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Dr Attya Bhatti Lecture 1 1 Course Outline THE BASICS OF DNA, CHROMOSOMES, CELLS, AND DEVELOPMENT • 1. Nucleic Acid Structure • 2. Chromosome Structure and Function • 3. Role of Chromatin Structure in Gene Control • 3. Genes in Pedigrees and Populations • 4. Cells and Cell-Cell Communication • 5. Principles of Development ANALYZING THE STRUCTURE AND EXPRESSION OF GENES AND GENOMES • 6. Amplifying DNA: Cell-based DNA Cloning and PCR • 7. Nucleic Acid Hybridization: Principles and Applications • 8. Analyzing the Structure and Expression of Genes and Genomes 2 INVESTIGATING THE HUMAN GENOME AND ITS RELATIONSHIP TO OTHER GENOMES • 9. Organization of the Human Genome • 10. Model Organisms, Comparative Genomics and Evolution • 11. Human Gene Expression • 12. Organization of the human, viral and bacterial genome • 13. Studying Gene Function in the Post-Genome Era HUMAN GENETIC VARIATION AND DISEASES • 13. Human Genetic Variability and its Consequences • 14. Genetic Mapping of Mendelian Characters • 15. Mapping Genes Conferring Susceptibility to Complex Disease • 16. Identifying Human Disease Genes and Susceptibility Factors • 17. Methylation - From DNA, RNA and Histones to Diseases and Treatment APPLIED HUMAN MOLECULAR GENETICS • 18. Genetic Testing of Individuals • 19. Pharmacogenetics, Personalized Medicine, and Population Screening • 20. Genetic Manipulation of Animals for Modeling Disease and Investigating Gene Function • 21. Genetic Approaches to Treating Disease • 22. Gene Regulation and Human Disease 3 Recommended Books (online available) From Genes to Genome; In Library 4 Assignments: Every Student will present seminar/ submit hard copy Quiz: Class Quizzes will held after two lecture Total Credits: 3(3-0) 5 Grading Policy 1st OHT + Assignment 2nd OHT + Assignment Final = 15% = 15% = 50% Attendance Class participation (Assignments, Quiz, Presentations) = 20% ……………………………………………………………………. Total =100 1st assignment (written) due on March 20, 2017by 5:00 pm 2nd assignment (written + presentation) due on April 25, 2017 by 5:00 pm 6 Class schedule and office timings Dr. Attya Bhatti Day Time Tuesday 1100 hrs1000hrs Contact Instructor 1.5 Dr. Attya Course title MVI-856 Method MG Room Class CR 6/305 Class CR 6/305 Office timings for students (only) 1000hs-1100hrs Wednesday Thursday Course 1100 hrs1000hrs 1.5 Dr. Attya MVI-856 MG Best way to reach me is via email Email address: [email protected] 7 What is Molecular Genetics? 8 Before we start – genetic data and the Internet 9 10 Useful Internet starting points for Human Molecular Genetics The National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov Links to many genetic resources including GenBank, OMIM, Medline etc. UK Human Genome Mapping Project Resource Centre: http://www.hgmp.mrc.ac.uk/ Select ‘Genome web sites' for many useful links. US Human Genome Mapping Project: http://www.ornl.gov/hgmis 11 Genome Database: http://www.gdb.org Medline: http://www.ncbi.nlm.nih.gov/PubMed OMIM:USA: http://www.ncbi.nlm.nih.gov/Omim/ UK: http://www.hgmp.mrc.ac.uk/omim/ Search engine: Yahoo, Google 12 DNA Structure and Chemistry a). Structure of DNA i). Structure of the bases, nucleosides, and nucleotides ii). Structure of the DNA double helix iii). Complementarity of the DNA strands b). Chemistry of DNA i). Forces contributing to the stability of the double helix ii). Denaturation of DNA 13 The Structure of DNA • The Primary Structure of DNA consists of a string of nucleotides joined together by phosphodiester linkages • Nucleotides – DNA are nucleotides, each comprising three parts: 1) a sugar, 2) a phosphate, 3) a nitrogen-containing base. Base Nucleoside Nucleotide +deoxyribose +phosphate 14 Primary structure of DNA • Consists of a string of nucleotides. • Each nucleotide consists of a five carbon sugar, a phosphate, and a base. Two types of DNA bases: Purines (adenine and guanine) Pyrimidines (thymine and cytosine) 15 Nitrogenous base, Purines consists of a six-sided ring attached to a five-sided ring Pyrimidines, consists of a six-sided ring only. DNA and RNA both contain two purines, adenine and guanine (A and G), cytosine (C), thymine (T), and uracil (U). Cytosine is present in both DNA and RNA; however, thymine is restricted to DNA, and uracil is found only in RNA. 16 Sugars 17 Phosphate group • Phosphate group, which consists of a phosphorus atom bonded to four oxygen atoms. • • Phosphate groups are found in every nucleotide and frequently carry a negative charge, which makes DNA acidic. • The phosphate is always bonded to the 5-carbon atom of the sugar in a nucleotide 18 Structure of DNA • The nucleotides of DNA are joined in polynucleotide strands by phosphodiester bonds that connect the 3 carbon atom of one nucleotide to the 5 phosphate group of the next. Each polynucleotide strand has polarity, with a 5 end and a 3 end. 19 Structure the DNA of the ii).ofStructure polynucleotide chain helix DNA double 5’ 3’ • polynucleotide chain • 3’,5’-phosphodiester bond 20 A-T base pair Hydrogen bonding of the bases G-C base pair Chargaff’s rule: The content of A equals the content of T, and the content of G equals the content of C in double-stranded DNA from any species 21 Secondary Structures of DNA • The double helix: DNA consists of two polynucleotide strands. The sugar–phosphate groups of each polynucleotide strand are on the outside of the molecule, and the bases are in the interior. • Hydrogen bonding joins the bases of the two strands: guanine pairs with cytosine, and adenine pairs with thymine. • The two polynucleotide strands of a DNA molecule are complementary and antiparallel. 22 23 Forms of DNA B-DNA: Alpha helix right-handed or clockwise, spiral. Predominate structure ; possesses approximately 10 base pairs (bp) per 360degree rotation of the helix; so each base pair is twisted 36 degrees relative to the adjacent bases . The base pairs are 0.34 nanometer (nm) apart; so each complete rotation of the molecule encompasses 3.4 nm. (Diameter of the helix is 2 nm) A relatively slim and elongated structure. Spiraling of the nucleotide strands creates major and minor grooves in the helix, features that are important for the binding of some DNA-binding proteins that regulate the expression of genetic information. 24 Forms of DNA A-DNA is an alpha (right-handed) helix, is shorter and wider than B-DNA and its bases are tilted away from the main axis of the molecule. Z-DNA: A radically different secondary structure forms a left-handed helix. Sugar–phosphate backbones zigzag back and forth, giving rise to the name Z-DNA (for zigzag). 25 26 local variation in DNA structure • Static, rigid structure that is invariant in its secondary structure. • Arises because of differences in local environmental conditions, – Proteins, – metals, and ions that may bind to the DNA. – The base sequence also influences DNA structure locally. 27 Special Structures in DNA and RNA • • • • A hairpin, consisting of a region of paired bases (which forms the stem) and a region of unpaired bases between the complementary sequences (which form a loop at the end of the stem). Results when sequences of nucleotides on the same strand are inverted complements. The sequence 5 TGCGAT 3 and 5 ATCGCA 3 are examples of inverted complements and, when these sequences are on the same nucleotide strand, they can pair and form a hairpin. A hairpin consists of a region of paired bases (the stem) and sometimes includes intervening unpaired bases (the loop). When the complementary sequences are contiguous, the hairpin has a stem but no loop 28 Special Structures in DNA and RNA In double-stranded DNA, sequences that are inverted replicas of each other are called inverted repeats. Inverted repeats are palindromes because the sequences on the two strands are the same but in reverse orientation. 29 Special Structures in DNA and RNA Cruciform, when a hairpin forms within each of the two single-stranded sequences 30 Special Structures in DNA and RNA RNA molecules may contain numerous hairpins, allowing them to fold up into complex structures 31 DNA Methylation • DNA methylation, in which methyl groups (–CH3) are added (by specific enzymes) to certain positions on the nucleotide bases. • In bacteria, adenine and cytosine are commonly methylated. 32 DNA Methylation • • In eukaryotic DNA, cytosine bases are often methylated to form 5-methylcytosine. The extent of cytosine methylation varies; – In most animal cells, about 5% of the cytosine bases are methylated – More than 50% of the cytosine bases in some plants are methylated – No methylation of cytosine has been detected in yeast cells – very low levels of methylation (about 1 methylated cytosine base per 12,500 nucleotides) are found in Drosophila. • Methylation is most frequent on cytosine nucleotides that sit next to guanine nucleotides on the same strand: • Related to gene expression. – Sequences that are methylated typically show low levels of transcription while sequences lacking methylation are actively being transcribed – Methylation can also affect the three-dimensional structure of the DNA molecule 33 Bends in DNA Some specific base sequences—such as a series of four or more adenine – thymine base pairs—cause the DNA double helix to bend. Bending affects how the DNA binds to certain proteins and important in controlling the transcription of some genes. • The SRY protein, which is encoded by a Y-linked gene and binds to certain DNA sequences and activates nearby genes that encode male traits. 34 Chemistry of DNA Forces affecting the stability of the DNA double helix • hydrophobic interactions - stabilize - hydrophobic inside and hydrophilic outside • stacking interactions - stabilize - relatively weak but additive van der Waals forces • hydrogen bonding - stabilize - relatively weak but additive and facilitates stacking • electrostatic interactions - destabilize - contributed primarily by the (negative) phosphates - affect intrastrand and interstrand interactions - repulsion can be neutralized with positive charges (e.g., positively charged Na+ ions or proteins) 35 Charge repulsion Stacking interactions Charge repulsion 36 Denaturation of DNA Double-stranded DNA Extremes in pH or high temperature Strand separation and formation of single-stranded random coils A-T rich regions denature first Cooperative unwinding of the DNA strands 37 Electron micrograph of partially melted DNA Double-stranded, G-C rich DNA has not yet melted A-T rich region of DNA has melted into a single-stranded bubble • A-T rich regions melt first, followed by G-C rich regions 38 Hyperchromicity Absorbance Absorbance maximum for single-stranded DNA Absorbance maximum for double-stranded DNA 220 260 300 The absorbance at 260 nm of a DNA solution increases when the double helix is melted into single strands. 39 Percent hyperchromicity DNA melting curve 100 50 0 50 70 90 Temperature oC • Tm is the temperature at the midpoint of the transition 40