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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
These small circular genomes . divided. a large body of work uses the term chromosome regardless of chromatin content. or it may unexpectedly evadeapoptosis leading to the progression of cancer. In prokaryotes. In practice "chromosome" is a rather loosely defined term. which is tightly coiled in on itself. circular DNA molecules called plasmids. sometimes accompanied by one or more smaller. Chromosomes may exist as either duplicated or unduplicated. the cell may undergo mitotic catastrophe and die. Chromosomal recombination plays a vital role in genetic diversity. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. If these structures are manipulated incorrectly. DNA is usually arranged as a circle. The structure of chromosomes and chromatin varies through the cell cycle. In eukaryotes. In prokaryotes and viruses. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. Also. through processes known as chromosomal instability and translocation. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). 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.defined nuclei) have smaller circular chromosomes. although there are many exceptions to this rule. cells may contain more than one type of chromosome. the term genophore is more appropriate when no chromatin is present. This allows the very long DNA molecules to fit into the cell nucleus. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. for example. However. Unduplicated chromosomes are single linear strands.
+21. An example of aneuploidy is trisomy 21. reflecting their bacterial origins. members of the same species have the same numbers of types of chromosomes (with the exception of sex chromosomes in males and females if sex is chromosomally determined).g. If the mutation involves only one or a few chromosomes in the genome (e. 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. Such individuals are called euploid and have the wild-type chromosome complement for the species. . The individual would have Down Syndrome and his/her karyotype would be written 47.+21.XX. a extra copy of human chromosome 21). copies of chromosome 21.XY or 47.3 MUTATIONS IN CHROMOSOME NUMBER Normally. 1. 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. XX (female) or 46 XY (male). in which an individual has 3. rather than 2.are also found in mitochondria and chloroplasts. the individual carrying the mutation is said to be aneuploid.
Fig 1. q-g "grande"). .4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). Once the cells have divided.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. along with special proteins. longer-lasting attachment in this region. In spite of their appearance. The shorter arms are called p arms (from the French petit. chromosomes are structurally highly condensed. The microtubules then pull the chromatids apart toward the centrosomes. This is the only natural context in which individual chromosomes are visible with an optical microscope. small) and the longer arms are called q arms (q follows p in the Latin alphabet. 1. and they form the classic four arm structure. A special DNA base sequence in the region of the kinetochores provides. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. This compact form makes the individual chromosomes visible. one of which is present on each sister chromatid. the chromatids are uncoiled and DNA can again be transcribed. During mitosis. which enables these giant DNA structures to be contained within a cell nucleus. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. 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. a pair of sister chromatids attached to each other at the centromere. the chromatin strands become more and more condensed.
bone marrow constitutes 4% of the total body mass of humans. In humans. bone marrow in large bones produces new blood cells. cytoplasm. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. On average.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.1.7 lbs). bone marrow accounts for approximately 2.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. producing the lymphocytes that support the body's immune system CHAPTER 2 2. which divides the nuclei. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. which use the bone marrow vasculature as a conduit to the body's systemic circulation.  Bone marrow is also a key component of the lymphatic system. in adults weighing 65 kg (143 lbs). in two separate nuclei.6 kg (5. . It is generally followed immediately by cytokinesis.
which lack a nucleus. The process of mitosis is fast and highly complex. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. forming single cells with multiple nuclei. This accounts for approximately 10% of the cell cycle. cytokinesis and mitosis may occur independently. prophase. anaphase and telophase. Mitosis occurs only in eukaryotic cells and the process varies in different species. The cell then divides in cytokinesis. . but is found in various different groups. to produce two identical daughter cells which are still diploid cells. For example. Even in animals. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. Because cytokinesis usually occurs in conjunction with mitosis.genetically identical to each other and to their parent cell. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. for instance during certain stages of fruit fly embryonic development. animals undergo an "open" mitosis. Prokaryotic cells. These stages are interphase. prometaphase. divide by a process called binary fission. This occurs most notably among the fungi and slime moulds. metaphase. However. where chromosomes divide within an intact cell nucleus. where the nuclear envelope breaks down before the chromosomes separate. "mitosis" is often used interchangeably with "mitotic phase". there are many cells where mitosis and cytokinesis occur separately.
This occurs during the S phase of interphase. . 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. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. Each chromosome now has an identical copy of itself. Because each resultant daughter cell should be genetically identical to the parent cell. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. the parent cell must make a copy of each chromosome before mitosis. and together the two are called sister chromatids. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs.
corresponding sister chromosomes are pulled toward opposite ends. each with a replica of the original genome. In animal cells. 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). A new nuclear envelope forms around the separated sister chromosomes. As mitosis completes.the cell begins cytokinesis. In plant cells. so they are renamed to sister chromosomes. the daughter cells will construct a new dividing cell wall between each other. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. As a matter of convention. separating the two developing nuclei. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. the parent cell will be split in half. because of the non-involvement of nuclear dynamics and lack of linear chromosomes.In most eukaryotes. the process of binary fission is very much different from the process of mitosis. Prokaryotic cells undergo a process similar to mitosis called binary fission. The chromosomes align themselves in a line spanning the cell. . As the cell elongates. each sister chromatid is now considered a chromosome. pulling apart the sister chromatids of each chromosome. giving rise to two daughter cells. However. Eventually.
Thus. the cell grows by producing proteins and cytoplasmic organelles. In highly vacuolated plant cells. a cell grows (G1).2. All these phases in the interphase are highly regulated. chromosomes are replicated only during the S phase. Interphase is divided into three phases: G1 (first gap). However. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . grows more and prepares for mitosis (G 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.1 Preprophase In plant cells only. prophase is preceded by a pre-prophase stage.2. where the cell prepares itself for cell division. S (synthesis). and G2 (second gap). continues to grow as it duplicates its chromosomes (S). and finally it divides (M) before restarting the cycle. the nucleus has to migrate into the center of the cell before mitosis can begin. This is achieved through the formation of a phragmosome. mainly via proteins. 2. It alternates with the much longer interphase. 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.
The cells of higher plants (such as the flowering plants) lack centrioles. . after the nuclear membrane breaks down. aligned at the metaphase plate. and microtubules have invaded the nuclear Prophase space.division. 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. 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. Cytokinesis has already begun. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. The chromosomes have chromatin has condensed. the pinched area is known as the cleavage furrow. This band marks the position where the cell will eventually divide. In addition to phragmosome formation. instead. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. degraded.
The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. which are made of a pair of centrioles found in most eukaryotic animal cells. giving a pair of centrosomes. which is replicated by the cell with the help of the nucleus before a new mitosis begins. chromatin condenses together into a highly ordered structure called a chromosome. the replicated chromosomes have two sister chromatids. bound together at the centromere by the cohesin protein complex. Chromosomes are typically visible at high magnification through a light microscope. the genetic material in the nucleus is in a loosely bundled coil called chromatin. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. Although centrioles help organize microtubule assembly. Close to the nucleus are structures called centrosomes. At the onset of prophase. The centrosome is the coordinating center for the cell's microtubules. they are not essential for the . Since the genetic material has already been duplicated earlier in S phase. A cell inherits a single centrosome at cell division.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally.
This motor activity. 2. and centrosomes are not always used in mitosis. Each chromosome forms two kinetochores at the centromere. on an average 20 ). it is known that it contains some form of molecular motor.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. When the spindle grows to sufficient length. kinetochore microtubules begin searching for kinetochores to attach to. This is called open mitosis. provides the pulling force necessary to later separate the chromosome's two chromatids. since they are absent from plants. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. When a microtubule connects with the kinetochore. the motor activates. . undergo a variation called closed mitosis where the spindle forms inside the nucleus. and it occurs in most multicellular organisms. Although the kinetochore structure and function are not fully understood. coupled with polymerisation and depolymerisation of microtubules. Fungi and some protists. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. one attached at each chromatid.2. or its microtubules are able to penetrate an intact nuclear envelope.formation of the spindle. such as algae or trichomonads. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Prometaphase is sometimes considered part of prophase. using energy from ATP to "crawl" up the tube toward the originating centrosome.
3 Metaphase A cell in late metaphase. an imaginary line that is equidistant from the two centrosome poles. All chromosomes (blue) but one have arrived at the metaphase plate. analogous to a tug-of-war between people of equal strength. in some sense.In the fishing pole analogy. convene along the metaphase plate or equatorial plane. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. . The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". In certain types of cells. Metaphase comes from the Greek meaning "after. 2. only roughly lining up along the midline. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. the chromosomes come under longitudinal tension from the two ends of the cell. the kinetochore would be the "hook" that catches a sister chromatid or "fish". The centromeres of the chromosomes." Microtubules find and attach to kinetochores in prometaphase. As a result. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.
” “against.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). Next. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. Two events then occur: first. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. allowing them to separate. The force that causes the centrosomes to move towards the ends of the cell is still unknown. At the end of anaphase. the cell proceeds to anaphase (from the Greek meaning “up. Early anaphase is usually defined as the separation of the sister chromatids. 2. These sister chromatids. which have now become distinct sister chromosomes. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. These two stages are sometimes called early and late anaphase. the proteins that bind sister chromatids together are cleaved.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate.” or “re-”). 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. the nonkinetochore microtubules elongate. . The signal creates the mitotic spindle checkpoint.” “back.
2. cell . unfold back into chromatin. using fragments of the parent cell's nuclear membrane. 2. Corresponding sister chromosomes attach at opposite ends of the cell. Mitosis is complete.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. but rather a separate process. the nonkinetochore microtubules continue to lengthen. but cell division is not yet complete. cytokinesis is a separate process that begins at the same time as telophase. necessary for completing cell division. In both animal and plant cells. It "cleans up" the after effects of mitosis. In animal cells. A new nuclear envelope. 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. At telophase. forms around each set of separated sister chromosomes. elongating the cell even more. Both sets of chromosomes. now surrounded by new nuclei. Cytokinesis is technically not even a phase of mitosis. pinching off the separated nuclei.5 Cytokinesis Cilliate undergoing cytokinesis. however.
1Significance Mitosis is important for the maintenance of the chromosomal set. skin and digestive tract. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. Following are the occasions in the lives of organism where mitosis happens: 2.3 Cell replacement In some parts of body.2 Development and growth The number of cells within an organism increases by mitosis.. which move along microtubules to the middle of the cell. zygote and also the basis of the growth of a multicellular body. This is the basis of the development of a multicellular body from a single cell i. cells are constantly sloughed off and replaced by new ones. New cells are formed by mitosis and so are exact copies of the cells being replaced.division is also driven by vesicles derived from the Golgi apparatus. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. The phragmoplast is a microtubule structure typical for higher plants.5.e. 2.5. 2. whereas some green algae use a phycoplast microtubule array during cytokinesis. Each daughter cell has a complete copy of the genome of its parent cell.5.g. separating the two nuclei. The end of cytokinesis marks the end of the M-phase. e. 2. Similarly.4 Regeneration .
a chromosome may fail to separate during anaphase.Some organisms can regenerate their parts of bodies. 2.7 Consequences of errors Although errors in mitosis are rare. 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. For example. the process may go wrong. The production of new cells is achieved by mitosis. especially during early cellular divisions in the zygote. the hydra reproduces asexually by budding. they fail to complete cell division and retain both nuclei in one cell. a condition often associated with cancer. These cells are considered aneuploid. resulting in binucleated cells.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. The same division happens during asexual reproduction or vegetative propagation in plants. Occasionally when cells experience nondisjunction. 2. a condition known as trisomy. sea star regenerates its lost arm through mitosis.5. . Mitosis continues in the cells of bud and it grows into a new individual. a condition known as monosomy. and the latter cell having only one chromosome (the homologous chromosome). The cells at the surface of hydra undergo mitosis and form a mass called bud.5. For example. In non-disjunction. One daughter cell will receive both sister chromosomes and the other will receive none.
causing inversion. Such tumours can send cancer cells to other parts in body where new tumours may form. Now what happens is that cell abnormally continue to divide at a single place. it may be treated erroneously as a separate chromosome. its organelles disintegrate and reform in a matter of hours. non-homologous chromosome. Errors in the control of mitosis may cause cancer. causing translocation. Benign tumours are not harmful as soon as they are not moving. It results in abnormal cell growth. This phenomenon is called metastasis or spreading of disease. . It may reattach to the original chromosome.Mitosis is a demanding process for the cell. The fragment may incorrectly reattach to another. sometimes mutuations occur in such genes and cells continue to divide. It results in the synthesis of execessive tissue growths. but in reverse orientation. As long as these tumours remain in their original location they are called benign tumours. When tissues more than the requirement are synthesized in a single organ. Occasionally. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. chromosomes may become damaged. An arm of the chromosome may be broken and the fragment lost. Or. All cells have genes that control the timing and number of mitosis. it results in the formation of Tumors. which goes through dramatic changes in ultrastructure. The effect of these genetic abnormalities depends on the specific nature of the error. and chromosomes are jostled constantly by probing microtubules. causing deletion. As soon as they start to move and invade other cells there are said to be malignant tumours. causing chromosomal duplication.
2. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. from the ancient Greek(between) and (stage). This process may also be referred to as endoreduplication and the cells as endoploid. analogous to a tug of war between equally strong people. 2. only roughly lining up along the middleline. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. align in the middle of the cell before being separated into each of the two daughter cells. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. 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. In certain types of cells. an imaginary line that is equidistant from the two centrosome poles.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. carrying genetic information. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Metaphase accounts for approximately 4% of the cell cycle's duration. An example of a cell that goes through endomitosis is the megakaryocyte. Preceded by events in prometaphase and followed by anaphase.7 Metaphase Metaphase. Early events of metaphase can .
which makes them most suitable for visual analysis. often with Giemsa (G banding) or Quinacrine. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. Chromosomes are condensed(Thickened) and highly coiled in metaphase. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Staining of the slides. when every kinetochore is properly attached to a bundle of microtubules. Such a signal creates the mitotic spindle checkpoint. 2. and separase.coincide with the later events of prometaphase. For classical cytogenetic analyses. produces a pattern of in total up to several hundred bands. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. does the cell enter anaphase. 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).8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. securin. 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. Normal metaphase spreads are used in . Metaphase chromosomes make the classical picture of chromosomes (karyotype). cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. Only after all chromosomes have become aligned at the metaphase plate. This would be accomplished by regulation of the anaphase-promoting complex.
which may lead to chimeric oncogenes. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. for example. losses of chromosomal segments or translocations. . Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. such as bcr-abl in chronic myelogenous leukemia.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments.
The study of whole sets of chromosomes is sometimes known as karyology. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). and what they look like under a light microscope. the position of the centromeres.  The preparation and study of karyotypes is part of cytogenetics. banding pattern. such as. autosomal chromosomes are present in two copies. and any other physical characteristics. . to study chromosomal aberrations. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. or an individual organism. Karyotypes can be used for many purposes. or may not. and the results may be used in evolutionary biology and medicine. Karyotypes describe the number of chromosomes. Attention is paid to their length.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. Thus. The term is also used for the complete set of chromosomes in a species. taxonomic relationships. ordered by size and position of centromere for chromosomes of the same size. cellular function. Karyogram of human male using Giemsa staining. and to gather information about past evolutionary events. in normal diploid organisms. There may. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. in humans 2n = 46. So. The study of karyotypes is important for cell biology and genetics. any differences between the sex chromosomes. be sex chromosomes.
which swells them and spreads the chromosomes . and he correctly insisted on humans having an XX/XY system. in 1882. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. in contrast to their genic contents. New techniques were needed to definitively solve the problem: 1. Pretreating cells in a hypotonic solution. The subsequent history of the concept can be followed in the works of Darlington and White. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Using cells in culture 2.3. The next stage took place after the development of genetics in the early 20th century. Considering their techniques. the discoverer of mitosis. at first favoring 46. The name was coined by another German anatomist. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Their behavior in animal (salamander) cells was described by Walther Flemming. von Waldeyer in 1888. 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 1912.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. these results were quite remarkable. He revised his opinion later from 46 to 48. concluding an XX/XO sex determination mechanism.
3. reducing the number.2. such as Giemsa. the great apes have 48 chromosomes. Rather interestingly. For humans. is applied after cells have been arrested during cell division by a solution of colchicine. Arresting mitosis in metaphase by a solution of colchicines 4.  Sometimes observations may be made on non-dividing (interphase) cells. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 3. Usually. a suitable dye. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.2.2 Observations on karyotypes 3.3. The sex of an unborn fetus can be determined by observation of interphase cells.1 Staining The study of karyotypes is made possible by staining. Human chromosome 2 was formed by a merger of ancestral chromosomes. 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: .
Heterochromatin stains darker than euchromatin. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. This feature probably reflects different amounts of DNA duplication. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). shape and banding of the chromosomes. Differences in the position of centromeres. indicating tighter packing. 3. both have six pairs of chromosomes (n=6) yet V. as well as other cytogenetic information. 6. type. permitting its loss without penalty to the organism (the dislocation hypothesis). A full account of a karyotype may therefore include the number. Humans have one pair fewer chromosomes than the great apes. Differences in absolute sizes of chromosomes. Differences in number and position of satellites. but the genes have been mostly translocated (added) to other chromosomes. This is brought about by translocations. which (when they occur) are small bodies attached to a chromosome by a thin thread. .1. faba chromosomes are many times larger. Differences in degree and distribution of heterochromatic regions. 5. 2. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. and mainly consists of genetically inactive repetitive DNA sequences. 4.
Between the germ-line and soma (between gametes and the rest of the body) 3. 3. Between the sexes 2. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. There is variation between species in chromosome number. the same cannot be said for their karyotypes. and in . Geographical variation between races 5.Variation is often found: 1. Normal karyotypes for females contain two X chromosomes and are denoted 46. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Any variation from the standard karyotype may lead to developmental abnormalities. XX. which are highly variable. males have both an X and a Y chromosome denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. Mosaics or otherwise abnormal individuals. Between members of a population (chromosome polymorphism) 4. XY.
But.1 Changes during development Instead of the usual gene repression. or other kinds of visible adjustment to the karyotype. Chromatin diminution (founding father: Theodor Boveri). it is quite unclear what the general significance might be. "We have a very poor understanding of the causes of karyotype evolution. used in conjunction with other phylogenetic data. despite many careful investigations. the general significance of karyotype evolution is obscure. some organisms go in for large-scale elimination of heterochromatin. In some cases there is even significant variation within species. . This variation provides the basis for a range of studies in evolutionary cytology. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. entire chromosomes are eliminated during development. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. which were previously inexplicable.. as in many sciarid flies. In some species. 3. In a review. found in some copepods and roundworms such as Ascaris suum. In this process. Chromosome elimination. In A... Although much is known about karyotypes at the descriptive level..detailed organization.3. despite their construction from the same macromolecules. Godfrey and Masters conclude: "In our view. portions of the chromosomes are cast away in particular cells.
. the inactivation is random as between the two Xs. The low record is held by the nematode Parascaris univalens.suum. 3.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. In marsupials it is always the paternal X which is inactivated. all the somatic cell precursors undergo chromatin diminution.3. The diploid number of the Chinese muntjac.. male = 7 chromosomes. Muntiacus muntjak. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. "They simply could not believe what they saw. Xinactivation. In placental mammals. where the haploid n = 1. Muntiacus reevesi. In human females some 15% of somatic cells escape inactivation. they were astonished to find it had female = 6. thus the mammalian female is a mosaic in respect of her X chromosomes. The existence of supernumerary or B chromosomes . with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. all telocentric. 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. was found to be 46. 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.. which was investigated by Kurt Benirschke and his colleague Doris Wurster. When they looked at the karyotype of the closely related Indian muntjac. the high record would be somewhere amongst the ferns.
Polyploidy in lower plants (ferns. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. Polyploidy in animals is much less common. Haplo-diploidy. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. Humans have FN = 82. FN ≤ 2n. 21 and 22).3. where one sex is diploid.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. horsetails and psilotales) is also common. but in grasses the average is much higher. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid.Endopolyploidy occurs when in adult differentiated . 14. 3. 15. 3. Polyploidy. though in this case they would not be regarded as normal members of the population. occurs mainly in plants. It is a common arrangement in the Hymenoptera. due to the presence of five acrocentric chromosome pairs (13. and the other haploid. and in some other groups. about 70%. It has been of major significance in plant evolution according to Stebbins. FN. and aneuploids are another example. Thus.means that chromosome number can vary even within one interbreeding population.3 Fundamental number The fundamental number. where there are more than two sets of homologous chromosomes in the cells. but it has been significant in some groups.
the daughter chromosomes separating from each other inside an intact nuclear membrane. Down syndrome and Turner syndrome are examples of this. Abnormalities in chromosome number usually cause a defect in development. . In many instances. 3. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). The cells do not always contain exact multiples (powers of two).tissues the cells have ceased to divide by mitosis. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. and serves differentiation and morphogenesis in many ways.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. See palaeopolyploidy for the investigation of ancient karyotype duplications. 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. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. but the nuclei contain more than the original somatic number of chromosomes. it is diverse and complex. 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.
500 sq mi (17. 3. the great apes have 24x2 chromosomes whereas humans have 23x2. 6. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.000 km2). Classic examples in plants are the genus Crepis. and 7. and Crocus. 4.  Closer to home. that the two chromosome morphs are adapted to different habitats. 5. where every number from x = 3 to x = 15 is represented by at least one species. In about 6. 3. Well-researched examples are the ladybird beetle Chilocorus stigma. the European shrew Sorex araneus. the chromosome number is variable from one individual to another. where the gametic (= haploid) numbers form the series x = 3.Aneuploidy may also occur within a group of closely related species. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. When this happens. Human chromosome 2 was formed by a merger of ancestral chromosomes. some mantids of the genus Ameles.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. living from rainforests to . reducing the number.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.
Chromosome rearrangements.subalpine meadows. Although it would be possible for a single gravid female to colonise an island. the present islands date from 0. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. but these are much less frequent. at least into the Cretaceous. show a clear "flow" of species from older to newer islands. make it possible to see which species are closely related. in the family Drosophilidae. probably 20 million years ago. The results are clear. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. The inversions. it is more likely to have been a group from the same species. There are also cases of colonization back to older islands. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. . especially inversions. Using K-Ar dating. The polytene banding of the 'picture wing' group. Drosophila and Scaptomyza. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. and skipping of islands. which can be dated to 30 mya.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). when plotted in tree form (and independent of all other information). In a sense. gene arrangements are visible in the banding patterns of each chromosome. the best-studied group of Hawaiian drosophilids.
7.1 Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes: • G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. • T-banding: visualize telomeres. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. 3. The light regions tend to be euchromatic. This method will normally produce 300-400 bands in a normal.7 Depiction of karyotypes 3. It yields a series of lightly and darkly stained bands .There are other animals and plants on the Hawaiian archipelago which have undergone similar. so it stains centromeres. early-replicating and GC rich. R-banding is the reverse of G-banding (the R stands for "reverse"). . The pattern of bands is very similar to that seen in G-banding. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).the dark regions tend to be heterochromatic. • • C-banding: Giemsa binds to constitutive heterochromatin. adaptive radiations. if less spectacular. late-replicating and AT rich. human genome.
respectively. Giemsa is specific for the phosphate groups of DNA. a dye. In the "classic" (depicted) karyotype. Quinacrine binds to the adeninethymine-rich regions. In addition. often Giemsa (G-banding).2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. and the long arm on the bottom. less frequently Quinacrine. 3. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. For example. denoting the activity of rRNA genes within the NOR.7. both chromosomes in a pair will have the same banding pattern. This yields a dark region where the silver is deposited. Karyotypes are arranged with the short arm of the chromosome on top. Cri du chat syndrome involves a deletion on the short arm of . Some karyotypes call the short and long arms p and q. Each chromosome has a characteristic banding pattern that helps to identify them. is used to stain bands on the chromosomes.
This method is also known as virtual karyotyping.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX. The critical region for this syndrome is deletion of 15.2.3 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. a combinatorial labeling method is used to generate many different colors. Image processing software then assigns a pseudo color to each spectrally different combination. 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.5p-. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. . 3. Because there are a limited number of spectrally-distinct fluorophores. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.XX. It is written as 46. allowing the visualization of the individually colored chromosomes.7.2) 3.chromosome 5. which is written as 46.del(5)(p15. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.
CHAPTER 4 .
otherwise known as 47.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. • • Patau syndrome is caused by trisomy of chromosome 13. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. translocations. Klinefelter syndrome. as in the presence of extra or missing chromosomes. including . Down syndrome. although they generally do not survive to birth.4. the most common male chromosomal disease. a common chromosomal disease. also known as aneuploidy. trisomy 9 and trisomy 16. often occur as a result of nondisjunction during meiosis in the formation of a gamete. X or 45. trisomies. are common numerical abnormalities. large-scale deletions or duplications. inversions. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. in which three copies of a chromosome are present instead of the usual two. or structural. Some disorders arise from loss of just a piece of one chromosome. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. Also documented are trisomy 8. Structural abnormalities often arise from errors in homologous recombination. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. as in derivative chromosome. is caused by trisomy of chromosome 21. X0). Numerical abnormalities. XXY is caused by an extra X chromosome.
from a truncated short arm on chromosome 5. They can be organized into two basic groups. from the loss of part of the short arm of chromosome 1. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. example of imprinting disorder.• Cri du chat (cry of the cat). 1p36 Deletion syndrome. numerical and structural anomalies. A chromosome anomaly. example of imprinting disorder. caused by abnormal formation of the larynx. There are many types of chromosome anomalies. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. one well-documented example is the Philadelphia chromosome. a deletion of the maternal genes. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. . abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. 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. The name comes from the babies' distinctive cry. a deletion of the paternal genes. A chromosome anomaly may be detected or confirmed in this manner.
segments from two different chromosomes have been exchanged. an entire chromosome has . Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. 4. In a Robertsonian translocation.). • • Translocations: When a portion of one chromosome is transferred to another chromosome. resulting in extra genetic material. etc. 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. also called the terminal 11q deletion disorder. Duplications: A portion of the chromosome is duplicated. In a reciprocal translocation. which is caused by partial deletion of the short arm of chromosome 4. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. Known disorders in humans include Wolf-Hirschhorn syndrome. rather than two). and Jacobsen syndrome. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy.3 Structural abnormalities When the chromosome's structure is altered.4.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). There are two main types of translocations. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. an X. Tetrasomy.
4. other cytogenetic banding techniques. 4. This can happen with or without loss of genetic material. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. They often lead to an increased tendency to develop certain types of malignancies.in humans these only occur with chromosomes 13.attached to another at the Centromere . therefore the genetic material is inverted. as well . and are therefore initially not inherited. 15. It includes routine analysis of G-Banded chromosomes. turned upside down and reattached.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. however. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. resulting in Mosaicism (where some cells have the anomaly and some do not). especially the chromosomes. can happen after conception. Some anomalies. Therefore. the anomaly is present in every cell of the body. 14. Chromosome anomalies can be inherited from a parent or be "de novo". • Inversions: A portion of the chromosome has broken off. 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. • Rings: A portion of a chromosome has broken off and formed a circle or ring. 21 and 22.
Their behavior in animal (salamander) cells was described by Walther Flemming. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. these results were quite remarkable. von Waldeyer in 1888.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). 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. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. and he correctly insisted on man having an XX/XY system. in 1882.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. He revised his opinion later from 46 to 48. Pre-treating cells in a hypotonic solution. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in contrast to their genic contents. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. 4. the discoverer of mitosis. concluding an XX/XO sex determination mechanism. New techniques were needed to definitively solve the problem: 1. Considering their techniques. The next stage took place after the development of genetics in the early 20th century. which swells them and spreads the chromosomes . Using cells in culture 2. at first favoring 46.
persimilis from wild populations in California and neighboring states. Rather interestingly.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Arresting mitosis in metaphase by a solution of colchicine 4. Using Painter's technique they studied the polytene . the great apes have 48 chromosomes. reducing the number. During her cytogenetic work. 4. In 1931. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).6 Applications in biology 4. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.6.2 Natural populations of Drosophila In the 1930s. a find which eventually led to her Nobel Prize in 1983. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Human chromosome 2 was formed by a merger of ancestral chromosomes.3. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D.6. McClintock discovered transposons. 4.
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. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. It was found that the various chromosome types do not fluctuate at random. In some congenital disorders. which enabled feeding. as they would if selectively neutral. 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. 4. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. Down syndrome is also referred to as trisomy 21.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. Evidence rapidly accumulated to show that natural selection was responsible. In 1959. Using a method invented by L'Heretier and Teissier. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. but adjust to certain frequencies at which they become stabilised. This had the benefit of eliminating migration as a possible explanation of the results. discoveries were quickly made related to aberrant chromosomes or chromosome number. as with most polymorphisms.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. . Dobzhansky bred populations in population cages. such as Down's syndrome. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. breeding and sampling whilst preventing escape.
Other numerical abnormalities discovered include sex chromosome abnormalities. which is required in normal females to compensate for having two copies of the chromosome. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). has Klinefelter's Syndrome. and XXXX. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. resulting in 47 total chromosomes. in addition to other tests. . with the development of more advanced techniques. Not all genes on the X Chromosome are inactivated. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. An individual with only one sex chromosome (the X) has Turner syndrome. Identification of the Philadelphia chromosome by cytogenetics. Pennsylvania. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. XYY. is used today as a diagnostic for CML. Thirteen years later.as both scientists were doing their research in Philadelphia. In 1960. Many other sex chromosome combinations are compatible with live birth including XXX. an additional X chromosome in a male. This abnormal chromosome was dubbed the Philadelphia chromosome .
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.8 Beginnings of molecular cytogenetics .FIG Advent of banding techniques In the late 1960s. 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. Deletion syndromes such as DiGeorge syndrome. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Caspersson developed banding techniques which differentially stain chromosomes. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. 4. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations.
This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. cloned and studied in ever greater detail. While radioisotope-labeled probes had been hybridized with DNA since 1969.In the 1980s. CHAPTER 5 Techniques 5. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). movement was now made in using fluorescent labeled probes. advances were made in molecular cytogenetics. 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 .
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.
5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •
bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations
5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,
you can solve technical computing problems faster than with traditional programming languages. CGH and Single nucleotide polymorphism-arrays. such as comparative genomic hybridization arrays. 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. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. C++. communications. test and measurement. including signal and image processing. control design. data visualization. and computational biology. financial modeling and analysis. Using MATLAB.generally between 200 and 1000 cells are counted and scored. such as C. and numerical computation. and FORTRAN. data analysis. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. You can use MATLAB in a wide range of applications. For congenital problems usually 20 metaphase cells are scored. .
6. such as declaring variables. specifying data types. MATLAB eliminates the need for ‘for’ loops. . These effects must be compensated to improve the results of the pairing algorithm. and allocating memory.MATLAB provides a number of features for documenting and sharing your work. With the MATLAB language.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. You can integrate your MATLAB code with other languages and applications. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. and distribute your MATLAB algorithms and applications. In many cases. It enables fast development and execution. As a result. The image processing step is composed of the following operations. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. one line of MATLAB code can often replace several lines of C or C++ code. 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.
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). 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. geometrical and dimensional differences must be removed. To compensate for this inhomogeneity.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. the spatially scaled images are histogram equalized. To compare chromosomes from a band pattern point of view. 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. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. 6. or at least attenuated. Therefore. 2) Geometrical compensation—The geometric compensation.2 Concepts used in this phase 1) Image conversion 2) Denoising .
the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. The resulting true color image has identical matrices for the red. For example. 6. You can perform certain conversions just using MATLAB syntax. and the results might not be meaningful. 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. so the image displays as shades of gray. . green. When you apply the filter to the true color image. you must first convert it to true color format. If you attempt to filter the indexed image. listed in the following table. as is appropriate. MATLAB filters the intensity values in the image.3) Edge detection 4) Two dimensional convolutions.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. For example. RGB = cat (3. there are other functions that return a different image type as part of the operation they perform. For example.I.I). and blue planes.I. In addition to these image type conversion functions.2.
If an image is being sent electronically from one place to another.2. we may expect errors to occur in the image signal. caused by external disturbance.parameters. We may use edges to measure the size of objects in an image. and hence the type of noise on the image. and we shall look at some of the more straightforward of them. Usually we know what type of errors to expect. hence we can choose the most appropriate method for reducing the effects. via satellite or wireless transmission. ) Where the parameters available depend on the method used 6. .6. to recognize or classify objects. Cleaning an image corrupted by noise is thus an important area of image restoration. to isolate particular objects from their background. The general Matlab command for finding edges is edge(image.2.5 Edge detection Edges contain some of the most useful information in an image. 6. or through networked cable. If one of these matrices describes a two-dimensional finite impulse response .'method'.B) computes the two-dimensional convolution of matrices A and B. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. There is a large number of edge finding algorithms in existence.3 Two dimensional convolutions C = conv2(A. .4 Denoising We may define noise to be any degradation in the image signal.
na+nb-1]. rgb2gray im2bw(im. imedfilt2(im1. nb]+1)/2). . minus one.'sobel'). edge(im1. That is.bmp')..nb].'shape') subsection of the two-dimensional convolution. 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('12345.0.7). The indices of the center element of B are defined as floor(([mb C = conv2(hcol.na] and the size of B is [mb. if the size of then the size of C is [ma+mb-1.(FIR) filter. The size of matrices..A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. this case is the same as C = conv2(hcol*hrow.hrow.A). If hcol is a column vector and hrow is a row vector.. C = conv2(. the other matrix is filtered in two dimensions.[3 3]). convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.
Msk conv2(double(BW). Index=1. bwlabel(B.j)~=0 for k=1:mx if L(i. n1(x1.2).1). for i=1:m for j=1:n if L(i.c] = find(L==22). for i=1:sx x1=rc(i.imy).imy]=size(BW). MODULE 2 clc [m. . L_number=zeros(mx. flag=0.n]=size(L). rc = [r c]. [r.[imx.y1)=255.1). mx=max(max(L)).j)==L_number(k) flag=1. nzeros(imx.double(msk)).8). [sx sy]=size(rc). y1=rc(i.
62. Index=Index+184.108.40.206.220.127.116.11.26. end.20.39. 36. Test_number=[3.). for x=1:46 [r.33.48.21.imy).1). n1(x1. n1=zeros(imx.28.2).29.j).41.45.9. for i=1:sx x1=rc(i. .18.104.22.168.22.214.171.124.6. rc = [r c].55.y1)=255. end %h=figure. y1=rc(i.30. end flag=0. end end L_number.imshow(n1.66].57. [sx sy]=size(rc).126.96.36.199.40.end end if flag~=1 L_number(Index)=L(i.188.8.131.52.59.19.4.c] = find(L==L_number((Test_number(x)))).
skel=im2double(f). skel=im2bw(skel. s1=bwmorph(s. BW=double(BW). BW=im2bw(f). Arm_length=zeros(46. [m n]=size(BW1). s=bwmorph(skel. end end end Circumference(i)=Circumference_sum.'.1.bmp')).'skel'. for i=1:46 f=imread(strcat(num2str(i).'spur'. Area=zeros(46.1).8). . Circumference_sum=0.1). f=imcomplement(f). for x=1:m for y=1:n if BW1(x.1).Inf).'canny'). BW1=edge(BW. Arm_length_sum=0.5*graythresh(skel)).y)==1 Circumference_sum=Circumference_sum+1.end Circumference=zeros(46.
end end end Area(i)=Area_sum. [m n]=size(BW). for x=1:m for y=1:n if s1(x. end Circumference. Arm_length. for x=1:m for y=1:n if BW(x. . BW=im2bw(f).[m n]=size(s1). end end end Arm_length(i)=Arm_length_sum.y)==1 Area_sum=Area_sum+1. Area_sum=0.y)==1 Arm_length_sum=Arm_length_sum+1.
1)=i.2)==46 Pair(46. Pair(i.2)=i+1. 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)). Pair(i.2)=j. end end end for i=1:45 if Pair(i.1)=i. .1)=46.Area. Pair(46. Pair(i. Pair(i.2). end end Pair. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)).
end end if flag~=1 if figure_flag~=47 subplot(23.1)==delete(j) flag=1. flag=0.2).1)).bmp')). end flag=0. end f2=imread(strcat(num2str(Pair(i.'.delete=zeros(46. figure_flag=1. figure_flag=figure_flag+1. imshow(f1).bmp')). delete(figure_flag)=Pair(i.figure_flag). for i=1:46 for j=1:46 if Pair(i.figure_flag).1). end f1=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1.'. .2.2)). imshow(f2).2. if figure_flag~=47 subplot(23.
to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. 2) feature extraction from the processed images . Copenhagen.end CONCLUTION In this paper. plus a new one. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. based on the MI. 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. and Philadelphia. The proposed algorithm is based on the traditional features extracted from the karyogram. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. in the scope of karyotyping process used in cytogentic analysis. 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. such as. dimensions and banding profiles.
from the chromosomes in the training set. 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. are processed in order to compensate for geometrical and intensity distortions. and finally. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. 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). shape. This normalization is needed to make it possible the band pattern comparison between chromosomes. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. Here. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. In the image processing step. and band pattern. the romosome images.characterizing the size. Tests using 19 karyograms based on bone marrow cells. and to normalize their dimensions. extracted from the unordered karyogram. The features extracted from the processed images discriminate each pair with respect to their size. shape and band pattern. Vectors of coefficients associated with each one of the 22 classes are computed and the distance between two arbitrary chromosomes is the minimum one among all distances obtained with these 22 vectors. achieves a 70.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms.working within an 8-D feature space.10% mean classification rate. 4) pairing. The training process consists in the estimation of each vector of coefficient .
The results presented in this paper are promising.10% classification ratewas obtained.performance of the classifier. In addition. presenting a uniform level of condensation. such as Edinburgh. centromere position. Using 27 karyograms andworking with a limited number of classes (≤ 8). Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. In fact. a 76. a new chromosome dataset with 9200 chromosomes from bone marrow cells. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes.. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. whose images are of significantly higher quality. This dataset was made publicly available . e. amean classification rate larger than 93% was obtained in all experiments. and from which it is possible to extract additional features. Copenhagen. called LK1 . Executing the algorithm on a higher quality dataset.g. or Philadelphia. despite the low quality of this type of chromosomes. REFERENCES .
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