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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
The structure of chromosomes and chromatin varies through the cell cycle. for example. Chromosomal recombination plays a vital role in genetic diversity. a large body of work uses the term chromosome regardless of chromatin content. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. through processes known as chromosomal instability and translocation.defined nuclei) have smaller circular chromosomes. the term genophore is more appropriate when no chromatin is present. However. circular DNA molecules called plasmids. although there are many exceptions to this rule. Chromosomes are the essential unit for cellular division and must be replicated. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). Chromosomes may exist as either duplicated or unduplicated. In eukaryotes. or it may unexpectedly evadeapoptosis leading to the progression of cancer. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. These small circular genomes . In prokaryotes and viruses. In practice "chromosome" is a rather loosely defined term. Unduplicated chromosomes are single linear strands. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. the cell may undergo mitotic catastrophe and die. which is tightly coiled in on itself. In prokaryotes. cells may contain more than one type of chromosome. If these structures are manipulated incorrectly. Also. DNA is usually arranged as a circle. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. sometimes accompanied by one or more smaller. divided.
XX. a extra copy of human chromosome 21). XX (female) or 46 XY (male). 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. Euploid human karyotypes are 46.+21. in which an individual has 3. copies of chromosome 21. Such individuals are called euploid and have the wild-type chromosome complement for the species. If the mutation involves only one or a few chromosomes in the genome (e. rather than 2. An example of aneuploidy is trisomy 21. The individual would have Down Syndrome and his/her karyotype would be written 47.g.+21.are also found in mitochondria and chloroplasts. 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). reflecting their bacterial origins. .XY or 47. the individual carrying the mutation is said to be aneuploid. 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.
During mitosis. a pair of sister chromatids attached to each other at the centromere. and they form the classic four arm structure. so that each daughter cell inherits one set of chromatids. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. along with special proteins. The microtubules then pull the chromatids apart toward the centrosomes. The shorter arms are called p arms (from the French petit. small) and the longer arms are called q arms (q follows p in the Latin alphabet. the chromatids are uncoiled and DNA can again be transcribed. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. one of which is present on each sister chromatid.Fig 1. 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. Once the cells have divided.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. which enables these giant DNA structures to be contained within a cell nucleus. This is the only natural context in which individual chromosomes are visible with an optical microscope. chromosomes are structurally highly condensed. . 1. longer-lasting attachment in this region. This compact form makes the individual chromosomes visible. In spite of their appearance.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). A special DNA base sequence in the region of the kinetochores provides.
6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. 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 accounts for approximately 2. cytoplasm. In humans. . bone marrow in large bones produces new blood cells. On average. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. bone marrow constitutes 4% of the total body mass of humans. which divides the nuclei. which use the bone marrow vasculature as a conduit to the body's systemic circulation.7 lbs).1. in adults weighing 65 kg (143 lbs). The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. 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.  Bone marrow is also a key component of the lymphatic system.
animals undergo an "open" mitosis. This accounts for approximately 10% of the cell cycle. anaphase and telophase. there are many cells where mitosis and cytokinesis occur separately. Mitosis occurs only in eukaryotic cells and the process varies in different species. The cell then divides in cytokinesis. cytokinesis and mitosis may occur independently. metaphase. divide by a process called binary fission. . prophase. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. for instance during certain stages of fruit fly embryonic development. The process of mitosis is fast and highly complex. This occurs most notably among the fungi and slime moulds. forming single cells with multiple nuclei. For example. where chromosomes divide within an intact cell nucleus. but is found in various different groups. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. Even in animals. to produce two identical daughter cells which are still diploid cells. Because cytokinesis usually occurs in conjunction with mitosis. However. These stages are interphase. Prokaryotic cells. which lack a nucleus. prometaphase. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. "mitosis" is often used interchangeably with "mitotic phase". The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next.genetically identical to each other and to their parent cell. where the nuclear envelope breaks down before the chromosomes separate.
. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. These two cells are identical and do not differ in any way from the original parent cell. Each chromosome now has an identical copy of itself. and together the two are called sister chromatids. This occurs during the S phase of interphase. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. Because each resultant daughter cell should be genetically identical to the parent cell. the parent cell must make a copy of each chromosome before mitosis. The sister chromatids are held together by a specialized region of the chromosome known as the centromere.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells.
giving rise to two daughter cells. pulling apart the sister chromatids of each chromosome. separating the two developing nuclei. the process of binary fission is very much different from the process of mitosis. In animal cells. As mitosis completes. each sister chromatid is now considered a chromosome. 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). the nuclear envelope which segregates the DNA from the cytoplasm disassembles. corresponding sister chromosomes are pulled toward opposite ends. . As a matter of convention. As the cell elongates. The chromosomes align themselves in a line spanning the cell. the parent cell will be split in half. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. In plant cells. Eventually. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. so they are renamed to sister chromosomes. Prokaryotic cells undergo a process similar to mitosis called binary fission.the cell begins cytokinesis. However. the daughter cells will construct a new dividing cell wall between each other.In most eukaryotes. A new nuclear envelope forms around the separated sister chromosomes.
a cell grows (G1). S (synthesis). and finally it divides (M) before restarting the cycle.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. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell .1 Preprophase In plant cells only. the cell grows by producing proteins and cytoplasmic organelles. The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another.2. the nucleus has to migrate into the center of the cell before mitosis can begin. This is achieved through the formation of a phragmosome. All these phases in the interphase are highly regulated.2. In highly vacuolated plant cells. continues to grow as it duplicates its chromosomes (S). Thus. During all three phases. grows more and prepares for mitosis (G 2). Interphase is divided into three phases: G1 (first gap). chromosomes are replicated only during the S phase. mainly via proteins. However. It alternates with the much longer interphase. where the cell prepares itself for cell division. and G2 (second gap). 2. prophase is preceded by a pre-prophase stage.
division. These microtubules can attach to kinetochores or they can interact with opposing microtubules. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. the pinched area is known as the cleavage furrow. This band marks the position where the cell will eventually divide. instead. The chromosomes have chromatin has condensed. 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. In addition to phragmosome formation. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. . after the nuclear membrane breaks down. aligned at the metaphase plate. degraded. Cytokinesis has already begun. The cells of higher plants (such as the flowering plants) lack centrioles. and microtubules have invaded the nuclear Prophase space. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes.
Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. the replicated chromosomes have two sister chromatids. they are not essential for the . Close to the nucleus are structures called centrosomes. The centrosome is the coordinating center for the cell's microtubules. chromatin condenses together into a highly ordered structure called a chromosome. which are made of a pair of centrioles found in most eukaryotic animal cells. the genetic material in the nucleus is in a loosely bundled coil called chromatin. At the onset of prophase. A cell inherits a single centrosome at cell division. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. Although centrioles help organize microtubule assembly. which is replicated by the cell with the help of the nucleus before a new mitosis begins. giving a pair of centrosomes. bound together at the centromere by the cohesin protein complex. Chromosomes are typically visible at high magnification through a light microscope. Since the genetic material has already been duplicated earlier in S phase. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell.
formation of the spindle. This is called open mitosis. on an average 20 ). When the spindle grows to sufficient length. Prometaphase is sometimes considered part of prophase. since they are absent from plants. When a microtubule connects with the kinetochore. Each chromosome forms two kinetochores at the centromere. and it occurs in most multicellular organisms. . 2. the motor activates.2. provides the pulling force necessary to later separate the chromosome's two chromatids. Fungi and some protists. kinetochore microtubules begin searching for kinetochores to attach to. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. or its microtubules are able to penetrate an intact nuclear envelope. one attached at each chromatid. This motor activity. Although the kinetochore structure and function are not fully understood. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. such as algae or trichomonads. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. coupled with polymerisation and depolymerisation of microtubules. and centrosomes are not always used in mitosis. using energy from ATP to "crawl" up the tube toward the originating centrosome. undergo a variation called closed mitosis where the spindle forms inside the nucleus.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. it is known that it contains some form of molecular motor.
The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". an imaginary line that is equidistant from the two centrosome poles." Microtubules find and attach to kinetochores in prometaphase.In the fishing pole analogy. in some sense. Metaphase comes from the Greek meaning "after.3 Metaphase A cell in late metaphase. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. In certain types of cells. convene along the metaphase plate or equatorial plane. 2. only roughly lining up along the midline. the kinetochore would be the "hook" that catches a sister chromatid or "fish". As a result. All chromosomes (blue) but one have arrived at the metaphase plate. The centromeres of the chromosomes. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. . the chromosomes come under longitudinal tension from the two ends of the cell. analogous to a tug-of-war between people of equal strength.
The force that causes the centrosomes to move towards the ends of the cell is still unknown. 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.” or “re-”). it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. allowing them to separate. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. Early anaphase is usually defined as the separation of the sister chromatids.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres).4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. Two events then occur: first. the nonkinetochore microtubules elongate. the proteins that bind sister chromatids together are cleaved. The signal creates the mitotic spindle checkpoint. These two stages are sometimes called early and late anaphase.” “against. At the end of anaphase. which have now become distinct sister chromosomes. These sister chromatids. 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. Next.” “back. 2. the cell proceeds to anaphase (from the Greek meaning “up. .
In animal cells. Cytokinesis is technically not even a phase of mitosis. unfold back into chromatin. using fragments of the parent cell's nuclear membrane. but cell division is not yet complete. however. Mitosis is complete.2. pinching off the separated nuclei. cell . At telophase. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. now surrounded by new nuclei. elongating the cell even more.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. Both sets of chromosomes. necessary for completing cell division.5 Cytokinesis Cilliate undergoing cytokinesis. In both animal and plant cells. Corresponding sister chromosomes attach at opposite ends of the cell. forms around each set of separated sister chromosomes. A new nuclear envelope. It "cleans up" the after effects of mitosis. the nonkinetochore microtubules continue to lengthen. 2. but rather a separate process. cytokinesis is a separate process that begins at the same time as telophase.
Similarly. Following are the occasions in the lives of organism where mitosis happens: 2.1Significance Mitosis is important for the maintenance of the chromosomal set.5.4 Regeneration .5..g. Each daughter cell has a complete copy of the genome of its parent cell. New cells are formed by mitosis and so are exact copies of the cells being replaced. zygote and also the basis of the growth of a multicellular body. e.5.5. which move along microtubules to the middle of the cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.division is also driven by vesicles derived from the Golgi apparatus. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. This is the basis of the development of a multicellular body from a single cell i. The phragmoplast is a microtubule structure typical for higher plants. 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. 2. cells are constantly sloughed off and replaced by new ones.e. The end of cytokinesis marks the end of the M-phase. whereas some green algae use a phycoplast microtubule array during cytokinesis.2 Development and growth The number of cells within an organism increases by mitosis. 2. skin and digestive tract.3 Cell replacement In some parts of body. separating the two nuclei.
the process may go wrong. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. One daughter cell will receive both sister chromosomes and the other will receive none. the hydra reproduces asexually by budding. and the latter cell having only one chromosome (the homologous chromosome).7 Consequences of errors Although errors in mitosis are rare. a condition often associated with cancer. they fail to complete cell division and retain both nuclei in one cell. These cells are considered aneuploid.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. The cells at the surface of hydra undergo mitosis and form a mass called bud. The production of new cells is achieved by mitosis. The same division happens during asexual reproduction or vegetative propagation in plants. 2.Some organisms can regenerate their parts of bodies. sea star regenerates its lost arm through mitosis. For example. a chromosome may fail to separate during anaphase. resulting in binucleated cells. a condition known as monosomy. In non-disjunction. Occasionally when cells experience nondisjunction. For example. especially during early cellular divisions in the zygote.5. a condition known as trisomy. 2. .5. Mitosis continues in the cells of bud and it grows into a new individual.
it results in the formation of Tumors. Occasionally. All cells have genes that control the timing and number of mitosis. causing inversion. non-homologous chromosome. but in reverse orientation. Such tumours can send cancer cells to other parts in body where new tumours may form. The effect of these genetic abnormalities depends on the specific nature of the error. Benign tumours are not harmful as soon as they are not moving. Or. As soon as they start to move and invade other cells there are said to be malignant tumours. It may reattach to the original chromosome. It results in abnormal cell growth. its organelles disintegrate and reform in a matter of hours.Mitosis is a demanding process for the cell. The fragment may incorrectly reattach to another. causing translocation. As long as these tumours remain in their original location they are called benign tumours. causing chromosomal duplication. which goes through dramatic changes in ultrastructure. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. it may be treated erroneously as a separate chromosome. Now what happens is that cell abnormally continue to divide at a single place. and chromosomes are jostled constantly by probing microtubules. sometimes mutuations occur in such genes and cells continue to divide. causing deletion. Errors in the control of mitosis may cause cancer. When tissues more than the requirement are synthesized in a single organ. . An arm of the chromosome may be broken and the fragment lost. chromosomes may become damaged. This phenomenon is called metastasis or spreading of disease. It results in the synthesis of execessive tissue growths.
microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. 2. Metaphase accounts for approximately 4% of the cell cycle's duration. Preceded by events in prometaphase and followed by anaphase.7 Metaphase Metaphase. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.2. Early events of metaphase can . This process may also be referred to as endoreduplication and the cells as endoploid. align in the middle of the cell before being separated into each of the two daughter cells. an imaginary line that is equidistant from the two centrosome poles. only roughly lining up along the middleline.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. In certain types of cells. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). resulting in cells with many copies of the same chromosome occupying a single nucleus. analogous to a tug of war between equally strong people. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. An example of a cell that goes through endomitosis is the megakaryocyte. from the ancient Greek(between) and (stage). chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. carrying genetic information.
produces a pattern of in total up to several hundred bands. Such a signal creates the mitotic spindle checkpoint. Staining of the slides. This would be accomplished by regulation of the anaphase-promoting complex.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. securin. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. Only after all chromosomes have become aligned at the metaphase plate. often with Giemsa (G banding) or Quinacrine. 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. Chromosomes are condensed(Thickened) and highly coiled in metaphase. does the cell enter anaphase. and separase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Metaphase chromosomes make the classical picture of chromosomes (karyotype). which makes them most suitable for visual analysis. when every kinetochore is properly attached to a bundle of microtubules. One of the cell cycle checkpoints occurs during prometaphase and metaphase. For classical cytogenetic analyses.coincide with the later events of prometaphase. Normal metaphase spreads are used in . 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). 2.
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. 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.
or an individual organism. autosomal chromosomes are present in two copies. Karyogram of human male using Giemsa staining. . the position of the centromeres. The study of whole sets of chromosomes is sometimes known as karyology. to study chromosomal aberrations. such as. and what they look like under a light microscope. The study of karyotypes is important for cell biology and genetics. Karyotypes can be used for many purposes. any differences between the sex chromosomes. or may not. and the results may be used in evolutionary biology and medicine.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. Attention is paid to their length. taxonomic relationships. in normal diploid organisms. Karyotypes describe the number of chromosomes. cellular function. There may. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). in humans 2n = 46. The term is also used for the complete set of chromosomes in a species.  The preparation and study of karyotypes is part of cytogenetics. ordered by size and position of centromere for chromosomes of the same size. Thus. So. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. and any other physical characteristics. and to gather information about past evolutionary events. be sex chromosomes. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. banding pattern.
New techniques were needed to definitively solve the problem: 1. Their behavior in animal (salamander) cells was described by Walther Flemming. Using cells in culture 2. Pretreating cells in a hypotonic solution. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. 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. 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. and he correctly insisted on humans having an XX/XY system. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. von Waldeyer in 1888. in contrast to their genic contents. The next stage took place after the development of genetics in the early 20th century. The name was coined by another German anatomist. Considering their techniques. these results were quite remarkable. which swells them and spreads the chromosomes .3. The subsequent history of the concept can be followed in the works of Darlington and White. in 1882. at first favoring 46. He revised his opinion later from 46 to 48.
Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Observations on karyotypes 3. The sex of an unborn fetus can be determined by observation of interphase cells. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Arresting mitosis in metaphase by a solution of colchicines 4.1 Staining The study of karyotypes is made possible by staining. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. a suitable dye. For humans. 3. 3. is applied after cells have been arrested during cell division by a solution of colchicine. Usually. the great apes have 48 chromosomes. reducing the number. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly. such as Giemsa.3.2. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.2 Observations Six different characteristics of karyotypes are usually observed and compared: .  Sometimes observations may be made on non-dividing (interphase) cells.2.
Differences in the position of centromeres. and mainly consists of genetically inactive repetitive DNA sequences. Heterochromatin stains darker than euchromatin. permitting its loss without penalty to the organism (the dislocation hypothesis). Humans have one pair fewer chromosomes than the great apes. This is brought about by translocations. A full account of a karyotype may therefore include the number. Differences in absolute sizes of chromosomes. 4. Differences in number and position of satellites. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. both have six pairs of chromosomes (n=6) yet V. 5. Differences in degree and distribution of heterochromatic regions. 2. but the genes have been mostly translocated (added) to other chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). as well as other cytogenetic information. shape and banding of the chromosomes. . 6. type. faba chromosomes are many times larger.1. This feature probably reflects different amounts of DNA duplication. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. indicating tighter packing. 3. which (when they occur) are small bodies attached to a chromosome by a thin thread.
3. the same cannot be said for their karyotypes. Mosaics or otherwise abnormal individuals. Normal karyotypes for females contain two X chromosomes and are denoted 46.Variation is often found: 1. XX. XY. males have both an X and a Y chromosome denoted 46. Between the sexes 2. and in . Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Between members of a population (chromosome polymorphism) 4.3 The human karyotype Most (but not all) species have a standard karyotype. There is variation between species in chromosome number. Any variation from the standard karyotype may lead to developmental abnormalities. Geographical variation between races 5. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. which are highly variable. Between the germ-line and soma (between gametes and the rest of the body) 3.
used in conjunction with other phylogenetic data. the general significance of karyotype evolution is obscure. Chromosome elimination. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. Although much is known about karyotypes at the descriptive level. In this process. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. .detailed organization.. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. In some cases there is even significant variation within species. In A. Chromatin diminution (founding father: Theodor Boveri). found in some copepods and roundworms such as Ascaris suum. portions of the chromosomes are cast away in particular cells. In some species. despite many careful investigations. 3.. as in many sciarid flies. some organisms go in for large-scale elimination of heterochromatin. In a review. entire chromosomes are eliminated during development.1 Changes during development Instead of the usual gene repression.3. or other kinds of visible adjustment to the karyotype.. "We have a very poor understanding of the causes of karyotype evolution. Godfrey and Masters conclude: "In our view. which were previously inexplicable. But. This variation provides the basis for a range of studies in evolutionary cytology. it is quite unclear what the general significance might be.. despite their construction from the same macromolecules.
where the haploid n = 1. Muntiacus reevesi. all telocentric.3. In human females some 15% of somatic cells escape inactivation. In marsupials it is always the paternal X which is inactivated. 3. was found to be 46.suum. the inactivation is random as between the two Xs. 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.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). thus the mammalian female is a mosaic in respect of her X chromosomes. they were astonished to find it had female = 6.. the high record would be somewhere amongst the ferns. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. Xinactivation. They kept quiet for two or three years because they thought something was wrong with their tissue culture. Muntiacus muntjak. In placental mammals. which was investigated by Kurt Benirschke and his colleague Doris Wurster.. all the somatic cell precursors undergo chromatin diminution. When they looked at the karyotype of the closely related Indian muntjac. The diploid number of the Chinese muntjac. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes... The existence of supernumerary or B chromosomes . The low record is held by the nematode Parascaris univalens. "They simply could not believe what they saw. male = 7 chromosomes.
where one sex is diploid. and in some other groups.means that chromosome number can vary even within one interbreeding population.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Humans have FN = 82. 15.3. FN ≤ 2n. 21 and 22).3 Fundamental number The fundamental number. Polyploidy in lower plants (ferns. It is a common arrangement in the Hymenoptera. Polyploidy in animals is much less common. where there are more than two sets of homologous chromosomes in the cells. It has been of major significance in plant evolution according to Stebbins. 3. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. about 70%. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. and the other haploid. due to the presence of five acrocentric chromosome pairs (13. Haplo-diploidy. but it has been significant in some groups. occurs mainly in plants. though in this case they would not be regarded as normal members of the population.Endopolyploidy occurs when in adult differentiated . and aneuploids are another example. Thus. FN. horsetails and psilotales) is also common. 3. Polyploidy. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. but in grasses the average is much higher. 14.
Abnormalities in chromosome number usually cause a defect in development.tissues the cells have ceased to divide by mitosis. but the nuclei contain more than the original somatic number of chromosomes. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). . it is diverse and complex. 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. The cells do not always contain exact multiples (powers of two). and serves differentiation and morphogenesis in many ways. the daughter chromosomes separating from each other inside an intact nuclear membrane. See palaeopolyploidy for the investigation of ancient karyotype duplications. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. 3. Down syndrome and Turner syndrome are examples of this. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. This 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.
and Crocus. 6. When this happens. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. Human chromosome 2 was formed by a merger of ancestral chromosomes. the chromosome number is variable from one individual to another. Well-researched examples are the ladybird beetle Chilocorus stigma. and 7. where the gametic (= haploid) numbers form the series x = 3. the European shrew Sorex araneus.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. In about 6. reducing the number. living from rainforests to . 3.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 4.Aneuploidy may also occur within a group of closely related species. the great apes have 24x2 chromosomes whereas humans have 23x2.  Closer to home. where every number from x = 3 to x = 15 is represented by at least one species. that the two chromosome morphs are adapted to different habitats. 3.000 km2). Classic examples in plants are the genus Crepis. some mantids of the genus Ameles. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. 5.500 sq mi (17.
the present islands date from 0. The polytene banding of the 'picture wing' group. Using K-Ar dating. it is more likely to have been a group from the same species. but these are much less frequent. show a clear "flow" of species from older to newer islands. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. when plotted in tree form (and independent of all other information). There are also cases of colonization back to older islands.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). gene arrangements are visible in the banding patterns of each chromosome. . The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. especially inversions. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. Chromosome rearrangements. in the family Drosophilidae. which can be dated to 30 mya. probably 20 million years ago. the best-studied group of Hawaiian drosophilids. at least into the Cretaceous.subalpine meadows. In a sense. Although it would be possible for a single gravid female to colonise an island. The results are clear. Drosophila and Scaptomyza. and skipping of islands. make it possible to see which species are closely related. The inversions. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain.
The light regions tend to be euchromatic.the dark regions tend to be heterochromatic. late-replicating and AT rich. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). • Q-banding is a fluorescent pattern obtained using quinacrine for staining. adaptive radiations.7.7 Depiction of karyotypes 3. • T-banding: visualize telomeres. The pattern of bands is very similar to that seen in G-banding. 3. so it stains centromeres.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. early-replicating and GC rich. if less spectacular. . R-banding is the reverse of G-banding (the R stands for "reverse"). human genome. This method will normally produce 300-400 bands in a normal.There are other animals and plants on the Hawaiian archipelago which have undergone similar. • • C-banding: Giemsa binds to constitutive heterochromatin. It yields a series of lightly and darkly stained bands .
and the long arm on the bottom.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. respectively. This yields a dark region where the silver is deposited. Some karyotypes call the short and long arms p and q.7. Cri du chat syndrome involves a deletion on the short arm of . less frequently Quinacrine.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. Giemsa is specific for the phosphate groups of DNA. a dye. is used to stain bands on the chromosomes. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. both chromosomes in a pair will have the same banding pattern. often Giemsa (G-banding). In addition. denoting the activity of rRNA genes within the NOR. Karyotypes are arranged with the short arm of the chromosome on top. Quinacrine binds to the adeninethymine-rich regions. In the "classic" (depicted) karyotype. For example. Each chromosome has a characteristic banding pattern that helps to identify them. 3.
chromosome 5. It is written as 46. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.7. Image processing software then assigns a pseudo color to each spectrally different combination.XX. . which is written as 46. 3. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.5p-.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Because there are a limited number of spectrally-distinct fluorophores.2. allowing the visualization of the individually colored chromosomes.2) 3. a combinatorial labeling method is used to generate many different colors. This method is also known as virtual karyotyping. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.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.XX. The critical region for this syndrome is deletion of 15. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.
CHAPTER 4 .
• • Patau syndrome is caused by trisomy of chromosome 13. or structural. also known as aneuploidy. trisomies. inversions. trisomy 9 and trisomy 16. Structural abnormalities often arise from errors in homologous recombination.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. the most common male chromosomal disease. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. Numerical abnormalities. although they generally do not survive to birth. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. otherwise known as 47. are common numerical abnormalities. as in the presence of extra or missing chromosomes. X0). X or 45. in which three copies of a chromosome are present instead of the usual two. Some disorders arise from loss of just a piece of one chromosome. large-scale deletions or duplications. translocations. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. is caused by trisomy of chromosome 21.4. a common chromosomal disease. including . as in derivative chromosome. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. XXY is caused by an extra X chromosome. Also documented are trisomy 8. Down syndrome. often occur as a result of nondisjunction during meiosis in the formation of a gamete. Klinefelter syndrome.
a deletion of the paternal genes. They can be organized into two basic groups. from a truncated short arm on chromosome 5. The name comes from the babies' distinctive cry. one well-documented example is the Philadelphia chromosome. 1p36 Deletion syndrome. A chromosome anomaly may be detected or confirmed in this manner. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. from the loss of part of the short arm of chromosome 1. . numerical and structural anomalies.• Cri du chat (cry of the cat). • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. 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. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. A chromosome anomaly. example of imprinting disorder. a deletion of the maternal genes. caused by abnormal formation of the larynx. There are many types of chromosome anomalies. example of imprinting disorder. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual.
etc. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. In a reciprocal translocation. which is caused by partial deletion of the short arm of chromosome 4. In a Robertsonian translocation. also called the terminal 11q deletion disorder. There are two main types of translocations. segments from two different chromosomes have been exchanged. 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. 4. rather than two).4.). and Jacobsen syndrome. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. Tetrasomy. Duplications: A portion of the chromosome is duplicated. • • Translocations: When a portion of one chromosome is transferred to another chromosome. an entire chromosome has . an X.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. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. resulting in extra genetic material. Known disorders in humans include Wolf-Hirschhorn syndrome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes).
21 and 22. the anomaly is present in every cell of the body. as well . This can happen with or without loss of genetic material. however.in humans these only occur with chromosomes 13. 4. Therefore. 14. and are therefore initially not inherited. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. 4. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. • Rings: A portion of a chromosome has broken off and formed a circle or ring. 15. other cytogenetic banding techniques.attached to another at the Centromere . This is why chromosome studies are often performed on parents when a child is found to have an anomaly. It includes routine analysis of G-Banded chromosomes. especially the chromosomes. can happen after conception. therefore the genetic material is inverted. resulting in Mosaicism (where some cells have the anomaly and some do not). • Inversions: A portion of the chromosome has broken off. They often lead to an increased tendency to develop certain types of malignancies. turned upside down and reattached. Chromosome anomalies can be inherited from a parent or be "de novo".3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. Some anomalies.
Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. The name was coined by another German anatomist. which swells them and spreads the chromosomes .5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. and he correctly insisted on man having an XX/XY system. Considering their techniques. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. in 1882. New techniques were needed to definitively solve the problem: 1. the discoverer of mitosis. He revised his opinion later from 46 to 48. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Using cells in culture 2. Their behavior in animal (salamander) cells was described by Walther Flemming. 4. concluding an XX/XO sex determination mechanism. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. von Waldeyer in 1888. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. at first favoring 46. in contrast to their genic contents. The next stage took place after the development of genetics in the early 20th century. these results were quite remarkable.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Pre-treating cells in a hypotonic solution.
reducing the number.3.6. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. During her cytogenetic work. McClintock discovered transposons. Using Painter's technique they studied the polytene . In 1931.2 Natural populations of Drosophila In the 1930s. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. 4. Rather interestingly. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Human chromosome 2 was formed by a merger of ancestral chromosomes.6 Applications in biology 4. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. the great apes have 48 chromosomes. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).6.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. persimilis from wild populations in California and neighboring states. Arresting mitosis in metaphase by a solution of colchicine 4. a find which eventually led to her Nobel Prize in 1983.
This had the benefit of eliminating migration as a possible explanation of the results. but adjust to certain frequencies at which they become stabilised. 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. as with most polymorphisms. . Evidence rapidly accumulated to show that natural selection was responsible. as they would if selectively neutral. Using a method invented by L'Heretier and Teissier. It was found that the various chromosome types do not fluctuate at random. Dobzhansky bred populations in population cages. breeding and sampling whilst preventing escape. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. 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.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. 4. Down syndrome is also referred to as trisomy 21. In 1959. In some congenital disorders. discoveries were quickly made related to aberrant chromosomes or chromosome number. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. 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. such as Down's syndrome.
In 1960. and XXXX. . Identification of the Philadelphia chromosome by cytogenetics. in addition to other tests. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. which is required in normal females to compensate for having two copies of the chromosome. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. with the development of more advanced techniques. An individual with only one sex chromosome (the X) has Turner syndrome. Many other sex chromosome combinations are compatible with live birth including XXX. an additional X chromosome in a male.as both scientists were doing their research in Philadelphia. has Klinefelter's Syndrome. Thirteen years later. XYY. Pennsylvania.Other numerical abnormalities discovered include sex chromosome abnormalities. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. is used today as a diagnostic for CML. This abnormal chromosome was dubbed the Philadelphia chromosome . the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. resulting in 47 total chromosomes. Not all genes on the X Chromosome are inactivated. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML).
FIG Advent of banding techniques In the late 1960s. and elongation techniques for all culture types that allow for higher resolution banding. Deletion syndromes such as DiGeorge syndrome. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Deletions within one chromosome could also now be more specifically named and understood. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.8 Beginnings of molecular cytogenetics . Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 4. Caspersson developed banding techniques which differentially stain chromosomes. 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.
advances were made in molecular cytogenetics.In the 1980s. CHAPTER 5 Techniques 5. While radioisotope-labeled probes had been hybridized with DNA since 1969. 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.1 Karyotyping . movement was now made in using fluorescent labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). cloned and studied in ever greater detail.
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,
and computational biology. and numerical computation. such as comparative genomic hybridization arrays. communications. . including signal and image processing. data analysis.generally between 200 and 1000 cells are counted and scored. control design. test and measurement. you can solve technical computing problems faster than with traditional programming languages. You can use MATLAB in a wide range of applications. and FORTRAN. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. 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. such as C. financial modeling and analysis. data visualization. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. CGH and Single nucleotide polymorphism-arrays. C++. For congenital problems usually 20 metaphase cells are scored. Using MATLAB.
and allocating memory. These effects must be compensated to improve the results of the pairing algorithm.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. It enables fast development and execution. such as declaring variables. You can integrate your MATLAB code with other languages and applications. one line of MATLAB code can often replace several lines of C or C++ code. 6. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. specifying data types. The image processing step is composed of the following operations. and distribute your MATLAB algorithms and applications. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. MATLAB eliminates the need for ‘for’ loops. As a result. .MATLAB provides a number of features for documenting and sharing your work. In many cases. With the MATLAB language. 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.
2) Geometrical compensation—The geometric compensation. 6. To compare chromosomes from a band pattern point of view. geometrical and dimensional differences must be removed. 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. 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). To compensate for this inhomogeneity. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. Therefore. 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.
so the image displays as shades of gray. In addition to these image type conversion functions. You can perform certain conversions just using MATLAB syntax.3) Edge detection 4) Two dimensional convolutions. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. listed in the following table.I. MATLAB filters the intensity values in the image. and the results might not be meaningful. there are other functions that return a different image type as part of the operation they perform.I). For example. For example.I. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. you must first convert it to true color format. green.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. and blue planes. MATLAB simply applies the filter to the indices in the indexed image matrix. if you want to filter a color image that is stored as an indexed image. When you apply the filter to the true color image. RGB = cat (3. as is appropriate. 6.2. For example. If you attempt to filter the indexed image. The resulting true color image has identical matrices for the red. .
Usually we know what type of errors to expect. caused by external disturbance. .4 Denoising We may define noise to be any degradation in the image signal. There is a large number of edge finding algorithms in existence.3 Two dimensional convolutions C = conv2(A. ) Where the parameters available depend on the method used 6. If an image is being sent electronically from one place to another. to recognize or classify objects. we may expect errors to occur in the image signal.2. hence we can choose the most appropriate method for reducing the effects. We may use edges to measure the size of objects in an image.'method'. and hence the type of noise on the image. via satellite or wireless transmission. These errors will appear on the image output in different ways depending on the type of disturbance in the signal.5 Edge detection Edges contain some of the most useful information in an image. 6.2. to isolate particular objects from their background. 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. or through networked cable.6. The general Matlab command for finding edges is edge(image.B) computes the two-dimensional convolution of matrices A and B.parameters.
'shape') subsection of the two-dimensional convolution.bmp').na] and the size of B is [mb.hrow. the other matrix is filtered in two dimensions.A).'sobel'). rgb2gray im2bw(im. If hcol is a column vector and hrow is a row vector. The size of matrices. ..(FIR) filter. nb]+1)/2). convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.na+nb-1].7). The indices of the center element of B are defined as floor(([mb C = conv2(hcol. 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. this case is the same as C = conv2(hcol*hrow..A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. That is. C = conv2(. minus one.nb]. if the size of then the size of C is [ma+mb-1. imedfilt2(im1.0..[3 3]). edge(im1.
MODULE 2 clc [m. rc = [r c].y1)=255. .[imx.j)==L_number(k) flag=1.imy]=size(BW).1). L_number=zeros(mx. [r.8). [sx sy]=size(rc).imy). y1=rc(i. nzeros(imx.j)~=0 for k=1:mx if L(i. Msk conv2(double(BW).double(msk)).1). flag=0.n]=size(L).2). Index=1. bwlabel(B. for i=1:sx x1=rc(i. for i=1:m for j=1:n if L(i. n1(x1.c] = find(L==22). mx=max(max(L)).
end flag=0.y1)=255.). rc = [r c].end end if flag~=1 L_number(Index)=L(i. end.7.32. n1(x1. end end L_number. for x=1:46 [r. end %h=figure.18.104.22.168.52. 36.56. [sx sy]=size(rc).8.38. Index=Index+1.21.9. .22.66].41.20.39.imy). y1=rc(i.59.j).22.214.171.124). for i=1:sx x1=rc(i.10. n1=zeros(imx.126.96.36.199.188.8.131.52.184.108.40.206.220.127.116.11.1).27.imshow(n1.26. Test_number=[3.c] = find(L==L_number((Test_number(x)))).18.104.22.168.
5*graythresh(skel)).bmp')). end end end Circumference(i)=Circumference_sum.1.'spur'. s1=bwmorph(s.1).end Circumference=zeros(46.'. [m n]=size(BW1). skel=im2bw(skel.1).Inf). BW1=edge(BW. f=imcomplement(f). Arm_length=zeros(46. BW=double(BW). for i=1:46 f=imread(strcat(num2str(i). s=bwmorph(skel. for x=1:m for y=1:n if BW1(x.1).'skel'. Area=zeros(46. Arm_length_sum=0.8). .'canny').y)==1 Circumference_sum=Circumference_sum+1. Circumference_sum=0. skel=im2double(f). BW=im2bw(f).
for x=1:m for y=1:n if BW(x.y)==1 Area_sum=Area_sum+1. end Circumference. end end end Arm_length(i)=Arm_length_sum. for x=1:m for y=1:n if s1(x.y)==1 Arm_length_sum=Arm_length_sum+1. Arm_length. BW=im2bw(f). [m n]=size(BW).[m n]=size(s1). . Area_sum=0. end end end Area(i)=Area_sum.
Pair(i. Pair(i. Pair(i. Pair(i. Pair(46.2).Area.1)=i. end end Pair.2)=i+1. end end end for i=1:45 if Pair(i. .1)=46.2)==46 Pair(46. Pair=zeros(46.2)=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)). 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)=j.1)=i.
.2. figure_flag=1. end end if flag~=1 if figure_flag~=47 subplot(23. imshow(f2). imshow(f1). figure_flag=figure_flag+1.2. end f2=imread(strcat(num2str(Pair(i.2)).1)).'. flag=0.figure_flag).delete=zeros(46.'.bmp')).1). end f1=imread(strcat(num2str(Pair(i.2). figure_flag=figure_flag+1.figure_flag).1)==delete(j) flag=1. for i=1:46 for j=1:46 if Pair(i.bmp')). if figure_flag~=47 subplot(23. end flag=0. delete(figure_flag)=Pair(i.
The proposed algorithm is based on the traditional features extracted from the karyogram. in the scope of karyotyping process used in cytogentic analysis.end CONCLUTION In this paper. Copenhagen. such as. 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. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. plus a new one. and Philadelphia. 2) feature extraction from the processed images . to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. dimensions and banding profiles. 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. based on the MI.
from the chromosomes in the training set. extracted from the unordered karyogram. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. achieves a 70. Tests using 19 karyograms based on bone marrow cells. and finally. 4) pairing. the romosome images. Here. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. are processed in order to compensate for geometrical and intensity distortions.10% mean classification rate. and to normalize their dimensions. and band pattern. The features extracted from the processed images discriminate each pair with respect to their 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).characterizing the size. 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. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. shape. 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 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. In the image processing step. shape and band pattern.working within an 8-D feature space. The training process consists in the estimation of each vector of coefficient .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.
presenting a uniform level of condensation. such as Edinburgh.. The results presented in this paper are promising. REFERENCES . was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. e. amean classification rate larger than 93% was obtained in all experiments. This dataset was made publicly available . whose images are of significantly higher quality. called LK1 . Copenhagen. or Philadelphia.g. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. and from which it is possible to extract additional features. centromere position. Using 27 karyograms andworking with a limited number of classes (≤ 8). In addition. a 76.performance of the classifier. a new chromosome dataset with 9200 chromosomes from bone marrow cells.10% classification ratewas obtained. In fact. Executing the algorithm on a higher quality dataset. despite the low quality of this type of chromosomes.
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