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Chromosome Document

Chromosome Document

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Published by: Pushpa Mohan Raj on Apr 07, 2013
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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

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

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. rather than 2. If the mutation involves only one or a few chromosomes in the genome (e. Euploid human karyotypes are 46. Such individuals are called euploid and have the wild-type chromosome complement for the species. XX (female) or 46 XY (male).3 MUTATIONS IN CHROMOSOME NUMBER Normally. reflecting their bacterial origins.XY or 47. The individual would have Down Syndrome and his/her karyotype would be written 47. copies of chromosome 21.g. 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).XX.+21. 1.are also found in mitochondria and chloroplasts. a extra copy of human chromosome 21). the individual carrying the mutation is said to be aneuploid. . The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.+21. An example of aneuploidy is trisomy 21.

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

bone marrow in large bones produces new blood cells. .7 lbs). The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. which use the bone marrow vasculature as a conduit to the body's systemic circulation. in adults weighing 65 kg (143 lbs). Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. In humans. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. On average. which divides the nuclei.1. [1] Bone marrow is also a key component of the lymphatic system. bone marrow accounts for approximately 2. It is generally followed immediately by cytokinesis.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. in two separate nuclei.6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2. cytoplasm. bone marrow constitutes 4% of the total body mass of humans.

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

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

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

chromosomes are replicated only during the S phase. All these phases in the interphase are highly regulated. However.1 Preprophase In plant cells only. Thus. S (synthesis). grows more and prepares for mitosis (G 2). It alternates with the much longer interphase.2.2. Interphase is divided into three phases: G1 (first gap). 2. During all three phases. This is achieved through the formation of a phragmosome. the cell grows by producing proteins and cytoplasmic organelles. continues to grow as it duplicates its chromosomes (S). 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.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. mainly via proteins. where the cell prepares itself for cell division. a cell grows (G1). a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . In highly vacuolated plant cells. the nucleus has to migrate into the center of the cell before mitosis can begin. and G2 (second gap). and finally it divides (M) before restarting the cycle. prophase is preceded by a pre-prophase stage.

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

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

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

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

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

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

RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. Following are the occasions in the lives of organism where mitosis happens: 2. e.5. 2. separating the two nuclei.5. whereas some green algae use a phycoplast microtubule array during cytokinesis. 2. The end of cytokinesis marks the end of the M-phase. New cells are formed by mitosis and so are exact copies of the cells being replaced. The phragmoplast is a microtubule structure typical for higher plants. Similarly.5.2 Development and growth The number of cells within an organism increases by mitosis.3 Cell replacement In some parts of body.5. This is the basis of the development of a multicellular body from a single cell i.e. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. zygote and also the basis of the growth of a multicellular body.1Significance Mitosis is important for the maintenance of the chromosomal set. which move along microtubules to the middle of the cell.g. 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.4 Regeneration . Each daughter cell has a complete copy of the genome of its parent cell. cells are constantly sloughed off and replaced by new ones. skin and digestive tract.division is also driven by vesicles derived from the Golgi apparatus..

5. especially during early cellular divisions in the zygote. Occasionally when cells experience nondisjunction. Mitosis continues in the cells of bud and it grows into a new individual.Some organisms can regenerate their parts of bodies. One daughter cell will receive both sister chromosomes and the other will receive none. 2.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. For example. The production of new cells is achieved by mitosis. For example. they fail to complete cell division and retain both nuclei in one cell. sea star regenerates its lost arm through mitosis. In non-disjunction. a chromosome may fail to separate during anaphase.7 Consequences of errors Although errors in mitosis are rare. a condition known as monosomy. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. The same division happens during asexual reproduction or vegetative propagation in plants. the process may go wrong. a condition often associated with cancer. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). a condition known as trisomy. 2.5. resulting in binucleated cells. These cells are considered aneuploid. the hydra reproduces asexually by budding. and the latter cell having only one chromosome (the homologous chromosome). The cells at the surface of hydra undergo mitosis and form a mass called bud. .

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

In certain types of cells. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. align in the middle of the cell before being separated into each of the two daughter cells. from the ancient Greek(between) and (stage). analogous to a tug of war between equally strong people. an imaginary line that is equidistant from the two centrosome poles. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. 2.2.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. Early events of metaphase can . resulting in cells with many copies of the same chromosome occupying a single nucleus. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). only roughly lining up along the middleline. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase.7 Metaphase Metaphase. Metaphase accounts for approximately 4% of the cell cycle's duration. carrying genetic information. An example of a cell that goes through endomitosis is the megakaryocyte. Preceded by events in prometaphase and followed by anaphase. This process may also be referred to as endoreduplication and the cells as endoploid.

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. Staining of the slides. securin. does the cell enter anaphase. Only after all chromosomes have become aligned at the metaphase plate. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. 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. Metaphase chromosomes make the classical picture of chromosomes (karyotype). Such a signal creates the mitotic spindle checkpoint. Normal metaphase spreads are used in . when every kinetochore is properly attached to a bundle of microtubules. often with Giemsa (G banding) or Quinacrine. 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). produces a pattern of in total up to several hundred bands. which makes them most suitable for visual analysis. 2. Chromosomes are condensed(Thickened) and highly coiled in metaphase. This would be accomplished by regulation of the anaphase-promoting complex. and separase. For classical cytogenetic analyses. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase.coincide with the later events of prometaphase.

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

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

The next stage took place after the development of genetics in the early 20th century. The name was coined by another German anatomist. He revised his opinion later from 46 to 48. at first favoring 46. these results were quite remarkable. and he correctly insisted on humans having an XX/XY system. Considering their techniques. Pretreating cells in a hypotonic solution. 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 1912.3. Using cells in culture 2. von Waldeyer in 1888. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in 1882. the discoverer of mitosis. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. The subsequent history of the concept can be followed in the works of Darlington and White. which swells them and spreads the chromosomes . New techniques were needed to definitively solve the problem: 1. concluding an XX/XO sex determination mechanism. in contrast to their genic contents. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.1 History of karyotype studies 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. Their behavior in animal (salamander) cells was described by Walther Flemming.

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

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

XX. Between the germ-line and soma (between gametes and the rest of the body) 3. Geographical variation between races 5. males have both an X and a Y chromosome denoted 46. 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. and in . XY. Between the sexes 2. There is variation between species in chromosome number. Mosaics or otherwise abnormal individuals. 3. the same cannot be said for their karyotypes.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. which are highly variable. Normal karyotypes for females contain two X chromosomes and are denoted 46. Between members of a population (chromosome polymorphism) 4.Variation is often found: 1.

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

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

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

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

Classic examples in plants are the genus Crepis.000 km2). the chromosome number is variable from one individual to another. Human chromosome 2 was formed by a merger of ancestral chromosomes. In about 6. the great apes have 24x2 chromosomes whereas humans have 23x2.500 sq mi (17. living from rainforests to .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. that the two chromosome morphs are adapted to different habitats. the European shrew Sorex araneus. some mantids of the genus Ameles. and 7. and Crocus. where every number from x = 3 to x = 15 is represented by at least one species. 6. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. reducing the number. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. 5. 3. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. where the gametic (= haploid) numbers form the series x = 3. [41] Closer to home. 3. Well-researched examples are the ladybird beetle Chilocorus stigma.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. 4. When this happens.Aneuploidy may also occur within a group of closely related species.

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

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

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

.7.2.XX. which is written as 46. This method is also known as virtual karyotyping. 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. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.2) 3. 3. It is written as 46.XX. The critical region for this syndrome is deletion of 15.3 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.chromosome 5. Because there are a limited number of spectrally-distinct fluorophores.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. allowing the visualization of the individually colored chromosomes.del(5)(p15.5p-. a combinatorial labeling method is used to generate many different colors. Image processing software then assigns a pseudo color to each spectrally different combination. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.

CHAPTER 4 .

otherwise known as 47. or structural. Structural abnormalities often arise from errors in homologous recombination. Some disorders arise from loss of just a piece of one chromosome.4. Down syndrome. trisomy 9 and trisomy 16. • • Patau syndrome is caused by trisomy of chromosome 13. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. a common chromosomal disease. Klinefelter syndrome. including . the most common male chromosomal disease. X0). inversions. X or 45. is caused by trisomy of chromosome 21. Also documented are trisomy 8. as in the presence of extra or missing chromosomes. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. in which three copies of a chromosome are present instead of the usual two. Numerical abnormalities. large-scale deletions or duplications. often occur as a result of nondisjunction during meiosis in the formation of a gamete. translocations. although they generally do not survive to birth. are common numerical abnormalities. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. also known as aneuploidy. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. as in derivative chromosome. XXY is caused by an extra X chromosome. trisomies.

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

In a reciprocal translocation. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Known disorders in humans include Wolf-Hirschhorn syndrome.4. etc. Duplications: A portion of the chromosome is duplicated. • • Translocations: When a portion of one chromosome is transferred to another chromosome. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.). 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. In a Robertsonian translocation. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. There are two main types of translocations. segments from two different chromosomes have been exchanged. also called the terminal 11q deletion disorder. rather than two). an entire chromosome has . Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. and Jacobsen syndrome. an X. 4.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). resulting in extra genetic material.3 Structural abnormalities When the chromosome's structure is altered. Tetrasomy. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. which is caused by partial deletion of the short arm of chromosome 4.

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

von Waldeyer in 1888. The name was coined by another German anatomist. 4. The next stage took place after the development of genetics in the early 20th century. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. these results were quite remarkable. Painter in 1922 was not certain whether the diploid number of man was 46 or 48.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. which swells them and spreads the chromosomes . New techniques were needed to definitively solve the problem: 1. Considering their techniques. in contrast to their genic contents. He revised his opinion later from 46 to 48. 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. at first favoring 46. and he correctly insisted on man having an XX/XY system. Using cells in culture 2. Pre-treating cells in a hypotonic solution. in 1882. concluding an XX/XO sex determination mechanism. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. the discoverer of mitosis.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Their behavior in animal (salamander) cells was described by Walther Flemming. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.

a find which eventually led to her Nobel Prize in 1983. reducing the number. McClintock discovered transposons. the great apes have 48 chromosomes.6.2 Natural populations of Drosophila In the 1930s.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. In 1931. 4. Human chromosome 2 was formed by a merger of ancestral chromosomes. During her cytogenetic work. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly.3. 4. Arresting mitosis in metaphase by a solution of colchicine 4. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. persimilis from wild populations in California and neighboring states. Using Painter's technique they studied the polytene .6. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).6 Applications in biology 4.

In some congenital disorders. breeding and sampling whilst preventing escape. as they would if selectively neutral. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. as with most polymorphisms. . Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. Dobzhansky bred populations in population cages. Using a method invented by L'Heretier and Teissier. Evidence rapidly accumulated to show that natural selection was responsible.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. This had the benefit of eliminating migration as a possible explanation of the results. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. 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. which enabled feeding. It was found that the various chromosome types do not fluctuate at random. discoveries were quickly made related to aberrant chromosomes or chromosome number. Down syndrome is also referred to as trisomy 21. but adjust to certain frequencies at which they become stabilised. such as Down's syndrome. In 1959. 4. 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. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy.

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

4. 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. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. and elongation techniques for all culture types that allow for higher resolution banding.8 Beginnings of molecular cytogenetics . Deletions within one chromosome could also now be more specifically named and understood. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. Deletion syndromes such as DiGeorge syndrome.FIG Advent of banding techniques In the late 1960s. Caspersson developed banding techniques which differentially stain chromosomes. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.

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

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,

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

one line of MATLAB code can often replace several lines of C or C++ code. and allocating memory. The image processing step is composed of the following operations. These effects must be compensated to improve the results of the pairing algorithm. and distribute your MATLAB algorithms and applications. MATLAB eliminates the need for ‘for’ loops. 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. .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. You can integrate your MATLAB code with other languages and applications. In many cases. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks.MATLAB provides a number of features for documenting and sharing your work. specifying data types. It enables fast development and execution. 6. With the MATLAB language. As a result. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems.

the spatially scaled images are histogram equalized.2 Concepts used in this phase 1) Image conversion 2) Denoising . 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 2) Geometrical compensation—The geometric compensation. Therefore. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. or at least attenuated. 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). 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. To compare chromosomes from a band pattern point of view. 6. geometrical and dimensional differences must be removed.

the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. For example. and blue planes. and the results might not be meaningful. so the image displays as shades of gray. green. you must first convert it to true color format.I). RGB = cat (3. If you attempt to filter the indexed image. .I. if you want to filter a color image that is stored as an indexed image. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. When you apply the filter to the true color image. For example. The resulting true color image has identical matrices for the red. MATLAB filters the intensity values in the image. You can perform certain conversions just using MATLAB syntax.3) Edge detection 4) Two dimensional convolutions. there are other functions that return a different image type as part of the operation they perform. In addition to these image type conversion functions. MATLAB simply applies the filter to the indices in the indexed image matrix.I. listed in the following table. 6. For example.2. as is appropriate.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.

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

A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. That is.. The size of matrices.'sobel'). imedfilt2(im1.(FIR) filter. C = conv2(. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. .na] and the size of B is [mb.. minus one.[3 3]). nb]+1)/2). edge(im1. this case is the same as C = conv2(hcol*hrow. rgb2gray im2bw(im.hrow.na+nb-1].0.nb]. if the size of then the size of C is [ma+mb-1. If hcol is a column vector and hrow is a row vector.'shape') subsection of the two-dimensional convolution. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.7). 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 other matrix is filtered in two dimensions.bmp').A)..

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

20. rc = [r c]. n1=zeros(imx.57. for x=1:46 [r.15. end %h=figure.imy).7.6.66].[]). end flag=0.27.60.52.10.48.30.62.c] = find(L==L_number((Test_number(x)))).59.38. for i=1:sx x1=rc(i.24.9. end end L_number.50.8.54.40.1).14.28.56. .j). [sx sy]=size(rc).imshow(n1.35.26. y1=rc(i.42.49.55. end.22.end end if flag~=1 L_number(Index)=L(i. Index=Index+1.11.21. Test_number=[3.65.51.31.y1)=255.4.29.41.19.39.43.32. n1(x1.45.33.2). 36.

y)==1 Circumference_sum=Circumference_sum+1.5*graythresh(skel)). . Arm_length_sum=0. Area=zeros(46. Circumference_sum=0.1. skel=im2double(f). BW1=edge(BW. BW=im2bw(f).'spur'.1). BW=double(BW). f=imcomplement(f).end Circumference=zeros(46.1).bmp')). skel=im2bw(skel.Inf). end end end Circumference(i)=Circumference_sum. s1=bwmorph(s.'canny').'skel'.'. s=bwmorph(skel. for x=1:m for y=1:n if BW1(x.1).8). [m n]=size(BW1). Arm_length=zeros(46. for i=1:46 f=imread(strcat(num2str(i).

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

2)=i. Pair(i.Area.2)=i+1. 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(46.1)=i.1)=46. 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)). . Pair(46. Pair(i.1)=i. Pair(i. Pair(i. end end Pair.2). Pair=zeros(46.2)=j. end end end for i=1:45 if Pair(i.

.'.bmp')).2)). figure_flag=1.figure_flag).bmp')).1)).delete=zeros(46.1)==delete(j) flag=1. delete(figure_flag)=Pair(i. flag=0.2. figure_flag=figure_flag+1. imshow(f2).2).1).figure_flag). if figure_flag~=47 subplot(23.2. end flag=0. for i=1:46 for j=1:46 if Pair(i. end f1=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1. end f2=imread(strcat(num2str(Pair(i.'. imshow(f1). end end if flag~=1 if figure_flag~=47 subplot(23.

based on the MI. and Philadelphia. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. in the scope of karyotyping process used in cytogentic analysis. such as. dimensions and banding profiles. plus a new one. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. The proposed algorithm is based on the traditional features extracted from the karyogram.end CONCLUTION In this paper. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. 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. 2) feature extraction from the processed images . 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. Copenhagen.

3) training of a classifier (performed once) where similarity among chromosomes are characterized.10% mean classification rate. and finally. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. are processed in order to compensate for geometrical and intensity distortions. 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 . the romosome images. shape. and band pattern.characterizing the size. 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). 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.working within an 8-D feature space. In the image processing step. 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. and to normalize their dimensions. This normalization is needed to make it possible the band pattern comparison between chromosomes. The training process consists in the estimation of each vector of coefficient . Tests using 19 karyograms based on bone marrow cells. Here. achieves a 70. extracted from the unordered karyogram. from the chromosomes in the training set. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. 4) pairing. shape and band pattern. The features extracted from the processed images discriminate each pair with respect to their size.

In addition. Copenhagen. or Philadelphia. a new chromosome dataset with 9200 chromosomes from bone marrow cells. centromere position. Executing the algorithm on a higher quality dataset. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements.. and from which it is possible to extract additional features. REFERENCES . it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. amean classification rate larger than 93% was obtained in all experiments. This dataset was made publicly available [29]. called LK1 . The results presented in this paper are promising. presenting a uniform level of condensation. whose images are of significantly higher quality. In fact. Using 27 karyograms andworking with a limited number of classes (≤ 8). was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. despite the low quality of this type of chromosomes. such as Edinburgh.performance of the classifier. a 76.10% classification ratewas obtained. e.g.

revised and enlarged. p. and Mulligan P. 6th ed. Evolution of genetic systems. A dictionary of genetics. 7th ed. Columbia University Press NY. Kiev.P Oxford & NY.A. The spermatogenesis of man. Plant Breed.C. Bull. 8. 1958.J. preconception and the counting of the human chromosomes. Etudes sur la spermatogenese humaine. 1922. 27. 1924. Anat. Arch.D. 465-502.. 7.D. Hist. 23.D. ^ Stebbins G. [in Russian] 6. Res. Genet. 1912. The morphology of chromosomes. biologie 27. Med. ^ Concise Oxford Dictionary London. 2006. The chromosomes. Edinburgh. ^ Darlington C. Oxford U. Chapter XII: The Karyotype. 3rd ed. 2nd ed.S. 3. 147–49. ^ Painter T. 1973. 1931. ^ Levitsky G. The material basis of heredity.1. ^ von Winiwarter H. 28 2. ^ a b c White M.J. Chapman & Hall. p242 5. 10. 48. ^ Kottler M.A. 1950. . 19-174. ^ a b White M. ^ King R. From 48 to 46: cytological technique. Applied Bot. 93. State Publication Office of the Ukraine. 4.D. Cambridge University Press. Oliver & Boyd. 1973. 1974. Stansfield W. 1939. Animal cytology and evolution. 9. Variation and evolution in plants. ^ Levitsky G. Cambridge University Press. 129. 11.K. Bull.L.

New York.fcgi?artid=34032 19. Bioessays18: 133–138. Chromosome elimination in sciarid flies. Human and mammalian cytogenetics: a historical perspective. 17. Barch. Kinetophore reproduction theory may explain rapid chromosome evolution. 21. Eosin Y and Azure-A. 2nd ed. ^ A preparation which includes the dyes Methylene Blue. 2000.^ a b Hsu T. Evolutionary genetics.gov/articlerender.12.L. London. The Association of Cytogenetic Technologists. ^ Godfrey L. ^ Maynard Smith J. 13. J. 1998. Zoology 37. ^ Stebbins G. Exp. 1956. Chromosome stains.^ a b Gustashaw K. and Masters J.C 16. Bernard V. . 2001. Arnold. ed. ^ Painter T. ^ Goday C. 9821– 9823. NY. Oxford. p218-9 20. Raven Press.C.S.R. 1923. 291-336. Hereditas 42. The spermatogenesis of humans. Studies in mammalian spermatogenesis II. Springer-Verlag. 14. PNAS 97. The chromosome number of man.H & Levan A. In The ACT Cytogenetics Laboratory Manual 2nd ed. 15.nih. M. 1971.R.http://www. Chromosomal evolution in higher plants. 1996.J. p85-6 18. 1979. Chromatin diminution in nematodes.M. 1-6.B. and Esteban M. Bioessays23: 242–250. & Tobler H.pubmedcentral.C. 1991. ^ Tjio J. ^ Müller F.

D. F. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. Botanical Journal of the Linnean Society 102: 205–217.S.. ^ Khandelwal S. 1364-1366. Park. Retrieved 2011-03-16. 2006. 28. A dictionary of genetics. 2001.A. Stamford CT. Oxford U.K. (2005). (1945-05-15). Retrieved 2008-03-18. 1990..K. R. ^ Matthey. and Benirschke K. ^ Gilbert S. ^ Wurster D.D. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). Developmental biology. Zool. Sinauer Associates.1007/BF02153623.A. Stansfield W.C.. Chromosome evolution in the genus Ophioglossum L. Y. ^ Kim. Nam. Chapter 9 24.. and Mulligan P.H. J.1007/s10228-004-0257-z. 291: 310–16. 2006.. Exp. 23. ^ King R. 25. J. . Muntiacus muntjak: a deer with a low diploid number. doi:10. Chapman.doi:10.H. Experientia (Basel) 1 (2): 50– 56. 1970. & Gregory T. Indian Muntjac. 7th ed.F.R.K. 8th ed. 27.22. ^ Wyngaard G. 26.P Oxford & NY. Science 168. C. Noh. "L'evolution de la formule chromosomiale chez les vertébrés". Ichthyological Research 52 (1): 97.

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