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During the Metaphase type of cell division in the Bone Marrow, Karyotyping method gives the visual representation of the 46 chromosomes paired and arranged in decreasing order of size. This representation is useful in leukemia purposes. This method is a difficult one because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges. So here, Karyotyping uses new mutual information method which is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. This algorithm is formulated as such a method of combinatorial optimization problem. Where the distances between homologous chromosomes are minimized and the distances between non homologous ones are maximized. It is solved by using an integer programming approach. In this project chromosome dataset Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes was used for this study.
Introduction 1.1 The study of chromosome morphology and its relation with some genetic diseases is the main goal of cytogenetic. Normal human cells have 23 classes of large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The chromosome contains approximately 30 000 genes (genotype) and large tracts of non coding sequences. The analysis of genetic material can involve the examination of specific chromosomal regions using DNA probes, e.g., fluorescent in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic hybridization (CGH) , or the morphological and pattern analysis of entire chromosomes, the conventional cytogenetic, which is the focus of this paper. These cytogenetic studies are very important in the detection of acquired chromosomal abnormalities, such as translocations, duplications, inversions, deletions, monosomies, or trisomies. These techniques are particularly useful in the diagnosis of cancerous diseases and are the preferred ones in the characterization of the different types of leukemia, which is the motivation of this paper . The pairing of chromosomes is one of the main steps in conventional cytogenetic analysis where a correctly ordered karyogram is produced for diagnosis of genetic diseases based on the patient karyotype. The karyogram is an image representation of the stained human chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) , where the chromosomes are arranged in 22 pairs of somatic homologous elements plus two sex-determinative chromosomes (XX for the female or XY for the male), displayed in decreasing order of size. A karyotype is the set of characteristics
extracted from the karyogram that may be used to detect chromosomal abnormalities. The metaphase is the step of the cellular division process where the chromosomes are in their most condensed state. This is the most appropriated moment to its visualization and abnormality recognition because the chromosomes appear well defined and clear. The pairing and karyotyping procedure, usually done manually by visual inspection, is time consuming and technically demanding. The application of the G-banding procedure to the chromosomes generates a distinct transverse banding pattern characteristic for each class, which is the most important feature for chromosome classification and pairing. The International System for Cytogenetic Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as for all the chromosomes of a normal human, and the clinical staff is trained to pair and interpret each specific karyogram according to the ISCN information. Other features, related to the chromosome dimensions and shape, are also used to increase the discriminative power of the manual or automatic classifiers. 1.2 CHROMOSOME A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes,regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Chromosomes vary widely between different organisms. The DNA molecule may be circular or linear, and can be composed of 100,000 to 10,000,000,000 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without
the term genophore is more appropriate when no chromatin is present. cells may contain more than one type of chromosome. In eukaryotes. a large body of work uses the term chromosome regardless of chromatin content. If these structures are manipulated incorrectly. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. The structure of chromosomes and chromatin varies through the cell cycle. although there are many exceptions to this rule. However. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). Unduplicated chromosomes are single linear strands. sometimes accompanied by one or more smaller. These small circular genomes . Chromosomes may exist as either duplicated or unduplicated. divided. or it may unexpectedly evadeapoptosis leading to the progression of cancer. DNA is usually arranged as a circle. the cell may undergo mitotic catastrophe and die. Also. This allows the very long DNA molecules to fit into the cell nucleus. Chromosomes are the essential unit for cellular division and must be replicated. In prokaryotes and viruses. Chromosomal recombination plays a vital role in genetic diversity. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. for example. which is tightly coiled in on itself. In prokaryotes. In practice "chromosome" is a rather loosely defined term. circular DNA molecules called plasmids.defined nuclei) have smaller circular chromosomes. through processes known as chromosomal instability and translocation. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny.
+21. The individual would have Down Syndrome and his/her karyotype would be written 47. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. If the mutation involves only one or a few chromosomes in the genome (e.XX. a extra copy of human chromosome 21). 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.g. copies of chromosome 21. Euploid human karyotypes are 46. . the individual carrying the mutation is said to be aneuploid. An example of aneuploidy is trisomy 21. Such individuals are called euploid and have the wild-type chromosome complement for the species. 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).+21. rather than 2.3 MUTATIONS IN CHROMOSOME NUMBER Normally.XY or 47. in which an individual has 3. XX (female) or 46 XY (male).are also found in mitochondria and chloroplasts. 1.
Once the cells have divided. A special DNA base sequence in the region of the kinetochores provides. 1. and they form the classic four arm structure.Fig 1. This is the only natural context in which individual chromosomes are visible with an optical microscope. longer-lasting attachment in this region. This compact form makes the individual chromosomes visible. During mitosis. along with special proteins. which enables these giant DNA structures to be contained within a cell nucleus. the chromatids are uncoiled and DNA can again be transcribed. The microtubules then pull the chromatids apart toward the centrosomes. so that each daughter cell inherits one set of chromatids. q-g "grande"). a pair of sister chromatids attached to each other at the centromere. small) and the longer arms are called q arms (q follows p in the Latin alphabet. The shorter arms are called p arms (from the French petit.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. .1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. the chromatin strands become more and more condensed. chromosomes are structurally highly condensed. In spite of their appearance. one of which is present on each sister chromatid.
6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2.  Bone marrow is also a key component of the lymphatic system. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. . which divides the nuclei. It is generally followed immediately by cytokinesis. In humans. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. in two separate nuclei. cytoplasm.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.7 lbs). The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. bone marrow in large bones produces new blood cells.1. On average. bone marrow accounts for approximately 2. in adults weighing 65 kg (143 lbs). bone marrow constitutes 4% of the total body mass of humans. which use the bone marrow vasculature as a conduit to the body's systemic circulation.
prometaphase. prophase. Because cytokinesis usually occurs in conjunction with mitosis. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. "mitosis" is often used interchangeably with "mitotic phase". The cell then divides in cytokinesis. . Prokaryotic cells. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. For example. This accounts for approximately 10% of the cell cycle. divide by a process called binary fission. metaphase. to produce two identical daughter cells which are still diploid cells. Even in animals. where the nuclear envelope breaks down before the chromosomes separate. However. cytokinesis and mitosis may occur independently. which lack a nucleus. but is found in various different groups. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. The process of mitosis is fast and highly complex. there are many cells where mitosis and cytokinesis occur separately. Mitosis occurs only in eukaryotic cells and the process varies in different species. animals undergo an "open" mitosis.genetically identical to each other and to their parent cell. anaphase and telophase. forming single cells with multiple nuclei. These stages are interphase. for instance during certain stages of fruit fly embryonic development. This occurs most notably among the fungi and slime moulds. where chromosomes divide within an intact cell nucleus. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer.
Because each resultant daughter cell should be genetically identical to the parent cell. the parent cell must make a copy of each chromosome before mitosis. . the 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. 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 genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. The sister chromatids are held together by a specialized region of the chromosome known as the centromere.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells.
As the cell elongates. separating the two developing nuclei. As mitosis completes. Prokaryotic cells undergo a process similar to mitosis called binary fission. corresponding sister chromosomes are pulled toward opposite ends. pulling apart the sister chromatids of each chromosome. However. the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). the daughter cells will construct a new dividing cell wall between each other. the parent cell will be split in half. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. A new nuclear envelope forms around the separated sister chromosomes.In most eukaryotes. In plant cells. so they are renamed to sister chromosomes. As a matter of convention. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. . the process of binary fission is very much different from the process of mitosis. Eventually. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. each sister chromatid is now considered a chromosome.the cell begins cytokinesis. In animal cells. The chromosomes align themselves in a line spanning the cell. giving rise to two daughter cells. each with a replica of the original genome.
chromosomes are replicated only during the S phase.2. grows more and prepares for mitosis (G 2). the cell grows by producing proteins and cytoplasmic organelles. In highly vacuolated plant cells. S (synthesis). Interphase is divided into three phases: G1 (first gap). All these phases in the interphase are highly regulated. This is achieved through the formation of a phragmosome.1 Preprophase In plant cells only. the nucleus has to migrate into the center of the cell before mitosis can begin. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . 2. and G2 (second gap). It alternates with the much longer interphase. mainly via proteins.2. 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. prophase is preceded by a pre-prophase stage. continues to grow as it duplicates its chromosomes (S). During all three phases. where the cell prepares itself for cell division. a cell grows (G1).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. and finally it divides (M) before restarting the cycle. However. Thus.
Cytokinesis has already begun. instead. The chromosomes have chromatin has condensed. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves.division. preprophase is characterized by the formation of a ring of microtubules and actin filaments (called preprophase band) underneath the plasma membrane around the equatorial plane of the future mitotic spindle. . These microtubules can attach to kinetochores or they can interact with opposing microtubules. degraded. after the nuclear membrane breaks down. In addition to phragmosome formation. aligned at the metaphase plate. and microtubules have invaded the nuclear Prophase space. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. This band marks the position where the cell will eventually divide. The cells of higher plants (such as the flowering plants) lack centrioles. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. the pinched area is known as the cleavage furrow.
A cell inherits a single centrosome at cell division. giving a pair of centrosomes. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. 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. Since the genetic material has already been duplicated earlier in S phase. the replicated chromosomes have two sister chromatids. Close to the nucleus are structures called centrosomes. which is replicated by the cell with the help of the nucleus before a new mitosis begins.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. they are not essential for the . The centrosome is the coordinating center for the cell's microtubules. Although centrioles help organize microtubule assembly. which are made of a pair of centrioles found in most eukaryotic animal cells. At the onset of prophase. bound together at the centromere by the cohesin protein complex. chromatin condenses together into a highly ordered structure called a chromosome.
undergo a variation called closed mitosis where the spindle forms inside the nucleus.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. Each chromosome forms two kinetochores at the centromere. since they are absent from plants. and centrosomes are not always used in mitosis. Fungi and some protists. provides the pulling force necessary to later separate the chromosome's two chromatids. Prometaphase is sometimes considered part of prophase. This motor activity. such as algae or trichomonads. When a microtubule connects with the kinetochore.2. This is called open mitosis. 2. When the spindle grows to sufficient length. . one attached at each chromatid. Although the kinetochore structure and function are not fully understood. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. on an average 20 ). it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. kinetochore microtubules begin searching for kinetochores to attach to. it is known that it contains some form of molecular motor. using energy from ATP to "crawl" up the tube toward the originating centrosome. coupled with polymerisation and depolymerisation of microtubules. and it occurs in most multicellular organisms.formation of the spindle. or its microtubules are able to penetrate an intact nuclear envelope. the motor activates.
in some sense. the kinetochore would be the "hook" that catches a sister chromatid or "fish". The centromeres of the chromosomes. In certain types of cells. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. As a result. only roughly lining up along the midline. 2. All chromosomes (blue) but one have arrived at the metaphase plate. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. convene along the metaphase plate or equatorial plane. the chromosomes come under longitudinal tension from the two ends of the cell. an imaginary line that is equidistant from the two centrosome poles. Metaphase comes from the Greek meaning "after.In the fishing pole analogy.3 Metaphase A cell in late metaphase. ." Microtubules find and attach to kinetochores in prometaphase. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. analogous to a tug-of-war between people of equal strength.
Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). Two events then occur: first. the nonkinetochore microtubules elongate. the cell has succeeded in separating identical copies of the genetic material into two distinct populations.” “against. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. At the end of anaphase.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. The signal creates the mitotic spindle checkpoint. These two stages are sometimes called early and late anaphase. the cell proceeds to anaphase (from the Greek meaning “up. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. . The force that causes the centrosomes to move towards the ends of the cell is still unknown. Next. 2. which have now become distinct sister chromosomes. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell.” “back. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart.” or “re-”). allowing them to separate. These sister chromatids. Early anaphase is usually defined as the separation of the sister chromatids. the proteins that bind sister chromatids together are cleaved.
A new nuclear envelope. forms around each set of separated sister chromosomes. At telophase. cell . a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. In animal cells.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. now surrounded by new nuclei. Cytokinesis is technically not even a phase of mitosis.5 Cytokinesis Cilliate undergoing cytokinesis. Corresponding sister chromosomes attach at opposite ends of the cell. unfold back into chromatin. cytokinesis is a separate process that begins at the same time as telophase. but rather a separate process.2. It "cleans up" the after effects of mitosis. Mitosis is complete. pinching off the separated nuclei. 2. but cell division is not yet complete. In both animal and plant cells. however. using fragments of the parent cell's nuclear membrane. the nonkinetochore microtubules continue to lengthen. elongating the cell even more. Both sets of chromosomes. necessary for completing cell division. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase.
g. 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. e. The phragmoplast is a microtubule structure typical for higher plants. New cells are formed by mitosis and so are exact copies of the cells being replaced. Similarly. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. The end of cytokinesis marks the end of the M-phase.4 Regeneration .5.1Significance Mitosis is important for the maintenance of the chromosomal set.3 Cell replacement In some parts of body.5. 2.division is also driven by vesicles derived from the Golgi apparatus. This is the basis of the development of a multicellular body from a single cell i. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. 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. cells are constantly sloughed off and replaced by new ones. which move along microtubules to the middle of the cell.e.5. zygote and also the basis of the growth of a multicellular body. skin and digestive tract. separating the two nuclei. Following are the occasions in the lives of organism where mitosis happens: 2..2 Development and growth The number of cells within an organism increases by mitosis. 2.5.
resulting in binucleated cells.5. For example. sea star regenerates its lost arm through mitosis. The production of new cells is achieved by mitosis.7 Consequences of errors Although errors in mitosis are rare. These cells are considered aneuploid. especially during early cellular divisions in the zygote. In non-disjunction.Some organisms can regenerate their parts of bodies. they fail to complete cell division and retain both nuclei in one cell.5. The same division happens during asexual reproduction or vegetative propagation in plants. Mitosis continues in the cells of bud and it grows into a new individual. One daughter cell will receive both sister chromosomes and the other will receive none. 2. a condition often associated with cancer.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. For example. The cells at the surface of hydra undergo mitosis and form a mass called bud. the hydra reproduces asexually by budding. 2. . Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. a condition known as trisomy. the process may go wrong. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). a chromosome may fail to separate during anaphase. and the latter cell having only one chromosome (the homologous chromosome). Occasionally when cells experience nondisjunction. a condition known as monosomy.
it may be treated erroneously as a separate chromosome. it results in the formation of Tumors. its organelles disintegrate and reform in a matter of hours. causing translocation. chromosomes may become damaged. Such tumours can send cancer cells to other parts in body where new tumours may form. Or. Benign tumours are not harmful as soon as they are not moving. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. Errors in the control of mitosis may cause cancer. When tissues more than the requirement are synthesized in a single organ. The fragment may incorrectly reattach to another. It results in the synthesis of execessive tissue growths. This phenomenon is called metastasis or spreading of disease. As long as these tumours remain in their original location they are called benign tumours. non-homologous chromosome. sometimes mutuations occur in such genes and cells continue to divide. As soon as they start to move and invade other cells there are said to be malignant tumours. Now what happens is that cell abnormally continue to divide at a single place. It may reattach to the original chromosome. All cells have genes that control the timing and number of mitosis. An arm of the chromosome may be broken and the fragment lost. causing chromosomal duplication. Occasionally. causing deletion. and chromosomes are jostled constantly by probing microtubules. The effect of these genetic abnormalities depends on the specific nature of the error. It results in abnormal cell growth.Mitosis is a demanding process for the cell. which goes through dramatic changes in ultrastructure. causing inversion. but in reverse orientation. .
An example of a cell that goes through endomitosis is the megakaryocyte. carrying genetic information. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. In certain types of cells. align in the middle of the cell before being separated into each of the two daughter cells. an imaginary line that is equidistant from the two centrosome poles. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. 2. Preceded by events in prometaphase and followed by anaphase. Early events of metaphase can . from the ancient Greek(between) and (stage).6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. resulting in cells with many copies of the same chromosome occupying a single nucleus.2. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). only roughly lining up along the middleline. Metaphase accounts for approximately 4% of the cell cycle's duration. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes.7 Metaphase Metaphase. analogous to a tug of war between equally strong people. This process may also be referred to as endoreduplication and the cells as endoploid. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.
cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. securin.coincide with the later events of prometaphase. 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.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. 2. Metaphase chromosomes make the classical picture of chromosomes (karyotype). Staining of the slides. which makes them most suitable for visual analysis. and separase. when every kinetochore is properly attached to a bundle of microtubules. This would be accomplished by regulation of the anaphase-promoting complex. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). produces a pattern of in total up to several hundred bands. often with Giemsa (G banding) or Quinacrine. For classical cytogenetic analyses. does the cell enter anaphase. 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. Only after all chromosomes have become aligned at the metaphase plate. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Normal metaphase spreads are used in . Such a signal creates the mitotic spindle checkpoint.
. losses of chromosomal segments or translocations.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. which may lead to chimeric oncogenes. such as bcr-abl in chronic myelogenous leukemia. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. for example.
in normal diploid organisms. or an individual organism. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). ordered by size and position of centromere for chromosomes of the same size. any differences between the sex chromosomes. and the results may be used in evolutionary biology and medicine. There may. taxonomic relationships. Attention is paid to their length. cellular function. The study of karyotypes is important for cell biology and genetics. Thus. autosomal chromosomes are present in two copies. and what they look like under a light microscope. Karyogram of human male using Giemsa staining. the position of the centromeres. in humans 2n = 46. to study chromosomal aberrations. . The term is also used for the complete set of chromosomes in a species. Karyotypes describe the number of chromosomes. such as. banding pattern.  The preparation and study of karyotypes is part of cytogenetics. or may not. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. be sex chromosomes. Karyotypes can be used for many purposes. So. and any other physical characteristics. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. and to gather information about past evolutionary events. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. The study of whole sets of chromosomes is sometimes known as karyology.
at first favoring 46. Their behavior in animal (salamander) cells was described by Walther Flemming. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. which swells them and spreads the chromosomes . in 1882. New techniques were needed to definitively solve the problem: 1. 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. The name was coined by another German anatomist.3. The next stage took place after the development of genetics in the early 20th century. concluding an XX/XO sex determination mechanism. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. He revised his opinion later from 46 to 48. in contrast to their genic contents. and he correctly insisted on humans having an XX/XY system. the discoverer of mitosis. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Considering their techniques. Pretreating cells in a hypotonic solution. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. The subsequent history of the concept can be followed in the works of Darlington and White. these results were quite remarkable. Using cells in culture 2.
3.2.2 Observations on karyotypes 3. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.1 Staining The study of karyotypes is made possible by staining. 3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Usually. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Observations Six different characteristics of karyotypes are usually observed and compared: . For humans. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. the great apes have 48 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. a suitable dye. is applied after cells have been arrested during cell division by a solution of colchicine. 3.  Sometimes observations may be made on non-dividing (interphase) cells.2. such as Giemsa. Rather interestingly. The sex of an unborn fetus can be determined by observation of interphase cells. reducing the number. Arresting mitosis in metaphase by a solution of colchicines 4.
This is brought about by translocations. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. permitting its loss without penalty to the organism (the dislocation hypothesis). Heterochromatin stains darker than euchromatin. . 2. 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. 4. Differences in absolute sizes of chromosomes. both have six pairs of chromosomes (n=6) yet V. A full account of a karyotype may therefore include the number. 6. indicating tighter packing.1. and mainly consists of genetically inactive repetitive DNA sequences. Differences in number and position of satellites. but the genes have been mostly translocated (added) to other chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). faba chromosomes are many times larger. 5. Humans have one pair fewer chromosomes than the great apes. shape and banding of the chromosomes. type. 3. as well as other cytogenetic information. Differences in degree and distribution of heterochromatic regions. which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in the position of centromeres.
XX. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes.Variation is often found: 1. Geographical variation between races 5. Between the sexes 2. which are highly variable. XY. Normal karyotypes for females contain two X chromosomes and are denoted 46. 3. Between members of a population (chromosome polymorphism) 4. and in . There is variation between species in chromosome number. Between the germ-line and soma (between gametes and the rest of the body) 3. Mosaics or otherwise abnormal individuals. the same cannot be said for their karyotypes. males have both an X and a Y chromosome denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. 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.
some organisms go in for large-scale elimination of heterochromatin. In some species. . In some cases there is even significant variation within species. portions of the chromosomes are cast away in particular cells. despite their construction from the same macromolecules. This variation provides the basis for a range of studies in evolutionary cytology. In this process. entire chromosomes are eliminated during development. as in many sciarid flies.. Chromatin diminution (founding father: Theodor Boveri)..detailed organization. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. 3. In A.3. or other kinds of visible adjustment to the karyotype. Although much is known about karyotypes at the descriptive level. Chromosome elimination.. In a review. Godfrey and Masters conclude: "In our view. it is quite unclear what the general significance might be. which were previously inexplicable. used in conjunction with other phylogenetic data. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed.1 Changes during development Instead of the usual gene repression. But. "We have a very poor understanding of the causes of karyotype evolution. found in some copepods and roundworms such as Ascaris suum.. the general significance of karyotype evolution is obscure. despite many careful investigations. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost.
In marsupials it is always the paternal X which is inactivated. They kept quiet for two or three years because they thought something was wrong with their tissue culture. which was investigated by Kurt Benirschke and his colleague Doris Wurster. When they looked at the karyotype of the closely related Indian muntjac.. 3.. 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 diploid number of the Chinese muntjac. Muntiacus reevesi. The existence of supernumerary or B chromosomes .. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). thus the mammalian female is a mosaic in respect of her X chromosomes. The low record is held by the nematode Parascaris univalens. all telocentric. male = 7 chromosomes. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. they were astonished to find it had female = 6. Muntiacus muntjak. "They simply could not believe what they saw.suum.3. the inactivation is random as between the two Xs. Xinactivation.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. In placental mammals. where the haploid n = 1. all the somatic cell precursors undergo chromatin diminution.. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. In human females some 15% of somatic cells escape inactivation. the high record would be somewhere amongst the ferns. was found to be 46.
3. horsetails and psilotales) is also common. due to the presence of five acrocentric chromosome pairs (13. Humans have FN = 82.means that chromosome number can vary even within one interbreeding population. 14. occurs mainly in plants. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. where there are more than two sets of homologous chromosomes in the cells. and the other haploid. It has been of major significance in plant evolution according to Stebbins.3 Fundamental number The fundamental number. FN. though in this case they would not be regarded as normal members of the population. where one sex is diploid. but it has been significant in some groups.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Polyploidy in animals is much less common. and in some other groups. Haplo-diploidy. It is a common arrangement in the Hymenoptera. Thus. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. 3. FN ≤ 2n.Endopolyploidy occurs when in adult differentiated . 21 and 22).3. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. 15. 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. and aneuploids are another example. but in grasses the average is much higher. about 70%. Polyploidy in lower plants (ferns. Polyploidy.
This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. The cells do not always contain exact multiples (powers of two). but the nuclei contain more than the original somatic number of chromosomes.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. it is diverse and complex. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. Abnormalities in chromosome number usually cause a defect in development. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. 3. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). Down syndrome and Turner syndrome are examples of this.tissues the cells have ceased to divide by mitosis. See palaeopolyploidy for the investigation of ancient karyotype duplications. the daughter chromosomes separating from each other inside an intact nuclear membrane. and serves differentiation and morphogenesis in many ways. . The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. In many instances. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate.
500 sq mi (17. 4. In about 6. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 6. 5.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. the great apes have 24x2 chromosomes whereas humans have 23x2.6 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. some mantids of the genus Ameles. 3.  Closer to home. Well-researched examples are the ladybird beetle Chilocorus stigma.000 km2). There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. living from rainforests to . the chromosome number is variable from one individual to another. Classic examples in plants are the genus Crepis. that the two chromosome morphs are adapted to different habitats. where the gametic (= haploid) numbers form the series x = 3. and 7.Aneuploidy may also occur within a group of closely related species. where every number from x = 3 to x = 15 is represented by at least one species. and Crocus. When this happens. Human chromosome 2 was formed by a merger of ancestral chromosomes. the European shrew Sorex araneus. reducing the number. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. 3.
subalpine meadows. The results are clear. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. especially inversions. Chromosome rearrangements. Drosophila and Scaptomyza. The polytene banding of the 'picture wing' group. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. the best-studied group of Hawaiian drosophilids. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. gene arrangements are visible in the banding patterns of each chromosome. make it possible to see which species are closely related. at least into the Cretaceous. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. but these are much less frequent. which can be dated to 30 mya. the present islands date from 0. In a sense. show a clear "flow" of species from older to newer islands. it is more likely to have been a group from the same species. when plotted in tree form (and independent of all other information). Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). and skipping of islands. Although it would be possible for a single gravid female to colonise an island. The inversions. in the family Drosophilidae. There are also cases of colonization back to older islands. . probably 20 million years ago. Using K-Ar dating.
3. human genome. adaptive radiations. . • T-banding: visualize telomeres.7. if less spectacular. early-replicating and GC rich.7 Depiction of karyotypes 3.the dark regions tend to be heterochromatic. The light regions tend to be euchromatic. late-replicating and AT rich.There are other animals and plants on the Hawaiian archipelago which have undergone similar. This method will normally produce 300-400 bands in a normal. It yields a series of lightly and darkly stained bands . so it stains centromeres. The pattern of bands is very similar to that seen in G-banding.1 Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes: • G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). R-banding is the reverse of G-banding (the R stands for "reverse"). • • C-banding: Giemsa binds to constitutive heterochromatin. • Q-banding is a fluorescent pattern obtained using quinacrine for staining.
less frequently Quinacrine.7. respectively. Each chromosome has a characteristic banding pattern that helps to identify them.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. This yields a dark region where the silver is deposited. Quinacrine binds to the adeninethymine-rich regions. In the "classic" (depicted) karyotype. both chromosomes in a pair will have the same banding pattern. Giemsa is specific for the phosphate groups of DNA. a dye. Karyotypes are arranged with the short arm of the chromosome on top. is used to stain bands on the chromosomes. Some karyotypes call the short and long arms p and q. denoting the activity of rRNA genes within the NOR. For example. 3. In addition. and the long arm on the bottom. Cri du chat syndrome involves a deletion on the short arm of . often Giemsa (G-banding).• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein.
2. Because there are a limited number of spectrally-distinct fluorophores. Image processing software then assigns a pseudo color to each spectrally different combination. a combinatorial labeling method is used to generate many different colors.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. . This method is also known as virtual karyotyping. which is written as 46. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.2) 3. The critical region for this syndrome is deletion of 15.chromosome 5.del(5)(p15. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.7. 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. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.XX. It is written as 46.5p-.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.XX. 3. allowing the visualization of the individually colored chromosomes.
CHAPTER 4 .
a common chromosomal disease. as in derivative chromosome. in which three copies of a chromosome are present instead of the usual two. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. XXY is caused by an extra X chromosome. X0). • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. • • Patau syndrome is caused by trisomy of chromosome 13. the most common male chromosomal disease. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Some disorders arise from loss of just a piece of one chromosome. often occur as a result of nondisjunction during meiosis in the formation of a gamete.4. large-scale deletions or duplications. trisomies.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. as in the presence of extra or missing chromosomes. is caused by trisomy of chromosome 21. are common numerical abnormalities. also known as aneuploidy. translocations. Down syndrome. Structural abnormalities often arise from errors in homologous recombination. or structural. including . trisomy 9 and trisomy 16. although they generally do not survive to birth. Klinefelter syndrome. otherwise known as 47. Numerical abnormalities. Also documented are trisomy 8. X or 45. inversions.
The name comes from the babies' distinctive cry. . 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. from the loss of part of the short arm of chromosome 1. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. a deletion of the paternal genes. They can be organized into two basic groups. example of imprinting disorder. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. numerical and structural anomalies. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. A chromosome anomaly may be detected or confirmed in this manner.• Cri du chat (cry of the cat). There are many types of chromosome anomalies. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. example of imprinting disorder. caused by abnormal formation of the larynx. from a truncated short arm on chromosome 5. one well-documented example is the Philadelphia chromosome. A chromosome anomaly. a deletion of the maternal genes. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. 1p36 Deletion syndrome.
rather than two). In a reciprocal translocation. Duplications: A portion of the chromosome is duplicated. There are two main types of translocations. 4. an X. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). Tetrasomy. and Jacobsen syndrome. • • Translocations: When a portion of one chromosome is transferred to another chromosome. In a Robertsonian translocation. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.3 Structural abnormalities When the chromosome's structure is altered. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. Known disorders in humans include Wolf-Hirschhorn syndrome. segments from two different chromosomes have been exchanged. resulting in extra genetic material. an entire chromosome has .). etc.4. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. also called the terminal 11q deletion disorder. 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.
• Rings: A portion of a chromosome has broken off and formed a circle or ring. 21 and 22. This is why chromosome studies are often performed on parents when a child is found to have an anomaly.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. can happen after conception. 4. Some anomalies. This can happen with or without loss of genetic material. therefore the genetic material is inverted. • Inversions: A portion of the chromosome has broken off. turned upside down and reattached. Therefore. however. 14. and are therefore initially not inherited. resulting in Mosaicism (where some cells have the anomaly and some do not). They often lead to an increased tendency to develop certain types of malignancies. Chromosome anomalies can be inherited from a parent or be "de novo".attached to another at the Centromere . as well . 15. It includes routine analysis of G-Banded chromosomes. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. especially the chromosomes.in humans these only occur with chromosomes 13. other cytogenetic banding techniques.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. 4. the anomaly is present in every cell of the body.
in contrast to their genic contents. Their behavior in animal (salamander) cells was described by Walther Flemming. at first favoring 46. Pre-treating cells in a hypotonic solution. von Waldeyer in 1888. Considering their techniques. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. He revised his opinion later from 46 to 48. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. concluding an XX/XO sex determination mechanism. and he correctly insisted on man having an XX/XY system. the discoverer of mitosis.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. New techniques were needed to definitively solve the problem: 1. which swells them and spreads the chromosomes . these results were quite remarkable. The name was coined by another German anatomist. Using cells in culture 2. 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. The next stage took place after the development of genetics in the early 20th century. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. in 1882.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.
Squashing the preparation on the slide forcing the chromosomes into a single plane 5.3. the great apes have 48 chromosomes.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. a find which eventually led to her Nobel Prize in 1983. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. In 1931. During her cytogenetic work. McClintock discovered transposons. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.6. Rather interestingly. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.6 Applications in biology 4. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Using Painter's technique they studied the polytene . reducing the number. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Human chromosome 2 was formed by a merger of ancestral chromosomes. 4. persimilis from wild populations in California and neighboring states. Arresting mitosis in metaphase by a solution of colchicine 4.6. 4.2 Natural populations of Drosophila In the 1930s.
. Dobzhansky bred populations in population cages. Using a method invented by L'Heretier and Teissier. such as Down's syndrome. 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. breeding and sampling whilst preventing escape.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. Down syndrome is also referred to as trisomy 21. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. This had the benefit of eliminating migration as a possible explanation of the results. as they would if selectively neutral. In 1959. Evidence rapidly accumulated to show that natural selection was responsible. which enabled feeding. discoveries were quickly made related to aberrant chromosomes or chromosome number. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. as with most polymorphisms. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. 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. In some congenital disorders. It was found that the various chromosome types do not fluctuate at random.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. but adjust to certain frequencies at which they become stabilised.
The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. has Klinefelter's Syndrome.as both scientists were doing their research in Philadelphia. Many other sex chromosome combinations are compatible with live birth including XXX. XYY. is used today as a diagnostic for CML. resulting in 47 total chromosomes. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. Identification of the Philadelphia chromosome by cytogenetics. In 1960. an additional X chromosome in a male. and XXXX. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). An individual with only one sex chromosome (the X) has Turner syndrome. . This abnormal chromosome was dubbed the Philadelphia chromosome . Pennsylvania. which is required in normal females to compensate for having two copies of the chromosome.Other numerical abnormalities discovered include sex chromosome abnormalities. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. in addition to other tests. Thirteen years later. with the development of more advanced techniques. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. Not all genes on the X Chromosome are inactivated.
Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid.FIG Advent of banding techniques In the late 1960s. Deletions within one chromosome could also now be more specifically named and understood. Caspersson developed banding techniques which differentially stain chromosomes. and elongation techniques for all culture types that allow for higher resolution banding.8 Beginnings of molecular cytogenetics . Deletion syndromes such as DiGeorge syndrome. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 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. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.
This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. 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. cloned and studied in ever greater detail. While radioisotope-labeled probes had been hybridized with DNA since 1969. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. CHAPTER 5 Techniques 5.In the 1980s.1 Karyotyping . 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,
For congenital problems usually 20 metaphase cells are scored. CGH and Single nucleotide polymorphism-arrays. test and measurement. . Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. control design. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. C++. You can use MATLAB in a wide range of applications. communications. you can solve technical computing problems faster than with traditional programming languages. data analysis. including signal and image processing. and FORTRAN. and numerical computation.generally between 200 and 1000 cells are counted and scored. and computational biology. 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. such as C. financial modeling and analysis. data visualization. Using MATLAB.
6.MATLAB provides a number of features for documenting and sharing your work.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. In many cases. These effects must be compensated to improve the results of the pairing algorithm. The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted. specifying data types. With the MATLAB language. . and allocating memory. It enables fast development and execution. As a result. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. MATLAB eliminates the need for ‘for’ loops. You can integrate your MATLAB code with other languages and applications. and distribute your MATLAB algorithms and applications. The image processing step is composed of the following operations. such as declaring variables. one line of MATLAB code can often replace several lines of C or C++ code.
or at least attenuated. 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. To compare chromosomes from a band pattern point of view. 6. 2) Geometrical compensation—The geometric compensation. 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 Concepts used in this phase 1) Image conversion 2) Denoising . the spatially scaled images are histogram equalized. 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. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. Therefore.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. To compensate for this inhomogeneity.
you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. 6. as is appropriate.I). For example.I. if you want to filter a color image that is stored as an indexed image.I. If you attempt to filter the indexed image. For example. MATLAB simply applies the filter to the indices in the indexed image matrix. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. For example. and the results might not be meaningful. . there are other functions that return a different image type as part of the operation they perform. so the image displays as shades of gray. When you apply the filter to the true color image. you must first convert it to true color format. listed in the following table. You can perform certain conversions just using MATLAB syntax.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. In addition to these image type conversion functions. MATLAB filters the intensity values in the image.3) Edge detection 4) Two dimensional convolutions. The resulting true color image has identical matrices for the red. RGB = cat (3. green.2. and blue planes.
. We may use edges to measure the size of objects in an image.3 Two dimensional convolutions C = conv2(A. via satellite or wireless transmission.5 Edge detection Edges contain some of the most useful information in an image. to recognize or classify objects. These errors will appear on the image output in different ways depending on the type of disturbance in the signal.4 Denoising We may define noise to be any degradation in the image signal.'method'. we may expect errors to occur in the image signal. caused by external disturbance. hence we can choose the most appropriate method for reducing the effects.parameters. Usually we know what type of errors to expect. 6. and we shall look at some of the more straightforward of them. . and hence the type of noise on the image. or through networked cable. If an image is being sent electronically from one place to another. ) Where the parameters available depend on the method used 6. to isolate particular objects from their background.2.2. If one of these matrices describes a two-dimensional finite impulse response . The general Matlab command for finding edges is edge(image.6.B) computes the two-dimensional convolution of matrices A and B. There is a large number of edge finding algorithms in existence. Cleaning an image corrupted by noise is thus an important area of image restoration.
If hcol is a column vector and hrow is a row vector. That is. nb]+1)/2)..na] and the size of B is [mb.'shape') subsection of the two-dimensional convolution. C = conv2(. .. the other matrix is filtered in two dimensions. rgb2gray im2bw(im.hrow. edge(im1.[3 3]).(FIR) filter. imedfilt2(im1.na+nb-1].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. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.A).. The size of matrices.0.nb].7). if the size of then the size of C is [ma+mb-1.'sobel'). as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. minus one.bmp').
n]=size(L).j)~=0 for k=1:mx if L(i.y1)=255.[imx. MODULE 2 clc [m.1). mx=max(max(L)).2). for i=1:sx x1=rc(i. Msk conv2(double(BW).imy).1). n1(x1.imy]=size(BW). y1=rc(i. [sx sy]=size(rc). [r. for i=1:m for j=1:n if L(i.c] = find(L==22).j)==L_number(k) flag=1. nzeros(imx. L_number=zeros(mx. Index=1. flag=0. bwlabel(B.double(msk)). rc = [r c].8). .
j).8. end flag=0.40.56.29.62.27. Index=Index+1. rc = [r c]. for x=1:46 [r.26. for i=1:sx x1=rc(i. end %h=figure.9.imshow(n188.8.131.52. [sx sy]=size(rc).184.108.40.206. 36.y1)=255. end.41.49.imy).39.11.1).22.45. Test_number=[3. n1(x1.54. y1=rc(i.220.127.116.11.).18.104.22.168.end end if flag~=1 L_number(Index)=L(i.c] = find(L==L_number((Test_number(x)))). .55. n1=zeros(imx.38.43.66].22.214.171.124.2).126.96.36.199. end end L_number.65.21.
'skel'.y)==1 Circumference_sum=Circumference_sum+1.'. Circumference_sum=0. for x=1:m for y=1:n if BW1(x. skel=im2bw(skel.bmp')).5*graythresh(skel)). Arm_length_sum=0.1.end Circumference=zeros(46.1). BW1=edge(BW.1). s1=bwmorph(s. for i=1:46 f=imread(strcat(num2str(i). Area=zeros(46.Inf). s=bwmorph(skel.1). Arm_length=zeros(46. . BW=im2bw(f). BW=double(BW). [m n]=size(BW1). f=imcomplement(f).8). skel=im2double(f).'spur'.'canny'). end end end Circumference(i)=Circumference_sum.
end end end Area(i)=Area_sum. Arm_length. BW=im2bw(f).y)==1 Area_sum=Area_sum+1. end end end Arm_length(i)=Arm_length_sum. Area_sum=0. . [m n]=size(BW). for x=1:m for y=1:n if s1(x.y)==1 Arm_length_sum=Arm_length_sum+1. for x=1:m for y=1:n if BW(x.[m n]=size(s1). end Circumference.
1)=46. end end Pair.2).2)=i+1. Pair(i. Pair(46. 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. Pair=zeros(46. . Pair(i.Area. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)).1)=i.2)=j.2)=i.2)==46 Pair(46. end end end for i=1:45 if Pair(i.1)=i. Pair(i.
end flag=0.1). end f2=imread(strcat(num2str(Pair(i.bmp')). if figure_flag~=47 subplot(23.2. figure_flag=figure_flag+1.1)). figure_flag=figure_flag+1.2).figure_flag). flag=0.2.bmp')). imshow(f2). end end if flag~=1 if figure_flag~=47 subplot(23.2)). . figure_flag=1. for i=1:46 for j=1:46 if Pair(i.figure_flag).'.delete=zeros(46. end f1=imread(strcat(num2str(Pair(i. delete(figure_flag)=Pair(i. imshow(f1).'.1)==delete(j) flag=1.
The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. Copenhagen. 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 proposed algorithm is based on the traditional features extracted from the karyogram. in the scope of karyotyping process used in cytogentic analysis. and Philadelphia. based on the MI. dimensions and banding profiles. such as.end CONCLUTION In this paper. 2) feature extraction from the processed images . plus a new one. 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. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern.
shape. and to normalize their dimensions. the romosome images. 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 .characterizing the size. by minimizing the overall distances between chromosomes of the same class (intraclass) and by maximizing the distances between chromosomes when at least one of them does not belong to that class (interclass). The training process consists in the estimation of each vector of coefficient . shape and band pattern. 4) pairing. are processed in order to compensate for geometrical and intensity distortions. Here. and band pattern. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. 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. In the image processing step. and finally. achieves a 70. extracted from the unordered karyogram. Tests using 19 karyograms based on bone marrow cells. This normalization is needed to make it possible the band pattern comparison between chromosomes. 3) training of a classifier (performed once) where similarity among chromosomes are characterized.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. The features extracted from the processed images discriminate each pair with respect to their size. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. from the chromosomes in the training set.working within an 8-D feature space.10% mean classification rate. 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.
such as Edinburgh. Executing the algorithm on a higher quality dataset. a new chromosome dataset with 9200 chromosomes from bone marrow cells. 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. a 76.10% classification ratewas obtained. The results presented in this paper are promising. called LK1 . amean classification rate larger than 93% was obtained in all experiments. This dataset was made publicly available . it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset.performance of the classifier. whose images are of significantly higher quality.. e. Copenhagen. centromere position. Using 27 karyograms andworking with a limited number of classes (≤ 8). or Philadelphia. and from which it is possible to extract additional features. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. In fact. In addition. despite the low quality of this type of chromosomes.g. REFERENCES .
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