Molecular Biology of Genes (BIOM335) Lecture Notes PDF

Welcome to Molecular Biology of Genes (BIOM335)! Dr. Fatima Labeed  Course structure Please see the syllabus document on Black Board (BB) for details of topics and assessments. There are 2 lectures (each around 60-70 minutes) per week. Lecture notes/slides are...

Welcome to Molecular Biology of Genes (BIOM335)! Dr. Fatima Labeed  Course structure Please see the syllabus document on Black Board (BB) for details of topics and assessments. There are 2 lectures (each around 60-70 minutes) per week. Lecture notes/slides are available on BB. However, please note these are being regularly updated and modified. Please ensure you check the latest versions of the lectures.  Course structure Assessments will be based on: Tests, assignment/presentations, midterm and final exams. Attendance: Your number (alongside your name) will be called 3 times. If no answer is given, you will be marked as absent. I cannot mark absent students as present.  Please arrive on time. Late arrivals may result in attendance being marked absent. Any mitigating circumstances causing absences need to be reported to the Advisory who will decide whether to excuse the absence or not. Decisions are not made by the instructor.  Genes Are DNA Genes and chromosomes Introduction  What is meant by “genome”? – Genome: A long sequence of DNA, the complete set of hereditary information carried by the organism as well as its individual cells. – The genome: – Chromosomal DNA (in humans, the genome consists of 23 pairs of chromosomes located in the nucleus, and mitochondrial DNA as circular chromosome in the mitochondria) – DNA in plasmids (a plasmid is a small circular double stranded DNA, distinct from chromosomal DNA. It is found in bacteria and some eukaryotes). – Organellar DNA (in eukaryotes), as found in mitochondria and chloroplasts  Genome We use the term information because the genome does not itself perform an active role in the development of the organism The products of expression its nucleotide* sequences within the genome determine development By a complex series of interactions, the DNA sequence produces all of the proteins of the organism at the appropriate time and within the appropriate cells. *Remember: Nucleotides are basic building blocks of nucleic acids (DNA & RNA) Proteins are so important, they are needed for development and functioning. Think of all cell structures, receptors, metabolism  Genome Physically, the Genome genome may be divided into a number Physically of different DNA Different DNA molecules, molecules or chromosomes (or chromosomes) Functionally Genome: is the sequence of DNA Genes (many or few depending on organism) carried by chromosomes Each gene encodes a single type of RNA  Genes and Chromosomes Each of the discrete chromosomes within the genome may contain a large number of genes. Genomes for living organisms may contain as few as ∼500 genes (for a mycoplasma, a type of bacterium), ∼20,000 to 25,000 for a human being, or as many as ∼50,000 to 60,000 for rice.  Genes and chromosomes Chromosome – A discrete unit of the genome carrying many genes. – Each chromosome consists of a very long molecule of duplex (double stranded) DNA and an approximately equal mass of proteins, and is visible as a morphological entity only during cell division. Genes in Chromosomes Human chromosomes - Karyotype  Genes Genes live in chromosomes Each gene has a particular location to call its home. The location is called a genetic locus* *Locus is one location but called loci if describing more than one location  Genes and Chromosomes Chromosome pair: Homologous chromosomes - Carry the same sequence of genes - Pair has genes at the same loci - Alleles (maybe the same or different) Remember: Alleles are sequences of DNA. They are versions of the same gene, e.g. the gene for eye colour.  Genes Genes determines our physical features and how we work on the inside Genes: The basic physical and functional unit of heredity.  A gene encodes an RNA, which may encode a polypeptide The first definition of the gene as a functional unit followed from the discovery that individual genes are responsible for the production of specific proteins (e.g. enzyme) Later, the chemical makeup between the DNA of the gene and its protein product led to the suggestion that a gene encodes a protein This in turn led to the discovery of the complex apparatus by which the DNA sequence of a gene determines the amino acid sequence of a polypeptide.  These contain DNA Genes Some genes instruct making proteins Some genes do not code for proteins They make the basis of the genome! It is reported that only 1% of DNA is made up of protein coding genes. The other, non coding DNA (ncDNA) do not provide instructions to make proteins. Some ncDNA is transcribed into functional but non coding RNA, e.g. tRNA, microRNA, rRNA and regulatory RNAs  Genes Functionally, the genome is divided into genes. They can be: – Structural genes: These encode proteins required for structural and functional needs. They code for any RNA or polypeptide other than a regulator – Regulator genes: These encode factors that control the expression of structural genes. Regulator genes carry our metabolic reactions  A gene encodes an RNA, which may encode a polypeptide Understanding the process by which a gene is expressed allows us to make a more rigorous definition of its nature A gene is a sequence of DNA that directly produces a single strand of another nucleic acid, RNA, with a sequence that is identical to one of the two polynucleotide strands of DNA. A gene encodes an RNA, which may encode a polypeptide  A gene encodes an RNA, which may encode a polypeptide In many cases, the RNA is in turn used to direct production of a polypeptide In other cases, such as rRNA and tRNA genes, the RNA transcribed from the gene is the functional end product Thus, a gene is a sequence of DNA that encodes an RNA, and in protein-coding genes, the RNA in turn encodes a polypeptide. Each gene is a sequence of DNA that encodes a single type of RNA and in many cases, ultimately a polypeptide.  DNA Is the Genetic Material of Bacteria and Viruses In Bacteria: Bacterial transformation provided the first support that DNA is the genetic material of bacteria. During transformation, genetic properties can be Both S & R are strains of s.pneumoniae. transferred from one S (Smooth) type killed mice by causing pneumonia. They produce a capsular polysaccharide that bacterial strain to another evades the host immune response by extracting DNA from R (Rough) type didn’t kill mice due to absence of the first strain and capsular polysaccharide adding it to the second strain.  DNA Is the Genetic Material of Bacteria and Viruses In Bacteria: Bacterial transformation provided the first support that DNA is the genetic material of bacteria. During transformation, genetic properties can be transferred from one bacterial strain to another by extracting DNA from the first strain and adding it to the second strain. The DNA of S-type bacteria can transform R-type bacteria into the same S-type DNA Is the Genetic Material of Bacteria and Viruses The Transforming principle – DNA that is taken up by a bacterium and whose expression then changes the properties of the recipient cell. DNA Is the Genetic Material of Bacteria and Viruses In viruses (Phage T2) Phage T2 infects E.Coli bacteria. They attach to outer surface. Some virus material enters a bacterial cell and causing bacterial cell to lyse.  When the DNA and protein components of bacteriophages are labelled with different radioactive isotopes (32P for DNA and 35S for protein), only the DNA is transmitted to the progeny phages produced by infecting bacteria. The infection showed that DNA is the genetic material of viruses. See video: The genetic material of phage T2 is DNA https://youtu.be/bNsSaxCsIIg?si=USQUdiP26yIPXLVd  DNA Is the Genetic Material of Eukaryotic Cells This was found thanks to scientific experiments. DNA can be added to eukaryotic cells in culture or from one species to another. The DNA enters the cell and result in a new protein produced. If an isolated gene is added to eukaryotic cells, its incorporation can lead to the production of a particular protein. This was called Transfection (typically done in animal cells)- a process similar to bacterial transformation. The DNA then becomes part of its genome and inherited as a result. The result is a new phenotype.  DNA Is the Genetic Material of Eukaryotic Cells To demonstrate this, experiments were done with thymidine kinase (TK)- important for the synthesis of DNA. By adding TK DNA, some cell colonies started producing TK and hence survived. Eukaryotic cells can acquire a new phenotype as the result of transfection  DNA Is the Genetic Material of Eukaryotic Cells The genetic material of all organisms and many viruses is DNA. Some viruses however, use RNA as the genetic material. So genetic material is always nucleic acid, principally DNA except in RNA viruses.  Polynucleotide Chains Have Nitrogenous Bases Linked to a Sugar–Phosphate Backbone A nucleoside consists of a purine or pyrimidine base linked to the 1′ carbon of a pentose sugar. The difference between DNA and RNA is in the group at the 2′ position of the sugar. – DNA has a deoxyribose sugar (2′–H); RNA has a ribose sugar (2′–OH).  Polynucleotide Chains Have Nitrogenous Bases Linked to a Sugar–Phosphate Backbone A nucleotide consists of a nucleoside linked to a phosphate group on either the 5′ or 3′ carbon of the (deoxy)ribose. DNA contains the four bases adenine, guanine, cytosine, and thymine; RNA has uracil instead of thymine.  Polynucleotide Chains Have Nitrogenous Bases Linked to a Sugar–Phosphate Backbone Successive (deoxy)ribose residues of a polynucleotide chain are joined by a phosphate group between the 3′ carbon of one sugar and the 5′ carbon of the next sugar. One end of the chain (conventionally written on the left) has a free 5′ end and the other end of the chain has a free 3′ end. A polynucleotide chain  DNA Is a Double Helix The B-form of DNA is a double helix consisting of two polynucleotide chains that run antiparallel. A polynucleotide chain  DNA Is a Double Helix The nitrogenous bases of each chain are flat purine or pyrimidine rings that face inward and pair with one another by hydrogen bonding to form only A-T or G- C pairs. Complementary – Base pairs that match up in the pairing reactions in double helical nucleic acids (A with T in DNA or with U in RNA, and C with G). Flat base pairs lie perpendicular to the sugar-phosphate backbone  DNA Is a Double Helix The diameter of the double helix is 20 Å (2nm), and there is a complete turn every 34 Å, with ten base pairs per turn (~10.4 base pairs per turn in solution). The double helix has a major (wide) groove and a minor (narrow) groove. Å= Ångström which equates to 10-10m  DNA Is a Double Helix Overwound – B-form DNA that has more than one turn per 10.5 base pairs of the helix. Underwound – B-form DNA that has less than one turn per 10.5 base pairs of the helix.  DNA Replication is Semiconservative Semiconservative replication – DNA replication accomplished by separation of the strands of a parental duplex (double stranded), each strand then acting as a template for synthesis of a complementary strand. The sequences of the daughter strands are determined by complementary base pairing with the separated parental strands. Base pairing provides the mechanism for replicating DNA  DNA Replication is Semiconservative The Meselson–Stahl experiment used “heavy” isotope labeling to show that the single polynucleotide strand is the unit of DNA that is conserved during replication. This complementary base pairing produced a first- generation duplex- made of one daughter strand and one parental strand. Replication of DNA is semiconservative Polymerases Act on Separated DNA Strands at the Replication Fork Replication of DNA is undertaken by a complex of enzymes that separate the parental strands and synthesize the daughter strands. Denaturation – In DNA, this involves the separation of the two strands due to breaking of hydrogen bonds between bases. Renaturation – The reassociation of denatured complementary single strands of a DNA double helix. Polymerases Act on Separated DNA Strands at the Replication Fork The replication fork is the point at which the parental strands are separated. The enzymes that synthesize DNA are called DNA polymerases. The replication fork Polymerases Act on Separated DNA Strands at the Replication Fork Nucleases are enzymes that degrade nucleic acids; they include DNases and RNases and can be categorized as endonucleases or exonucleases. An endonuclease cleaves a bond within a nucleic acid An exonuclease removes bases one at a time  Genetic Information Can Be Provided by DNA or RNA Cellular genes are DNA, but viruses may have genomes of RNA. DNA is converted into RNA by transcription, and RNA may be converted into DNA by reverse transcription. RNA polymerase – An enzyme that synthesizes RNA using a DNA template (formally described as DNA- dependent RNA polymerases).  Genetic Information Can Be Provided by DNA or RNA Central dogma – Information cannot be transferred from protein to protein or protein to nucleic acid, but can be transferred between nucleic acids and from nucleic acid to protein. The translation of RNA into protein is unidirectional. The central dogma  Nucleic Acids Hybridize by Base Pairing Heating causes the two strands of a DNA duplex to separate. The melting temperature (Tm) is the midpoint of the temperature range for denaturation. Complementary single strands can renature or anneal when the temperature is reduced. Denatured single strands of DNA can renature to give the duplex form  Nucleic Acids Hybridize by Base Pairing Denaturation and renaturation/hybridization can occur with DNA–DNA, DNA–RNA, or RNA– RNA combinations. – Hybridization can be intermolecular or intramolecular. Base pairing  Nucleic Acids Hybridize by Base Pairing The ability of two single- stranded nucleic acids to hybridize is a measure of their complementarity. Filter hybridization  Mutations Change the Sequence of DNA All mutations are changes in the sequence of DNA. Mutations may occur spontaneously or may be induced by mutagens. Mutation rates  Mutations May Affect Single Base Pairs or Longer Sequences A point mutation changes a single base pair. Point mutations can be caused by the chemical conversion of one base into another or by errors that occur during replication. Mutations can be induced by chemical modification of a base  Mutations May Affect Single Base Pairs or Longer Sequences A point mutation can be: – Transition replaces a G-C base pair with an A-T base pair or vice versa. – Transversion replaces a purine with a pyrimidine, such as changing A-T to T-A. Insertions and/or deletions can result from the movement of transposable elements.  The Effects of Mutations Can Be Reversed Forward mutations alter the function of a gene, and back mutations (or revertants) reverse their effects. Insertions can revert by deletion of the inserted material, but deletions cannot revert. Point mutations can revert: Either by restoring the original mutation or by compensation elsewhere in the gene. Point mutations and insertions can revert, but deletions cannot revert  The Effects of Mutations Can Be Reversed True reversion – A mutation that restores the original sequence of the DNA. Second-site reversion – A second mutation suppressing the effect of a first mutation within the same gene. Suppression occurs when a mutation in a second gene bypasses the effect of mutation in the first gene.  Mutations Are Concentrated at Hotspots The frequency of mutation at any particular base pair is statistically equivalent, except for hotspots, where the frequency is increased by at least an order of magnitude. Spontaneous mutations are concentrated at a hotspot Many Hotspots Result from Modified Bases A common cause of hot spots is chemical modifications of cytosine, where it’s deaminated generating uracil (causing G-U pairing). Also, modification of 5- methylcytosine, which is spontaneously deaminated to thymine causing G-T pairing. A hotspot can result from the high frequency of change in copy number of a short, tandemly repeated sequence. Deamination  Some Hereditary Agents Are Extremely Small Some very small hereditary agents do not code for polypeptide, but consist of RNA or protein with heritable properties. Viroid – A small infectious subviral nucleic acid (circular RNA) infecting plants, that does not have a protein coat. PSTV RNA (PSTV: Potato Spindle Tuber viroid). Mutations make them less infectious  Some Hereditary Agents Are Extremely Small Prion – A proteinaceous infectious agent that behaves as an inheritable trait even though it contains no nucleic acid. – One example is PrPSc (which cannot be degraded by proteases), the agent of scrapie in sheep and bovine spongiform encephalopathy. Similar to the human version (C-JD). Remember which way round is the mutation Wikipedia