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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.
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 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without
a large body of work uses the term chromosome regardless of chromatin content. In eukaryotes. In practice "chromosome" is a rather loosely defined term. DNA is usually arranged as a circle. sometimes accompanied by one or more smaller. Chromosomal recombination plays a vital role in genetic diversity. Chromosomes may exist as either duplicated or unduplicated. the term genophore is more appropriate when no chromatin is present. the cell may undergo mitotic catastrophe and die. Also. divided. Unduplicated chromosomes are single linear strands. circular DNA molecules called plasmids. However. The structure of chromosomes and chromatin varies through the cell cycle. This allows the very long DNA molecules to fit into the cell nucleus. If these structures are manipulated incorrectly. Chromosomes are the essential unit for cellular division and must be replicated. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. for example. In prokaryotes and viruses. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). which is tightly coiled in on itself. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. although there are many exceptions to this rule. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. These small circular genomes . through processes known as chromosomal instability and translocation. cells may contain more than one type of chromosome. In prokaryotes.defined nuclei) have smaller circular chromosomes. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. or it may unexpectedly evadeapoptosis leading to the progression of cancer.
Euploid human karyotypes are 46. 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. The individual would have Down Syndrome and his/her karyotype would be written 47. rather than 2. . An example of aneuploidy is trisomy 21. a extra copy of human chromosome 21). XX (female) or 46 XY (male).g. the individual carrying the mutation is said to be aneuploid. copies of chromosome 21. If the mutation involves only one or a few chromosomes in the genome (e.+21.are also found in mitochondria and chloroplasts. 1. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.3 MUTATIONS IN CHROMOSOME NUMBER Normally. Such individuals are called euploid and have the wild-type chromosome complement for the species.+21. in which an individual has 3.XX.XY or 47. reflecting their bacterial origins. 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).
longer-lasting attachment in this region. a pair of sister chromatids attached to each other at the centromere. In spite of their appearance.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. the chromatids are uncoiled and DNA can again be transcribed. along with special proteins. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. A special DNA base sequence in the region of the kinetochores provides.Fig 1. so that each daughter cell inherits one set of chromatids. . Once the cells have divided. one of which is present on each sister chromatid. The shorter arms are called p arms (from the French petit.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). and they form the classic four arm structure. 1. The microtubules then pull the chromatids apart toward the centrosomes. which enables these giant DNA structures to be contained within a cell nucleus. small) and the longer arms are called q arms (q follows p in the Latin alphabet. This is the only natural context in which individual chromosomes are visible with an optical microscope. During mitosis. the chromatin strands become more and more condensed. 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. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. This compact form makes the individual chromosomes visible.
cytoplasm.1. bone marrow constitutes 4% of the total body mass of humans. bone marrow in large bones produces new blood cells. producing the lymphocytes that support the body's immune system CHAPTER 2 2. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. 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 is also a key component of the lymphatic system. On average. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. in adults weighing 65 kg (143 lbs).6 kg (5.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones.7 lbs). in two separate nuclei.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. bone marrow accounts for approximately 2. . It is generally followed immediately by cytokinesis. In humans. which use the bone marrow vasculature as a conduit to the body's systemic circulation. which divides the nuclei.
The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. The cell then divides in cytokinesis. animals undergo an "open" mitosis. Because cytokinesis usually occurs in conjunction with mitosis. forming single cells with multiple nuclei. cytokinesis and mitosis may occur independently. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. for instance during certain stages of fruit fly embryonic development. "mitosis" is often used interchangeably with "mitotic phase". This occurs most notably among the fungi and slime moulds. .genetically identical to each other and to their parent cell. prophase. which lack a nucleus. Mitosis occurs only in eukaryotic cells and the process varies in different species. For example. However. prometaphase. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. These stages are interphase. metaphase. The process of mitosis is fast and highly complex. where the nuclear envelope breaks down before the chromosomes separate. This accounts for approximately 10% of the cell cycle. there are many cells where mitosis and cytokinesis occur separately. where chromosomes divide within an intact cell nucleus. Even in animals. Prokaryotic cells. divide by a process called binary fission. but is found in various different groups. to produce two identical daughter cells which are still diploid cells. anaphase and telophase.
This occurs during the S phase of interphase. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. These two cells are identical and do not differ in any way from the original 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. Because each resultant daughter cell should be genetically identical to the 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 parent cell must make a copy of each chromosome before mitosis. Each chromosome now has an identical copy of itself. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. . and together the two are called sister chromatids.
separating the two developing nuclei. As a matter of convention.the cell begins cytokinesis. A new nuclear envelope forms around the separated sister chromosomes. each with a replica of the original genome. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. each sister chromatid is now considered a chromosome. In animal cells. the parent cell will be split in half. the daughter cells will construct a new dividing cell wall between each other. Prokaryotic cells undergo a process similar to mitosis called binary fission. As the cell elongates. pulling apart the sister chromatids of each chromosome. In plant cells.In most eukaryotes. corresponding sister chromosomes are pulled toward opposite ends. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. . The chromosomes align themselves in a line spanning the cell. 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). As mitosis completes. Eventually. giving rise to two daughter cells. the process of binary fission is very much different from the process of mitosis. so they are renamed to sister chromosomes. However.
chromosomes are replicated only during the S phase. Interphase is divided into three phases: G1 (first gap). continues to grow as it duplicates its chromosomes (S).2. and G2 (second gap). In highly vacuolated plant cells. mainly via proteins. 2. a cell grows (G1). and finally it divides (M) before restarting the cycle. the cell grows by producing proteins and cytoplasmic organelles. It alternates with the much longer interphase. grows more and prepares for mitosis (G 2). a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . During all three phases.2. where the cell prepares itself for cell division.1 Preprophase In plant cells only. All these phases in the interphase are highly regulated. prophase is preceded by a pre-prophase stage. However. Thus. S (synthesis). the nucleus has to migrate into the center of the cell before mitosis can begin. This is achieved through the formation of a phragmosome.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. 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.
Telophase: The decondensing chromosomes are surrounded by nuclear membranes. aligned at the metaphase plate. and microtubules have invaded the nuclear Prophase space. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. This band marks the position where the cell will eventually divide. These microtubules can attach to kinetochores or they can interact with opposing microtubules. Cytokinesis has already begun. the pinched area is known as the cleavage furrow. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. after the nuclear membrane breaks down.division. . The cells of higher plants (such as the flowering plants) lack centrioles. degraded. instead. In addition to phragmosome formation. The chromosomes have chromatin has condensed. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. 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.
giving a pair of centrosomes. Since the genetic material has already been duplicated earlier in S phase. chromatin condenses together into a highly ordered structure called a chromosome. Chromosomes are typically visible at high magnification through a light microscope. Close to the nucleus are structures called centrosomes. they are not essential for the . the genetic material in the nucleus is in a loosely bundled coil called chromatin. the replicated chromosomes have two sister chromatids. bound together at the centromere by the cohesin protein complex. A cell inherits a single centrosome at cell division.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. At the onset of prophase. Although centrioles help organize microtubule assembly. which are made of a pair of centrioles found in most eukaryotic animal cells. The centrosome is the coordinating center for the cell's microtubules. which is replicated by the cell with the help of the nucleus before a new mitosis begins.
such as algae or trichomonads. kinetochore microtubules begin searching for kinetochores to attach to. When a microtubule connects with the kinetochore. since they are absent from plants. Prometaphase is sometimes considered part of prophase. or its microtubules are able to penetrate an intact nuclear envelope. and centrosomes are not always used in mitosis. This is called open mitosis. This motor activity. coupled with polymerisation and depolymerisation of microtubules. and it occurs in most multicellular organisms. on an average 20 ). it is known that it contains some form of molecular motor. When the spindle grows to sufficient length. undergo a variation called closed mitosis where the spindle forms inside the nucleus. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Although the kinetochore structure and function are not fully understood. the motor activates. Each chromosome forms two kinetochores at the centromere. one attached at each chromatid.formation of the spindle. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space.2. using energy from ATP to "crawl" up the tube toward the originating centrosome. 2. Fungi and some protists. provides the pulling force necessary to later separate the chromosome's two chromatids. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. .
This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. The centromeres of the chromosomes. As a result. .3 Metaphase A cell in late metaphase.In the fishing pole analogy. Metaphase comes from the Greek meaning "after. In certain types of cells. convene along the metaphase plate or equatorial plane. 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. an imaginary line that is equidistant from the two centrosome poles. the chromosomes come under longitudinal tension from the two ends of the cell." Microtubules find and attach to kinetochores in prometaphase. All chromosomes (blue) but one have arrived at the metaphase plate. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. in some sense. 2. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". analogous to a tug-of-war between people of equal strength. only roughly lining up along the midline.
the proteins that bind sister chromatids together are cleaved.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres).” “against.” “back. Two events then occur: first. Next.” or “re-”). 2. These sister chromatids. The force that causes the centrosomes to move towards the ends of the cell is still unknown. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. the cell proceeds to anaphase (from the Greek meaning “up. allowing them to separate. the nonkinetochore microtubules elongate.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. 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. At the end of anaphase. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. . These two stages are sometimes called early and late anaphase. which have now become distinct sister chromosomes. Early anaphase is usually defined as the separation of the sister chromatids. The signal creates the mitotic spindle checkpoint.
but rather a separate process. now surrounded by new nuclei. In both animal and plant cells.2. however.5 Cytokinesis Cilliate undergoing cytokinesis. At telophase. cell . a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. cytokinesis is a separate process that begins at the same time as telophase. A new nuclear envelope. In animal cells. Mitosis is complete. but cell division is not yet complete. necessary for completing cell division. the nonkinetochore microtubules continue to lengthen. 2. pinching off the separated nuclei. Cytokinesis is technically not even a phase of mitosis. elongating the cell even more. Both sets of chromosomes. using fragments of the parent cell's nuclear membrane. forms around each set of separated sister chromosomes.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. It "cleans up" the after effects of mitosis. unfold back into chromatin. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. Corresponding sister chromosomes attach at opposite ends of the cell.
In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.4 Regeneration .5. 2. zygote and also the basis of the growth of a multicellular body.g.division is also driven by vesicles derived from the Golgi apparatus.5. e. New cells are formed by mitosis and so are exact copies of the cells being replaced. Following are the occasions in the lives of organism where mitosis happens: 2.1Significance Mitosis is important for the maintenance of the chromosomal set. which move along microtubules to the middle of the cell. The phragmoplast is a microtubule structure typical for higher plants.5. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. cells are constantly sloughed off and replaced by new ones. 2.3 Cell replacement In some parts of body. Each daughter cell has a complete copy of the genome of its parent cell. Similarly. This is the basis of the development of a multicellular body from a single cell i..5. skin and digestive tract.2 Development and growth The number of cells within an organism increases by mitosis. The end of cytokinesis marks the end of the M-phase. whereas some green algae use a phycoplast microtubule array during cytokinesis. 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. separating the two nuclei.e.
the hydra reproduces asexually by budding. . resulting in binucleated cells.7 Consequences of errors Although errors in mitosis are rare. Occasionally when cells experience nondisjunction. One daughter cell will receive both sister chromosomes and the other will receive none. a condition known as monosomy. The same division happens during asexual reproduction or vegetative propagation in plants. especially during early cellular divisions in the zygote. a condition often associated with cancer. Mitosis continues in the cells of bud and it grows into a new individual. a chromosome may fail to separate during anaphase. a condition known as trisomy.Some organisms can regenerate their parts of bodies. 2. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). the process may go wrong. In non-disjunction.5. sea star regenerates its lost arm through mitosis.5. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. For example. The production of new cells is achieved by mitosis. 2. and the latter cell having only one chromosome (the homologous chromosome). These cells are considered aneuploid. For example. The cells at the surface of hydra undergo mitosis and form a mass called bud. they fail to complete cell division and retain both nuclei in one cell.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction.
it results in the formation of Tumors.Mitosis is a demanding process for the cell. This phenomenon is called metastasis or spreading of disease. causing translocation. sometimes mutuations occur in such genes and cells continue to divide. causing deletion. Or. The effect of these genetic abnormalities depends on the specific nature of the error. Occasionally. It results in the synthesis of execessive tissue growths. Now what happens is that cell abnormally continue to divide at a single place. Benign tumours are not harmful as soon as they are not moving. but in reverse orientation. As long as these tumours remain in their original location they are called benign tumours. An arm of the chromosome may be broken and the fragment lost. and chromosomes are jostled constantly by probing microtubules. It may reattach to the original chromosome. causing inversion. As soon as they start to move and invade other cells there are said to be malignant tumours. its organelles disintegrate and reform in a matter of hours. It results in abnormal cell growth. causing chromosomal duplication. non-homologous chromosome. which goes through dramatic changes in ultrastructure. Such tumours can send cancer cells to other parts in body where new tumours may form. When tissues more than the requirement are synthesized in a single organ. chromosomes may become damaged. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. The fragment may incorrectly reattach to another. . All cells have genes that control the timing and number of mitosis. it may be treated erroneously as a separate chromosome. Errors in the control of mitosis may cause cancer.
Early events of metaphase can . is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. from the ancient Greek(between) and (stage). resulting in cells with many copies of the same chromosome occupying a single nucleus. an imaginary line that is equidistant from the two centrosome poles. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. This process may also be referred to as endoreduplication and the cells as endoploid. only roughly lining up along the middleline. An example of a cell that goes through endomitosis is the megakaryocyte.7 Metaphase Metaphase.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. 2. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. align in the middle of the cell before being separated into each of the two daughter cells. Preceded by events in prometaphase and followed by anaphase. carrying genetic information.2. Metaphase accounts for approximately 4% of the cell cycle's duration. In certain types of cells. analogous to a tug of war between equally strong people.
when every kinetochore is properly attached to a bundle of microtubules. Staining of the slides. Metaphase chromosomes make the classical picture of chromosomes (karyotype). For classical cytogenetic analyses. Chromosomes are condensed(Thickened) and highly coiled in metaphase. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Such a signal creates the mitotic spindle checkpoint. 2.coincide with the later events of prometaphase.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. 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. which makes them most suitable for visual analysis. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. often with Giemsa (G banding) or Quinacrine. 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. This would be accomplished by regulation of the anaphase-promoting complex. does the cell enter anaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Normal metaphase spreads are used in . produces a pattern of in total up to several hundred bands. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. securin.
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. such as bcr-abl in chronic myelogenous leukemia. .methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. 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. for example.
in humans 2n = 46. Attention is paid to their length. and to gather information about past evolutionary events.  The preparation and study of karyotypes is part of cytogenetics. Thus. or an individual organism. cellular function. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). or may not. and what they look like under a light microscope. The term is also used for the complete set of chromosomes in a species. any differences between the sex chromosomes. to study chromosomal aberrations. be sex chromosomes. . The study of whole sets of chromosomes is sometimes known as karyology. in normal diploid organisms. and any other physical characteristics. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. the position of the centromeres. such as. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. and the results may be used in evolutionary biology and medicine. There may. So. Karyogram of human male using Giemsa staining. Karyotypes describe the number of chromosomes. Karyotypes can be used for many purposes. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. taxonomic relationships. ordered by size and position of centromere for chromosomes of the same size. autosomal chromosomes are present in two copies. banding pattern. The study of karyotypes is important for cell biology and genetics.
Their behavior in animal (salamander) cells was described by Walther Flemming. New techniques were needed to definitively solve the problem: 1. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Considering their techniques. The subsequent history of the concept can be followed in the works of Darlington and White. 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. and he correctly insisted on humans having an XX/XY system. von Waldeyer in 1888.3. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. which swells them and spreads the chromosomes . Using cells in culture 2. The next stage took place after the development of genetics in the early 20th century. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. He revised his opinion later from 46 to 48. Pretreating cells in a hypotonic solution. concluding an XX/XO sex determination mechanism. the discoverer of mitosis. in 1882. 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. The name was coined by another German anatomist. these results were quite remarkable. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. at first favoring 46.
is applied after cells have been arrested during cell division by a solution of colchicine. 3. For humans. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. such as Giemsa. Rather interestingly. The sex of an unborn fetus can be determined by observation of interphase cells. a suitable dye.3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . Cutting up a photomicrograph and arranging the result into an indisputable karyogram.1 Staining The study of karyotypes is made possible by staining. reducing the number. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. Usually.2 Observations on karyotypes 3. the great apes have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. 3.2.2.  Sometimes observations may be made on non-dividing (interphase) cells. Arresting mitosis in metaphase by a solution of colchicines 4.
Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. Heterochromatin stains darker than euchromatin. which (when they occur) are small bodies attached to a chromosome by a thin thread. 6. . Differences in the position of centromeres. faba chromosomes are many times larger. Differences in absolute sizes of chromosomes. 4. This feature probably reflects different amounts of DNA duplication. but the genes have been mostly translocated (added) to other chromosomes.1. This is brought about by translocations. type. permitting its loss without penalty to the organism (the dislocation hypothesis). shape and banding of the chromosomes. Differences in number and position of satellites. 3. and mainly consists of genetically inactive repetitive DNA sequences. A full account of a karyotype may therefore include the number. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). 2. as well as other cytogenetic information. indicating tighter packing. both have six pairs of chromosomes (n=6) yet V. Humans have one pair fewer chromosomes than the great apes. Differences in degree and distribution of heterochromatic regions. 5.
XY. 3. Between members of a population (chromosome polymorphism) 4. Between the sexes 2. Any variation from the standard karyotype may lead to developmental abnormalities. Geographical variation between races 5. Between the germ-line and soma (between gametes and the rest of the body) 3. There is variation between species in chromosome number. and in . Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. males have both an X and a Y chromosome denoted 46.Variation is often found: 1.3 The human karyotype Most (but not all) species have a standard karyotype. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Normal karyotypes for females contain two X chromosomes and are denoted 46. which are highly variable. Mosaics or otherwise abnormal individuals. the same cannot be said for their karyotypes. XX.
Although much is known about karyotypes at the descriptive level. "We have a very poor understanding of the causes of karyotype evolution. portions of the chromosomes are cast away in particular cells. But. Godfrey and Masters conclude: "In our view.1 Changes during development Instead of the usual gene repression.. as in many sciarid flies. In a review. or other kinds of visible adjustment to the karyotype. despite many careful investigations. the general significance of karyotype evolution is obscure. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. Chromosome elimination. some organisms go in for large-scale elimination of heterochromatin.. used in conjunction with other phylogenetic data. In some cases there is even significant variation within species. found in some copepods and roundworms such as Ascaris suum. In this process. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. entire chromosomes are eliminated during development. In some species.. . and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species.3.. This variation provides the basis for a range of studies in evolutionary cytology. Chromatin diminution (founding father: Theodor Boveri). it is quite unclear what the general significance might be.detailed organization. 3. In A. despite their construction from the same macromolecules. which were previously inexplicable.
The diploid number of the Chinese muntjac. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. The low record is held by the nematode Parascaris univalens. Muntiacus muntjak. 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 existence of supernumerary or B chromosomes .suum. They kept quiet for two or three years because they thought something was wrong with their tissue culture. In marsupials it is always the paternal X which is inactivated.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. all telocentric. was found to be 46. male = 7 chromosomes. they were astonished to find it had female = 6. where the haploid n = 1. thus the mammalian female is a mosaic in respect of her X chromosomes. "They simply could not believe what they saw. the high record would be somewhere amongst the ferns. 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. Muntiacus reevesi. In placental mammals. Xinactivation.3.. 3. which was investigated by Kurt Benirschke and his colleague Doris Wurster. When they looked at the karyotype of the closely related Indian muntjac. In human females some 15% of somatic cells escape inactivation... the inactivation is random as between the two Xs.
and aneuploids are another example. 3. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. about 70%. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. 14. horsetails and psilotales) is also common. It is a common arrangement in the Hymenoptera. It has been of major significance in plant evolution according to Stebbins. FN ≤ 2n. where there are more than two sets of homologous chromosomes in the cells.Endopolyploidy occurs when in adult differentiated .3. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. where one sex is diploid. due to the presence of five acrocentric chromosome pairs (13. Polyploidy. but it has been significant in some groups. 15. Polyploidy in animals is much less common. Humans have FN = 82. occurs mainly in plants. and in some other groups. Haplo-diploidy. 21 and 22). and the other haploid. FN. though in this case they would not be regarded as normal members of the population.means that chromosome number can vary even within one interbreeding population. Thus.3 Fundamental number The fundamental number. Polyploidy in lower plants (ferns.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. 3. but in grasses the average is much higher. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present.
This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. it is diverse and complex. but the nuclei contain more than the original somatic number of chromosomes. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. and serves differentiation and morphogenesis in many ways. the daughter chromosomes separating from each other inside an intact nuclear membrane. 3. 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. . The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. Abnormalities in chromosome number usually cause a defect in development.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. The cells do not always contain exact multiples (powers of two). Down syndrome and Turner syndrome are examples of this.tissues the cells have ceased to divide by mitosis. In many instances. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. See palaeopolyploidy for the investigation of ancient karyotype duplications. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted).
3. Human chromosome 2 was formed by a merger of ancestral chromosomes. 3.  Closer to home. reducing the number. When this happens. 6. and Crocus. and 7. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 4. the chromosome number is variable from one individual to another. 5.6 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.000 km2). There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. the European shrew Sorex araneus.500 sq mi (17. that the two chromosome morphs are adapted to different habitats. Well-researched examples are the ladybird beetle Chilocorus stigma. Classic examples in plants are the genus Crepis. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. In about 6. the great apes have 24x2 chromosomes whereas humans have 23x2. 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. some mantids of the genus Ameles.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations.Aneuploidy may also occur within a group of closely related species.
it is more likely to have been a group from the same species. and skipping of islands. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. Although it would be possible for a single gravid female to colonise an island. show a clear "flow" of species from older to newer islands. Using K-Ar dating. The results are clear. Chromosome rearrangements. especially inversions. gene arrangements are visible in the banding patterns of each chromosome. in the family Drosophilidae. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. probably 20 million years ago. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll.subalpine meadows. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. make it possible to see which species are closely related. The inversions. the best-studied group of Hawaiian drosophilids. the present islands date from 0. Drosophila and Scaptomyza. . The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. but these are much less frequent. when plotted in tree form (and independent of all other information). at least into the Cretaceous. The polytene banding of the 'picture wing' group. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. In a sense.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). There are also cases of colonization back to older islands. which can be dated to 30 mya.
The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). The light regions tend to be euchromatic. adaptive radiations.7 Depiction of karyotypes 3. human genome.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. . • Q-banding is a fluorescent pattern obtained using quinacrine for staining. This method will normally produce 300-400 bands in a normal.the dark regions tend to be heterochromatic. so it stains centromeres.7. The pattern of bands is very similar to that seen in G-banding. It yields a series of lightly and darkly stained bands . R-banding is the reverse of G-banding (the R stands for "reverse"). if less spectacular. 3. • T-banding: visualize telomeres. late-replicating and AT rich.There are other animals and plants on the Hawaiian archipelago which have undergone similar. • • C-banding: Giemsa binds to constitutive heterochromatin. early-replicating and GC rich.
often Giemsa (G-banding). a dye. 3. In the "classic" (depicted) karyotype.7. Giemsa is specific for the phosphate groups of DNA. Quinacrine binds to the adeninethymine-rich regions.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. Cri du chat syndrome involves a deletion on the short arm of . the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. is used to stain bands on the chromosomes. For example. less frequently Quinacrine. and the long arm on the bottom. In addition. both chromosomes in a pair will have the same banding pattern. Some karyotypes call the short and long arms p and q. Karyotypes are arranged with the short arm of the chromosome on top. respectively. Each chromosome has a characteristic banding pattern that helps to identify them. This yields a dark region where the silver is deposited. denoting the activity of rRNA genes within the NOR.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH.
8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. 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. The critical region for this syndrome is deletion of 15. 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. .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.del(5)(p15. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. It is written as 46.2. which is written as 46.2) 3. Image processing software then assigns a pseudo color to each spectrally different combination. 3.7. This method is also known as virtual karyotyping. Because there are a limited number of spectrally-distinct fluorophores. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. a combinatorial labeling method is used to generate many different colors.chromosome 5.5p-.XX.XX.
CHAPTER 4 .
Klinefelter syndrome. is caused by trisomy of chromosome 21. Structural abnormalities often arise from errors in homologous recombination. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. or structural. including . Some disorders arise from loss of just a piece of one chromosome. X0). trisomy 9 and trisomy 16. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. Down syndrome. in which three copies of a chromosome are present instead of the usual two. X or 45. otherwise known as 47. trisomies. are common numerical abnormalities. also known as aneuploidy. a common chromosomal disease. often occur as a result of nondisjunction during meiosis in the formation of a gamete. the most common male chromosomal disease. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. although they generally do not survive to birth. inversions. as in derivative chromosome. large-scale deletions or duplications. Numerical abnormalities. • • Patau syndrome is caused by trisomy of chromosome 13. Also documented are trisomy 8. translocations. XXY is caused by an extra X chromosome. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. as in the presence of extra or missing chromosomes.4.
The name comes from the babies' distinctive cry. numerical and structural anomalies. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. caused by abnormal formation of the larynx. 1p36 Deletion syndrome. example of imprinting disorder. a deletion of the paternal genes. from a truncated short arm on chromosome 5. There are many types of chromosome anomalies.• Cri du chat (cry of the cat). A chromosome anomaly may be detected or confirmed in this manner. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. example of imprinting disorder. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. a deletion of the maternal genes. from the loss of part of the short arm of chromosome 1. one well-documented example is the Philadelphia chromosome. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. . 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. They can be organized into two basic groups. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. A chromosome anomaly.
2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). In a reciprocal translocation. Tetrasomy. Known disorders in humans include Wolf-Hirschhorn syndrome. 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. rather than two).4.3 Structural abnormalities When the chromosome's structure is altered. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. an X. segments from two different chromosomes have been exchanged. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21.). and Jacobsen syndrome. etc. • • Translocations: When a portion of one chromosome is transferred to another chromosome. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. an entire chromosome has . There are two main types of translocations. Duplications: A portion of the chromosome is duplicated. In a Robertsonian translocation. which is caused by partial deletion of the short arm of chromosome 4. 4. resulting in extra genetic material. also called the terminal 11q deletion disorder.
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. 14. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. especially the chromosomes.attached to another at the Centromere . 21 and 22. Therefore. • Inversions: A portion of the chromosome has broken off. turned upside down and reattached. therefore the genetic material is inverted. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.in humans these only occur with chromosomes 13. the anomaly is present in every cell of the body. however. resulting in Mosaicism (where some cells have the anomaly and some do not). Chromosome anomalies can be inherited from a parent or be "de novo". Some anomalies. 4.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. can happen after conception. as well . It includes routine analysis of G-Banded chromosomes. • Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material. They often lead to an increased tendency to develop certain types of malignancies. 15. 4. and are therefore initially not inherited. other cytogenetic banding techniques.
Pre-treating cells in a hypotonic solution. the discoverer of mitosis.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. 4. 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. in 1882. The next stage took place after the development of genetics in the early 20th century. concluding an XX/XO sex determination mechanism. von Waldeyer in 1888. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. in contrast to their genic contents. Their behavior in animal (salamander) cells was described by Walther Flemming. Using cells in culture 2. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. Considering their techniques. and he correctly insisted on man having an XX/XY system. these results were quite remarkable. at first favoring 46. which swells them and spreads the chromosomes .5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. He revised his opinion later from 46 to 48. The name was coined by another German anatomist. New techniques were needed to definitively solve the problem: 1.
It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.6. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.3. 4. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Natural populations of Drosophila In the 1930s. In 1931. McClintock discovered transposons. reducing the number.6. Arresting mitosis in metaphase by a solution of colchicine 4. Rather interestingly. persimilis from wild populations in California and neighboring states. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.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. During her cytogenetic work. the great apes have 48 chromosomes. 4.6 Applications in biology 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). a find which eventually led to her Nobel Prize in 1983. Using Painter's technique they studied the polytene .
In some congenital disorders. It was found that the various chromosome types do not fluctuate at random.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. Evidence rapidly accumulated to show that natural selection was responsible. such as Down's syndrome. discoveries were quickly made related to aberrant chromosomes or chromosome number. Dobzhansky bred populations in population cages. as with most polymorphisms. This had the benefit of eliminating migration as a possible explanation of the results. 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. . 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. Down syndrome is also referred to as trisomy 21. which enabled feeding. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. as they would if selectively neutral. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. In 1959. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. 4. but adjust to certain frequencies at which they become stabilised. breeding and sampling whilst preventing escape. Using a method invented by L'Heretier and Teissier.
as both scientists were doing their research in Philadelphia. Not all genes on the X Chromosome are inactivated. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. Identification of the Philadelphia chromosome by cytogenetics.Other numerical abnormalities discovered include sex chromosome abnormalities. and XXXX. In 1960. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. resulting in 47 total chromosomes. an additional X chromosome in a male. in addition to other tests. which is required in normal females to compensate for having two copies of the chromosome. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). Pennsylvania. This abnormal chromosome was dubbed the Philadelphia chromosome . An individual with only one sex chromosome (the X) has Turner syndrome. with the development of more advanced techniques. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. has Klinefelter's Syndrome. Thirteen years later. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. . is used today as a diagnostic for CML. XYY. Many other sex chromosome combinations are compatible with live birth including XXX.
Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 4. 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. Deletions within one chromosome could also now be more specifically named and understood.FIG Advent of banding techniques In the late 1960s.8 Beginnings of molecular cytogenetics . 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. Deletion syndromes such as DiGeorge syndrome. 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 change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. 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. cloned and studied in ever greater detail. CHAPTER 5 Techniques 5. advances were made in molecular cytogenetics. While radioisotope-labeled probes had been hybridized with DNA since 1969.In the 1980s.1 Karyotyping . Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).
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,
generally between 200 and 1000 cells are counted and scored. and FORTRAN. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. including signal and image processing. 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. financial modeling and analysis. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. You can use MATLAB in a wide range of applications. such as comparative genomic hybridization arrays. and numerical computation. For congenital problems usually 20 metaphase cells are scored. . communications. control design. test and measurement. data visualization. data analysis. such as C. C++. you can solve technical computing problems faster than with traditional programming languages. CGH and Single nucleotide polymorphism-arrays. Using MATLAB. and computational biology.
. As a result. With the MATLAB language. You can integrate your MATLAB code with other languages and applications. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. 6. These effects must be compensated to improve the results of the pairing algorithm. 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. The image processing step is composed of the following operations. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. In many cases. specifying data types.MATLAB provides a number of features for documenting and sharing your work. MATLAB eliminates the need for ‘for’ loops. such as declaring variables. and distribute your MATLAB algorithms and applications. one line of MATLAB code can often replace several lines of C or C++ code. It enables fast development and execution. 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.
or at least attenuated. Therefore.2 Concepts used in this phase 1) Image conversion 2) Denoising . 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). geometrical and dimensional differences must be removed. the spatially scaled images are histogram equalized. To compensate for this inhomogeneity. 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. 2) Geometrical compensation—The geometric compensation. To compare chromosomes from a band pattern point of view. 6. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram.
as is appropriate. and the results might not be meaningful. When you apply the filter to the true color image.2. In addition to these image type conversion functions. if you want to filter a color image that is stored as an indexed image. You can perform certain conversions just using MATLAB syntax. For example. and blue planes. 6. If you attempt to filter the indexed image. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension.3) Edge detection 4) Two dimensional convolutions. you must first convert it to true color format.I. so the image displays as shades of gray. .I). MATLAB filters the intensity values in the image. For example. listed in the following table. MATLAB simply applies the filter to the indices in the indexed image matrix. 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. green. RGB = cat (3.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. there are other functions that return a different image type as part of the operation they perform.I. The resulting true color image has identical matrices for the red.
and hence the type of noise on the image. hence we can choose the most appropriate method for reducing the effects. or through networked cable. via satellite or wireless transmission. .3 Two dimensional convolutions C = conv2(A. We may use edges to measure the size of objects in an image. . Usually we know what type of errors to expect.5 Edge detection Edges contain some of the most useful information in an image. If one of these matrices describes a two-dimensional finite impulse response . we may expect errors to occur in the image signal. caused by external disturbance. and we shall look at some of the more straightforward of them. There is a large number of edge finding algorithms in existence.parameters. ) Where the parameters available depend on the method used 6.2. If an image is being sent electronically from one place to another. The general Matlab command for finding edges is edge(image. 6. to recognize or classify objects. to isolate particular objects from their background. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. Cleaning an image corrupted by noise is thus an important area of image restoration.6.4 Denoising We may define noise to be any degradation in the image signal.B) computes the two-dimensional convolution of matrices A and B.2.'method'.
A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.0.hrow. The size of matrices.7). convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. edge(im1.na] and the size of B is [mb.'sobel').[3 3]).nb]. . C = conv2(. imedfilt2(im1.'shape') subsection of the two-dimensional convolution.A)... That is. this case is the same as C = conv2(hcol*hrow. If hcol is a column vector and hrow is a row vector.bmp').na+nb-1].(FIR) filter. minus one. nb]+1)/2).. rgb2gray im2bw(im. the other matrix is filtered in two dimensions. if the size of then the size of C is [ma+mb-1. 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.
[imx. [sx sy]=size(rc).n]=size(L). Index=1.j)==L_number(k) flag=1.y1)=255. mx=max(max(L)).j)~=0 for k=1:mx if L(i. n1(x1.imy]=size(BW).8). for i=1:sx x1=rc(i. bwlabel(B. flag=0. y1=rc(i. MODULE 2 clc [m. rc = [r c].2). for i=1:m for j=1:n if L(i.1).double(msk)). L_number=zeros(mx.c] = find(L==22). Msk conv2(double(BW).imy). nzeros(imx.1). [r. .
imy).imshow(n1.48. 36. [sx sy]=size(rc).26.62.10. n1(x1.35.52. end flag=0.1).43.14.19.end end if flag~=1 L_number(Index)=L(i.18.104.22.168.4.j).30.57. y1=rc(i.22.60.38. end.y1)=255.32. .9.c] = find(L==L_number((Test_number(x)))).15.55.27. n1=zeros(imx. rc = [r c]. Test_number=[3.20. end end L_number. for i=1:sx x1=rc(i.22.214.171.124.2).33.8. Index=Index+1.39.50.).126.96.36.199]. end %h=figure.188.8.131.52. for x=1:46 [r.28.40.65.
1). skel=im2bw(skel. Area=zeros(46. . BW=im2bw(f). s=bwmorph(skel.'canny').'skel'.5*graythresh(skel)). BW=double(BW).1). Arm_length_sum=0. f=imcomplement(f). BW1=edge(BW. Arm_length=zeros(46. end end end Circumference(i)=Circumference_sum.1. for i=1:46 f=imread(strcat(num2str(i).Inf).8).'.'spur'.end Circumference=zeros(46. Circumference_sum=0. for x=1:m for y=1:n if BW1(x.y)==1 Circumference_sum=Circumference_sum+1. skel=im2double(f). s1=bwmorph(s.1).bmp')). [m n]=size(BW1).
for x=1:m for y=1:n if BW(x.y)==1 Area_sum=Area_sum+1. end Circumference. BW=im2bw(f). [m n]=size(BW). Arm_length. . end end end Arm_length(i)=Arm_length_sum.[m n]=size(s1). for x=1:m for y=1:n if s1(x. end end end Area(i)=Area_sum. Area_sum=0.y)==1 Arm_length_sum=Arm_length_sum+1.
2)=i.1)=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)=j. Pair(i. Pair(i.Area. 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)). Pair(i. end end end for i=1:45 if Pair(i.2)==46 Pair(46.2). Pair(46. end end Pair. .1)=i.2)=i+1. Pair=zeros(46.1)=46.
imshow(f2).figure_flag). flag=0.'.1). if figure_flag~=47 subplot(23. .2).1)==delete(j) flag=1. for i=1:46 for j=1:46 if Pair(i. imshow(f1).'.2.1)). end end if flag~=1 if figure_flag~=47 subplot(23. figure_flag=figure_flag+1. delete(figure_flag)=Pair(i. end f2=imread(strcat(num2str(Pair(i. figure_flag=1. end f1=imread(strcat(num2str(Pair(i.2.2)). end flag=0.delete=zeros(46.figure_flag). figure_flag=figure_flag+1.bmp')).bmp')).
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. based on the MI. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. 2) feature extraction from the processed images . Copenhagen. and Philadelphia. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. 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. dimensions and banding profiles. The proposed algorithm is based on the traditional features extracted from the karyogram.end CONCLUTION In this paper. in the scope of karyotyping process used in cytogentic analysis. such as. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure.
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.10% mean classification rate. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. 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). are processed in order to compensate for geometrical and intensity distortions. 4) pairing. The features extracted from the processed images discriminate each pair with respect to their size. 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. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. and band pattern. Here. shape. and finally. from the chromosomes in the training set. and to normalize their dimensions.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. 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 . In the image processing step. the romosome images. achieves a 70. The training process consists in the estimation of each vector of coefficient . extracted from the unordered karyogram. shape and band pattern.working within an 8-D feature space.characterizing the size. Tests using 19 karyograms based on bone marrow cells.
was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes.. a new chromosome dataset with 9200 chromosomes from bone marrow cells. Copenhagen. This dataset was made publicly available . Using 27 karyograms andworking with a limited number of classes (≤ 8). or Philadelphia. whose images are of significantly higher quality.10% classification ratewas obtained. centromere position.g. e. a 76. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. In addition. Executing the algorithm on a higher quality dataset.performance of the classifier. 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. REFERENCES . In fact. called LK1 . The results presented in this paper are promising. such as Edinburgh. and from which it is possible to extract additional features. despite the low quality of this type of chromosomes. presenting a uniform level of condensation.
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