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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
although there are many exceptions to this rule. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. In prokaryotes. The structure of chromosomes and chromatin varies through the cell cycle. Chromosomal recombination plays a vital role in genetic diversity. through processes known as chromosomal instability and translocation. Chromosomes may exist as either duplicated or unduplicated. a large body of work uses the term chromosome regardless of chromatin content. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. However. the cell may undergo mitotic catastrophe and die. These small circular genomes . circular DNA molecules called plasmids. divided. sometimes accompanied by one or more smaller. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). or it may unexpectedly evadeapoptosis leading to the progression of cancer. Also. In prokaryotes and viruses. for example. In eukaryotes. This allows the very long DNA molecules to fit into the cell nucleus. cells may contain more than one type of chromosome. DNA is usually arranged as a circle. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes.defined nuclei) have smaller circular chromosomes. 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. If these structures are manipulated incorrectly. In practice "chromosome" is a rather loosely defined term. Unduplicated chromosomes are single linear strands. the term genophore is more appropriate when no chromatin is present. which is tightly coiled in on itself.
in which an individual has 3.3 MUTATIONS IN CHROMOSOME NUMBER Normally.XY or 47. rather than 2. 1.+21. Euploid human karyotypes are 46. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. The individual would have Down Syndrome and his/her karyotype would be written 47.are also found in mitochondria and chloroplasts. An example of aneuploidy is trisomy 21. If the mutation involves only one or a few chromosomes in the genome (e. Such individuals are called euploid and have the wild-type chromosome complement for the species. 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. copies of chromosome 21. XX (female) or 46 XY (male). a extra copy of human chromosome 21). 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). .+21.XX.g. the individual carrying the mutation is said to be aneuploid. reflecting their bacterial origins.
This is the only natural context in which individual chromosomes are visible with an optical microscope. In spite of their appearance. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. which enables these giant DNA structures to be contained within a cell nucleus. so that each daughter cell inherits one set of chromatids. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. This compact form makes the individual chromosomes visible. A special DNA base sequence in the region of the kinetochores provides. chromosomes are structurally highly condensed. one of which is present on each sister chromatid. and they form the classic four arm structure. q-g "grande"). 1. a pair of sister chromatids attached to each other at the centromere.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II.Fig 1. along with special proteins. longer-lasting attachment in this region. 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). Once the cells have divided. The microtubules then pull the chromatids apart toward the centrosomes. the chromatids are uncoiled and DNA can again be transcribed. the chromatin strands become more and more condensed. small) and the longer arms are called q arms (q follows p in the Latin alphabet. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. During mitosis. .
 Bone marrow is also a key component of the lymphatic system. producing the lymphocytes that support the body's immune system CHAPTER 2 2. organelles and cell membrane into two cells containing roughly equal shares of these cellular components.7 lbs). cytoplasm. In humans. bone marrow constitutes 4% of the total body mass of humans.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. It is generally followed immediately by cytokinesis. bone marrow in large bones produces new blood cells.1.6 kg (5. which use the bone marrow vasculature as a conduit to the body's systemic circulation. bone marrow accounts for approximately 2. which divides the nuclei. in adults weighing 65 kg (143 lbs). . The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. in two separate nuclei. On average.
forming single cells with multiple nuclei. cytokinesis and mitosis may occur independently. where the nuclear envelope breaks down before the chromosomes separate. there are many cells where mitosis and cytokinesis occur separately. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. which lack a nucleus. where chromosomes divide within an intact cell nucleus. This accounts for approximately 10% of the cell cycle. metaphase. "mitosis" is often used interchangeably with "mitotic phase". Mitosis occurs only in eukaryotic cells and the process varies in different species. . but is found in various different groups. The process of mitosis is fast and highly complex. prometaphase. The cell then divides in cytokinesis. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. This occurs most notably among the fungi and slime moulds. to produce two identical daughter cells which are still diploid cells. animals undergo an "open" mitosis. For example. divide by a process called binary fission. prophase. for instance during certain stages of fruit fly embryonic development. Even in animals. Because cytokinesis usually occurs in conjunction with mitosis.genetically identical to each other and to their parent cell. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. These stages are interphase. anaphase and telophase. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. However. Prokaryotic cells.
Each chromosome now has an identical copy of itself. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. and together the two are called sister chromatids. 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 period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. . This occurs during the S phase of interphase. the parent cell must make a copy of each chromosome before mitosis.
In most eukaryotes. The chromosomes align themselves in a line spanning the cell. the daughter cells will construct a new dividing cell wall between each other. As a matter of convention.the cell begins cytokinesis. . corresponding sister chromosomes are pulled toward opposite ends. so they are renamed to sister chromosomes. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. each sister chromatid is now considered a chromosome. separating the two developing nuclei. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. 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). Eventually. In animal cells. In plant cells. As the cell elongates. As mitosis completes. Prokaryotic cells undergo a process similar to mitosis called binary fission. However. giving rise to two daughter cells. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. each with a replica of the original genome. pulling apart the sister chromatids of each chromosome. the process of binary fission is very much different from the process of mitosis. the parent cell will be split in half. A new nuclear envelope forms around the separated sister chromosomes.
1 Preprophase In plant cells only. This is achieved through the formation of a phragmosome. a cell grows (G1). However. continues to grow as it duplicates its chromosomes (S). Thus. Interphase is divided into three phases: G1 (first gap). where the cell prepares itself for cell division. prophase is preceded by a pre-prophase stage. grows more and prepares for mitosis (G 2). In highly vacuolated plant cells. and G2 (second gap). 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. During all three phases. It alternates with the much longer interphase.2.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. 2. mainly via proteins.2. and finally it divides (M) before restarting the cycle. chromosomes are replicated only during the S phase. All these phases in the interphase are highly regulated. S (synthesis). a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . the cell grows by producing proteins and cytoplasmic organelles. the nucleus has to migrate into the center of the cell before mitosis can begin.
aligned at the metaphase plate. Cytokinesis has already begun. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. .division. 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. and microtubules have invaded the nuclear Prophase space. The cells of higher plants (such as the flowering plants) lack centrioles. This band marks the position where the cell will eventually divide. the pinched area is known as the cleavage furrow. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. These microtubules can attach to kinetochores or they can interact with opposing microtubules. instead. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. The chromosomes have chromatin has condensed. degraded. In addition to phragmosome formation. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. after the nuclear membrane breaks down.
At the onset of prophase. which is replicated by the cell with the help of the nucleus before a new mitosis begins. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. the replicated chromosomes have two sister chromatids. Since the genetic material has already been duplicated earlier in S phase. Chromosomes are typically visible at high magnification through a light microscope. which are made of a pair of centrioles found in most eukaryotic animal cells. chromatin condenses together into a highly ordered structure called a chromosome.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. The centrosome is the coordinating center for the cell's microtubules. bound together at the centromere by the cohesin protein complex. they are not essential for the . Although centrioles help organize microtubule assembly. A cell inherits a single centrosome at cell division. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. Close to the nucleus are structures called centrosomes. giving a pair of centrosomes. the genetic material in the nucleus is in a loosely bundled coil called chromatin.
A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Prometaphase is sometimes considered part of prophase. Although the kinetochore structure and function are not fully understood. Each chromosome forms two kinetochores at the centromere. kinetochore microtubules begin searching for kinetochores to attach to. coupled with polymerisation and depolymerisation of microtubules. When a microtubule connects with the kinetochore. or its microtubules are able to penetrate an intact nuclear envelope.formation of the spindle. such as algae or trichomonads. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. undergo a variation called closed mitosis where the spindle forms inside the nucleus. This is called open mitosis. and centrosomes are not always used in mitosis. . When the spindle grows to sufficient length. on an average 20 ). since they are absent from plants. Fungi and some protists. and it occurs in most multicellular organisms. one attached at each chromatid. 2. provides the pulling force necessary to later separate the chromosome's two chromatids. the motor activates.2. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. it is known that it contains some form of molecular motor. using energy from ATP to "crawl" up the tube toward the originating centrosome. This motor activity.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space.
convene along the metaphase plate or equatorial plane. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. an imaginary line that is equidistant from the two centrosome poles. . The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". the chromosomes come under longitudinal tension from the two ends of the cell. As a result. All chromosomes (blue) but one have arrived at the metaphase plate. 2.In the fishing pole analogy. in some sense. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores." Microtubules find and attach to kinetochores in prometaphase. only roughly lining up along the midline. The centromeres of the chromosomes. Metaphase comes from the Greek meaning "after. In certain types of cells. analogous to a tug-of-war between people of equal strength. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. the kinetochore would be the "hook" that catches a sister chromatid or "fish".3 Metaphase A cell in late metaphase.
the cell has succeeded in separating identical copies of the genetic material into two distinct populations.” or “re-”). pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. At the end of anaphase. allowing them to separate.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. which have now become distinct sister chromosomes. the nonkinetochore microtubules elongate. The signal creates the mitotic spindle checkpoint. the proteins that bind sister chromatids together are cleaved. The force that causes the centrosomes to move towards the ends of the cell is still unknown. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. These two stages are sometimes called early and late anaphase. .” “back. 2. These sister chromatids.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. Two events then occur: first. Next. Early anaphase is usually defined as the separation of the sister chromatids. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned.” “against. the cell proceeds to anaphase (from the Greek meaning “up.
cytokinesis is a separate process that begins at the same time as telophase. using fragments of the parent cell's nuclear membrane. forms around each set of separated sister chromosomes. A new nuclear envelope. Both sets of chromosomes. but rather a separate process. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. but cell division is not yet complete.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. Corresponding sister chromosomes attach at opposite ends of the cell. the nonkinetochore microtubules continue to lengthen. It "cleans up" the after effects of mitosis. Mitosis is complete. pinching off the separated nuclei. unfold back into chromatin. 2. In both animal and plant cells. Cytokinesis is technically not even a phase of mitosis.2. necessary for completing cell division. At telophase. however. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be.5 Cytokinesis Cilliate undergoing cytokinesis. now surrounded by new nuclei. In animal cells. cell . elongating the cell even more.
New cells are formed by mitosis and so are exact copies of the cells being replaced. 2. which move along microtubules to the middle of the cell. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. e.3 Cell replacement In some parts of body.division is also driven by vesicles derived from the Golgi apparatus.g.e. The phragmoplast is a microtubule structure typical for higher plants. 2.. 2. zygote and also the basis of the growth of a multicellular body. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.5.4 Regeneration . Similarly.2 Development and growth The number of cells within an organism increases by mitosis.5. separating the two nuclei.1Significance Mitosis is important for the maintenance of the chromosomal set. 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. skin and digestive tract. The end of cytokinesis marks the end of the M-phase. Each daughter cell has a complete copy of the genome of its parent cell.5.5. This is the basis of the development of a multicellular body from a single cell i. whereas some green algae use a phycoplast microtubule array during cytokinesis. Following are the occasions in the lives of organism where mitosis happens: 2.
a condition known as trisomy.Some organisms can regenerate their parts of bodies. The production of new cells is achieved by mitosis. Mitosis continues in the cells of bud and it grows into a new individual. 2. and the latter cell having only one chromosome (the homologous chromosome). These cells are considered aneuploid. The same division happens during asexual reproduction or vegetative propagation in plants. resulting in binucleated cells. 2. they fail to complete cell division and retain both nuclei in one cell. the process may go wrong. .6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). One daughter cell will receive both sister chromosomes and the other will receive none. For example.5. sea star regenerates its lost arm through mitosis. For example. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. a condition known as monosomy.7 Consequences of errors Although errors in mitosis are rare.5. In non-disjunction. The cells at the surface of hydra undergo mitosis and form a mass called bud. a condition often associated with cancer. Occasionally when cells experience nondisjunction. the hydra reproduces asexually by budding. especially during early cellular divisions in the zygote. a chromosome may fail to separate during anaphase.
causing chromosomal duplication. it results in the formation of Tumors. Occasionally. chromosomes may become damaged. Now what happens is that cell abnormally continue to divide at a single place. causing inversion. . Benign tumours are not harmful as soon as they are not moving. As long as these tumours remain in their original location they are called benign tumours. its organelles disintegrate and reform in a matter of hours. it may be treated erroneously as a separate chromosome. but in reverse orientation. sometimes mutuations occur in such genes and cells continue to divide. Errors in the control of mitosis may cause cancer. Or. and chromosomes are jostled constantly by probing microtubules. Such tumours can send cancer cells to other parts in body where new tumours may form. non-homologous chromosome. When tissues more than the requirement are synthesized in a single organ. This phenomenon is called metastasis or spreading of disease. The fragment may incorrectly reattach to another. It results in abnormal cell growth. An arm of the chromosome may be broken and the fragment lost. It results in the synthesis of execessive tissue growths. which goes through dramatic changes in ultrastructure. causing deletion. causing translocation. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. As soon as they start to move and invade other cells there are said to be malignant tumours.Mitosis is a demanding process for the cell. The effect of these genetic abnormalities depends on the specific nature of the error. It may reattach to the original chromosome. All cells have genes that control the timing and number of mitosis.
7 Metaphase Metaphase. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). This process may also be referred to as endoreduplication and the cells as endoploid. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. analogous to a tug of war between equally strong people. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. Early events of metaphase can .6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.2. align in the middle of the cell before being separated into each of the two daughter cells. only roughly lining up along the middleline. resulting in cells with many copies of the same chromosome occupying a single nucleus. from the ancient Greek(between) and (stage). Metaphase accounts for approximately 4% of the cell cycle's duration. carrying genetic information. 2. Preceded by events in prometaphase and followed by anaphase. In certain types of cells. an imaginary line that is equidistant from the two centrosome poles. An example of a cell that goes through endomitosis is the megakaryocyte.
Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). produces a pattern of in total up to several hundred bands. Only after all chromosomes have become aligned at the metaphase plate. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. 2. For classical cytogenetic analyses. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Chromosomes are condensed(Thickened) and highly coiled in metaphase. Staining of the slides. and separase. often with Giemsa (G banding) or Quinacrine. Such a signal creates the mitotic spindle checkpoint. This would be accomplished by regulation of the anaphase-promoting complex. Metaphase chromosomes make the classical picture of chromosomes (karyotype).8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. does the cell enter anaphase. Normal metaphase spreads are used in .coincide with the later events of prometaphase. when every kinetochore is properly attached to a bundle of microtubules. securin. 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. 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.
Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. such as bcr-abl in chronic myelogenous leukemia. for example. . Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. losses of chromosomal segments or translocations.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. which may lead to chimeric oncogenes.
such as. cellular function. Karyogram of human male using Giemsa staining. Thus. autosomal chromosomes are present in two copies. or may not. The term is also used for the complete set of chromosomes in a species. So. . and any other physical characteristics. be sex chromosomes. in normal diploid organisms. The study of karyotypes is important for cell biology and genetics. and to gather information about past evolutionary events. The study of whole sets of chromosomes is sometimes known as karyology. taxonomic relationships. banding pattern. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. ordered by size and position of centromere for chromosomes of the same size. or an individual organism. and what they look like under a light microscope. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. in humans 2n = 46.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). any differences between the sex chromosomes. Attention is paid to their length.  The preparation and study of karyotypes is part of cytogenetics. There may. to study chromosomal aberrations. Karyotypes describe the number of chromosomes. Karyotypes can be used for many purposes. the position of the centromeres. and the results may be used in evolutionary biology and medicine.
Pretreating cells in a hypotonic solution. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. The subsequent history of the concept can be followed in the works of Darlington and White. in 1882. and he correctly insisted on humans having an XX/XY system. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in contrast to their genic contents. 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. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48.3. these results were quite remarkable.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. the discoverer of mitosis. Considering their techniques. Using cells in culture 2. New techniques were needed to definitively solve the problem: 1. concluding an XX/XO sex determination mechanism. The name was coined by another German anatomist. von Waldeyer in 1888. which swells them and spreads the chromosomes . Their behavior in animal (salamander) cells was described by Walther Flemming. at first favoring 46. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. The next stage took place after the development of genetics in the early 20th century. He revised his opinion later from 46 to 48.
It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. For humans. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.1 Staining The study of karyotypes is made possible by staining. the great apes have 48 chromosomes.3. Arresting mitosis in metaphase by a solution of colchicines 4. Usually. 3. a suitable dye.2 Observations Six different characteristics of karyotypes are usually observed and compared: .2 Observations on karyotypes 3. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.  Sometimes observations may be made on non-dividing (interphase) cells.2. reducing the number. 3.2. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Human chromosome 2 was formed by a merger of ancestral chromosomes. Rather interestingly. The sex of an unborn fetus can be determined by observation of interphase cells. is applied after cells have been arrested during cell division by a solution of colchicine. such as Giemsa.
Heterochromatin stains darker than euchromatin. . Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. 4. Humans have one pair fewer chromosomes than the great apes. Differences in the position of centromeres.1. which (when they occur) are small bodies attached to a chromosome by a thin thread. This feature probably reflects different amounts of DNA duplication. faba chromosomes are many times larger. 2. but the genes have been mostly translocated (added) to other chromosomes. type. This is brought about by translocations. as well as other cytogenetic information. 3. indicating tighter packing. permitting its loss without penalty to the organism (the dislocation hypothesis). Differences in number and position of satellites. 6. and mainly consists of genetically inactive repetitive DNA sequences. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Differences in absolute sizes of chromosomes. shape and banding of the chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). Differences in degree and distribution of heterochromatic regions. A full account of a karyotype may therefore include the number. 5. both have six pairs of chromosomes (n=6) yet V.
the same cannot be said for their karyotypes. Normal karyotypes for females contain two X chromosomes and are denoted 46. Any variation from the standard karyotype may lead to developmental abnormalities. Between the sexes 2. There is variation between species in chromosome number. Geographical variation between races 5. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. XX. Between the germ-line and soma (between gametes and the rest of the body) 3. Between members of a population (chromosome polymorphism) 4. males have both an X and a Y chromosome denoted 46. 3. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. XY.3 The human karyotype Most (but not all) species have a standard karyotype. which are highly variable. Mosaics or otherwise abnormal individuals.Variation is often found: 1. and in .
found in some copepods and roundworms such as Ascaris suum. In some cases there is even significant variation within species. 3.. as in many sciarid flies. "We have a very poor understanding of the causes of karyotype evolution. used in conjunction with other phylogenetic data.. which were previously inexplicable. In this process. entire chromosomes are eliminated during development.detailed organization. Chromatin diminution (founding father: Theodor Boveri).1 Changes during development Instead of the usual gene repression. the general significance of karyotype evolution is obscure.. despite their construction from the same macromolecules. In a review. But. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species.. Chromosome elimination. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. In A. . Godfrey and Masters conclude: "In our view. portions of the chromosomes are cast away in particular cells. or other kinds of visible adjustment to the karyotype.3. In some species. despite many careful investigations. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. This variation provides the basis for a range of studies in evolutionary cytology. Although much is known about karyotypes at the descriptive level. it is quite unclear what the general significance might be. some organisms go in for large-scale elimination of heterochromatin. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed.
thus the mammalian female is a mosaic in respect of her X chromosomes. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. all the somatic cell precursors undergo chromatin diminution. Muntiacus muntjak. all telocentric.3.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. The diploid number of the Chinese muntjac.suum. In human females some 15% of somatic cells escape inactivation. The low record is held by the nematode Parascaris univalens. When they looked at the karyotype of the closely related Indian muntjac. 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. The existence of supernumerary or B chromosomes . the high record would be somewhere amongst the ferns. male = 7 chromosomes.. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. "They simply could not believe what they saw.. Muntiacus reevesi. In placental mammals. was found to be 46. 3. which was investigated by Kurt Benirschke and his colleague Doris Wurster. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). 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. Xinactivation... where the haploid n = 1. the inactivation is random as between the two Xs. they were astonished to find it had female = 6.
3.3 Fundamental number The fundamental number. about 70%. due to the presence of five acrocentric chromosome pairs (13. and the other haploid.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. but it has been significant in some groups. horsetails and psilotales) is also common. It is a common arrangement in the Hymenoptera. Polyploidy. It has been of major significance in plant evolution according to Stebbins. Thus. Polyploidy in lower plants (ferns. of a karyotype is the number of visible major chromosomal arms per set of chromosomes.3. 14. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. 21 and 22). FN. 15. occurs mainly in plants. Humans have FN = 82. but in grasses the average is much higher. FN ≤ 2n.Endopolyploidy occurs when in adult differentiated . 3. where there are more than two sets of homologous chromosomes in the cells. and in some other groups. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present.means that chromosome number can vary even within one interbreeding population. and aneuploids are another example. where one sex is diploid. Haplo-diploidy. Polyploidy in animals is much less common. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. though in this case they would not be regarded as normal members of the population.
. the daughter chromosomes separating from each other inside an intact nuclear membrane. it is diverse and complex. The cells do not always contain exact multiples (powers of two). but the nuclei contain more than the original somatic number of chromosomes.tissues the cells have ceased to divide by mitosis. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. In many instances. and serves differentiation and morphogenesis in many ways. Abnormalities in chromosome number usually cause a defect in development. See palaeopolyploidy for the investigation of ancient karyotype duplications. 3.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. 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. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. Down syndrome and Turner syndrome are examples of this. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man.
In about 6.5 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.500 sq mi (17. 6. 4. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. where the gametic (= haploid) numbers form the series x = 3.6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson. the great apes have 24x2 chromosomes whereas humans have 23x2. reducing the number. living from rainforests to . Well-researched examples are the ladybird beetle Chilocorus stigma.Aneuploidy may also occur within a group of closely related species. Human chromosome 2 was formed by a merger of ancestral chromosomes. When this happens. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world.000 km2). that the two chromosome morphs are adapted to different habitats. the European shrew Sorex araneus. 3. 3. and 7. Classic examples in plants are the genus Crepis. and Crocus. 5. some mantids of the genus Ameles. where every number from x = 3 to x = 15 is represented by at least one species.  Closer to home. the chromosome number is variable from one individual to another.
but these are much less frequent. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. it is more likely to have been a group from the same species.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). in the family Drosophilidae. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. the best-studied group of Hawaiian drosophilids.subalpine meadows. The results are clear. Drosophila and Scaptomyza. Chromosome rearrangements. make it possible to see which species are closely related. and skipping of islands. at least into the Cretaceous. 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. the present islands date from 0. Although it would be possible for a single gravid female to colonise an island. There are also cases of colonization back to older islands. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. especially inversions. In a sense. The inversions. . gene arrangements are visible in the banding patterns of each chromosome. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. when plotted in tree form (and independent of all other information). Using K-Ar dating. probably 20 million years ago. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. The polytene banding of the 'picture wing' group. which can be dated to 30 mya. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches.
• • C-banding: Giemsa binds to constitutive heterochromatin. so it stains centromeres.the dark regions tend to be heterochromatic. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. The light regions tend to be euchromatic. 3.7 Depiction of karyotypes 3. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). late-replicating and AT rich. R-banding is the reverse of G-banding (the R stands for "reverse").There are other animals and plants on the Hawaiian archipelago which have undergone similar. • T-banding: visualize telomeres. if less spectacular. early-replicating and GC rich. The pattern of bands is very similar to that seen in G-banding.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.7. . human genome. This method will normally produce 300-400 bands in a normal. adaptive radiations. It yields a series of lightly and darkly stained bands .
In addition.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. Giemsa is specific for the phosphate groups of DNA. Some karyotypes call the short and long arms p and q. Karyotypes are arranged with the short arm of the chromosome on top. and the long arm on the bottom. both chromosomes in a pair will have the same banding pattern. less frequently Quinacrine.7.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. a dye. Cri du chat syndrome involves a deletion on the short arm of . denoting the activity of rRNA genes within the NOR. Each chromosome has a characteristic banding pattern that helps to identify them. 3. is used to stain bands on the chromosomes. For example. Quinacrine binds to the adeninethymine-rich regions. This yields a dark region where the silver is deposited. respectively. often Giemsa (G-banding). In the "classic" (depicted) karyotype.
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.chromosome 5. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. Image processing software then assigns a pseudo color to each spectrally different combination.2. It is written as 46.XX.del(5)(p15.3 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. This method is also known as virtual karyotyping. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.7. Because there are a limited number of spectrally-distinct fluorophores. . 3. 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.5p-.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. which is written as 46. The critical region for this syndrome is deletion of 15. a combinatorial labeling method is used to generate many different colors.2) 3.XX.
CHAPTER 4 .
1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. or structural. are common numerical abnormalities. the most common male chromosomal disease. otherwise known as 47. trisomy 9 and trisomy 16. Numerical abnormalities. in which three copies of a chromosome are present instead of the usual two. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Down syndrome. including . is caused by trisomy of chromosome 21. a common chromosomal disease. Also documented are trisomy 8. inversions.4. Klinefelter syndrome. trisomies. X or 45. although they generally do not survive to birth. X0). • • Patau syndrome is caused by trisomy of chromosome 13. Some disorders arise from loss of just a piece of one chromosome. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. XXY is caused by an extra X chromosome. translocations. Structural abnormalities often arise from errors in homologous recombination. large-scale deletions or duplications. also known as aneuploidy. as in derivative chromosome. often occur as a result of nondisjunction during meiosis in the formation of a gamete. as in the presence of extra or missing chromosomes. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45.
caused by abnormal formation of the larynx. 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. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. a deletion of the paternal genes. A chromosome anomaly. . example of imprinting disorder. There are many types of chromosome anomalies. from the loss of part of the short arm of chromosome 1. from a truncated short arm on chromosome 5. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. 1p36 Deletion syndrome. a deletion of the maternal genes. example of imprinting disorder. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes.• Cri du chat (cry of the cat). Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. The name comes from the babies' distinctive cry. numerical and structural anomalies. They can be organized into two basic groups. one well-documented example is the Philadelphia chromosome. A chromosome anomaly may be detected or confirmed in this manner. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia.
There are two main types of translocations. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy.4. which is caused by partial deletion of the short arm of chromosome 4. resulting in extra genetic material.). also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. 4. Duplications: A portion of the chromosome is duplicated. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.3 Structural abnormalities When the chromosome's structure is altered. In a Robertsonian translocation. • • Translocations: When a portion of one chromosome is transferred to another chromosome. an entire chromosome has . also called the terminal 11q deletion disorder. and Jacobsen syndrome. In a reciprocal translocation. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. Tetrasomy. 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. an X. segments from two different chromosomes have been exchanged. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). etc. rather than two). Known disorders in humans include Wolf-Hirschhorn syndrome.
Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. This can happen with or without loss of genetic material. This is why chromosome studies are often performed on parents when a child is found to have an anomaly.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. therefore the genetic material is inverted. 4. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.in humans these only occur with chromosomes 13. 21 and 22. Chromosome anomalies can be inherited from a parent or be "de novo". as well . other cytogenetic banding techniques. • Inversions: A portion of the chromosome has broken off. It includes routine analysis of G-Banded chromosomes. Therefore.attached to another at the Centromere . can happen after conception. resulting in Mosaicism (where some cells have the anomaly and some do not). 4. • Rings: A portion of a chromosome has broken off and formed a circle or ring. especially the chromosomes. turned upside down and reattached. Some anomalies. and are therefore initially not inherited. however. 14. the anomaly is present in every cell of the body.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. They often lead to an increased tendency to develop certain types of malignancies. 15.
Pre-treating cells in a hypotonic solution. The next stage took place after the development of genetics in the early 20th century. Using cells in culture 2. von Waldeyer in 1888. in contrast to their genic contents. which swells them and spreads the chromosomes . 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. these results were quite remarkable.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). the discoverer of mitosis. Their behavior in animal (salamander) cells was described by Walther Flemming. at first favoring 46. and he correctly insisted on man having an XX/XY system. concluding an XX/XO sex determination mechanism. New techniques were needed to definitively solve the problem: 1. in 1882. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. The name was coined by another German anatomist. Considering their techniques. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. He revised his opinion later from 46 to 48. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. 4.
reducing the number.2 Natural populations of Drosophila In the 1930s. Arresting mitosis in metaphase by a solution of colchicine 4. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 4. Using Painter's technique they studied the polytene . a find which eventually led to her Nobel Prize in 1983. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). 4.3. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. McClintock discovered transposons. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. the great apes have 48 chromosomes.6. Rather interestingly.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. In 1931.6.6 Applications in biology 4. During her cytogenetic work. persimilis from wild populations in California and neighboring states. Human chromosome 2 was formed by a merger of ancestral chromosomes.
7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. which enabled feeding. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. In some congenital disorders.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. such as Down's syndrome. This had the benefit of eliminating migration as a possible explanation of the results. It was found that the various chromosome types do not fluctuate at random. as with most polymorphisms. breeding and sampling whilst preventing escape. Evidence rapidly accumulated to show that natural selection was responsible. as they would if selectively neutral. discoveries were quickly made related to aberrant chromosomes or chromosome number. Dobzhansky bred populations in population cages. Abnormalities arising from nondisjunction events can cause cells with aneuploidy (additions or deletions of entire chromosomes) in one of the parents or in the fetus. 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. Using a method invented by L'Heretier and Teissier. but adjust to certain frequencies at which they become stabilised. 4. In 1959. . 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. Down syndrome is also referred to as trisomy 21.
is used today as a diagnostic for CML. XYY. Not all genes on the X Chromosome are inactivated. an additional X chromosome in a male. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. Identification of the Philadelphia chromosome by cytogenetics. and XXXX. which is required in normal females to compensate for having two copies of the chromosome. Pennsylvania.as both scientists were doing their research in Philadelphia. This abnormal chromosome was dubbed the Philadelphia chromosome . which is why there is a phenotypic effect seen in individuals with extra X chromosomes. In 1960. An individual with only one sex chromosome (the X) has Turner syndrome. . resulting in 47 total chromosomes. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). with the development of more advanced techniques. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. has Klinefelter's Syndrome. Many other sex chromosome combinations are compatible with live birth including XXX. Thirteen years later. in addition to other tests.Other numerical abnormalities discovered include sex chromosome abnormalities.
Deletion syndromes such as DiGeorge syndrome. 4. and elongation techniques for all culture types that allow for higher resolution banding. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material.FIG Advent of banding techniques In the late 1960s.8 Beginnings of molecular cytogenetics . Caspersson developed banding techniques which differentially stain chromosomes. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 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. 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.
In the 1980s. 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.1 Karyotyping . Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. CHAPTER 5 Techniques 5. movement was now made in using fluorescent labeled probes. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. While radioisotope-labeled probes had been hybridized with DNA since 1969. advances were made in molecular cytogenetics.
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.
5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •
bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations
5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,
including signal and image processing. you can solve technical computing problems faster than with traditional programming languages. CGH and Single nucleotide polymorphism-arrays. such as comparative genomic hybridization arrays. and numerical computation. financial modeling and analysis. test and measurement. data visualization.generally between 200 and 1000 cells are counted and scored. and FORTRAN. You can use MATLAB in a wide range of applications. communications. Using MATLAB. control design. data analysis. and computational biology. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. C++. . such as C. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. For congenital problems usually 20 metaphase cells are scored.
These effects must be compensated to improve the results of the pairing algorithm. It enables fast development and execution. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. In many cases. . specifying data types. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. and allocating memory. such as declaring variables. MATLAB eliminates the need for ‘for’ loops. 6. The image processing step is composed of the following operations.MATLAB provides a number of features for documenting and sharing your work. As a result. With the MATLAB language. one line of MATLAB code can often replace several lines of C or C++ code. You can integrate your MATLAB code with other languages and applications.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. 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. and distribute your MATLAB algorithms and applications.
4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. To compensate for this inhomogeneity. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis.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). 2) Geometrical compensation—The geometric compensation. geometrical and dimensional differences must be removed. the spatially scaled images are histogram equalized. or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. To compare chromosomes from a band pattern point of view. 6. 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.
the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations.3) Edge detection 4) Two dimensional convolutions.2.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.I. For example. as is appropriate. If you attempt to filter the indexed image.I). 6. you must first convert it to true color format. In addition to these image type conversion functions. RGB = cat (3.I. if you want to filter a color image that is stored as an indexed image. The resulting true color image has identical matrices for the red. For example. there are other functions that return a different image type as part of the operation they perform. and blue planes. MATLAB simply applies the filter to the indices in the indexed image matrix. . When you apply the filter to the true color image. and the results might not be meaningful. For example. You can perform certain conversions just using MATLAB syntax. listed in the following table. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. so the image displays as shades of gray. green. MATLAB filters the intensity values in the image.
2.2. caused by external disturbance. We may use edges to measure the size of objects in an image. and hence the type of noise on the image. we may expect errors to occur in the image signal. Usually we know what type of errors to expect. 6. The general Matlab command for finding edges is edge(image. to recognize or classify objects. ) Where the parameters available depend on the method used 6.parameters. If an image is being sent electronically from one place to another.6. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. . If one of these matrices describes a two-dimensional finite impulse response .B) computes the two-dimensional convolution of matrices A and B.'method'. via satellite or wireless transmission.4 Denoising We may define noise to be any degradation in the image signal. .3 Two dimensional convolutions C = conv2(A. There is a large number of edge finding algorithms in existence.5 Edge detection Edges contain some of the most useful information in an image. Cleaning an image corrupted by noise is thus an important area of image restoration. to isolate particular objects from their background. hence we can choose the most appropriate method for reducing the effects. and we shall look at some of the more straightforward of them. or through networked cable.
if the size of then the size of C is [ma+mb-1. nb]+1)/2).[3 3]).nb]. The size of matrices..A).0. edge(im1.(FIR) filter. . the other matrix is filtered in two dimensions... this case is the same as C = conv2(hcol*hrow.'shape') subsection of the two-dimensional convolution. imedfilt2(im1.7).'sobel'). If hcol is a column vector and hrow is a row vector.bmp'). rgb2gray im2bw(im. 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. That is.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.na] and the size of B is [mb. minus one. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.hrow.na+nb-1]. C = conv2(.
mx=max(max(L)). rc = [r c]. [r. for i=1:m for j=1:n if L(i. flag=0.y1)=255.j)==L_number(k) flag=1.[imx.n]=size(L). Msk conv2(double(BW). for i=1:sx x1=rc(i. nzeros(imx. n1(x1.j)~=0 for k=1:mx if L(i.imy). MODULE 2 clc [m. . bwlabel(B.c] = find(L==22).double(msk)).8).2). y1=rc(i. [sx sy]=size(rc). L_number=zeros(mx.imy]=size(BW). Index=1.1).1).
for i=1:sx x1=rc(i.38. Index=Index+1.22.28. end.18.104.22.168.22.214.171.124.29.imshow(n1.c] = find(L==L_number((Test_number(x)))).27. Test_number=[3.54.40. for x=1:46 [r.126.96.36.199.9.43. [sx sy]=size(rc).19.62.2). end %h=figure.65.33.30.j).188.8.131.52. end end L_number.). 36. rc = [r c].66]. n1(x1. end flag=0. .60.48.y1)=255.11.39. y1=rc(i.24.4. n1=zeros(imx.imy).184.108.40.206.end end if flag~=1 L_number(Index)=L(i.42.1).50.6.
y)==1 Circumference_sum=Circumference_sum+1.'.'skel'. Circumference_sum=0.end Circumference=zeros(46. f=imcomplement(f).bmp')). BW=double(BW). s=bwmorph(skel.1. s1=bwmorph(s. Arm_length_sum=0.5*graythresh(skel)).'canny').1).1). skel=im2bw(skel. skel=im2double(f).1). for x=1:m for y=1:n if BW1(x.8). BW1=edge(BW. Area=zeros(46.Inf).'spur'. BW=im2bw(f). . for i=1:46 f=imread(strcat(num2str(i). end end end Circumference(i)=Circumference_sum. [m n]=size(BW1). Arm_length=zeros(46.
for x=1:m for y=1:n if s1(x. Area_sum=0. end end end Arm_length(i)=Arm_length_sum.y)==1 Arm_length_sum=Arm_length_sum+1. for x=1:m for y=1:n if BW(x.y)==1 Area_sum=Area_sum+1.[m n]=size(s1). BW=im2bw(f). end Circumference. Arm_length. . [m n]=size(BW). end end end Area(i)=Area_sum.
2)=i.1)=i.Area. Pair=zeros(46.1)=46. Pair(i. Pair(46.2)==46 Pair(46. end end end for i=1:45 if Pair(i.2)=i+1. 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)).1)=i. end end Pair. Pair(i. . 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). Pair(i.2)=j.
delete(figure_flag)=Pair(i.bmp')). imshow(f1).delete=zeros(46.bmp')).1).figure_flag).1)==delete(j) flag=1.1)). end f2=imread(strcat(num2str(Pair(i.2)). end flag=0.'. .'.2. end end if flag~=1 if figure_flag~=47 subplot(23. figure_flag=figure_flag+1. for i=1:46 for j=1:46 if Pair(i. end f1=imread(strcat(num2str(Pair(i.2. figure_flag=figure_flag+1. if figure_flag~=47 subplot(23. figure_flag=1.2). imshow(f2). flag=0.figure_flag).
The proposed algorithm is based on the traditional features extracted from the karyogram. 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. and Philadelphia. 2) feature extraction from the processed images . based on the MI. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. such as. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. plus a new one.end CONCLUTION In this paper. dimensions and banding profiles. in the scope of karyotyping process used in cytogentic analysis. The images of these chromosomes present less quality and level of detail than the ones usually used in traditional genetic analysis using datasets such as Edinburgh. Copenhagen. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure.
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 . Tests using 19 karyograms based on bone marrow cells. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. 4) pairing. 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. and to normalize their dimensions. The features extracted from the processed images discriminate each pair with respect to their size. are processed in order to compensate for geometrical and intensity distortions. and finally. achieves a 70. Here.10% mean classification rate. and band pattern.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. 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). extracted from the unordered karyogram.working within an 8-D feature space. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. shape. shape and band pattern. The training process consists in the estimation of each vector of coefficient . 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.characterizing the size. This normalization is needed to make it possible the band pattern comparison between chromosomes. the romosome images. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. from the chromosomes in the training set.
The results presented in this paper are promising. such as Edinburgh. REFERENCES . centromere position. whose images are of significantly higher quality. In fact. In addition. a new chromosome dataset with 9200 chromosomes from bone marrow cells. Copenhagen. and from which it is possible to extract additional features. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. or Philadelphia.performance of the classifier. amean classification rate larger than 93% was obtained in all experiments.g. Executing the algorithm on a higher quality dataset. e. This dataset was made publicly available . presenting a uniform level of condensation. 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. Using 27 karyograms andworking with a limited number of classes (≤ 8). a 76. despite the low quality of this type of chromosomes..10% classification ratewas obtained. called LK1 .
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