BIOL 3110 B - Molecular Biology I: Nucleic Acid Metabolism - 2024Fall - York University PDF

Copyright and Academic Misconduct Reminder: This material is for your personal use only. It is not to be downloaded, shared or distributed in anyway. Thank you. All material associated with this course, including lecture recordings, activities, quizzes, laboratories, are to be used for personal st...

Copyright and Academic Misconduct Reminder: This material is for your personal use only. It is not to be downloaded, shared or distributed in anyway. Thank you. All material associated with this course, including lecture recordings, activities, quizzes, laboratories, are to be used for personal study purposes only. Unauthorized distribution in any form can lead to a violation under Canadian Copyright Law and Academic Misconduct charges under York University Senate Policy. Unauthorized distribution includes sharing and/or uploading of material.  WELCOME to BIOL 3110 B – 2024Fall MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM Instructor: Dr. Yi Sheng Departments of Biology York University Office: LSB 327B Course email: [email protected] https://eclass.yorku.ca/course/view.php?id=121158 BIOL 3110 B - MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM https://eclass.yorku.ca/course/view.php?id=121158 BIOL 3110 B - MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM Textbooks & lecture resources No specific text required Optional: Molecular Biology of the Gene, 7th Ed, Watson et al Optional: Nelson and Cox (2021). Lehninger Principles of Biochemistry. 8th Edition. Macmillan Learning. BIOL 3110 B - MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM Evaluation Percentage of the Dates overall grade Test 1 25 % Oct 8, 2024 Test 2 25 % Nov 12, 2024 Final Exam 40 % Exam period Activity 10% Throughout If you miss one of the midterms, the weight of the missed midterm will be automatically transferred to the final exam. No makeup exam will be provided. The final exam is cumulative but weighted (usually > 65% of questions is on material post midterm 2 ) Activities, worth 10% of the overall mark (e.g. iClicker, online eClass quizzes, refer to details posted on your course eClass site) Any question about this course outline and schedule? 5  Create REEF Student Account iClick Reef set up – option 1 (eClass) Go to your eClass course website and click link for iclicker set up. Students must enter their first name, last name, and email address. We recommend that they use their York email address. IMPORTANT !!! - You need to provide Although it says “Optional,” students must provide student number to get your activity grades York University Student Number in the Student ID imported to your gradebook on eClass field to syn eClass gradebook. York University email address York University Student Number 7  iClick Reef set up F24-BIOL 3110secB Yi Sheng York University iClick Reef set up Congratulations! You are done iclick reef set up.  BIOL3110 B Lecture 1 – Journey to the Discovery of DNA as the Molecule of Inheritance The Twisting Path to the Double Helix  LECTURE 1 - KEY POINTS Describe Paul Berg’s experiment in 1971. Explain the experimental design and significance of the experiment. [Comprehension] Describe Mendel’s Laws of inheritance. [Comprehension] Describe Miescher’s experiment à the first to “isolate” “nuclein”. Explain the procedures and principles behind the DNA purification. Explain the chemical composition of nucleic acid discovered by Miescher’s research. [Comprehension] Describe the criteria for the molecule/the carrier of inheritance. Reason why DNA was not considered to be the carrier of genetic information in Miescher’s time. [Knowledge] Describe how Fleming’s findings about chromatin/chromosome contribute to the discovery of DNA as the molecule of inheritance. [Comprehension] Describe Thomas Morgan’s research approaches and findings. Explain how Morgan’s discovery contradicts Mendel’s first Law. [Comprehension] Describe Hermann Muller’s research approaches and findings. Explain how Muller’s research contributes to the discovery of DNA as the molecule of inheritance. [Comprehension] For Beadle and Tatum’s experiment, explain the choice of the model organism, the hypothesis of the experiment, the experimental design, and interpret the observation and conclusion of the experiment. Explain the significance of Beadle and Tatum’s research. [Comprehension, Analysis] Compare and contrast Forward and Reverse genetic screen. Explain why Beatle and Tatum’s experiment is a type of Forward genetic screen. [Comprehension, Analysis] 12 What is Molecular Biology? = Recombinant DNA (rDNA) 8technology * * * * * * * * * 13  14 https://www.sciencehistory.org/historical-profile/paul-berg  A Big Question: What is inheritance? John Lennon Julian Lennon The concept of “likeness” between generations has fascinated philosophers and scientists since the ancient times. There must be “information” that is passed from one generation to the next. 15 13 What is the molecule of inheritance? Criteria must be fulfilled by the carrier of inheritance 16 What is the molecule of inheritance? The Story Starts with Gregor Mendel and Johann Friedrich Miescher in the 19th Century *From Wellcome Images 1866 - Gregor Mendel 1869 - Johann Friedrich Miescher Discovered the chemical17 substance nuclein, Discovered Laws of inheritance later called deoxyribonucleic acid (DNA) Images licensed through CC-BY-NC with attribution to XBio What is the molecule of inheritance? Gregor Mendel (Austria): Spent 8 years meticulously studying, recording and quantifying cross- breeding of 22 varieties of peas. Published paper titled "Experiments in Plant Hybridisation" in 1866. 18 What is the molecule of inheritance? Mendel’s Laws of inheritance and Inheritance factors : 1. Law of independent assortment: Specific traits (e.g. stem length or seed colour) determined by Mendel’s Inheritance factors operate independently of one another. 2. Law of independent segregation: Inherited characteristics exist in alternative forms (e.g tall vs short) – known as alleles For each characteristic, an individual possesses two paired alleles – one inherited from each parent These pairs segregate in germ cells (gametes) and recombine during reproduction (fertilization) Each parent transmits one allele to each offspring 19 What is the molecule of inheritance? Mendel’s Laws of inheritance: 3. Law of dominance: For each characteristic, one factor is dominant and appears more often, in a 3:1 ratio The alternative form is recessive The constant 3:1 ratio represents the random combination of alleles during reproduction. Any combination that includes the dominant allele will express the dominant trait 20 Punnett square What is the molecule of inheritance? Gregor Mendel (Austria): In modern terms, Mendel’s factors that carried the traits = genes Also in 1866, Ernst Haeckel (embryologist in Germany) proposed that the nucleus contained the factors responsible for the transmission of hereditary traits The nucleus has to take care of the inheritance of the heritable characters, while the surrounding cytoplasm is concerned with accommodation or adaptation to the environment. - Ernst Haeckel 21 What is the molecule of inheritance? Friedrich Miescher (Switzerland/Germany): First to “isolate” ”nuclein”, what we now know as DNA (1869), studying leukocytes collected from pus on fresh surgical bandages. In pus, he found the ideal base material for his analyses, and its “histological purity” allowed him to purify the chemical building blocks that constitute cells. 22 http://www.dnaftb.org/ 16 What is the molecule of inheritance? Friedrich Miescher (Switzerland/Germany): Nuclein from pus Nuclein from Salmon sperm Later discovered that salmon sperm Through simple extraction of cells proved to be the ideal source using alkaline solution à nuclei material for isolating large precipitated out of solution à used quantities of pure nuclein. to isolate chemical substance within nuclei. Found that nuclein contained mostly carbon, nitrogen, hydrogen, He named the material found inside oxygen, and phosphorous atoms. the nuclei “nuclein”. Modern day He was lucky to have picked these By chemical analyses, Miescher cell types for his studies because picture of determined that collecting both leukocytes and spermatozoa 1. Nuclein does not contain proteins, salmon sperm are easily purified, and contained since it does not contain any sulfur mostly nuclei (small cytoplasm) à (found in proteins). facilitated enrichment of the nuclear 2. Nuclein contains large amounts of components phosphorous. 23 What is the molecule of inheritance? The Journey to show Miescher’s “nuclein” is the Mendel’s “inheritance factor” Inheritance Nuclein factor *From Wellcome Images 1866 - Gregor Mendel 1869 - Johann Friedrich Miescher Discovered units of heredity, later Discovered the chemical24 substance nuclein, called genes later called deoxyribonucleic acid (DNA) What is the molecule of inheritance? Inheritance Nuclein factor 1866 - Gregor Mendel Discovered units of heredity 1869 - Johann Friedrich Miescher 1879 - Walther Flemming Discovered nuclein, made up of C, H, N, O, P, but NO S. Observed Chromosome in mitosis 1880 - Albrecht Kossel (Germany): 1905 - 1909 - William Bateson (USA) and Wilhelm Discovered that nucleic acids contain four nitrogen- Johannsen (Denmark): containing groups: Genes, genotype and phenotype cytosine, thymine, adenine, and guanine. 1907 - Thomas Morgan: concept of genetic linkage and sex-linked genes 1927- Hermann Muller: genes can be inherited 1941- George Beatle and Edward Tatum: One gene one enzyme 25 What is the molecule of inheritance? Albrecht Kossel (Germany): Worked on the constitution of the cell nucleus – in 1880s, showed that nuclein consisted of a protein component (which he termed “histon”, i.e. modern day histones), as well as a non-protein component (nucleic acids). Discovered that nucleic acids contain four nitrogen-containing groups: cytosine, thymine, adenine, and guanine. 26 Proteins were made up of 20 amino acids whereas nucleic acids only has 4 different bases à at the time, thought to be “not complex” enough to be molecule of inheritance What is the molecule of inheritance? Walther Flemming (Germany): In the 19th Century, scientists started to use microscopy to study cells, cell functions and structures. However, resolution of the microscopes was poor and the cells’ inner structures are mostly grey-on-grey shapes. In 1879, Flemming found a basophilic dye that specifically stained the material inside the cell nucleus, which he termed “chromatin” (Greek word for colour). Using this dye to stain salamander embryos going through cell division, Flemming saw the stained material (chromatin) collect into thread-like structures (chromosomes, from Greek word meaning coloured bodies). 27 What is the molecule of inheritance? Walther Flemming (Germany): He also coined the term “mitosis” (Greek word for threads) to describe the behaviour of chromosomes during cell division. During mitosis, he saw that each individual chromosome break into two, thus doubling the number of chromosomes. He also saw that identical sets of chromosomes being pulled apart and separately go to the two dividing cells. Therefore, each daughter cell contained a set of chromosomes identical to the parental cell 28 What is the molecule of inheritance? William Bateson (USA) and Wilhelm Johannsen (Denmark): In 1905, Bateson first coined the term “genetics” (Greek word meaning to give birth) to describe the study of inheritance. In 1909, Johannsen introduced the basic terminology for genetics: Genes = units of hereditary information Genotype = genetic constitution of an organism Phenotype = an organism’s totality of inherited characteristics 29 Relationship between genotype and phenotype What is the molecule of inheritance? Thomas Hunt Morgan (USA): Embryologist turned heredity researcher– starting from 1907, pioneered the breeding of the common fruit fly (Drosophila melanogaster) for genetic studies. Found that while almost all Drosophila has red eyes, very rarely there are a few that have white eyes (in one breeding experiment, out of 1,237 first generation offsprings, 3 had white eyes). Noticed that all white-eye flies were male, and proposed that association of eye color and sex in fruit flies had a physical and mechanistic basis in the chromosomes. Red- and White-eyed Drosophila 28 What is the molecule of inheritance? Thomas Hunt Morgan (USA): Discovered sex-linked genes, and developed the concept of genetic linkage (i.e. genes that are close together on the chromosomes are often inherited together during meiosis, which contradicts Mendel’s law). He also used physical, chemical, or radiological means to mutate Drosophila and used breeding experiments to follow heritable mutations. The physical basis of genetic linkage is how close the genes are to one another on a chromosome (or on different chromosomes) What is the molecule of inheritance? Hermann Muller (USA): Worked with Morgan to use X rays to induce mutations in Drosophila genome (1927). Found that genes can be mutated by chemical or radiological insults, and more importantly, the mutated genes can be passed on from one generation to the next! I.e. genes can be inherited What do genes do? 39 What is the molecule of inheritance? George Beadle and Edward Tatum (USA): Used bread mould, Neurospora crassa, to determine if and how genes control known biochemical reactions. Neurospora was chosen because a) it was easy to grow, and reproduce by crossing b) it was easy to produce mutation effects (since haploid state ie. spore only has one set of unpaired chromosomes), and c) the biochemistry of amino acid synthesis was already known. 33 Lab cultures of Neurospora in flasks What is the molecule of inheritance? George Beadle and Edward Tatum (USA): the classic experiment (published in 1941) Goal: test the function of a gene ➔ Perform genetic screen experiment: ➔ Mutate genome and screen for specific phenotype à loss of ability to synthesize a specific amino acid Complementary experiment + AAs Minimal medium plus the amino acid indicated Lacking AAs 34 Types of genetic screens: Common approaches to identify gene functions: Random Targeted mutagenesis mutagenesis Select for mutant Known gene mutation phenotype Identify Observe for responsible genes phenotypic changes 35 Neurospora (bread mould) life cycle: Mut Spore WT Wildtype spores: -AA +AA ➔ can synthesize all amino acids (AAs) ➔ can grow on complete medium or minimal medium (without AAs) Mutant spores: ➔ select for strains that have mutations that disrupted Mut WT AA biosynthesis pathway ➔ cannot grow in minimum medium unless supplemented with AA in growth medium What is the molecule of inheritance? George Beadle and Edward Tatum (USA): By this method they isolated four strains of Neurospora that only grew on minimal medium if supplemented with arginine. Genetic analyses showed that each strain differ from the normal wild type by one gene Suggested that loss of multiple genes resulted in same phenotype à loss of the production of a single amino acids (e.g. arginine). One gene one enzyme hypothesis: (Srb & Horowitz 1944) DNA Protein The one gene, one enzyme (or later one gene, one polypeptide) concept is considered by some as the pre-cursor of the Central Dogma. 35 Beadle and Tatum experiment: Isolated 4 mutant strains that grew on minimal medium only when supplemented with arginine At that time, knew that arginine biosynthetic pathway = multi-step process involving different enzymes  To test hypothesis: First lecture ends 40