[BIO 130 LAB] 1 Cell Division & Karyotyping.pdf

CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 Repair tissue (such as regeneration/wound healing)...

CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 Repair tissue (such as regeneration/wound healing) TABLE OF CONTENTS Importance of Meiosis: I. Cell Cycle ○ production of gamete/sex cells for reproduction A. Interphase B. Mitosis (M) phase II. Mitosis A. Prophase B. Metaphase C. Anaphase D. Telophase III. Meiosis A. Interphase B. Meiosis I a. Prophase I b. Metaphase I Figure 1: Phases of the cell cycle c. Anaphase I d. Telophase I C. Meiosis II INTERPHASE a. Prophase II b. Metaphase II NOTE! not part of mitosis (only part of the cell cycle) c. Anaphase II Overview d. Telophase II ○ Distinct nucleus, intact nuclear membrane IV. Introduction to Karyotyping ○ Chromatin A. Karyogram Genetic materials inside the nucleus appear as thin, thread-like structures a. Normal Male ○ Within the nucleus, there are 1-2 nucleoli (dense, b. Normal Female darkly stained bodies formed by several B. Nomenclature of Chromosomes chromosomal materials that code for certain a. Causes of Chromosomal RNAs) Aberrations ○ Centrosome C. Some Chromosomal Abnormalities Near the nucleus V. Karyotyping Preparation and Analysis Contains centrioles A. Amniocentesis B. Procedure I. CELL CYCLE Series of events in a cell leading to its division and duplication Figure 2: Whitefish blastula in interphase stage. 80X. Can be divided into two periods: ○ Interphase - cell grows, accumulating nutrients G0 phase (Gap/Growth 0) _______________________________________________________ and duplicating its DNA Refers to both quiescent and senescent cells ○ Mitosis (M) phase - cell splits itself into 2 distinct ○ Quiescent cells → not actively proliferating and thus cells must temporarily halt their progression through Importance of Mitosis: the cell cycle ○ Replication of somatic cells to help organism: ○ Senescent cells → cell ages and permanently stops Grow dividing but does not die Develop Resting phase where the cell has left the cycle and has BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 1 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 stopped dividing ○ Metaphase Some cell undergo this phase semi-permanently (e.g. ○ Anaphase liver and kidney cells) ○ Telophase G 1 phase (Gap/Growth 1) ______________________________________________________ Starts from the end of the previous M phase until the beginning of DNA synthesis Biosynthetic activities of the cell resume at a high rate Variable per cell type Proceeds to S-phase after: ○ Cell is appropriate size ○ Synthesized all enzymes and proteins required for DNA synthesis Figure 3.1: Stages of Mitotic somatic cells of whitefish ○ No mutations (checkpoint of G1) blastula. 40X. S phase (Synthesis) ______________________________________________________ DNA synthesis When completed, all of the chromosomes have been replicated ○ 2 daughter DNA molecules G 2 phase (Gap/Growth 2) ______________________________________________________ Lasts until the cell enter mitosis Significant biosynthesis occurs during this phase ○ production of microtubules (required during the Figure 3.2: Phases of Mitosis process of mitosis) Inhibition of protein synthesis during G2 phase prevents the cell from undergoing mitosis PROPHASE Proceeds to M-phase after: ○ Completed synthesis Start of disappearance of nuclear membrane and ○ No mutations nucleolus in a cell Nucleus of cell is darker due to condensation or MITOSIS (M) PHASE thickening of chromatin materials in the nucleoplasm to form dark- stained string-like chromosomes Mitosis & Cytokinesis Centrioles → seen in the opposite poles Process by which a cell separates the chromosomes in Asters its nucleus into two identical sets ○ ray-like microtubule bodies Mitosis ○ radiating around each centriole and mitotic ○ Nuclear division spindles forming between centrioles ○ Multinucleated cell → nuclear division without Each chromosome is composed of 2 chromatids cytokinesis Cytokinesis ○ Divides the nuclei, cytoplasm, organelles, and cell membrane into 2 cells II. MITOSIS Occurs in somatic cells Produces 2 daughter cells that are genetically identical Figure 4.1: Whitefish blastula in prophase stage. 80X. Can be divided into 4 phases (PMAT): ○ Prophase BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 2 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 Figure 4.2: Early Prophase Figure 5.2: Metaphase Figure 4.3: Late Prophase Figure 5.3: Metaphase METAPHASE ANAPHASE Complete absence of nuclear membrane and 2 chromatids from each chromosome separate and nucleolus in a cell migrate to the opposite poles of the cell Centrioles have moved to the opposite poles of the cell anaphase = away and form spindle fibers Once the chromatids separate, these are considered Chromosomes as chromosomes reaching the opposite poles ○ Alignment in the equatorial plane/metaphase Early Anaphase plate of the cell (middle) ○ the area of separation of the chromatids is narrow ○ much shorter and more condensed ○ chromatids are located halfway between the ○ readily recognized equatorial plane and the opposite poles ○ made up of chromatids joined together at their Late Anaphase central region called centromere ○ the chromatids are already at the opposite poles Kinetochore of the cell ○ protein coat found in each of the sister chromatid Spindle fibers ○ attached at the centromere of each chromosome metaphase = middle Figure 6.1: Whitefish blastula in the early (left) and late (right) anaphase stage. 80X. Figure 5.1: Whitefish blastula in metaphase stage. 80X. BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 3 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 Figure 6.2: Anaphase Figure 7.2: Early Telophase Figure 6.3: Anaphase Figure 7.3: Early Telophase Late Telophase & Cytokinesis TELOPHASE ○ Cell membrane that formed the cleavage furrow completely (more constricted) ○ Separates the mitotic cell into 2 smaller daughter cells lying adjacent to each other ○ Each daughter cells presents the following: Smaller than the mother cell Distinct nucleus, nuclear membrane, and nucleolus Chromatids have dispersed to form light-stained granular chromatin material in Figure 7.1: Whitefish blastula in the early (left) and late the nucleoplasm (right) telophase stage. 80X. Disappearance of spindle fibers ○ Cytokinesis → division of the cytoplasm Early Telophase ○ Appearance of cleavage furrow on the cell membrane at the equatorial position cleavage furrow → constriction of the plasma membrane at the region of the equatorial plate ○ Spindle fibers are still visible ○ Chromatids are already at the opposite poles of the cell ○ Nuclear membrane and the nucleolus start to reappear ○ Asters and mitotic spindles disappear Figure 7.4: Late Telophase III. MEIOSIS Special type of cell division, specifically of germ cells, necessary for sexual reproduction in eukaryotes ○ Produces gametes (sperm and egg cells) BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 4 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 PROPHASE I _______________________________________________________ One diploid (2n) cell’s chromosomes undergoes recombination to form four haploid (n) cells OCCURS only in gonads ○ Spermatogenesis in testes ○ Oogenesis in ovaries INTERPHASE Figure 9: Meiotic Prophase Stage Longest phase of Meiosis ○ DNA exchange between homologous chromosomes results in chromosomal crossover ○ Paired chromosomes are called bivalents or tetrads, having 2 chromosomes and 4 chromatids through synapsis 1) LEPTONEMA Figure 8: Meiotic Interphase Stage GROWTH 1 (G1) PHASE ➔ Protein and enzyme synthesis for growth ➔ Chromosomes consists of a single molecule of DNA (identical to somatic cells) Figure 10: Leptonema Stage SYNTHESIS (S) PHASE ➔ Greek word for “thin threads” ➔ DNA Replication (chromosomes become a ➔ Individual chromosomes condense into visible complex of 2 identical sister chromatids) strands within the nucleus ➔ Considered diploid due to the same number of ➔ Along these are chromomeres or nucleosomes centromeres ◆ Localized condensations resembling beads on a string GROWTH 2 (G2) PHASE ➔ Very short duration of coiling chromosome fibers ➔ NOT PRESENT but corresponds to Prophase I ➔ Chromosomes consists of a single molecule of 2) ZYGONEMA DNA (identical to somatic cells) MEIOSIS I Referred as reductional division where 2 haploid (n) cells are produced with 46 chromatids or 23 Figure 11: Zygonema Stage chromosomes each ○ Segregates homologous chromosomes and sorts ➔ Greek word for “paired/yoked threads” them randomly, resulting in the reduction of the ➔ Chromosomes approx. line up with each other total chromosome number into half into homologous rough pairs ○ NOTE: chromosomes do not divide in this stage ➔ Synapsis takes place (zipperlike pairing) to form since a single centromere holds each pair of sister bivalents chromatids together BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 5 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 3) PACHYNEMA ➔ Greek word for “two threads” ➔ Distinct split appears between 2 chromosomes in each bivalent and chromatids become distinguishable ➔ Homologous chromosomes remain tightly bound at the chiasma Figure 12: Pachynema Stage 5) DIAKINESIS ➔ Greek word for “thick threads” ➔ Chromosomal crossover occurs and bivalents are formed ◆ Each bivalent appears to contain 4 members from the 2 pairs of sister chromatids referred as tetrad Figure 15: Diakinesis Stage ➔ Sex chromosomes are not wholly identical and only exchange info over a small region of ➔ Greek word for “moving through” homology ➔ First point in meiosis where 4 pair of tetrads are visible Homologous Chromosomes ➔ Chromatids further shorten and condense ○ Pair of chromosomes (maternal and paternal) that ➔ Terminalization occurs are similar in shape and size ◆ Chiasmata move towards the end of the ○ Tetrads (homologous pairs) carry genes controlling tetrad as the chromosomes separate the same inherited traits ➔ Nucleoli disappears, nuclear membrane ○ Each locus (position of gene) is in the same disintegrates into vesicles, and meiotic spindle position on homologues begins to form Crossing Over ○ Variation may occur between non-sister METAPHASE I _______________________________________________________ chromatids at the chiasmata (sites or crossing over) ○ Segments break and reattach to the other chromatid Sex Chromosomes ○ XX - female ○ XY - male Figure 16: Meiotic Metaphase Stage Chromosomes are maximally shortened and thickened Figure 13: Sex chromosomes Chiasmata becomes the only point connecting the non-sister chromatids together 4) DIPLONEMA Homologous pairs move together along the metaphase plate ➔ As the microtubules attach to the kinetochores, the homologous chromosomes align along an equatorial plane, bisecting the spindle Figure 14: Diplonema Stage BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 6 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 ANAPHASE I _______________________________________________________ PROPHASE II _______________________________________________________ Figure 19: Meiotic Prophase II Stage Figure 17: Meiotic Anaphase Stage Nucleoli and nuclear envelopes disappears Whole chromosomes (1 half of each tetrad) are pulled ➔ Chromatids shorten and thicken toward opposing poles, forming 2 haploid sets/ dyad ➔ Centrioles move to the polar regions and arrange ➔ Each chromosome still contains a pair of sister its spindle fibers chromatids ➔ Cell elongates in preparation for division METAPHASE II _______________________________________________________ TELOPHASE I _______________________________________________________ Figure 20: Meiotic Metaphase II Stage Centromeres contain 2 kinetochore that attach to Figure 18: Meiotic Telophase Stage spindle fibers from the centrosomes at each pole ➔ New equatorial metaphase plate is rotated by 90° Nuclear membrane forms around the dyads First meiotic division ends when chromosomes arrive ANAPHASE II _______________________________________________________ at the poles ➔ Each daughter cell now has half the number of chromosomes but each chromosome consists a pair of chromatids MEIOSIS II Also known as the equatorial/equational division where each dyad (2 haploid cells) is essential to achieve haploidy (4 haploid cells) Genetic result is fundamentally different from mitosis Figure 21: Meiotic Anaphase II Stage Centromeres are cleaved as the microtubules pull the sister chromatids apart, forming monads ➔ Sister chromosomes move toward opposing poles BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 7 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 ○ 22 pairs of autosomal chromosomes TELOPHASE II _______________________________________________________ ○ 1 pair of sex chromosomes Male: XY Female: XX Figure 22: Meiotic Telophase II Stage Marked by uncoiling and lengthening of the chromosomes as the spindle fibers disappear ➔ Nuclear envelope reform and cleavage produces Figure 23: Normal Male (Left) and Normal Female (Right) 4 daughter cells, each with a haploid set of Karyotype chromosomes NOMENCLATURE OF CHROMOSOMES IV. INTRODUCTION TO KARYOTYPING Karyotyping ○ A test to examine chromosomes in a sample of cells ○ Can detect some abnormalities associated with chromosome structure and number Figure 25: Parts of a metaphase chromosome Karyotype ○ can show prospective parents whether they have Metaphase Chromosome certain abnormalities that can be passed on to ○ sister chromatid their offspring ○ centromere ○ may be used to learn the cause of a child’s constriction point disability divides the chromosome into two sections or ○ can reveal the gender of a fetus “arms” ○ test for certain defects through examination of ○ p-arm cells from uterine fluid (amniocentesis) or short arm of the chromosome through sampling of placental membranes. ○ q-arm A part of cytogenetics long arm of the chromosome ○ Cytogenetics → study of the structure and ○ location of the centromere on each chromosome properties of chromosomes, chromosomal gives the chromosome its characteristic shape behavior during mitosis and meiosis, Metacentric → center of the chromosome chromosomal influence on the phenotype and the Human chromosomes 1, 3, 16, 19, 20 factors that cause chromosomal changes Submetacentric (L-shaped) → non-centrally located so that one arm is longer than the KARYOGRAM other Human chromosomes 2, 4, 12, 17, 18, and X Picture representation of all of the chromosomes in a Acrocentric → near the end of a cell organized into their homologous pairs chromosome Reveals genetic information for that cell Human chromosomes 13, 14, 15, 21, 22, and Y Normal Male and Female Human Karyotype _______________________________________________________ Telocentric → end or telomere region In normal diploid organisms, autosomal chromosomes not present in normal healthy human are present in two copies chromosome Total pairs of chromosomes: 23 BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 8 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 Figure 26: Types of chromosome based on centromere location Causes of Chromosomal Aberrations _______________________________________________________ Chromosomal Aberrations Figure 28: Comparison of Normal Karyotype (Left) and ○ changes to the structure or number of Karyotype of Trisomy 21 (Right) chromosomes ○ caused by errors during cell division Chromosomal Aberrations be categorized as: ○ Structural SOME CHROMOSOMAL ABNORMALITIES ○ Numerical (Aneuploidy) Structural Chromosomal Aberration Cri-du Chat Syndrome (46, X? 5p-) _______________________________________________________ ○ Deletion portion of the chromosome is removed ○ Duplication part of the chromosome is duplicated resulting in extra genetic material ○ Inversion genetic material is inverted ○ Translocation a piece of one chromosome has broken off from its original location and attached to another chromosome Numerical Chromosomal Aberration (Aneuploidy) Figure 29: Karyotype of Cri-du Chat Syndrome ○ Monosomy only one chromosome of a pair is present Also known as “cry of the cat syndrome” ○ Trisomy ➔ Cry sounds like a cat in distress due to the infant’s three copies of a chromosome instead of larynx/voice box being improperly developed a pair ➔ Cri-du chat babies are severely mentally retarded and have a small cranium Chromosome Affected: Partial chromosome no. 5 (damaged p) (Deletion) Number of Chromosomes: 45 1/2 Incidence Rate: 1/100,000 live births Death in infancy or early childhood Patau Syndrome (47, X? +13) _______________________________________________________ Figure 27: Causes of Normal Aberrations Figure 30: Karyotype of Patau Syndrome BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 9 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 Produces severe mental retardation ➔ Highly characteristic pattern of malformations: Elongated skull, narrow pelvis, rocker bottom feet, grasping 2 central fingers by thumb & middle ➔ Ears are often low set, mouth and teeth are small Chromosome Affected: no. 13 (Trisomy) Number of Chromosomes: 47 Incidence Rate: 1/5,000 live births Death in early infancy Figure 33: Incidence of Down Syndrome Births Edward’s Syndrome _______________________________________________________ Turner’s Syndrome _______________________________________________________ Figure 31: Karyotype of Edward’s Syndrome Figure 34: Karyotype of Turner’s Syndrome Also produces severe mental retardation ➔ Very characteristic malformations of the skull, pelvis, and feet Missing an X chromosome but visibly female Chromosome Affected: no. 18 (Trisomy) ➔ Shorter, chunky build, and does not develop Number of Chromosomes: 47 secondary sex characteristics (no menstruation or Death in early infancy breast development) ➔ Thick fold of skin on either side of the neck at birth ➔ infertile Down Syndrome (47, X? +21) _______________________________________________________ Chromosome Affected: Single X in female (XO) (Monosomy) Number of Chromosomes: 45 Frequency: 1/2500 live female births Triple X Syndrome _______________________________________________________ Figure 32: Karyotype of Down Syndrome One of the most common causes of mental retardation ➔ Marked by a short stature, broad hands, stubby fingers/toes, wide rounded face, large protruding tongue - makes speech difficult Figure 35: Karyotype of Trisomy X Syndrome High incidence of respiratory infections, heart defects, and leukemia Sterility sometimes occur Chromosome Affected: 21 (Trisomy) Normal mental ability Number of Chromosomes: 47 ➔ Distinguished from normal XX females only through Average risk of having it: 1/750 live births karyotyping Death in early infancy Chromosome Affected: Extra X in female (XXX) BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 10 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 (Trisomy) Number of Chromosomes: 47 Klinefelter’s Syndrome (47, XXY) _______________________________________________________ V. KARYOTYPING PREPARATION AND ANALYSIS Matching a chromosome with its homolog considers: ○ LENGTH ○ CENTROMERE LOCATION ○ BANDING PATTERN (thru staining) Misconception: Each dark band represents a single gene Fact: One thin band can contain hundreds of genes Figure 36: Karyotype of Klinefelter Syndrome AMNIOCENTESIS Tall and sterile, with small testicles and low testosterone Type of prenatal testing used to determine genetic ➔ Infertile and display poor sexual development abnormality within the fetus ➔ Some have female characteristics like breast Sampling of placental membranes wherein cells are development and may display learning and removed from the mother at approx. 16 weeks’ behavior problems gestation ➔ Subnormal intelligence Chromosome Affected: Extra X in male (XXY) PROCEDURE (Trisomy) Number of Chromosomes: 47 Occurs in about 1/1000 male births Jacob’s Syndrome (47, XYY) _______________________________________________________ Figure 38: Overview of Karyotyping 1) In a growth medium, a blood tissue sample is added and incubated for 2-3 days to stimulate mitosis until the metaphase stage. This is important for its chromosomes to be replicated, condensed, and visible to a microscope. Figure 37: Karyotype of Jacobʼs Syndrome 2) Cells are transferred to a tube, centrifuged, and stained to enhance the chromosomes before Chromosome aberration in which individuals are not photographing. markedly affected 3) After printing, arrange the chromosomes in Affected individuals tend to be taller as a group and homologous pairs (same length and centromere have a slightly higher risk for behavioral problems location) from largest to smallest. ➔ Normal intelligence 4) Use the Denver System of Classification for Chromosome Affected: Extra Y in male (XYY) Human Chromosomes for banding patterns Number of Chromosomes: 47 assisting. Incidence rate: 1/1000 live male births BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 11 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC CELL DIVISION AND KARYOTYPING BIO 130 LAB INTARMED 2030 | Dr. Vitor | LU2 SEM 1 | SY. 2024-2025 Figure 39: Denver System of Classification for Human Chromosomes 5) Use completed normal chromosome spread for males and females to compare it with the unknown chromosome spread. 6) Analyze the child’s karyogram if there are abnormalities present and provide a diagnosis. BIO 130 LAB LU 2 SEM 1 | IMED 2030 Page 12 of 12 DE LUNA, MD; ALMARIO, JMM; BISCOCHO, TMA; REYES, JCDC