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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
This allows the very long DNA molecules to fit into the cell nucleus. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. Chromosomes may exist as either duplicated or unduplicated. which is tightly coiled in on itself. In prokaryotes and viruses. Unduplicated chromosomes are single linear strands. DNA is usually arranged as a circle. In prokaryotes. or it may unexpectedly evadeapoptosis leading to the progression of cancer. divided. for example. The structure of chromosomes and chromatin varies through the cell cycle.defined nuclei) have smaller circular chromosomes. In practice "chromosome" is a rather loosely defined term. These small circular genomes . cells may contain more than one type of chromosome. circular DNA molecules called plasmids. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. through processes known as chromosomal instability and translocation. In eukaryotes. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. although there are many exceptions to this rule. Also. the term genophore is more appropriate when no chromatin is present. Chromosomes are the essential unit for cellular division and must be replicated. If these structures are manipulated incorrectly. sometimes accompanied by one or more smaller. the cell may undergo mitotic catastrophe and die. a large body of work uses the term chromosome regardless of chromatin content. Chromosomal recombination plays a vital role in genetic diversity. However.
Chromosomal Mutations are substantial changes in chromosome structure that are large enough to be visible by karyotyping (see lab manual) and thus typically affect more than one gene. reflecting their bacterial origins.+21. copies of chromosome 21. XX (female) or 46 XY (male).3 MUTATIONS IN CHROMOSOME NUMBER Normally. . in which an individual has 3.XY or 47. An example of aneuploidy is trisomy 21.are also found in mitochondria and chloroplasts.g. Such individuals are called euploid and have the wild-type chromosome complement for the species. 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). The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. a extra copy of human chromosome 21). Euploid human karyotypes are 46.+21. rather than 2. The individual would have Down Syndrome and his/her karyotype would be written 47. 1. the individual carrying the mutation is said to be aneuploid.XX. If the mutation involves only one or a few chromosomes in the genome (e.
During mitosis. Once the cells have divided. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. The microtubules then pull the chromatids apart toward the centrosomes. . longer-lasting attachment in this region. and they form the classic four arm structure. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. which enables these giant DNA structures to be contained within a cell nucleus. so that each daughter cell inherits one set of chromatids. 1. small) and the longer arms are called q arms (q follows p in the Latin alphabet. one of which is present on each sister chromatid. the chromatin strands become more and more condensed. along with special proteins. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. a pair of sister chromatids attached to each other at the centromere. the chromatids are uncoiled and DNA can again be transcribed. q-g "grande").Fig 1.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). chromosomes are structurally highly condensed. This is the only natural context in which individual chromosomes are visible with an optical microscope.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. In spite of their appearance. A special DNA base sequence in the region of the kinetochores provides. The shorter arms are called p arms (from the French petit. This compact form makes the individual chromosomes visible.
6 kg (5. In humans.1.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. bone marrow in large bones produces new blood cells. bone marrow accounts for approximately 2. which divides the nuclei. On average. . cytoplasm.7 lbs).5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. which use the bone marrow vasculature as a conduit to the body's systemic circulation. in adults weighing 65 kg (143 lbs). The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. bone marrow constitutes 4% of the total body mass of humans.  Bone marrow is also a key component of the lymphatic system. It is generally followed immediately by cytokinesis. in two separate nuclei. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. 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.
which lack a nucleus. Prokaryotic cells. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. Mitosis occurs only in eukaryotic cells and the process varies in different species. for instance during certain stages of fruit fly embryonic development. prometaphase. Because cytokinesis usually occurs in conjunction with mitosis. but is found in various different groups. The cell then divides in cytokinesis. there are many cells where mitosis and cytokinesis occur separately. These stages are interphase. prophase. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. where the nuclear envelope breaks down before the chromosomes separate. Even in animals. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. . anaphase and telophase.genetically identical to each other and to their parent cell. The process of mitosis is fast and highly complex. This accounts for approximately 10% of the cell cycle. forming single cells with multiple nuclei. For example. cytokinesis and mitosis may occur independently. to produce two identical daughter cells which are still diploid cells. "mitosis" is often used interchangeably with "mitotic phase". metaphase. animals undergo an "open" mitosis. This occurs most notably among the fungi and slime moulds. However. where chromosomes divide within an intact cell nucleus. divide by a process called binary fission.
This occurs during the S phase of interphase. and together the two are called sister chromatids. 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. 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. These two cells are identical and do not differ in any way from the original parent cell. the parent cell must make a copy of each chromosome before mitosis. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. .Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells.
As a matter of convention. However. A new nuclear envelope forms around the separated sister chromosomes. 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). In animal cells. each sister chromatid is now considered a chromosome. the daughter cells will construct a new dividing cell wall between each other. separating the two developing nuclei. In plant cells. so they are renamed to sister chromosomes. Eventually. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. the parent cell will be split in half. corresponding sister chromosomes are pulled toward opposite ends. giving rise to two daughter cells. each with a replica of the original genome. the nuclear envelope which segregates the DNA from the cytoplasm disassembles.In most eukaryotes. . because of the non-involvement of nuclear dynamics and lack of linear chromosomes. Prokaryotic cells undergo a process similar to mitosis called binary fission. As the cell elongates. pulling apart the sister chromatids of each chromosome. The chromosomes align themselves in a line spanning the cell. the process of binary fission is very much different from the process of mitosis. As mitosis completes.the cell begins cytokinesis.
chromosomes are replicated only during the S phase. the nucleus has to migrate into the center of the cell before mitosis can begin. 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. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . continues to grow as it duplicates its chromosomes (S). 2. grows more and prepares for mitosis (G 2). mainly via proteins. During all three phases. S (synthesis). the cell grows by producing proteins and cytoplasmic organelles. prophase is preceded by a pre-prophase stage. All these phases in the interphase are highly regulated. This is achieved through the formation of a phragmosome. where the cell prepares itself for cell division. Interphase is divided into three phases: G1 (first gap). In highly vacuolated plant cells.2. Thus.1 Preprophase In plant cells only.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. and finally it divides (M) before restarting the cycle. However. a cell grows (G1). and G2 (second gap). It alternates with the much longer interphase.
Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Cytokinesis has already begun. This band marks the position where the cell will eventually divide. 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. and microtubules have invaded the nuclear Prophase space. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. The chromosomes have chromatin has condensed. In addition to phragmosome formation.division. . instead. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. aligned at the metaphase plate. The cells of higher plants (such as the flowering plants) lack centrioles. degraded. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. the pinched area is known as the cleavage furrow. 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.
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. A cell inherits a single centrosome at cell division. chromatin condenses together into a highly ordered structure called a chromosome. giving a pair of centrosomes. Since the genetic material has already been duplicated earlier in S phase. which is replicated by the cell with the help of the nucleus before a new mitosis begins. The centrosome is the coordinating center for the cell's microtubules. Chromosomes are typically visible at high magnification through a light microscope.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. they are not essential for the . bound together at the centromere by the cohesin protein complex. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. Close to the nucleus are structures called centrosomes. Although centrioles help organize microtubule assembly. the genetic material in the nucleus is in a loosely bundled coil called chromatin. which are made of a pair of centrioles found in most eukaryotic animal cells. At the onset of prophase.
Prometaphase is sometimes considered part of prophase. coupled with polymerisation and depolymerisation of microtubules. Fungi and some protists. it is known that it contains some form of molecular motor. When a microtubule connects with the kinetochore. . A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. kinetochore microtubules begin searching for kinetochores to attach to. and centrosomes are not always used in mitosis. Although the kinetochore structure and function are not fully understood. using energy from ATP to "crawl" up the tube toward the originating centrosome. one attached at each chromatid. This motor activity.formation of the spindle. provides the pulling force necessary to later separate the chromosome's two chromatids. since they are absent from plants. and it occurs in most multicellular organisms. 2. on an average 20 ). When the spindle grows to sufficient length. This is called open mitosis. undergo a variation called closed mitosis where the spindle forms inside the nucleus. Each chromosome forms two kinetochores at the centromere.2. such as algae or trichomonads. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. the motor activates. or its microtubules are able to penetrate an intact nuclear envelope. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number.
Metaphase comes from the Greek meaning "after. only roughly lining up along the midline. an imaginary line that is equidistant from the two centrosome poles. In certain types of cells. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. in some sense. analogous to a tug-of-war between people of equal strength. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.In the fishing pole analogy. . The centromeres of the chromosomes. the chromosomes come under longitudinal tension from the two ends of the cell. All chromosomes (blue) but one have arrived at the metaphase plate.3 Metaphase A cell in late metaphase. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. 2. As a result. the kinetochore would be the "hook" that catches a sister chromatid or "fish"." Microtubules find and attach to kinetochores in prometaphase. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. convene along the metaphase plate or equatorial plane.
the cell proceeds to anaphase (from the Greek meaning “up. These sister chromatids. Next. The signal creates the mitotic spindle checkpoint. The force that causes the centrosomes to move towards the ends of the cell is still unknown.” “back. Two events then occur: first. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). the cell has succeeded in separating identical copies of the genetic material into two distinct populations. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. Early anaphase is usually defined as the separation of the sister chromatids. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell.” or “re-”).4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. the proteins that bind sister chromatids together are cleaved. At the end of anaphase. allowing them to separate. which have now become distinct sister chromosomes. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. the nonkinetochore microtubules elongate. . 2. These two stages are sometimes called early and late anaphase. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned.” “against.
the nonkinetochore microtubules continue to lengthen. but cell division is not yet complete.2. using fragments of the parent cell's nuclear membrane. In animal cells. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. Corresponding sister chromosomes attach at opposite ends of the cell.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. Both sets of chromosomes. cell . however. 2. At telophase. now surrounded by new nuclei. It "cleans up" the after effects of mitosis. cytokinesis is a separate process that begins at the same time as telophase. pinching off the separated nuclei. Mitosis is complete. necessary for completing cell division. elongating the cell even more.5 Cytokinesis Cilliate undergoing cytokinesis. Cytokinesis is technically not even a phase of mitosis. but rather a separate process. A new nuclear envelope. In both animal and plant cells. unfold back into chromatin. forms around each set of separated sister chromosomes. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be.
2 Development and growth The number of cells within an organism increases by mitosis.1Significance Mitosis is important for the maintenance of the chromosomal set. Each daughter cell has a complete copy of the genome of its parent cell.4 Regeneration .5. This is the basis of the development of a multicellular body from a single cell i. e.g.division is also driven by vesicles derived from the Golgi apparatus. skin and digestive tract.e. which move along microtubules to the middle of the cell. Following are the occasions in the lives of organism where mitosis happens: 2. separating the two nuclei. 2. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.3 Cell replacement In some parts of body. whereas some green algae use a phycoplast microtubule array during cytokinesis. zygote and also the basis of the growth of a multicellular body. Similarly. 2. cells are constantly sloughed off and replaced by new ones.5. The phragmoplast is a microtubule structure typical for higher plants. 2. The end of cytokinesis marks the end of the M-phase. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall..5. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. New cells are formed by mitosis and so are exact copies of the cells being replaced.
and the latter cell having only one chromosome (the homologous chromosome). These cells are considered aneuploid.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. 2. a condition known as monosomy. The cells at the surface of hydra undergo mitosis and form a mass called bud. The same division happens during asexual reproduction or vegetative propagation in plants. .Some organisms can regenerate their parts of bodies.7 Consequences of errors Although errors in mitosis are rare. 2. The production of new cells is achieved by mitosis. resulting in binucleated cells.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. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). they fail to complete cell division and retain both nuclei in one cell. especially during early cellular divisions in the zygote. One daughter cell will receive both sister chromosomes and the other will receive none. a chromosome may fail to separate during anaphase. the hydra reproduces asexually by budding. a condition often associated with cancer. For example. a condition known as trisomy. Occasionally when cells experience nondisjunction. the process may go wrong. In non-disjunction.5. Mitosis continues in the cells of bud and it grows into a new individual.
sometimes mutuations occur in such genes and cells continue to divide. Or. Such tumours can send cancer cells to other parts in body where new tumours may form. causing chromosomal duplication. When tissues more than the requirement are synthesized in a single organ. The fragment may incorrectly reattach to another. but in reverse orientation. Benign tumours are not harmful as soon as they are not moving. it results in the formation of Tumors. causing translocation. This phenomenon is called metastasis or spreading of disease. It may reattach to the original chromosome. All cells have genes that control the timing and number of mitosis. causing deletion. causing inversion. its organelles disintegrate and reform in a matter of hours. Errors in the control of mitosis may cause cancer. Now what happens is that cell abnormally continue to divide at a single place. . Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours.Mitosis is a demanding process for the cell. As long as these tumours remain in their original location they are called benign tumours. Occasionally. An arm of the chromosome may be broken and the fragment lost. It results in abnormal cell growth. which goes through dramatic changes in ultrastructure. and chromosomes are jostled constantly by probing microtubules. As soon as they start to move and invade other cells there are said to be malignant tumours. non-homologous chromosome. It results in the synthesis of execessive tissue growths. chromosomes may become damaged. it may be treated erroneously as a separate chromosome. The effect of these genetic abnormalities depends on the specific nature of the error.
An example of a cell that goes through endomitosis is the megakaryocyte.7 Metaphase Metaphase. an imaginary line that is equidistant from the two centrosome poles. carrying genetic information. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). Preceded by events in prometaphase and followed by anaphase. resulting in cells with many copies of the same chromosome occupying a single nucleus. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. Early events of metaphase can . analogous to a tug of war between equally strong people. Metaphase accounts for approximately 4% of the cell cycle's duration. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. This process may also be referred to as endoreduplication and the cells as endoploid.2. only roughly lining up along the middleline. from the ancient Greek(between) and (stage). 2. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. align in the middle of the cell before being separated into each of the two daughter cells. In certain types of cells.
Such a signal creates the mitotic spindle checkpoint. often with Giemsa (G banding) or Quinacrine. produces a pattern of in total up to several hundred bands.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. Chromosomes are condensed(Thickened) and highly coiled in metaphase. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. securin. which makes them most suitable for visual analysis. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. 2. 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.coincide with the later events of prometaphase. Normal metaphase spreads are used in . It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Staining of the slides. This would be accomplished by regulation of the anaphase-promoting complex. Metaphase chromosomes make the classical picture of chromosomes (karyotype). Only after all chromosomes have become aligned at the metaphase plate. does the cell enter anaphase. For classical cytogenetic analyses. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). and separase. when every kinetochore is properly attached to a bundle of microtubules.
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. which may lead to chimeric oncogenes.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. . 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.
Karyotypes can be used for many purposes. The study of whole sets of chromosomes is sometimes known as karyology. The study of karyotypes is important for cell biology and genetics. taxonomic relationships. to study chromosomal aberrations. and to gather information about past evolutionary events. or an individual organism. Thus. banding pattern. the position of the centromeres. So. or may not. in normal diploid organisms. The term is also used for the complete set of chromosomes in a species. such as. and what they look like under a light microscope. Karyogram of human male using Giemsa staining. ordered by size and position of centromere for chromosomes of the same size. and the results may be used in evolutionary biology and medicine. cellular function. any differences between the sex chromosomes. . Attention is paid to their length. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). There may. Karyotypes describe the number of chromosomes.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell.  The preparation and study of karyotypes is part of cytogenetics. in humans 2n = 46. and any other physical characteristics. autosomal chromosomes are present in two copies. be sex chromosomes. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies.
New techniques were needed to definitively solve the problem: 1. Using cells in culture 2. von Waldeyer in 1888. 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. which swells them and spreads the chromosomes . The name was coined by another German anatomist.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. in contrast to their genic contents. concluding an XX/XO sex determination mechanism. the discoverer of mitosis. Pretreating cells in a hypotonic solution. and he correctly insisted on humans having an XX/XY system. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. these results were quite remarkable. The next stage took place after the development of genetics in the early 20th century. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. at first favoring 46. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in 1882. Their behavior in animal (salamander) cells was described by Walther Flemming. He revised his opinion later from 46 to 48. Considering their techniques.3.
2.1 Staining The study of karyotypes is made possible by staining. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. a suitable dye. Usually. For humans. 3. such as Giemsa. Rather interestingly. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. reducing the number.2. The sex of an unborn fetus can be determined by observation of interphase cells. Human chromosome 2 was formed by a merger of ancestral chromosomes. 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . is applied after cells have been arrested during cell division by a solution of colchicine.3.2 Observations on karyotypes 3. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.  Sometimes observations may be made on non-dividing (interphase) cells. the great apes have 48 chromosomes. Arresting mitosis in metaphase by a solution of colchicines 4.
permitting its loss without penalty to the organism (the dislocation hypothesis). Humans have one pair fewer chromosomes than the great apes. Differences in the position of centromeres. and mainly consists of genetically inactive repetitive DNA sequences. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. but the genes have been mostly translocated (added) to other chromosomes. both have six pairs of chromosomes (n=6) yet V. This feature probably reflects different amounts of DNA duplication. 3. 4. 6. Differences in degree and distribution of heterochromatic regions. as well as other cytogenetic information. 5. type. shape and banding of the chromosomes. This is brought about by translocations. Heterochromatin stains darker than euchromatin. 2. Differences in number and position of satellites. Differences in absolute sizes of chromosomes. indicating tighter packing. faba chromosomes are many times larger. A full account of a karyotype may therefore include the number.1. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. which (when they occur) are small bodies attached to a chromosome by a thin thread. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). .
Variation is often found: 1. XY. XX. Between the sexes 2. which are highly variable. Any variation from the standard karyotype may lead to developmental abnormalities.3 The human karyotype Most (but not all) species have a standard karyotype. the same cannot be said for their karyotypes. males have both an X and a Y chromosome denoted 46. Normal karyotypes for females contain two X chromosomes and are denoted 46. and in . Mosaics or otherwise abnormal individuals. There is variation between species in chromosome number. Geographical variation between races 5. Between the germ-line and soma (between gametes and the rest of the body) 3. 3. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Between members of a population (chromosome polymorphism) 4.
some organisms go in for large-scale elimination of heterochromatin.. used in conjunction with other phylogenetic data. In this process. as in many sciarid flies. In A. which were previously inexplicable. found in some copepods and roundworms such as Ascaris suum. In some cases there is even significant variation within species. Godfrey and Masters conclude: "In our view. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. . "We have a very poor understanding of the causes of karyotype evolution. In a review.. But. despite many careful investigations.3. it is quite unclear what the general significance might be. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost.detailed organization. entire chromosomes are eliminated during development. Although much is known about karyotypes at the descriptive level. 3. the general significance of karyotype evolution is obscure. portions of the chromosomes are cast away in particular cells.1 Changes during development Instead of the usual gene repression. In some species. Chromosome elimination. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. despite their construction from the same macromolecules.. or other kinds of visible adjustment to the karyotype.. This variation provides the basis for a range of studies in evolutionary cytology. Chromatin diminution (founding father: Theodor Boveri).
. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. the high record would be somewhere amongst the ferns. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. The low record is held by the nematode Parascaris univalens. When they looked at the karyotype of the closely related Indian muntjac. In marsupials it is always the paternal X which is inactivated. 3. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. was found to be 46. The diploid number of the Chinese muntjac.3. Xinactivation. thus the mammalian female is a mosaic in respect of her X chromosomes.suum. male = 7 chromosomes. Muntiacus reevesi. "They simply could not believe what they saw. which was investigated by Kurt Benirschke and his colleague Doris Wurster. The existence of supernumerary or B chromosomes . In human females some 15% of somatic cells escape inactivation.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. They kept quiet for two or three years because they thought something was wrong with their tissue culture.. the inactivation is random as between the two Xs. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation)... In placental mammals. all the somatic cell precursors undergo chromatin diminution. Muntiacus muntjak. where the haploid n = 1. they were astonished to find it had female = 6. all telocentric.
Endopolyploidy occurs when in adult differentiated . Thus. It is a common arrangement in the Hymenoptera. and aneuploids are another example. 3.means that chromosome number can vary even within one interbreeding population. 14. occurs mainly in plants. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. where there are more than two sets of homologous chromosomes in the cells. FN. 15.3. Haplo-diploidy. 3. FN ≤ 2n.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. Polyploidy in lower plants (ferns. and in some other groups. 21 and 22). and the other haploid.3 Fundamental number The fundamental number. due to the presence of five acrocentric chromosome pairs (13. though in this case they would not be regarded as normal members of the population. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. It has been of major significance in plant evolution according to Stebbins. where one sex is diploid. Polyploidy. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. about 70%. Humans have FN = 82. Polyploidy in animals is much less common. but it has been significant in some groups.
This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). the daughter chromosomes separating from each other inside an intact nuclear membrane. Abnormalities in chromosome number usually cause a defect in development. 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. Down syndrome and Turner syndrome are examples of this. it is diverse and complex. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species.tissues the cells have ceased to divide by mitosis. but the nuclei contain more than the original somatic number of chromosomes. . which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. See palaeopolyploidy for the investigation of ancient karyotype duplications. 3. The cells do not always contain exact multiples (powers of two). In many instances.
Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.Aneuploidy may also occur within a group of closely related species.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. 4. where every number from x = 3 to x = 15 is represented by at least one species. the great apes have 24x2 chromosomes whereas humans have 23x2. that the two chromosome morphs are adapted to different habitats.000 km2). some mantids of the genus Ameles. the European shrew Sorex araneus. In about 6. and 7. 5. 3.500 sq mi (17. 6. reducing the number. 3. where the gametic (= haploid) numbers form the series x = 3. living from rainforests to . the chromosome number is variable from one individual to another. When this happens. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world.  Closer to home. Well-researched examples are the ladybird beetle Chilocorus stigma. and Crocus. Classic examples in plants are the genus Crepis.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. Human chromosome 2 was formed by a merger of ancestral chromosomes.
The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. and skipping of islands. probably 20 million years ago. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. in the family Drosophilidae. There are also cases of colonization back to older islands. Drosophila and Scaptomyza.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). show a clear "flow" of species from older to newer islands. which can be dated to 30 mya. gene arrangements are visible in the banding patterns of each chromosome. The results are clear. The polytene banding of the 'picture wing' group. Chromosome rearrangements. but these are much less frequent. it is more likely to have been a group from the same species. . The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. when plotted in tree form (and independent of all other information). Although it would be possible for a single gravid female to colonise an island. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. make it possible to see which species are closely related. In a sense. Using K-Ar dating. especially inversions. at least into the Cretaceous.subalpine meadows. the present islands date from 0. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. The inversions. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. the best-studied group of Hawaiian drosophilids. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain.
The pattern of bands is very similar to that seen in G-banding. early-replicating and GC rich. human genome. It yields a series of lightly and darkly stained bands . • T-banding: visualize telomeres. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. This method will normally produce 300-400 bands in a normal. R-banding is the reverse of G-banding (the R stands for "reverse").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. if less spectacular. 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. so it stains centromeres. late-replicating and AT rich. • • C-banding: Giemsa binds to constitutive heterochromatin. adaptive radiations. .7 Depiction of karyotypes 3.7. 3.There are other animals and plants on the Hawaiian archipelago which have undergone similar. The light regions tend to be euchromatic.
Each chromosome has a characteristic banding pattern that helps to identify them. and the long arm on the bottom. often Giemsa (G-banding). This yields a dark region where the silver is deposited. less frequently Quinacrine. Karyotypes are arranged with the short arm of the chromosome on top. Some karyotypes call the short and long arms p and q. a dye. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. both chromosomes in a pair will have the same banding pattern. 3. In the "classic" (depicted) karyotype. For example. respectively.7. Quinacrine binds to the adeninethymine-rich regions. denoting the activity of rRNA genes within the NOR.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. In addition. is used to stain bands on the chromosomes.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. Giemsa is specific for the phosphate groups of DNA. Cri du chat syndrome involves a deletion on the short arm of .
allowing the visualization of the individually colored chromosomes.7. Image processing software then assigns a pseudo color to each spectrally different combination. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.XX.chromosome 5.2) 3. Because there are a limited number of spectrally-distinct fluorophores. a combinatorial labeling method is used to generate many different colors. 3.XX. .5p-. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. It is written as 46. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. The critical region for this syndrome is deletion of 15. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors.del(5)(p15. This method is also known as virtual karyotyping.2. which is written as 46.
CHAPTER 4 .
trisomies. as in the presence of extra or missing chromosomes. Numerical abnormalities.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. Some disorders arise from loss of just a piece of one chromosome. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. are common numerical abnormalities. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. translocations. • • Patau syndrome is caused by trisomy of chromosome 13. Structural abnormalities often arise from errors in homologous recombination. although they generally do not survive to birth. otherwise known as 47. X or 45. often occur as a result of nondisjunction during meiosis in the formation of a gamete. is caused by trisomy of chromosome 21. as in derivative chromosome. Down syndrome. inversions. trisomy 9 and trisomy 16. also known as aneuploidy. XXY is caused by an extra X chromosome. the most common male chromosomal disease. including . Also documented are trisomy 8. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. in which three copies of a chromosome are present instead of the usual two. large-scale deletions or duplications. Klinefelter syndrome.4. or structural. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. a common chromosomal disease. X0).
They can be organized into two basic groups. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. 1p36 Deletion syndrome. a deletion of the maternal genes. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. 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. example of imprinting disorder. numerical and structural anomalies. . from the loss of part of the short arm of chromosome 1. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. A chromosome anomaly. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. There are many types of chromosome anomalies. a deletion of the paternal genes. A chromosome anomaly may be detected or confirmed in this manner. caused by abnormal formation of the larynx. one well-documented example is the Philadelphia chromosome. The name comes from the babies' distinctive cry. example of imprinting disorder. from a truncated short arm on chromosome 5.• Cri du chat (cry of the cat). abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes.
In a reciprocal translocation. There are two main types of translocations. an X. which is caused by partial deletion of the short arm of chromosome 4. Known disorders in humans include Wolf-Hirschhorn syndrome. Duplications: A portion of the chromosome is duplicated. and Jacobsen syndrome. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. • • Translocations: When a portion of one chromosome is transferred to another chromosome. In a Robertsonian translocation. resulting in extra genetic material.4. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. an entire chromosome has . Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. rather than two).). Tetrasomy. also called the terminal 11q deletion disorder. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. 4. etc. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes).3 Structural abnormalities When the chromosome's structure is altered. segments from two different chromosomes have been exchanged.
resulting in Mosaicism (where some cells have the anomaly and some do not). especially the chromosomes. 14. It includes routine analysis of G-Banded chromosomes. 21 and 22. other cytogenetic banding techniques. and are therefore initially not inherited. 15.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. as well . Therefore. This can happen with or without loss of genetic material. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. They often lead to an increased tendency to develop certain types of malignancies. • Rings: A portion of a chromosome has broken off and formed a circle or ring.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. the anomaly is present in every cell of the body. can happen after conception. 4. turned upside down and reattached.in humans these only occur with chromosomes 13.attached to another at the Centromere . This is why chromosome studies are often performed on parents when a child is found to have an anomaly. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. 4. therefore the genetic material is inverted. however. • Inversions: A portion of the chromosome has broken off. Some anomalies. Chromosome anomalies can be inherited from a parent or be "de novo".
The next stage took place after the development of genetics in the early 20th century. these results were quite remarkable.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. The name was coined by another German anatomist. Their behavior in animal (salamander) cells was described by Walther Flemming. He revised his opinion later from 46 to 48. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. in 1882. concluding an XX/XO sex determination mechanism. New techniques were needed to definitively solve the problem: 1.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). the discoverer of mitosis. Considering their techniques. von Waldeyer in 1888. Pre-treating cells in a hypotonic solution. at first favoring 46. and he correctly insisted on man having an XX/XY system. 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. 4. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. which swells them and spreads the chromosomes . Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Using cells in culture 2. in contrast to their genic contents.
6 Applications in biology 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. 4. Human chromosome 2 was formed by a merger of ancestral chromosomes. Arresting mitosis in metaphase by a solution of colchicine 4.6.3. reducing the number. McClintock discovered transposons. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Using Painter's technique they studied the polytene . In 1931. During her cytogenetic work.2 Natural populations of Drosophila In the 1930s. a find which eventually led to her Nobel Prize in 1983. persimilis from wild populations in California and neighboring states.6. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. 4. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Rather interestingly. the great apes have 48 chromosomes.
In 1959. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. 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. breeding and sampling whilst preventing escape. Down syndrome is also referred to as trisomy 21. such as Down's syndrome.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. Evidence rapidly accumulated to show that natural selection was responsible. discoveries were quickly made related to aberrant chromosomes or chromosome number. . It was found that the various chromosome types do not fluctuate at random. 4. but adjust to certain frequencies at which they become stabilised. Dobzhansky bred populations in population cages. as they would if selectively neutral. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. Using a method invented by L'Heretier and Teissier. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. This had the benefit of eliminating migration as a possible explanation of the results. which enabled feeding. In some congenital disorders.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. as with most polymorphisms.
Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML).as both scientists were doing their research in Philadelphia. and XXXX. . resulting in 47 total chromosomes. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. with the development of more advanced techniques. in addition to other tests. Not all genes on the X Chromosome are inactivated. An individual with only one sex chromosome (the X) has Turner syndrome.Other numerical abnormalities discovered include sex chromosome abnormalities. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. an additional X chromosome in a male. is used today as a diagnostic for CML. has Klinefelter's Syndrome. Thirteen years later. XYY. which is required in normal females to compensate for having two copies of the chromosome. Pennsylvania. Identification of the Philadelphia chromosome by cytogenetics. Many other sex chromosome combinations are compatible with live birth including XXX. This abnormal chromosome was dubbed the Philadelphia chromosome . the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. In 1960.
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. and elongation techniques for all culture types that allow for higher resolution banding. Caspersson developed banding techniques which differentially stain chromosomes. Deletion syndromes such as DiGeorge syndrome. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Deletions within one chromosome could also now be more specifically named and understood. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material.8 Beginnings of molecular cytogenetics .FIG Advent of banding techniques In the late 1960s. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 4. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.
1 Karyotyping . This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. cloned and studied in ever greater detail. movement was now made in using fluorescent labeled probes. While radioisotope-labeled probes had been hybridized with DNA since 1969. CHAPTER 5 Techniques 5. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).In the 1980s. 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,
you can solve technical computing problems faster than with traditional programming languages. such as comparative genomic hybridization arrays. including signal and image processing.generally between 200 and 1000 cells are counted and scored. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. data visualization. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. Using MATLAB. C++. You can use MATLAB in a wide range of applications. . test and measurement. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. such as C. and computational biology. data analysis. and numerical computation. financial modeling and analysis. communications. control design. For congenital problems usually 20 metaphase cells are scored. and FORTRAN. CGH and Single nucleotide polymorphism-arrays.
MATLAB provides a number of features for documenting and sharing your work. such as declaring variables. . you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks.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. These effects must be compensated to improve the results of the pairing algorithm. 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. With the MATLAB language. 6. 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. MATLAB eliminates the need for ‘for’ loops. As a result. In many cases. The image processing step is composed of the following operations. It enables fast development and execution. and allocating memory. and distribute your MATLAB algorithms and applications. specifying data types.
geometrical and dimensional differences must be removed. 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. To compensate for this inhomogeneity. 6. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast.2 Concepts used in this phase 1) Image conversion 2) Denoising . 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 spatially scaled images are histogram equalized. Therefore. or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. To compare chromosomes from a band pattern point of view.
You can perform certain conversions just using MATLAB syntax. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. In addition to these image type conversion functions. When you apply the filter to the true color image. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. if you want to filter a color image that is stored as an indexed image. there are other functions that return a different image type as part of the operation they perform.I. 6. so the image displays as shades of gray. For example.2. and blue planes.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. green. MATLAB filters the intensity values in the image. RGB = cat (3. The resulting true color image has identical matrices for the red. If you attempt to filter the indexed image. and the results might not be meaningful. For example. listed in the following table. as is appropriate.3) Edge detection 4) Two dimensional convolutions.I. you must first convert it to true color format. For example.I). MATLAB simply applies the filter to the indices in the indexed image matrix. .
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 .6. . . The general Matlab command for finding edges is edge(image.B) computes the two-dimensional convolution of matrices A and B. caused by external disturbance.2. Usually we know what type of errors to expect. or through networked cable. Cleaning an image corrupted by noise is thus an important area of image restoration. There is a large number of edge finding algorithms in existence. and we shall look at some of the more straightforward of them. via satellite or wireless transmission.'method'. to recognize or classify objects.4 Denoising We may define noise to be any degradation in the image signal. to isolate particular objects from their background. we may expect errors to occur in the image signal. We may use edges to measure the size of objects in an image.3 Two dimensional convolutions C = conv2(A. 6. ) Where the parameters available depend on the method used 6.5 Edge detection Edges contain some of the most useful information in an image.parameters.2. hence we can choose the most appropriate method for reducing the effects. and hence the type of noise on the image. If an image is being sent electronically from one place to another.
rgb2gray im2bw(im..nb].A)..'shape') subsection of the two-dimensional convolution. if the size of then the size of C is [ma+mb-1. nb]+1)/2). .0.7).na] and the size of B is [mb.(FIR) filter.[3 3]). this case is the same as C = conv2(hcol*hrow. the other matrix is filtered in two dimensions. imedfilt2(im1. minus one. 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.bmp'). C = conv2(.na+nb-1].hrow.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. edge(im1.'sobel'). The indices of the center element of B are defined as floor(([mb C = conv2(hcol. The size of matrices.. If hcol is a column vector and hrow is a row vector. That is.
[sx sy]=size(rc). for i=1:m for j=1:n if L(i. mx=max(max(L)). [r.j)==L_number(k) flag=1.imy). L_number=zeros(mx.y1)=255.1).c] = find(L==22). nzeros(imx. Msk conv2(double(BW).1). for i=1:sx x1=rc(i.j)~=0 for k=1:mx if L(i.double(msk)). n1(x1. bwlabel(B.n]=size(L). rc = [r c].2).[imx. y1=rc(i. .8). flag=0. MODULE 2 clc [m. Index=1.imy]=size(BW).
66].51. .43.6.imy).7.33. n1=zeros(imx. rc = [r c].28.57.40. Test_number=[188.8.131.52.11.imshow(n1.49.41. for x=1:46 [r.184.108.40.206. end flag=0.15.).220.127.116.11. n1(x1. y1=rc(i.18.104.22.168.8.j).9.65.38. for i=1:sx x1=rc(i. Index=Index+22.214.171.124.1).27.42. end end L_number.2).21. end.y1)=255.45. [sx sy]=size(rc).end end if flag~=1 L_number(Index)=L(i. end %h=figure.35.19. 36.c] = find(L==L_number((Test_number(x)))).39.50.59.
for i=1:46 f=imread(strcat(num2str(i).Inf).1). s1=bwmorph(s.1. . Area=zeros(46.1). BW=im2bw(f).'. skel=im2bw(skel. BW=double(BW).'spur'. for x=1:m for y=1:n if BW1(x.'canny'). skel=im2double(f). f=imcomplement(f). [m n]=size(BW1).8).'skel'. s=bwmorph(skel.y)==1 Circumference_sum=Circumference_sum+1. BW1=edge(BW. end end end Circumference(i)=Circumference_sum. Circumference_sum=0. Arm_length=zeros(46.1).5*graythresh(skel)). Arm_length_sum=0.bmp')).end Circumference=zeros(46.
end end end Area(i)=Area_sum. Arm_length. BW=im2bw(f).y)==1 Area_sum=Area_sum+1. end Circumference. end end end Arm_length(i)=Arm_length_sum.[m n]=size(s1). [m n]=size(BW). Area_sum=0. for x=1:m for y=1:n if BW(x. .y)==1 Arm_length_sum=Arm_length_sum+1. for x=1:m for y=1:n if s1(x.
. Pair(46. Pair(i. Pair=zeros(46. 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)).1)=i.2)=i+1. end end end for i=1:45 if Pair(i. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)).2)==46 Pair(46.Area. end end Pair.1)=i.2)=i.2). Pair(i.1)=46.2)=j. Pair(i. Pair(i.
1)). imshow(f2).2. end flag=0. end f2=imread(strcat(num2str(Pair(i.bmp')).2.2)).'. if figure_flag~=47 subplot(23.'.bmp')). flag=0.2).figure_flag). for i=1:46 for j=1:46 if Pair(i. figure_flag=1. . imshow(f1).figure_flag). end f1=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1. delete(figure_flag)=Pair(i.1)==delete(j) flag=1.delete=zeros(46. figure_flag=figure_flag+1.1). end end if flag~=1 if figure_flag~=47 subplot(23.
to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. plus a new one. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure.end CONCLUTION In this paper. in the scope of karyotyping process used in cytogentic analysis. based on the MI. 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. such as. dimensions and banding profiles. Copenhagen. 2) feature extraction from the processed images . The images of these chromosomes present less quality and level of detail than the ones usually used in traditional genetic analysis using datasets such as Edinburgh. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. The proposed algorithm is based on the traditional features extracted from the karyogram.
Here. 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 . and band pattern. from the chromosomes in the training set. Tests using 19 karyograms based on bone marrow cells. Vectors of coefficients associated with each one of the 22 classes are computed and the distance between two arbitrary chromosomes is the minimum one among all distances obtained with these 22 vectors. The training process consists in the estimation of each vector of coefficient . shape. and finally. The features extracted from the processed images discriminate each pair with respect to their size.working within an 8-D feature space. and to normalize their dimensions.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. are processed in order to compensate for geometrical and intensity distortions. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. the romosome images. 4) pairing. This normalization is needed to make it possible the band pattern comparison between chromosomes. shape and band pattern.characterizing the size. In the image processing step. extracted from the unordered karyogram.10% mean classification rate. achieves a 70. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. 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. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. 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).
Executing the algorithm on a higher quality dataset. a 76.performance of the classifier. amean classification rate larger than 93% was obtained in all experiments. or Philadelphia. 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. such as Edinburgh. In addition. centromere position. Copenhagen. Using 27 karyograms andworking with a limited number of classes (≤ 8). e.. The results presented in this paper are promising. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. despite the low quality of this type of chromosomes. and from which it is possible to extract additional features.10% classification ratewas obtained. In fact.g. 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. REFERENCES . whose images are of significantly higher quality. called LK1 . This dataset was made publicly available .
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