BMS240 Human Molecular Genetics
BMS240 Human Molecular Genetics Contents Topic 6a: DNA replication ........................................................................................................ 5 Define and use the terms: template, semiconservative, bidirectional, replication fork, leading strand, lagging strand, proofreading, homologous recombination ..... 5 Describe and understand the ‘Meselson and Stahl’ experiment in detail ................. 5 Describe the replication direction and the differences between the leading- and lagging strand synthesis. ....................................................................................................... 7 Describe the basic apparatus for DNA replication in prokaryotic cells....................... 8 Initiation ............................................................................................................................... 8 Unwinding............................................................................................................................ 8 Elongation ........................................................................................................................... 9 Termination.......................................................................................................................... 9 Understand the high fidelity of the replication system and understand in detail the function of basepairing in achieving this. ......................................................................... 9 Describe the replication model at the end of chromosomes using the terms telomere and telomerase .................................................................................................. 10 Topic 6b: Transcription ............................................................................................................ 12 Describe and use the terms: transcription, mRNA, tRNA, rRNA, template strand, coding strand, promoter, initiation, elongation, termination, transcription start site ................................................................................................................................................ 12 Describe the transcription process in both eukaryotes and prokaryotes in detail and explain the differences between eukaryotes and prokaryotes ......................... 12 Prokaryotes........................................................................................................................ 13 Describe the basal apparatus for transcription in pro and eukaryotic cells ............. 14 Prokaryotic RNA polymerase ......................................................................................... 14 Eukaryotic RNA polymerase ........................................................................................... 15 Appreciate the function of basepairing in transcription .............................................. 15 Topic 7: RNA molecules and RNA processing .................................................................... 16 Define and describe the terms: RNA interference, lncRNA, miRNA, siRNA, colinearity, exon intron, splice site branchpoint, lariat ................................................. 16 Appreciate the principles of colinearity and non-colinearity ..................................... 16 Understand and describe the process of RNA processing in detail including the addition of the 5' cap, the poly-A tail and splicing ....................................................... 17 1
Addition of a 5’ cap ........................................................................................................ 18 Addition of a poly-A tail .................................................................................................. 18 RNA splicing ...................................................................................................................... 19 Appreciate the existence of introns and exons and the implications this has on gene expression ................................................................................................................... 20 Outline two alternative RNA splicing pathways ............................................................. 20 Outline the structure of tRNA ............................................................................................. 21 Appreciate and define the concept of RNA interference .......................................... 21 Define two types of interfering RNA ................................................................................. 21 Describe the production of miRNA and siRNA and key differences between miRNA and siRNA and their role in gene expression .................................................................. 22 Define the importance of long non-coding RNAs for gene expression..................... 23 Topic 8: The genetic code and translation......................................................................... 23 Describe and use the terms: translation, peptide bond, genetic code, codon, anticodon, wobble .................................................................................................................... 23 Describe the experiments performed by ‘Beadle and Tatum’ and ‘Srb and Horowitz’ ................................................................................................................................ 24 Beadle and Tatum ........................................................................................................... 24 Srb and Horowitz .............................................................................................................. 25 Describe one method used to crack the genetic code.............................................. 25 Appreciate the role of the reading frame and the initiation codon in translation . 26 Describe the basic machinery for translation in pro and eukaryotic cells (from initiation through to termination) ...................................................................................... 26 Describe the process of translation in pro- and eukaryotic cells (from initiation through to termination) ...................................................................................................... 27 1/ Charging of tRNA ........................................................................................................ 27 2/ Initiation......................................................................................................................... 28 3/ Elongation .................................................................................................................... 29 4/ Termination ................................................................................................................... 30 Describe the differences in translation process between eukaryotes and prokaryotes and how this can be used in human medicine ....................................... 30 Appreciate the function of basepairing in translation ................................................. 30 Describe and understand the role of ribosomes and tRNA in translation ................. 30 Topic 9: Gene Mutations and DNA Repair ......................................................................... 31 Define and describe the terms: mutation, mutagen, substitution mutation, frameshift mutation, missense mutation, nonsense mutation, silent mutation, neutral mutation, insertion, mutagenesis, carcinogen, unequal cross over ............. 31
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Describe in detail the different types of mutations (focus on substitution, insertion, deletion, frameshift, missense, nonsense and silent mutations and expanding nucleotide repeats) described in the chapter .............................................................. 31 Base substitution ............................................................................................................... 31 Insertions and deletions .................................................................................................. 32 Expanding nucleotide repeats ...................................................................................... 32 Appreciate and be able to explain the difference between germline and somatic cell mutations ....................................................................................................................... 33 Understand the difference between spontaneous and induced mutations ........... 33 Spontaneous Replication Errors ..................................................................................... 33 Spontaneous Chemical Changes ................................................................................ 33 Chemically Induced Mutations ..................................................................................... 34 Radiation ........................................................................................................................... 34 Describe and understand how mutations get fixated in the genome ...................... 34 Describe the various DNA repair mechanisms (Mismatch repair, Nucleotide excision repair, Base excision repair and Direct repair)................................................ 35 Mismatch Repair .............................................................................................................. 35 Direct repair ...................................................................................................................... 35 Base-excision repair ......................................................................................................... 36 Nucleotide-Excision Repair............................................................................................. 37 Appreciate the role of DNA repair mechanisms in inherited disorders and know two examples of these diseases and how they are caused ....................................... 38 Topic 10: Genomics and proteomics and epigenetics .................................................... 38 Define and describe the terms: microarray, genome wide association study, haplotype, SNP, STS, EST, proteomics, epigenetics, DNA methylation, paramutation, genomic imprinting and epigenome. ............................................................................. 38 Understand the difference between genetic and physical maps and how it can be explained ........................................................................................................................ 39 Describe the difference between map based sequencing and shotgun sequencing ........................................................................................................................... 39 Describe microarray technology using an example..................................................... 41 Describe the 3 molecular mechanisms that underlie many epigenetic phenotypes, use the honeybee as an example of one of these ....................................................... 41 Changes in patterns of DNA methylation ................................................................... 41 Chemical modification of histone proteins ................................................................. 42 RNA molecules that affect chromatin structure and gene expression ................. 42 Describe the role RNA can play in paramutations using Kit mice as an example .. 42
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Understand that epigenetic changes can have multigenerational effects using mice as an example ........................................................................................................... 42 Explain the role of epigenetics in X-inactivation ............................................................ 43 Explain the difference between the genome and epigenetics using monozygotic twins as an example ........................................................................................................... 43 Describe the epigenome and information gained from the epigenome ................ 43 Topic 10: Cancer genetics..................................................................................................... 44 Understand that cancer is a genetic disease ................................................................ 44 Describe Knudson’s multistep model of cancer ............................................................ 44 Understand what is meant by clonal selection .............................................................. 45 Describe in detail the difference between oncogenes and tumour suppressor genes ..................................................................................................................................... 45 Understand how a mutation of a normal gene results in conversion to oncogenes or inactivation of tumour suppressor genes .................................................................... 46 Describe the difference between familial and sporadic cancer, giving examples 46 Understand the role of miRNA in cancer ........................................................................ 46 Briefly describe the role of epigenetics in cancer ......................................................... 47 Be familiar with gene-environment interactions ............................................................ 47
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Topic 6a: DNA replication Define and use the terms: template, semiconservative, bidirectional, replication fork, leading strand, lagging strand, proofreading, homologous recombination Template - Two strands of DNA separate, each becoming a ______ for replication Semiconservative - Replication in which the two nucleotide strands of DNA separate, each serving as a template for the synthesis of a new strand. Bidirectional - Replication at both ends of a replication bubble. Replication fork - Point at which a double-stranded DNA molecule separates into two single strands that serve as templates for replication. Leading strand - DNA strand that is replicated continuously. Lagging strand - DNA strand that is replicated discontinuously. Proofreading - Ability of DNA polymerases to remove and replace incorrectly paired nucleotides during replication. Homologous recombination - Exchange of genetic information between homologous DNA molecules.
Describe and understand the ‘Meselson and Stahl’ experiment in detail This experiment was used to support Watson and Cricks theory that DNA replicated through semiconservative replication. At the time, there were three theories:
To prove that DNA used semiconservative replication, they:
Raised E. coli in a heavy isotope of nitrogen, 15N, for several generations, and took out their first sample. They then incorporated the E. coli into a medium containing the regular nitrogen isotope, 14N. They took samples 20 minutes apart (the generation time of E. coli). All the samples were placed into a centrifuge in a caesium chloride (CsCl) solution, a similar density as DNA. This created an equilibrium density gradient. 5
These were the results:
The first round of replication ruled out conservative replication as there was only one band, if it was conservative there would be two bands. The second round of replication ruled out dispersive replication as there was two bands, if it was dispersive there would only be one band, but each generation would be slightly higher in the test tube than the previous generation.
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Describe the replication direction and the differences between the leadingand lagging strand synthesis. DNA Polymerase can only add nucleotides in the 5’3’ direction. Because the two strands of DNA are antiparallel one strand can elongate continuously along the leading strand, while the other going in the opposite direction can only lay down nucleotides discontinuously along the lagging strand. These discontinuous segments are known as Okizaki fragments.
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Describe the basic apparatus for DNA replication in prokaryotic cells Initiation An initiator protein binds to the origin of replication (oriC) and causes a short section of DNA to unwind. The origin of replication is an area rich in A and T nucleotides, these nucleotides only have 2 H bonds compared to C and G which have 3.
Unwinding DNA helicase binds to the lagging-strand template at each replication fork and moves in the 5’3’ direction along this strand, moving the replication fork. Helicase breaks the hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule. Single-strand-binding proteins (SSBs) attach tightly to the exposed single-stranded DNA. These proteins protect the single-stranded nucleotide chains and prevent the strands from snapping back together or formation of secondary structures such as hairpins DNA gyrase, a topoisomerase, reduces the torsional strain (torque) that builds up ahead of the replication fork because of unwinding by making a double-stranded break in one segment of the DNA helix, passing another segment of the helix through the break, and then resealing the broken ends of the DNA.
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Elongation Primase synthesizes short stretches of RNA nucleotides which provide a 3’-OH group to which DNA polymerase can attach DNA nucleotides. DNA polymerase III is a large multiprotein complex that acts as the main workhorse of replication. It synthesises nucleotide strands by adding new nucleotides to the 3’ end of a growing DNA molecule. This enzyme has two enzymatic activities:
Its 5’3’ polymerase activity allows it to add new nucleotides in the 5’3’ direction. Its 3’5’ exonuclease activity allows it to remove nucleotides in the 3’5’ direction, enabling it to correct errors.
DNA polymerase I has:
5’3’ polymerase activity allows it to add new nucleotides in the 5’3’ direction. 3’5’ exonuclease activity allows it to remove nucleotides in the 3’5’ direction, enabling it to correct errors 5’3’ exonuclease activity, which is used to remove the primers laid down by primase and to replace them with DNA nucleotides by synthesizing in a 5’3’ direction.
DNA polymerase I has lower processivity than DNA polymerase III. DNA ligase joins Okazaki fragments by sealing breaks in the sugar–phosphate backbone of newly synthesized DNA
Termination Tus binds to specific termination sequences which blocks the movement of helicase, thus stalling the replication fork and preventing further DNA replication.
Understand the high fidelity of the replication system and understand in detail the function of basepairing in achieving this. Replication is extremely accurate, with less than one error per billion nucleotides. The high level of accuracy in DNA replication is produced by 1. Nucleotide selection 2. Proofreading 3. Mismatch repair
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Describe the replication model at the end of chromosomes using the terms telomere and telomerase In circular DNA, there’s no issue because as nucleotides are added to the exposed 3’-OH and eventually they meet up with of the first laid nucleotide.
In linear DNA, once the primer is removed there is a small gap which becomes exposed, in some cells, such as somatic cells, chromosomes shorten every time replication occurs. This happens in most somatic cells and is a normal sign of aging.
Certain cells, such as germ cells and early embryonic cells are able to overcome this issue with telomerase. The telomeres are filled with a G rich overhang, this overhang can be extended by telomerase, a ribonucleoprotein (containing RNA and protein). Telomerase is complimentary to this G strand (contains C’s). This extends the chromosome temporarily and allows for further elongation. It continues to move down the chromosome so more nucleotides can be added. The complimentary strand can then be filled in.
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Addition of a 5’ cap ........................................................................................................ 18 Addition of a poly-A tail .................................................................................................. 18 RNA splicing ...................................................................................................................... 19 Appreciate the existence of introns and exons and the implications this has on gene expression ................................................................................................................... 20 Outline two alternative RNA splicing pathways ............................................................. 20 Outline the structure of tRNA ............................................................................................. 21 Appreciate and define the concept of RNA interference .......................................... 21 Define two types of interfering RNA ................................................................................. 21 Describe the production of miRNA and siRNA and key differences between miRNA and siRNA and their role in gene expression .................................................................. 22 Define the importance of long non-coding RNAs for gene expression..................... 23 Topic 8: The genetic code and translation......................................................................... 23 Describe and use the terms: translation, peptide bond, genetic code, codon, anticodon, wobble .................................................................................................................... 23 Describe the experiments performed by ‘Beadle and Tatum’ and ‘Srb and Horowitz’ ................................................................................................................................ 24 Beadle and Tatum ........................................................................................................... 24 Srb and Horowitz .............................................................................................................. 25 Describe one method used to crack the genetic code.............................................. 25 Appreciate the role of the reading frame and the initiation codon in translation . 26 Describe the basic machinery for translation in pro and eukaryotic cells (from initiation through to termination) ...................................................................................... 26 Describe the process of translation in pro- and eukaryotic cells (from initiation through to termination) ...................................................................................................... 27 1/ Charging of tRNA ........................................................................................................ 27 2/ Initiation......................................................................................................................... 28 3/ Elongation .................................................................................................................... 29 4/ Termination ................................................................................................................... 30 Describe the differences in translation process between eukaryotes and prokaryotes and how this can be used in human medicine ....................................... 30 Appreciate the function of basepairing in translation ................................................. 30 Describe and understand the role of ribosomes and tRNA in translation ................. 30 Topic 9: Gene Mutations and DNA Repair ......................................................................... 31 Define and describe the terms: mutation, mutagen, substitution mutation, frameshift mutation, missense mutation, nonsense mutation, silent mutation, neutral mutation, insertion, mutagenesis, carcinogen, unequal cross over ............. 31
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Describe in detail the different types of mutations (focus on substitution, insertion, deletion, frameshift, missense, nonsense and silent mutations and expanding nucleotide repeats) described in the chapter .............................................................. 31 Base substitution ............................................................................................................... 31 Insertions and deletions .................................................................................................. 32 Expanding nucleotide repeats ...................................................................................... 32 Appreciate and be able to explain the difference between germline and somatic cell mutations ....................................................................................................................... 33 Understand the difference between spontaneous and induced mutations ........... 33 Spontaneous Replication Errors ..................................................................................... 33 Spontaneous Chemical Changes ................................................................................ 33 Chemically Induced Mutations ..................................................................................... 34 Radiation ........................................................................................................................... 34 Describe and understand how mutations get fixated in the genome ...................... 34 Describe the various DNA repair mechanisms (Mismatch repair, Nucleotide excision repair, Base excision repair and Direct repair)................................................ 35 Mismatch Repair .............................................................................................................. 35 Direct repair ...................................................................................................................... 35 Base-excision repair ......................................................................................................... 36 Nucleotide-Excision Repair............................................................................................. 37 Appreciate the role of DNA repair mechanisms in inherited disorders and know two examples of these diseases and how they are caused ....................................... 38 Topic 10: Genomics and proteomics and epigenetics .................................................... 38 Define and describe the terms: microarray, genome wide association study, haplotype, SNP, STS, EST, proteomics, epigenetics, DNA methylation, paramutation, genomic imprinting and epigenome. ............................................................................. 38 Understand the difference between genetic and physical maps and how it can be explained ........................................................................................................................ 39 Describe the difference between map based sequencing and shotgun sequencing ........................................................................................................................... 39 Describe microarray technology using an example..................................................... 41 Describe the 3 molecular mechanisms that underlie many epigenetic phenotypes, use the honeybee as an example of one of these ....................................................... 41 Changes in patterns of DNA methylation ................................................................... 41 Chemical modification of histone proteins ................................................................. 42 RNA molecules that affect chromatin structure and gene expression ................. 42 Describe the role RNA can play in paramutations using Kit mice as an example .. 42
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Understand that epigenetic changes can have multigenerational effects using mice as an example ........................................................................................................... 42 Explain the role of epigenetics in X-inactivation ............................................................ 43 Explain the difference between the genome and epigenetics using monozygotic twins as an example ........................................................................................................... 43 Describe the epigenome and information gained from the epigenome ................ 43 Topic 10: Cancer genetics..................................................................................................... 44 Understand that cancer is a genetic disease ................................................................ 44 Describe Knudson’s multistep model of cancer ............................................................ 44 Understand what is meant by clonal selection .............................................................. 45 Describe in detail the difference between oncogenes and tumour suppressor genes ..................................................................................................................................... 45 Understand how a mutation of a normal gene results in conversion to oncogenes or inactivation of tumour suppressor genes .................................................................... 46 Describe the difference between familial and sporadic cancer, giving examples 46 Understand the role of miRNA in cancer ........................................................................ 46 Briefly describe the role of epigenetics in cancer ......................................................... 47 Be familiar with gene-environment interactions ............................................................ 47
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Topic 6a: DNA replication Define and use the terms: template, semiconservative, bidirectional, replication fork, leading strand, lagging strand, proofreading, homologous recombination Template - Two strands of DNA separate, each becoming a ______ for replication Semiconservative - Replication in which the two nucleotide strands of DNA separate, each serving as a template for the synthesis of a new strand. Bidirectional - Replication at both ends of a replication bubble. Replication fork - Point at which a double-stranded DNA molecule separates into two single strands that serve as templates for replication. Leading strand - DNA strand that is replicated continuously. Lagging strand - DNA strand that is replicated discontinuously. Proofreading - Ability of DNA polymerases to remove and replace incorrectly paired nucleotides during replication. Homologous recombination - Exchange of genetic information between homologous DNA molecules.
Describe and understand the ‘Meselson and Stahl’ experiment in detail This experiment was used to support Watson and Cricks theory that DNA replicated through semiconservative replication. At the time, there were three theories:
To prove that DNA used semiconservative replication, they:
Raised E. coli in a heavy isotope of nitrogen, 15N, for several generations, and took out their first sample. They then incorporated the E. coli into a medium containing the regular nitrogen isotope, 14N. They took samples 20 minutes apart (the generation time of E. coli). All the samples were placed into a centrifuge in a caesium chloride (CsCl) solution, a similar density as DNA. This created an equilibrium density gradient. 5
These were the results:
The first round of replication ruled out conservative replication as there was only one band, if it was conservative there would be two bands. The second round of replication ruled out dispersive replication as there was two bands, if it was dispersive there would only be one band, but each generation would be slightly higher in the test tube than the previous generation.
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Describe the replication direction and the differences between the leadingand lagging strand synthesis. DNA Polymerase can only add nucleotides in the 5’3’ direction. Because the two strands of DNA are antiparallel one strand can elongate continuously along the leading strand, while the other going in the opposite direction can only lay down nucleotides discontinuously along the lagging strand. These discontinuous segments are known as Okizaki fragments.
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Describe the basic apparatus for DNA replication in prokaryotic cells Initiation An initiator protein binds to the origin of replication (oriC) and causes a short section of DNA to unwind. The origin of replication is an area rich in A and T nucleotides, these nucleotides only have 2 H bonds compared to C and G which have 3.
Unwinding DNA helicase binds to the lagging-strand template at each replication fork and moves in the 5’3’ direction along this strand, moving the replication fork. Helicase breaks the hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule. Single-strand-binding proteins (SSBs) attach tightly to the exposed single-stranded DNA. These proteins protect the single-stranded nucleotide chains and prevent the strands from snapping back together or formation of secondary structures such as hairpins DNA gyrase, a topoisomerase, reduces the torsional strain (torque) that builds up ahead of the replication fork because of unwinding by making a double-stranded break in one segment of the DNA helix, passing another segment of the helix through the break, and then resealing the broken ends of the DNA.
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Elongation Primase synthesizes short stretches of RNA nucleotides which provide a 3’-OH group to which DNA polymerase can attach DNA nucleotides. DNA polymerase III is a large multiprotein complex that acts as the main workhorse of replication. It synthesises nucleotide strands by adding new nucleotides to the 3’ end of a growing DNA molecule. This enzyme has two enzymatic activities:
Its 5’3’ polymerase activity allows it to add new nucleotides in the 5’3’ direction. Its 3’5’ exonuclease activity allows it to remove nucleotides in the 3’5’ direction, enabling it to correct errors.
DNA polymerase I has:
5’3’ polymerase activity allows it to add new nucleotides in the 5’3’ direction. 3’5’ exonuclease activity allows it to remove nucleotides in the 3’5’ direction, enabling it to correct errors 5’3’ exonuclease activity, which is used to remove the primers laid down by primase and to replace them with DNA nucleotides by synthesizing in a 5’3’ direction.
DNA polymerase I has lower processivity than DNA polymerase III. DNA ligase joins Okazaki fragments by sealing breaks in the sugar–phosphate backbone of newly synthesized DNA
Termination Tus binds to specific termination sequences which blocks the movement of helicase, thus stalling the replication fork and preventing further DNA replication.
Understand the high fidelity of the replication system and understand in detail the function of basepairing in achieving this. Replication is extremely accurate, with less than one error per billion nucleotides. The high level of accuracy in DNA replication is produced by 1. Nucleotide selection 2. Proofreading 3. Mismatch repair
9
Describe the replication model at the end of chromosomes using the terms telomere and telomerase In circular DNA, there’s no issue because as nucleotides are added to the exposed 3’-OH and eventually they meet up with of the first laid nucleotide.
In linear DNA, once the primer is removed there is a small gap which becomes exposed, in some cells, such as somatic cells, chromosomes shorten every time replication occurs. This happens in most somatic cells and is a normal sign of aging.
Certain cells, such as germ cells and early embryonic cells are able to overcome this issue with telomerase. The telomeres are filled with a G rich overhang, this overhang can be extended by telomerase, a ribonucleoprotein (containing RNA and protein). Telomerase is complimentary to this G strand (contains C’s). This extends the chromosome temporarily and allows for further elongation. It continues to move down the chromosome so more nucleotides can be added. The complimentary strand can then be filled in.
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