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During the Metaphase type of cell division in the Bone Marrow, Karyotyping method gives the visual representation of the 46 chromosomes paired and arranged in decreasing order of size. This representation is useful in leukemia purposes. This method is a difficult one because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges. So here, Karyotyping uses new mutual information method which is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. This algorithm is formulated as such a method of combinatorial optimization problem. Where the distances between homologous chromosomes are minimized and the distances between non homologous ones are maximized. It is solved by using an integer programming approach. In this project chromosome dataset Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes was used for this study.
Introduction 1.1 The study of chromosome morphology and its relation with some genetic diseases is the main goal of cytogenetic. Normal human cells have 23 classes of large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The chromosome contains approximately 30 000 genes (genotype) and large tracts of non coding sequences. The analysis of genetic material can involve the examination of specific chromosomal regions using DNA probes, e.g., fluorescent in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic hybridization (CGH) , or the morphological and pattern analysis of entire chromosomes, the conventional cytogenetic, which is the focus of this paper. These cytogenetic studies are very important in the detection of acquired chromosomal abnormalities, such as translocations, duplications, inversions, deletions, monosomies, or trisomies. These techniques are particularly useful in the diagnosis of cancerous diseases and are the preferred ones in the characterization of the different types of leukemia, which is the motivation of this paper . The pairing of chromosomes is one of the main steps in conventional cytogenetic analysis where a correctly ordered karyogram is produced for diagnosis of genetic diseases based on the patient karyotype. The karyogram is an image representation of the stained human chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) , where the chromosomes are arranged in 22 pairs of somatic homologous elements plus two sex-determinative chromosomes (XX for the female or XY for the male), displayed in decreasing order of size. A karyotype is the set of characteristics
extracted from the karyogram that may be used to detect chromosomal abnormalities. The metaphase is the step of the cellular division process where the chromosomes are in their most condensed state. This is the most appropriated moment to its visualization and abnormality recognition because the chromosomes appear well defined and clear. The pairing and karyotyping procedure, usually done manually by visual inspection, is time consuming and technically demanding. The application of the G-banding procedure to the chromosomes generates a distinct transverse banding pattern characteristic for each class, which is the most important feature for chromosome classification and pairing. The International System for Cytogenetic Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as for all the chromosomes of a normal human, and the clinical staff is trained to pair and interpret each specific karyogram according to the ISCN information. Other features, related to the chromosome dimensions and shape, are also used to increase the discriminative power of the manual or automatic classifiers. 1.2 CHROMOSOME A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes,regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Chromosomes vary widely between different organisms. The DNA molecule may be circular or linear, and can be composed of 100,000 to 10,000,000,000 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without
This allows the very long DNA molecules to fit into the cell nucleus. Unduplicated chromosomes are single linear strands. or it may unexpectedly evadeapoptosis leading to the progression of cancer. sometimes accompanied by one or more smaller. If these structures are manipulated incorrectly. Chromosomal recombination plays a vital role in genetic diversity. In practice "chromosome" is a rather loosely defined term. These small circular genomes . although there are many exceptions to this rule. The structure of chromosomes and chromatin varies through the cell cycle. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. Also. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. through processes known as chromosomal instability and translocation. which is tightly coiled in on itself. DNA is usually arranged as a circle. Chromosomes are the essential unit for cellular division and must be replicated. the cell may undergo mitotic catastrophe and die. In eukaryotes. a large body of work uses the term chromosome regardless of chromatin content. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. In prokaryotes and viruses. In prokaryotes. circular DNA molecules called plasmids. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). cells may contain more than one type of chromosome. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin.defined nuclei) have smaller circular chromosomes. the term genophore is more appropriate when no chromatin is present. for example. Chromosomes may exist as either duplicated or unduplicated. divided. However.
XX (female) or 46 XY (male). The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.XX. The individual would have Down Syndrome and his/her karyotype would be written 47. copies of chromosome 21. . in which an individual has 3.are also found in mitochondria and chloroplasts. Such individuals are called euploid and have the wild-type chromosome complement for the species. rather than 2. reflecting their bacterial origins.g.+21. 1. 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). An example of aneuploidy is trisomy 21.XY or 47. Euploid human karyotypes are 46. a extra copy of human chromosome 21).+21. the individual carrying the mutation is said to be aneuploid. 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.3 MUTATIONS IN CHROMOSOME NUMBER Normally. If the mutation involves only one or a few chromosomes in the genome (e.
along with special proteins. The microtubules then pull the chromatids apart toward the centrosomes. During mitosis. a pair of sister chromatids attached to each other at the centromere. which enables these giant DNA structures to be contained within a cell nucleus. q-g "grande"). and they form the classic four arm structure. . Once the cells have divided. the chromatids are uncoiled and DNA can again be transcribed. longer-lasting attachment in this region. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form.Fig 1. small) and the longer arms are called q arms (q follows p in the Latin alphabet. so that each daughter cell inherits one set of chromatids. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. the chromatin strands become more and more condensed. A special DNA base sequence in the region of the kinetochores provides. one of which is present on each sister chromatid.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). In spite of their appearance. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. chromosomes are structurally highly condensed. This is the only natural context in which individual chromosomes are visible with an optical microscope. This compact form makes the individual chromosomes visible. The shorter arms are called p arms (from the French petit. 1.
On average. .1. which divides the nuclei. It is generally followed immediately by cytokinesis. which use the bone marrow vasculature as a conduit to the body's systemic circulation.7 lbs).1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. bone marrow in large bones produces new blood cells. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. producing the lymphocytes that support the body's immune system CHAPTER 2 2. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. cytoplasm.  Bone marrow is also a key component of the lymphatic system. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. in adults weighing 65 kg (143 lbs). bone marrow constitutes 4% of the total body mass of humans. bone marrow accounts for approximately 2. In humans.6 kg (5. in two separate nuclei.
"mitosis" is often used interchangeably with "mitotic phase". there are many cells where mitosis and cytokinesis occur separately. . Prokaryotic cells. This accounts for approximately 10% of the cell cycle.genetically identical to each other and to their parent cell. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. Because cytokinesis usually occurs in conjunction with mitosis. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. metaphase. This occurs most notably among the fungi and slime moulds. forming single cells with multiple nuclei. Mitosis occurs only in eukaryotic cells and the process varies in different species. Even in animals. animals undergo an "open" mitosis. cytokinesis and mitosis may occur independently. The cell then divides in cytokinesis. However. where the nuclear envelope breaks down before the chromosomes separate. to produce two identical daughter cells which are still diploid cells. The process of mitosis is fast and highly complex. but is found in various different groups. which lack a nucleus. prometaphase. where chromosomes divide within an intact cell nucleus. anaphase and telophase. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. prophase. For example. for instance during certain stages of fruit fly embryonic development. These stages are interphase. divide by a process called binary fission. 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. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. . These two cells are identical and do not differ in any way from the original parent cell. the parent cell must make a copy of each chromosome before mitosis. and together the two are called sister chromatids. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. This occurs during the S phase of interphase. Because each resultant daughter cell should be genetically identical to the parent cell. Each chromosome now has an identical copy of itself.
The chromosomes align themselves in a line spanning the cell. In plant cells. . the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). each sister chromatid is now considered a chromosome. so they are renamed to sister chromosomes. Eventually. As mitosis completes. pulling apart the sister chromatids of each chromosome. As the cell elongates. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. each with a replica of the original genome. the daughter cells will construct a new dividing cell wall between each other. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. However. the process of binary fission is very much different from the process of mitosis. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. the parent cell will be split in half. giving rise to two daughter cells. In animal cells.the cell begins cytokinesis. A new nuclear envelope forms around the separated sister chromosomes. separating the two developing nuclei.In most eukaryotes. Prokaryotic cells undergo a process similar to mitosis called binary fission. corresponding sister chromosomes are pulled toward opposite ends. As a matter of convention.
Interphase is divided into three phases: G1 (first gap). where the cell prepares itself for cell division.1 Preprophase In plant cells only. 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. continues to grow as it duplicates its chromosomes (S). the nucleus has to migrate into the center of the cell before mitosis can begin. a cell grows (G1). chromosomes are replicated only during the S phase. However. and finally it divides (M) before restarting the cycle.2. In highly vacuolated plant cells. All these phases in the interphase are highly regulated. the cell grows by producing proteins and cytoplasmic organelles. grows more and prepares for mitosis (G 2). 2. It alternates with the much longer interphase. and G2 (second gap). a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . mainly via proteins. prophase is preceded by a pre-prophase stage. During all three phases. This is achieved through the formation of a phragmosome.2.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. Thus. S (synthesis).
aligned at the metaphase plate. These microtubules can attach to kinetochores or they can interact with opposing microtubules. instead. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. Cytokinesis has already begun. The cells of higher plants (such as the flowering plants) lack centrioles. The chromosomes have chromatin has condensed. 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. degraded. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. . Telophase: The decondensing chromosomes are surrounded by nuclear membranes. and microtubules have invaded the nuclear Prophase space. the pinched area is known as the cleavage furrow. This band marks the position where the cell will eventually divide. after the nuclear membrane breaks down. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. In addition to phragmosome formation.division. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten.
bound together at the centromere by the cohesin protein complex. Although centrioles help organize microtubule assembly. giving a pair of centrosomes. which are made of a pair of centrioles found in most eukaryotic animal cells.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. chromatin condenses together into a highly ordered structure called a chromosome. A cell inherits a single centrosome at cell division. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. Chromosomes are typically visible at high magnification through a light microscope. which is replicated by the cell with the help of the nucleus before a new mitosis begins. Since the genetic material has already been duplicated earlier in S phase. the genetic material in the nucleus is in a loosely bundled coil called chromatin. The centrosome is the coordinating center for the cell's microtubules. 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. they are not essential for the . At the onset of prophase. the replicated chromosomes have two sister chromatids.
. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. or its microtubules are able to penetrate an intact nuclear envelope. undergo a variation called closed mitosis where the spindle forms inside the nucleus. the motor activates. kinetochore microtubules begin searching for kinetochores to attach to. 2. using energy from ATP to "crawl" up the tube toward the originating centrosome. provides the pulling force necessary to later separate the chromosome's two chromatids. on an average 20 ).2. This motor activity. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. coupled with polymerisation and depolymerisation of microtubules. and centrosomes are not always used in mitosis. When the spindle grows to sufficient length.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. one attached at each chromatid. When a microtubule connects with the kinetochore. Each chromosome forms two kinetochores at the centromere. Fungi and some protists. Although the kinetochore structure and function are not fully understood. since they are absent from plants. Prometaphase is sometimes considered part of prophase.formation of the spindle. it is known that it contains some form of molecular motor. and it occurs in most multicellular organisms. such as algae or trichomonads. This is called open mitosis. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number.
convene along the metaphase plate or equatorial plane. an imaginary line that is equidistant from the two centrosome poles.3 Metaphase A cell in late metaphase. analogous to a tug-of-war between people of equal strength. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. only roughly lining up along the midline. Metaphase comes from the Greek meaning "after. . In certain types of cells. the kinetochore would be the "hook" that catches a sister chromatid or "fish". As a result. in some sense. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. the chromosomes come under longitudinal tension from the two ends of the cell.In the fishing pole analogy. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. 2." Microtubules find and attach to kinetochores in prometaphase. All chromosomes (blue) but one have arrived at the metaphase plate. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. The centromeres of the chromosomes.
the cell proceeds to anaphase (from the Greek meaning “up.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. Early anaphase is usually defined as the separation of the sister chromatids. allowing them to separate. Two events then occur: first. . pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached.” “back. The force that causes the centrosomes to move towards the ends of the cell is still unknown. These two stages are sometimes called early and late anaphase. These sister chromatids. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. 2. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. the nonkinetochore microtubules elongate.” “against.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). which have now become distinct sister chromosomes. Next. the proteins that bind sister chromatids together are cleaved. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement.” or “re-”). the cell has succeeded in separating identical copies of the genetic material into two distinct populations. At the end of anaphase.
cytokinesis is a separate process that begins at the same time as telophase. At telophase. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. the nonkinetochore microtubules continue to lengthen. 2. Cytokinesis is technically not even a phase of mitosis. however.2. In animal cells. now surrounded by new nuclei. forms around each set of separated sister chromosomes. elongating the cell even more. necessary for completing cell division. but cell division is not yet complete. pinching off the separated nuclei.5 Cytokinesis Cilliate undergoing cytokinesis. Both sets of chromosomes. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. Corresponding sister chromosomes attach at opposite ends of the cell. Mitosis is complete.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. In both animal and plant cells. using fragments of the parent cell's nuclear membrane. unfold back into chromatin. A new nuclear envelope. It "cleans up" the after effects of mitosis. but rather a separate process. cell .
. Similarly. which move along microtubules to the middle of the cell. Each daughter cell has a complete copy of the genome of its parent cell.1Significance Mitosis is important for the maintenance of the chromosomal set. The end of cytokinesis marks the end of the M-phase.e. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.division is also driven by vesicles derived from the Golgi apparatus. The phragmoplast is a microtubule structure typical for higher plants. skin and digestive tract. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.5. Following are the occasions in the lives of organism where mitosis happens: 2. cells are constantly sloughed off and replaced by new ones.5. zygote and also the basis of the growth of a multicellular body. whereas some green algae use a phycoplast microtubule array during cytokinesis.5.3 Cell replacement In some parts of body. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.4 Regeneration . 2.g.2 Development and growth The number of cells within an organism increases by mitosis. 2. 2. This is the basis of the development of a multicellular body from a single cell i.5. separating the two nuclei. e. New cells are formed by mitosis and so are exact copies of the cells being replaced.
For example. resulting in binucleated cells. One daughter cell will receive both sister chromosomes and the other will receive none. The same division happens during asexual reproduction or vegetative propagation in plants. a condition often associated with cancer. In non-disjunction. 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. they fail to complete cell division and retain both nuclei in one cell.Some organisms can regenerate their parts of bodies. For example. a condition known as monosomy. the hydra reproduces asexually by budding. and the latter cell having only one chromosome (the homologous chromosome). a condition known as trisomy. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. 2.5. These cells are considered aneuploid. . Mitosis continues in the cells of bud and it grows into a new individual. sea star regenerates its lost arm through mitosis.5. Occasionally when cells experience nondisjunction. The cells at the surface of hydra undergo mitosis and form a mass called bud. a chromosome may fail to separate during anaphase.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. the process may go wrong. 2. The production of new cells is achieved by mitosis.7 Consequences of errors Although errors in mitosis are rare.
causing chromosomal duplication. causing deletion. This phenomenon is called metastasis or spreading of disease. It may reattach to the original chromosome. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. non-homologous chromosome. As soon as they start to move and invade other cells there are said to be malignant tumours. it results in the formation of Tumors. The fragment may incorrectly reattach to another. An arm of the chromosome may be broken and the fragment lost. Benign tumours are not harmful as soon as they are not moving. and chromosomes are jostled constantly by probing microtubules.Mitosis is a demanding process for the cell. its organelles disintegrate and reform in a matter of hours. Errors in the control of mitosis may cause cancer. . It results in the synthesis of execessive tissue growths. it may be treated erroneously as a separate chromosome. sometimes mutuations occur in such genes and cells continue to divide. The effect of these genetic abnormalities depends on the specific nature of the error. Or. chromosomes may become damaged. All cells have genes that control the timing and number of mitosis. It results in abnormal cell growth. As long as these tumours remain in their original location they are called benign tumours. Such tumours can send cancer cells to other parts in body where new tumours may form. Now what happens is that cell abnormally continue to divide at a single place. When tissues more than the requirement are synthesized in a single organ. causing inversion. but in reverse orientation. Occasionally. which goes through dramatic changes in ultrastructure. causing translocation.
chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. This process may also be referred to as endoreduplication and the cells as endoploid. 2. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate).2. carrying genetic information. analogous to a tug of war between equally strong people. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. an imaginary line that is equidistant from the two centrosome poles. Preceded by events in prometaphase and followed by anaphase. An example of a cell that goes through endomitosis is the megakaryocyte. Early events of metaphase can . only roughly lining up along the middleline. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. align in the middle of the cell before being separated into each of the two daughter cells. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. resulting in cells with many copies of the same chromosome occupying a single nucleus. from the ancient Greek(between) and (stage). In certain types of cells.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division.7 Metaphase Metaphase. Metaphase accounts for approximately 4% of the cell cycle's duration.
cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. This would be accomplished by regulation of the anaphase-promoting complex. Only after all chromosomes have become aligned at the metaphase plate. 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. produces a pattern of in total up to several hundred bands. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Staining of the slides. For classical cytogenetic analyses. Metaphase chromosomes make the classical picture of chromosomes (karyotype).8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. when every kinetochore is properly attached to a bundle of microtubules.coincide with the later events of prometaphase. Normal metaphase spreads are used in . does the cell enter anaphase. often with Giemsa (G banding) or Quinacrine. and separase. securin. Such a signal creates the mitotic spindle checkpoint. Chromosomes are condensed(Thickened) and highly coiled in 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). which makes them most suitable for visual analysis. 2.
which may lead to chimeric oncogenes. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. for example. such as bcr-abl in chronic myelogenous leukemia.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. . losses of chromosomal segments or translocations. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations.
cellular function. and any other physical characteristics. The term is also used for the complete set of chromosomes in a species. and what they look like under a light microscope. in humans 2n = 46. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. banding pattern. taxonomic relationships. autosomal chromosomes are present in two copies. be sex chromosomes. or an individual organism. So. Thus. the position of the centromeres. . Karyogram of human male using Giemsa staining. Attention is paid to their length. or may not.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. The study of karyotypes is important for cell biology and genetics. Karyotypes describe the number of chromosomes.  The preparation and study of karyotypes is part of cytogenetics. The study of whole sets of chromosomes is sometimes known as karyology. to study chromosomal aberrations. any differences between the sex chromosomes. and the results may be used in evolutionary biology and medicine. such as. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). and to gather information about past evolutionary events. Karyotypes can be used for many purposes. in normal diploid organisms. There may. ordered by size and position of centromere for chromosomes of the same size.
The next stage took place after the development of genetics in the early 20th century. Using cells in culture 2. von Waldeyer in 1888. which swells them and spreads the chromosomes . and he correctly insisted on humans having an XX/XY system. in contrast to their genic contents. Their behavior in animal (salamander) cells was described by Walther Flemming. at first favoring 46. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. New techniques were needed to definitively solve the problem: 1. 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. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. The name was coined by another German anatomist. in 1882. these results were quite remarkable.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Pretreating cells in a hypotonic solution. the discoverer of mitosis. 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. He revised his opinion later from 46 to 48. concluding an XX/XO sex determination mechanism.3. Considering their techniques.
2. a suitable dye. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. 3. reducing the number.2.1 Staining The study of karyotypes is made possible by staining. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.  Sometimes observations may be made on non-dividing (interphase) cells. Arresting mitosis in metaphase by a solution of colchicines 4. 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . For humans. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. such as Giemsa. the great apes have 48 chromosomes.3. Human chromosome 2 was formed by a merger of ancestral chromosomes. is applied after cells have been arrested during cell division by a solution of colchicine. Usually. The sex of an unborn fetus can be determined by observation of interphase cells. Rather interestingly.2 Observations on karyotypes 3.
Differences in degree and distribution of heterochromatic regions. 3. 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. . type. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. and mainly consists of genetically inactive repetitive DNA sequences. Differences in absolute sizes of chromosomes. shape and banding of the chromosomes. 6. Humans have one pair fewer chromosomes than the great apes. as well as other cytogenetic information. 4. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). both have six pairs of chromosomes (n=6) yet V. Differences in number and position of satellites. A full account of a karyotype may therefore include the number. This feature probably reflects different amounts of DNA duplication. Heterochromatin stains darker than euchromatin. 5. but the genes have been mostly translocated (added) to other chromosomes. faba chromosomes are many times larger. which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in the position of centromeres. This is brought about by translocations.1. indicating tighter packing. permitting its loss without penalty to the organism (the dislocation hypothesis).
Any variation from the standard karyotype may lead to developmental abnormalities. Between the sexes 2. Between the germ-line and soma (between gametes and the rest of the body) 3. Normal karyotypes for females contain two X chromosomes and are denoted 46. Between members of a population (chromosome polymorphism) 4. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes.3 The human karyotype Most (but not all) species have a standard karyotype.Variation is often found: 1. Geographical variation between races 5. males have both an X and a Y chromosome denoted 46. XY. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. and in . the same cannot be said for their karyotypes. which are highly variable. There is variation between species in chromosome number. Mosaics or otherwise abnormal individuals. 3. XX.
"We have a very poor understanding of the causes of karyotype evolution. despite many careful investigations. In some cases there is even significant variation within species. used in conjunction with other phylogenetic data. 3. But. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. In this process. In some species. entire chromosomes are eliminated during development. as in many sciarid flies. which were previously inexplicable. portions of the chromosomes are cast away in particular cells. In a review. or other kinds of visible adjustment to the karyotype. Chromatin diminution (founding father: Theodor Boveri). Chromosome elimination. .. found in some copepods and roundworms such as Ascaris suum. it is quite unclear what the general significance might be.. This variation provides the basis for a range of studies in evolutionary cytology. In A. some organisms go in for large-scale elimination of heterochromatin.detailed organization.1 Changes during development Instead of the usual gene repression.3. Although much is known about karyotypes at the descriptive level. the general significance of karyotype evolution is obscure.. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. despite their construction from the same macromolecules. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed.. 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 low record is held by the nematode Parascaris univalens. they were astonished to find it had female = 6. male = 7 chromosomes. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. thus the mammalian female is a mosaic in respect of her X chromosomes.3.. In marsupials it is always the paternal X which is inactivated... the inactivation is random as between the two Xs. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). When they looked at the karyotype of the closely related Indian muntjac.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. Muntiacus muntjak.suum. "They simply could not believe what they saw. the high record would be somewhere amongst the ferns. all telocentric. all the somatic cell precursors undergo chromatin diminution. Xinactivation. The diploid number of the Chinese muntjac. In placental mammals. which was investigated by Kurt Benirschke and his colleague Doris Wurster. was found to be 46. 3.. Muntiacus reevesi. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. 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. In human females some 15% of somatic cells escape inactivation. The existence of supernumerary or B chromosomes . where the haploid n = 1. They kept quiet for two or three years because they thought something was wrong with their tissue culture.
and the other haploid. and aneuploids are another example. FN ≤ 2n.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. 3. and in some other groups. but in grasses the average is much higher. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. It is a common arrangement in the Hymenoptera.3 Fundamental number The fundamental number. Haplo-diploidy. Thus. FN. occurs mainly in plants. where there are more than two sets of homologous chromosomes in the cells. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. Humans have FN = 82. about 70%. horsetails and psilotales) is also common. Polyploidy in lower plants (ferns.3. though in this case they would not be regarded as normal members of the population. Polyploidy in animals is much less common.means that chromosome number can vary even within one interbreeding population. 21 and 22). the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. 15. It has been of major significance in plant evolution according to Stebbins.Endopolyploidy occurs when in adult differentiated . but it has been significant in some groups. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. where one sex is diploid. Polyploidy. 3. 14. due to the presence of five acrocentric chromosome pairs (13.
. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. See palaeopolyploidy for the investigation of ancient karyotype duplications. The cells do not always contain exact multiples (powers of two). The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. the daughter chromosomes separating from each other inside an intact nuclear membrane. Abnormalities in chromosome number usually cause a defect in development. it is diverse and complex. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). In many instances. 3. and serves differentiation and morphogenesis in many ways. 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. but the nuclei contain more than the original somatic number of chromosomes.tissues the cells have ceased to divide by mitosis. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. Down syndrome and Turner syndrome are examples of this.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species.
5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. some mantids of the genus Ameles. that the two chromosome morphs are adapted to different habitats. When this happens.Aneuploidy may also occur within a group of closely related species. and Crocus.500 sq mi (17. where the gametic (= haploid) numbers form the series x = 3. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.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. and 7. the chromosome number is variable from one individual to another. Classic examples in plants are the genus Crepis.000 km2). the great apes have 24x2 chromosomes whereas humans have 23x2. the European shrew Sorex araneus. Human chromosome 2 was formed by a merger of ancestral chromosomes. Well-researched examples are the ladybird beetle Chilocorus stigma. 3. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 3. 5. 4. 6.  Closer to home. In about 6. where every number from x = 3 to x = 15 is represented by at least one species. reducing the number. living from rainforests to .
In a sense. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. Although it would be possible for a single gravid female to colonise an island. when plotted in tree form (and independent of all other information). Using K-Ar dating.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). especially inversions. probably 20 million years ago. Drosophila and Scaptomyza. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. it is more likely to have been a group from the same species. at least into the Cretaceous. which can be dated to 30 mya. the present islands date from 0. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. the best-studied group of Hawaiian drosophilids. The inversions. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. and skipping of islands. gene arrangements are visible in the banding patterns of each chromosome. . The results are clear. The polytene banding of the 'picture wing' group. There are also cases of colonization back to older islands. in the family Drosophilidae.subalpine meadows. make it possible to see which species are closely related. show a clear "flow" of species from older to newer islands. but these are much less frequent. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. Chromosome rearrangements. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches.
• Q-banding is a fluorescent pattern obtained using quinacrine for staining. 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. This method will normally produce 300-400 bands in a normal. . 3. • T-banding: visualize telomeres. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). The light regions tend to be euchromatic.7 Depiction of karyotypes 3. R-banding is the reverse of G-banding (the R stands for "reverse"). It yields a series of lightly and darkly stained bands . • • C-banding: Giemsa binds to constitutive heterochromatin. if less spectacular. early-replicating and GC rich. adaptive radiations. human genome. so it stains centromeres.the dark regions tend to be heterochromatic.There are other animals and plants on the Hawaiian archipelago which have undergone similar. late-replicating and AT rich.7.
denoting the activity of rRNA genes within the NOR. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. In the "classic" (depicted) karyotype.7. 3. This yields a dark region where the silver is deposited. and the long arm on the bottom. Some karyotypes call the short and long arms p and q. Karyotypes are arranged with the short arm of the chromosome on top. For example. In addition.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. Giemsa is specific for the phosphate groups of DNA. often Giemsa (G-banding). Cri du chat syndrome involves a deletion on the short arm of . respectively. both chromosomes in a pair will have the same banding pattern. Quinacrine binds to the adeninethymine-rich regions. is used to stain bands on the chromosomes. Each chromosome has a characteristic banding pattern that helps to identify them. less frequently Quinacrine.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. a dye.
2) 3. It is written as 46.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. . The critical region for this syndrome is deletion of 15.chromosome 5.7. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination. 3. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. 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.XX.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. which is written as 46. a combinatorial labeling method is used to generate many different colors. allowing the visualization of the individually colored chromosomes.5p-.XX. Because there are a limited number of spectrally-distinct fluorophores.2. This method is also known as virtual karyotyping.del(5)(p15. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.
CHAPTER 4 .
the most common male chromosomal disease. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Klinefelter syndrome. Numerical abnormalities. trisomies. inversions. as in derivative chromosome. including . a common chromosomal disease. Also documented are trisomy 8.4. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. X0).1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. in which three copies of a chromosome are present instead of the usual two. as in the presence of extra or missing chromosomes. large-scale deletions or duplications. Down syndrome. translocations. are common numerical abnormalities. Structural abnormalities often arise from errors in homologous recombination. • • Patau syndrome is caused by trisomy of chromosome 13. although they generally do not survive to birth. Some disorders arise from loss of just a piece of one chromosome. also known as aneuploidy. XXY is caused by an extra X chromosome. trisomy 9 and trisomy 16. is caused by trisomy of chromosome 21. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. or structural. otherwise known as 47. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. often occur as a result of nondisjunction during meiosis in the formation of a gamete. X or 45.
A chromosome anomaly. numerical and structural anomalies. 1p36 Deletion syndrome. caused by abnormal formation of the larynx. from the loss of part of the short arm of chromosome 1. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. A Karyotype refers to a full set of chromosomes from an individual which can be compared to a "normal" Karyotype for the species via genetic testing. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. They can be organized into two basic groups. example of imprinting disorder. one well-documented example is the Philadelphia chromosome. A chromosome anomaly may be detected or confirmed in this manner. example of imprinting disorder. a deletion of the paternal genes. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual.• Cri du chat (cry of the cat). There are many types of chromosome anomalies. . a deletion of the maternal genes. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. from a truncated short arm on chromosome 5. The name comes from the babies' distinctive cry. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis.
Duplications: A portion of the chromosome is duplicated. Tetrasomy. In a reciprocal translocation. and Jacobsen syndrome. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. Known disorders in humans include Wolf-Hirschhorn syndrome. • • Translocations: When a portion of one chromosome is transferred to another chromosome. etc. There are two main types of translocations. also called the terminal 11q deletion disorder. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.3 Structural abnormalities When the chromosome's structure is altered. 4. 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. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. rather than two). an X. resulting in extra genetic material.4. In a Robertsonian translocation.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. segments from two different chromosomes have been exchanged.). which is caused by partial deletion of the short arm of chromosome 4. an entire chromosome has .
other cytogenetic banding techniques. turned upside down and reattached. especially the chromosomes. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 4. and are therefore initially not inherited. Some anomalies. Chromosome anomalies can be inherited from a parent or be "de novo".3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. • Inversions: A portion of the chromosome has broken off. 4. 15. Therefore. This can happen with or without loss of genetic material. • Rings: A portion of a chromosome has broken off and formed a circle or ring. however. as well . resulting in Mosaicism (where some cells have the anomaly and some do not). It includes routine analysis of G-Banded chromosomes.attached to another at the Centromere .4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. the anomaly is present in every cell of the body. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. 21 and 22. 14.in humans these only occur with chromosomes 13. can happen after conception. They often lead to an increased tendency to develop certain types of malignancies. therefore the genetic material is inverted.
concluding an XX/XO sex determination mechanism. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Pre-treating cells in a hypotonic solution. The next stage took place after the development of genetics in the early 20th century. The name was coined by another German anatomist. these results were quite remarkable. the discoverer of mitosis. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. 4. von Waldeyer in 1888. He revised his opinion later from 46 to 48. Their behavior in animal (salamander) cells was described by Walther Flemming. and he correctly insisted on man having an XX/XY system. which swells them and spreads the chromosomes . Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. in 1882. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Considering their techniques. Using cells in culture 2.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. in contrast to their genic contents. at first favoring 46.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). New techniques were needed to definitively solve the problem: 1.
Arresting mitosis in metaphase by a solution of colchicine 4. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.6 Applications in biology 4.3. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). 4. McClintock discovered transposons. Using Painter's technique they studied the polytene . Rather interestingly. During her cytogenetic work.6. the great apes have 48 chromosomes. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 4. persimilis from wild populations in California and neighboring states. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.6. Human chromosome 2 was formed by a merger of ancestral chromosomes. In 1931. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.2 Natural populations of Drosophila In the 1930s. a find which eventually led to her Nobel Prize in 1983. reducing the number.
It was found that the various chromosome types do not fluctuate at random. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. 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.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. In 1959. Evidence rapidly accumulated to show that natural selection was responsible. such as Down's syndrome. This had the benefit of eliminating migration as a possible explanation of the results.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. breeding and sampling whilst preventing escape. . 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. discoveries were quickly made related to aberrant chromosomes or chromosome number. but adjust to certain frequencies at which they become stabilised. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. Dobzhansky bred populations in population cages. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. Using a method invented by L'Heretier and Teissier. Down syndrome is also referred to as trisomy 21. In some congenital disorders. as with most polymorphisms. as they would if selectively neutral. 4. which enabled feeding.
Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. . which is why there is a phenotypic effect seen in individuals with extra X chromosomes.as both scientists were doing their research in Philadelphia. Pennsylvania.Other numerical abnormalities discovered include sex chromosome abnormalities. Not all genes on the X Chromosome are inactivated. XYY. is used today as a diagnostic for CML. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). Many other sex chromosome combinations are compatible with live birth including XXX. resulting in 47 total chromosomes. Identification of the Philadelphia chromosome by cytogenetics. an additional X chromosome in a male. with the development of more advanced techniques. An individual with only one sex chromosome (the X) has Turner syndrome. which is required in normal females to compensate for having two copies of the chromosome. This abnormal chromosome was dubbed the Philadelphia chromosome . Thirteen years later. has Klinefelter's Syndrome. in addition to other tests. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. and XXXX. In 1960. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22.
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. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material.8 Beginnings of molecular cytogenetics . Deletion syndromes such as DiGeorge syndrome. and elongation techniques for all culture types that allow for higher resolution banding. 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. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Caspersson developed banding techniques which differentially stain chromosomes. Deletions within one chromosome could also now be more specifically named and understood. 4. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.
While radioisotope-labeled probes had been hybridized with DNA since 1969. advances were made in molecular cytogenetics.In the 1980s. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).1 Karyotyping . CHAPTER 5 Techniques 5. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. cloned and studied in ever greater detail. movement was now made in using fluorescent labeled probes.
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.
5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •
bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations
5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,
including signal and image processing. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development.generally between 200 and 1000 cells are counted and scored. CGH and Single nucleotide polymorphism-arrays. 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. test and measurement. data analysis. and numerical computation. data visualization. For congenital problems usually 20 metaphase cells are scored. Using MATLAB. such as comparative genomic hybridization arrays. . you can solve technical computing problems faster than with traditional programming languages. communications. such as C. and computational biology. C++. 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. control design. You can use MATLAB in a wide range of applications. financial modeling and analysis.
such as declaring variables. As a result. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. and allocating memory. The image processing step is composed of the following operations. With the MATLAB language. one line of MATLAB code can often replace several lines of C or C++ code. It enables fast development and execution. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems.MATLAB provides a number of features for documenting and sharing your work. These effects must be compensated to improve the results of the pairing algorithm. specifying data types. 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.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. . 6. You can integrate your MATLAB code with other languages and applications. and distribute your MATLAB algorithms and applications. In many cases. MATLAB eliminates the need for ‘for’ loops.
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). the spatially scaled images are histogram equalized.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. To compare chromosomes from a band pattern point of view. geometrical and dimensional differences must be removed.2 Concepts used in this phase 1) Image conversion 2) Denoising . performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. 6. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 2) Geometrical compensation—The geometric compensation. or at least attenuated. Therefore. To compensate for this inhomogeneity.
so the image displays as shades of gray. you must first convert it to true color format. 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. 6. The resulting true color image has identical matrices for the red.I). For example. as is appropriate. When you apply the filter to the true color image. You can perform certain conversions just using MATLAB syntax. and the results might not be meaningful. and blue planes. MATLAB simply applies the filter to the indices in the indexed image matrix. For example. In addition to these image type conversion functions. green.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. 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. listed in the following table. RGB = cat (3. there are other functions that return a different image type as part of the operation they perform. . MATLAB filters the intensity values in the image.I.I.3) Edge detection 4) Two dimensional convolutions. if you want to filter a color image that is stored as an indexed image.2.
6.4 Denoising We may define noise to be any degradation in the image signal. via satellite or wireless transmission. These errors will appear on the image output in different ways depending on the type of disturbance in the signal.B) computes the two-dimensional convolution of matrices A and B. There is a large number of edge finding algorithms in existence. .3 Two dimensional convolutions C = conv2(A. If an image is being sent electronically from one place to another.'method'. Usually we know what type of errors to expect. The general Matlab command for finding edges is edge(image. we may expect errors to occur in the image signal. Cleaning an image corrupted by noise is thus an important area of image restoration.6.2. and hence the type of noise on the image. and we shall look at some of the more straightforward of them. 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 recognize or classify objects. If one of these matrices describes a two-dimensional finite impulse response . caused by external disturbance. to isolate particular objects from their background. ) Where the parameters available depend on the method used 6. or through networked cable.parameters. . We may use edges to measure the size of objects in an image.
(FIR) filter.hrow. .[3 3]). nb]+1)/2).A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.7)..na+nb-1]. imedfilt2(im1. 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. If hcol is a column vector and hrow is a row vector. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.'sobel').nb]. The size of matrices. C = conv2(.na] and the size of B is [mb. minus one. rgb2gray im2bw(im.. edge(im1.bmp'). this case is the same as C = conv2(hcol*hrow.. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. the other matrix is filtered in two dimensions. That is.0.A).'shape') subsection of the two-dimensional convolution. if the size of then the size of C is [ma+mb-1.
. Msk conv2(double(BW).imy]=size(BW). flag=0. [sx sy]=size(rc). [r. bwlabel(B. n1(x1.n]=size(L). L_number=zeros(mx.imy).8).[imx. MODULE 2 clc [m.j)==L_number(k) flag=1.double(msk)). Index=1.1). nzeros(imx.c] = find(L==22).y1)=255.j)~=0 for k=1:mx if L(i.2). for i=1:sx x1=rc(i.1). for i=1:m for j=1:n if L(i. y1=rc(i. mx=max(max(L)). rc = [r c].
Index=Index+126.96.36.199. end end L_number. end.7.32. for i=1:sx x1=rc(i.59.c] = find(L==L_number((Test_number(x)))).188.8.131.52.184.108.40.206.220.127.116.11. for x=1:46 [r.31. end %h=figure.18.104.22.168. end flag=0.65.41.8.42.52. y1=rc(i.9.2).29.66].19.55.10.end end if flag~=1 L_number(Index)=L(i.1).imy).). .35.49. 36. n1=zeros(imx.22.214.171.124.51. rc = [r c].43.40. n1(x1. [sx sy]=size(rc). Test_number=[3.j).imshow(n1.y1)=255.48.
Area=zeros(46. Circumference_sum=0.Inf). s=bwmorph(skel.'. BW=im2bw(f).end Circumference=zeros(46. Arm_length=zeros(46.1. BW=double(BW). f=imcomplement(f).5*graythresh(skel)). Arm_length_sum=0. s1=bwmorph(s. for i=1:46 f=imread(strcat(num2str(i).1).8).'spur'. BW1=edge(BW.bmp')). for x=1:m for y=1:n if BW1(x. [m n]=size(BW1). .'skel'. skel=im2double(f). end end end Circumference(i)=Circumference_sum.y)==1 Circumference_sum=Circumference_sum+1.1).1). skel=im2bw(skel.'canny').
y)==1 Arm_length_sum=Arm_length_sum+1. Arm_length.y)==1 Area_sum=Area_sum+1. end end end Area(i)=Area_sum. BW=im2bw(f). Area_sum=0. for x=1:m for y=1:n if BW(x. .[m n]=size(s1). [m n]=size(BW). end end end Arm_length(i)=Arm_length_sum. end Circumference. for x=1:m for y=1:n if s1(x.
2)=j. end end end for i=1:45 if Pair(i.1)=i.Area. Pair(46.2)=i+1.2).1)=i.1)=46. . Pair(i. end end Pair. Pair(i. Pair(i. Pair=zeros(46.2)==46 Pair(46.2)=i. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)). 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.
figure_flag). delete(figure_flag)=Pair(i. figure_flag=figure_flag+1.1). end end if flag~=1 if figure_flag~=47 subplot(23.bmp')). figure_flag=1. imshow(f1). end f2=imread(strcat(num2str(Pair(i. for i=1:46 for j=1:46 if Pair(i. imshow(f2). if figure_flag~=47 subplot(23.2).2.'.bmp')).1)==delete(j) flag=1. .2)).2. end f1=imread(strcat(num2str(Pair(i.delete=zeros(46.figure_flag).1)). figure_flag=figure_flag+1. end flag=0. flag=0.'.
Copenhagen. The proposed algorithm is based on the traditional features extracted from the karyogram. 2) feature extraction from the processed images . based on the MI. The main goal of this paper is to provide useful contributions toward the design of a fully automatic chromosomes pairing algorithm of bone marrow cells to be used in the diagnosis of leukemia. such as. dimensions and banding profiles. and Philadelphia. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. plus a new one. in the scope of karyotyping process used in cytogentic analysis.end CONCLUTION In this paper. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. 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. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern.
This normalization is needed to make it possible the band pattern comparison between chromosomes. extracted from the unordered karyogram. and finally. Here. 4) pairing. shape. and to normalize their dimensions. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. The training process consists in the estimation of each vector of coefficient .working within an 8-D feature space. Vectors of coefficients associated with each one of the 22 classes are computed and the distance between two arbitrary chromosomes is the minimum one among all distances obtained with these 22 vectors. The addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the . In the image processing step. shape and band pattern. Tests using 19 karyograms based on bone marrow cells. are processed in order to compensate for geometrical and intensity distortions. 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. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.10% mean classification rate. from the chromosomes in the training set. The features extracted from the processed images discriminate each pair with respect to their 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).working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. achieves a 70. the romosome images.characterizing the size. and band pattern.
Executing the algorithm on a higher quality dataset..performance of the classifier. and from which it is possible to extract additional features. centromere position. e. In addition. In fact. amean classification rate larger than 93% was obtained in all experiments. Using 27 karyograms andworking with a limited number of classes (≤ 8). presenting a uniform level of condensation.g.10% classification ratewas obtained. despite the low quality of this type of chromosomes. a 76. a new chromosome dataset with 9200 chromosomes from bone marrow cells. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. called LK1 . it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. The results presented in this paper are promising. REFERENCES . This dataset was made publicly available . Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. or Philadelphia. whose images are of significantly higher quality. such as Edinburgh. Copenhagen.
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Studies in mammalian spermatogenesis II. ^ Müller F. Zoology 37. Hereditas 42.R. Bioessays18: 133–138. The spermatogenesis of humans. & Tobler H.http://www. 1923. ^ Painter T. . 2000. 14. ^ Tjio J. 1998. Evolutionary genetics. 17.L.fcgi?artid=34032 19. p85-6 18.B.C 16.nih. 1979. Raven Press. 2001.S. ^ Goday C. Exp.R. J. 2nd ed.J. Chromatin diminution in nematodes. NY. 1-6. PNAS 97. ed. M. 21. ^ Stebbins G. 291-336.^ a b Hsu T.pubmedcentral. The chromosome number of man. New York. Human and mammalian cytogenetics: a historical perspective. Barch.C.C. In The ACT Cytogenetics Laboratory Manual 2nd ed. 1971. 15. ^ Maynard Smith J. Chromosomal evolution in higher plants. Bernard V. 1996. Eosin Y and Azure-A.^ a b Gustashaw K. ^ A preparation which includes the dyes Methylene Blue. London. The Association of Cytogenetic Technologists. 1991.12.gov/articlerender. p218-9 20. and Masters J. 1956. Chromosome stains. 13.H & Levan A. Chromosome elimination in sciarid flies. 9821– 9823. Kinetophore reproduction theory may explain rapid chromosome evolution.M. ^ Godfrey L. Oxford. and Esteban M. Bioessays23: 242–250. Arnold. Springer-Verlag.
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