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Unit 3: Unit 3: Introduction Where are we heading? Unit 1: What is cancer and why should we care? Unit 2: What does it mean to be a 'normal' cell? Unit 3: How do normal cells become cancerous? Unit 4: How does cancer make us sick? Unit 5: How is cancer diagnosed and treated? ______________________________________ In Unit 3 we'll zoom in on how cell function is disrupted in cancer. Lesson 3.1 will give you the opportunity to apply your understanding about DNA replication and protein synthesis to an investigation of how DNA is organized, and how mutations can affect gene function. Lesson 3.2 examines how DNA mutations affect protein synthesis and how this may promote development of cancer. Lesson 3.3 investigates the internal clock that allows a cell to age normally, and how this clock is disrupted in cancer. Lesson 3.4 explores how cancer cells become immortal by cheating normal cell death. Lesson 3.5 grapples with the idea that each cancer cell evolves independently, so that each tumor has different characteristics. 79 LESSON 3.1 WORKBOOK What is in our DNA sequence? DEFINITIONS OF TERMS For a complete list of defined terms, see the Glossary. Gene expression – the process by which information from a gene-coding region of DNA is used to make a protein. Somatic cells – All cells that aren’t eggs or sperm Autosome – one of 22 chromosomes in human DNA that is found in females and males Zygote – Fertilized egg Wo r k b o o k Lesson 3.1 The previous Unit described the regulatory mechanisms that keep normal cells normal. We learned that these mechanisms occur through the activity of proteins, which are encoded in our genomic DNA. Tumors and cancer occur when DNA is mutated so that those regulatory proteins can no longer control cellular behavior. This lesson explains how genomic DNA is organized, and how that organization can dynamically regulate gene expression to impact cell behavior. What is in our DNA sequence? We have learned in other modules, as well as in Unit 2, how the activity of the proteins a cell synthesizes determines that cell’s behavior. So to understand cell behavior we need to understand those proteins and how they work. Each protein is encoded by a DNA sequence called a gene. The gene’s DNA sequence is first transcribed into an RNA sequence, and then the RNA sequence is then translated into an amino acid sequence that forms a protein. As the protein matures it folds into a characteristic 3-dimensional shape that permits it to perform its function. For example receptor proteins always contain a binding ‘pocket’ where their specific ligand can fit. Generating proteins from gene sequences is also known as gene expression and altering gene expression, for instance following DNA mutation, can have serious effects on cell behavior. For example, as we learned in Unit 1, proto-oncogenes are genes that normally make sure that cell proliferation is tailored to tissue requirements. However, when proto-oncogenes mutate to form oncogenes, proliferation is no longer regulated and tumors form. Understanding how DNA is organized will give us a better idea of how and when the important functional mutations to genes occur. Most somatic cells have 23 pairs of chromosomes. 22 pairs of chromosomes are found in both male and female cells, these chromosomes are called autosomes, while the 23rd pair, called sex chromosomes, can take one of two forms. Female cells usually have the XY form, whereas males usually have YY. Germ cells (eggs and sperm) only have one of each chromosome rather than a pair, because they fuse together during fertilization to produce the zygote which then has 23 pairs of chromosomes. MC Questions: ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ 1. True or false: Gene expression refers to generating proteins from DNA. aa. True. bb. False. ________________________________ ________________________________ ________________________________ ________________________________ 2. Which of the following does the genome contain? aa. Autosomes; bb. Introns; cc. Exons; dd. All the above. ________________________________ ________________________________ ________________________________ 80 LESSON READINGS DEFINITIONS OF TERMS Genome – The complete sequence of all the chromosomes Exons – the nucleotide sequence of a gene that codes for protein sequence. Introns – Parts of a gene between exons that do not contain sequence necessary for protein coding, and are removed prior to translation. Non-coding DNA – DNA sequences that do not make protein. They may make RNA molecules that are necessary for gene expression. Transposon – a segment of DNA that is capable of moving into another position in a genome. Retrotransposon – a segment of DNA that is capable of copying itself into RNA and undergoing reverse transcription to form a new DNA segment that can move into another position in the genome. Wo r k b o o k Lesson 3.1 Human chromosomes contain 32 billion nucleotides. All of these nucleotides together make the human genome. Figure 1 shows how the genome is divided into various types of functions. When the human genome was first fully sequenced in 2001, it was clear that only a very small proportion, maybe 2%, contains DNA that actually codes for genes. People found this very surprising because at the time it was not at all obvious what the rest of the genome was for. For a long time the remaining 98% was dismissed as ‘junk’ DNA Figure 1: Genes compose only 2% of all the that we had somehow acquired during evoluDNA sequence in our genome. The majority tion, but that had no functional significance. In of sequence is composed of retrotransposons, DNA transposons, and noncoding DNA. fact, the non-protein encoding DNA sequence turns out not to be ‘junk’ at all; instead it is composed of important sequences that can regulate how and when gene expression occurs. The DNA sequences that directly code for amino acids are called exons (called genes in Figure 1). Interspersed within exons are sequences called introns. Introns are non-coding DNA that play important roles in regulating how exons are expressed. Yet other non-coding DNA sequences (the green wedge in Figure 1) can be transcribed into RNA but are not translated into protein. Some of these RNA sequences, like transfer RNA (tRNA) or microRNAs help protein translation. Another large component are regulatory sequences that also affect gene expression without making RNA. Finally a major chunk of non-coding DNA sequences are called DNA transposons and retrotransposons. These sequences can actually jump around the genome from one location to another. Obviously, if they land in a gene sequence they can affect gene expression. Thus even though most of the genome does not code for protein directly it does play an indirect role in regulating protein expression. Genome organization: gene sequences. As we learned in Lesson 2.4, DNA is packaged around histones, with the tightness of the packaging determining whether or not DNA will be transcribed. We learned in Lesson 2.5 DNA can be transcribed when it is loosely packed and that it is transcribed when a transcription factor binds to a promoter sequence (also known as a regulatory sequence) a stretch of non-coding DNA located just in front of the coding sequence. When the transcription factor is bound to the regulatory sequence, RNA polymerase can bind to the coding sequence of the DNA and transcribe the gene into RNA. Up to now we only learned about transcription factors that promote gene expression, but inhibitory transcription factors MC Questions: 3. Which of the following DNA sequences are most prevalent in the genome? aa. Genes; bb. Introns; cc. Non-coding DNA; dd. Retrotransposons. ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ 4. True or false: Most of our DNA contains sequence involved in protein expression. aa. True. bb. False. ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ 81 LESSON READINGS also exist. When they bind to the regulatory sequence, they block it and prevent gene expression. Regulatory sequences therefore play multiple roles to stimulate and inhibit gene expression some of which are extremely complex DEFINITIONS OF TERMS Transcription factor – Protein that binds to a DNA sequence at the start of a gene and either promotes or inhibits transcription. Each gene is composed of two regions – the region that codes for the amino acids in the protein, called the exon, and non-coding regions interspersed among the exons called introns. As a result the DNA sequence of a gene in the genome is often considerably longer than the sequence of the protein that will result at the end of translation (remember that each amino acid is coded for by three nucleotides). The progression from DNA to RNA to protein is illustrated graphically in Figure 2, which represents the gene sequence for a protein with 10 exons, each colored differently. We can see how, in the DNA sequence, each exon is separated from another by a log stretch of non-coding introns (represented by the dotted line). Both the exons (colored) and the introns (black line) are transcribed into RNA, but when the RNA is translated into protein the introns are removed, so that the protein is now a single molecule composed of the different introns. The process of removing the introns from RNA as it is translated into protein is called splicing. As a result the size of the protein is much smaller than the size of the RNA and much, much smaller than the size of the gene. Regulatory sequence – a segment of DNA that is responsible for increasing or decreasing the expression of specific genes in the cell. Figure 2: Genes are composed of short coding sequences called exons, which are separated by longer, non-coding sequences called introns. When a gene is transcribed into RNA, both introns and exons are included in the sequence. Introns are removed from mRNA sequence, leaving only exon sequence to make proteins. Wo r k b o o k Lesson 3.1 What is the purpose of introns and exons? Dividing the protein up into chunks like this provides an opportunity to create protein diversity. Sometimes when an intron is removed an exon will be removed along with it. For example a protein without the yellow exon could potentially be very different from the protein with it, if the yellow exon provided the protein with an important function – for example the ability to respond to signals from the environment. MC Questions: 5. What is the best description of regulatory sequence? aa. DNA sequence encoding proteins that regulate cell behavior; bb. DNA sequence that regulates gene expression; cc. DNA sequence that regulates cell cycle; dd. DNA sequence that regulates translation. 6. Which of the following best describes the purpose of splicing? aa. Removal of intron DNA sequence to allow for translation of exon sequence; bb. Removal of intron RNA sequence to allow for translation of exon sequence; cc. Removal of exon DNA sequence to allow for translation of intron sequence; dd. Removal of exon RNA sequence to allow for translation of intron sequence. 82 LESSON READINGS Introns perform two important functions in a gene: First, having large stretches of intron DNA can act as a kind of ‘insulation’ for the coding sequences in the exon. If most of the DNA in a gene is not involved in coding for protein, random mutations to the gene are less likely to affect an important region, even if the gene is unfolded ready for transcription and therefore vulnerable to carcinogens. Second it has recently been discovered that introns actually contain regulatory sequences that can control how much RNA is transcribed from the gene, therefore regulating protein levels. This can be Non-gene sequences in the genome DEFINITIONS OF TERMS Transposable element – the collective term for transposons and retrotransposons, or any DNA sequence that can change its position within a genome. Reverse transcriptase – the enzyme responsible for copying an RNA template into a doublestranded DNA sequence. This is used in retroviruses and in retrotransposons. Wo r k b o o k Lesson 3.1 Maybe surprisingly, over half of the genome sequence is not fixed in place. Instead it is composed of moveable segments of DNA collectively called transposable elements. These moveable elements are divided into two groups DNA transposons and retrotransposons that move around differently. DNA transposons can actually cut themselves out of the genomic DNA and paste themselves into another region – not surprisingly this is called ‘cut and paste’ because the DNA sequence is no longer in its usual place. On the other hand retrotransposons are transcribed into RNA and then re-converted back into DNA by an enzyme called reverse transcriptase. You may remember from the ID module that reverse transcriptase is essential to the replication cycle of HIV. Reverse transcriptase, which is encoded within the retrotransposon sequence itself is able to convert single-strand RNA sequences into double-stranded DNA sequences, which can then insert themselves somewhere else in the DNA. In the case of retrotransposons there are therefore now 2 copies in the genome – one in the original location and the second somewhere else in the genome. Genomic DNA is clearly a dynamic flexible molecule rather than an inert sequence. The moveable sequences of DNA allows for diversity. Having DNA that can move around can disrupt gene Figure 3: Transposon movement occurs through a “cut and paste” manner, where the transposon is removed from the original DNA sequence and inserts into a new location. Retrotransposons move through a “copy and paste” method, where the RNA copy of the retrotransposon is converted into DNA, which then inserts into another site of DNA. MC Questions: 7. Which of the following is a useful function of introns? (Circle all correct) aa. Promoting cell survival. bb. Providing diversity of gene expression. cc. Regulating gene expression. dd. Spacing of exon sequence. 8. True or false: Transposable elements do not express proteins. aa. True. bb. False. ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ 83 LESSON READINGS expression when they insert inappropriately into genes. Having reverse transcriptase could lead to duplication of actual genes if the reverse transcriptase transcribed and then inserted them somewhere else. This could clearly be a problem if that gene is a proto-oncogene, and in fact there are several examples of proto-oncogenes to which this has happened. DEFINITIONS OF TERMS Epigenetic silencing – the silencing of expression of certain regions of DNA through modification of its DNA sequence. Wo r k b o o k Lesson 3.1 Fortunately, most transposable elements are tightly wrapped around histones, which inhibits their function in the same way that tight wrapping prevents gene expression. In the case of transposable elements, this is called epigenetic silencing. However, as we learned in Unit 2 if tumor formation disrupts the packaging of DNA it can also stop the epigenetic silencing of genes involved in transposon/retrotransposon movement. As a result the activity of transposons and retrotransposons is increased significantly in cancer, which, as we will see in the next lesson, increases the chance that genes important for keeping cells normal will become mutated. MC Questions: 9. How do transposable elements affect gene expression? (Circle all correct.) aa. Disrupt a gene sequence by insertion. bb. Induce epigenetic silencing of genes. cc. Reverse transcription of gene RNA sequences. dd. Decrease length of introns. 10. Which of the following explains why there are more retrotransposons than transposons in our genome? aa. Retrotransposons use a “copy and paste” strategy to move. bb. Retrotransposons use a “cut and paste” strategy to move. cc. Retrotransposons are normally highly active in the cell. dd. Transposons are more inactive in the cell than retrotransposons. 84 STUDENT RESPONSES Describe 2-3 reasons why the 98% of our DNA that does not encode proteins should not be called 'junk' DNA. _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ ____________________________________________________________________________________________________ Remember to identify your sources _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ Wo r k b o o k Lesson 3.1 _____________________________________________________________________________________________________ ___________________________________________________________________________________________ 85 TERMS TERM For a complete list of defined terms, see the Glossary. Wo r k b o o k Lesson 3.1 DEFINITION Autosome One of 22 chromosomes in human DNA that is found in females and males Complementary A sequence of DNA or RNA that contains the appropriate sequence of nucleotides to pair with another strand of DNA or RNA. Diploid Any cell that has a pair of chromosomes, or two sets of chromosomal DNA. Epigenetic silencing The silencing of expression of certain regions of DNA through modification of its DNA sequence. Exons The nucleotide sequence of a gene that codes for protein sequence. Gene expression The process by which information from a gene coding region of DNA is used to make a protein. Genome The complete sequence of all the chromosomes Haploid Any cell that only has a single set chromosomal DNA. Introns Parts of a gene between exons that do not contain sequence necessary for protein coding, and are removed prior to translation. Meiosis The process of cell division in sexually reproducing organisms that reduces the number of chromosomes from diploid to haploid. Non-coding DNA DNA sequences that do not make protein. They may make RNA molecules that are necessary for gene expression. Regulatory sequence A segment of DNA that is responsible for increasing or decreasing the expression of specific genes in the cell. Retrotransposon A segment of DNA that is capable of copying itself into RNA and undergoing reverse transcription to form a new DNA segment that can move into another position in the genome. Reverse transcriptase The enzyme responsible for copying an RNA template into a double-stranded DNA sequence. This is used in retroviruses and in retrotransposons. Somatic cells All cells that aren’t eggs or sperm Transcription factor Protein that binds to a DNA sequence at the start of a gene and either promotes or inhibits transcription. 86 TERMS TERM DEFINITION Transposable element The collective term for transposons and retrotransposons, or any DNA sequence that can change its position within a genome. Transposon A segment of DNA that is capable of moving into another position in a genome. Zygote Fertilized egg For a complete list of defined terms, see the Glossary. Wo r k b o o k Lesson 3.1 87