereas induced mutations arise as a result of environmental agents such as chemicals or radiation that increases the chances of changes to nucleotide sequences.

Mutagens

Environmental Agents that cause mutations are termed mutagens and research has shown that exposure to these substances over a long period of time increases their harmful effects (more so that simply giving a larger dose). The process of inducing a mutation is termed mutagenesis. There are many mutagens known, including:

- Chemical mutagens:

o Ingested chemicals: Alcohol; tar in tobacco smoke, food additives and preservatives.

o Irritants and poisons: Organic solvents such as benzene, cleaning products, medications.

- Biological Mutagens: Some viruses and micro-organisms (e.g. hepatitis B virus, HIV, Epstein- Barr Virus) directly influence the genetic material in cells, changing the functioning of genes and triggering cancers

- Mutagenic radiation:

o Ionising Radiation: Radioactive materials from nuclear reactions

o Ultraviolet (UV) radiation

Extra: When a causative agent cannot be identified, a mutation is said to the spontaneous mutation. When a causative agent can be identified, the mutation is said to be and induced mutation.

Mutations occur in all organisms. A mutation that occurs in a body cell of an organism is a somatic mutation. Only that cell and daughter cells that it produces by mitosis will have the mutation. Somatic mutations are not passed onto the next generation.

A mutation that occurs in a cell that produces gametes by meiosis is a germline mutation. Germline mutations are heritable.

• Discuss evidence for the Mutagenic nature of radiation

During the late 1800s and early 1900s many scientists were involved in studying radiation. Since the harmful effects of radiation were unknown then, scientists, such as Marie Curie, who were exposed to large amounts of radiation over prolonged periods of time, developed various illnesses. Marie Curie worked with ionising radiation for most of her career and died from leukemia due to overexposure to radioactive emissions.

Many years later, survivors of the 1945 bombing of Hiroshima suffered physical mutations as a result of the radioactive output form the nuclear explosion. More recently, victims of the nuclear meltdown in Chernobyl have suffered high levels of infertility and genetic mutations as well as non-cancerous side effects such as cardiovascular and respiratory conditions.

The link between exposure to ionising radiation and an increase in the occurrence of certain illnesses such as leukemia and other cancers was identified in the early 1900’s but further evidence was needed to accept that radiation was directly causing these cancers.

Between 1925 and 1940, experimental research provided evidence of the Mutagenic nature of radiation. Advances in cell studies also provided support [look at printed sheets].

• Explain how mutations in DNA may lead to the generation of new alleles

Mutations alter genes by changing the nucleotide sequence in the DNA. As a result one or genes may be affected. If a gene is altered from its original form, the two variations of the gene are termed alleles of that gene. These changes to the genes may result in the production of new proteins. Most new proteins have little effect on the organism, but few will lead to genetic disorders and inherited diseases.

Inheritable mutations pass to future generations

Changes to genetic material in somatic cells are not passed on to offspring – the new allele may cause a defect in an individual, but will not affect future generators. However, mutations in germ-line cells (gametic mutations) produce alleles that can be inherited and may therefore have significant effects on populations and so are important in evolution.

Effects of mutations at the gene and chromosome level

Changes to genetic material arise during replication and they may result in a change to a single gene – termed gene mutations. Other mutations involve the rearrangement of a block of genes or whole chromosomes – chromosomes mutations.

Gene Mutations

On a molecular level, mutations may involve:

- Base substitution (point mutation): One pair of nucleotides (e.g. A-T) is substituted for another pair (e.g. G-C).

- Frame shift (macromutation): Extra bases or added or delete from a strand of DNA, changing the whole sequence of nucleotides.

- A sequence within a gene may be duplicated or translocated (moved).

Chromosomal Mutations

On a molecular level, it whole chromosomes become re-arranged (deleted duplicated or translocated and attached to one another) a change in chromosome number may arise. This usually occurs as a result of chromosomes not separating out correctly during meiosis (non-disjunction). The resulting cells may have one chromosome less than normal or one extra chromosome.

The effect of a chromosomal mutation could result in disorders such as Down syndrome, where individuals have three copies of chromosome 21.

Most gene mutations produce recessive alleles because they prevent the gene from producing a functional protein. Because of this, we could all be carrying large numbers of mutations in our genome and be completely unaware of them. If the changed recessive alleles occur in the homozygous form in individuals (or a dominant allele is present in even one copy) the mutation can affect the phenotype of the individual. This phenotypic change may be of a advantage to the organism or it may be harmful.

Some phenotypic changes that do not affect the individual adversely or in a positive way may accumulate. They may become beneficial or harmful to the organism if selective pressure arises as a result of sudden environmental change.

• Explain how an understanding of the source of variation in organisms has provided support for Darwin’s theory of evolution by natural selection.

Mutations are the basic source of all variation. Understanding mutagenesis allows us to explain how variations arise. The understanding that mutations affect the base sequence of DNA allows us to understand how they can be passed from one generation to the next. It supports Darwin’s theory of evolution because it provides a mechanism to explain how heritable variation arises.

New Alleles lead to change in phenotype

A mutation will result in a change in phenotype that may be negligible in its effect, or it may confer some advantage or disadvantage to the organism. Mutations therefore provide a diversity of genetic material that result in variation in phenotype. If mutations can be inherited, they provide the variation on which natural selection acts, for evolution to occur.

For evolutionary purposes, a mutation can be redefined as a heritable change in the genetic material.

Hence our understanding of the cause of mutation allows us to identify a mechanism through which natural selection (chance) acts and creates variation in the phenotype of individuals. Our understanding of the biochemical nature of mutation as a source of variation provides evidence that natural processes cause variations (that may be inheritable) within a population. This allows those organisms that have favourable characteristics to survive and pass on their favourable characteristics to future generations. Furthermore, mitosis, meiosis and sexual reproduction all have mechanisms (such as crossing over and mutation) that ensure that variation occurs from one generation to the next. This variation is natural – corresponding to the Darwin-Wallace theory of evolution by natural selection.

• process and analyse information from secondary sources to explain a modern example of ‘natural’ selection

LOOK AT SECTION 1 – The peppered moth

• process information from secondary sources to describe and analyse the relative importance of the work of:

o James Watson

o Francis Crick

o Rosalind Franklin

o Maurice Wilkins

In determining the structure of DNA and the impact of the quality of collaboration and communication on their scientific research

Rosalind Franklin:

Rosalind Franklin’s work was imperative in determining the structure of DNA. Rosalind Franklin made marked advances in x-ray diffraction techniques with DNA. She adjusted her equipment to produce an extremely fine beam of x-rays. She extracted finer DNA fibres than ever before and arranged them in parallel bundles. And she studied the fibres' reactions to humid conditions. Without Rosalind Franklin’s DNA excellent ability to produce high quality images of the DNA structure using crystallography, Watson and Crick had no evidence for their DNA model. Infact, it was not until the famous X-shaped photo, that Franklin produced, was shown to both Watson and Crick that they realised that DNA was in the form of a double helix. Thus Rosalind Franklins work was extremely important.

Francis Crick and James Watson:

Francis Crick was a physicist and biologist who worked with Watson in the race to uncover the structure of DNA. Watson and Crick were responsible for developing many models of DNA structure. This began with the three chain helix, which was ultimately proved to be incorrect. The idea that DNA may be a code for making proteins was proposed by Crick – the four letters corresponding to the four nitrogen bases. Crick suggested that the order of these bases creates the code. Crick also confirmed that the codon is three bases long and some of these codons code for the same amino acid.

Watson and Crick were able to identify that the structure of DNA was a double helix (with the help of Rosalind Franklin). However, their genius was shown when they discovered the internal nitrogen base structure of the DNA. Watson proposed many models including identical base pairing, however none survived close scrutiny. However, using Chargoff’s rule (where the number of adenine bases was equal to the number of thymine bases and the number of guanine was equal to the number of cytosine bases) the answer suddenly hit Watson – that the adenine base was internally connected to the thymine and the guanine to the cytosine. He realised that this would mean that the A-T bond would be exactly the same size as the G-C bond (they knew that hydrogen bonds connected the bases). Thus they finally discovered the double helix structure of DNA.

Maurice Wilkins

Maurice Wilkins was part of the team, which included Rosalind Franklin that focused on X-ray crystallography. The discovery and demonstrations of the DNA image taken by Franklin with the aid of Wilkins inspired Watson and Crick. Using a 1952 Wilkins/Franklin X-ray diffraction picture of the DNA molecule, Crick and Watson were able in 1953 to build their correct and detailed model of the DNA molecule. Thus his work was of extreme importance in the development of the DNA double helix.

Collaboration and Communication

As for the discovery of the DNA structure, indeed all scientific discoveries, it is rarely the work of one person or team. Instead, breakthroughs come via a series of conclusions, over a period of years, often with unconnected teams working on slightly related topics.

There is no doubt about the contribution of Wilkins and Franklin to the discovery of DNA. They gave Crick and Watson their data. Data that was unavailable to anyone else in the world. Watson and Crick were partners that equally contributed to the model and the functioning of DNA - hence emphasising the importance of collaboration in teamwork in major scientific investigations/research.

There was several struggles and strained relationships between the above four individuals during the development of the DNA model. However, it was their collaboration that ultimately led to the development of the helix. Watson and Crick would have never been able to develop the double-helix model if Wilkins had not shown them [without the permission of Franklin], the image obtained from crystallography. Thus despite ongoing conflict between Maurice Wilkins, Rosalind Franklin and the Watson/Crick duo, it was their joint effort that was essential in the development of the double-helix model for DNA.

• process information to construct a flow chart that shows that changes in DNA sequences can result in changes in cell activity

Extra:

Change in Phenotype reflects changes in DNA Structure

Importance of advances in scientific understanding on the direction of scientific thinking

Understanding the one gene-one polypeptide hypothesis and the more recently discovered structure of DNA and its role in protein synthesis brings us to an understanding of the basics of gene expression – that the instructions encoded in DNA are converted into the characteristics of an organism (its phenotype). This can be explained genetically as follows:

- The specific sequence of nucleotide pairs in a DNA molecule is responsible for the sequence of amino acids in the polypeptide chain.

- The sequence of amino acids in a polypeptide determines the chemical bonding capabilities and therefore the physical shape of the resulting protein.

- The shape and chemical nature of the protein influences the phenotype of the organism

- Mutations alter the specific sequence of nucleotides in the DNA, which may affect the phenotype, often in an adverse manner. Therefore it is important to conserve the integrity of the nucleotide sequence of DNA.

- Mutations are important to geneticists researching biochemical pathways in organisms and functions of genes – mutants lacking the ability to perform a process reveal the nucleotide sequence essential to that process.

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