Cytogenetics Lecture Notes PDF

CYTOGENETICS LECTURE: Introduction to Cytogenetics DR. SARAH DY HINOGUIN-CASIO PRELIM 1ST SEMESTER – A.Y 2022-2023 INTRODUCTION TO CYTOGENETICS The Big Bang Theory proposes the followi...

CYTOGENETICS LECTURE: Introduction to Cytogenetics DR. SARAH DY HINOGUIN-CASIO PRELIM 1ST SEMESTER – A.Y 2022-2023 INTRODUCTION TO CYTOGENETICS The Big Bang Theory proposes the following: - The universe began as an extremely hot Branches of Clinical Pathology and dense dot only a few millimeters 1. Hematology – study of blood wide. 2. Blood Bank – blood transfusion - It since grew over 13.7 billion years into 3. Serology / Immunology – deals with the vast and cooler expanding cosmos antigen, antibody reactions 4. Microbiology – bacteriology etc. THE CREATION STORY 5. Clinical Microscopy – study of parasites and the rest of the cells 1st day heaven, Earth, light 6. Clinical Chemistry – chemical testing of all 2nd day firmament the fluids 3rd day water, dry land, grass, 7. Special Chemistry (CYTOGENETICS) herb-yielding seed, fruit Science was confirming that everything in the bible tree is really true. 4th day sun and moon (two great lights), stars GENETICS 5th day water and flying Genetics creatures - comes from the word genesis or beginning 6th day land creatures, male and - From Ancient Greek genetikos (genitive) & female genesis (origin) 7th day ended and rested. God - A discipline in biology blessed the seventh day - The science of heredity and variation in living and sanctified it. organisms. Origin & Significance THE EVOLUTION HYPOTHESIS  An explanation of how the genes came into being. 1. The fossil known as Java Man, discovered  Not uncommon to hear people responding in 1891, and that given the name Pekin to this question with false explanations Man, discovered in 1923, have both been which have roots in evolution and other non- exposed as false intermediate forms scientific teaching. 2. A single tooth discovered in 1922 and given  How it produced different races maybe the name Nebraska Man was puzzling… subsequently, in 1927, determined to have belonged to a wild boar CLINICAL LABORATORY MEDICINE 3. The skull discovered in 1912 and known as Focused on 2 subspecialties of Genetics: Piltdown Man was exposed as hoax in  Human Genetics – the study of heredity in 1953, have been placed on display for the man previous 40 years. Smith Wood  Medical Genetics – the study of human reconstructed the skull fragments and genetic variation of medical significance hypothesized that they belonged to a THE BIG BANG THEORY human. Georges Lemaitre 4. fossil referred to as Zinjanthropus, - A Belgian priest who first suggested the Big Bang discovered in 1956, was realized to have Theory in the 1920s when he theorized that the been just an ordinary ape universe began from a single primordial atom 5. Ramapithecus discovered in the 1930s The and exhibited as an intermediate form ALYSSA JENNIFER S. EGAR  for the next 50 years, was declassified with  In 1910, Thomas Hunt Morgan, argued the realization in 1981 that it was just an that genes are on chromosomes based on ordinary species of baboon observations of a sex-linked white eye 6. Research conducted in 1999-2000 showed mutation in fruit flies. that Lucy, discovered in Africa in 1974, was  In 1913, his student Alfred Sturtevant used also invalid the phenomenon in genetic linkage to show 7. When it was realized that the fossil skull that genes are arranged linearly on the known as the Taung Child, discovered in chromosome 1924, was that of a young gorilla, this was similarly removed from the list in 1954 CYTOGENETICS COMPARISON OF THE EVOLUTIONARY  The science that combines the methods AND CREATIONARY ORIGIN THEORIES and findings of cytology and genetics to allow the investigation of heredity at the Creation Hypothesis Evolution Hypothesis cellular level, through careful evaluation of Intelligent design Ape-like creature the chromosomes. The cell, in its complexity Darwin himself said that  Mendel established Genetics as a new field discloses a well-planed, “if the cell were more of science, around 16 years before the and well-programmed complex that it appears, discovery of chromosomes (1866), individual & collaborative then, his whole theory is making cytogenetics as one of the oldest system wrong” fields of genetics. Consistent in its With missing links  After the turn of 19th century, the developmental theory importance of sex chromosomes was Death has to be Denies origin in order to overcome deny death established. Creator of life takes over Uses genetic studies to  1959 - Cytogenetic studies were first utilized creature’s sad fate avoid death in clinical laboratory studies, making a Assigned billions of years instead of days major advancement in clinical diagnosis: 1. The ability to detect changes in Francisco Ayala chromosome structure, - “On the basis of only 6.7% heterosity, the 2. Direct correlation of these changes to average human couple could have 10 children disease & phenotypic anomalies in before they would have to have one child identical individuals. to another  Many of tests have become the “gold standard” for diagnosis MEDICAL GENETICS Clinical Fields  There is a growing number of disease genes being cloned. There - Clinical Genetics is increasing emphasis on - Genetic Counseling developing molecular direct mutation analyses. Laboratory Sciences - Cytogenetics  These analytical studies work well - Molecular Genetics when a diagnosis is known or - Biochemical Genetics suspected.  In the absence of a known disorder, MODERN SCIENCE OF GENETICS cytogenetics retains its position as the only clinical laboratory test to be  Seeks to understand the process of able to survey genetic cellular inheritance. constitution of an individual with a  Began with the work of Gregor Johann single assay. Mendel, a German-Czech Augustinian  Clinical applications for cytogenetic monk & scientist (mid-nineteenth century). analysis = range from prenatal  Mendel “observed that organisms inherit diagnosis to cancer evaluation traits via discrete units of inheritance, which are now called genes.” ALYSSA JENNIFER S.EGAR  CYTOGENETICS LECTURE: Cell and Cell Division DR. SARAH DY HINOGUIN-CASIO PRELIM 1ST SEMESTER – A.Y 2022-2023 THE CELL Protoplasm – the living substance of the cell - subdivided into 2 compartments: a) cytoplasm b) karyoplasm Cytoplasm – the bulk is water - suspended & or dissolved therein are various inorganic & organic chemicals Cytosol – the fluid suspension or undissolved portion in the cytoplasm - contains organelles Cytoskeleton a) microtubules b) microfilaments - this system maintains the: a) shapes of cells b) ability to move c) intracellular pathways within cell Organelles CELLS – basic functional unit of any living thing. - metabolically active cellular structures that TISSUES - similar cells that are grouped together, execute or perform specific & distinctive and function to serve a common purpose. functions Inclusions Four Basic Tissues: - by-products of metabolism a) Epithelium c) Muscle - storage forms of: b) Connective Tissues d) Nervous a) various nutrients b) inert crystals These four basic tissues are assembled to form c) pigments ORGANS. Membranes - serves as boundary of the cell (cell Organs are collected into ORGAN SYSTEMS, to membrane) perform specific task, performing a collection of - partitions the cell into compartments: associated a) nucleus b) organelles functions: digestion, reproduction, respiration - made up of phospholipid bilayer - provides large surface areas for human body composed of >200 different types biochemical of cells - reactions essential for maintenance of life All cells possess certain unifying & common characteristics: every cell FUNCTIONS A. Surrounded by a bilipid plasma membrane CELL MEMBRANE B. Possesses functioning organelles - also called plasma membrane or C. Synthesizes macromolecules for its own plasmalemma use or for export Cell Membrane Functions: D. Produces energy a) maintaining structural integrity of cell E. Capable of communicating with other cells. ALYSSA JENNIFER S.EGAR b) acts as interface between cytoplasm & external observed only during interphase milieu because itdissipates during cell b) controlling movements of substances in & out of division. the cell (selective permeability) - no more than 2-3 nucleoli in each cell c) regulating cell-cell interactions - contains only small amount of DNA, d) recognition (via receptors) of antigens, and which is also inactive foreign cells as well as altered cells - become hypertrophic in malignancy - located in the pale-staining fibrillar Protein Synthetic and Packaging Machinery of center: the cell: - tips of chromosomes 13, 14, 15, 21, 22 a) ribosomes (in humans), containing the nucleolar- b) rough endoplasmic reticulum (RER) organizing regions (NORs), where gene c) Golgi apparatus loci that encode rRNA are located Smooth Endoplasmic Reticulum: - abundant only in cells whose active THE CELL CYCLE functions are: - series of events that prepare the cell for a) synthesis of steroids dividing into 2 daughter cells b) synthesis of cholesterol & triglycerides  2 major events: c) detoxification of toxic materials (alcohol a) interphase = occupies most of the cell’s &barbiturates) “life”; a longer period of time during which Lysosomes the cell: - have an acidic pH & contain hydrolytic b.1) increases its size & content enzymes b.2) replicates its genetic material Mitochondria b) mitosis - the short period of time during - possess their own DNA and perform which the cell divides its nucleus & oxidative phosphorylation and lipid cytoplasm may last only 10 - 20 hours synthesis Cytoskeleton 3 major components: a) filaments: thin - cellular movement intermediate - maintenance of 3-dimensional framework of cell b) microtubules - act as intracellular pathways Centrioles - small cylindrical structures - composed of 9 microtubule triplets - usually paired, arranged perpendicular to each other - located in the microtubule organizing center, known as centrosome Nucleus Interphase - largest organelle of the cell - subdivided into 3 phases: - houses 3 major components: a. G1 (gap) phase a) chromatin, the genetic material of the - when synthesis of macromolecules cell essential for DNA duplication begins. b) nucleolus, center for ribosomal RNA - time when the cells are performing their (rRNA) synthesis assigned tasks, metabolizing, c) Nucleoplasm synthesizing, etc. Nucleolus triggers the cell to begin a cell division event: - deeply staining a) To replace dead or dying cells, - non-membrane bound structure b) To produce more cells to enlarge the organism (growth and development) ALYSSA JENNIFER S.EGAR c) Reproduction, i.e., to increase the number of unicellular organisms. b. S (synthetic) phase - when the DNA is duplicated - therefore, from single stranded DNA in G1 phase to double stranded DNA in G2. c. G2 phase Pro Metaphase - preparations for mitosis - begins when nuclear envelope - e.g., tubulin is synthesized, the protein disappear used to manufacture & microtubules of - microtubules attached to kinetochore, the spindle apparatus in prophase of called mitotic spindle microtubules mitosis. - microtubules that do not become incorporated into spindle apparatus, the Cells that become highly differentiated after the polar microtubules. last mitotic event, may cease to undergo - mitotic spindle microtubules assist in mitosis, leave the cycle to enter the G0 phase migration of the chromosomes so that (resting stage / outside or stable phase) either: they become oriented into an alignment a) permanently – neurons, muscles with the mitotic spindle. b) temporarily – peripheral lymphocytes Metaphase - begins when newly duplicated Mitosis (M) chromosomes align themselves on the - occurs at the conclusion of G2 phase equator of the mitotic spindle – and thus completes the cell cycle. metaphase plate configuration. 1. Karyokinesis - nuclear division - chromosomes become maximally 2. Cytokinesis - cytoplasmic division condensed - spindle microtubules attached to the Mitosis 5 distinct stages: kinetochores a) prophase d) anaphase b) prometaphase e) telophase c) metaphase Prophase - chromosomes condensed to become visible microscopically. - chromosome consists of 2 parallel sister chromatids, joined together at 1 Anaphase point (centromere) - begins when sister chromatids separate - as chromosomes condense, nucleolus & begin to migrate to opposite poles disappears spindle – kinetochore attachment site - centrosome divides, each contains a leads the way with arms of pair of centrioles & a microtubule- chromosome simple trailing, organizing center (MTOC) contributing nothing to the migration or - from each MOTC, the following its pathway. develop: - as result of shortening of microtubules a) astral rays – assist in orienting the via depolymerization at the kinetochore MOTC at the pole end. b) spindle fibers – assist in directing - Late anaphase – cleavage furrow the chromosome migration to the pole. begins to form at the plasmalemma. - at the centromere region of each chromatid, new MOTC develops, “kinetochore” - spindle fibers attach to kinetochore in preparation for migration ALYSSA JENNIFER S.EGAR Telophase - lasts a long time & subdivided into: - terminal phase of mitosis 1) Leptotene – individual chromosomes, - characterized by: composed of 2 chromatids joined at a) cytokinesis – division of cytoplasm into 2 centromere, begin to condense, forming equal parts long strands in nucleus. b) reconstitution of the nucleus & nuclear 2) Zygotene – homologous pairs of envelope chromosomes approximate each other, c) disappearance of mitotic spindle lining up in register (gene locus to gene d) unwinding of chromosomes into locus), and make synapses via chromatin synaptonemal complex, forming a tetrad e) nucleolus developed from the NORs (nucleolar organizing region) on each of the five pairs of chromosomes. 3) Pachytene - chromosomes continue to condense, becoming thicker and shorter, chiasmata (crossing over sites) are formed as random exchange of genetic material occurs between homologous chromosomes. 4) Diplotene – chromosomes continue to condense and then begin to separate, revealing chiasmata. 5) Diakinesis – chromosomes condensed maximally; nucleolus & nuclear envelope disappears, freeing the chromosomes. MEIOSIS - special type of cell division resulting in the Metaphase I formation of gametes or germ cells – ova & - homologous chromosomes align as pairs, spermatozoa. on the equatorial plate of the spindle - 2 crucial results: apparatus in random order, ensuring a a) Reduction of chromosomal number from the subsequent reshuffling of maternal & diploid (2n) to the haploid (1n) number; paternal chromosomes. ensuring that each gamete carries the Anaphase I haploid amount of DNA - homologous pairs of chromosomes begin to & Chromosomes pull apart, & migrate to opposite poles. b) recombination of genes, ensuring genetic - each chromosome still consists of 2 variability & diversity of the gene pool. chromatids Telophase I Two separate events / steps in Meiosis: - similar to Telophase of Mitosis. a) Meiosis I (Reductional Division) 1) homologous pairs of chromosomes line up 2) members of each pair separate and go to opposite poles 3) cell divides 4) daughter cell receives half the number of chromosomes In gametogenesis, when the germ cells are in the S phase of the cell cycle preceding meiosis, - the amount of DNA is doubled to 4n, and the chromosome number is also doubled to 4n. Prophase I - the commencement of meiosis, begins after DNA has been doubled to 4n Two separate events / steps in Meiosis: ALYSSA JENNIFER S.EGAR b) Meiosis II (Equatorial Division) - occurs without DNA synthesis - proceeds rapidly through four phases & cytokinesis, to form 4 daughter cells, each with the haploid chromosome number. COMPLETE CYCLE ALYSSA JENNIFER S.EGAR  CYTOGENETICS LECTURE: Inheritance DR. SARAH DY HINOGUIN-CASIO PRELIM 1ST SEMESTER – A.Y 2022-2023 INHERITANCE d. The two genes for each character segregate - The patterns governing how genetic during gamete production information is transmitted from generation to generation are collectively known as the 3. Law of Independent Assortment principles of inheritance - Alleles of different genes pass randomly to - Inheritance is not subtyping offspring - In the gametes , alleles of one gene Austrian Monk, creationist Gregor Mendel (father separate independently of those another of Cytogenetics) gene , and thus all possible combinations of - Mendel’s Laws (1865) went unnoticed until alleles are equally probable. 35 years later , when they were - The emergence of one trait will not affect simultaneously and independently the emergence of another discovered by Hugo De Vries in the - Mendel concluded that each organism Netherlands , Erich Von Tschermak in carries two copies of its expressed Austria , and Carl Correns in Germany phenotype. If one differs from the other on the same phenotype one will inevitably Mendel’s three laws or Basic Principles of dominate the other. Heredity: This law was later found to have important 1. Law of Dominance (Unit Inheritance) exceptions: If two genes are very close to each - Each trait is determined by two factors other on a chromosome, they tend to be passed (alleles), inherited one from each parent. down together. Each of these factors exhibit a characteristic dominant, that are dominant will mask the GENOTYPE expression of those that are recessive - The genetic make-up of an organism 2. Law of Segregation - The two alleles of a gene are never PHENOTYPE transmitted together from one parent to - The external appearance of an organism an offspring. This means that , in humans , caused by genotype an individual egg or sperm is form with only one allele of each gene GENE POOL - Each of the two inherited factors (alleles) - All of the genes and their alleles present in possessed by the parent will segregate a population and pass during meiosis into separate gamete (eggs or sperm), which will carry GENOME only one of the factors. - Entire genetic material of an organism This specific law has four parts, which are: HYBRID a. Alternative versions of genes, or alleles - Receive different alleles for a trait from each account for variations in inherited parent characteristics b. For each characteristic or trait an organism GENES inherits two alternative forms of that gene, - Composed of DNA (deoxyribonucleic acid), one from each parent whose building blocks , the nucleotides , c. If the two alleles differ , then one , the code for the multitude of proteins in the dominant allele , is fully expressed in the human body , including enzymes and organism’s appearance. The other , the structural proteins recessive allele has no noticeable effect on the organism’s appearance. ALYSSA JENNIFER S.EGAR  - In 2001 , estimates place the number of human protein-coding genes between 25,000 and 35,000 GENOTYPE - Subset of alleles of an individual PHENOTYPE - Characteristics of an individual Female reproductive cell or ovum bringing the haploid then the other cell comes from the Male reproductive cell or the father forming a zygote making an individual who has a mixture of two alternative alleles ALLELES - Different forms of a trait; one form of a gene - Example of traits: eye color, skin color, height, hair texture (most traits are determined by multiple genes with multiple alleles) - Examples of alleles: brown or blue , albino In figuring out what the offspring “look-like” or pigmented , tall or short, curly or straight 1. Dominant Why are genes and how are they passed along? - the fully expressed gene, has - Coiled until they become “sausage-like” control on the phenotype structures called chromosomes - denoted by a capital letter 2. Recessive - gene is completely masked in the phenotype - Locus: location of a specific gene on a - denoted by a lower-case letter chromosome - Allele: alternative version of a gene An easier way to figure out what the offspring “look-like” 1. Punnett Square ALYSSA JENNIFER S.EGAR  75% - attached earlobes 25% - unattached earlobes Proportion: 3:1 Example: - Mother (Aa) x Father (Aa) 1. What are the genotypes of the a. Genotype offspring? - Two of the same is called 2. If the dominant gene is attached homozygous earlobes and the recessive gene - Different is called heterozygous is unattached earlobes , what are the phenotype/s of the APPLICATION TO BLOOD TYPES offspring? Antigen on surface – gives the person his blood phenotype Floating around – antibodies Group A Group B Group AB Group O Red Blood A B AB O Cell Type Antigens A antigen B antigen A and AB None in Red antigen Blood Cell Antibodies Anti-B Anti-A None Anti-A in Plasma and Anti- B ABO BLOODTYPES: - O – Recessive - A, B – dominant - AB – co-dominant Genotype Phenotype Antigen Anti-bodies AO Type A A Anti-B (Heterozygous A) AA(Homozygous Type A A Anti-B A) BO(Heterozygous Type B B Anti-A B) BB(Homozygous Type B B Anti-A B) OO Type O - Anti-A & Anti-B AB Type AB A&B - Additional concepts about inheritance that need to be added to cover other traits: - A gene on one of the 22 pairs of autosomes that is, the non-sex- determining chromosomes – is called an autosomal gene ALYSSA JENNIFER S.EGAR  - Similarly, a trait or disease associated with that gene is an autosomal trait - Autosomal conditions are the most common and are equally likely to occur in males or females. Autosomal traits are further classified as either dominant or recessive. 1. Autosomal Dominant Inheritance - Autosomal Dominant Traits are 5. X-linked recessive inheritance those in which a single copy of an - Expressed in allele is enough for the trait to be a. Males (who are necessarily expressed or shown in the hemizygous for the one gene phenotype of the animal mutation because they have - Dominant conditions are those that only one X chromosome ; are expressed in heterozygotes b. Females who are homozygous 2. Autosomal Recessive Inheritance for the mutation - Autosomal recessive traits require - Carrier females do not usually that individual have 2 copies of express phenotype the trait to express the phenotype 6. Mitochondrial Inheritance - The genes for autosomal-recessive - Refers to the additional genes in traits are also located on the cell’s mitochondria autosomes , but mutant , disease- - Mitochondria are almost exclusively causing alleles are recessive to the passed from parent to child in the normal alleles; thus, if one normal egg and not in the sperm , a allele is present , it is usually hallmark of mitochondrial sufficient to prevent any expression inheritance is transmission from an of the disease affected woman to all of her children 3. X-Linked Inheritance - Females have two X chromosomes and males have an X and Y (XY) - Smaller Y chromosomes has only a very few genes as compared to the larger X - Often referred as sex-linked- inheritance - A distinguishing feature is the lack of male-to-male transmission, because a father transmits only his Y chromosome, and not his X to his sons 4. X-linked Dominant Inheritance - Works differently depending upon whether the mother or father is the carrier of a gene that causes disease or disorder - All daughters of an affected father will also be affected but none of his sons will be affected (unless the mother is also affected) - In addition , the mother of an affected son is also affected (but not necessarily the other way around) ALYSSA JENNIFER S.EGAR  CYTOGENETICS LECTURE: DNA-RNA DR. SARAH DY HINOGUIN-CASIO PRELIM 1ST SEMESTER – A.Y 2022-2023 DNA-RNA the right place, another to get rid of the protein when it’s done its job, and the list DEOXYRIBONUCLEICACID (ACID) goes on and on. Contained in the nucleus - Proteins to be packaged are synthesized on - Found in every cell of our body and contains the RER surface, whereas proteins all the information that makes you, “you”. destined for the cytosol are manufactured - If extracted and stretched out, a person’s within the cytosol. entire DNA would be enough to get to the - The information for the primary structure of moon and back 13 times a protein (sequence of amino acids) is - In each cell there are 46 chromosomes housed in the DNA of the nucleus. - Tightly coiled DNA form chromosomes NUCLEUS - they like to hang around in pairs – you get half from your mother and half from your - Contains nearly all of the DNA possessed by the cell, as well as the mechanisms for father. RNA synthesis. - Chromosomes can be divided into sections known as genes. CHROMATIN - DNA is made up of two (2) strands that stick - Complex of DNA and proteins together to make a double helix. - Represents the relaxed, uncoiled - The information coming from the DNA is chromosomes of the interphase nucleus. transcribed into a strand of mRNA - The sequence of codons of the mRNA Types of Chromatins: represents the chain of amino acids, in 1) Heterochromatin which each codon is composed of 3 - Condensed inactive form; visible with light consecutive nucleotides. microscope - Periphery of the nucleus 2) Euchromatin - Lighter, non-visible portions of the nucleus - Represents the active area, where the genetic material of the DNA molecules is being transcribed into RNA - When euchromatin is examined under the electron microscope, if unwound, looks like beads on a string of pearls on a necklace ✓ Beads – nucleosomes ✓ String – DNA molecule: filament with GENES 2nm in diameter - are like recipes for the cell (they tell it how to make proteins) PROTEIN - you have a protein to do everything in your body - for example, one to make a colored pigment for your eyes, another to protect that protein so that it can do its job, another to direct it ALYSSA JENNIFER S.EGAR DNA  Each represents a specific segment of the - Acts as a template for RNA transcription DNA molecule that codes for the synthesis - Double-stranded polynucleotide chain of a particular protein. wound into a double helix  The sequential arrangement of bases - A double helix is established by the constituting the gene represents the formation of hydrogen bonds between sequence of amino acids of the protein. complimentary bases on each strand of DNA molecule GENETIC CODE - Is designed in such a manner that a triplet NUCLEOTIDE of consecutive bases – codon – denotes a - Are linked to one another by particular amino acid. phosphodiester bonds (a type of covalent - Each amino acid is represented by a bond) formed between the sugar molecules. different codon - More or less 100,000 genes & 3 billion Each nucleotide is composed of: nucleotide bases. a) A nitrogenous base b) A deoxyribose sugar [pentose (5 C sugar)]; DEOXYRIBONUCLEIC ACID (ACID) and - Contained in the nucleus c) A phosphate molecule - Single-stranded NITROGENOUS BASES - The sequence of the 5 – nitrogenous bases, determines unique structure of DNA and RNA. - Nitrogenous base sticks out of the strand - N-bases connect with another sugar- phosphate backbone strand Two (2) types of Bases: Pyrimidines (6-member ring) - A pyrimidine always connects to a purine - Adenine with thymine (A – T) [2 bonds] - they are complementary to each other (5’ end – 3’ end) - Guanine with cytosine (G – C) [3 hydrogen bonds] ✓ Cytosine ✓ Thymine (DNA only) ✓ Uracil (RNA only) Purines (6-member ring + 5 ring) - Adenine - Guanine POLYNUCLEOTIDE - Formed by bonds between sugar and phosphate (sugar-phosphate backbone) GENES -Biological information that is passed from one cell generation to the next “units of RIBONUCLEIC ACID (RNA) heredity” - Like DNA: polynucleotide with nitrogenous bases sticking out of sugar-phosphate  Located in specific loci (locus for singular) backbone strand.  Located at specific regions on DNA - RNA exists as a single sugar phosphate molecule backbone strand. ALYSSA JENNIFER S.EGAR  - Uracil, replaces thymine, and is - A 5s rRNA molecule – synthesized in the complimentary to adenine nucleus + ribosomal proteins, synthesized - Sugar is ribose instead of deoxyribose. in the cytoplasm a. Transported into the nucleolus b. Associate with 45s rRNA molecule, Synthesis of the 3 types of RNA is catalyzed by forming a very large ribonucleoprotein 3 different RNA polymerases: particle. a. Messenger RNA (mRNA) by RNA polymerase II b. Transfer RNA (tRNA) by RNA DNA polymerase III - Acts as template for RNA transcription c. Ribosomal RNA (rRNA) by RNA polymerase I Note: mechanism of transcription is generally the same for all 3 types of RNA. Messenger RNA (mRNA) - Carries the genetic code from the nucleus to the cytoplasm to act as a template for protein synthesis - Each mRNA is a complimentary copy the region of the DNA molecule that constitute 1 gene - Consists of a series of codons - Also has: a) Start codon (AUG) b) Stop codons (UAA, UAG, UGA) - Once formed, transported to cytoplasm and translated to protein. Transfer RNA (tRNA) - Ferries activated amino acids to the ribosome/mRNA complex, resulting in the formation of protein - About 80 nucleotides in length - Folded upon itself to resemble a cloverleaf with base pairing between some nucleotides. Two (2) significant regions in the tRNA: a) Anticodon – recognizes the codon of mRNA b) Amino acid-bearing region – 3’ end of molecule RIBOSOMAL RNA (Rrna) - Forms associations with proteins and enzymes in the nucleus to form ribosomes - Synthesized in the fibrillar (pars fibrosa) region of the nucleolus - The primary transcript is called 45S rRNA (pre-RNA), a huge molecule of about 13,000 nucleotides. ALYSSA JENNIFER S.EGAR  CYTOGENETICS LECTURE: DNA Replication DR. SARAH CASIO PRELIM 1ST SEMESTER – A.Y 2022-2023 DNA REPLICATION chains, each of them with parts of both - One major question for the human mind is parent and daughter molecules how does life continues - One of the most important mechanisms for  The correct model is the all life cells to give off springs is semiconservative DNA Replication undoubtedly the DNA which was proved by the experiment - Prior to cell division, the DNA material in the of Meselson-Stahl. original cell must be duplicated so that after cell division, each new cell contains the full RECAP amount of DNA material 1. Nucleotides are monomers that are made - The process of DNA duplication is usually of a phosphate, a sugar (deoxyribose) called replication and a heterocyclic base (Thymine, - DNA Replication answers to the question: Cytosine – pyrimidines – Adenine, “When a cell divides, where does the Guanine-purine) extra DNA come from?” 2. The combination of a sugar and a - DNA Replication is the process whereby an heterocyclic base gives a nucleoside. entire double-stranded DNA is copied to When a phosphate is added to the produce a second, identical DNA double molecule, a nucleotide is created. helix 3. The phosphate of a nucleotide binds both - This is how DNA Replication or DNA to the “5 carbon of one deoxyribose and Synthesis succeeds. the 3 carbon of next deoxyribose. This is - Replication of DNA begins at a replication how the nucleotides create the strand. origin 4. The strands of a DNA double helix are - Bacterial and viral DNA have only one antiparallel which means that one chain replication origin, whereas eukaryotic DNA runs in 5’-3’ and the other runs 3’-5’ has many sited where DNA synthesis 5. The two strands are complementary begins simultaneously  Connection happens between the - Replication origins are spaced between adenines and thymine (2 hydrogen 30,000 to 300,000 nucleotides from each bond) and between cytosines and other. guanines (3 hydrogen bond) 6. And thus, each of them can be a matrix There are three possible models that describe for the creation of a new complementary the accurate creation of the daughter chains: and antiparallel strand 7. The DNA Double Helix makes a 1. Semiconservative Replication complete turn in over 10 nucleotide - DNA replication would create two pairs. molecules. Each of them would be a 8. About 25 hydrogen bonds are created complex of an old (parental and a daughter in this complete turn. strand) 9. The power of these 25 bonds is equal to 2. Conservative Replication 1 covalent bond (bond between carbon - According to this model, the DNA and oxygen) Replication process would create a brand new DNA double helix made of two DNA REPLICATION daughter strands while the parental chains - The original polynucleotide strand of DNA would stay together. serves as a temple to guide the synthesis of 3. Dispersive Replication the new complementary polynucleotide of - According to this model the replication DNA process would create two DNA double- ALYSSA JENNIFER S.EGAR  - DNA replication is an intricate process requiring the concerted action of many different proteins & several enzymes - The replication proteins are clustered together in particular locations in the cell Enzymes of DNA Replication 1. Helicase – Unwound a portion of the DNA Double Helix 2. RNA Primase – Attaches RNA primers 2. One of the most important steps of DNA to the replicating strands Replication is the binding of RNA Primase 3. DNA Polymerase delta (ä) – Binds to in the initiation point of the 3’-5’ parent the 5’ and 3’ strand in order to bring chain. nucleotides and create the daughter  RNA Primase can attract RNA leading strand nucleotides which bind to the DNA 4. DNA Polymerase epsilon (å) – Binds nucleotides of the 3’-5’ strand due to the to the 3’-5’ strand in order to create hydrogen bonds between the bases discontinuous segments starting from  RNA nucleotides are the primers for different RNA primers binding DNA nucleotides 5. Exonuclease (DNA Polymerase I) - Finds and removes the RNA Primers 6. DNA Ligase – adds phosphate in the remaining gaps of the phosphate-sugar backbone 7. Nucleases – remove wrong nucleotides from the daughter strand Steps in DNA Replication 3. The elongation process is different for the 5’-3’ and 3’-5’ template  5’3 template: The 3’-5’ proceeding daughter strand that uses a 5’-3’ template is called leading strand Because DNA Polymerase ä can “read” the template and continuously adds nucleotides complementary to the nucleotides of the template 1. The first major step in DNA Synthesis is the breaking of hydrogen bonds between bases of the two antiparallel strands  The splitting happens in places of the chains which are rich in A-T, because there are only two hydrogen bonds  Helicase is the enzyme that splits the two strands  The initiation points where the splitting starts is called “origin of replication”.  3’-5’ template: The 3’-5’ template The structure that is created is known as cannot be “read” by DNA Polymerase ä “Replication Fork”  The new strand is called lagging strand ALYSSA JENNIFER S.egAR   In the lagging strand the RNA  So, the end of the parental strand Primase adds more RNA Primers where the last primer binds isn’t  DNA Polymerase å reads the replicated. template and lengthens the bursts.  These ends of linear (chromosomal) The gap between two RNA DNA consists of noncoding DNA that primers is call “Okazaki Fragments” contains repeat sequences and are  The RNA Primers are necessary called telomeres for DNA Polymerase å to bind  As a result , a part of the telomere is nucleotides to the 3’ end of them removed in every cycle of DNA  The daughter strand is elongated Replication with the binding of more DNA nucleotides 6. The DNA Replication is not completed before a mechanism of repair fixes possible errors caused during replication  Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps 4. In the lagging strand, the DNA Pol I – exonuclease – reads the fragments and removes the RNA Primers.  The gaps are closed with the action of DNA Polymerase (adds complementary nucleotides to the gaps) and DNA Ligase (adds phosphate in the Speed of DNA Replication remaining gaps of the phosphate -sugar backbone) - The genome of complex eukaryotes is  Each new double helix is consisted of huge and the process of DNA Replication one old and one new chain. This is what should be incredibly fast we call semiconservative replication. - A chromosome of 250 million pair of bases can be replicated in several hours - The speed of DNA replication for the humans is about 50 nucleotides per second per replication fork - The human genome can be copied only in a few hours because many replication forks take place at the same time (multiple initiation sites) - But the speed of DNA replication in bacteria is much longer (about 1000 nucleotides per 5. The last step of DNA Replication is the second) Termination - That is a reason why during the process of  This process happens when the DNA bacterial replication the rate of errors is Polymerase reaches to an end of the much higher strand  In the last section of the lagging strand, when the RNA Primer is removed, it is not possible for DNA Polymerase to seal the gap (because there is no primer) ALYSSA JENNIFER S.egAR ALYSSA JENNIFER S.egAR  CYTOGENETICS LECTURE: DNA Transcription DR. SARAH DY HINOGUIN-CASIO MIDTERM 1ST SEMESTER – A.Y 2022-2023 DNA TRANSCRIPTION Proteins are important molecules in During transcription, a DNA sequence is organisms because they carry out so many read by RNA polymerase, which different jobs, including the production of produces a complementary, antiparallel the other molecules. RNA strand. As opposed to DNA The information for making proteins is Replication, transcription results in an carried in each cell’s DNA, where the RNA complement that includes uracil(U) sequence of bases in a gene encodes what instead of thymine(T). sequence of amino acids should be joined to make a protein The sequence of amino acids joined together folds into a specific shape, which allows the protein to carry out its specific - although RNA polymerase performs job. transcription, the enzyme needs assisting Protein Synthesis is a two-step process: proteins to help initiate and produces the transcript 1. Transcription: a temporary copy of - These factors either associate directly with information in RNA is made using DNA as RNA Polymerase or add in building the a template actual transcription complex apparatus to 2. Translation: the base sequence of mRNA is help regulate the appropriate transcript decoded to make a sequence of amino - The general term for such associated acids proteins are Transcription Factors  To save energy, transcription and - The stretch of DNA transcribed into an translation of a particular gene (information RNA molecule is called a transcription for a protein in DNA) are only carried out unit and encodes at least one gene when a particular protein is needed. - If the gene transcribed encodes for a protein, the result of transcription is messenger RNA (mRNA) which will then be used to create that protein via the process of translation - Alternatively, the transcribed gene may encode for either ribosomal RNA (rRNA) or transfer RNA (tRNA) , other components of the protein-assembly process, or other ribosomes. - A DNA Transcription unit encoding for a protein contains not only the sequence that will eventually be directly translated into the protein ( the coding sequence) TRANSCRIPTION - But also regulatory sequences that - The synthesis of RNA from a DNA template directly and regulate the synthesis of that - The process of creating an equivalent RNA protein copy of a sequence of DNA - The regulatory sequence before - The first step leading to gene expression (upstream) the coding sequence is called the coding sequence is called the five prime untranslated region (5’UTR) Alyssa Jennifer Egar | MEDICAL LABORATORY SCIENCE  - The sequence following (downstream) the - RNA polymerase is able to bind to core coding sequence is called the three prime promoters in the presence of various untranslated region (3’UTR) specific transcription factors - As in DNA Replication , DNA is read from - The most common type of core promoter: 3’-5’ during transcription, while the TATA box (short DNA sequence), found - complementary RNA is created from the 30 base pairs from the start site of 5’-3’ direction transcription - Although DNA is arranged as two - The TATA box, as a core promoter, is the antiparallel strands in a double helix , only binding site for a transcription factor known one of the two DNA Strands, called the as TATA Binding Protein (TBP), which is template strand , is used for itself a subunit of another transcription transcription factor, called Transcription Factor IID - The other DNA strand is called the coding (TFIID). strand, because its sequence is the same - After TFIID binds to the TATA Box via as the newly created RNA transcript the TBP, five more transcription factors (except for the substitution of uracil for and RNS polymerase combine around the thymine) TATA box in a series of stages to form a - The use of only the 3’-5’ strand eliminates pre-initiation complex the need for the Okazaki fragments seen in - One transcription factor, DNA Helicase, is DNA replication involved in the separation of opposing strands of double-stranded DNA to provide Transcription is divided into five stages: access to a single-stranded DNA template. 1. Pre-initiation - However, only a low, or basal, rate of 2. Initiation transcription is driven by the pre-initiation 3. Promoter clearance complex alone 4. Elongation - Other proteins known as activators and 5. Termination repressors, along with any associated coactivators or corepressors, are responsible for modulating transcription rate. INITIATION - Binding of the RNA polymerase (RNAP) and association of the initiation complex to promoter and enhancer regions on double- MAJOR STEPS stranded DNA - RNA polymerase does not directly PRE-INITIATION recognize the core promoter sequences - RNA polymerase requires the presence of - Transcription factors mediate the binding a core promoter sequence in the DNA for of RNA polymerase and the initiation of the initiation of transcription transcription - Promoters are found at -30,-75, and -90 - Only after certain transcription factors are base pairs upstream from the start site of attached to the promoter does the RNA transcription polymerase bind to it. The completed - core promoters = sequences within the assembly form the transcription initiation promoter essential for transcription complex initiation  PROMOTER CLEARANCE appropriate RNA editing factors to bind; - After the first bond is synthesized , the may intrinsic to the RNA polymerase or due RNA polymerase must clear the promoter to chromatin structure. - During this time there is a tendency to TERMINATION release the RNA transcript and produce - Recognition of the transcription truncated transcripts , abortive initiation. termination sequence and the release of - Abortive initiation continues to occur until RNA polymerase and the newly formed the sigma factor rearranges, resulting in RNA polymer. the transcription elongation complex(which gives 35 base pairs moving footprint) - The sigma factor is released before 80 nucleotides of mRNA are synthesized. - Once the transcript reaches approximately - Bacteria use two different strategies for 23 nucleotides, it no longer slips and transcription termination elongation can occur. - In Rho-independent transcription - This , like most of the remainder of termination, RNA transcription stops when transcription , is an energy-dependent the newly synthesized RNA molecule forms process, consuming adenosine a G-C rich hairpin loop followed by a run triphosphate (ATP) of US, which makes it detach from the DNA - Process coincides with phosphorylation by template. TFIIH of serine 5 on the carboxy terminal - In the “RHO-dependent” type of domain of RNAP. termination, a protein factor called “RHO” destabilizes the interaction between the ELONGATION template and the mRNA, thus releasing the - Covalent addition of ribonucleic bases to newly synthesized mRNA from the the 3’ end of a growing chain pairing with a elongation complex. single stranded DNA template. The transcription termination in - eukaryotes is less understood but involves cleavage of the new transcript followed by template-independent addition - of As at its new 3’ end, in - process called - Using the template strand (or noncoding polyadenylation. strand) as a template, RNA molecule is produced from 5’ to 3’, an exact copy of the coding strand MEASURING AND DETECTING - Difference: Thymine is replaced with TRANSCRIPTION Uracil, and the nucleotides are composed - electron micrograph of the ribosomal of a ribose(5-carbon) sugar where DNA transcription process has deoxyribose (one less oxygen atom in - the forming of mRNA strands are visible as its sugar-phosphate backbone) branches from the main DNA strand - Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases Transcription can be measured and detected in on a single DNA template and multiple s variety of ways: rounds of transcription (amplification of 1. Nuclear Run-on Essay: measures the particular mRNA), so many mRNA relative abundance of newly formed molecules can be rapidly produced from a transcripts single copy of a gene 2. RNase protection assay & Chip-Chip of - Elongation also involves a proofreading RNAP: detective active transcription sites mechanism that can replace incorrectly 3. RT-PCR: measures the absolute incorporated bases – short pause; allow abundance of total or nuclear RNA levels,  which may however differ from transcription - An associated enzyme, ribonuclease H, rates digests the RNA strand, and reverse 4. DNA microarrays: measures the relative transcriptase synthesizes a complementary abundance of the global total or nuclear strand of DNA to form a double helix DNA RNA levels; however, these may differ from structure transcription rates - This cDNA is integrated into the host cell’s 5. In Situ Hybridization: detects the genome via another enzyme (integrase) presence of a transcript causing the host cell to generate viral 6. MS2 tagging: by incorporating RNA stem proteins which reassemble into new viral loops, such as MS2, into a gene, these particles become incorporated into newly - Subsequently, the host cell undergoes synthesized RNA. The stem loops can programmed cell death, apoptosis. then be detected using a fusion of GFP and - Some eukaryotic cells contain an enzyme the MS2 coat protein, which has a high with reverse transcription activity called affinity, sequence specific interaction with telomerase the MS2 stem loops. The recruitment of - Telomerase is a reverse is a transcriptase GFP to the site of transcription is visualized that lengthens the ends of a linear as a single fluorescent spot. chromosomes, and carries and RNA 7. Northern blot: the traditional method, and template from which it synthesizes DNA until the advent of RNA-sequence, the repeating sequence , or “junk” DNA most quantitative - This repeated sequence of DNA is 8. RNA-sequence: applies next-generation important because every time a linear sequencing techniques to sequence whole chromosome is duplicated it is shortened in transcriptomes, which allows the length. measurement of relative abundance of - With “junk” DNA at the ends of RNA , as well as the detection of additional chromosomes, the shortening eliminates variations such as fusion genes, post- some of the non-essential, repeated translational edits and novel splice sites sequence rather than the protein-coding REVERSE TRANSCRIPTION DNA sequence farther away from the chromosome end. - Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes indefinitely without losing important protein-coding DNA sequence - Activation of telomerase could be part of the process that allows cancer cells to become technically immortal. However, the true in vivo significance of telomerase has Scheme of Reverse Transcription still not been empirically proven. - Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA; main enzyme responsible for synthesis: reverse transcriptase - HIV has an RNA genome that is duplicated into DNA - The resulting DNA can be merged with the DNA genome of the host cell - In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome  CYTOGENETICS LECTURE: DNA Translation DR. SARAH DY HINOGUIN-CASIO MIDTERM 1ST SEMESTER – A.Y 2022-2023 DNA TRANSLATION and it also carries an anticodon. The - DNA provides the roadmap for making anticodon is the complementary proteins. The DNA is translated to nucleotides sequence to a given codon. messenger RNA (mRNA) which in turn - The tRNA will pick up the appropriate directs the building of the protein, one amino acid the cytoplasm that is coded for amino acid at a time. by the mRNA codon and to which its anticodon matches. In translation the mRNA will run through the - Translation occurs when the mRNA rRNA from the 5’ end (with A U G) to the strand moves out of the nucleus and termination codon at the 3’ end. into the cytoplasm. - The first codon, A U G, will start in the A - At this point mRNA, rRNA and tRNA all site. come together. - There, the tRNA with the appropriate - The rRNA is the factory of translation, while anticodon, U A C, will meet up with the the tRNA becomes the worker start codon bringing with it the appropriate amino acid, methionine. - The rRNA consists of two parts, the large ribosomal unit and the small ribosomal unit. On the large ribosomal unit are two sites- the A site and the P site. - These will be the sites of polypeptide synthesis and elongation. - The tRNA molecules have an amino acid (the monomer of proteins) attachment site  Once this is complete, the complex will An example of an mRNA codon chart used to move over to the P site. determine amino acids. - The next codon will move in, connect with its tRNA and appropriate amino acid. The two amino acids in the rRNA will then form a peptide bond. - At this point, the first tRNA will disconnect from its U A C amino acid and go back into the cytoplasm. - The second tRNA with the smell, elongating polypeptide attached will move into the P site. - The third codon will enter the rRNA and the process will happen again as before. - This will continue in assembly-line fashion with the polypeptide chain elongating all the time until a stop or termination codon enters the A site. - At this point the translation will stop and the completed polypeptide chain will disconnect to be folded into a complete protein. - The rRNA will float off to be used in other translations and the mRNA in other transcriptions. Each amino acid is specified by a sequence of 3 nucleotides, a word in the vocabulary of the genome: Amide Asparagine (Asn, N) = MW: 114.11 Glutamine (Gln, Q) = MW: 128.14  CYTOGENETICS LECTURE: Chromosomes DR. SARAH DY HINOGUIN-CASIO CHROMOSOMES - Chromosomes – first observed in tumor cells by Walther Fleming in 1882, 16 years after establishment of genetics. Why in tumor cells? - Because in tumor the cells here are fast divide and the mass or tumor is fast growing it’s because of the multiplying cells. If the cells is actively dividing it means we could see a lot active nuclei. Compare and contrast prokaryotic and eukaryotic chromosomes. Eukaryotic chromosomes EUKARYOTIC CHROMOSOMES  More than one - human chromosomes: 46 (44 autosomes; 2  Linear rather than circular sex chromosomes)  Sequestered within a nucleus  other eukaryotes:  Composed of DNA and globular - fruit fly (2x: 8) proteins (histones) - kingfisher (2x: 132)  Nucleosomes (10 nm diameter beads) - tobacco (4x: 48)  Chromatic (30 nm diameter). A very thin - adder’s tongue fern (2x: 1260) material. Extranuclear DNA: Eukaryotes  Active genes- EUCHROMATIN - Mitochondria, chloroplasts  Inactive DNA – HETEROCHOMATIN - DNAs are circular - Codes for 5% RNA and polypeptides (nonfunctional) required for the organelle replication and function Plasmids  Fungi and protozoa - S. cerevisiae – contains 70 copies of a plasmid (2μm circle) PROKARYOTIC CHROMOSOMES - A prokaryotic chromosome consists of a single molecule of DNA in the form of a closed loop E.g. 1. agrobacterium tumefaciens = 1 linear, 1 circular 2. Vibrio cholerae = 2 circular 3. Bacterial – circular molecule of DNA associated with proteins and RNA - Located in NUCLEOID - Folded into loops that are 50,000 – 100,000 bp  The parts of a chromosomes:  Telomere – the ends of the chromosomes  Centromere – the primary constriction of the chromosomes; it also divides the chromosome into a short arm (p) and a long arm (q)  Chromatid – a single molecule of DNA PLASMIDS Classification of Chromosomes: - Small, circular molecules of DNA - By size and position of centromere. - Few thousand bp to million bp - Carries information required for replication, Metacentric – centromere in the cellular traits middle of the chromosome Submetacentric – centromere divides the chromosome into 1/3 and 2/3 Acrocentric – centromere near the end of the chromosome  Scientists called cytogeneticists can Types of Plasmids recognize and identify many of these gross 1. Fertility (F) factors – chromosomal abnormalities by examining conjugation, transfer of genes chromosomes through a microscope. 2. Resistance (R) factors – carry Cytogeneticists use three things to tell genes for resistance to one or more antimicrobial drugs, heavy chromosomes apart: metals or toxins. 1. Chromosome size 3. Bacteriocin factors – carry 2. The position of the centromere genes for proteinaceous toxins 3. Characteristic banding patterns of (bacteriocin). alternating light and dark bands (caused by 4. Virulence plasmids staining the chromosomes with dyes).  combination, allowing the visualization of the individually colored chromosomes. DETECTION In the “classic” (depicted) karyotype, a dye, 3. Digital karyotyping often Giemsa (G-banding), less frequently - Digital karyotyping is a technique used Quinacrine, is used to stain bands on the to quantify the DNA copy number on a chromosomes. genomic scale. - Giemsa is specific for the phosphate - Short sequences of DNA from specific groups of DNA. Quinacrine binds to the loci all over the genome are isolated adenine-thymine rich regions. and enumerated. - Each chromosome has a characteristic - This is also known as virtual banding pattern that helps to identify them; karyotyping. both chromosomes in a pair will have the same banding pattern. - Karyotypes are arranged with the short arm of the chromosome on top, and the long arm on the bottom. - The short and long arms are called p and q, respectively. - In addition, the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. - Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. - Fluorescently labeled probes of each chromosome are made by labeling chromosome-specific DNA with different fluorophores. - Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling method is used to generate many different colors. - Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. - Image processing software then assigns a pseudo color to each spectrally different  CYTOGENETICS LECTURE: Chromosomes DR. SARAH DY HINOGUIN-CASIO SEMI-FINALS CHROMOSOMES - Chromosomes – first observed in tumor cells by Walther Fleming in 1882, 16 years after establishment of genetics. Why in tumor cells? - Because in tumor the cells here are fast divide and the mass or tumor is fast growing it’s because of the multiplying cells. If the cells is actively dividing it means we could see a lot active nuclei. Compare and contrast prokaryotic and eukaryotic chromosomes. Eukaryotic chromosomes EUKARYOTIC CHROMOSOMES  More than one - human chromosomes: 46 (44 autosomes; 2  Linear rather than circular sex chromosomes)  Sequestered within a nucleus  other eukaryotes:  Composed of DNA and globular - fruit fly (2x: 8) proteins (histones) - kingfisher (2x: 132)  Nucleosomes (10 nm diameter beads) - tobacco (4x: 48)  Chromatic (30 nm diameter). A very thin - adder’s tongue fern (2x: 1260) material. Extranuclear DNA: Eukaryotes  Active genes- EUCHROMATIN - Mitochondria, chloroplasts  Inactive DNA – HETEROCHOMATIN - DNAs are circular - Codes for 5% RNA and polypeptides (nonfunctional) required for the organelle replication and function Plasmids  Fungi and protozoa - S. cerevisiae – contains 70 copies of a plasmid (2μm circle) PROKARYOTIC CHROMOSOMES - A prokaryotic chromosome consists of a single molecule of DNA in the form of a closed loop E.g. 1. agrobacterium tumefaciens = 1 linear, 1 circular 2. Vibrio cholerae = 2 circular 3. Bacterial – circular molecule of DNA associated with proteins and RNA - Located in NUCLEOID - Folded into loops that are 50,000 – 100,000 bp AJSE  The parts of a chromosomes:  Telomere – the ends of the chromosomes  Centromere – the primary constriction of the chromosomes; it also divides the chromosome into a short arm (p) and a long arm (q)  Chromatid – a single molecule of DNA PLASMIDS Classification of Chromosomes: - Small, circular molecules of DNA - By size and position of centromere. - Few thousand bp to million bp - Carries information required for replication, Metacentric – centromere in the cellular traits middle of the chromosome Submetacentric – centromere divides the chromosome into 1/3 and 2/3 Acrocentric – centromere near the end of the chromosome  Scientists called cytogeneticists can Types of Plasmids recognize and identify many of these gross 1. Fertility (F) factors – chromosomal abnormalities by examining conjugation, transfer of genes chromosomes through a microscope. 2. Resistance (R) factors – carry Cytogeneticists use three things to tell genes for resistance to one or more antimicrobial drugs, heavy chromosomes apart: metals or toxins. 1. Chromosome size 3. Bacteriocin factors – carry 2. The position of the centromere genes for proteinaceous toxins 3. Characteristic banding patterns of (bacteriocin). alternating light and dark bands (caused by 4. Virulence plasmids staining the chromosomes with dyes).  combination, allowing the visualization of the individually colored chromosomes. DETECTION In the “classic” (depicted) karyotype, a dye, 3. Digital karyotyping often Giemsa (G-banding), less frequently - Digital karyotyping is a technique used Quinacrine, is used to stain bands on the to quantify the DNA copy number on a chromosomes. genomic scale. - Giemsa is specific for the phosphate - Short sequences of DNA from specific groups of DNA. Quinacrine binds to the loci all over the genome are isolated adenine-thymine rich regions. and enumerated. - Each chromosome has a characteristic - This is also known as virtual banding pattern that helps to identify them; karyotyping. both chromosomes