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1 Introduction to Histology and Basic Histological Techniques 2 Definition

Histology:
  

branch of anatomy study of tissues does not deal only with tissues but also with functions

3 Tissue Preparation
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Steps in Tissue Preparation  Fixation  Dehydration  Clearing  Embedding  Sectioning  Mounting  Staining

4 Fixation
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Fixation


refers to the treatment of tissue with chemical agents that retards the alteration of the tissue subsequent to death or removal from the body maintains normal structure of the tissue most common fixatives used in light microscopy are: Both of these fixatives cross-link proteins, thus maintaining a life-like image of the object.  neutral buffered formalin  Bouins fluid

  

5 Dehydration
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Dehydration

 

process of removing water from the tissues graded series of alcohol baths beginning with 50% to 100% alcohol are

used to remove the water 6 Clearing


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Clearing
  

process of treating the tissues with xylene xylene is a chemical that is miscible with melted paraffin the tissue becomes transparent with xylene

7 Embedding
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Embedding  done to distinguish the overlapping cells in a tissue and extracellular matrix from one another  usual embedding medium is melted paraffin  the tissue is placed in a suitable container of melted paraffin until the tissue is completely infiltrated

8 Trimming and Sectioning


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Trimming


removal of excess paraffin wax from the tissue block cutting the tissue with the use of a microtome ideal thickness of tissue is about 5 to 10 um Can also be performed on specimens frozen in liquid nitrogen or on the rapid freeze bar of a cryostat

Sectioning
  

9 Mounting and Staining


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Mounting


placing tissue sections on adhesive-coated glass slide provides contrast to the tissue with the use of stains stains that are used are grouped into 3 classes:  stains that differentiate between acidic and basic components of the cell  specialized stains that differentiate the fibrous components of the extracellular matrix

Staining
 

metallic salts that precipitate on tissues, forming metal deposits on them

10 Staining
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Most commonly used stain in histology:  Hematoxylin and Eosin (H & E)  Hematoxylin is a base that colors the acidic components of the cells a bluish tint  DNA and RNA, nucleus and cytoplasm rich in  ribosomes have acidic pH and stain dark blue  (basophilic)  Eosin is an acid that dyes the basic components of the cell a pinkish color  regions of the cytoplasm have basic pH and  stain pink (acidophilic)


11 Light Microscope
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Compound Microscopes  composed of a specific arrangement of lenses


 

permits a high magnification and good resolution of being viewed several types of light microscopes are distinguished by:  type of light used as a light source  manner in which they use the light source

12 Digital Imaging techniques


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Digital Imaging Technique  utilize computer technology to capture and manipulate histologic techniques  images are stored in digital format and therefore are easy to archived and retrieval is almost instantaneous and thus allows electronic transmission of images  advantages  immediate visualization of acquired image  digital modification of the image  capability of enhancing the image by the use of commercially

available software

13 Advanced Visualization Procedure: Histochemistry


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Histochemistry  method of staining  provides information concerning the presence and location of intracellular and extracellular macromolecules  tissue structures are colored when a chemical group (e.g. carboxyl, phosphoric or aldehyde) reacts with the stain  suitable for diagnosis of pathologic conditions  not suitable for routine tissue staining

14 Advanced Visualization Procedure: Histochemistry


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Histochemistry  capitalize on enzyme activity, chemical reactivity or other physicochemical phenomena associated with the constituent of interest  permits relatively good localization of some enzymes and macromolecules  reactions of interest are monitored by the formation of an insoluble precipitate that takes on a certain color  performed on frozen tissues and can be applied to both light and electron microscopy  Ex. Periodic Acid Schiff Stain

15 Advanced Visualization Procedure: Histochemistry 16 Advanced Visualization Procedure: Immunocytochemistry


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Immunocytochemistry  uses fluoresceinated antibodies and antiantinbodies to provide more precise intracellular and extracellular localization of macromolecules than is possible with histochemistry


there are two methods of antibody labeling:  Direct: antibody against the macromolecule is labeled with a fluorescent dye; antibody is made to react with the macromolecule and the resultant complex is viewed with a fluorescent microscope

Indirect: fluorescent-labeled antibodies are prepared against an antibody that reacts with a particular antigen ; when viewed with a fluorescent microscope, the region of fluorescence represents the location of the antibody.

17 Advanced Visualization Procedure: Immunocytochemistry


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Antibody Labeling in Immunocytochemistry

18 Advanced Visualization Procedure: Autoradiography


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Autoradiography  uses the incorporation of radioactive isotopes into macromolecules, which are then visualized by the use of an overlay of film emulsion

19 Advanced Visualization Procedure: Autoradiography


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Autoradiography

20 Electron Microscopy
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makes use of electron beams instead of light rays and magnetic fields instead of lenses 2 types:
 

Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM)

21 Electron Microscopy: Transmission Electron Microscope


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Transmission Electron Microscope:  uses much thinner sections


 

requires heavy metal precipitation techniques Fixatives used include buffered solutions of glutaraldehyde, paraformaldehyde, osmium tetroxide and potassium permanganate ( these does not only preserve fine structural details but also act as electron dense

stains) 22 Electron Microscopy: Transmission Electron Microscope


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Transmission Electron Microscope:

23 Electron Microscopy: Transmission Electron Microscope


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Transmission Electron Microscope:

24 Electron Microscopy: Transmission Electron Microscope


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Transmission Electron Microscope:

25 Electron Microscopy: Scanning Electron Microscope


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Scanning Electron Microscope:  provides a 3-D image of the specimen


 

used to view the surface of a solid specimen specimen is prepared in a special manner that permits a heavy metal such as gold or palladium, to be deposited on the specimens surface

26 Electron Microscopy: Scanning Electron Microscope 27 Electron Microscopy: Freeze-Fracture Technique


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Freeze-Fracture Technique  reveals internal aspects of membranes  tissue does not suffer mechanical damage  quick-frozen specimens are treated with cryopreservatives that do not develop ice crystals during the freezing process  replica of the surface is generated as the specimen is hit by a supercooled razor blade that fractures along cleavage planes, which are regions of least molecular bonding  fracture face is coated at an angle by evaporated platinum and carbon, forming accumulations of platinum on one side and no accumulation on

the opposite side next to the projection, thus generating a replica the replica is then examined by transmission electron microscopy

28 Electron Microscopy: Freeze-Fracture Technique


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Freeze-Fracture Technique

Slide Show Outline 1 2 3 Introduction


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All cells possess certain unifying characteristics and thus can be described in general terms:

4 Introduction
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Cells are the basic functional units of complex organisms. The human body is composed of more than 200 different types of cells, each performing a different function

5 Introduction
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Protoplasm, the living substance of the cell is divided into two compartments:

6 Cell Membrane: A Selectively Permeable Barrier The cell membrane forms a selectively permeable barrier between the cytoplasm and the extracellular fluid

7 Cell Membrane: A Selectively Permeable Barrier


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Functions
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o o o

maintains the structural integrity of the cell controls movements of substances in and out of the cell (selective permeability) regulates cell to cell interactions recognition via receptors, antigens, foreign cells and altered cells acts as an interface between the cytoplasm and the extracellular fluid

8 Cell Membrane: A Selectively Permeable Barrier


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Cell Membrane The cross section of the cell membrane shows two different structures:
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The phospholipids are the round yellow structures with the blue tails. The proteins are the lumpy structures that are scattered among the phospholipids.

9 Cell Membrane Structure: The Phospholipid Cell Membrane - Structure


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This is a representation of a phospholipid The yellow structure represents the polar head, which is the hydrophillic or water loving section of the phospholipid. The blue tails that come off the sphere represent the non-polar tails which are the hydrophobic or water fearing ends of the phospholipid.

10 Cell Membrane Structure: Model of a Phospholipid


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Structural Model of a Phospholipid


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The two long chains coming off the bottom of this molecule are made up of carbon and hydrogen. These chains are not attracted to water. These are the non-polar tails. At the other end of the phospholipid is a phosphate group and several double bonded oxygens. The atoms at this end of the molecule are not shared equally. This end of the molecule has a charge and is attracted to water. This is the polar head. If you mix phospholipids in water they will form these double layered structures. The hydrophillic ends will be in contact with water. The hydrophobic ends will face inwards touching each other.

11 Cell Membrane Structure: Proteins Components Cell Membrane Structure


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Floating around in the cell membrane are different kinds of proteins. These are generally globular proteins. They are not held in any fixed pattern but instead float around in the phospholipid layer. Generally these proteins structurally fall into three catagories:
  

carrier proteins marker proteins receptor proteins

12 Cell Membrane Structure: Carrier Proteins


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Cell Membrane Structure: Carrier proteins


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They are multipass membrane transport proteins that possess binding sites or molecules on both sides of the lipid bilayer. Transport may be passive, along an electrochemical concentration gradient; or active against a gradient.

They do not extend through the membrane and they bond and drag molecules through the bilipid layer and release them on the opposite side.

13 Cell Membrane Structure: Cholesterol


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Cell Membrane Structure: Cholesterol


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Steroids are sometimes a component of cell membranes in the form of cholesterol. When it is present, it reduces the fluidity of the membrane. Not all membranes contain cholesterol.

o o

14 Cell Membrane Function


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The cell membrane's function, in general, revolves around its membrane proteins. General classification of membrane proteins include:  receptor proteins which allow cells to communicate  transport proteins which regulate what enters or leaves the cell  marker proteins which identify the cell

15 Cell Membrane Function: Receptor Proteins


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Cell Membrane Function: Receptor Proteins


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These proteins are used in intercellular communication. This causes the receptor protein to release a signal to perform some action.

16 Cell Membrane Function: Membrane Transport Proteins


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Cell Membrane Function: Membrane Transport Proteins

Shown is a carrier protein that can transport substances across an

electrochemical gradient or utilize ATP-driven transport mechanisms to ferry specific substances across the cell membrane against the concentration gradient 17 Cell Membrane Function: Membrane Transport Proteins
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Cell Membrane Function: Membrane Transport Proteins

Transport may be uniport , a single molecule moving in one direction

18 Cell Membrane Function: Membrane Transport Proteins


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Cell Membrane Function: Membrane Transport Proteins

Transport may be coupled, two different molecules moving in the same direction Symports also use the process of diffusion. In this case, a molecule that is moving naturally into the cell through diffusion is used to drag another molecule into the cell. In this example glucose hitches a ride with sodium

o o

19 Cell Membrane Function: Membrane Transport Proteins


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Cell Membrane Function: Membrane Transport Proteins Mechanism of Symport


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Symport coupled transport is the simultaneous transport of two different molecules or ions across a lipid bilayer membrane in the same direction.

20 Cell Membrane Function: Membrane Transport Proteins

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Cell Membrane Function: Membrane Transport Proteins Mechanism of Antiport


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Antiport coupled transport involves two different molecules or ions across a lipid bilayer membrane in opposite directions.

21 Cell Membrane Function: Marker Proteins


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Cell Membrane Function: Marker Proteins

Marker proteins extend across the cell membrane and serve to identify the cell. They are as unique as fingerprints. They play an important role in organ transplants.

o o

22 Cell Membrane Function: Membrane Transport Proteins


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Cell Membrane Function: Membrane Transport Proteins

Channel proteins are also membrane transport proteins that may be gated or ungated They are incapable of transporting substances against a concentration gradient. Channel proteins extend through the bilipid layer. They form a pore through the membrane that can move molecules in several ways. In some cases the channel proteins simply act as a passive pore. Molecules will randomly move through the opening in a process called diffusion. This requires no energy, molecules move from an area of high concentration to an area of low concentration.

23 Cell Membrane Function: Proteins Using ATP


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Cell Membrane Function: Proteins Using ATP


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Some proteins actively use energy from the ATPs in the cell to drag molecules from areas of low concentration to areas of high concentration (working directly against the concentration gradient). The sodium/potassium pump is an example in which the energy of a phosphate is used to exchange sodium ions for potassium ions.

24 Cytoplasm The term cytoplasm refers to everything between the cell membrane and the nuclear envelope. It consists primarily of water and contains various organelles as well as salts, dissolved gases and nutrients.

25 Organelles in the Cytoplasm: Golgi Apparatus


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Golgi Apparatus

Functions in synthesis of carbohydrates and in the modification of and sorting of proteins manufactured in the rough endoplasmic reticulum

26 Organelles in the Cytoplasm: Lysosomes


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Lysosomes

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Suicide sacs Involved in intracellular digestion

27 Organelles in the Cytoplasm Smooth Endoplasmic Reticulum

Smooth Endoplasmic Reticulum (SER)


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Not studded with ribosomes For carbohydrate and lipid synthesis as well as detoxification

28 Organelles in the Cytoplasm: Rough Endoplasmic Reticulum


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Rough Endoplasmic Reticulum (RER)


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Studded with ribosomes Vesicles associated with the RER and golgi possess a protein coat and surface markers

29 Organelles in the Cytoplasm: Mitochondrion


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Mitochondrion

Possess their own DNA and performs oxidative phosphorylation and lipid synthesis. Note that the inner membrane is thrown into folds called cristae.

30 Organelles in the Cytoplasm: Centrioles


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Centrioles
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These are small cylindrical structures consisting of nine microtubule triplets. They constitute the core of the microtubule organizing center or the centrosome.

31 Organelles in the Cytoplasm: Peroxisomes


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Peroxisomes

These are self-replicating organelles that contain oxidative enzymes.

32 Ribosomes in the Cytoplasm


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Ribosomes o They are small particles that provide cell surface for protein synthesis.
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Each ribosome is composed of a small subunit (40S) and a large subunit (60S), both of which are manufactured or assembled in the nucleolus and released separately in the cytosol. The 40S subunit is composed of 33 proteins and an 18S rRNA. The 60S subunit has 49 proteins and 3 rRNAs.

o o o

33 Nucleus
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Nucleus
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This is the largest organelle of the cell. It contains nearly all the DNAs possessed by the cell and all the mechanisms for RNA synthesis. It is surrounded by a nuclear envelope which is composed of two parallel unit membranes that fuse with each other at certain regions to form perforations known as nuclear pores. It houses three major components  chromatin  nucleolus  nucleoplasm

34 Nucleus
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Chromatin

Chromatin is the complex of DNA and protein found inside the nuclei of eukaryotic cells The nucleic acids are in the form of double-stranded DNA. The major proteins involved in chromatin are histone proteins. The functions of chromatin are:  to package DNA into a smaller volume to fit in the cell  to strengthen the DNA to allow mitosis and meiosis  and to serve as a mechanism to control expression

o o o

35 Nucleus
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Nucleolus

This is a deeply staining non-membrane bounded structure within the nucleus that is involved in the rRNA synthesis and in the assembly of small and large ribosomal subunits

Slide Show Outline 1 CELL CYCLE and REPLICATION 2 The Cell Cycle 3 Mitosis
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Mitosis o When a eukaryotic cell divides into two, each daughter or progeny cell must receive a complete set of genes (for diploid cells, this means 2 complete set of genomes=2n)
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In human cells there are 23 pairs of chromosomes, 22 pairs are autosomes and 1 pair constitute the sex chromosomes (XX for

females and XY for males)

4 Mitosis
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Mitosis is preceded by an exact duplication of the DNA and associated proteins in the nucleus This occurs during interphasethe period between actual mitotic divisions. After this doubling, the chromosomes are organized, complete division and are then equally distributed to the two daughter cells during mitosis.

5 Mitosis
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All somatic cells pass through the mitotic and interphase periods at one time or another. The duration of the mitotic period is usually about 0.5 to 2 hours, while the intermitotic period can vary from a few hours to many years.

6 Mitosis
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The mitotic index (proportion of cells undergoing division at any one time) varies greatly depending upon the type of tissue and the physiological needs of the organism. A high mitotic index is observed in: o all growing embryonic tissues o certain adult tissues such as :  bone marrow  intestinal crypts  cancer cells

7 Interphase
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Interphase o Interphase is not a stage of mitosis o The chromosomes cannot be distinguished and appear as scattered granules connected by a network of pale-staining strands within a distinct nuclear membrane. o A nucleolus is usually present

8 Stages of Mitosis

Mitosis is divided into 4 stages for convenience of study: o Prophase o Metaphase o Anaphase o Telophase

9 Interphase
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Interphase o is not a stage of mitosis o Longest period in the cell cycle o Divided into 3 stages called into order:  G1 phase  S phase  G2 phase o The chromosomes cannot be distinguished and appear as scattered granules connected by a network of pale-staining strands within a distinct nuclear membrane. o A nucleolus is usually present

10 Prophase
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Prophase o Chromatin materials which are made up of DNA and associated proteins condense into chromosomes o Nucleolus fades o Chromosomes split and form 2 sister chromatids o The 2 sister chromatids remain attach at a region called centromere

11 Prometaphase
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Prometaphase
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Dissolution of nuclear envelope Spindle fibers come in contact with the chromosomes

12 Metaphase
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Metaphase o chromosomes become arranged at the equatorial plate which is midway between the two pairs of centrioles

the spindle is now fully formed and does not stain (achromatic) and consists of fine microtubules, some of which are attached to the chromosomes. During this period the centromeres begin to show indications of splitting

13 Anaphase
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Anaphase o each chromosome pair completes its splitting, and the two daughter chromosomes move toward opposite poles o spindle microtubules may be seen between the retreating chromosomes o spindle fibers shorten through a process known as depolymerization (removal of tubulin proteins)

14 Telophase
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Telophase o telophase, the chromosomes have reached the spindle poles and appear as a dense, basophilic mass within which individual chromosomes cannot be defined. o the nuclear membrane reappears, and the outlines of the chromosomes disappear leaving scattered chromatin granules connected by a pale-staining network. o nucleoli reappear and seem to be associated with a particular chromosome.

15 Cytokinesis
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Cytokinesis o the division of the cytoplasm, usually occurs during the telophase, but the synchrony between nuclear and cytoplasmic telophase, is not constant o cytokinesis may begin as early as late anaphase or be delayed beyond the nuclear reconstruction of telophase

16 Mitotic Figures in Tissue Sections


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Cells in mitosis are easy to spot. Instead of a nucleus, the chromosomes are visible as tangled, dark-staining threads.

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We call these "mitotic figures." Counting mitotic figures sometimes helps the pathologist tell benign from malignant. If you see a nuclear membrane, it is not a mitotic figure

17 Meiosis
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Meiosis is the type of cell division by which germ cells (eggs and sperm) are produced. Meiosis involves a reduction in the amount of genetic material Meiosis comprises two successive nuclear divisions with only one round of DNA replication. Four stages can be described for each nuclear division Interphase: Before meiosis begins, genetic material is duplicated

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18 First Meiotic Division


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First division of meiosis: Reductional

2n

Prophase 1 o Duplicated chromatin condenses. o Each chromosome consists of two, closely associated sister chromatids o Crossing-over can occur during the latter part of this stage Metaphase 1 o Homologous chromosomes align at the equatorial plate

Anaphase 1 o Homologous pairs separate with sister chromatids remaining together

Telophase 1 o Two daughter cells are formed with each daughter containing only one chromosome of the homologous pair.

19 Second Meiotic Division

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Second division of meiosis: n n o Gamete formation Prophase II o chromosomes shorten, condense and thicken Metaphase II o Chromosomes align at the equatorial plate Anaphase II o Centromeres divide and sister chromatids migrate separately towards opposite pole Telophase II o Cell division is complete. o Four haploid daughter cells are formed

20 Gametogenesis
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Meiosis is studied in relation to gametogenesis Gametogenesis is the general process of gamete formation in both males and females. Female gametogenesis: oogenesis Male gametogenesis: spermatogenesis

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21 Oogenesis
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Oogenesis is the process of meiosis in female organisms from an oogonium to a primary oocyte, to a secondary oocyte, and then to an ovum. Oogenesis begins soon after fertilization, as primordial germ cells travel from the yolk sac to the gonads, where they begin to proliferate mitotically. They become oocytes once they enter the stages of meiosis several months after birth. Now called primordial follicles, they are made up of oogenic cells from the primordial germ cells surrounded by follicle cells from the somatic line.

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The oocyte is then arrested in the first meiotic prophase until puberty.

22 Oogenesis
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At puberty, between 4 to 10 follicles begin to develop, although only 1-2 are actually released. Surrounding each oocyte is a zona pellucida, membrana granulosa, and theca cell layer. Each oocyte finishes its first meiotic division, creating a secondary oocyte and polar body, which serves no further function. It begins the next meiosis cycle and is arrested in its second metaphase, at which point it is released from the ovary in ovulation. It will not finish the meiosis cycle until it is fertilized by a sperm.

23 Oogenesis 24 Spermatogenesis
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Spermatogenesis: the process by which stem cells develop into mature spermatozoa. There are three phases: o Spermatocytogenesis (Mitosis) o Meiosis o Spermiogenesis

25 Spermatogenesis
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Spermatocytogenesis o Also called mitosis o Stem cells  Type A spermatogonia divide mitotically to produce cells that begin differentiation into Type B spermatogonia  Spermatogonia have spherical or oval nuclei, and rest on the basement membrane

26 Spermatogenesis 27 Spermatogenesis
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Meiosis: o Cells in prophase of the first meiotic division are primary spermatocytes. o They are characterized by highly condensed chromosomes giving the nucleus a coarse chromatin pattern and an intermediate position

o o

in the seminiferous epithelium. This is a long stage, so many primary spermatocytes can be seen. Primary spermatocytes go through the first meiotic division and become secondary spermatocytes. The cells quickly proceed through this stage and complete the second meiotic division. Because this stage is short there are few secondary spermatocytes to be seen in sections. is the process by which the diploid number of chromosomes present in spermatogonia (the stem cells) is reduced to the haploid number present in mature spermatozoa

28 Spermatogenesis 29 Spermatogenesis
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Meiosis o The products of the second meiotic division are called spermatids. o They are spherical cells with interphase nuclei, positioned high in the epithelium. o Since spermatids go through a metamorphosis into spermatozoa, they occur in early through late stages. o All of these progeny cells remain attached to each other by cytoplasmic bridges. o The bridges remain until sperm cells are fully differentiated

30 Spermatogenesis
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Spermiogenesis o This is the metamorphosis of spherical spermatids into elongated spermatozoa. o No further mitosis or meiosis occurs. o During spermiogenesis, the acrosome forms, the flagellar apparatus forms, and most excess cytoplasm (the residual body) is separated and left in the Sertoli cell. o Spermatozoa are released into the lumen of the seminiferous tubule. o A small amount of excess cytoplasm (the cytoplasmic droplet) is shed later in the epididymis.

31 Spermatogenesis 32 Spermatogenesis

Spermiogenesis

a process of metamorphosis from a round cell with typical organelles to a highly specialized, elongated cell well adapted for traversing the male and female reproductive tracts and achieving fertilization of an egg

33 Spermiogenesis 34 Spermatogenesis 35 Spermatogenesis

Slide Show Outline 1BASIC TERMINOLOGY IN GENETICS 2CYTOGENETICS


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Cytogenetics is the study of chromosomes and the related diseased states caused by numerical and structural chromosome abnormalities. A variety of cell or tissue types can be used to perform these studies.

3GENE

A segment of DNA that codes for a polypeptide

4GENOME
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The entire set of genes possessed by an individual

5GENE POOL
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The entire set of genes possessed by an individual

6HOMOLOGOUS CHROMOSOMES
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Two physically identical chromosomes with the same gene loci but not necessarily the same alleles; one is of maternal origin and the other paternal

7SEX CHROMOSOMES
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Two chromosomes (XX or XY) that determines a persons sex

8AUTOSOMES
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All chromosomes, except the sex chromosomes; occur in 22 homologous pairs

9GENE LOCUS
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The point in a chromosome where a particular gene is located

1ALLELE 0 y Any member of a given pair of gene; any alternative form that a particular gene can take 1GENOTYPE 1 y The genetic make-up of an individual; alleles that a person has for a particular trait 1PHENOTYPE 2 y A detectable trait, such as eye color, blood type, appearance of fingers, etc.

1RECESSIVE ALLELE 3 y An allele that is hidden/not expressed in the presence of a dominant gene 1DOMINANT ALLELE 4 y Allele that is always expressed whenever present 1HOMOZYGOUS 5 y Pure individual; with identical alleles for a given gene 1HETEROZYGOTE 6 y An individual with two different alleles for a given gene 1CARRIER 7 y A person who carries a recessive allele but does not phenotypically express it 1CODOMINANCE 8 y A condition in which both alleles are fully expressed when present in the same individual 1INCOMPLETE DOMINANCE 9 y A condition in which two alleles are both expressed when present in the same individual and the phenotype is intermediate between those which each allele would produce alone 2PLEIOTROPY 0 y A condition in which a single phenotype results from the combined action of genes at two or more different loci, as in eye color 2SEX LINKAGE 1 y Inheritance of a gene on the x or y chromosome so that the associated phenotype is expressed more in one sex than in the other

2PENETRANCE 2 y The percentage of individuals with a given phenotype who actually exhibit the phenotype predicted from it 2CHROMOSOMES 3 y Three dimensional structure in the cell that is chemically made up of DNA 2G BANDING CHROMOSOMES 4 y Characteristic banding patterns caused by staining the chromosomes with dye called Giemsa 2FISH 5 y A molecular cytogenetic technique in which fluorescent gene probes are used to determine the presence or absence of chromosomes, DNA specific sequences or genes 2M-FISH 6 y Multi-Color Fluorescent in situ hybridization. y All chromosomes are distinguished by a specific color 2CGH 7 y Comparative Genomic Hybridization Comparative genomic hybridization (CGH) is a molecular-cytogenetic method for the analysis of copy number changes (gains /losses) in the DNA content of tumor cells. The method is based on the hybridization of fluorescently labeled tumor DNA (frequently Fluorescein - FITC) and normal DNA (frequently Rhodamine or Texas Red) to normal human metaphase preparations. 2HISTONE 8 y http://images.search.yahoo.com/search/images/view?back=http%3A%2F%2 Fimages.search.yahoo.com%2Fsearch%2Fimages%3Fp%3DHistones%26ei %3DUTF-8%26fr%3Dyfp-t482%26fp_ip%3DPH%26x%3Dwrt&w=417&h=986&imgurl=www.unc.ed u%2Fdepts%2Fmarzluff%2Fhistone.jpg&rurl=http%3A%2F%2Fwww.unc. edu%2Fdepts%2Fmarzluff%2Fhistone.html&size=52.9kB&name=histone.j

pg&p=Histones&type=jpeg&no=2&tt=356&oid=e0e737270c654730&ei=U TF-8 2HISTONE 9 y Histones o combine with DNA to form nucleosomes, the fundamental structural units of chromatin. o Nucleosomes pack DNA in a stable coiled form in eukaryotic nuclei. o The total DNA complement of the nucleus, with associated histone and non-histone proteins, is broken into individual lengths; these are the chromosomes.
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3NUCLEOSOME 0 y The coiling of DNA around nucleosomes and the further winding of nucleosomes into chromatin fibers greatly compact the DNA of the eukaryotic nuclei. y Winding the DNA into nucleosomes and chromatin fibers is estimated to shorten its length by at least a factor of 10,000. y Because packing of nucleosomes in chromatin fibers probably prevents access to DNA by the enzymes of transcription and replication, the fibers probably unwind for these activities to take place. 3NUCLEOSOME 1 y The coiling of DNA around nucleosomes and the further winding of nucleosomes into chromatin fibers greatly compact the DNA of the eukaryotic nuclei.
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Winding the DNA into nucleosomes and chromatin fibers is estimated to shorten its length by at least a factor of 10,000. Because packing of nucleosomes in chromatin fibers probably prevents access to DNA by the enzymes of transcription and replication, the fibers probably unwind for these activities to take place.

3NUCLEOSOME 2 y Retrieved from: http://www.zoology.ubc.ca/~bio463/images/nucleosome.jpg 3SOLENOID

3
y

Retrieved from: http://www.benbest.com/lifeext/solenoid.gif

3MITOSIS 4 y Retrieved from: http://images.search.yahoo.com/search/images/view?back=http%3A%2F%2 Fimages.search.yahoo.com%2Fsearch%2Fimages%3Fp%3DMITOSIS%26e i%3DUTF-8%26fr%3Dyfp-t482%26fp_ip%3DPH%26x%3Dwrt&w=500&h=600&imgurl=www.dartmo uth.edu%2F%7Ecbbc%2Fcourses%2Fbio4%2Fbio4lectures%2Fimages%2Fmitosis.JPG&rurl=http%3A%2F%2Fwarren.dusd.n et%2F%7Ecrobinson%2FCoordinated_Past_Web_Pages%2Fmitosis_meiosi s.htm&size=17.1kB&name=mitosis.JPG&p=MITOSIS&type=jpeg&no=12 &tt=15,737&oid=09d38de0a2885188&ei=UTF-8 3 TRISOMY 21 5 3TRISOMY 21 6 y Retrieved from:http://www.millerandlevine.com/genome/trisomy-21.jpg y http://www.woodbinehouse.com/images/large/down_syndrome_the_first_18 _months.jpg y http://www.down-syndrom.ch/Fotos/Welcom1.jpg 3CRI-DU CHAT 7 y http://images.search.yahoo.com/search/images/view?back=http%3A%2F%2 Fimages.search.yahoo.com%2Fsearch%2Fimages%3Fp%3DCRI%2BDU% 2BCHAT%2Bsyndrome%26ei%3DUTF-8%26fr%3Dyfp-t482%26fp_ip%3DPH%26x%3Dwrt&w=925&h=916&imgurl=www.busine ss.unet.com%2F%7Ecridchat%2Fgallery%2Fj%2Fjanel_1.jpg&rurl=http%3A% 2F%2Fwww.business.unet.com%2F%7Ecridchat%2Fgallery%2Fj%2Fjanel_1.htm&size=150.8kB &name=janel_1.jpg&p=CRI+DU+CHAT+syndrome&type=jpeg&no=2&tt =394&oid=6bea37bb9482898a&ei=UTF-8 3KARYOTYPE 8 y Retrieved from http://fig.cox.miami.edu/~cmallery/255/255hist/mcb10.0.karyotype.jpg

Organization of the chromosomes of an individual, lined up from largest to smallest according to the location of the centromere.

Slide Show

Outline 1 CHROMOSOMAL STRUCTURE 2 Introduction


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Cytogenetic analyses are almost always based on examination of chromosomes fixed during mitotic metaphase. During that phase of the cell cycle, DNA has been replicated and the chromatin is highly condensed. The two daughter DNAs are encased in chromosomal proteins forming sister chromatids, which are held together at their centromere. The centromere is the structure where the mitotic spindle attaches prior to segregation

3 Metaphase Chromosome
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Metaphase chromosomes differ from one another in size and shape, and the absolute length of any one chromosome varies depending on the stage of mitosis in which it was fixed. However, the relative position of the centromere is constant, which means that that the ratio of the lengths of the two arms is constant for each chromosome. This ratio is an important parameter for chromosome identification.

4 Metaphase Chromosome
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The ratio of lengths of the two arms allows classification of chromosomes into several basic morphologic types

5 Chromosomal Banding
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Centromere position and arm ratios can assist in identifying specific pairs of chromosomes, but inevitably several or many pairs of chromosomes appear identical by these criteria. The ability to identify specific chromosomes with certainty was revolutionized by discovery that certain dyes would produce reproducible patterns of bands when used to stain chromosomes.

Chromosome banding has since become a standard and indispensible tool for cytogenetic analysis., and several banding techniques have been developed: o Q banding: chromosomes are stained with a fluorescent dye such as quinacrine o G banding: produced by staining with Giemsa after digesting the chromosomes with trypsin o C banding: chromosomes are treated with acid and base, then stained with Giemsa stain

6 Chromosome Banding
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Each of these techniques produces a pattern of dark and light (or fluorescent versus non-fluorescent) bands along the length of the chromosomes. Importantly, each chromosome displays a unique banding pattern, analagous to a "bar code", which allows it to be reliably differentiated from other chromosomes of the same size and centromeric position.

7 Chromosome Banding
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In the following figure, human chromosome pairs 1, 2 and 3 are seen with and without G banding.

Slide Show Outline 1 CLASSIFICATION OF CHROMOSOMES FOR KARYOPTYPING 2 Introduction


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Chromosomes are arranged into seven groups based on size and centromere location. The centromeres can be found in the: o Middle of the chromosome (median) o Near one end (acrocentric) o Between the middle and end: submedian

3 Group A Chromosomes

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Chromosomes 1 to 3 Largest Median centromere

4 Group B Chromosomes
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Chromosomes 4 to 5 Large Submedian centromere

5 Group C Chromosomes
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Chromosomes 6 to 12 Medium sized Submedian centromere

6 Group D Chromosomes
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Chromosomes 13 to 15 Medium-sized Acrocentric centromere

7 Group E Chromosomes
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Chromosomes 16 to 18 Short Median or submedian centromere

8 Group F Chromosomes
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Chromosomes 19-20

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Short Median centromere

9 Group G Chromosomes
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Chromosomes 21 to 22 Very short Acrocentric centromere

Slide Show 10 Sex Chromosomes


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Chromosome X o Similar to Group C Chromosome Y o Similar to Group G

Outline 1 CHROMOSOMAL DISORDERS 2 Autosomal Disorders


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Trisomy 21 o Down Syndrome Trisomy 18 o Edward Syndrome

Trisomy 13 o Patau Syndrome

3 Chromosomal Aberrations 4 Autosomal Aberration


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Cri du Chat Syndrome o deletion in chromosome no. 5 o defective larynx o mishapen ears o small head

5 Sex Chromosomal Aberrations


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Klinefelter Syndrome o XXY o Phenotypically male Turner Syndrome o XO o Only viable human monosomy Metafemale XYY syndrome

6 Sex Chromosomal Abberations (Klinefelter Syndrome)


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Klinefelter syndrome (KS) is a disorder that occurs in some men who have more than one X chromosome (XXY). Virtually every cell in the body contains chromosomes, which carry the genes that determine many of our physical, intellectual, and emotional characteristics.

7 Sex Chromosomal Aberrations (Klinefelter Syndrome)


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Males usually inherit a single X chromosome from their mother and a single Y chromosome from their father. Males with KS get at least one extra X chromosome from either their mother or their father. In most cases there is only one extra chromosome. The extra X chromosome is caused by a biological accident.

Normally, an egg has one X chromosome and a sperm has one X or one Y chromosome.

8 Sex Chromosomal Aberrations (Klinefelter Syndrome) Risk Factors for Klinefelter Syndrome A risk factor is something that increases your chances of getting a disease or condition. Women over age 35 have a slightly increased chance of having a child with KS. There are no other known risk factors for this disorder.

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9 Sex Chromosomal Aberrations (Klinefelter Syndrome) Although the chromosome variation XXY occurs in approximately 1 out of 1,000 live male births, many men with it do not develop KS. When KS does develop, it usually goes undetected until puberty and sometimes much later. It may present in childhood as learning problems, adolescence as excessive breast development, in adult life as infertility concerns and as an unexpected finding on an amniocentesis.

10 Sex Chromosomal Aberrations (Klinefelter Syndrome) Symptoms/Characteristics may include: o Tallness with extra long arms and legs o Abnormal body proportions (long legs, short trunk) o Enlarged breasts o Lack of facial and body hair o Small firm testes o Small penis o Lack of ability to produce sperm

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Diminished sex drive Sexual dysfunction Learning disabilities Personality impairment

11 Sex Chromosomal Aberrations (Klinefelter Syndrome) 12 Sex Chromosomal Aberrations (Klinefelter Syndrome) 13 Sex Chromosomal Aberrations (Klinefelter Syndrome) 14 Sex Chromosomal Aberrations (Klinefelter Syndrome) 15 Sex Chromosomal Aberrations (Klinefelter Syndrome) 16 Sex Chromosomal Aberrations (Klinefelter Syndrome)
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Treatment o Testosterone o When boys with KS are 10 to 12 years old, their hormone levels are checked yearly. o If their testosterone levels are low, then treatment with testosterone may be very helpful. o Men diagnosed in adulthood may also benefit from taking testosterone. o However, testosterone cannot reverse infertility.

17 Sex Chromosomal Aberrations (Klinefelter Syndrome)


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Treatment Testosterone is most often given through regular shots in the form of depotestosterone. The benefits of this treatment include: o Increased strength o More muscular, male appearance o Growth of facial and body hair o Better self-esteem o Modulation of mood

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Increased energy Increased ability to concentrate Greater sex drive

18 Sex Chromosomal Aberrations (Turner Syndrome) 19 Sex Chromosomal Aberrations (Turner Syndrome) 20 Sex Chromosomal Aberrations (Turner Syndrome) 21 Sex Chromosomal Aberrations (Turner Syndrome) 22 Sex Chromosomal Aberrations (Turner Syndrome) 23 Sex Chromosomal Aberrations (Turner Syndrome) 24 Sex Chromosomal Aberrations (XYY Syndrome) 25 Sex Chromosomal Aberrations (XYY Syndrome)

Slide Show Outline 1 ANEUPLOIDY and DELETIONS 2 EUPLOIDY


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Euploidy is the condition of having a normal number of structurally normal chromosomes. Euploid human females have 46 chromosomes (44 autosomes and two X chromosomes), and euploid bulls have 60 chromosomes (58 autosomes plus an X and a Y chromosome).

3 ANEUPLOIDY

Aneuploidy o is the condition of having less than or more than the normal diploid number of chromosomes, and is the most frequently observed type of cytogenetic abnormality. o abnormality in chromosome number o in other words, it is any deviation from euploidy, although many authors restrict use of this term to conditions in which only a small number of chromosomes are missing or added.

4 ANEUPLOIDY
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Aneuploidy o is recognized as a small deviation from euploidy for the simple reason that major deviations are rarely compatible with survival, and such individuals usually die prenatally

5 ANEUPLOIDY
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The two most commonly observed forms of aneuploidy are monosomy and trisomy: o Monosomy  2n-1  is lack of one of a pair of chromosomes  an individual having only one chromosome 6 is said to have monosomy 6.  A common monosomy seen in many species is X chromosome monosomy, also known as Turner's syndrome.  XO is the only viable human monosomy  Monosomy is most commonly lethal during prenatal development o Trisomy  2n + 1  is having three chromosomes of a particular type  a common autosomal trisomy in humans is Down syndrome, or trisomy 21, in which a person has three instead of the normal two chromosome 21s.  Trisomy is a specific instance of polysomy, a more general term that indicates having more than two of any given chromosome.

6 TRIPLOIDY

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triploid individual has three of every chromosome presence of three haploid sets of chromosomes a triploid human would have 69 chromosomes (3 haploid sets of 23) a triploid dog 117 chromosomes production of triploids seems to be relatively common and can occur by, for example, fertilization by two sperm cells. however, birth of a live triploid is extraordinarily rare and such individuals are quite abnormal the rare triploid that survives for more than a few hours after birth is almost certainly a mosaic, having a large proportion of diploid cells.

7 DELETION
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chromosome deletion occurs when the chromosome breaks and a piece is lost involves loss of genetic information and results in what could be considered "partial monosomy" for that chromosome

8 INVERSION
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A related abnormality is a chromosome inversion. a break or breaks occur and that fragment of chromosome is inverted and rejoined rather than being lost inversions are thus rearrangements that do not involve loss of genetic material and, unless the breakpoints disrupt an important gene, individuals carrying inversions have a normal phenotype

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