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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
In prokaryotes. which is tightly coiled in on itself. Chromosomes are the essential unit for cellular division and must be replicated. The structure of chromosomes and chromatin varies through the cell cycle. In prokaryotes and viruses. In practice "chromosome" is a rather loosely defined term. DNA is usually arranged as a circle. circular DNA molecules called plasmids. cells may contain more than one type of chromosome. Chromosomal recombination plays a vital role in genetic diversity. Also. Chromosomes may exist as either duplicated or unduplicated. However. although there are many exceptions to this rule. the term genophore is more appropriate when no chromatin is present. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. the cell may undergo mitotic catastrophe and die. If these structures are manipulated incorrectly. Unduplicated chromosomes are single linear strands. through processes known as chromosomal instability and translocation. In eukaryotes. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. a large body of work uses the term chromosome regardless of chromatin content.defined nuclei) have smaller circular chromosomes. This allows the very long DNA molecules to fit into the cell nucleus. These small circular genomes . for example. or it may unexpectedly evadeapoptosis leading to the progression of cancer. 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. divided. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). sometimes accompanied by one or more smaller.
copies of chromosome 21. 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.+21. in which an individual has 3. 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). XX (female) or 46 XY (male).g. Euploid human karyotypes are 46.are also found in mitochondria and chloroplasts.3 MUTATIONS IN CHROMOSOME NUMBER Normally. . The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. The individual would have Down Syndrome and his/her karyotype would be written 47.+21. 1.XY or 47. reflecting their bacterial origins. a extra copy of human chromosome 21). An example of aneuploidy is trisomy 21. Such individuals are called euploid and have the wild-type chromosome complement for the species. rather than 2. 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.
The microtubules then pull the chromatids apart toward the centrosomes. The shorter arms are called p arms (from the French petit. 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. Once the cells have divided. In spite of their appearance. so that each daughter cell inherits one set of chromatids. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. q-g "grande"). microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores.Fig 1. This is the only natural context in which individual chromosomes are visible with an optical microscope. one of which is present on each sister chromatid. and they form the classic four arm structure. . This compact form makes the individual chromosomes visible. 1. which enables these giant DNA structures to be contained within a cell nucleus. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. the chromatids are uncoiled and DNA can again be transcribed. the chromatin strands become more and more condensed. A special DNA base sequence in the region of the kinetochores provides. chromosomes are structurally highly condensed. longer-lasting attachment in this region.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). along with special proteins. During mitosis.
 Bone marrow is also a key component of the lymphatic system. producing the lymphocytes that support the body's immune system CHAPTER 2 2. . Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. cytoplasm. On average.7 lbs). The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.6 kg (5. which use the bone marrow vasculature as a conduit to the body's systemic circulation. bone marrow in large bones produces new blood cells. in two separate nuclei.1. in adults weighing 65 kg (143 lbs).5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. bone marrow constitutes 4% of the total body mass of humans.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. It is generally followed immediately by cytokinesis. which divides the nuclei. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. bone marrow accounts for approximately 2. In humans.
where chromosomes divide within an intact cell nucleus. However. prometaphase. . forming single cells with multiple nuclei. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. cytokinesis and mitosis may occur independently. The process of mitosis is fast and highly complex.genetically identical to each other and to their parent cell. 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. there are many cells where mitosis and cytokinesis occur separately. animals undergo an "open" mitosis. divide by a process called binary fission. for instance during certain stages of fruit fly embryonic development. This occurs most notably among the fungi and slime moulds. where the nuclear envelope breaks down before the chromosomes separate. Even in animals. prophase. For example. metaphase. Because cytokinesis usually occurs in conjunction with mitosis. The cell then divides in cytokinesis. Mitosis occurs only in eukaryotic cells and the process varies in different species. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. This accounts for approximately 10% of the cell cycle. but is found in various different groups. anaphase and telophase. "mitosis" is often used interchangeably with "mitotic phase". These stages are interphase. to produce two identical daughter cells which are still diploid cells. which lack a nucleus. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell.
Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. 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. This occurs during the S phase of interphase. Each chromosome now has an identical copy of itself. . The sister chromatids are held together by a specialized region of the chromosome known as the centromere. and together the two are called sister chromatids. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. Because each resultant daughter cell should be genetically identical to the parent cell.
giving rise to two daughter cells.the cell begins cytokinesis. each with a replica of the original genome. As the cell elongates. separating the two developing nuclei. A new nuclear envelope forms around the separated sister chromosomes. In animal cells. However. corresponding sister chromosomes are pulled toward opposite ends. the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). the nuclear envelope which segregates the DNA from the cytoplasm disassembles. so they are renamed to sister chromosomes. As mitosis completes. 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. Prokaryotic cells undergo a process similar to mitosis called binary fission. The chromosomes align themselves in a line spanning the cell. the parent cell will be split in half. pulling apart the sister chromatids of each chromosome. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten.In most eukaryotes. each sister chromatid is now considered a chromosome. In plant cells. Eventually. the daughter cells will construct a new dividing cell wall between each other.
grows more and prepares for mitosis (G 2). the nucleus has to migrate into the center of the cell before mitosis can begin.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. prophase is preceded by a pre-prophase stage. chromosomes are replicated only during the S phase.1 Preprophase In plant cells only. and finally it divides (M) before restarting the cycle. During all three phases. However. This is achieved through the formation of a phragmosome.2. the cell grows by producing proteins and cytoplasmic organelles.2. and G2 (second gap). 2. Thus. It alternates with the much longer interphase. The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. Interphase is divided into three phases: G1 (first gap). continues to grow as it duplicates its chromosomes (S). a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . a cell grows (G1). In highly vacuolated plant cells. mainly via proteins. All these phases in the interphase are highly regulated. S (synthesis). where the cell prepares itself for cell division.
aligned at the metaphase plate. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. These microtubules can attach to kinetochores or they can interact with opposing microtubules. after the nuclear membrane breaks down. degraded. 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 chromosomes have chromatin has condensed. The cells of higher plants (such as the flowering plants) lack centrioles. instead. . In addition to phragmosome formation. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. the pinched area is known as the cleavage furrow. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes.division. This band marks the position where the cell will eventually divide. Cytokinesis has already begun. and microtubules have invaded the nuclear Prophase space.
which are made of a pair of centrioles found in most eukaryotic animal cells. Close to the nucleus are structures called centrosomes. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. Chromosomes are typically visible at high magnification through a light microscope. the replicated chromosomes have two sister chromatids. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. bound together at the centromere by the cohesin protein complex. Since the genetic material has already been duplicated earlier in S phase.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. giving a pair of centrosomes. At the onset of prophase. they are not essential for the . The centrosome is the coordinating center for the cell's microtubules. chromatin condenses together into a highly ordered structure called a chromosome. Although centrioles help organize microtubule assembly. which is replicated by the cell with the help of the nucleus before a new mitosis begins. A cell inherits a single centrosome at cell division. the genetic material in the nucleus is in a loosely bundled coil called chromatin.
2. undergo a variation called closed mitosis where the spindle forms inside the nucleus. Although the kinetochore structure and function are not fully understood. or its microtubules are able to penetrate an intact nuclear envelope. using energy from ATP to "crawl" up the tube toward the originating centrosome. 2. Fungi and some protists. . and it occurs in most multicellular organisms. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. kinetochore microtubules begin searching for kinetochores to attach to. provides the pulling force necessary to later separate the chromosome's two chromatids. This is called open mitosis. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. on an average 20 ). and centrosomes are not always used in mitosis. This motor activity. one attached at each chromatid. Each chromosome forms two kinetochores at the centromere. coupled with polymerisation and depolymerisation of microtubules. the motor activates. When a microtubule connects with the kinetochore. Prometaphase is sometimes considered part of prophase. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. When the spindle grows to sufficient length. since they are absent from plants. it is known that it contains some form of molecular motor. such as algae or trichomonads.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space.formation of the spindle.
The centromeres of the chromosomes. Metaphase comes from the Greek meaning "after. in some sense. As a result. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. 2. analogous to a tug-of-war between people of equal strength. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.3 Metaphase A cell in late metaphase.In the fishing pole analogy. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". . All chromosomes (blue) but one have arrived at the metaphase plate. an imaginary line that is equidistant from the two centrosome poles. only roughly lining up along the midline." Microtubules find and attach to kinetochores in prometaphase. the kinetochore would be the "hook" that catches a sister chromatid or "fish". 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. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. the chromosomes come under longitudinal tension from the two ends of the cell. convene along the metaphase plate or equatorial plane.
are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. Two events then occur: first. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. Early anaphase is usually defined as the separation of the sister chromatids. These two stages are sometimes called early and late anaphase. The force that causes the centrosomes to move towards the ends of the cell is still unknown.” or “re-”). These sister chromatids. allowing them to separate. . The signal creates the mitotic spindle checkpoint. 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. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell.” “back. the proteins that bind sister chromatids together are cleaved. the cell proceeds to anaphase (from the Greek meaning “up. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. 2. which have now become distinct sister chromosomes. Next. the nonkinetochore microtubules elongate.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres).” “against.
with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. In both animal and plant cells. the nonkinetochore microtubules continue to lengthen. Both sets of chromosomes. pinching off the separated nuclei. 2. In animal cells. forms around each set of separated sister chromosomes. 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. however. but cell division is not yet complete. necessary for completing cell division. using fragments of the parent cell's nuclear membrane.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. Cytokinesis is technically not even a phase of mitosis.5 Cytokinesis Cilliate undergoing cytokinesis. A new nuclear envelope. cell . Corresponding sister chromosomes attach at opposite ends of the cell. unfold back into chromatin. elongating the cell even more. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. Mitosis is complete. but rather a separate process.2.
division is also driven by vesicles derived from the Golgi apparatus. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.5.5.g. The phragmoplast is a microtubule structure typical for higher plants.3 Cell replacement In some parts of body. This is the basis of the development of a multicellular body from a single cell i.5.4 Regeneration . Similarly. 2. 2.1Significance Mitosis is important for the maintenance of the chromosomal set. cells are constantly sloughed off and replaced by new ones.5. e. skin and digestive tract.. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. zygote and also the basis of the growth of a multicellular body. Each daughter cell has a complete copy of the genome of its parent cell. separating the two nuclei. The end of cytokinesis marks the end of the M-phase.2 Development and growth The number of cells within an organism increases by mitosis. Following are the occasions in the lives of organism where mitosis happens: 2. which move along microtubules to the middle of the cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. 2.e. whereas some green algae use a phycoplast microtubule array during cytokinesis. New cells are formed by mitosis and so are exact copies of the cells being replaced.
Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. and the latter cell having only one chromosome (the homologous chromosome). The same division happens during asexual reproduction or vegetative propagation in plants. a condition often associated with cancer. Occasionally when cells experience nondisjunction. the hydra reproduces asexually by budding. For example.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. they fail to complete cell division and retain both nuclei in one cell. 2. the process may go wrong. These cells are considered aneuploid. a condition known as trisomy. sea star regenerates its lost arm through mitosis. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). especially during early cellular divisions in the zygote. One daughter cell will receive both sister chromosomes and the other will receive none. For example. In non-disjunction. Mitosis continues in the cells of bud and it grows into a new individual.7 Consequences of errors Although errors in mitosis are rare. a chromosome may fail to separate during anaphase. 2.5. .Some organisms can regenerate their parts of bodies. The production of new cells is achieved by mitosis. resulting in binucleated cells.5. The cells at the surface of hydra undergo mitosis and form a mass called bud. a condition known as monosomy.
Benign tumours are not harmful as soon as they are not moving. Now what happens is that cell abnormally continue to divide at a single place. . It results in abnormal cell growth. causing deletion. non-homologous chromosome. Or. Errors in the control of mitosis may cause cancer.Mitosis is a demanding process for the cell. which goes through dramatic changes in ultrastructure. An arm of the chromosome may be broken and the fragment lost. The fragment may incorrectly reattach to another. chromosomes may become damaged. It may reattach to the original chromosome. causing chromosomal duplication. As long as these tumours remain in their original location they are called benign tumours. It results in the synthesis of execessive tissue growths. and chromosomes are jostled constantly by probing microtubules. causing inversion. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. its organelles disintegrate and reform in a matter of hours. This phenomenon is called metastasis or spreading of disease. Occasionally. As soon as they start to move and invade other cells there are said to be malignant tumours. causing translocation. When tissues more than the requirement are synthesized in a single organ. All cells have genes that control the timing and number of mitosis. but in reverse orientation. sometimes mutuations occur in such genes and cells continue to divide. Such tumours can send cancer cells to other parts in body where new tumours may form. it results in the formation of Tumors. The effect of these genetic abnormalities depends on the specific nature of the error. it may be treated erroneously as a separate chromosome.
7 Metaphase Metaphase. In certain types of cells. Preceded by events in prometaphase and followed by anaphase. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. An example of a cell that goes through endomitosis is the megakaryocyte. from the ancient Greek(between) and (stage). resulting in cells with many copies of the same chromosome occupying a single nucleus. This process may also be referred to as endoreduplication and the cells as endoploid.2.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. carrying genetic information. an imaginary line that is equidistant from the two centrosome poles. 2. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. Metaphase accounts for approximately 4% of the cell cycle's duration. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). analogous to a tug of war between equally strong people. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Early events of metaphase can . align in the middle of the cell before being separated into each of the two daughter cells. only roughly lining up along the middleline.
Metaphase chromosomes make the classical picture of chromosomes (karyotype). Staining of the slides. 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). Such a signal creates the mitotic spindle checkpoint. securin.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. when every kinetochore is properly attached to a bundle of microtubules. Normal metaphase spreads are used in . and separase. This would be accomplished by regulation of the anaphase-promoting complex. 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. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. produces a pattern of in total up to several hundred bands. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. often with Giemsa (G banding) or Quinacrine.coincide with the later events of prometaphase. Only after all chromosomes have become aligned at the metaphase plate. For classical cytogenetic analyses. Chromosomes are condensed(Thickened) and highly coiled in metaphase. which makes them most suitable for visual analysis. does the cell enter anaphase. 2. One of the cell cycle checkpoints occurs during prometaphase and metaphase.
which may lead to chimeric oncogenes. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. for example.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. such as bcr-abl in chronic myelogenous leukemia. losses of chromosomal segments or translocations. .
In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). banding pattern. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. or an individual organism. in humans 2n = 46. or may not. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies.  The preparation and study of karyotypes is part of cytogenetics. Thus. and what they look like under a light microscope. in normal diploid organisms. the position of the centromeres. be sex chromosomes. The term is also used for the complete set of chromosomes in a species. Karyotypes describe the number of chromosomes. Attention is paid to their length. ordered by size and position of centromere for chromosomes of the same size.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. such as. So. to study chromosomal aberrations. autosomal chromosomes are present in two copies. . taxonomic relationships. The study of whole sets of chromosomes is sometimes known as karyology. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. Karyogram of human male using Giemsa staining. There may. The study of karyotypes is important for cell biology and genetics. cellular function. any differences between the sex chromosomes. Karyotypes can be used for many purposes. and to gather information about past evolutionary events. and the results may be used in evolutionary biology and medicine. and any other physical characteristics.
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. concluding an XX/XO sex determination mechanism. Their behavior in animal (salamander) cells was described by Walther Flemming. in contrast to their genic contents. The subsequent history of the concept can be followed in the works of Darlington and White.3. Using cells in culture 2. Considering their techniques. and he correctly insisted on humans having an XX/XY system. the discoverer of mitosis. which swells them and spreads the chromosomes . The next stage took place after the development of genetics in the early 20th century. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. at first favoring 46. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. He revised his opinion later from 46 to 48. von Waldeyer in 1888. Pretreating cells in a hypotonic solution. New techniques were needed to definitively solve the problem: 1. in 1882. these results were quite remarkable. 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. 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.
Human chromosome 2 was formed by a merger of ancestral chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.2 Observations on karyotypes 3. such as Giemsa.1 Staining The study of karyotypes is made possible by staining. a suitable dye.3. the great apes have 48 chromosomes.2. reducing the number.2. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. 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 Observations Six different characteristics of karyotypes are usually observed and compared: . Arresting mitosis in metaphase by a solution of colchicines 4. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. For humans. Usually. 3. The sex of an unborn fetus can be determined by observation of interphase cells. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Rather interestingly.
both have six pairs of chromosomes (n=6) yet V. 3. A full account of a karyotype may therefore include the number. 4. indicating tighter packing. faba chromosomes are many times larger. Differences in degree and distribution of heterochromatic regions. but the genes have been mostly translocated (added) to other chromosomes. Humans have one pair fewer chromosomes than the great apes. as well as other cytogenetic information. This feature probably reflects different amounts of DNA duplication.1. 2. 6. Differences in the position of centromeres. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). . This is brought about by translocations. Heterochromatin stains darker than euchromatin. permitting its loss without penalty to the organism (the dislocation hypothesis). shape and banding of the chromosomes. Differences in number and position of satellites. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. Differences in absolute sizes of chromosomes. type. and mainly consists of genetically inactive repetitive DNA sequences. which (when they occur) are small bodies attached to a chromosome by a thin thread. 5.
3 The human karyotype Most (but not all) species have a standard karyotype. and in . Between the sexes 2. Mosaics or otherwise abnormal individuals. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Between members of a population (chromosome polymorphism) 4. which are highly variable. XY. Any variation from the standard karyotype may lead to developmental abnormalities. males have both an X and a Y chromosome denoted 46. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Geographical variation between races 5.Variation is often found: 1. Normal karyotypes for females contain two X chromosomes and are denoted 46. Between the germ-line and soma (between gametes and the rest of the body) 3. the same cannot be said for their karyotypes. XX. 3. There is variation between species in chromosome number.
"We have a very poor understanding of the causes of karyotype evolution. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. Godfrey and Masters conclude: "In our view. the general significance of karyotype evolution is obscure.3. portions of the chromosomes are cast away in particular cells. In a review. as in many sciarid flies. used in conjunction with other phylogenetic data.1 Changes during development Instead of the usual gene repression. Although much is known about karyotypes at the descriptive level. it is quite unclear what the general significance might be. In some cases there is even significant variation within species. Chromatin diminution (founding father: Theodor Boveri).. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. which were previously inexplicable.. This variation provides the basis for a range of studies in evolutionary cytology... despite many careful investigations. In A. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. In this process. But. 3. entire chromosomes are eliminated during development. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species.detailed organization. . found in some copepods and roundworms such as Ascaris suum. despite their construction from the same macromolecules. some organisms go in for large-scale elimination of heterochromatin. or other kinds of visible adjustment to the karyotype. Chromosome elimination. In some species.
3.. thus the mammalian female is a mosaic in respect of her X chromosomes. was found to be 46. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. the inactivation is random as between the two Xs. The diploid number of the Chinese muntjac. In placental mammals. 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. Muntiacus muntjak. all telocentric. 3. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation).2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. Xinactivation.. the high record would be somewhere amongst the ferns.. "They simply could not believe what they saw. In marsupials it is always the paternal X which is inactivated. they were astonished to find it had female = 6. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.. all the somatic cell precursors undergo chromatin diminution. They kept quiet for two or three years because they thought something was wrong with their tissue culture. Muntiacus reevesi. The existence of supernumerary or B chromosomes . which was investigated by Kurt Benirschke and his colleague Doris Wurster. In human females some 15% of somatic cells escape inactivation.suum. When they looked at the karyotype of the closely related Indian muntjac. male = 7 chromosomes. The low record is held by the nematode Parascaris univalens. where the haploid n = 1.
Endopolyploidy occurs when in adult differentiated . 21 and 22). FN ≤ 2n. It is a common arrangement in the Hymenoptera. due to the presence of five acrocentric chromosome pairs (13. where there are more than two sets of homologous chromosomes in the cells.means that chromosome number can vary even within one interbreeding population. Thus. Haplo-diploidy. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. 3. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present.3 Fundamental number The fundamental number. occurs mainly in plants. where one sex is diploid. Polyploidy. but it has been significant in some groups. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. horsetails and psilotales) is also common. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. 14. and aneuploids are another example. It has been of major significance in plant evolution according to Stebbins. Polyploidy in lower plants (ferns. about 70%. though in this case they would not be regarded as normal members of the population. 3. Humans have FN = 82. but in grasses the average is much higher.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. and the other haploid.3. FN. and in some other groups. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. Polyploidy in animals is much less common. 15.
The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. See palaeopolyploidy for the investigation of ancient karyotype duplications. 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 daughter chromosomes separating from each other inside an intact nuclear membrane. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. it is diverse and complex. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. In many instances. but the nuclei contain more than the original somatic number of chromosomes. . Down syndrome and Turner syndrome are examples of this. Abnormalities in chromosome number usually cause a defect in development. and serves differentiation and morphogenesis in many ways.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. 3. The cells do not always contain exact multiples (powers of two).tissues the cells have ceased to divide by mitosis. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted).
5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. and Crocus. Human chromosome 2 was formed by a merger of ancestral chromosomes. where every number from x = 3 to x = 15 is represented by at least one species. and 7. the European shrew Sorex araneus. In about 6.500 sq mi (17. 4.000 km2).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. where the gametic (= haploid) numbers form the series x = 3. 6. that the two chromosome morphs are adapted to different habitats. 3. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 3. 5. When this happens. Well-researched examples are the ladybird beetle Chilocorus stigma. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups.  Closer to home. the great apes have 24x2 chromosomes whereas humans have 23x2. Classic examples in plants are the genus Crepis. the chromosome number is variable from one individual to another.Aneuploidy may also occur within a group of closely related species. reducing the number. living from rainforests to . some mantids of the genus Ameles.
The inversions. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. in the family Drosophilidae. at least into the Cretaceous. Chromosome rearrangements. make it possible to see which species are closely related. The results are clear. especially inversions. and skipping of islands. The polytene banding of the 'picture wing' group. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. Using K-Ar dating. but these are much less frequent. it is more likely to have been a group from the same species.subalpine meadows. 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). gene arrangements are visible in the banding patterns of each chromosome. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. probably 20 million years ago. Drosophila and Scaptomyza. There are also cases of colonization back to older islands. show a clear "flow" of species from older to newer islands. the best-studied group of Hawaiian drosophilids. Although it would be possible for a single gravid female to colonise an island. which can be dated to 30 mya. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. In a sense. . the present islands date from 0. enabled Carson to work out the evolutionary tree long before genome analysis was practicable.
adaptive radiations. 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. • T-banding: visualize telomeres. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).7. This method will normally produce 300-400 bands in a normal. if less spectacular.the dark regions tend to be heterochromatic. 3. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. . late-replicating and AT rich.There are other animals and plants on the Hawaiian archipelago which have undergone similar.7 Depiction of karyotypes 3. The light regions tend to be euchromatic. human genome. so it stains centromeres. early-replicating and GC rich. R-banding is the reverse of G-banding (the R stands for "reverse"). • • C-banding: Giemsa binds to constitutive heterochromatin. It yields a series of lightly and darkly stained bands .
Karyotypes are arranged with the short arm of the chromosome on top. Giemsa is specific for the phosphate groups of DNA. a dye. 3.7. Some karyotypes call the short and long arms p and q. Quinacrine binds to the adeninethymine-rich regions. often Giemsa (G-banding). both chromosomes in a pair will have the same banding pattern. Each chromosome has a characteristic banding pattern that helps to identify them. denoting the activity of rRNA genes within the NOR. Cri du chat syndrome involves a deletion on the short arm of . In the "classic" (depicted) karyotype. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. For example. less frequently Quinacrine. is used to stain bands on the chromosomes.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. and the long arm on the bottom. respectively. In addition.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. This yields a dark region where the silver is deposited.
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. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different 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. which is written as 46.5p-. . 3.2) 3.7. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. It is written as 46.3 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. allowing the visualization of the individually colored chromosomes.XX.chromosome 5.XX. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. The critical region for this syndrome is deletion of 15.2. 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. Because there are a limited number of spectrally-distinct fluorophores.
CHAPTER 4 .
as in derivative chromosome. Numerical abnormalities. the most common male chromosomal disease. although they generally do not survive to birth.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. are common numerical abnormalities. large-scale deletions or duplications. translocations. XXY is caused by an extra X chromosome. is caused by trisomy of chromosome 21. including . X or 45. Klinefelter syndrome. trisomy 9 and trisomy 16. often occur as a result of nondisjunction during meiosis in the formation of a gamete. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. as in the presence of extra or missing chromosomes. also known as aneuploidy. trisomies. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. X0). inversions. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. or structural. Down syndrome. Structural abnormalities often arise from errors in homologous recombination. Also documented are trisomy 8. 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. Some disorders arise from loss of just a piece of one chromosome. in which three copies of a chromosome are present instead of the usual two.4. otherwise known as 47. • • Patau syndrome is caused by trisomy of chromosome 13.
Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual.• Cri du chat (cry of the cat). They can be organized into two basic groups. caused by abnormal formation of the larynx. There are many types of chromosome anomalies. . • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. example of imprinting disorder. a deletion of the paternal genes. example of imprinting disorder. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. one well-documented example is the Philadelphia chromosome. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. The name comes from the babies' distinctive cry. from the loss of part of the short arm of chromosome 1. 1p36 Deletion syndrome. 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. A chromosome anomaly. from a truncated short arm on chromosome 5. a deletion of the maternal genes. numerical and structural anomalies. A chromosome anomaly may be detected or confirmed in this manner.
In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. an X. 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 17.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). rather than two). Tetrasomy. and Jacobsen syndrome. In a Robertsonian translocation.4. There are two main types of translocations. 4.3 Structural abnormalities When the chromosome's structure is altered. etc. an entire chromosome has . also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. Known disorders in humans include Wolf-Hirschhorn syndrome. also called the terminal 11q deletion disorder. Duplications: A portion of the chromosome is duplicated. In a reciprocal translocation. segments from two different chromosomes have been exchanged.). This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. which is caused by partial deletion of the short arm of chromosome 4. • • Translocations: When a portion of one chromosome is transferred to another chromosome. resulting in extra genetic material.
14. Therefore. It includes routine analysis of G-Banded chromosomes. can happen after conception. Chromosome anomalies can be inherited from a parent or be "de novo". • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. 21 and 22. This can happen with or without loss of genetic material. the anomaly is present in every cell of the body. other cytogenetic banding techniques. • Rings: A portion of a chromosome has broken off and formed a circle or ring.attached to another at the Centromere . therefore the genetic material is inverted. resulting in Mosaicism (where some cells have the anomaly and some do not). and are therefore initially not inherited. • Inversions: A portion of the chromosome has broken off. They often lead to an increased tendency to develop certain types of malignancies. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. as well . however. 4. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. Some anomalies. turned upside down and reattached. 15.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. especially the chromosomes.in humans these only occur with chromosomes 13. 4.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell.
the discoverer of mitosis. these results were quite remarkable. He revised his opinion later from 46 to 48.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. and he correctly insisted on man having an XX/XY system. Considering their techniques. at first favoring 46. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. The name was coined by another German anatomist. von Waldeyer in 1888. New techniques were needed to definitively solve the problem: 1. in 1882. in contrast to their genic contents. 4. Their behavior in animal (salamander) cells was described by Walther Flemming. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. Pre-treating cells in a hypotonic solution. concluding an XX/XO sex determination mechanism. Painter in 1922 was not certain whether the diploid number of man was 46 or 48.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. which swells them and spreads the chromosomes . The next stage took place after the development of genetics in the early 20th century. Using cells in culture 2. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.
the great apes have 48 chromosomes. 4. Arresting mitosis in metaphase by a solution of colchicine 4. 4. In 1931. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). reducing the number.6. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.2 Natural populations of Drosophila In the 1930s. persimilis from wild populations in California and neighboring states.3. McClintock discovered transposons. Rather interestingly.6 Applications in biology 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.6. Human chromosome 2 was formed by a merger of ancestral chromosomes. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. During her cytogenetic work. Using Painter's technique they studied the polytene . Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. a find which eventually led to her Nobel Prize in 1983.
7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. as they would if selectively neutral. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. Using a method invented by L'Heretier and Teissier. Down syndrome is also referred to as trisomy 21. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. discoveries were quickly made related to aberrant chromosomes or chromosome number. . but adjust to certain frequencies at which they become stabilised. such as Down's syndrome. Evidence rapidly accumulated to show that natural selection was responsible. This had the benefit of eliminating migration as a possible explanation of the results. In some congenital disorders. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. In 1959. It was found that the various chromosome types do not fluctuate at random. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. breeding and sampling whilst preventing escape. Dobzhansky bred populations in population cages. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. which enabled feeding.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. 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. as with most polymorphisms. 4.
Not all genes on the X Chromosome are inactivated. This abnormal chromosome was dubbed the Philadelphia chromosome .Other numerical abnormalities discovered include sex chromosome abnormalities. Many other sex chromosome combinations are compatible with live birth including XXX. with the development of more advanced techniques. which is required in normal females to compensate for having two copies of the chromosome. Pennsylvania. An individual with only one sex chromosome (the X) has Turner syndrome. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. resulting in 47 total chromosomes. Identification of the Philadelphia chromosome by cytogenetics.as both scientists were doing their research in Philadelphia. has Klinefelter's Syndrome. an additional X chromosome in a male. XYY. Thirteen years later. . and XXXX. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). In 1960. in addition to other tests. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. is used today as a diagnostic for CML.
and elongation techniques for all culture types that allow for higher resolution banding. Deletions within one chromosome could also now be more specifically named and understood. 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. Caspersson developed banding techniques which differentially stain chromosomes. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid.FIG Advent of banding techniques In the late 1960s. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Deletion syndromes such as DiGeorge syndrome.8 Beginnings of molecular cytogenetics . 4.
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 5.1 Karyotyping . While radioisotope-labeled probes had been hybridized with DNA since 1969. cloned and studied in ever greater detail. movement was now made in using fluorescent labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). advances were made in molecular cytogenetics.In the 1980s. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.
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,
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. test and measurement. For congenital problems usually 20 metaphase cells are scored. and FORTRAN. . Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. and numerical computation. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. including signal and image processing. such as C. data visualization. and computational biology. communications. Using MATLAB. control design. such as comparative genomic hybridization arrays. financial modeling and analysis. you can solve technical computing problems faster than with traditional programming languages. C++.generally between 200 and 1000 cells are counted and scored. data analysis. CGH and Single nucleotide polymorphism-arrays. You can use MATLAB in a wide range of applications.
The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. The image processing step is composed of the following operations. It enables fast development and execution.MATLAB provides a number of features for documenting and sharing your work. and distribute your MATLAB algorithms and applications. . 6. With the MATLAB language.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. one line of MATLAB code can often replace several lines of C or C++ code. As a result. and allocating memory. 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. 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. You can integrate your MATLAB code with other languages and applications. In many cases. These effects must be compensated to improve the results of the pairing algorithm. MATLAB eliminates the need for ‘for’ loops.
or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. 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. geometrical and dimensional differences must be removed. Therefore. To compensate for this inhomogeneity. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. the spatially scaled images are histogram equalized. 2) Geometrical compensation—The geometric compensation. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 6. 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 . To compare chromosomes from a band pattern point of view.
I. and blue planes. if you want to filter a color image that is stored as an indexed image. The resulting true color image has identical matrices for the red. For example. MATLAB filters the intensity values in the image.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. green. 6. 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. You can perform certain conversions just using MATLAB syntax.I). you must first convert it to true color format.3) Edge detection 4) Two dimensional convolutions. When you apply the filter to the true color image. For example. For example. RGB = cat (3. .I. as is appropriate. In addition to these image type conversion functions. and the results might not be meaningful. MATLAB simply applies the filter to the indices in the indexed image matrix. listed in the following table. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. If you attempt to filter the indexed image. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations.2.
via satellite or wireless transmission. to recognize or classify objects. If an image is being sent electronically from one place to another. ) Where the parameters available depend on the method used 6. The general Matlab command for finding edges is edge(image. and hence the type of noise on the image. 6.3 Two dimensional convolutions C = conv2(A. If one of these matrices describes a two-dimensional finite impulse response . We may use edges to measure the size of objects in an image. . and we shall look at some of the more straightforward of them. Cleaning an image corrupted by noise is thus an important area of image restoration. hence we can choose the most appropriate method for reducing the effects.5 Edge detection Edges contain some of the most useful information in an image.2. to isolate particular objects from their background. caused by external disturbance. Usually we know what type of errors to expect. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. . or through networked cable.6.'method'. There is a large number of edge finding algorithms in existence.4 Denoising We may define noise to be any degradation in the image signal.2.parameters. we may expect errors to occur in the image signal.B) computes the two-dimensional convolution of matrices A and B.
imedfilt2(im1.7).na+nb-1].(FIR) filter. 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 other matrix is filtered in two dimensions.. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.'shape') subsection of the two-dimensional convolution.A).. edge(im1.'sobel').na] and the size of B is [mb. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. C = conv2(. If hcol is a column vector and hrow is a row vector. this case is the same as C = conv2(hcol*hrow.nb].[3 3]). . That is. The size of matrices. rgb2gray im2bw(im. minus one.0.hrow. if the size of then the size of C is [ma+mb-1. nb]+1)/2).A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.bmp')..
1). rc = [r c]. . Msk conv2(double(BW).j)~=0 for k=1:mx if L(i.y1)=255.double(msk)). for i=1:m for j=1:n if L(i.n]=size(L). flag=0.2).imy]=size(BW). [r.[imx.1).imy). mx=max(max(L)). bwlabel(B. L_number=zeros(mx.8). y1=rc(i. nzeros(imx. for i=1:sx x1=rc(i. [sx sy]=size(rc).c] = find(L==22). Index=1. n1(x1.j)==L_number(k) flag=1. MODULE 2 clc [m.
15.imshow(n1. end.39.27.24.imy). [sx sy]=size(rc).18.104.22.168. end flag=0.14.6.51. Index=Index+1.2).22.214.171.124.38.52.54.y1)=255.62. .).43.57.c] = find(L==L_number((Test_number(x)))).1).9.65. end end L_number. for i=1:sx x1=rc(i.126.96.36.199.31. for x=1:46 [r.32.55. end %h=figure. 36.66].10.19. Test_number=[188.8.131.52.42.45. n1=zeros(imx.end end if flag~=1 L_number(Index)=L(i.41.59. rc = [r c].j). n1(x1. y1=rc(i.184.108.40.206.
BW1=edge(BW.y)==1 Circumference_sum=Circumference_sum+1.1.'canny').5*graythresh(skel)). BW=im2bw(f). skel=im2double(f).bmp')). s1=bwmorph(s.1). f=imcomplement(f). for x=1:m for y=1:n if BW1(x. [m n]=size(BW1).1).end Circumference=zeros(46. skel=im2bw(skel.'. . Circumference_sum=0. BW=double(BW).'skel'. for i=1:46 f=imread(strcat(num2str(i). Arm_length_sum=0.Inf).'spur'.1). s=bwmorph(skel. Arm_length=zeros(46.8). Area=zeros(46. end end end Circumference(i)=Circumference_sum.
Area_sum=0. end Circumference.y)==1 Area_sum=Area_sum+1. BW=im2bw(f).[m n]=size(s1).y)==1 Arm_length_sum=Arm_length_sum+1. [m n]=size(BW). Arm_length. end end end Arm_length(i)=Arm_length_sum. for x=1:m for y=1:n if BW(x. end end end Area(i)=Area_sum. for x=1:m for y=1:n if s1(x. .
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(46.1)=46. Pair(i. Pair(i.2)=i.Area.2)=i+1.1)=i. Pair(46. end end Pair.2)=j. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)). Pair(i. end end end for i=1:45 if Pair(i. .1)=i. Pair(i.2). Pair=zeros(46.
.bmp')). figure_flag=figure_flag+1. end f1=imread(strcat(num2str(Pair(i. flag=0.figure_flag). end f2=imread(strcat(num2str(Pair(i. end end if flag~=1 if figure_flag~=47 subplot(23.'.figure_flag).2.bmp')). imshow(f1). end flag=0. if figure_flag~=47 subplot(23. figure_flag=1. imshow(f2). figure_flag=figure_flag+1. delete(figure_flag)=Pair(i.1)).2)).1)==delete(j) flag=1. for i=1:46 for j=1:46 if Pair(i.2).2.'.delete=zeros(46.1).
2) feature extraction from the processed images . 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. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. The proposed algorithm is based on the traditional features extracted from the karyogram. such as. plus a new one. in the scope of karyotyping process used in cytogentic analysis.end CONCLUTION In this paper. Copenhagen. and Philadelphia. based on the MI. dimensions and banding profiles. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. 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.
shape and band pattern. achieves a 70. from the chromosomes in the training set. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. Here.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. 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. shape. are processed in order to compensate for geometrical and intensity distortions. the romosome images. extracted from the unordered karyogram. 4) pairing. and band pattern. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. and finally. and to normalize their dimensions. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. In the image processing step. 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.10% mean classification rate.working within an 8-D feature space. Tests using 19 karyograms based on bone marrow cells. The training process consists in the estimation of each vector of coefficient . This normalization is needed to make it possible the band pattern comparison between chromosomes. 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 . 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 features extracted from the processed images discriminate each pair with respect to their size.characterizing the size.
In fact. called LK1 . amean classification rate larger than 93% was obtained in all experiments. such as Edinburgh. e. The results presented in this paper are promising. Copenhagen. This dataset was made publicly available . despite the low quality of this type of chromosomes. Executing the algorithm on a higher quality dataset.10% classification ratewas obtained. In addition. Using 27 karyograms andworking with a limited number of classes (≤ 8). Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. whose images are of significantly higher quality. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset.g. presenting a uniform level of condensation. a 76. REFERENCES . or Philadelphia. centromere position.. and from which it is possible to extract additional features. a new chromosome dataset with 9200 chromosomes from bone marrow cells.performance of the classifier.
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