Introduction to Cytogenetics PDF

INTRODUCTION TO CYTOGENETICS KAMILLE SAMSON Rmt2023 RMT 2023 CYTOGENETICS: RE...

INTRODUCTION TO CYTOGENETICS KAMILLE SAMSON Rmt2023 RMT 2023 CYTOGENETICS: REVIEW OF RELATED TERMINOLOGIES Transfer RNA (tRNA) GENETICS AND GENES RNA that delivers amino acids to the sites of protein GENETICS synthesis the study of inherited traits and their variation HEREDITY The sum of all biological processes by which particular characteristics are transmitted from parents to their offspring. GENES the units of heredity, which is the transmission of inherited traits. Genes can be found on the nucleic acid DNA that is usually GENES, CHROMOSOMES AND GENOMES located in the chromosomes that controls the development GENOME of one or more traits and it is the basic unit of genetic it is the complete set of genetic instructions characteristic of information is passed from parents to offspring. an organism, including protein-encoding genes and other DNA sequences THE NUCLEIC ACIDS in humans, a copy of entire genomes is more than 3 billion There are four different nucleotides present in a DNA DNA pairs that is contained in all the cells that contain molecule. nucleus The various sequence combinations of these bases ultimately CHROMOSOMES encode genetic information. structures that are a product of DNA coiling in association with proteins DEOXYRIBONUCLEIC ACID (DNA) a molecule whose function is to store and transfer genetic ✓ the segment of DNA strand that encodes for the production information of different proteins, may have different variants. These are make the proteins that living things needed to grow called alleles. Adenine, Thymine, Guanine, Cytosine (CATG) ✓ Alleles-pair of genes that occupy a certain location in the genes; alternate form of genes; product of mutation RIBONUCLEIC ACID (RNA) Important molecule in protein synthesis Directly codes for amino acids Acts as a messenger between DNA and Ribosomes to make proteins Adenine, Uracil, Guanine, Cytosine (GUAC) ✓ Both DNA and RNA are polymers of repeating called nucleotides CENTRAL DOGMA DNA sequence-builds the genes of an organism which in turn as a process in which the information in the DNA is converted encode for particular proteins into a functional product which is the proteins. MUTATIONS, PHENOTYPE, AND GENOTYPE TRANSCRIPTION Alleles are products of mutations. MUTATIONS defined as any heritable change in the DNA sequence and are the source of all genetic variation phenomenon of change occurring in DNA sequences PHENOTYPE observable traits or features of an organism (alleles that are expressed) Synthesis of RNA copy of a segment of a DNA. RNA will physical traits; eye color synthesize by the enzyme RNA polymerase GENOTYPE TRANSLATION set of alleles for a given trait carried by an organism (alleles Process of translating the sequence of a mRNA molecule of a that are present) sequence of amino acids during protein synthesis. responsible for unique traits/characteristics: diseases The RNAs MITOSIS AND MEIOSIS Messenger RNA (mRNA) MITOSIS carries the instructions for protein synthesis to the sites of a type of cell division in which one somatic cell give rise to two protein synthesis new ones Ribosomal RNA (rRNA) cell duplication/reproduction; 2 genetically identical daughter combines with proteins to form ribosomes cells MEIOSIS cell division involved in the production of gametes specialized type of cell division that reduces chromosome numbers by half and creating 4 haploid cells. Each genetically distinct to parent cells that give rise to them. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I IMMUNOSERO 1  BRANCHES OF GENETICS gamete formation (the formation of egg cells and sperm) CLASSICAL GENETICS CHROMOSOME THEORY OF INHERITANCE refers to the study of the laws of hereditary transmission in living organisms. It began with Mendel’s study of Heredity is dependent on the genes contained in the inheritance in garden peas. structures called chromosomes. The chromosomes were Mainly based on phenotype characteristics: size, shape contributed to the individual by the gametes (ova and POPULATION GENETICS sperm cells) The study of genes in populations of animals, plants, and Diploid number (2n)- the characteristic number of microbes provide information on past migrations, chromosomes a eukaryote has in most of its cells evolutionary relationships and extents of mixing among Chromosomes in diploid cells exist in pairs called different varieties and species, and methods of adaptation homologous chromosomes to the environment. Haploid chromosomes- one copy of each chromosomes CYTOGENETICS (non-homologous)- gametes and each cell carries of one branch of genetics that study the structure of the DNA copy coming from the mother and father within the cell nucleus. It studies the number and Chromosomes behave differently during the two forms of morphology of the chromosomes. cell division, mitosis and meiosis. MOLECULAR GENETICS In mitosis (duplication), the chromosomes are copied and the study of the molecular structure of DNA, its cellular distributed to each daughter cell. Both cells obtain a diploid activities (including its replication), and its influence in set of chromosomes. determining the overall makeup of an organism. In meiosis (division), the cells receive only one Study of genes using DNA techniques: DNA amplification chromosome from each chromosome pair, and the resulting Purpose: further the knowledge in genes and their behavior number of chromosomes is called the haploid number (n). CHROMOSOMAL THEORY OF INHERITANCE states that EARLY HISTORY OF GENETICS AND THEORIES OF “inherited traits are controlled by genes residing on INHERITANCE chromosomes faithfully transmitted through gametes, Epigenesis- way of genes to change in the phase of the maintaining genetic continuity from generation to environmental influence (+-) generation. ARISTOTLE proposed that “humors” served as bearers of traits humors-blood, yellow bile, black bile and phlegm first to publish the theory of epigenesis WILLIAM HARVEY proposed the theory of epigenesis C.F. WOLFF German physician that formulated the theory of epigenesis PREFORMATIONISM states that the fertilized egg contains a complete miniature adult called a homunculus Homunculus-fully formed individual that existed within the germ cell of one of its parents prior to fertilization and grow in size until it is ready to be born CELL THEORY VS SPONTANEOUS GENERATION Cell theory- only cells are capable to produce more CHEMICAL NATURE OF GENES Spontaneous generation- idea that organisms can develop independently of cells The major chemical component of chromosomes were o It isn’t supported by cell theory DNA and proteins. o the living organisms develop from the non-living matter STRUCTURE OF DNA AND RNA CHARLES DARWIN proposed that existing species arose by descent with modification from ancestral species formulated theory of Natural Selection ✓ Natural selection states that individuals with heritable traits that allow them to adapt to their environment are better able to survive and reproduce than those with less adaptive traits. GREGOR MENDEL published a paper describing how traits are passed from one generation to the other, utilizing pea plants as models proposed that traits are passed from parents to offspring in a predictable manner (punnette square) further concluded that each trait in pea plants is controlled by a pair of factors (which we now call genes) and that DNA is a long, ladder-like macromolecule that twists to form members of a gene pair separate from each other during a double helix. Each strand of the molecule is made up of nucleotides. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I IMMUNOSERO 1  The four types of nucleotides found in DNA are: A The cloned DNA fragments can be isolated from the (adenine), G (guanine), C (cytosine), T (thymine) bacterial host cells. The DNA ladder are exact complements of each other, so Such fragments can be used to isolate genes, study their that the double helix consists of A=T and G=C base pairs. organization and expression, and to study their nucleotide The RNA is also made up of nucleotides but contains a sequence and evolution. different sugar than DNA. (ribose) It is a single-stranded molecule that contains uracil (U) in place of thymine. A=U, C=G GENE EXPRESSION: FROM DNA TO PHENOTYPE The genetic information in the DNA is expressed to form a functional gene product, which in most cases, a protein. In eukaryotic cells, the process begins in the nucleus with transcription. The mRNA produced then moves to the cytoplasm and migrates to the ribosomes. The synthesis of protein under the direction of the mRNA is called translation. Eukaryotic cells- found in humans, plants, animals, fungi, and insects and it has a nucleus Prokaryotic cells- found in the bacteria, much less complicated and no nucleus Information encoded in mRNA (the genetic code) consists BIOTECHNOLOGY of linear series of nucleotide triplets (codon). Genetic code- set of rules by which the information The use of recombinant DNA technology and other encoded in genetic material from DNA to RNA sequences molecular techniques to make products is called are translated into produce by living cells. Genes that codes biotechnology-technology that utilizes biological system of for proteins are called as codons. a living organism to develop different products. Each codon is complementary to the information stored in The use of recombinant DNA technology to genetically DNA and specifies the insertion of a specific amino acid into modify crop plants has revolutionized agriculture. a protein. Biotechnology has also changed the way human proteins Protein assemble is accomplished with the aid of tRNAs. for medical use are produced. Biotechnology-derived genetic testing is now available PROTEINS AND BIOLOGICAL FUNCTION to perform prenatal diagnosis of heritable disorders and to test parents for their status as heterozygous carriers of Proteins perform diverse biological functions more than 100 inherited disorders. Enzymes, the largest category of proteins, serve as biological catalysts GENOMICS, PROTEOMICS AND BIOINFORMATICS Enzymes- biological molecule that speed up the rate of chemical reactions that takes place within cells. There have been efforts to decode each gene in the Other types are critical components of cells and organisms genome and establish its function Some carry essential molecules (hemoglobin), regulate Genomics-study of genome. It studies the structure, body processes (protein hormones e.g., insulin), take part function, and evolution of genes and genomes. Uses of in muscle (actin and myosin) and connective tissue combination recombinant DNA, DNA sequencing method (collagen) and bioinformatics to sequence, assemble and anlyze the A protein’s shape and chemical behavior are determined structure of genome by its linear sequence of amino acids, which in turn is Genome- complete set of instructions of an organism, dictated by the stored information in the DNA of a gene that stored in chromosomes is transferred to RNA, which then directs the protein’s Proteomics-identifies the set of proteins present in a cell synthesis. under a given set of conditions, and studies their functions Once a protein is made, its biochemical or structural and interactions properties play a role in producing a phenotype. o Proteome- entire set of proteins When mutation alters a gene, it may modify or even Bioinformatics- subfield of information technology used to eliminate the encoded protein’s usual function and cause store, retrieve and analyze the massive amount of data an altered phenotype. generated by genomics and proteomics RECOMBINANT DNA TECHNOLOGY MODEL ORGANISMS IN GENETIC STUDIES Researchers discovered restriction enzymes/ Principles of inheritance described by Mendel were endonuclease that could be used to cut any organism’s universal among plants and animals DNA at specific nucleotide sequences, therefore producing Geneticists gradually came to focus attention on small a reproducible set of DNA fragments. number of organisms, including the fruit fly (Drosophila Soon researchers discovered ways on how to insert the melanogaster) and the mouse (Mus musculus) DNA fragments into carrier DNA molecules (vectors) to Reasons for using small number of organisms: form recombinant DNA molecules using DNA ligases o genetic mechanisms were the same in most (connect 2 strands of DNA together forming a bondo on organisms phosphate and deoxyribose). o these organisms had characteristics that made The recombinant DNA will be transferred into bacterial cells them especially suitable for genetic research. to produce thousands of copies, or clones. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I IMMUNOSERO 2  They were easy to grow, had relatively short life cycles, produced many offspring, and their genetic analysis was fairly straightforward. They were called model organisms GENETICS, ETHICS AND SOCIETY Genetics and its applications in biotechnology are developing much faster than the social conventions, public policies, and laws required to regulate their use. There are many genetics related issues, including concerns about prenatal testing, genetic discrimination, ownership of genes, access to and safety of gene therapy, and genetic privacy. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I IMMUNOSERO 3  CELLS AND ORGANELLES PROFESSOR: MAAM KAMILLE SAMSON Rmt2023 CELLS CELL STRUCTURES AND ORGANELLES Cell is the basic unit of life Cells are highly varied and highly organized structures Their forms and functions are dependent on the genetic expression by each cell type Bone, blood, nerve and muscle cells are somatic cells, also called as body cells Somatic cells have two copies of the genome and are said to be diploid Sperm and egg cells have only one copy of the genome and are said to be haploid All cells are surrounded by a plasma membrane, a covering that defines cell boundary PROKARYOTES VS EUKARYOTES o The plasma membrane actively controls the movement of materials in and out of the cell Prokaryotes lack nucleus as opposed to the nucleated cells of o Support and protect the cell. the eukaryotes o Semi-permeable Specialized organelles are also present in eukaryotic cells Most animal cells have glycocalyx or cell coat Eukaryotes and prokaryotes both have cell membrane, o The glycocalyx provides biochemical identity at the cytoplasm, ribosomes, DNA (eu:nucleus, pro: not in nucleus) surface of cells, and the components of the coat that Eukaryote has membrane bound organelles while prokaryote establish cellular identity are under genetic control has none (eukaryotic) CHEMICAL CONSTITUENTS OF CELLS o Prokaryotic: it serves as a protecting factor against host factor Cells are composed of macromolecules important in The nucleus is a membrane bound structure that houses the biological processes. The major groups of these substances DNA, which is complex with protein into thin fibers are: carbohydrates, lipids, proteins and nucleic acids. o During the nondivisional phases of the cell cycle, Carbohydrates provide energy the fibers are uncoiled and dispersed into chromatin Lipids form membranes and hormones, provide insulation and (contains DNA and proteins) store energy; building blocks of the structure and functions of o During mitosis and meiosis, chromatin fibers coil living cells (fats, oils, wax) and condense into chromosomes (humans has 23 Proteins have many diverse functions in the body, and are pairs of chromosomes) important in blood clotting, nerve transmission, muscle o The nucleolus, present inside the nucleus, is where contraction, and immunity, while others serve and catalysts ribosomal RNA is synthesized Most important in genetics are the nucleic acids DNA and In prokaryotes, the genetic material is compacted into an RNA unenclosed region called the nucleoid (nucleus like) o Nucleoid- irregularly shape region within the cells of the prokaryotes contain the genetic materials of the cells The DNA in prokaryotes is not associated with proteins as is the case with eukaryotes OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 4  The remainder of the cell within the plasma membrane, MICROTUBULES excluding the nucleus, is called as the cytoplasm and includes a variety of organelles Microtubules are long and o Cytoplasm: thick and jelly like substance present in hollow the cell membrane It provides many cellular o Most of chemical reaction takes place movements The endoplasmic reticulum appears smooth in places Composed of a pair of where it serves as site for synthesis of fatty acids and protein called tubulin phospholipids, and in other places, appears rough as it is They form the cilia, which studded with ribosomes (bound ribosomes) are hair-like structures o Responsible for transportation of substances throughout the cells MICROFILAMENTS o Plays a primary role in metabolism of carbohydrates, synthesis of lipids, steroids and proteins. These are long, thin rods composed of many molecules of the Ribosomes are sites of protein synthesis, guided by the protein actin information contained in the mRNA Solid and narrower than microtubules, they enable cells to The mitochondria provide energy by breaking down nutrients withstand stretching and compression from food. The energy liberated from food is captured and They also help anchor one cell to another stored in the bonds present in a molecule called adenosine triphosphate (ATP) INTERMEDIATE FILAMENTS o Powerhouse of the cell The centrioles are a pair of complex structure that are located They have diameters intermediate between those of in a specialized region called the centrosome (found only in microtubules and microfilaments animal cells; plays a major role in organizing microtubules). It has a protein dimer o These are associated with the organization of spindle They are abundant in skin and nerve cells fibers that function in mitosis and meiosis. In actively dividing skin cells, it forms a strong inner o The organization of spindle fibers by the centrioles framework that firmly attaches cells to each other and to the plays an important role in the movement of underlying tissue chromosomes during cell division Notes: Modern Cell theory CYTOSKELETON ✓ Cell is the smallest unit in all organisms ✓ All living things are made of cells The cytoskeleton is a meshwork of protein rods and tubules ✓ All cells come from pre-existing cells that molds the distinctive structures of a cell, positioning organelles and providing three-dimensional shape. o Network of fibers forming the eukaryotic cell, prokaryotic cells and Archaean’s (single cell prokaryotes) o Provides shape and support to the cell and organizes the cell, facilitate the transport of molecules, cell division and cell signaling. The cytoskeleton includes three major types of elements microtubules, microfilaments, and intermediate filaments. They are distinguished by protein type, diameter, and how they aggregate into larger structures. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 5  MEIOSIS AND CHROMOSOME MORPHOLOGY CYTOGENETICS WEEK 8 Rmt2023 MEIOSIS In the first meiotic anaphase, duplicated homologs are pulled As in mitosis, meiosis apart and segregated into the two daughter nuclei. is preceded by a Only in the second division (meiosis II) are the sister process of DNA replication that converts each chromosome into two sister chromatids. Meiosis is the form of eukaryotic cell division that produces haploid sex cells or gametes (which contain a single copy of each chromosome) chromatids pulled apart and segregated (as in mitosis) to from diploid cells produce haploid daughter nuclei. (which contain two produces four haploid nuclei, each of which contains either copies of each the maternal or paternal copy of each chromosome, but chromosome). not both The process takes the form of one DNA PROPHASE I: replication followed by subdivided into the following five phases based on two successive chromosomal behaviour nuclear and cellular o Leptotene, Zygotene, Pachytene, Diplotene and divisions (Meiosis I Diakinesis. and Meiosis II). take hours in yeasts, days in mice, and weeks in higher In these species, the reproductive cycle ends when a sperm and plants egg fuse to form a diploid zygote, which has the potential to form It is during early prophase I that the homologs begin to a new individual. associate along their length in a process THE KEY FEATURES OF MEIOSIS ARE AS FOLLOWS called pairing Meiosis involves two sequential cycles of nuclear and cell As prophase division called meiosis I and meiosis II but only a single cycle progresses, the of DNA replication. homologs become Meiosis I is initiated after the parental chromosomes have more closely replicated to produce identical sister chromatids at the S juxtaposed, forming phase. a four-chromatid Meiosis involves pairing of homologous chromosomes and structure called a recombination between them. bivalent DNA double-strand Meiosis I-separates the pair of homologous breaks are formed chromosomes at several locations in each sister chromatid, resulting in large o often called the reduction division numbers of DNA recombination events between the homologs Meiosis II- separates each chromosomes into two lead to reciprocal DNA exchanges called crossovers, chromatids where the DNA of a chromatid crosses over to become Four haploid cells are formed at the end of meiosis II. continuous with the DNA of a homologous chromatid MEIOTIC EVENTS CAN BE GROUPED UNDER THE o During the prophase I, homologous chromosome FOLLOWING PHASES pairs and exchange of DNA to form recombinant MEIOSIS INCLUDES T WO ROUNDS OF CHROMOSOME chromosomes SEGREGATION The first of these divisions (meiosis I) segregates the homologs. The duplicated paternal and maternal homologs pair up alongside each other and become physically linked by the process of genetic recombination. o Chiasma-cross over of the chromatids OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 6  homologous chromosomes are completed by the end of pachytene, leaving the chromosomes linked at the sites of crossing over. The final stage of meiotic prophase I is diakinesis. This is marked by decriminalization of chiasmata. During this phase the chromosomes are fully condensed and the meiotic spindle is assembled to prepare the homologous chromosomes for separation. By the end of diakinesis, the nucleolus disappears and the nuclear envelope also breaks down. Diakinesis represents transition to metaphase. Prometaphase I- chromosome attach to spindle fibers by the kinetochores Leptotene: the homologs condense and pair METAPHASE I o chromatin material begins to condense; The bivalent chromosome becomes visible; sister chromatids are chromosomes align on tightly associated and not separately visible. the equatorial plate Zygotene: chromosomes continue to shorten and thicken The microtubules from the synaptonemal complex begins to assemble at sites where the opposite poles of the homologs are closely associated and recombination the spindle attach to the events are occurring. pair of homologous The complex formed by a pair of synapsed homologous chromosomes. chromosomes is called a bivalent or a tetrad. Homologous pairs of the chromosomes in bivalent arranged Pachytene: the assembly process is complete, and the as a double row along in the metaphase plate. The homologs are synapsed along their entire lengths. During this arrangement of the paired chromosomes with the spindle stage bivalent chromosomes now clearly appears as tetrads. apparatus is random along with the metaphase plate. o Crossing over of homologous chromosome to produce chiasmata ANAPHASE I Diplotene: the disassembly of the synaptonemal complexes The homologous chromosomes and the concomitant condensation and shortening of the separate, while sister chromatids chromosomes remain associated at their o Homologous chromosomes start to separate but centromeres remain attach to chiasmata Homologous pairs of the o within each tetrad, each pair of sister chromosomes in bivalent are chromatids begins to separate; synaptonemal separated and moves to the complex can no longer be seen; chromosomes opposite poles of the cell continue to condense, and all four chromatids of the tetrad can be clearly see crossover events between nonsister chromatids can be seen TELOPHASE I as inter-homolog connections called chiasmata (singular The nuclear membrane and chiasma) nucleolus reappear, cytokinesis Diakinesis: Homologous chromosome will continue to follows and this is called as diad separate, the chiasma will move to the ends of chromosomes of cells. o chromosomes reach their most condensed state; The stage between the two nucleolus and nuclear envelope break down. meiotic divisions is called Diakinesis represents transition to metaphase interkinesis and is generally CROSSING OVER short lived. Crossing over is Interkinesis is followed by the exchange of prophase II, a much simpler prophase than prophase I. genetic material Telophase I-The chromosomes will become more diffuse and between two the nuclear membrane will form homologous Cytokinesis-final cellular division to form new cells and this is chromosomes. followed by meiosis II Crossing over is o Physical process of cell division which divides the also an enzyme- cytoplasm or the parental cell into 2 daughter cells mediated process Meiosis I-reduction division; the original diploid cells had two and the enzyme copies of each chromosomes involved is Newly formed haploid cells-one copy of each chromosome called Interkinesis (Interphase II)- period of rest that cells of some recombinase. species enter during meiosis bet meiosis I and meiosis II Recombination o No DNA replication will occur between o Replication does occur in interphase I OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 7  such chromosome segregation errors during egg development are the most common cause of both MEIOSIS II spontaneous abortion (miscarriage) and mental Separates chromosomes into two chromatids retardation in humans PROPHASE II: When homologs fail to separate properly—a phenomenon called nondisjunction— the result is that some of the Meiosis II is initiated immediately resulting haploid gametes lack a particular chromosome, after cytokinesis while others have more than one copy of it. meiosis II resembles a normal Upon fertilization, these gametes form abnormal embryos, mitosis most of which die. The nuclear membrane Segregation errors during meiosis I increase greatly with disappears by the end of advancing maternal age. prophase II. The chromosomes again become compact. “mother” cells are Chromosome will begin to condense, nuclear membrane will be dissolve, spindle fibers will be formed diploid (2n) METAPHASE II At this stage the chromosomes align at the equator and the microtubules from opposite poles of the spindle get attached to the kinetochores of sister chromatids. ANAPHASE II It begins with the simultaneous splitting of the centromere of each chromosome (which was holding the sister chromatids together), allowing them to move toward opposite poles of the cell Centromeres will divide and sister chromatids move to opposite ends of the cells as spindle fibers will be shortened TELOPHASE II Meiosis ends with telophase II, in which the two groups of chromosomes once again get enclosed by a nuclear envelope; cytokinesis follows resulting in the formation of tetrad of cells i.e., four haploid daughter cells. Chromosomes reach the opposite ends and the nuclear membrane will be formed SIGNIFICANCE OF MEIOSIS Meiosis is the mechanism by which conservation of specific chromosome number of each species is achieved across generations in sexually reproducing organisms, even though the process, per se, paradoxically, results in reduction of chromosome number by half. It also increases the genetic variability in the population of organisms from one generation to the next. Variations are very important for the process of evolution. MEIOSIS FREQUENTLY GOES WRONG Mistakes are especially common in human female meiosis, which arrests for years after diplotene: meiosis I is completed only at ovulation, and meiosis II only after the egg is fertilized. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 8  CHROMOSOME MORPHOLOGY prokaryotic cells (cells without defined nuclei) have smaller circular chromosomes, although there are many exceptions to this rule. CHROMOSOME NOMENCLATURE International System for Human Cytogenetic Nomenclature (ISCN) has been developed by the Standing Committee on Human Cytogenetic Nomenclature The pair of non-sex chromosomes (autosomes) are serially numbered, 1 to 22, as nearly as possible in descending order of length. (longest chromosomes will be the first in line) Identification of the chromosomes is based on size, position of centromere and other morphological features. Chromosome short arms are called p (petit) and long arms q (queue). THERE ARE FOUR T YPES OF CHROMOSOMES BASED UPON THE POSITION OF THE CENTROMERE METACENTRIC In this type of chromosome the centromere occurs in the center and all the four chromatids are of equal length SUBMETACENTRIC In this type of chromosome the centromere is a little away from the center and therefore chromatids of one side are slightly longer than the other side. ACROCENTRIC In this type of chromosome the centromere is located closer to one end of chromatid therefore the chromatids on opposite side are very long. A small round structure, attached by a very thin thread is observed on the side of Chromosomes shorter chromatid. There are 46 chromosomes in every somatic cell of a human The small round structure being. Of which 22 pairs (44) are autosomes 23rd pair (XX that is a part of the or XY) are sex chromosomes. chromatid is termed as The DNA molecule may be circular or linear, and can be satellite. composed of 100,000 to 10,000,000,000 nucleotides in a long chain. TELOCENTRIC Typically, eukaryotic cells (cells with nuclei) have large linear In this type of chromosome the centromere is chromosomes placed at one end of the chromatid and hence only one arm. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 9  Such telocentric chromosomes are not seen in human cells * Telomeres: long regions of repetitive non-coding DNA that cap chromosomes to stop replication; undergo partial degradation (i.e., become shorter) each time a cell undergoes division TWO WAYS IN REPRESENTING CHROMOSOMES SEM: Chromosomes (uncondensed in nucleus, upper right) OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 10  MODEL ORGANISM Yeast Eukaryotic system. Signaling molecules and cell A model organism is a non-human species that is cycle are nearly similar. extensively studied to understand basic biological Good model system to phenomena, with the expectation that discoveries made in the understand many human model organism can be extrapolated to other species, diseases including cancer including humans. and Werner’s syndrome Study of model organisms is for understanding human health Ease of genetic manipulation and diseases are one of many ways that genetics and allows its use for analyzing and functionally dissecting gene biotechnology changing in everyday life products from other eukaryotes. Can be a plant, animal or microbe that can be used to study Last decade four Nobel prizes were awarded for discoveries certain biological processes involving yeast. Baker’s yeast commonly used for bread Group of species that have been studied extensively since they making - Simplest eukaryotic organism are easy to maintain on their controlled laboratory conditions Studying its biology has enabled scientists to work out the but greatly because they possess a number of experimental connection bet. genes, proteins and functions that they carry advantages out in our cells Model is a simplified system that is accessible and easily Example of a yeast species – Saccharomyces cerevisiae manipulated (budding yeast) is a fungus, eukaryote, single-celled Ex: Testing effectiveness of vaccine on lab mice organism that grows haploid or diploid, has sexual/asexual reproduction, small in size at least in 5-10 um diameter, easy Typical Characteristics of Model Organisms to grow in lab, easily cultured, stored and manipulated Small adult size to easily contain genetically Rapid development with short life cycles Cab be breed in large numbers Worm (Caenorhabditis elegans) Readily available and inexpensive maintenance One of the best characterized Similar genes or similar-sized genomes to humans multicellular animals at the level of Tractability to experimental methodology easily genomics, genetics, embryology managed/controlled Its genome is fully sequenced C. elegans is unique in that it can be Model Organisms grown and genetically manipulated with the speed and ease of Bacteria (Escherichia coli) a microorganism while offering the features of a real animal Yeast C. elegans has a full set of organ systems, has complex Plant - Thale cress (Arabidopsis thaliana) sensory systems, shows coordinated behavior, and it is Worm - Caenorhabditis elegans possible to trace the lineage of every one of its approximately Fruit fly (Drosophila melanogaster) 1000 constituent cells Zebra fish (Danio rerio) hermaphrodite, parasite (nematode), eukaryote, small in size Chick Embryo at least 1 mm in length, diploid, easy and fast growth, short life Mouse (Mus musculus) cycle and stores at least 1000 eggs C. elegans Life Cycle and Research Bacteria Developmental and Cell biology Bacteria under the microscope Neurobiology Aging Human disease studies Bacteria as a Model Organism The foundations of molecular biology were based on studies of bacteria. Antibiotics Recombinant DNA technologies Ex: E. coli in intestine, prokaryote, single-celled organism, easily grown in lab and can be grown into millions of copies due to rapid growth, easily cultured, stored and manipulated, small size 2 um long and at least 0.5 um wide OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 11  Young, South-African scientist named Sydney Brenner simpler than the mouse so that he could study the worked with Francis Crick looking at the function of DNA development and function of the nervous system and it is clear for them that the future of molecular biology Development in ex vivo. Experiments or measurements done was to be investigating development and the nervous system on tissues from an organism in an external environment w/ → discovered C. elegans because its characteristics fitted on minimal alteration of natural conditions Sydney’s experimental criteria → considered model Entire initial development is transparent. organism 48hrs is enough for the development of most of the organ systems. Fruit fly (Drosophila melanogaster) Currently showing real promise in heart failure research due A versatile model organism that has to their unique ability to repair their own heart muscles → been used extensively for biomedical hope that some principles can be applied to humans research. Lays 200 eggs per week compared to 15 pups of mouse in Easy-to-manipulate genetic system and 21 days; used to identify genes and pathways underlying a can be used to study development, broad range of human diseases from cardiovascular and physiology and behavior. musculoskeletal diseases to death and cancer Biological complexity comparable to that of a mammal Many organ systems in mammals have well-conserved Chick Embryo homologues in Drosophila The chick embryo provides an excellent model system for Has provided new insights into forms of cancer, studying the development of higher vertebrates wherein neurodegenerative diseases, behavior, immunity, aging, growth accompanies morphogenesis biological process multigenic inheritance, and development causing a tissue/organ to develop its shape by controlling its At least 3 mm long, the females live for about 1 month at spatial distributor of our cell during the embryonic room temp. but can increase up to 2 months at lower temp. development and can lay 30-50 eggs per day, they also breed and feed on fermenting fruit or other sources of fermenting sugars such as wastes in drains (low maintenance) 75% of the genes that caused diseases on humans are also found in fruit fly Has short, simple reproductive cycle occurs within 8-14 days depending on the environmental temp and fertilization stage Simple diet, sources of carbs and proteins (inexpensive) Life Cycle of Drosophila Mouse (Mus musculus) lab mice preferred mammalian model for genetic research Closest mammalian model organism to humans Genes that code for proteins responsible for carrying out vital Zebra fish (Danio rerio) biological processes in both the Small size, short life cycle, ease of human and the mouse share a high degree of similarity. culture, and ability to readily produce Therefore, the mouse has already proven extremely useful in mutations relevant to human health development, genetic, and immunology studies and disease Transgenics and KO’s possible The embryonic development can be Knockout organism is used to study gene function usually by seen through its transparent egg and closely resembles that investigating the effect of gene loss of higher vertebrates A great system for studying and understanding human Other shared features with humans include blood, kidney, disease, as well as a mechanism for investigating new and optical systems treatment strategies in ways that cannot be done in humans In addition, its genome is half the size of the mouse and Cost-effective, cheap to look after, multiply quickly, human genomes, which is valuable in identification of key reproduce every 3 weeks, the time bet. the mice being born vertebrate genes. and giving birth is short usually around 10 weeks meaning George Streisinger (tropical fish enthusiast, University of several generations can be observed at once Oregon) – used a vertebrae model that was physiologically OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 12  1 mouse year = 30 human years laser material interrelations Thale cress (Arabidopsis thaliana) C. elegans Excellent genetics Limited Small flowering plant Hermaphrodites or external Has a small genome relative to other self-fertilization morphology plants and is easily grown under Fast generation times Less similar to laboratory conditions Powerful molecular human Amenable to some genetics techniques (cloning - Some particularly generation of transgenics process of taking embryological Allows insight into numerous features genetic info from one manipulations of plant biology, including those of living thing and difficult significant value to agriculture, energy, generating identical environment, and human health copies of it, RNAi - Eukaryote, diploid, at least 20-25 cm or biological process in higher in height, occupies small space, w/c RNA molecules short life cycle, easy to cross, seeds inhibit gene can be stored, popular tool for understanding molecular expression/translation biology of many plant traits, useful for genetic mapping and by neutralizing sequencing targeted mRNA) Genome sequence Relative Advantages and Limitations of Model Organisms complete ORGANISMS ADVANTAGES LIMITATIONS Few cells: 959 cells Yeast Excellent genetics Unicellular and lineages known Genes can be easily No distinct Morphology fully cloned tissues characterized Powerful molecular Embryological Zebra fish Simplest vertebrate Not yet trivial to techniques manipulations Good genetics clone genes Possess all basic difficult Transparent embryos Transgenics eukaryotic cell Targeted gene Embryo not trivial organelles disruption still manipulations No targeted Cell cycle control difficult possible gene disruption similar to animals although Organ systems Genome sequenced possible similar to other Excellent genetics vertebrates (e.g., RNAi effective eyes, heart, blood, Fast generation time gastrointestinal tract) Second site Rapid vertebrate suppressor or development enhancer screens Thale cress Universal model plant Embryological Powerful molecular Small size manipulations techniques Relatively short life non trivial Genes can be easily cycle cloned Small, sequenced Transgenic animals genome easily generated - Transformed easily mostly in mice, they Transgenics have foreign genes Chick/chick Availability, low-cost Limited deliberately inserted embryo Accessibility, outside genetics into their genome of mother Genome Targeted Great for sequenced misexpression of embryological genes in space and manipulation; time transplants of tissue Mosaic analysis: Easily transfected by determine where avian retroviruses gene acts - mosaic is Mouse Mammals, brains Classic an individual in w/c similar to human, all genetics diff. cells have diff. homologous difficult genotypes areas/cell types Early-acting Laser ablation of “Reverse” genetics: mutant single cells possible - targeted Kos phenotypes used genetically to Developmental difficult to study describe explosive overview same as for Embryonic all mammals OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 13  Large mutant manipulations collection difficult Construction of Development chimeric embryos and life cycle possible relatively slow Availability of material at all stages Source of primary cells for culture Table 1.2 Model Organisms Used to Study Human Diseases ORGANISMS HUMAN DISEASE E. coli Colon cancer and other cancers S. cerevisiae Cancer, Werner syndrome D. melanogaster Nervous system disorders, cancer C. elegans Diabetes D. rerio CVD M. musculus Lesch-Nyhan disease, CF, fragile-X syndrome and many other disease OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 14  Mendelian GENETICS (part 1) Figure 3-1. 7 pairs of contrasting traits and the results of mendel’s seven monohybrid crosses of the garden pea (Pisum sativum). In each case, MENDELIAN GENETICS pollen derived from plants exhibiting one trait was Gregor Johann Mendel (1856-1868) used to fertilize the ova of the plants ehbiting the 1st set or hybrid experimentations of other trait. In the F1 generation, 1 of the 2 traits was garden peas launching his career exhibited by all plants. The contrasting trait major postulates of transmission reappeared in approx.. ¼ of the F2 plants. genetics Filial 1 (F1) – first generation Filial 2 (F2) – second garden pea (Pisum sativum) generation discrete units of inheritance exist and predicted their behavior during Mendel’s simplest crosses involved only one pair of the formation of gametes (elementen) contrasting traits. (monohybrid cross made by mating Mendel’s postulates were accepted as through breeding individuals from two parent strains), the basis for the study of what is known from two parent strains, each exhibiting one of the two as transmission genetics contrasting forms of the character under study. GARDEN PEAS (Pisum sativum) Initially, we examine the first generation of offspring of easy to grow and hybridize artificially such a cross, and then we consider the results of selfing, self-fertilizing in nature but easy to the offspring of self-fertilizing individuals from this crossbreed experimentally, first generation. reproduces well and grows in The original parents constitute the P1 or parental, maturity within a single season only generation, their offspring are the F1 or first filial seven visible features (unit generation, and the individuals resulting from the characters), each represented by selfed F1 generation are the F2 or second filial two contrasting forms, or traits generation. Character: stem height: traits: tall When Mendel crossed tall plants with dwarf plants, the and dwarf resulting F1 generation consisted only of tall plants. He selected six other visibly (gibberellin) contrasting pairs of traits involving seed shape and When members of the F1 generation were selfed, color, pod shape and color, and pod and flower Mendel observed that 787 of 1064 F2 plants were tall, arrangement. while the remaining 277 were dwarf. Note that in this From local seed merchants, Mendel obtained two cross the dwarf trait disappears in the F1 only to reappear breeding strains those in which each trait appears in the F2 generation. in change generation after generation in self- To explain these results, Mendel fertilizing plants proposed the existence of particular In addition to his choice of a suitable organism, he unit factors for each trait. restricted his examination to one or very few pairs He suggested that these factors serve of contrasting traits in each experiment and kept as the basic units of heredity and accurate quantitative records are passed unchanged from From analysis of data, Mendel derived certain generation to generation, postulates that have become principles of determining the various traits transmission genetics expressed by each individual plant. Therefore, Mendel hypothesized precisely how such factors could account for the results of the monohybrid crosses “round” and “wrinkled” peas arise from the R gene. MENDELS’ FIRST THREE POSTULATES Using the consistent pattern of results from monohybrid causes, Mendel derived the following three postulates or the principles of inheritance UNIT FACTORS IN PAIRS Genetic characters are controlled by unit factors that exist in pairs in individual organisms. In each diploid, individual receives one factor from each parent ✓ Because the factors occur in pairs, three combinations are possible: two factors for tall, two factors for dwarf, or one factor for each trait. Every individual possesses one of these three combinations which determines the stem’s height OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 15  DOMINANCE/RECESSIVENESS When two unlike unit factors responsible for a single character are present in a single individual, one unit factor is dominant to the other, which is said to be recessive. ✓ the trait expressed in the F1 generation is controlled by the dominant unit factor ✓ the trait not expressed is controlled by the recessive unit factor ✓ pertains only when unlike unit factors are present in pairs Dominant and recessive are also used to designate traits. TERMINOLOGIES In this case, tall stems are dominant over recessive dwarf Genotype: the genetic constitution of an individual stems (PP, Pp, pp) Phenotype: the outward appearance of an individual SEGREGATION (purple, white) During the formation of gametes, the paired unit factors Dominant phenotype: the phenotype seen when two separate or segregate randomly so that each gamete alternative alleles are present together (Pp: purple) receives one or the other with equal likelihood. Dominant allele: the form of the gene that is expressed If a pair contains like units of factors, ex: both specific for tall, when two alternative alleles are present together (P>p) then all its gametes receive one of the same sides of unit of Recessive allele: the form of the gene that is not factor (in this case, tall trait) expressed when two alternative alleles are present If an individual contains unlike unit of factors, ex: one for tall together. and one for dwarf, then each gamete has a 50% probability Recessive phenotype: the phenotype that is only seen of receiving either tall or dwarf trait when two identical alleles are found together (pp: white) Homozygous: having two identical alleles (PP or pp) MODERN GENETIC TERMINOLOGY Heterozygous: having two different alleles (Pp) The physical appearance of PUNNETT SQUARES a trait is the phenotype of Genotypes and phenotypes resulting from combining the individual. Physical gametes by fertilization can be easily visualized by expressions of the info constructing a diagram called as Punnett square contained in unit factors This method of analysis Mendel’s unit factors illustrates F1 to F1 represent units of monohybrid cross inheritance called genes by Punnett square, named modern geneticists. after Reginald C. the phenotype is Punnett. determined by alternative the vertical column forms of a single gene represents those of the called alleles. female parent, and the When alleles are written in horizontal row pairs to represent the two- represents those of the unit factors (DD, Dd, or dd), male parent. these symbols are called the This process lists all genotype. Designates possible random genetic makeup of an fertilization events. individual for traits it The genotypes and describes, whether haploid phenotypes of all or diploid potential offspring are By reading genotype → ascertained by reading phenotype will be known the combinations in the When both alleles are the boxes same (DD or dd), the individual is homozygous or a homozygote; when the alleles are different (Dd), we use the term heterozygous or a heterozygote. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 16  THE TEST CROSS: ONE CHARACTER To distinguish the genotype, Mendel devised the testcross method. The organism expressing the dominant phenotype, but of unknown genotype, is crossed to a known homozygous recessive individual. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 17  Mendelian GENETICS (part 2) In the dihybrid cross: at least 12/16 in 2nd filial plants are yellow while 4/16 are green exhibiting the ratio of 3:1 12/16 of all the 2nd filial plants that have round seeds while 4/16 have wrinkled skids exhibiting the 3:1 ratio MENDEL’S DIHYBRID CROSS GENERATED A UNIQUE F2 RATIO Such a cross, involving two pairs of contrasting traits, is a dihybrid cross, or two factor cross. The F1 offspring will all be yellow and round. It is therefore apparent that yellow is dominant to green and that round is dominant to wrinkled. examined 2 characters simultaneously (2 pairs of contrasting traits)→dihybrid cross Sample: pea plant is having yellow seeds that are round breed with those having green seeds which are wrinkled→ Predict the frequenting of all possible 2nd filial phenotypes 1st filial offspring: all yellow and round (yellow is dominant by applying the product law of probabilities to green and round is dominant to wrinkled) 1st fililal offspring: 9/16 filial plants expresseed the yellow Probability of 2 or more independent events occurring and round traits; 3/16 yellow, wrinkled; 3/16 green, round; simultaneously is equal to the product of their individual 1/16 green, wrinkled probabilities Variation: 1st filial and 2nd filial results will remain unchanged → clear o E.g. the probability of 2nd filial plant in having yellow and round seeds (3/4) or (9/16) as it has ¾ of round and ¾ yellow INDEPENDENT ASSORTMENT 2 monohydrate cross separately: 2 sets of pairs as being inherited independently of each other→ chance of any In both process, the 1st filial genotypes are identical → 2nd filial generation are also identical in both cross exponents plant having yellow or green seeds is not influenced by the chance that this plant will have a round or wrinkled seeds thus because yellow is dominant to green seeds On the basis of similar results in numerous dihybrid the first filial plant in 1st theoretical cross, would have crosses, Mendel proposed a fourth postulate called yellow seeds independent assortment 2nd theoretical cross→all 1st filial plants would have a During gamete formation, segregating pairs of unit round skids (round is dominant to wrinkled) factors assort independently of each other. 1st filial plant in dihybrid cross → yellow, round This postulate stipulates that segregation of any pair of The predicted second filial generation results of the first unit factors occurs independently of all others. As a cross are ¾ of yellow, ¼ green and in the 2nd cross: ¾ result of random segregation, each gamete receives one round, ¼ wrinkled member of every pair of unit factors. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 18  o For one pair, whichever unit factor is received, does not influence the outcome of the segregation of any other Such a family tree is called a pedigree. By analyzing a parent thus acc to independent assortment→ all possible pedigree, we may be able to predict how the trait under combination of gametes should be formed in equal study is inherited—for example, is it due to a dominant or frequency recessive allele? o Each 1st filial generation fertilization event, each zygote has an equal probability of receiving one of When many pedigrees for the same trait are studied, we the four combinations from each parent can often ascertain the mode of inheritance. o If many offsprings are produced: 9/16 have yellow and round while 3/16 have yellow and wrinkled and 3/16 Forked-Line Method or Branch Diagram-much less green round seeds, 1/16 green wrinkled seeds = 9:3:3:1 difficult; simple application of the laws of probability (ideal ratio) that is established for the dihybrid cross o Small number of offspring: highly unlikely to match the ratio of 9:3:3:1 o MENDEL’S POSTULATE IN INDEPENDENT ASSORMENT: each pair of unit factors segregates independently: formed in equal probability Thus, according to the postulate of independent assortment, all possible combinations of gametes are formed in equal frequency The testcross can also be applied to individuals that express two dominant traits but whose genotypes are unknown. THE TRIHYBRID CROSS DEMONSTRATES THAT MENDEL’S PRINCIPLES APPLY TO INHERITANCE OF MULTIPLE TRAITS three pairs of contrasting traits, in what is called a trihybrid cross, or three-factor cross. When F1 individuals serve as parents, each produces eight different gametes in equal frequencies. At this point, we could construct a Punnett square with 64 separate boxes and read out the phenotypes. The results are easily calculated if the principles of segregation and independent assortment are followed 1st filial individuals→ heterozygous for all 3 pairs→ results in the phenotypic expression of the dominance of those capital letters PEDIGREES REVEAL PATTERNS OF INHERITANCE OF HUMAN TRAITS Pedigree/family tree: determine the mode of inheritance The traditional way to study inheritance has been to construct a family tree, indicating the presence or absence of the trait in question for each member of each generation. OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 19  hemophilia→ bleeding disorder; blood does not clot properly female: carrier OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CYTOGENETICS 20 PEDIGREE ANALYSIS -placed in birth order from left to right and labelled with Arabic numerals, from the eldest to youngest. PEDIGREE ANALYSIS -each generation is indicated by roman numerals. PEDIGREE REVEALS PATTERNS OF INHERITANCE OF MONOZYGOTIC HUMAN TRAITS for identical twins The traditional way to study inheritance has been to construct a diagonal lines are linked by a horizontal line identical twins family tree, indicating the presence or absence of the trait in (sex must be the same) question for each member of each generation. Such a family tree DIZYGOTIC is called a pedigree. By analyzing a pedigree, we may be able to for fraternal twins predict how the trait under study is inherited same as monozygotic but lacks a connecting line (sex must PEDIGREE CONVENTIONS be the same or different) -a PROBAND this is a person serving as the starting point for the genetic study of a family; first affected family member who seeks medical attention for genetics disorder individual whose phenotype first brought attention to the family indicated by an arrow connected to the designation p can be applied to either male or female PEDIGREE ANALYSIS