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Brought to you by American Society of Cytopathology Core Curriculum in Molecular Biology Copyright 2010 American Society of Cytopathology Brought to you by American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 2 Molecular Science Nucleic Acid Biochemistry Stephanie A. Hamilton, EdD, SCT, MB(ASCP)CM MD Anderson Cancer Center Houston, Texas Copyright 2010 American Society of Cytopathology Molecular Pathology Brought to you by • Molecular pathology is the study of nucleic acids (DNA and RNA) from patient specimens mainly to diagnose genetic diseases, direct choice of therapy, detect presence of pathogens and for identity testing Copyright 2010 American Society of Cytopathology Brought to you by Genome and Cells • Each organism has a unique genome • A genome includes all of the genes of the organism • All cells in a person’s body carry the same genome or genetic information in the nucleus. The expression of different genes is what determines cell function thus producing a nerve cell, muscle cell or skin cell Copyright 2010 American Society of Cytopathology Cell, Chromosome and DNA Copyright 2010 American Society of Cytopathology Brought to you by http://members.cox.net/chromosome3/ Accessed 10/19/2010 Brought to you by Chromosomes, Genes and DNA • A gene can be defined as a region of DNA that controls a hereditary characteristic • Genes are a sequence of nucleotides in DNA helix • Human genome includes all of the genes (about 30,000 genes) located on 23 pairs of chromosomes • There are 22 pairs of autosomes and 1 pair of sex chromosomes, XX or XY • The human genome contains about 3 billion nucleotide pairs Copyright 2010 American Society of Cytopathology Brought to you by Genetic Information • Genotype: the DNA nucleotide sequence responsible for a phenotype • Phenotype: a trait or group of traits resulting from transcription and translation of genes • Position effect: A gene inserted or moved into a different chromosomal location, it may be expressed differently than it was in its original position Copyright 2010 American Society of Cytopathology Brought to you by Watson-Crick Model Crick-Crick Model • In 1953, James Watson and Francis Crick proposed structure for DNA • Watson‐Crick model: a DNA molecule consists of two polynucleotide strands coiled around each other in a helical "twisted ladder" structure • Two sugar‐phosphate backbones are on the outside of the double helix forming the two sides of the ladder • Bases are on the inside forming the rungs of the ladder. • Bases follow a specific base pairing rule: Adenine pairing with Thymine and Guanine pairing with Cytosine • How the bases are arranged in the DNA determines the genetic code Copyright 2010 American Society of Cytopathology Nucleic Acids Brought to you by • The nucleic acids are polynucleotide chains since the units from which nucleic acids are constructed are called nucleotides. • Each nucleotide consists of three components: – a nitrogenous base, either a purine or a pyrimidine – a pentose sugar, deoxyribose (DNA) or ribose (RNA) – a phosphate group Copyright 2010 American Society of Cytopathology Brought to you by Nucleotides • A nucleotide may have one of more phosphate groups – Example: adenosine with one phosphate is adenosine monophosphate (AMP); adenosine with 3 phosphates is adenosine triphosphate (ATP) • Nucleotides can be converted to nucleosides by hydrolysis Copyright 2010 American Society of Cytopathology Brought to you by Copyright 2010 American Society of Cytopathology Brought to you by Bases • The nitrogenous bases are of two kinds: purines and pyrimidines • Purines: – Two ring structures: fused five (pentamer)‐ and six (hexamer)‐membered rings • Adenine (amino purine) • Guanine (amino‐oxypurine) • Pyrimidines: – Single six‐membered ring structures • Cytosine (oxy‐aminopyrimidine) • Uracil (di‐oxypyrimidine) • Thymine (di‐oxy‐methylpyrimidine) Copyright 2010 American Society of Cytopathology Brought to you by Bases in DNA and RNA In DNA 2 oxy groups (ketone groups) Replaces thymine in RNA Pyrimindines Copyright 2010 American Society of Cytopathology Purines Nitrogenous Bases in DNA NH2 Brought to you by Double bond O NH2 group CH3 methyl group Copyright 2010 American Society of Cytopathology Nucleotide and Nucleoside Brought to you by • A base linked to a sugar and a phosphate is called a nucleotide – Ex. ATP (adenosine triphosphate); ADP (adenosine diphosphate)—energy is released when phosphates are removed • A base linked to a sugar is called a nucleoside – Attached sugar may be ribose (in ribonucleic acid or RNA) – Attached sugar may be deoxyribose in deoxyribose acid or DNA) Copyright 2010 American Society of Cytopathology Sugars in DNA and RNA Brought to you by • Both DNA and RNA have five‐carbon sugar • Deoxyribose in DNA or Ribose in RNA • Deoxyribose: – a monosaccharide containing five carbon atoms – includes an aldehyde functional group in its linear structure – it is a deoxy sugar derived from the pentose sugar ribose by the replacement of the hydroxyl group at the 2 position with hydrogen, leading to the net loss of an oxygen atom. Copyright 2010 American Society of Cytopathology Brought to you by Ribose and Deoxyribose Sugars Has one oxygen atom less; thus “deoxy” Copyright 2010 American Society of Cytopathology Brought to you by What are the differences between DNA and RNA? DNA RNA Base: Thymine Base: Uracil Sugar: Deoxyribose Sugar: Ribose Strands: Double stranded Strands: Single stranded Copyright 2010 American Society of Cytopathology Brought to you by How are the three subunits linked? • The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms in the sugar rings. • The point of attachment of the sugar to the base is the hydroxyl group on C‐1’ carbon atom. Copyright 2010 American Society of Cytopathology Brought to you by Each nucleotide consists of a five-carbon sugar, the first carbon of which is covalently joined to a nitrogen base and the fifth carbon to a triphosphate moiety Copyright 2010 American Society of Cytopathology Brought to you by DNA Complementary Base Pairing • Each type of base on one strand forms a bond with just one type of base on the other strand, called complementary base pairing • Purines form hydrogen bonds to pyrimidines – A bonding only to T – C bonding only to G • AT and GC are called base pairs • How the bases are arranged in the DNA determines the genetic code Copyright 2010 American Society of Cytopathology Brought to you by Structure of DNA • In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand • The asymmetric ends of a strand of DNA bases are referred to as the 5’ (five prime) and 3’(three prime) ends • The DNA double helix is held together by hydrogen bonds between the bases of the two strands – AT forms two hydrogen bonds – GC forms three hydrogen bonds Copyright 2010 American Society of Cytopathology Brought to you by Hydrogen Bonds Between Bases • Both the percentage of GC base pairs and overall length of a DNA helix determine the strength of the association between the two strands – Double‐stranded sequences rich in G and C hold together more strongly (thus, have a higher “melting point”) than sequences rich in A and T • Hydrogen bonds are not covalent, so they can be broken and rejoined relatively easily • The two strands of DNA can therefore be pulled apart like a zipper, either by a mechanical force or high temperature Copyright 2010 American Society of Cytopathology Brought to you by DNA Structure http://www.bae.uky.edu/~snokes/BAE549thermo/microbio/nucleicacids.htm. Accessed 6/23/10 Copyright 2010 American Society of Cytopathology DNA Double Helix Brought to you by Major groove Minor groove • As a result of asymmetries in base pairing, longer antiparallel DNA double strands will begin to twist • Appears to have alternating wide and narrow grooves called “major” and “minor” grooves • There are different 3‐D forms of DNA Buckingham, L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. Philadelphia: F.A. Davis Company, 2007. Copyright 2010 American Society of Cytopathology Brought to you by Forms of DNA Structure • B form – – – – – – Twists to the right, clockwise Helix makes a turn every 3.4 nm Neighboring base pair is 0.34 nm Intertwined strands make 2 groove based on widths Has major and minor grooves Usually found in vivo or in solution • A form – Twists to the right – Turns at 2.3 nm – Exists in alcohol or salt solution (dehydrated solutions) • Z form – – – – – Twists to the left Turns at 4.6 nm Exists in high salt or alcohol solution Has high CG contents Still controversial if it does exist in living cells Copyright 2010 American Society of Cytopathology Units of Measurement Brought to you by • The following abbreviations are commonly used to describe the length of a DNA molecule: – – – – bp = base pair(s) kb (= kbp) = kilo base pairs = 1,000 bp mb = mega base pairs = 1,000,000 bp gb = giga base pairs = 1,000,000,000 bp • When DNA is single stranded or in RNA the measurement is in number of nucleotides‐nt (base pair, pentose sugar & phosphate group) Copyright 2010 American Society of Cytopathology Brought to you by Chromosomes • A gamete has 1 copy of the genome, and is called haploid • Diploid organisms, inherit a haploid set of all their genes (23 chromosomes) from each parent; thus, humans have 2 copies of every gene except X & Y chromosomes • Euploid: Cell with a normal complement of chromosomes • Aneuploid: Cell with an abnormal complement of chromosomes (such as an increased number of chromosomes) Copyright 2010 American Society of Cytopathology Brought to you by http://picsdigger.com/image/a0bb3308/ Accessed 10/19/2010 Copyright 2010 American Society of Cytopathology Chromosome Brought to you by • A chromosome has the following components: – Centromere ‐ A constricted chromosome region to which spindle fibers attach during cell division – Telomere ‐ The ends of linear chromosomes that are required for replication and stability – p arm is the short arm of a chromosome – q arm is the long arm of a chromosome Copyright 2010 American Society of Cytopathology Brought to you by Histones and Nonhistones • If uncoiled, the DNA contained by each chromosome would be very long and difficult to fit into a cell • Chromosomal DNA is packaged into a compact structure with the help of specialized proteins called histones • The DNA plus histones together is called chromatin • The DNA double helix wraps around a central core of eight histone protein molecules (an octamer) to form a single nucleosome • The nonhistone protein molecules help the DNA molecule to condense further to fit into the chromosomes Copyright 2010 American Society of Cytopathology Brought to you by How the DNA is kept compact? http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/lamar/histoneh1.htm Accessed 10/19/2010 Copyright 2010 American Society of Cytopathology Brought to you by Histones • Five types of histones: – H1 (H5), H2A, H2B, H3, H4 • H1 and its homologous protein H5 are involved in higher order structures • Other 4 types of histones along with DNA forms nucleosomes • Each nucleosome consists of 146 bp DNA and 8 histones (2 pairs of each) • DNA is wrapped around the histone core • These histones contain lysine residues which provide a positive charge—which interacts well with the negative charged DNA creating a opposite bond • One way to weaken this bond is through acetylation. This may neutralize the positive charge and weaken the binding force between histones and DNA (This step does occur during transcription) Copyright 2010 American Society of Cytopathology Brought to you by Heterochromatin and Euchromatin • Heterochromatin: – – – – Tightly coiled, highly‐compacted DNA Less available for RNA transcription Genetically inactive Found only in eukaryotic (with nucleus) cells • Euchromatin: – – – – – Lightly packed, more open form of chromatin DNA not bound to a histone complex Rich in gene concentration Often under active transcription Found in eukaryotes and prokaryotes (cells without nuclei) Copyright 2010 American Society of Cytopathology Brought to you by Extrachromosomal Structures: Bacteriophage • • • • Bacteriophage genomes are mosaic Genome of any one phage species appears to be composed of numerous individual modules These modules may be found in other phage species in different arrangements Mycobacteriophages, bacteriophages with mycobacterial hosts, are excellent examples of mosaicism http://en.wikipedia.org/wiki/Bacteriophage#Genome_structure Copyright 2010 American Society of Cytopathology Brought to you by Extrachromosomal Structures: Plasmids • Plasmids are: – Extra circular genetic that can be passed from bacteria to bacteria (bacterial conjugation) – In biotechnology, plasmids are often used in recombination work • Some other organism’s gene is inserted into bacteria plasmid • Then, bacterial multiply and transcribe inserted gene into many useful products (vector) http://wiki.answers.com/Q/What_is_the_function_of_plasmid Copyright 2010 American Society of Cytopathology Example of Plasmid: Ti stands for Tumor inducing http://arabidopsis.info/students/paaras/t_dna.htm Brought to you by Extrachromosomal Structures: Mitochondrial • • • • • • • • Copyright 2010 American Society of Cytopathology Mitochondrial DNA (mt DNA or mDNA) Circular, covalently closed, double‐ stranded DNA located in mitochondria Converts the chemical energy from food into ATP Can be regarded as smallest chromosome (15,000‐17,000 bp Is inherited solely from mother Derived from circular genomes of bacteria that were engulfed by eukaryotic cells (endosymbiotic theory) Each mitochondrion estimated to contain 2‐10 mtDNA copies Brought to you by Extrachromosomal Structures: Yeast Artificial Chromosomes (YACs) • YACs are vectors • Used to clone DNA fragments larger than 100 kb and up to 3000 kb • Useful for cloning large genes and for mapping complex genomes http://www.accessexcellence.org/RC/VL/GG/YAC.php Copyright 2010 American Society of Cytopathology http://en.wikipedia.org/wiki/Yeast_artificial_chromosome Brought to you by Mass Spectrometry • Is an analytical technique • Measures the mass‐to‐charge ratio of charged particles • Used to determine: – masses of particles – Elemental composition of a sample or molecule – Chemical structures of molecules such as peptides and other chemical compounds Copyright 2010 American Society of Cytopathology Brought to you by Mass Spectrometry • How it works: – Ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass‐to‐charge ratios • MS instruments consist of: – An ion source – A mass analyzer (sorts ions by their masses by applying electromagnetic fields) – A detector (measures the value of an indicator quantity) Copyright 2010 American Society of Cytopathology