Cytogenetics Topic Notes PDF

CYTOGENETICS COMPILED TOPIC NOTES TOPIC 1: ORIGIN AND IMPORTANCE OF CYTOGENETICS 1.1: Cytogenetics and Its Branches What is Cytogenetics? Cytogenetics is the study of chromosomes and the related disease states cause by abnormal chromosome number and/or structure. The cell is the basic structural...

CYTOGENETICS COMPILED TOPIC NOTES TOPIC 1: ORIGIN AND IMPORTANCE OF CYTOGENETICS 1.1: Cytogenetics and Its Branches What is Cytogenetics? Cytogenetics is the study of chromosomes and the related disease states cause by abnormal chromosome number and/or structure. The cell is the basic structural and functional unit of all organisms. The chromosomes that cell contain are the containers of the hereditary factors which we call as the “gene”. Chromosomes in cells that are abnormal in number and size have an abnormal effect to what the organism will eventually become. Cytogenetics if dissected is from two branches of science: Cytology- study of cells Genetics- study of heredity Therefore, CYTOGENETICS tries to understand how cells produce what organism will eventually look like including the genetic disorders it has. In modern times, cytogenetics or genetics itself has reached the molecular level. This resulted to biochemical and molecular genetics. What are the fields of Genetics? 1. Transmission Genetics- A scientist working in the field of transmission genetics examines the relationship between the transmission of genes from parent to offspring and the outcome of the offspring’s traits. a. Example: How can two brown-eyed parents produce a blue-eyed child? b. The following questions are under transmission genetics: i. How are chromosomes transmitted during cell division and gamete formation? ii. What are the common patterns of inheritance for genes? 2. Molecular Genetics- The goal of molecular genetics is to understand how the genetic material works at the molecular level. It is understanding the molecular features of DNA and how these features underlie the expression of genes. a. The following questions are under Molecular genetics: i. What is the composition and conformation of chromosomes? ii. How is the genetic material copied? iii. How is gene expression regulated so it occurs under the appropriate conditions, in the appropriate cell type, and the correct stage of development? iv. What is the molecular nature of mutation? 3. Population Genetics- this field helps us understand how process such as natural selection have resulted in the prevalence of individuals that carry particular alleles. Population geneticists are particularly interested in genetic variation and how that variation is related to an organism’s environment. In this field, the frequencies of alleles are of central importance. a. The following are questions under Population Genetics: i. Why are two or more different alleles of a gene maintained in a population? ii. What factors alter the prevalence of alleles within a population? iii. What are the contributions of genetics and environment in the outcome of a trait? iv. How do genetics and environment influence quantitative traits, such as size and weight? *Natural Selection- the process whereby organism better adapted to their environment tend to survive and produce more offspring. The theory of its action was first fully expounded by Charles Darwin and is now believed to be the main process that brings about evolution. (Definition from Oxford Dictionary) 1.2: The History of Cytogenetics Historical Development By the middle of the 19th century, the universality of cell division as the central phenomenon in the reproduction of organisms was established, and Virchow expressed it in the famous aphorism “Omnis cellula e cellula”. From this time on, the study of cells and of heredity and evolution converged, as was stated by Wilson: “Heredity appears as a consequence of genetic continuity of the cells by division” CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 1 of 46 Notable Persons and Discoveries: Van Beneden, Flemming, Strasburger, Boveri and others- observed germ cells which supported the theory of the continuity of the germ plasm proposed by Weisman in 1883. o GERM THEORY states o that the transference of hereditary factors from one generation to the next takes place through the continuity of what he called ‘germ plasm’, located on the sex elements (Sperm and Egg), and not through somatic cells. Discovery of Fertilization in Animals- foreseen by O. Hertwig but observed directly by H. Fol (1879) Discovery of Fertilization in Plants- observed by Strasburger.; both discovery led to the theory that the cell nucleus is the bearer of the physical basis of heredity Roux- postulated that chromatin, the substance of the nucleus that constitutes the chromosome, must have a linear organization Weismann- stated that hereditary units are disposed along the chromosomes in an orderly manner Gregor Mendel- discovered the Fundamental Laws of Heredity in 1865, but at that time cytologic changes produced in the sex cells were not sufficiently known to permit an interpretation of the independent segregation of hereditary characters. For this and other reasons, little attention was paid to Mendel’s work until the botanists Correns, Tschermack and De Vries in 1901 independently rediscovered Mendel’s Law 1865/ 1866 Gregor Mendel published his investigations into inheritance of pea plants 1890 Theodor Boveri suggested that chromosomes are involved with inheritance 1900 Walter Sutton observed chromosomes in grasshopper cells 1900/1901 Mendel's work was rediscovered by three scientists: Hugo De Vries, Erich von Tschermack, and Carl Correns 1902 Archibald Garrod discovered that some diseases must be inherited 1903 Sutton and Boveri, working independently, suggested that each egg of sperm cell contains only one of each chromosome pair 1905 Edmund Beecher Wilson and Nettie Stevens, working independently, proposed that certain chromosomes determine sex. They show that a single Y chromosome determines maleness, and two copies of the X chromosome determine femaleness 1906 Bateson gave the term ‘genetics’ 1909 Wilhelm Johannsen used the term 'gene' to describe the carrier of heredity, 'genotype' to describe an organism's genetic make-up, and 'phenotype' to describe an organism's outward appearance 1910 Thomas Hunt Morgan proved that genes are carried on chromosomes. He also showed that some characteristics are carried on the sex chromosome 1911/1913 Alfred Henry Sturtevant mapped the genes o the fruit fly’s sex chromosome 1912 Sir William Henry Bragg and his son discover that X-rays can be used to study the molecular structure of simple crystals, such as salt 1926 Morgan published the ‘Theory of the Gene’ 1928 Frederick Griffith discovered 'transformation' in bacteria 1944 Oswald Avery, Colin MacLeod, and Maclyn Mccatty used bacteria to show that DNA is the hereditary material 1949 Erwin Chargaff finds that the amounts of adenine and thymine in DNA are about the same, as area the amounts of guanine and cytosine 1953 James Watson and Francis Crick proposed that the DNA molecule is a double- stranded helix 1963-1966 Marshall Nirenberg and Heinrich Matthaei work out the genetic code 1977 DNA from virus is sequenced for the first time by Frederick Sanger, Walter Gilbert and Allan Maxam, working independently 1983 Kary Mullis discovered the Polymerase Chain Reaction (PCR), enabling lengths of DNA to be multiplied 1987 Rebecca Cann, Mark Stoneking, and Allan Wilson analyze mitochondrial DNA in different human races. They declared that humans have a common ancestor who lived 200,000 years ago CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 2 of 46  1989 The first Human gene is sequences by Francis Collins and Lap-chee Tsui. It is the gene that cause cystic fibrosis 1990 The Human Genome Project is launched 1993 Cystic fibrosis became the first genetic disease to be treated using gene therapy 1995 The genome of H.influenzae is sequenced. This is the first complete genome of an organism 2000 First draft sequences of human genome are released at the same time by the Human Genome Project and Celera genomics 2003 The Human Genome Project is successfully completed on 14th of April Reference: Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space Browser Publishing p.4-5 TOPIC 2: CYTOLOGICAL BASIS OF HEREDITY 2.1: Origin and Importance of Cytogenetics The body of all living organisms (bacteria, blue green algae, plants and animals) except viruses has cellular organizations and may contain one or many cells. The organisms with only one cell in their body are called unicellular organisms (e.g bacteria, blue green algae, some algae, Protozoa). The organisms having many cells in their body are called multicellular organism (e.g most plants and animals). Any cellular organisms may contain only one type of cell from the following types of cells: A. Prokaryotic cells; B. Eukaryotic Cells *The term prokaryotic and Eukaryotic were suggested by Hans Ris in the 1960’s. PROKARYOTIC CELLS: From Gr. pro=primitive or before; karyon=nucleus; they are small, simple and most primitive. They are probably the first to come into existence about 3.5 billion years ago; essentially a one- envelop system organized in depth. It consists of central nuclear components (viz., DNA molecule, RNA Molecule and nuclear proteins) surround by cytoplasmic ground substance; the cytoplasm of a prokaryotic cell lacks in well defined cytoplasmic organelles; nuclear envelope; nucleoli, cytoskeleton, centrioles and basal bodies. Ex. Bacteria EUKARYOTIC CELLS: From Gr., eu=well; karyon= nucleus; they have evolved from the prokaryotic cells and the first eukaryotic (nucleated) cells may have arisen 1.4 billion years ago (Vidal, 1983); essentially two envelope systems and they are much larger than prokaryotic cells. Secondary membranes envelop the nucleus and other internal organelles; The eukaryotic cells are true cells which occur in the plants and animals. CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 3 of 46 Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54 2.2: CELL STRUCTURE AND ITS FUNCTION The cell is the basic unit of organizations or structure of all living matter. Within a selective and retentive semipermeable membrane, it contains a complete set of different kinds of units necessary to permit its own growth and reproduction from simple nutrients. All forms of life, except viruses, consist of cells; Consists of two distinct areas which in living cells, are in constant motion: Cytoplasm and Nucleus Cell Structure Structure & Organelles Function Cytoplasm -major portion of the protoplasmic substance contained in the cell membrane - several organelles (functionally important for the survival of the cell and their presence or size may vary between different organisms and different tissues) are found esp. in eukaryotes Endoplasmic Reticulum -an organelle where lipid production and some protein translation occurs -Rough ER has attached ribosomes -Smooth ER has no attached ribosomes; functions in lipid metabolism (both catabolism and anabolism; they synthesize a variety of phospholipids, cholesterol and steroids); glycogenolysis and drug detoxfication Golgi Apparatus -a cup-shaped organelle which is located near the nucleus in many cell; -consists of a set of cisternae (i.e, closed fluid- filled flattened membranous sacs or vesicles) - Function: 1. packaging of secretory materials that are to be discharged from cell 2. The processing of proteins 3. The synthesis of certain polysaccharides and glycolipid 4. The sorting of proteins destined for various location 5. Proliferation of membranous element for the plasma membrane 6. Formation of acrosome of the spermatozoa Ribosome – small particles which may be free floating in the cytoplasm or attached to the ER - play an important role in the synthesis of protein -May exist either in the free state in the cytosol or attached to RER Mitochondria -structures where most of the cellular energy is produced in the form of ATP Chloroplast -plastids in plant cell which contain chlorophyll and serve as the photosynthetic factory of the plants * both mitochondria and chloroplasts are found in plants, algae and some protozoans * they also posses their own genomic DNA which are circular and are not complexed with proteins unlike the nuclear genome CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 4 of 46 Centrosome –unlike organelles, not bound by membranes to separate it from the surrounding cytosol - found in most animals and lower plant cells -consists of two cylindrical structures called centrioles * The centrosome serves as the organizing unit for microtubules Microtubule -hollow tubes of dynamic protein polymers composed of subunits that contain 1 mol of α tubulin and 1 mol of β tubulin; -extend and retract to provide shape and structure to eukaryotic cell -they form the network that internal components move along to their proper destination within the cell Spindle Fibers -attach to chromosomes during the early stages of mitosis and meiosis are also composed of microtubules *Thus, centrosome is essential for the correct formation of spindle fibers and the proper movement of eukaryotic chromosomes during mitosis and meiosis. *In some organisms, such as fungi, the spindle pole body serves the function of the centrosome. Nucleus – the dark staining body within the cytoplasm - contains the primary genome of the cell which is organized as linear, double-stranded DNA that is complexed w/ protein(nucleoprotein) - primary director of cellular activity and inheritance - surrounded by a double membrane that appears in active contact w/ ER and the cell membrane. - nuclear content consists of a dark network or chromatin (during cell division becomes distinct bodies or chromosomes) Nucleoli/Nucleolus -1 or more spherical bodies may be found attached to specific chromosome regions -the site where ribosomes are manufactured; where ribosomal DNA transcribes most of rRNA molecules Nuclear Envelope -comprises two nuclear membranes- inner nuclear membrane which is lined by nuclear lamina and an outer nuclear membrane which is continuous with RER -the nuclear envelope is interrupted by structures called pores or nucleopores which contains pore complexes that regulates the exchange between nucleus and cytoplasm. COMPARISON OF PROKAYORTIC AND EUKARYOTIC CELLS Prokaryotes Eukaryotes Typical organisms bacteria, archaea protists, fungi, plants, animals CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 5 of 46  ~ 10-100 µm (sperm cells, apart Typical size ~ 1-10 µm from the tail, are smaller) real nucleus with double Type of nucleus nucleoid region; no real nucleus membrane linear molecules (chromosomes) DNA circular (usually) with histone proteins RNA-synthesis inside the nucleus RNA-/protein-synthesis coupled in cytoplasm protein synthesis in cytoplasm Ribosomes 50S+30S 60S+40S highly structured by Cytoplasmatic structure very few structures endomembranes and a cytoskeleton flagella and cilia containing Cell movement flagella made of flagellin microtubules; lamellipodia and filopodia containing actin one to several thousand (though Mitochondria none some lack mitochondria) Chloroplasts none in algae and plants single cells, colonies, higher Organization usually single cells multicellular organisms with specialized cells Mitosis (fission or budding) Cell division Binary fission (simple division) Meiosis DNA Content 1 × 106 to 5 × 106 1.5 × 107 to 5 × 109 Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54 Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C 2.3: THE CHROMOSOME Chromosomes are complex structures located in the cell nucleus. They are composed of DNA, histone and non-histone proteins, RNA, and polysaccharides. They are basically the “packages” that contain the DNA. Normally chromosomes cannot be seen with a light microscope but during cell division, they become condensed enough to be easily analyzed at 1000x. To collect cells with their chromosomes in this condensed state they are exposed to a mitotic inhibitor which blocks formation of the spindle and arrests cell division at the metaphase stage. Discovered by Karl von Nageli (1842) after staining techniques were developed that made them visible Heinrich Wilhelm Waldeyer (1888)- coined the term chromosome (means “colored body”) Genes are located on the chromosomes which exist as chromatin network in the non dividing cells Chromatin - linear eukaryotic chromosomes are composed of a complex double-stranded DNA and protein; Exist in two forms: a. Euchromatin – found in a loosely packed state; involved in gene duplication, gene transcription and phenogenesis or phenotypic expression of gene through some type of protein synthesis CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 6 of 46  b. Heterochromatin- highly condensed and readily visible organization; exist both in the region of centromere and in sex chromatin and is late replicating one What does chromosomes do? The unique structure of chromosomes keeps DNA tightly wrapped around spool-like protein called “histones”. Without such packaging, DNA molecules would be too long to fit inside cells. For example, if all of the DNA is a single human cell were unwound from their histones and placed end-to-end, they would stretch 6 feet; they are a key part of cell division that ensures DNA is accurately copied and distributed in the vast majority of cell divisions. The chromosome structure Chromosomes Under the microscope, chromosome appear as thin, thread-like structures. They all have a short arm designated as “P” meaning petite and a long arm designated as “Q”, they are separated by a primary constriction called the “centromere” The centromere is the location of spindle attachment and is an integral part of the chromosome; essential for the normal movement and segregation of chromosomes during cell division Chromosomes maintain constant size and shape at specific stages of the cell cycle Condensed chromosomes may be as short as 0.25 μ (in fungi and birds) or as long as 30 μ in Trillium sp. Kinetochore - the proteinaceous structure on the surface of the centromere to which the microtubules attach Each DNA Molecule That Forms a Linear Chromosome Must Contain a Centromere, Two Telomeres, and Replication Origins CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 7 of 46 Figure 4-22. The three DNA sequences required to produce a Eukaryotic chromosome that can be replicated and then segregated at mitosis. Each chromosome has multiple origins of replication, one centromere, and two telomeres. Shown here is the sequence of events a typical chromosome follows during the cell cycle. The DNA replicates in interphase beginning at the origins of replication and proceeding bidirectionally from the origins across the chromosome. In M phase, the centromere attaches the duplicated chromosomes to the mitotic spindle so that one copy is distributed to each daughter cell during mitosis. The centromere also helps to hold the duplicated chromosomes together until they are ready to be moved apart. The telomeres form special caps at each chromosome end. -Secondary Constriction - further constriction may be observed in some chromosomes; this may include pinching off of a small chromosomal section called the satellite -these secondary constrictions are often associated with regions where the nucleolus is formed or attached -Nucleolus-forming regions - the organization of the nucleolus is the function of a specific point on a particular chromosome. When a nucleolus is visible, it can be seen to be attached to this nucleolus-organizing region. The chromosome where this region is located is known as the nucleolus organizer. Chromomeres and knobs -When a mitotic chromosome is stretched out it would be observed to consist of a string of characteristic particles of unequal sizes at unequal distances apart. The smaller “beads on the string” are called chromomeres (visible dark bands); the larger ones are called knobs. TYPES OF CHROMOSOME Human metaphase chromosomes come in three basic shapes and can be categorized according to the length of the short and long arms and also the centromere location: 1. Metacentric- these chromosomes have short and long arms of equal length with centromere in the middle. 2. Sub-metacentric- these chromosomes have short and long arms of unequal length with the centromere more towards one side 3. Acrocentric- These chromosomes have a centromere very near to one end and have very small short arms. They frequently have a secondary constrictions on the short arms that connect very small pieces of DNA, called “Stalks” and “Satellites”, to the centromere. The stalks contain aenes which code for ribosomal RNA. CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 8 of 46 Chromosome Complement - a particular species possesses a constant number of chromosomes. Cells may be either: 1. Diploid – contain two sets of chromosomes - found in most cells of eukaryotic organisms - 2n=46 (humans) *humans have 23 homologous chromosome pairs Homologous chromosomes or homologs -members of the same chromosome pair 2. Haploid – contain one set of chromosomes - include reproductive cells or gametes - eggs and sperm are haploid cells that contain that contain one member of each homologous pair (n=23) Species 2n Human being (Homo sapiens) 46 Garden pea (Pisum sativum) 14 Fruit fly (Drosophila melanogaster) 8 House mouse (Mus musculus) 40 Roundworm (Ascaris sp.) 2 Pigeon (Columba livia) 80 Boa constrictor (Constrictor constrictor) 36 Cricket (Gryllus domesticus) 22 Lily (Lily longiflorum) 24 Indian fern (Ophioglossum reticulatum) 1260 1. Homomorphic chromosome pairs - homologous pairs are made up of identical partners 2. Heteromorphic chromosome pair - have unequal size and composition (eg. Sex chromosomes) a. homogametic sex (females in mammals) - because all their gametes contain an X chromosome CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 9 of 46  b. heterogametic sex (males in mammals) - because their gametes contain either an X or Y. Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54 Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space Browser Publishing p.4-5 Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C 2.4: CELL CYCLE Cell cycle refers to the regular and repetitive physical and chemical process taking place within the cell. A cell cycle is a cycle which means there is no fixed starting point. The first major phase of the cell is the interphase. Then, the second major phase- the M Phase. Cell cycle is simply cell reproduction. A. Interphase 1. G1 phase 2. S-phase 3. G2 phase B. Mitosis phase Interphase -cells spends most of their lives in interphase. In this phase of the cell cycle, cells are not actively dividing.; chromosomes are uncondensed throughout interphase. During G1, cells undergo a period of rapid growth, and the chromosomes are unduplicated. During the S phase, cells begin to prepare for division during interphase by duplicating its chromosomes. At the end of S Phase, all the chromosomes are therefore duplicated chromosomes. During G2, the cell again grows and complete the preparation for division (mitosis or M Phase). 1. G1 (First Gap Phase) First stage of interphase The protein synthesis and RNA synthesis within the cell resumes that was interrupted during the process of mitosis Growth and young cell maturation occurs, which accomplish the physiological function The phase during which the cell cycle starts with synthesis of RNA and protein required by the young cells for their growth and maturity. G1 phase is usually termed as the prior to DNA Synthesis phase. 2. S Phase The second stage of Interphase DNA synthesis takes place; S stands for synthesis CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 10 of 46  Soon after the G1 phase, DNA checking and subsequent repair occurs during the variable pause phase before the transition of the cell cycle to the S phase The S phase of the interphase deals with the semi-conservative synthesis of DNA occurs. Replication of cellular DNA begins with the S phase, which when gets duplicated with the cell containing nearly double the amount of chromosomes, the cells from the S phase move into the G2 Phase 3. G2 Phase (Second Gap Phase) The third stage of interphase There is an increase in the synthesis of the RNA and the protein, which is followed by another round of proof reading and subsequent repair among the newly synthesized DNA sequences before the cell cycle transits to the mitotic cycle The mitotic spindle formed from the cytokinetic fibers start forming and the cell ensures the number of chromosomes and the organelles present, which further leads the cell cycle from the interphase to the mitotic phase. Typical Cell Cycle in a cell culture INITIATION OF MITOSIS Mitosis Promoting Factor o protein complex which initiates the mitotic phase of the cell cycle o made of two proteins: 1. Cyclin B - one that oscillates in quantity during the cell cycle 2. CDC2- one whose quantity is constant - encoded by the cdc2 gene - a kinase (an enzyme that transfers a phosphate group on to a protein by phosphorylation) *CDC2 kinase is only functional when it is combined with cyclin which is referred as cyclin-dependent kinase (CDK) *Once mitosis has been initiated, anaphase-promoting complex (APC) or cyclosome: 1. degrades the Cyclin B protein of MPF 2. permits the separation of the sister chromatids at the start of anaphase CELL CYCLE CHECKPOINTS Checkpoints refers some points in the cell cycle which allow the cell to make sure that various events have been properly completed before the next phase begins CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 11 of 46  1. G1/S Checkpoint- a. If the G1/S checkpoint detects damage, the p53 protein targets the cell for regulated death (apoptosis) 2. Spindle Attachment Checkpoint a. ensures that spindle fibers are attached to every kinetochore before the sister chromatids attempt to separate b. involves several proteins including mitotic arrest-deficient protein 2 (MAD2) (binds to kinetochore) MITOSIS Mitosis only occurs among somatic cells or body cells. It is a form of eukaryotic cell division that produces two daughter cells with the same genetic component as the parent cell. (- 2n = 2n) In some single-celled organisms mitosis forms the basis of asexual reproduction. In diploid multicellular organisms sexual reproduction involves the fusion of two haploid gametes to produce a diploid zygote. Mitotic divisions of the zygote and daughter cells are then responsible for the subsequent growth and development of the organism. In the adult organism, mitosis plays a role in cell replacement, wound healing and tumor formation. Mitosis, although a continuous process, is conventionally divided into five stages: prophase, prometaphase, metaphase, anaphase and telophase. 1. Prophase a. -Chromatin in the nucleus begins to condense and becomes visible in the light microscope as chromosomes b. The nucleolus disappears c. Centrioles begin moving to opposite ends of the cell and fibers extend from the centromeres; some fibers cross the cell to form the mitotic spindle d. The number of microtubules that attach to each kinetochore differs in different species; 1 microtubule attaches per kinetochore in yeast 4- 7/kinetochore in cells of rat fetus 70 to 150 attach in the plant Haemanthus katherinae 2. Prometaphase a. The nuclear membrane dissolves, marking the beginning of prometaphase b. Proteins attach to the centromeres creating the kinetochores c. Microtubules attach at the kinetochores and the chromosomes begin moving 3. Metaphase CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 12 of 46  a. Spindle fibers align the chromosomes along the middle of the cell nucleus; line is referred to as the metaphase plate; this organization helps to ensure that in the next phase, when the chromosomes are separated each new nucleus will receive one copy of each chromosome 4. Anaphase a. The paired chromosomes separate at the kinetochores and move to opposite side of the cell. Motion results from a combination of kinetochore movement along the spindle microtubules and through the physical interaction of polar microtubules 5. Telophase a. Chromatids arrive at opposite poles of cell, and new membranes form around the daughter nuclei b. The chromosomes disperse and are no longer visible under the light microscope c. The spindle fibers disperse, and cytokinesis or the partitioning of the cell may also begin during this stage CYTOKINESIS In animal cells, cytokinesis results when a fiber ring composed of protein called actin around the center of the cell contracts pinching the cell into two daughter cells, each with one nucleus. In plant cells, the rigid wall requires that a cell plate be synthesized between the two daughter cells. This separation of the genetic material in a mitotic nuclear division (or karyokinesis) is followed by a separation of the cell cytoplasm in a cellular division (or cytokinesis) to produce two daughter cells. SIGNIFICANCE OF MITOSIS Mitosis is important for the maintenance of the chromosomal set; each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. *With this stability ensured, single-celled organisms could thrive and multicellular organisms could evolve. PROPHASE ME CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 13 of 46 METAPHASE TELOPHASE ANAPHASE MEIOSIS The main goal of mitosis is to preserve the chromosomes of the parent cells to the daughter cells in order to attain genetic continuity. This means that the information of one kind of skin cell is the same in all skin cells. This kind of cell division happens in all cells except to sex cells. Sex cells (gametes), which are haploid cells, undergo a special type of cell division which is properly called “meiosis”. The goal of meiosis is to reduce the number of chromosomes in half, so that when fertilization occurs, the number of chromosomes will be reestablished. PHASES OF MEIOSIS Two successive nuclear divisions occur, Meiosis I (Reduction) and Meiosis II (Division). Meiosis produces 4 haploid cells. Mitosis produces 2 diploid cells. The old name for meiosis was reduction/division. Meiosis I reduces the ploidy level from 2n to n (reduction) whole Meiosis II divides the remaining set of chromosomes in a mitosis-like process (division). MEIOSIS I 1. Prophase I a. Has a unique event- the pairing (by an as yet undiscovered mechanism) of homologous chromosomes. b. Synapsis – is the process of linking of the replicated homologous chromosomes. The resulting chromosome is termed tetrad, being composed of two chromatids from each chromosome, forming a thick (4-strand) structure c. Crossing over- may occur at this point; during crossing-over chromatids break and may be reattached to a different homologous chromosomes; crossing over between homologous chromosomes produce chromosomes with new association of genes and alleles d. Prophase I is further subdivided: CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 14 of 46  i. Leptotene- means thin thread; chromosomes first become visible as long, thread- like structures; initial phase of condensation of the chromosomes ii. Zygotene- means paired thread; marked by lateral pairing, or synapsis, of homologous chromosomes, beginning at the chromosome tips iii. Pachytene- means thick thread; condensation of the chromosome continues; throughout this period, the chromosomes continue to shorten and thicken; genetic exchange or crossing over happens here; but crossing over is not apparent until the transition to diplotene iv. Diplotene- means double thread; the synapsed chromosomes begin to separate, and the diplotene chromosomes begin to double; each cross connection called chiasma, is formed by a breakage and rejoining between nonsister chromatids v. Diakenesis- final period; means moving apart; the homologous chromosomes seem to repel each other and the segments not connected by chiasmata move apart; near the end of Diakenesis, the formation of a spindle is initiated and the nuclear envelope breaks down. 2. METAPHASE I a. Tetrads line-up along the equator of the spindle b. Spindle fibers attach to the centromere region of each homologous chromosome pair; other metaphase events as in mitosis 3. ANAPHASE I a. when tetrads separate, and are drawn to opposite poles by the spindle fibers. The centromeres in Anaphase I remain intact 4. TELOPHASE I a. Similar to telophase of mitosis, except that only one set of (replicated) chromosomes is in each “cell” b. Depending on species, new nuclear envelopes may or may not form; some animal cells may have division of the centrioles during this phase *At the beginning of Telophase I, each half of the cell has a complete haploid set of chromosomes, but each chromosome is still composed of two sister chromatids *Cytokinesis usually occurs simultaneously with Telophase I forming two hapoloid daughter cells *No chromosome replication occurs between the end of meiosis I and the beginning of meiosis II, as the chromosomes are already replicated MEISOSIS II 1. Prophase II a. A spindle apparatus forms b. In late prophase II, chromosomes, each still composed of two chromatids, move toward the opposite plate 2. Metaphase II a. Chromosomes are positioned in the metaphase plate as in mitosis b. Because of crossing over in meiosis I, the two sister chromatids of each chromosomes are “not” genetically identical c. The kinetochores of sister chromatids are attached to microtubules extending from opposite poles 3. Anaphase II a. The centromeres of each chromosome finally separate, and the sister chromatids come apart b. The sister chromatids of each chromosome now move as two individual chromosome toward opposite poles 4. Telophase and Cytokinesis a. Nuclei form, the chromosome begin decondensing, and cytokinesis occurs b. Meiotic division of one parent cell produces four daughter cells, each with a haploid set of (unreplicated) chromosome c. Each of the four daughter cells is genetically distinct from the other daughter cells and from the parent cell CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 15 of 46 TOPIC 3: Inheritance; Mendelian Principle 3.1: Mendelian Genetics Gregor Mendel The pioneer of classical geneticist Austrian Monk, born in what is now Czech Republic in 1822 Son of peasant farmer, studied Theology and was ordained priest Order St. Augustine. Went to the university of Vienna, where he studied botany and learned the Scientific Method Worked with pure lines of peas for eight years Prior to Mendel, heredity was regarded as a "blending" process and the offspring were essentially a "dilution" of the different parental characteristics Mendel was the first biologist to use Mathematics – to explain his results quantitatively. Mendel predicted o The concept of genes o That genes occur in pairs o That one gene of each pair is present in the gametes Mendel’s Pea Experiment 1. Choosing true-breeding pea plants Mendel chose a pea plants that is true breeding for trait like round seeds. He chose a pea plant that is also true breeding for the contrasting trail like a wrinkled seed. (True breeding means all of the offspring look like the parents, i.e all round or all wrinkled) 2. Cross pollinating the parent plants. He cross-pollinated the true-breeding round-seed pea plant to the true-breeding wrinkled seeded pea plant. These pea plants can to be known as the ‘parental generation’ 3. The result of cross-pollination of P is F1. The parents (parental generation, or simple ‘P’) are allowed to self-fertilize. These generation produced progenies, or offspring. The offspring of the cross is called first filial generation, or simple F1. In the case round vs. wrinkled, it was observed that the F1 is round seeded and not-wrinkled-seeded. In short, the round seeded trait is expressed; the wrinkled-seeded trait is masked. 4. Allowing self-fertilization of F1 producing F2 Mendel allowed the first filial generation to self-fertilize. The result of the self-fertilization is a new generation of progenies called second filial-generation or simple F2. In the case of the round-seeded F1, some of its progenies are usually round-seeded and a few are wrinkled- seeded. In short, the round-seeded trait still appeared; the wrinkled-seeded trait reappeared. CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 16 of 46 MENDEL’s POSTULATE Mendel, after his experiments, arrived to some generalizations which are known as postulates. Mendel did not actually state them as postulates, nor did he propose a theory of inheritance. 1. Traits are controlled by heritable factors 2. Factors are passed from parent to offspring in reproductive cells 3. Each individual contains pairs of factors in every cell except reproductive cells 4. Paired factors segregate during the formation of reproductive cells so that each reproductive cell gets one of the factors of a pair. 5. There is an equal chance that a reproductive cell will get one or the other factor of a pair. 6. Each factors from one parent has an equal chance of combining either with the identical factor or with the other factor from the other parent during fertilization. 7. Sometimes one factor dominates the other factor; in such cases, the dominant factor controls the feature of the plant 8. When two or more traits are under consideration, the factors for each trait assort independently to the reproductive cells. MENDEL’S LAW OF INHERITANCE/ MENDEL’S PRINCIPLE 1. Principle of Dominance a. IF two contrasting traits (round vs. wrinkled) code for the same character (Seed shape), one trait s dominant while the other is recessive. The dominant trait is always expressed in F1; while the recessive trait is masked only to reappear in the F2 generation. The round and wrinkled are called alleles, the alternative versions of a gene. 2. Law of Segregation a. The two alleles for a heritable character separate or segregate during gamete formation, and end up in different gametes. Thus, an egg or a sperm gets only one of the two alleles that are present in the body cells of the organism 3. Law of Independent Assortment a. Each pair of alleles segregate independently of other pairs of alleles during gamete formation. This follows at least tracing the inheritance of two heritable characters; Genes do not influence each other with regard to the sorting of alleles into gametes; every possible combination of alleles for every gene is equally likely to occur; Genes get shuffled – these many combinations are one of the advantages of sexual reproduction b. Example in dihybrid crosses 3.2: Monohybrid and Dihybrid cross MONOHYBRID CROSS Is a genetic mix between two individuals who have a homozygous genotypes or genotypes that have completely dominant or completely recessive alleles, which result in opposite phenotypes of certain genetic trait. PUNNETT SQUARE A useful tool to do genetic crosses For a monohybrid cross, you need a square divided by four that looks like a window pane We use the Punnett square to predict the genotypes and phenotypes of the offspring. Steps in Using the PUNNETT SQUARE STEPS: 1. determine the genotypes of the parent organisms 2. write down your "cross" (mating) CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 17 of 46  3. draw a p-square 4. Parent genotypes: TT and t t Cross TT  tt 5. "split" the letters of the genotype for each parent & put them "outside" the p-square 6. determine the possible genotypes of the offspring by filling in the p-square 7. summarize results (genotypes & phenotypes of offspring) T- Dominant allele Tall Plant t- recessive allele Short Plant Parents TT- Homozygous Tall plant tt- heterozygous short plant TTxtt T T Genotype: t Tt Tt 100% Heterozygous Tall Plants Phenotype: 100% Tall Plants Tt Tt t Another Example: Heterozygous Cross Tt x TT T T T TT TT t Tt Tt Genotype: 50% Homozygous Tall Plants (TT, TT) 50% Heterozygous Tall Plants (Tt, Tt) Phenotype 100% Tall Plants (T or the dominant allele is expressed) DIHYBRID CROSS is a cross between two different lines/genes that differ in two observed traits CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 18 of 46  a cross that shows the possible offspring for two traits Studying the inheritance of two characters simultaneously How are Dihybrid Crosses Possible? Mendel Concluded that due to the Law of Independent Assortment, different traits are not linked and have equal probability of showing up in offspring. Example: Tall Purple plants, Tall White plant, short purple plants and short white plants. Keep in mind, dominance will skew the ratios, but all of these outcomes are POSSIBLE! Is the inheritance of one character affected by the inheritance of another? THE LAW OF INDEPENDENT ASSORTMENT  It appears that the inheritance of seed shape has no influence over the inheritance of seed colour  The two characters are inherited INDEPENDENTLY  The pairs of alleles that control these two characters assort themselves independently Yellow (Y) is dominant to green (y). Round (R) is dominant to wrinkled (r). Steps to solving dihybrid crosses: 1) Make gametes for each parent. 2) Put gametes of one parent on top of square and the other parent on the side of square. 3) Fill in squares. R r Y y 4) Determine ratio of offspring. R r Example: RrYy x RrYy Y y WRONG WAY OF DOING IT! F1 -> F2 Dihybrid When the YyRr plant is mated, it makes 4 kinds of gamete. Each gamete gets 1 copy of each gene, so 1/2 are Y and 1/2 are y. Independently, 1/2 are R and 1/2 are r. Thus, 1/4 of the gametes are YR, 1/4 are Yr, 1/4 are yR, and 1/4 are yr. This happens for both male and female gametes. CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 19 of 46  The gametes combine randomly. The combinations are shown in a 16 cell Punnett square. Law of independent assortment: each pair of alleles segregates into gametes independently 4 sets of gametes are produced in equal probability YR, Yr, yR, yr Only true for genes on separate chromosomes! How come? Mendel's second law. For unlinked genes, the alleles from each gene segregate into the gametes independently of one another. Some genes are linked, which means that they don't segregate independently of each other and thus don't give the 9:3:3:1 ratio of F2 offspring. EXAMPLE: Fur Color: Coat Texture: B: Black R: Rough b: White r: Smooth In this example, we will cross a heterozygous individual with another heterozygous individual. Their genotypes will be: BbRr x BbRr 1. First, you must find ALL possible gametes that can be made from each parent. 2. Remember, each gamete must have one B and one R. 3. Possible gametes: a. BR Next, arrange all possible gametes for one b. Br parent along the top of your Punnett Square, and all possible gametes for the c. bR other parent down the side of your Punnett d. br Square… CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 20 of 46 CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 21 of 46 3.3: Non-Mendelian Patterns of Inheritance It is no doubt that Mendel was able to explain what, in many years, other people could not explain. However, there are traits which do not conform to the discovered laws of Mendel. 1. Incomplete Dominance a. In incomplete dominance, one allele of a pair is not fully dominant over its partner, so a heterozygous phenotype somewhere in between the two homozygous phenotypes emerges. An example is the case of snapdragons (starr et al., 160p) b. When an organism is heterozygous for a trait, it will show a third phenotype; the third phenotype is a blend of the other two c. Example: cross between white and red flower the offspring becomes pink 2. Codominance/Multiple Alleles a. A pair of non-identical alleles specific two phenotypes, which are both expressed at the same time in heterozygotes. b. Example is the AB blood type; Both A and B antigen is expressed 3. Pleiotropy a. Most genes have multiple phenotypic effects, a property called pleiotropy (Gr. pleion=more). b. For example- pleiotropic alleles are responsible for the multiple symptoms associated with certain heredity diseases in humans, such as cystic fibrosis and sickle-cell disease 4. Epistasis a. (Gr. word meaning “stopping”) a gene at one locus alters the phenotypic expression of a gene at a second locus b. Example- in mice and many other mammals; black coat is dominant to brown. Let’s designate B and b as the two alleles for this character. For a mouse to have a brown fur, its genotype must be bb. But there is more to the story. A second gene determines whether or not pigment will be deposited in the hair. The dominant allele symbolized as C (for color), results in the deposition of either black or brown pigment, depending on the genotype at the first locus. But if the mouse is homozygous recessive for the second locus (cc, then the coat is white (albino), regardless of the genotype of the black/brown locus. The gene for pigment deposition is sai to be epistatic to the gene that codes for black or brown pigment (Reexe et al., 262p) 5. Polygenic Inheritance a. Mendel studied characters that could be classified on an either-or-basis, such as purple versus white flower color. But many characters like skin color and height, vary in the population along a continuum (in gradations). These are called quantitative variations usually indicate polygenic inheritance, an additive effect of two or more genes on a single phenotypic character (the converse of pleiotropy, where a single gene affects several phenotypic characters) Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54 Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space Browser Publishing p.4-5 Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C TOPIC 4: DNA REPLICATION AND DNA SYNTHESIS DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid), the principal genetic materials of living organisms, are chemically called nucleic acids and are complex molecules largen than most proteins and contains Carbon, Oxygen, Hydrogen, Nitrogen and Phosphorous. 4.1: The DNA and RNA DNA (Deoxyribonucleic Acid) CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 22 of 46 The 3-Dimensional structure of DNA was discovered in 1953 by Watson and Crick in Cambridge, using the experimental data of Wilkins and Franklin in London, for which they won a Nobel prize. Ms. Franklin however died before the award and the Nobel Prize is never awarded posthumously. The main features of the structure are: 1. DNA is a double-stranded, so there are two polynucleotide stands alongside each other. 2. The strands are antiparallel, i.e they run in opposite directions thus 5’ to 3’ is parallel to 3’ to 5’ 3. The two strands are wound round each other to form a double helix (not a spiral) 4. The two strands are joined together by hydrogen bonds 5. The bases therefore form base pairs, which are like rungs of ladder. 6. The base pairs are specific. A only binds to T (and T to A); and C only binds to G (G with C) 7. These are called complementary base pairs (or sometimes Watson-Crick base pairs). (A-T and G- C) 8. This means that whatever the sequence of bases along one strand, the sequence of bases on the other strand must be complementary to it. RNA (Ribonucleic Acid) Like DNA, RNA is polymeric nucleic acid of four monomeric robotids or ribonucleotids. Each ribonucleotide contains a pentose sugar (D-ribose); a molecule of phosphate group and nitrogen base. The nitrogen bases of RNA are two purines; The four ribonucleotides also occur freely in nucleoplasm but in the form of triphosphate of ribonucleotisides such as ATP and uridine triphosophate (UTP) RNA molecule may be either single stranded or double stranded but not helical like DNA molecules 3 Different Types of RNA 1. mRNA- Messenger RNA- this carries information from the nucleus to the ribosomes which are sites for protein synthesis. The coding sequence of the mRNA determines the amino acid sequence in the protein. The mRNA is a straight molecule extends from 5’ to 3’ end. It is transcribed from a DNA template. On the mRNA nucleotides are arranged into codons consisting of 3 bases each. Each such codon specifies an amino acid. 2. tRNA- transfer RNA- this RNA type is a small chain of about 80 nucleotides; tRNA transfers specific amino acid molecules to a growing polypeptide chain. The tRNA has a clover lead model with 5 arms each with a specific function. The tRNA also has an anticodon region that can base pair with the codon region on the mRNA. 3. rRNA- Ribosomal RNA- synthesized in the nucleolus. In the cytoplasm, ribosomal RNA and protein combine together to form a nucleoprotein called a ribosomes. The ribosome and mRNA bind to carry out protein synthesis DNA Replication The Enzymes used in DNA Replication Enzymes are proteins which are created in order to complete a certain task within the body, and in most cases catalyze chemical reactions. In this case, enzymes carry out the process of DNA Replication. 1. DNA Helicase- Unzips strand of DNA by breaking hydrogen bonds between base pairs of nucleotide. In short, the DNA is cut down in the middle into two strands 2. Single site bonding proteins- clasps onto both DNA strands to prevent the re-zipping of DNA 3. DNA Polymerase- adds nucleotides to DNA Strands. Also serves as the corrector for mismatched nucleotides. 4. DNA Ligase- Binds Okazaki fragments together within the lagging strand, with forms one complete DNA strand Lagging vs. Leading Strand DNA has two sides that are coiled up. One side has a 5’ end, and the complementary side has a 3’ end. This means that on one side, the sequence begins with a Phosphorus, and the other side begins with a sugar. Because both strands are built from the 5’ end to the 3’ end, both strands must be built in a different way. The leading strand is the strand that is continuously built throughout DNA Replication. The lagging CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 23 of 46 strand must be built differently because both strands built from the 5’ to 3’ direction. The lagging strand is built piece, and those pieces are later connected into one full DNA strand with the help of DNA Ligase. Leading Strand runs 3’ to 5’ (left -> right) Lagging Strand runs 5’ to 3’ The Process of DNA Replication from Beginning to End 1. DNA Replication begins with one strand of DNA 2. DNA helicase then runs throughout the DNA strand and breaks it apart into two halves 3. Single sit bonding proteins hold back what has been unzipped to ensure that the sides do not rebind. 4. After, DNA Polymerase attaches the primer (where DNA attaches to), and begins to lengthen the new strands of DNA by adding nucleotide sequences to it 5. While this is occurring, on the lagging strand (5’ to 3’) the process is happening in the same fashion, but the new DNA on this strand is being built in small segments called the “Okazaki Fragments” which will then be bind by the DNA Ligase into one complete strand of new DNA 6. DNA Replication has now been completed. 4.2: From Gene to Protein (Transcription and Translation) The Flow of Genetic Information Overview The information content of DNA is in the form of specific sequences of nucleotides The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins Proteins are the links between genotype and phenotype Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation How was the fundamental relationship between genes and proteins discovered? 1.Evidence from the study of Metabolic defects In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway 2. Nutritional Mutants in Neurospora: Scientific Inquiry George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal medium as a result of inability to synthesize certain molecules Using crosses, they identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme 3. The Products of Gene Expression: A Developing Story Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one protein Many proteins are composed of several polypeptides, each of which has its own gene Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 24 of 46 BASIC PRINCIPLES OF TRANSCRIPTION AND TRANSLATION RNA is the intermediate between genes and the proteins for which they code Transcription is the synthesis of RNA under the direction of DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA Ribosomes are the sites of translation In prokaryotes, mRNA produced by transcription is immediately translated without more processing In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA A primary transcript is the initial RNA transcript from any gene The central dogma is the concept that cells are governed by a cellular chain of command: DNA → RNA → protein The Genetic Code There are 20 amino acids, but there are only four nucleotide bases in DNA How many bases correspond to an amino acid? Codons: Triplets of Bases The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words These triplets are the smallest units of uniform length that can code for all the amino acids Example: AGT at a particular position on a DNA strand results in the placement of the amino acid serine at the corresponding position of the polypeptide to be produced During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction Each codon specifies the addition of one of 20 amino acids CRACKING THE CODE All 64 codons were deciphered by the mid-1960s Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 25 of 46 Evolution of the Genetic Code The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transplanted from one species to another TRANSCRIPTION Transcription, the first stage of gene expression, can be examined in more detail RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DNA that is transcribed is called a transcription unit CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 26 of 46 Synthesis of RNA Transcript The three stages of transcription: – Initiation – Elongation – Termination RNA Polymerase Binding and Initiation of Transcription Promoters signal the initiation of RNA synthesis Transcription factors mediate the binding of RNA polymerase and the initiation of transcription The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes Termination of Transcription The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator In eukaryotes, the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain; the polymerase eventually falls off the DNA Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm During RNA processing, both ends of the primary transcript are usually altered Also, usually some interior parts of the molecule are cut out, and the other parts spliced together Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way: – The 5 end receives a modified nucleotide 5 cap – The 3 end gets a poly-A tail These modifications share several functions: – They seem to facilitate the export of mRNA – They protect mRNA from hydrolytic enzymes – They help ribosomes attach to the 5 end Split Genes and RNA Splicing Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions These noncoding regions are called intervening sequences, or introns The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence RIBOZYMES Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 27 of 46 TRANSLATION The translation of mRNA to protein can be examined in more detail A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) Molecules of tRNA are not identical: Each carries a specific amino acid on one end Each has an anticodon on the other end; the anticodon base- pairs with a complementary codon on mRNA Accurate translation requires two steps: – First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl- tRNA synthetase – Second: a correct match between the tRNA anticodon and an mRNA codon Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon RIBOSOMES Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) A ribosome has three binding sites for tRNA: 1. The P site holds the tRNA that carries the growing polypeptide chain 2. The A site holds the tRNA that carries the next amino acid to be added to the chain 3. The E site is the exit site, where discharged tRNAs leave the ribosome CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 28 of 46 The three stages of translation: – Initiation – Elongation – Termination Ribosome Association and Initiation of Translation The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits First, a small ribosomal subunit binds with mRNA and a special initiator tRNA Then the small subunit moves along the mRNA until it reaches the start codon (AUG) Proteins called initiation factors bring in the large subunit that completes the translation initiation complex Elongation of Polypeptide Chain During the elongation stage, amino acids are added one by one to the preceding amino acid Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation Termination of Translation Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome The A site accepts a protein called a release factor The release factor causes the addition of a water molecule instead of an amino acid This reaction releases the polypeptide, and the translation assembly then comes apart Completing and Targeting the Functional Protein Often translation is not enough to make a functional protein Polypeptide chains are modified after translation Completed proteins are targeted to specific sites in the cell 4.3: Mutations Mutations are changes in the genetic material of a cell or virus Point mutations are chemical changes in just one base pair of a gene The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein Types of Point Mutations Point mutations within a gene can be divided into two general categories – Base-pair substitutions – Base-pair insertions or deletions *If you have at least access to the internet please download the powerpoint to see pictures* 1.Base- pair Substitutions A base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 29 of 46  Missense mutations still code for an amino acid, but not necessarily the right amino acid Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein 2.Base-pair Insertion and Deletion Insertions and deletions are additions or losses of nucleotide pairs in a gene These mutations have a disastrous effect on the resulting protein more often than substitutions do Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation MUTAGENS Spontaneous mutations can occur during DNA replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations ADDITIONAL NOTES & REVIEW: Archaea are prokaryotes, but share many features of gene expression with eukaryotes Bacteria and eukarya differ in their RNA polymerases, termination of transcription and ribosomes; archaea tend to resemble eukarya in these respects Bacteria can simultaneously transcribe and translate the same gene In eukarya, transcription and translation are separated by the nuclear envelope In archaea, transcription and translation are likely coupled WHAT IS GENE? The idea of the gene itself is a unifying concept of life We have considered a gene as: – A discrete unit of inheritance – A region of specific nucleotide sequence in a chromosome – A DNA sequence that codes for a specific polypeptide chain In summary, a gene can be defined as a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule Reference: Powerpoint Lecture from Biology by Neil Campbell and Jane Reece CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 30 of 46 TOPIC 5: STEM CELLS 5.1: Stem Cells and its Characteristics Stem Cells -A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues -every cell in body can be traced back to a fertilized egg that came into existence from the union of egg and sperm. But the body is made up of over 200 different types of cells. Not just one. All of these cell types come from a pool of stem cells in the early embryo. -During early development, as well as later in life, various types of stem cells give rise to the specialized or differentiated cells that carry out the specific functions of the body, such as skin, blood, muscles and nerve cell. Stem Cell Characteristics ‘Blank cells’ (unspecialized) Capable of dividing and renewing themselves for long periods of time (proliferation and renewal) Have the potential to give rise to specialized cell types (differentiation) TYPES OF STEM CELLS 1. Embryonic Stem Cells- a. come from a five to six-day-old embryo. They have the ability to form virtually any type of cell found in the human body b. are derived from the part of a human embryo or fetus that will ultimately produce eggs or sperm (gametes). 2. Adult Stem Cells- a. are undifferentiated cells found among specialized or differentiated cells in a tissue or organ after birth. Based on current research they appear to have a more restricted ability to produce different cell types and to self-renew. STEM CELL TYPE Stem cell type Description Examples Totipotent Each cell can develop into a new Cells from early (1-3 days) individual embryos Pluripotent Cells can form any (over 200) cell types Some cells of blastocyst (5 to 14 days) Multipotent Cells differentiated, but can form a Fetal tissue, cord blood, and number of other tissues adult stem cells 5.2: Application of Stem Cells Disease Diabetes, Spinal cord injury, Parkinson’s disease, heart disease Genetic based Disease Cystic fibrosis, Huntington’s HOW THEY CAN COULD TREAT CERTAIN TYPES OF DISEASE? 1. Tissue Repair a. Regenerate spinal cord, heart tissue, or any other major tissues in the body 2. Heart Disease CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 31 of 46  a. Adult bone marrow stem cells injected into the heart arteries are believed to improve cardiac function in victims of heart failure or heart attack 3. Leukemia and cancer a. Studies show that leukemia patients treated with stem cells emerge free of disease b. Injections of stem cells have also reduced pancreatic cancers in some patients 4. Rheumatoid Arthritis a. Adult stem cells may be helpful in jumpstarting repair of eroded cartilage 5. Type I Diabetes a. Pancreatic cells do not produce insulin b. Basic research focused on understanding how embryonic stem cells might be trained to become pancreatic islet cells needed to secrete insulin Unknowns in Stem Cell/Cloning Research It is uncertain that human embryonic stem cells in vitro can give rise to all the different cell types of the adult body. It is unknown if stem cells cultured in vitro (apart from the embryo) will function as the cells do when they are part of the developing embryo Challenges to Stem Cell/Cloning Research Stem cells need to be differentiated to the appropriate cell type(s) before they can be used clinically. Recently, abnormalities in chromosome number and structure were found in three human ESC lines. Stem cell development or proliferation must be controlled once placed into patients. Possibility of rejection of stem cell transplants as foreign tissues is very high. Contamination by viruses, bacteria, fungi, and Mycoplasma possible. The use of mouse “feeder” cells to grow ESC could result in problems due to xenotransplantation (complicating FDA requirements for clinical use). Ethics Reference: Understanding Stem Cells: An overview of the science and issues from the national academies retrieved on July 11, 2020 from https://www.nap.edu/resource/11278/Understanding_Stem_cells.pdf Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C TOPIC 6: VARIATION IN CHROMOSOME STRUCTURE 6.1: Variation in Genetic Structure New Chromosomes Normal human somatic cells have 46 chromosomes: 22 pairs, or homologs, of autosomes (Chromosome 1-22) and two sex chromosomes. This is called the diploid number. Females carry two X-chromosomes (46, XX) while males have an X and Y (46, XY). Germ cells (egg and sperm) have 23 chromosomes: one copy of each autosome plus a single six chromosomes. This is referred to as the haploid number. One chromosome from each autosomal pair plus on sex chromosomes is inherited from each parent. Mothers can contribute only an X chromosome to their children while fathers can contribute either an X or a Y. Genetic Variation Genetic variation refers to differences between members of the same species or those of different species Allelic variations are due to mutations in particular genes Chromosomal aberrations are substantial changes in chromosome structure ▪ These typically affect more than one gene ▪ They are also called chromosomal mutations CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 32 of 46 I.ALTERATION IN CHROMOSOME STRUCTURE There are two primary ways in which the structure of chromosomes can be altered: 1.The total amount of genetic information in the chromosome change: Decrease: Deficiencies/Deletion Increase: Duplication and Insertion 2. Genetic material may remain the same in number, but is re arranged a. Inversions b. Translocations Structural abnormalities involve changes in the structure of one or more chromosomes. TOTAL AMOUNT OF GENETIC INFORMATION 1.DELETION – involve loss of material from a single chromosome. The effects are typically severe since there is a loss of genetic material - deletion may be spontaneous of may be induced -Mis-division of the centromere -Radiation, UV, Chemicals, viruses may increase breakage -Deletions do not revert because the DNA is gone (degraded) -the effect of a deletion depends on what was deleted TYPES OF DELETION 1.Terminal Deletion- It involved a single break and the terminal part of the chromosome is lost. 2.Interstitial Deletion- Deletion that does not involve the terminal parts of a chromosome A deletion in one allele of a homozygous wildtype organism may give a normal phenotype While the same deletion in the wild-type allele of a heterozygote would produce a mutant phenotype. Deletion of the centromere results in an acentric chromosome that is lost, usually with serious or lethal consequences. No known living human has an entire autosome deleted from the genome. Example of human disorders caused by large chromosomal deletions: o Cri-du-chat (“cry of the cat”) syndrome (OMIM 123450), resulting from deletion of part of the short arm of chromosome 5 Facial Dysmorphisms Including microcephaly, round face, hypertelorism, epicanthal folds, low-set ears, and micrognathia. CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 33 of 46  Severe psychomotor and mental retardation Other health problems associated with CdC: Poor-suck, hypotonia, respiratory and heart defects, growth retardation, and cleft palate and/or lip. CdC patients are generally very sociable, but may exhibit maladaptive behaviors such as inattentiveness, hyperactivity, temper-tantrums, and self injury. 2.DUPLICATION - Duplications result from doubling of chromosomal segments, and occur in a range of sizes and locations TYPES OF DUPLICATION Tandem duplications are adjacent to each other. Reverse tandem duplications result in genes arranged in the opposite order of the original. Tandem duplication at the end of a chromosome is a terminal tandem duplication An example is the Drosophila eye shape allele, Bar, that reduces the number of eye facets, giving the eye a slit-like rather than oval appearance Another example is the Charcot–Marie–Tooth Disease o The foot of a person with Charcot–Marie–Tooth disease: CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 34 of 46  o The lack of muscle, a high arch, and claw toes are signs of this genetic disease. o caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. TOTAL AMOUNT OF GENETIC INFORMATION IS THE SAME BUT REARRANGED 1. INVERSIONS -occur when there are two breaks within a single chromosome and the broken segment flips 180 degrees (inverts) and reattaches to form a chromosome that is structurally out-of-sequence. -There is usually no risk for problems to an individual if the inversion is of familial origin (has been inherited from a parent) -There is a slightly increased risk if it is a de novo (new) mutation due possible to an interruption of a key gene sequence; although an inversion carrier ay be completely normal, they are slightly increased risk for producing chromosomally unbalanced embryo. This is because an inverted chromosome has difficulty pairing with its normal homolog during meiosis, which can result if gametes containing unbalanced derivative chromosomes if an unequal cross-over event occurs. TWO TYPES OF INVERSION How do inversions behave genetically? Crossing-over within the inversion loop of a paracentric inversion connects homologous centromeres in a dicentric bridge while also producing an acentric fragment—a fragment without a centromere. Then, as the chromosomes separate in anaphase I, the centromeres remain linked by the bridge, which orients the centromeres so that the noncrossover chromatids lie farthest apart. The acentric fragment cannot align itself or move and is, consequently, lost. Tension eventually breaks the bridge, forming two chromosomes with terminal deletions The gametes containing such deleted chromosomes may be inviable but, even if viable, the zygotes that they eventually form are inviable. Hence, a crossover event, which normally generates the recombinant class of meiotic products, instead produces lethal products. The overall result is a lower recombinant frequency. In fact, for genes within the inversion, the RF is zero. For genes flanking the inversion, the RF is reduced in proportion to the relative size of the inversion. Inversions affect recombination in another way too. Inversion heterozygotes often have mechanical pairing problems in the region of the inversion these pairing problems reduce the frequency of crossing-over and hence the recombinant frequency in the region. The net genetic effect of a pericentric inversion is the same as that of a paracentric one… In a pericentric inversion, because the centromeres are contained within the inverted region, the chromosomes that have crossed over disjoin in the normal fashion, without the creation of a bridge. However, the crossover produces chromatids that contain a duplication and a deficiency for different parts of the chromosome CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 35 of 46  In this case, if a nucleus carrying a crossover chromosome is fertilized, the zygote dies because of its genetic imbalance. Again, the result is the selective recovery of noncrossover chromosomes in viable progeny. NOTE: Two mechanisms reduce the number of recombinant products among the progeny of inversion heterozygotes: elimination of the products of crossovers in the inversion loop and inhibition of pairing in the region of the inversion. What about homozygous inversions?  In such cases the homologous inverted chromosomes pair and cross over normally, there are no bridges, and the meiotic products are viable. CONSEQUENCIES OF CHROMOSOME INVERSION IN HUMANS: lowered fertility due to production of unbalanced gametes 2.TRANSLOCATION -Translocation involve exchange of material between two or more chromosomes. If a translocation is reciprocal (balanced) the risk for problems to an individual is similar to that with inversions: usually none if familial and slightly increased if de novo. Problems arise with translocations when gametes from a balanced parent are formed which do not contain both translocation products. When such a gamete combines with a normal gamete from the other parent result is an unbalanced embryo which is partially monosomic for one chromosome and partially trisomic for the other. There are two main types of Translocation: 1. Reciprocal (Balanced) Translocation 2.Robertsonian (unbalanced) Translocation *Both types are capable of causing disease in human ROBERTSONIAN TRANSLOCATIONS -the transfer of genetic material occurs in only one direction -are associated with phenotypic abnormalities or even lethality -Example: Familial Down Syndrome -In this condition, the majority of chromosome 21 is attached to chromosome 14 -The individual would have three copies of genes found on a large segment of chromosome 21; Therefore, they exhibit the characteristic of Down Syndrome -This translocation occurs as follows: Breaks occur at the extreme ends of the short arms of two non-homologous acrocentric chromosomes CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 36 of 46  The larger fragments fuse at their centromeic regions to form a single chromosome The small acrocentric fragments are subsequently lost This type of translocation is the most common type of chromosomal rearrangement in humans -Robertsonian Translocations are confined to chromosomes 13, 14, 15 21 (the acrocentric chromosomes) CHROMOSOMAL MUTATIONS AND HUMAN TUMORS Most human malignant tumors have chromosomal mutations Most common are translocations There is much variation in chromosome abnormalities, however, and they include simple rearrangements to complex changes in chromosome structure and number Many tumor types show a variety of mutations Some, however, are associated with specific chromosomal abnormalities  Follicular lymphoma is a type of non-Hodgkin lymphoma.  It develops when the body makes abnormal B-lymphocytes – the lymphoma cells. (B-lymphocytes are white blood cells that fight infection).  The lymphoma cells build up in lymph nodes. The most common symptom is a painless swelling in the neck, armpit or groin. CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 37 of 46 Burkitt’s Lymphoma vs. Small Noncleaved Non-Burkitt’s Burkitt’s Lymphoma High grade tumor Uniform appearance of abnormal cells t(8;14); t(8;22) or t(8;2) Endemic in equatorial Africa Small noncleaved non-Burkitt’s High-grade tumor Variability in size and shape of abnormal cells t(8;14); t(8:22) or t(8;2) ISOCHROMES a structure where a chromosome has lost one of its arms, and the replacement arm is an exact mirror image of the remaining arm Example: Pallister-Killian mosaic syndrome o a developmental disorder that affects many parts of the body. o characterized by extremely weak muscle tone (hypotonia) in infancy and early childhood, intellectual disability, distinctive facial features, sparse hair, areas of unusual skin coloring (pigmentation), and other birth defects. II. ALTERATION IN CHROMOSOME NUMBER Numerical abnormalities involve the loss and/or gain of a whole chromosome or chromosomes and can include both autosome and sex chromosomes. Examples include: Down Syndrome, Edward’s Syndrome and the likes Each species has a cha