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Chromosome Document

Chromosome Document

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Sections

  • 1.2 CHROMOSOME
  • 1.3 MUTATIONS IN CHROMOSOME NUMBER
  • 1.4 Metaphase chromatin and division
  • 1.5 BONE MARROW
  • 2.2.1 Preprophase
  • 2.2.2 Prometaphase
  • 2.3 Metaphase
  • 2.4 Anaphase
  • 2.5.1Significance
  • 2.5.2 Development and growth
  • 2.5.3 Cell replacement
  • 2.5.4 Regeneration
  • 2.5.6 Asexual reproduction
  • 2.5.7 Consequences of errors
  • 2.6 Endomitosis
  • 2.7 Metaphase
  • 2.8 Metaphase in cytogenetics and cancer studies
  • 3.1 History of karyotype studies
  • 3.2.1 Staining
  • 3.2.2 Observations
  • 3.3.1 Changes during development
  • 3.3.2 Number of chromosomes in a set
  • 3.3.3 Fundamental number
  • 3.4 Ploidy
  • 3.5 Aneuploidy
  • 3.6 Species trees
  • 3.7.1 Types of banding
  • 3.7.2 Classic karyotype cytogenetics
  • 3.7.3 Spectral karyotype (SKY technique)
  • 3.8 Digital karyotyping
  • 4.1 CHROMOSOMAL ABNORMALITIES
  • 4.2 Numerical Disorders
  • 4.3 Inheritance
  • 4.4 Cytogenetics
  • 4.5 Early years
  • 4.6.1 McClintock's work on maize
  • 4.6.2 Natural populations of Drosophila
  • 4.7 Human abnormalities and medical applications
  • 4.8 Beginnings of molecular cytogenetics
  • 5.1 Karyotyping
  • 5.2 Slide preparation
  • 5.3 Analysis
  • 5.4 Fluorescent in situ hybridization
  • 5.5 Slide preparation
  • 5.7 Analysis
  • 6.1 Image Processing
  • 6.2.1 Image conversion
  • 6.2.4 Denoising
  • 6.2.5 Edge detection
  • 6.3 Two dimensional convolutions

ABSTRACT

During the Metaphase type of cell division in the Bone Marrow, Karyotyping method gives the visual representation of the 46 chromosomes paired and arranged in decreasing order of size. This representation is useful in leukemia purposes. This method is a difficult one because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges. So here, Karyotyping uses new mutual information method which is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. This algorithm is formulated as such a method of combinatorial optimization problem. Where the distances between homologous chromosomes are minimized and the distances between non homologous ones are maximized. It is solved by using an integer programming approach. In this project chromosome dataset Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes was used for this study.

CHAPTER 1

Introduction 1.1 The study of chromosome morphology and its relation with some genetic diseases is the main goal of cytogenetic. Normal human cells have 23 classes of large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The chromosome contains approximately 30 000 genes (genotype) and large tracts of non coding sequences. The analysis of genetic material can involve the examination of specific chromosomal regions using DNA probes, e.g., fluorescent in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic hybridization (CGH) , or the morphological and pattern analysis of entire chromosomes, the conventional cytogenetic, which is the focus of this paper. These cytogenetic studies are very important in the detection of acquired chromosomal abnormalities, such as translocations, duplications, inversions, deletions, monosomies, or trisomies. These techniques are particularly useful in the diagnosis of cancerous diseases and are the preferred ones in the characterization of the different types of leukemia, which is the motivation of this paper . The pairing of chromosomes is one of the main steps in conventional cytogenetic analysis where a correctly ordered karyogram is produced for diagnosis of genetic diseases based on the patient karyotype. The karyogram is an image representation of the stained human chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) , where the chromosomes are arranged in 22 pairs of somatic homologous elements plus two sex-determinative chromosomes (XX for the female or XY for the male), displayed in decreasing order of size. A karyotype is the set of characteristics

extracted from the karyogram that may be used to detect chromosomal abnormalities. The metaphase is the step of the cellular division process where the chromosomes are in their most condensed state. This is the most appropriated moment to its visualization and abnormality recognition because the chromosomes appear well defined and clear. The pairing and karyotyping procedure, usually done manually by visual inspection, is time consuming and technically demanding. The application of the G-banding procedure to the chromosomes generates a distinct transverse banding pattern characteristic for each class, which is the most important feature for chromosome classification and pairing. The International System for Cytogenetic Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as for all the chromosomes of a normal human, and the clinical staff is trained to pair and interpret each specific karyogram according to the ISCN information. Other features, related to the chromosome dimensions and shape, are also used to increase the discriminative power of the manual or automatic classifiers. 1.2 CHROMOSOME A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes,regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Chromosomes vary widely between different organisms. The DNA molecule may be circular or linear, and can be composed of 100,000 to 10,000,000,000[1] nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without

In practice "chromosome" is a rather loosely defined term. although there are many exceptions to this rule. The structure of chromosomes and chromatin varies through the cell cycle. or it may unexpectedly evadeapoptosis leading to the progression of cancer. a large body of work uses the term chromosome regardless of chromatin content. which is tightly coiled in on itself. In prokaryotes and viruses. the term genophore is more appropriate when no chromatin is present. Chromosomes may exist as either duplicated or unduplicated. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). If these structures are manipulated incorrectly. sometimes accompanied by one or more smaller. through processes known as chromosomal instability and translocation. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. Chromosomes are the essential unit for cellular division and must be replicated. However. the cell may undergo mitotic catastrophe and die. cells may contain more than one type of chromosome. Unduplicated chromosomes are single linear strands. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. Also. divided. These small circular genomes . In eukaryotes. DNA is usually arranged as a circle. Chromosomal recombination plays a vital role in genetic diversity. In prokaryotes. for example.defined nuclei) have smaller circular chromosomes. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. This allows the very long DNA molecules to fit into the cell nucleus. circular DNA molecules called plasmids.

copies of chromosome 21.are also found in mitochondria and chloroplasts. Such individuals are called euploid and have the wild-type chromosome complement for the species.3 MUTATIONS IN CHROMOSOME NUMBER Normally. 1. Chromosomal Mutations are substantial changes in chromosome structure that are large enough to be visible by karyotyping (see lab manual) and thus typically affect more than one gene. in which an individual has 3.+21. a extra copy of human chromosome 21). If the mutation involves only one or a few chromosomes in the genome (e. The individual would have Down Syndrome and his/her karyotype would be written 47. Euploid human karyotypes are 46. XX (female) or 46 XY (male). reflecting their bacterial origins. .g.XX. the individual carrying the mutation is said to be aneuploid. members of the same species have the same numbers of types of chromosomes (with the exception of sex chromosomes in males and females if sex is chromosomally determined). rather than 2.+21. An example of aneuploidy is trisomy 21.XY or 47. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.

one of which is present on each sister chromatid. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. The microtubules then pull the chromatids apart toward the centrosomes. chromosomes are structurally highly condensed. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. the chromatin strands become more and more condensed. . and they form the classic four arm structure.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. A special DNA base sequence in the region of the kinetochores provides.Fig 1. This is the only natural context in which individual chromosomes are visible with an optical microscope. the chromatids are uncoiled and DNA can again be transcribed. The shorter arms are called p arms (from the French petit. a pair of sister chromatids attached to each other at the centromere. This compact form makes the individual chromosomes visible. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. longer-lasting attachment in this region. During mitosis. 1. small) and the longer arms are called q arms (q follows p in the Latin alphabet. q-g "grande"). which enables these giant DNA structures to be contained within a cell nucleus. along with special proteins.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). Once the cells have divided. In spite of their appearance. so that each daughter cell inherits one set of chromatids.

6 kg (5.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. bone marrow accounts for approximately 2. in two separate nuclei. which divides the nuclei. [1] Bone marrow is also a key component of the lymphatic system. bone marrow in large bones produces new blood cells. In humans. which use the bone marrow vasculature as a conduit to the body's systemic circulation.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. . in adults weighing 65 kg (143 lbs). It is generally followed immediately by cytokinesis.1. cytoplasm.7 lbs). On average. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. producing the lymphocytes that support the body's immune system CHAPTER 2 2. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. bone marrow constitutes 4% of the total body mass of humans.

metaphase. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. "mitosis" is often used interchangeably with "mitotic phase".[1] Prokaryotic cells. anaphase and telophase. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. where chromosomes divide within an intact cell nucleus. Even in animals. but is found in various different groups. prophase. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. The process of mitosis is fast and highly complex. there are many cells where mitosis and cytokinesis occur separately. These stages are interphase. This accounts for approximately 10% of the cell cycle. However. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. . animals undergo an "open" mitosis. cytokinesis and mitosis may occur independently. For example.genetically identical to each other and to their parent cell. divide by a process called binary fission. The cell then divides in cytokinesis. Because cytokinesis usually occurs in conjunction with mitosis. This occurs most notably among the fungi and slime moulds. for instance during certain stages of fruit fly embryonic development. Mitosis occurs only in eukaryotic cells and the process varies in different species. to produce two identical daughter cells which are still diploid cells. which lack a nucleus. where the nuclear envelope breaks down before the chromosomes separate. prometaphase. forming single cells with multiple nuclei.

and together the two are called sister chromatids. the parent cell must make a copy of each chromosome before mitosis. These two cells are identical and do not differ in any way from the original parent cell. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. Because each resultant daughter cell should be genetically identical to the parent cell. .Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. This occurs during the S phase of interphase. Each chromosome now has an identical copy of itself.

In plant cells. As a matter of convention. pulling apart the sister chromatids of each chromosome. The chromosomes align themselves in a line spanning the cell. . A new nuclear envelope forms around the separated sister chromosomes. Eventually. giving rise to two daughter cells. each sister chromatid is now considered a chromosome. the process of binary fission is very much different from the process of mitosis. the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). because of the non-involvement of nuclear dynamics and lack of linear chromosomes. However.In most eukaryotes. so they are renamed to sister chromosomes. As mitosis completes. the parent cell will be split in half. each with a replica of the original genome. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. separating the two developing nuclei. In animal cells.the cell begins cytokinesis. the daughter cells will construct a new dividing cell wall between each other. Prokaryotic cells undergo a process similar to mitosis called binary fission. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. As the cell elongates. corresponding sister chromosomes are pulled toward opposite ends.

1 Preprophase In plant cells only. mainly via proteins. Thus. the cell grows by producing proteins and cytoplasmic organelles. The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. a cell grows (G1). where the cell prepares itself for cell division. It alternates with the much longer interphase. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . During all three phases. However. prophase is preceded by a pre-prophase stage.2. 2. All these phases in the interphase are highly regulated. the nucleus has to migrate into the center of the cell before mitosis can begin. In highly vacuolated plant cells. continues to grow as it duplicates its chromosomes (S). S (synthesis). and finally it divides (M) before restarting the cycle. and G2 (second gap). chromosomes are replicated only during the S phase. Interphase is divided into three phases: G1 (first gap).2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. grows more and prepares for mitosis (G 2).2. This is achieved through the formation of a phragmosome.

after the nuclear membrane breaks down. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. The chromosomes have chromatin has condensed. Cytokinesis has already begun. preprophase is characterized by the formation of a ring of microtubules and actin filaments (called preprophase band) underneath the plasma membrane around the equatorial plane of the future mitotic spindle. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. The cells of higher plants (such as the flowering plants) lack centrioles. These microtubules can attach to kinetochores or they can interact with opposing microtubules. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. the pinched area is known as the cleavage furrow. This band marks the position where the cell will eventually divide. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. aligned at the metaphase plate. and microtubules have invaded the nuclear Prophase space. .division. In addition to phragmosome formation. instead. degraded.

Close to the nucleus are structures called centrosomes. they are not essential for the . Since the genetic material has already been duplicated earlier in S phase. A cell inherits a single centrosome at cell division. The centrosome is the coordinating center for the cell's microtubules. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. At the onset of prophase. chromatin condenses together into a highly ordered structure called a chromosome. Although centrioles help organize microtubule assembly.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. giving a pair of centrosomes. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. which are made of a pair of centrioles found in most eukaryotic animal cells. the replicated chromosomes have two sister chromatids. the genetic material in the nucleus is in a loosely bundled coil called chromatin. bound together at the centromere by the cohesin protein complex. Chromosomes are typically visible at high magnification through a light microscope. which is replicated by the cell with the help of the nucleus before a new mitosis begins.

and centrosomes are not always used in mitosis. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. on an average 20 ). such as algae or trichomonads. using energy from ATP to "crawl" up the tube toward the originating centrosome. Prometaphase is sometimes considered part of prophase. 2. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. coupled with polymerisation and depolymerisation of microtubules. one attached at each chromatid. This is called open mitosis. kinetochore microtubules begin searching for kinetochores to attach to. Each chromosome forms two kinetochores at the centromere. or its microtubules are able to penetrate an intact nuclear envelope. When a microtubule connects with the kinetochore. it is known that it contains some form of molecular motor.2. Although the kinetochore structure and function are not fully understood. This motor activity. the motor activates.formation of the spindle. When the spindle grows to sufficient length. undergo a variation called closed mitosis where the spindle forms inside the nucleus.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Fungi and some protists. provides the pulling force necessary to later separate the chromosome's two chromatids. since they are absent from plants. and it occurs in most multicellular organisms. .

All chromosomes (blue) but one have arrived at the metaphase plate. the kinetochore would be the "hook" that catches a sister chromatid or "fish". an imaginary line that is equidistant from the two centrosome poles. the chromosomes come under longitudinal tension from the two ends of the cell. in some sense. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. In certain types of cells. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. As a result. Metaphase comes from the Greek meaning "after.3 Metaphase A cell in late metaphase. The centromeres of the chromosomes. 2. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. . convene along the metaphase plate or equatorial plane." Microtubules find and attach to kinetochores in prometaphase.In the fishing pole analogy. only roughly lining up along the midline. analogous to a tug-of-war between people of equal strength.

Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). which have now become distinct sister chromosomes. the proteins that bind sister chromatids together are cleaved. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. Two events then occur: first.” “back. . although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. allowing them to separate. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. These sister chromatids. These two stages are sometimes called early and late anaphase. The force that causes the centrosomes to move towards the ends of the cell is still unknown. the nonkinetochore microtubules elongate. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. Next.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate.” or “re-”). the cell proceeds to anaphase (from the Greek meaning “up. At the end of anaphase. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned.” “against. 2. Early anaphase is usually defined as the separation of the sister chromatids. The signal creates the mitotic spindle checkpoint.

but cell division is not yet complete.2. pinching off the separated nuclei. cytokinesis is a separate process that begins at the same time as telophase. At telophase. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. the nonkinetochore microtubules continue to lengthen. Mitosis is complete. A new nuclear envelope. forms around each set of separated sister chromosomes. however. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. using fragments of the parent cell's nuclear membrane. Cytokinesis is technically not even a phase of mitosis. cell . now surrounded by new nuclei.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. Both sets of chromosomes. In both animal and plant cells. 2. In animal cells. It "cleans up" the after effects of mitosis. unfold back into chromatin. but rather a separate process. elongating the cell even more.5 Cytokinesis Cilliate undergoing cytokinesis. Corresponding sister chromosomes attach at opposite ends of the cell. necessary for completing cell division.

Each daughter cell has a complete copy of the genome of its parent cell.. which move along microtubules to the middle of the cell. e. The phragmoplast is a microtubule structure typical for higher plants.g. zygote and also the basis of the growth of a multicellular body. 2. New cells are formed by mitosis and so are exact copies of the cells being replaced. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. Similarly. cells are constantly sloughed off and replaced by new ones.5.3 Cell replacement In some parts of body.division is also driven by vesicles derived from the Golgi apparatus. skin and digestive tract.5. This is the basis of the development of a multicellular body from a single cell i. The end of cytokinesis marks the end of the M-phase.4 Regeneration . Following are the occasions in the lives of organism where mitosis happens: 2. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. whereas some green algae use a phycoplast microtubule array during cytokinesis. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.2 Development and growth The number of cells within an organism increases by mitosis.5. 2. separating the two nuclei.5.1Significance Mitosis is important for the maintenance of the chromosomal set. 2.e.

the hydra reproduces asexually by budding. One daughter cell will receive both sister chromosomes and the other will receive none. For example. Occasionally when cells experience nondisjunction. a condition often associated with cancer. 2. These cells are considered aneuploid. The cells at the surface of hydra undergo mitosis and form a mass called bud. resulting in binucleated cells. Mitosis continues in the cells of bud and it grows into a new individual. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). The same division happens during asexual reproduction or vegetative propagation in plants. the process may go wrong.5. a condition known as trisomy. The production of new cells is achieved by mitosis. they fail to complete cell division and retain both nuclei in one cell.5. a condition known as monosomy. .Some organisms can regenerate their parts of bodies. sea star regenerates its lost arm through mitosis. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. 2. a chromosome may fail to separate during anaphase. For example. and the latter cell having only one chromosome (the homologous chromosome). In non-disjunction. especially during early cellular divisions in the zygote.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction.7 Consequences of errors Although errors in mitosis are rare.

causing translocation. its organelles disintegrate and reform in a matter of hours. Errors in the control of mitosis may cause cancer. As long as these tumours remain in their original location they are called benign tumours. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. Or. This phenomenon is called metastasis or spreading of disease. sometimes mutuations occur in such genes and cells continue to divide. The effect of these genetic abnormalities depends on the specific nature of the error. causing inversion. it may be treated erroneously as a separate chromosome. It results in the synthesis of execessive tissue growths. All cells have genes that control the timing and number of mitosis. but in reverse orientation. non-homologous chromosome. Occasionally. chromosomes may become damaged. Benign tumours are not harmful as soon as they are not moving. Such tumours can send cancer cells to other parts in body where new tumours may form. It may reattach to the original chromosome.Mitosis is a demanding process for the cell. and chromosomes are jostled constantly by probing microtubules. It results in abnormal cell growth. . it results in the formation of Tumors. causing deletion. The fragment may incorrectly reattach to another. As soon as they start to move and invade other cells there are said to be malignant tumours. causing chromosomal duplication. which goes through dramatic changes in ultrastructure. An arm of the chromosome may be broken and the fragment lost. Now what happens is that cell abnormally continue to divide at a single place. When tissues more than the requirement are synthesized in a single organ.

An example of a cell that goes through endomitosis is the megakaryocyte.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. Preceded by events in prometaphase and followed by anaphase. This process may also be referred to as endoreduplication and the cells as endoploid. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. In certain types of cells. only roughly lining up along the middleline. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Metaphase accounts for approximately 4% of the cell cycle's duration. an imaginary line that is equidistant from the two centrosome poles. analogous to a tug of war between equally strong people. resulting in cells with many copies of the same chromosome occupying a single nucleus. align in the middle of the cell before being separated into each of the two daughter cells. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. 2. from the ancient Greek(between) and (stage).7 Metaphase Metaphase. Early events of metaphase can . carrying genetic information.2.

securin. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. produces a pattern of in total up to several hundred bands.coincide with the later events of prometaphase. Staining of the slides. often with Giemsa (G banding) or Quinacrine. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. which makes them most suitable for visual analysis. as chromosomes with connected kinetochores will start the events of metaphase individually before other chromosomes with unconnected kinetochores that are still lingering in the events of prometaphase. For classical cytogenetic analyses. 2. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). and separase. even if most of the kinetochores have been attached and most of the chromosomes have been aligned.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. Only after all chromosomes have become aligned at the metaphase plate. This would be accomplished by regulation of the anaphase-promoting complex. Chromosomes are condensed(Thickened) and highly coiled in metaphase. does the cell enter anaphase. when every kinetochore is properly attached to a bundle of microtubules. Normal metaphase spreads are used in . Metaphase chromosomes make the classical picture of chromosomes (karyotype). Such a signal creates the mitotic spindle checkpoint.

methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. . for example. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. losses of chromosomal segments or translocations. which may lead to chimeric oncogenes. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. such as bcr-abl in chronic myelogenous leukemia.

cellular function. such as. any differences between the sex chromosomes. [4] The preparation and study of karyotypes is part of cytogenetics. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. Karyogram of human male using Giemsa staining. The study of karyotypes is important for cell biology and genetics. There may. be sex chromosomes. in humans 2n = 46. and to gather information about past evolutionary events. Karyotypes describe the number of chromosomes. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. taxonomic relationships. banding pattern. The term is also used for the complete set of chromosomes in a species. and any other physical characteristics. The study of whole sets of chromosomes is sometimes known as karyology. Thus. autosomal chromosomes are present in two copies. .CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). in normal diploid organisms. Karyotypes can be used for many purposes. Attention is paid to their length. the position of the centromeres. or may not. to study chromosomal aberrations. So. or an individual organism. and what they look like under a light microscope. ordered by size and position of centromere for chromosomes of the same size. and the results may be used in evolutionary biology and medicine. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n.

when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Considering their techniques. The subsequent history of the concept can be followed in the works of Darlington and White. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Pretreating cells in a hypotonic solution. the discoverer of mitosis. Using cells in culture 2. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Their behavior in animal (salamander) cells was described by Walther Flemming. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. New techniques were needed to definitively solve the problem: 1.3. concluding an XX/XO sex determination mechanism. in 1882. The name was coined by another German anatomist. which swells them and spreads the chromosomes . von Waldeyer in 1888. in contrast to their genic contents.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. The next stage took place after the development of genetics in the early 20th century. these results were quite remarkable. at first favoring 46. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. He revised his opinion later from 46 to 48. and he correctly insisted on humans having an XX/XY system.

such as Giemsa.3. 3.2 Observations on karyotypes 3. For humans. [16] Sometimes observations may be made on non-dividing (interphase) cells. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. Arresting mitosis in metaphase by a solution of colchicines 4. 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . Human chromosome 2 was formed by a merger of ancestral chromosomes. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Rather interestingly. reducing the number. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. the great apes have 48 chromosomes. The sex of an unborn fetus can be determined by observation of interphase cells. is applied after cells have been arrested during cell division by a solution of colchicine.2. a suitable dye.1 Staining The study of karyotypes is made possible by staining. Usually.2.

shape and banding of the chromosomes. indicating tighter packing. Humans have one pair fewer chromosomes than the great apes. type. 3. A full account of a karyotype may therefore include the number. 5. This feature probably reflects different amounts of DNA duplication. Differences in the position of centromeres. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. Differences in absolute sizes of chromosomes. . Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. 4. as well as other cytogenetic information. This is brought about by translocations. 6. and mainly consists of genetically inactive repetitive DNA sequences. Differences in degree and distribution of heterochromatic regions. 2. permitting its loss without penalty to the organism (the dislocation hypothesis). both have six pairs of chromosomes (n=6) yet V.1. which (when they occur) are small bodies attached to a chromosome by a thin thread. but the genes have been mostly translocated (added) to other chromosomes. Heterochromatin stains darker than euchromatin. faba chromosomes are many times larger. Differences in number and position of satellites.

3. the same cannot be said for their karyotypes. Mosaics or otherwise abnormal individuals. and in . Any variation from the standard karyotype may lead to developmental abnormalities. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Between the germ-line and soma (between gametes and the rest of the body) 3. XX. which are highly variable. males have both an X and a Y chromosome denoted 46. There is variation between species in chromosome number. Normal karyotypes for females contain two X chromosomes and are denoted 46. Between the sexes 2.3 The human karyotype Most (but not all) species have a standard karyotype. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Between members of a population (chromosome polymorphism) 4. Geographical variation between races 5.Variation is often found: 1. XY.

. used in conjunction with other phylogenetic data.. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed.. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. despite their construction from the same macromolecules. found in some copepods and roundworms such as Ascaris suum. as in many sciarid flies. But. In some cases there is even significant variation within species. This variation provides the basis for a range of studies in evolutionary cytology. portions of the chromosomes are cast away in particular cells.3. it is quite unclear what the general significance might be. or other kinds of visible adjustment to the karyotype.1 Changes during development Instead of the usual gene repression. entire chromosomes are eliminated during development. the general significance of karyotype evolution is obscure. . This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. "We have a very poor understanding of the causes of karyotype evolution. In A. some organisms go in for large-scale elimination of heterochromatin. In some species. Although much is known about karyotypes at the descriptive level. 3.detailed organization. Chromosome elimination. which were previously inexplicable. In a review. despite many careful investigations. Godfrey and Masters conclude: "In our view.. In this process. Chromatin diminution (founding father: Theodor Boveri).

. male = 7 chromosomes. where the haploid n = 1.. which was investigated by Kurt Benirschke and his colleague Doris Wurster. They kept quiet for two or three years because they thought something was wrong with their tissue culture. The diploid number of the Chinese muntjac. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). the high record would be somewhere amongst the ferns. was found to be 46. In placental mammals.. The low record is held by the nematode Parascaris univalens. Muntiacus reevesi. In marsupials it is always the paternal X which is inactivated. "They simply could not believe what they saw.suum. Muntiacus muntjak.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. In human females some 15% of somatic cells escape inactivation. the inactivation is random as between the two Xs. all telocentric. all the somatic cell precursors undergo chromatin diminution. When they looked at the karyotype of the closely related Indian muntjac.. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.3. thus the mammalian female is a mosaic in respect of her X chromosomes. they were astonished to find it had female = 6. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. Xinactivation. The existence of supernumerary or B chromosomes . 3.

horsetails and psilotales) is also common. It is a common arrangement in the Hymenoptera. Polyploidy in animals is much less common. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. occurs mainly in plants.means that chromosome number can vary even within one interbreeding population. but in grasses the average is much higher. due to the presence of five acrocentric chromosome pairs (13. 14. 21 and 22). It has been of major significance in plant evolution according to Stebbins. where there are more than two sets of homologous chromosomes in the cells. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. but it has been significant in some groups. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. about 70%.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Humans have FN = 82. 15. and aneuploids are another example. though in this case they would not be regarded as normal members of the population. 3. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. and the other haploid. where one sex is diploid. and in some other groups. FN. FN ≤ 2n.Endopolyploidy occurs when in adult differentiated . Haplo-diploidy. Thus. Polyploidy. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. Polyploidy in lower plants (ferns.3. 3.3 Fundamental number The fundamental number.

In many instances. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. but the nuclei contain more than the original somatic number of chromosomes.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. Abnormalities in chromosome number usually cause a defect in development. . 3. the daughter chromosomes separating from each other inside an intact nuclear membrane. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. Down syndrome and Turner syndrome are examples of this. it is diverse and complex. The cells do not always contain exact multiples (powers of two). and serves differentiation and morphogenesis in many ways.tissues the cells have ceased to divide by mitosis. See palaeopolyploidy for the investigation of ancient karyotype duplications. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost.

Human chromosome 2 was formed by a merger of ancestral chromosomes. 6.000 km2). When this happens. 3.6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson. and 7. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 3.Aneuploidy may also occur within a group of closely related species. that the two chromosome morphs are adapted to different habitats. the European shrew Sorex araneus. Well-researched examples are the ladybird beetle Chilocorus stigma. and Crocus. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.500 sq mi (17.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. Classic examples in plants are the genus Crepis. the chromosome number is variable from one individual to another. 5. reducing the number. where the gametic (= haploid) numbers form the series x = 3. living from rainforests to . where every number from x = 3 to x = 15 is represented by at least one species. the great apes have 24x2 chromosomes whereas humans have 23x2. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. 4. In about 6. some mantids of the genus Ameles. [41] Closer to home.

make it possible to see which species are closely related. The polytene banding of the 'picture wing' group. Chromosome rearrangements. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. .4 million years ago (mya) (Mauna Kea) to 10mya (Necker). but these are much less frequent. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. show a clear "flow" of species from older to newer islands.subalpine meadows. probably 20 million years ago. and skipping of islands. In a sense. the best-studied group of Hawaiian drosophilids. in the family Drosophilidae. The results are clear. Using K-Ar dating. Although it would be possible for a single gravid female to colonise an island. it is more likely to have been a group from the same species. which can be dated to 30 mya. gene arrangements are visible in the banding patterns of each chromosome. at least into the Cretaceous. especially inversions. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The inversions. the present islands date from 0. when plotted in tree form (and independent of all other information). There are also cases of colonization back to older islands. Drosophila and Scaptomyza. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain.

R-banding is the reverse of G-banding (the R stands for "reverse"). The light regions tend to be euchromatic. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). . • • C-banding: Giemsa binds to constitutive heterochromatin. if less spectacular. It yields a series of lightly and darkly stained bands .7 Depiction of karyotypes 3. adaptive radiations. • Q-banding is a fluorescent pattern obtained using quinacrine for staining.1 Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes: • G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. late-replicating and AT rich.There are other animals and plants on the Hawaiian archipelago which have undergone similar.7. early-replicating and GC rich.the dark regions tend to be heterochromatic. so it stains centromeres. This method will normally produce 300-400 bands in a normal. • T-banding: visualize telomeres. The pattern of bands is very similar to that seen in G-banding. human genome. 3.

a dye. denoting the activity of rRNA genes within the NOR. Each chromosome has a characteristic banding pattern that helps to identify them. Giemsa is specific for the phosphate groups of DNA. often Giemsa (G-banding). respectively.7. In the "classic" (depicted) karyotype. Some karyotypes call the short and long arms p and q. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. This yields a dark region where the silver is deposited. less frequently Quinacrine. is used to stain bands on the chromosomes. Quinacrine binds to the adeninethymine-rich regions.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. For example.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. both chromosomes in a pair will have the same banding pattern. Karyotypes are arranged with the short arm of the chromosome on top. 3. In addition. Cri du chat syndrome involves a deletion on the short arm of . and the long arm on the bottom.

XX. Because there are a limited number of spectrally-distinct fluorophores. The critical region for this syndrome is deletion of 15.5p-. which is written as 46.chromosome 5. It is written as 46. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.2) 3.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Image processing software then assigns a pseudo color to each spectrally different combination.7.del(5)(p15. . Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. allowing the visualization of the individually colored chromosomes. This method is also known as virtual karyotyping. a combinatorial labeling method is used to generate many different colors. 3.XX.2. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.

CHAPTER 4 .

inversions. in which three copies of a chromosome are present instead of the usual two. as in derivative chromosome. also known as aneuploidy. large-scale deletions or duplications. XXY is caused by an extra X chromosome. • • Patau syndrome is caused by trisomy of chromosome 13. X0).4.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. although they generally do not survive to birth. including . the most common male chromosomal disease. is caused by trisomy of chromosome 21. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. X or 45. otherwise known as 47. are common numerical abnormalities. Also documented are trisomy 8. a common chromosomal disease. translocations. trisomy 9 and trisomy 16. Down syndrome. or structural. Structural abnormalities often arise from errors in homologous recombination. often occur as a result of nondisjunction during meiosis in the formation of a gamete. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. trisomies. Klinefelter syndrome. Some disorders arise from loss of just a piece of one chromosome. Numerical abnormalities. as in the presence of extra or missing chromosomes. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18.

A chromosome anomaly. a deletion of the maternal genes. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. from a truncated short arm on chromosome 5. one well-documented example is the Philadelphia chromosome. numerical and structural anomalies. from the loss of part of the short arm of chromosome 1. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. They can be organized into two basic groups.• Cri du chat (cry of the cat). A chromosome anomaly may be detected or confirmed in this manner. caused by abnormal formation of the larynx. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. The name comes from the babies' distinctive cry. . example of imprinting disorder. There are many types of chromosome anomalies. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. example of imprinting disorder. A Karyotype refers to a full set of chromosomes from an individual which can be compared to a "normal" Karyotype for the species via genetic testing. 1p36 Deletion syndrome. a deletion of the paternal genes. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual.

an entire chromosome has . In a reciprocal translocation.4. an X. and Jacobsen syndrome.). • • Translocations: When a portion of one chromosome is transferred to another chromosome. Tetrasomy. Known disorders in humans include Wolf-Hirschhorn syndrome. also called the terminal 11q deletion disorder. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. segments from two different chromosomes have been exchanged. rather than two). 4. There are two main types of translocations. which is caused by partial deletion of the short arm of chromosome 4. resulting in extra genetic material.3 Structural abnormalities When the chromosome's structure is altered. etc. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. Duplications: A portion of the chromosome is duplicated. In a Robertsonian translocation. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes).

Therefore. resulting in Mosaicism (where some cells have the anomaly and some do not). It includes routine analysis of G-Banded chromosomes. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. can happen after conception. however.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. especially the chromosomes. 21 and 22. • Rings: A portion of a chromosome has broken off and formed a circle or ring. 14. • Inversions: A portion of the chromosome has broken off. other cytogenetic banding techniques. therefore the genetic material is inverted. turned upside down and reattached. the anomaly is present in every cell of the body. as well . 4. Some anomalies.in humans these only occur with chromosomes 13. and are therefore initially not inherited. This can happen with or without loss of genetic material. 15. Chromosome anomalies can be inherited from a parent or be "de novo". This is why chromosome studies are often performed on parents when a child is found to have an anomaly. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm.attached to another at the Centromere . They often lead to an increased tendency to develop certain types of malignancies. 4.

He revised his opinion later from 46 to 48. New techniques were needed to definitively solve the problem: 1. Using cells in culture 2. at first favoring 46. and he correctly insisted on man having an XX/XY system. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. the discoverer of mitosis.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. concluding an XX/XO sex determination mechanism. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. Their behavior in animal (salamander) cells was described by Walther Flemming. 4. von Waldeyer in 1888.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). The name was coined by another German anatomist. Considering their techniques. which swells them and spreads the chromosomes . Painter in 1922 was not certain whether the diploid number of man was 46 or 48. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Pre-treating cells in a hypotonic solution. in 1882. in contrast to their genic contents. The next stage took place after the development of genetics in the early 20th century. these results were quite remarkable.

McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).6 Applications in biology 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. the great apes have 48 chromosomes. Using Painter's technique they studied the polytene . Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Natural populations of Drosophila In the 1930s. a find which eventually led to her Nobel Prize in 1983. reducing the number. 4. Arresting mitosis in metaphase by a solution of colchicine 4. In 1931. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.3. persimilis from wild populations in California and neighboring states. During her cytogenetic work. 4.6.6. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Rather interestingly. McClintock discovered transposons.

but adjust to certain frequencies at which they become stabilised. Evidence rapidly accumulated to show that natural selection was responsible.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. 4. Down syndrome is also referred to as trisomy 21. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. . Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. which enabled feeding. It was found that the various chromosome types do not fluctuate at random. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. discoveries were quickly made related to aberrant chromosomes or chromosome number. Using a method invented by L'Heretier and Teissier. breeding and sampling whilst preventing escape. as they would if selectively neutral. In some congenital disorders. such as Down's syndrome. Dobzhansky bred populations in population cages. Abnormalities arising from nondisjunction events can cause cells with aneuploidy (additions or deletions of entire chromosomes) in one of the parents or in the fetus. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. as with most polymorphisms. This had the benefit of eliminating migration as a possible explanation of the results. In 1959. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism.

which is why there is a phenotypic effect seen in individuals with extra X chromosomes. Not all genes on the X Chromosome are inactivated. Pennsylvania. which is required in normal females to compensate for having two copies of the chromosome. In 1960. and XXXX.Other numerical abnormalities discovered include sex chromosome abnormalities. Identification of the Philadelphia chromosome by cytogenetics. This abnormal chromosome was dubbed the Philadelphia chromosome . has Klinefelter's Syndrome. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. An individual with only one sex chromosome (the X) has Turner syndrome. Thirteen years later. an additional X chromosome in a male. resulting in 47 total chromosomes. Peter Nowell and David Hungerford [17] discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML).as both scientists were doing their research in Philadelphia. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. XYY. with the development of more advanced techniques. . is used today as a diagnostic for CML. in addition to other tests. Many other sex chromosome combinations are compatible with live birth including XXX. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome.

Caspersson developed banding techniques which differentially stain chromosomes. 4.8 Beginnings of molecular cytogenetics . Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Deletion syndromes such as DiGeorge syndrome.FIG Advent of banding techniques In the late 1960s. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Deletions within one chromosome could also now be more specifically named and understood. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. and elongation techniques for all culture types that allow for higher resolution banding. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.

CHAPTER 5 Techniques 5. While radioisotope-labeled probes had been hybridized with DNA since 1969. cloned and studied in ever greater detail.In the 1980s. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. movement was now made in using fluorescent labeled probes. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.1 Karyotyping . Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). advances were made in molecular cytogenetics.

Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.

5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization

Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •

bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations

5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,

. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. test and measurement. Using MATLAB. you can solve technical computing problems faster than with traditional programming languages. CGH and Single nucleotide polymorphism-arrays. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. data visualization. control design. and FORTRAN. and numerical computation. including signal and image processing. For congenital problems usually 20 metaphase cells are scored. financial modeling and analysis. data analysis. such as C. such as comparative genomic hybridization arrays.generally between 200 and 1000 cells are counted and scored. You can use MATLAB in a wide range of applications. C++. communications. and computational biology.

The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted. such as declaring variables. You can integrate your MATLAB code with other languages and applications. . MATLAB eliminates the need for ‘for’ loops. With the MATLAB language. specifying data types. and allocating memory.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. 6. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. and distribute your MATLAB algorithms and applications. These effects must be compensated to improve the results of the pairing algorithm. It enables fast development and execution.MATLAB provides a number of features for documenting and sharing your work. one line of MATLAB code can often replace several lines of C or C++ code. The image processing step is composed of the following operations. In many cases. As a result.

performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. the spatially scaled images are histogram equalized. To compare chromosomes from a band pattern point of view. Therefore. 6. geometrical and dimensional differences must be removed. a dimensional scaling is performed before the pattern features is extracted to make all the chromosome with the same size and aspect ratio by interpolating the original images. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. To compensate for this inhomogeneity.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding). or at least attenuated. 2) Geometrical compensation—The geometric compensation.2 Concepts used in this phase 1) Image conversion 2) Denoising .

MATLAB simply applies the filter to the indices in the indexed image matrix. and the results might not be meaningful.3) Edge detection 4) Two dimensional convolutions. green. In addition to these image type conversion functions. and blue planes. so the image displays as shades of gray. If you attempt to filter the indexed image. The resulting true color image has identical matrices for the red. When you apply the filter to the true color image. You can perform certain conversions just using MATLAB syntax.I. 6.I. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. you must first convert it to true color format. there are other functions that return a different image type as part of the operation they perform. . listed in the following table.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. if you want to filter a color image that is stored as an indexed image. For example. MATLAB filters the intensity values in the image. For example.I). RGB = cat (3.2. For example. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. as is appropriate.

to isolate particular objects from their background. caused by external disturbance. and hence the type of noise on the image.4 Denoising We may define noise to be any degradation in the image signal. Usually we know what type of errors to expect. There is a large number of edge finding algorithms in existence. or through networked cable. . Cleaning an image corrupted by noise is thus an important area of image restoration.B) computes the two-dimensional convolution of matrices A and B. If an image is being sent electronically from one place to another.parameters. via satellite or wireless transmission. We may use edges to measure the size of objects in an image. and we shall look at some of the more straightforward of them. to recognize or classify objects.5 Edge detection Edges contain some of the most useful information in an image.3 Two dimensional convolutions C = conv2(A. ) Where the parameters available depend on the method used 6. hence we can choose the most appropriate method for reducing the effects.2. we may expect errors to occur in the image signal. .6.2. If one of these matrices describes a two-dimensional finite impulse response . 6. The general Matlab command for finding edges is edge(image. These errors will appear on the image output in different ways depending on the type of disturbance in the signal.'method'.

[3 3]). if the size of then the size of C is [ma+mb-1. the other matrix is filtered in two dimensions. C = conv2(. If hcol is a column vector and hrow is a row vector.7). minus one. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.bmp'). .'shape') subsection of the two-dimensional convolution.. imedfilt2(im1.nb]. rgb2gray im2bw(im. nb]+1)/2).0. edge(im1..hrow. The size of matrices.'sobel'). That is.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.na+nb-1].na] and the size of B is [mb.. as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.A).(FIR) filter. this case is the same as C = conv2(hcol*hrow.

8). Index=1.imy). for i=1:sx x1=rc(i. [r.1).1).2). flag=0.c] = find(L==22). rc = [r c].y1)=255. L_number=zeros(mx.n]=size(L). n1(x1.j)~=0 for k=1:mx if L(i.double(msk)). for i=1:m for j=1:n if L(i.j)==L_number(k) flag=1.imy]=size(BW). . nzeros(imx. y1=rc(i.[imx. [sx sy]=size(rc). MODULE 2 clc [m. Msk conv2(double(BW). mx=max(max(L)). bwlabel(B.

62. [sx sy]=size(rc).43.2).11.7.y1)=255.26.50.imshow(n1.32. n1(x1.51.6.20.31.[]).66].45.4.42.end end if flag~=1 L_number(Index)=L(i. end flag=0.41.10.28.38.40.8. end end L_number.21.56.65. y1=rc(i.29. n1=zeros(imx. Index=Index+1.49.15.19.imy). 36.22.59.j).48.27.57.30. Test_number=[3. end %h=figure.39. for x=1:46 [r.33.c] = find(L==L_number((Test_number(x)))). .1). end.54.9.55.60.14. for i=1:sx x1=rc(i. rc = [r c].24.35.52.

1).1). f=imcomplement(f).1. Circumference_sum=0. BW1=edge(BW.'skel'.bmp')). Arm_length=zeros(46. for i=1:46 f=imread(strcat(num2str(i).5*graythresh(skel)). Area=zeros(46. s=bwmorph(skel. skel=im2double(f). s1=bwmorph(s.1).Inf). BW=double(BW). end end end Circumference(i)=Circumference_sum.y)==1 Circumference_sum=Circumference_sum+1. Arm_length_sum=0. for x=1:m for y=1:n if BW1(x. [m n]=size(BW1). skel=im2bw(skel.'spur'. .'canny').8).'. BW=im2bw(f).end Circumference=zeros(46.

for x=1:m for y=1:n if BW(x. [m n]=size(BW).[m n]=size(s1). end Circumference. end end end Arm_length(i)=Arm_length_sum. Area_sum=0.y)==1 Area_sum=Area_sum+1.y)==1 Arm_length_sum=Arm_length_sum+1. . Arm_length. end end end Area(i)=Area_sum. for x=1:m for y=1:n if s1(x. BW=im2bw(f).

Pair=zeros(46. end end end for i=1:45 if Pair(i.Area.2)=i. Pair(i. Pair(i. . Pair(i. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)).2). Pair(i.1)=46. end end Pair. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)).2)==46 Pair(46.2)=j.1)=i.2)=i+1.1)=i. Pair(46.

end end if flag~=1 if figure_flag~=47 subplot(23.2). figure_flag=figure_flag+1. end f2=imread(strcat(num2str(Pair(i. for i=1:46 for j=1:46 if Pair(i.bmp')). end f1=imread(strcat(num2str(Pair(i. delete(figure_flag)=Pair(i. imshow(f1). figure_flag=figure_flag+1.1)). imshow(f2).delete=zeros(46.figure_flag). flag=0. figure_flag=1.'.'.1)==delete(j) flag=1. end flag=0.2)). if figure_flag~=47 subplot(23.figure_flag).2. .2.bmp')).1).

in the scope of karyotyping process used in cytogentic analysis. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. The main goal of this paper is to provide useful contributions toward the design of a fully automatic chromosomes pairing algorithm of bone marrow cells to be used in the diagnosis of leukemia. dimensions and banding profiles. The images of these chromosomes present less quality and level of detail than the ones usually used in traditional genetic analysis using datasets such as Edinburgh. plus a new one.end CONCLUTION In this paper. The proposed algorithm is based on the traditional features extracted from the karyogram. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. based on the MI. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. such as. 2) feature extraction from the processed images . Copenhagen. and Philadelphia.

10% mean classification rate. and to normalize their dimensions. In the image processing step. achieves a 70. shape and band pattern. Vectors of coefficients associated with each one of the 22 classes are computed and the distance between two arbitrary chromosomes is the minimum one among all distances obtained with these 22 vectors. 4) pairing. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. are processed in order to compensate for geometrical and intensity distortions. the romosome images. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. extracted from the unordered karyogram.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.characterizing the size. from the chromosomes in the training set. The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram. Here. This normalization is needed to make it possible the band pattern comparison between chromosomes. The addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the .working within an 8-D feature space. and band pattern. shape. by minimizing the overall distances between chromosomes of the same class (intraclass) and by maximizing the distances between chromosomes when at least one of them does not belong to that class (interclass). The training process consists in the estimation of each vector of coefficient . The features extracted from the processed images discriminate each pair with respect to their size. Tests using 19 karyograms based on bone marrow cells. and finally.

In addition. e. whose images are of significantly higher quality. In fact. Using 27 karyograms andworking with a limited number of classes (≤ 8).. presenting a uniform level of condensation. Executing the algorithm on a higher quality dataset. called LK1 . Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. despite the low quality of this type of chromosomes. centromere position. such as Edinburgh. Copenhagen. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes.g. and from which it is possible to extract additional features.10% classification ratewas obtained. This dataset was made publicly available [29]. or Philadelphia. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. The results presented in this paper are promising. a new chromosome dataset with 9200 chromosomes from bone marrow cells. amean classification rate larger than 93% was obtained in all experiments. a 76.performance of the classifier. REFERENCES .

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