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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
a large body of work uses the term chromosome regardless of chromatin content. divided. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. which is tightly coiled in on itself. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. These small circular genomes . Unduplicated chromosomes are single linear strands. sometimes accompanied by one or more smaller. through processes known as chromosomal instability and translocation. Chromosomes may exist as either duplicated or unduplicated. for example. Also. The structure of chromosomes and chromatin varies through the cell cycle. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). the cell may undergo mitotic catastrophe and die. Chromosomal recombination plays a vital role in genetic diversity. cells may contain more than one type of chromosome. or it may unexpectedly evadeapoptosis leading to the progression of cancer. although there are many exceptions to this rule. In prokaryotes. circular DNA molecules called plasmids. This allows the very long DNA molecules to fit into the cell nucleus. If these structures are manipulated incorrectly. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. In eukaryotes. Chromosomes are the essential unit for cellular division and must be replicated. In practice "chromosome" is a rather loosely defined term. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin.defined nuclei) have smaller circular chromosomes. DNA is usually arranged as a circle. the term genophore is more appropriate when no chromatin is present. However. In prokaryotes and viruses.
The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. rather than 2. reflecting their bacterial origins. 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. XX (female) or 46 XY (male). Euploid human karyotypes are 46. in which an individual has 3. The individual would have Down Syndrome and his/her karyotype would be written 47.+21.XX.+21. the individual carrying the mutation is said to be aneuploid. An example of aneuploidy is trisomy 21. If the mutation involves only one or a few chromosomes in the genome (e.are also found in mitochondria and chloroplasts.3 MUTATIONS IN CHROMOSOME NUMBER Normally.XY or 47. copies of chromosome 21. 1. Such individuals are called euploid and have the wild-type chromosome complement for the species.g. . 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). a extra copy of human chromosome 21).
1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. The microtubules then pull the chromatids apart toward the centrosomes. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. a pair of sister chromatids attached to each other at the centromere. and they form the classic four arm structure. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. This compact form makes the individual chromosomes visible. the chromatin strands become more and more condensed. the chromatids are uncoiled and DNA can again be transcribed. chromosomes are structurally highly condensed. one of which is present on each sister chromatid. In spite of their appearance. 1. which enables these giant DNA structures to be contained within a cell nucleus. A special DNA base sequence in the region of the kinetochores provides. During mitosis. longer-lasting attachment in this region. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. q-g "grande"). Once the cells have divided. so that each daughter cell inherits one set of chromatids. The shorter arms are called p arms (from the French petit. . along with special proteins. small) and the longer arms are called q arms (q follows p in the Latin alphabet.Fig 1.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). This is the only natural context in which individual chromosomes are visible with an optical microscope.
The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. which use the bone marrow vasculature as a conduit to the body's systemic circulation. bone marrow in large bones produces new blood cells. . in adults weighing 65 kg (143 lbs). bone marrow accounts for approximately 2. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones.7 lbs). In humans. It is generally followed immediately by cytokinesis.  Bone marrow is also a key component of the lymphatic system.6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2.1. On average. cytoplasm. 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. in two separate nuclei. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. which divides the nuclei.
prophase. animals undergo an "open" mitosis. Because cytokinesis usually occurs in conjunction with mitosis. cytokinesis and mitosis may occur independently. Prokaryotic cells. Mitosis occurs only in eukaryotic cells and the process varies in different species. anaphase and telophase. forming single cells with multiple nuclei. metaphase. there are many cells where mitosis and cytokinesis occur separately. where chromosomes divide within an intact cell nucleus. The cell then divides in cytokinesis. However. . For example. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. for instance during certain stages of fruit fly embryonic development. These stages are interphase. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. but is found in various different groups. "mitosis" is often used interchangeably with "mitotic phase". where the nuclear envelope breaks down before the chromosomes separate. divide by a process called binary fission. prometaphase. to produce two identical daughter cells which are still diploid cells. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. Even in animals. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. The process of mitosis is fast and highly complex. This occurs most notably among the fungi and slime moulds.genetically identical to each other and to their parent cell. which lack a nucleus. This accounts for approximately 10% of the cell cycle.
Each chromosome now has an identical copy of itself.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. and together the two are called sister chromatids. . the parent cell must make a copy of each chromosome before mitosis. Because each resultant daughter cell should be genetically identical to the parent cell. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. This occurs during the S phase of interphase. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. These two cells are identical and do not differ in any way from the original parent cell.
In plant cells. the parent cell will be split in half. As mitosis completes. However. A new nuclear envelope forms around the separated sister chromosomes. Eventually. each with a replica of the original genome.In most eukaryotes. The chromosomes align themselves in a line spanning the cell. so they are renamed to sister chromosomes. the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). . the process of binary fission is very much different from the process of mitosis. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. As the cell elongates. As a matter of convention. the daughter cells will construct a new dividing cell wall between each other. giving rise to two daughter cells.the cell begins cytokinesis. pulling apart the sister chromatids of each chromosome. Prokaryotic cells undergo a process similar to mitosis called binary fission. corresponding sister chromosomes are pulled toward opposite ends. In animal cells. separating the two developing nuclei. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. each sister chromatid is now considered a chromosome.
1 Preprophase In plant cells only. a cell grows (G1). 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. In highly vacuolated plant cells. grows more and prepares for mitosis (G 2). It alternates with the much longer interphase. the cell grows by producing proteins and cytoplasmic organelles. chromosomes are replicated only during the S phase. continues to grow as it duplicates its chromosomes (S). mainly via proteins. All these phases in the interphase are highly regulated. However. S (synthesis). where the cell prepares itself for cell division. and finally it divides (M) before restarting the cycle. Thus. and G2 (second gap). 2. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . This is achieved through the formation of a phragmosome.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. Interphase is divided into three phases: G1 (first gap). During all three phases. prophase is preceded by a pre-prophase stage.2. the nucleus has to migrate into the center of the cell before mitosis can begin.2.
microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. instead. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. These microtubules can attach to kinetochores or they can interact with opposing microtubules. aligned at the metaphase plate. degraded. In addition to phragmosome formation. This band marks the position where the cell will eventually divide. The cells of higher plants (such as the flowering plants) lack centrioles. the pinched area is known as the cleavage furrow. and microtubules have invaded the nuclear Prophase space. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. after the nuclear membrane breaks down. 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. Telophase: The decondensing chromosomes are surrounded by nuclear membranes.division. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. Cytokinesis has already begun. The chromosomes have chromatin has condensed. .
Although centrioles help organize microtubule assembly. the replicated chromosomes have two sister chromatids. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. At the onset of prophase. the genetic material in the nucleus is in a loosely bundled coil called chromatin. chromatin condenses together into a highly ordered structure called a chromosome. they are not essential for the . giving a pair of centrosomes. Chromosomes are typically visible at high magnification through a light microscope. Close to the nucleus are structures called centrosomes. 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. which are made of a pair of centrioles found in most eukaryotic animal cells. A cell inherits a single centrosome at cell division. The centrosome is the coordinating center for the cell's microtubules. which is replicated by the cell with the help of the nucleus before a new mitosis begins.
one attached at each chromatid. such as algae or trichomonads. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. since they are absent from plants. and it occurs in most multicellular organisms.formation of the spindle. provides the pulling force necessary to later separate the chromosome's two chromatids. . it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. When a microtubule connects with the kinetochore. coupled with polymerisation and depolymerisation of microtubules. This is called open mitosis. the motor activates. kinetochore microtubules begin searching for kinetochores to attach to. using energy from ATP to "crawl" up the tube toward the originating centrosome. and centrosomes are not always used in mitosis. undergo a variation called closed mitosis where the spindle forms inside the nucleus. 2. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook.2. Fungi and some protists. Each chromosome forms two kinetochores at the centromere.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. When the spindle grows to sufficient length. it is known that it contains some form of molecular motor. This motor activity. Prometaphase is sometimes considered part of prophase. or its microtubules are able to penetrate an intact nuclear envelope. Although the kinetochore structure and function are not fully understood. on an average 20 ).
an imaginary line that is equidistant from the two centrosome poles. convene along the metaphase plate or equatorial plane. Metaphase comes from the Greek meaning "after.In the fishing pole analogy. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. . The centromeres of the chromosomes.3 Metaphase A cell in late metaphase. the kinetochore would be the "hook" that catches a sister chromatid or "fish". Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". 2. analogous to a tug-of-war between people of equal strength." Microtubules find and attach to kinetochores in prometaphase. in some sense. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. All chromosomes (blue) but one have arrived at the metaphase plate. the chromosomes come under longitudinal tension from the two ends of the cell. As a result. In certain types of cells. only roughly lining up along the midline.
allowing them to separate.” “back. which have now become distinct sister chromosomes. the proteins that bind sister chromatids together are cleaved. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres).” “against. Two events then occur: first. The force that causes the centrosomes to move towards the ends of the cell is still unknown. The signal creates the mitotic spindle checkpoint. Next. Early anaphase is usually defined as the separation of the sister chromatids.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. These sister chromatids. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. At the end of anaphase. These two stages are sometimes called early and late anaphase.” or “re-”). the nonkinetochore microtubules elongate. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. 2. . although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. the cell proceeds to anaphase (from the Greek meaning “up.
pinching off the separated nuclei. A new nuclear envelope. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. using fragments of the parent cell's nuclear membrane. but cell division is not yet complete. Cytokinesis is technically not even a phase of mitosis. 2. It "cleans up" the after effects of mitosis. now surrounded by new nuclei. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. In animal cells. cytokinesis is a separate process that begins at the same time as telophase. At telophase.5 Cytokinesis Cilliate undergoing cytokinesis. necessary for completing cell division. forms around each set of separated sister chromosomes. elongating the cell even more.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. unfold back into chromatin. however. the nonkinetochore microtubules continue to lengthen. In both animal and plant cells. Mitosis is complete.2. Both sets of chromosomes. but rather a separate process. cell . Corresponding sister chromosomes attach at opposite ends of the cell.
which move along microtubules to the middle of the cell. 2.5.2 Development and growth The number of cells within an organism increases by mitosis. zygote and also the basis of the growth of a multicellular body. 2.division is also driven by vesicles derived from the Golgi apparatus.e.5. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.g..1Significance Mitosis is important for the maintenance of the chromosomal set. The phragmoplast is a microtubule structure typical for higher plants. Similarly. New cells are formed by mitosis and so are exact copies of the cells being replaced. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. 2. cells are constantly sloughed off and replaced by new ones. whereas some green algae use a phycoplast microtubule array during cytokinesis.3 Cell replacement In some parts of body. This is the basis of the development of a multicellular body from a single cell i. e. separating the two nuclei. skin and digestive tract.4 Regeneration .5.5. Following are the occasions in the lives of organism where mitosis happens: 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. The end of cytokinesis marks the end of the M-phase. Each daughter cell has a complete copy of the genome of its parent cell.
Some organisms can regenerate their parts of bodies. 2. For example. the process may go wrong. One daughter cell will receive both sister chromosomes and the other will receive none.5. These cells are considered aneuploid.7 Consequences of errors Although errors in mitosis are rare. a condition known as trisomy. a condition known as monosomy. Mitosis continues in the cells of bud and it grows into a new individual. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. The same division happens during asexual reproduction or vegetative propagation in plants. and the latter cell having only one chromosome (the homologous chromosome). sea star regenerates its lost arm through mitosis. the hydra reproduces asexually by budding. they fail to complete cell division and retain both nuclei in one cell. . This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). a condition often associated with cancer. 2. resulting in binucleated cells.5. The cells at the surface of hydra undergo mitosis and form a mass called bud. Occasionally when cells experience nondisjunction. a chromosome may fail to separate during anaphase. In non-disjunction. The production of new cells is achieved by mitosis. For example. especially during early cellular divisions in the zygote.
The fragment may incorrectly reattach to another. causing inversion. Benign tumours are not harmful as soon as they are not moving. Or. It may reattach to the original chromosome. Occasionally. and chromosomes are jostled constantly by probing microtubules. it results in the formation of Tumors. An arm of the chromosome may be broken and the fragment lost. causing translocation. This phenomenon is called metastasis or spreading of disease. non-homologous chromosome. Errors in the control of mitosis may cause cancer. It results in the synthesis of execessive tissue growths. The effect of these genetic abnormalities depends on the specific nature of the error. Now what happens is that cell abnormally continue to divide at a single place.Mitosis is a demanding process for the cell. its organelles disintegrate and reform in a matter of hours. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. causing deletion. Such tumours can send cancer cells to other parts in body where new tumours may form. but in reverse orientation. . All cells have genes that control the timing and number of mitosis. causing chromosomal duplication. it may be treated erroneously as a separate chromosome. It results in abnormal cell growth. When tissues more than the requirement are synthesized in a single organ. chromosomes may become damaged. sometimes mutuations occur in such genes and cells continue to divide. As soon as they start to move and invade other cells there are said to be malignant tumours. which goes through dramatic changes in ultrastructure. As long as these tumours remain in their original location they are called benign tumours.
analogous to a tug of war between equally strong people.2. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). This process may also be referred to as endoreduplication and the cells as endoploid. only roughly lining up along the middleline.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. In certain types of cells. carrying genetic information.7 Metaphase Metaphase. from the ancient Greek(between) and (stage). chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. align in the middle of the cell before being separated into each of the two daughter cells. An example of a cell that goes through endomitosis is the megakaryocyte. an imaginary line that is equidistant from the two centrosome poles. resulting in cells with many copies of the same chromosome occupying a single nucleus. 2. Early events of metaphase can . microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. Metaphase accounts for approximately 4% of the cell cycle's duration. Preceded by events in prometaphase and followed by anaphase.
and separase. Chromosomes are condensed(Thickened) and highly coiled in metaphase. 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. Such a signal creates the mitotic spindle checkpoint. One of the cell cycle checkpoints occurs during prometaphase and metaphase. 2. Normal metaphase spreads are used in . cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. This would be accomplished by regulation of the anaphase-promoting complex.coincide with the later events of prometaphase. does the cell enter anaphase. which makes them most suitable for visual analysis. Metaphase chromosomes make the classical picture of chromosomes (karyotype). when every kinetochore is properly attached to a bundle of microtubules. For classical cytogenetic analyses. Only after all chromosomes have become aligned at the metaphase plate. Staining of the slides. often with Giemsa (G banding) or Quinacrine. 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. 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). 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. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase.
such as bcr-abl in chronic myelogenous leukemia. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. . Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. losses of chromosomal segments or translocations. which may lead to chimeric oncogenes. for example.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments.
cellular function. Thus.  The preparation and study of karyotypes is part of cytogenetics. Karyotypes describe the number of chromosomes. banding pattern. taxonomic relationships. any differences between the sex chromosomes. be sex chromosomes. . the position of the centromeres. Attention is paid to their length. ordered by size and position of centromere for chromosomes of the same size. to study chromosomal aberrations.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. such as. Karyogram of human male using Giemsa staining. Karyotypes can be used for many purposes. The study of whole sets of chromosomes is sometimes known as karyology. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. There may. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. in normal diploid organisms. autosomal chromosomes are present in two copies. or an individual organism. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. So. and the results may be used in evolutionary biology and medicine. and any other physical characteristics. or may not. in humans 2n = 46. The study of karyotypes is important for cell biology and genetics. and to gather information about past evolutionary events. and what they look like under a light microscope. The term is also used for the complete set of chromosomes in a species.
The subsequent history of the concept can be followed in the works of Darlington and White.3. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. which swells them and spreads the chromosomes . He revised his opinion later from 46 to 48. Considering their techniques. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Their behavior in animal (salamander) cells was described by Walther Flemming.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. concluding an XX/XO sex determination mechanism. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. and he correctly insisted on humans having an XX/XY system. Using cells in culture 2. 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. at first favoring 46. 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. Pretreating cells in a hypotonic solution. in 1882. in contrast to their genic contents. The name was coined by another German anatomist. the discoverer of mitosis. these results were quite remarkable. The next stage took place after the development of genetics in the early 20th century. von Waldeyer in 1888.
2 Observations on karyotypes 3.2. Rather interestingly.  Sometimes observations may be made on non-dividing (interphase) cells. reducing the number. 3. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.2 Observations Six different characteristics of karyotypes are usually observed and compared: . The sex of an unborn fetus can be determined by observation of interphase cells. such as Giemsa. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. 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. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.3. the great apes have 48 chromosomes. 3. a suitable dye. For humans. Arresting mitosis in metaphase by a solution of colchicines 4.1 Staining The study of karyotypes is made possible by staining.2. Usually.
as well as other cytogenetic information. both have six pairs of chromosomes (n=6) yet V. 6. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). 2. . shape and banding of the chromosomes. permitting its loss without penalty to the organism (the dislocation hypothesis). Differences in degree and distribution of heterochromatic regions. 3. 4. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. which (when they occur) are small bodies attached to a chromosome by a thin thread. This feature probably reflects different amounts of DNA duplication. but the genes have been mostly translocated (added) to other chromosomes. and mainly consists of genetically inactive repetitive DNA sequences. Differences in number and position of satellites. 5. indicating tighter packing. Differences in absolute sizes of chromosomes. Heterochromatin stains darker than euchromatin. faba chromosomes are many times larger. Humans have one pair fewer chromosomes than the great apes. This is brought about by translocations.1. Differences in the position of centromeres. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. type. A full account of a karyotype may therefore include the number.
Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. males have both an X and a Y chromosome denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. XY. which are highly variable.Variation is often found: 1. Geographical variation between races 5. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. 3. There is variation between species in chromosome number. XX. Normal karyotypes for females contain two X chromosomes and are denoted 46. Any variation from the standard karyotype may lead to developmental abnormalities. Between the sexes 2. the same cannot be said for their karyotypes. Mosaics or otherwise abnormal individuals. and in . Between the germ-line and soma (between gametes and the rest of the body) 3. Between members of a population (chromosome polymorphism) 4.
Godfrey and Masters conclude: "In our view. Chromosome elimination. 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. In some species.. But.. In A. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. despite many careful investigations. In some cases there is even significant variation within species.3.detailed organization. In a review.. which were previously inexplicable. This variation provides the basis for a range of studies in evolutionary cytology. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. some organisms go in for large-scale elimination of heterochromatin. found in some copepods and roundworms such as Ascaris suum. or other kinds of visible adjustment to the karyotype. In this process. 3. as in many sciarid flies. "We have a very poor understanding of the causes of karyotype evolution. Chromatin diminution (founding father: Theodor Boveri). portions of the chromosomes are cast away in particular cells. .1 Changes during development Instead of the usual gene repression. it is quite unclear what the general significance might be. used in conjunction with other phylogenetic data. entire chromosomes are eliminated during development.. the general significance of karyotype evolution is obscure. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. Although much is known about karyotypes at the descriptive level.
Muntiacus reevesi. male = 7 chromosomes. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). Xinactivation. In marsupials it is always the paternal X which is inactivated. the inactivation is random as between the two Xs. 3. they were astonished to find it had female = 6. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. When they looked at the karyotype of the closely related Indian muntjac. The existence of supernumerary or B chromosomes . The low record is held by the nematode Parascaris univalens. Muntiacus muntjak. 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.3. all telocentric. "They simply could not believe what they saw. the high record would be somewhere amongst the ferns. was found to be 46.. thus the mammalian female is a mosaic in respect of her X chromosomes. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. where the haploid n = 1.suum. all the somatic cell precursors undergo chromatin diminution.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. They kept quiet for two or three years because they thought something was wrong with their tissue culture.. In human females some 15% of somatic cells escape inactivation... which was investigated by Kurt Benirschke and his colleague Doris Wurster. The diploid number of the Chinese muntjac. In placental mammals.
FN. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. 21 and 22). It is a common arrangement in the Hymenoptera. occurs mainly in plants. due to the presence of five acrocentric chromosome pairs (13. but it has been significant in some groups. Thus. 3.Endopolyploidy occurs when in adult differentiated . though in this case they would not be regarded as normal members of the population. but in grasses the average is much higher. Haplo-diploidy. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. horsetails and psilotales) is also common. and aneuploids are another example. and in some other groups.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell.3. 15. It has been of major significance in plant evolution according to Stebbins. where one sex is diploid. where there are more than two sets of homologous chromosomes in the cells. 14. Humans have FN = 82. 3.means that chromosome number can vary even within one interbreeding population. about 70%. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid.3 Fundamental number The fundamental number. FN ≤ 2n. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. Polyploidy in lower plants (ferns. and the other haploid. Polyploidy in animals is much less common. Polyploidy.
and serves differentiation and morphogenesis in many ways. Down syndrome and Turner syndrome are examples of this.tissues the cells have ceased to divide by mitosis. but the nuclei contain more than the original somatic number of chromosomes. The cells do not always contain exact multiples (powers of two). In many instances. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). 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. it is diverse and complex. Abnormalities in chromosome number usually cause a defect in development. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. the daughter chromosomes separating from each other inside an intact nuclear membrane. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. See palaeopolyploidy for the investigation of ancient karyotype duplications. 3. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. .
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. that the two chromosome morphs are adapted to different habitats. and Crocus. When this happens. Classic examples in plants are the genus Crepis. where every number from x = 3 to x = 15 is represented by at least one species. and 7. 4. 6.  Closer to home. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. reducing the number. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups.500 sq mi (17.Aneuploidy may also occur within a group of closely related species.000 km2). living from rainforests to . There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. In about 6. Human chromosome 2 was formed by a merger of ancestral chromosomes. where the gametic (= haploid) numbers form the series x = 3. 3. the great apes have 24x2 chromosomes whereas humans have 23x2. the chromosome number is variable from one individual to another.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. Well-researched examples are the ladybird beetle Chilocorus stigma. 5. 3. the European shrew Sorex araneus. some mantids of the genus Ameles.
The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. In a sense. at least into the Cretaceous. Using K-Ar dating. The inversions. in the family Drosophilidae. Chromosome rearrangements. show a clear "flow" of species from older to newer islands. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. when plotted in tree form (and independent of all other information). All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. make it possible to see which species are closely related. and skipping of islands. 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. the present islands date from 0. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. There are also cases of colonization back to older islands. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. Although it would be possible for a single gravid female to colonise an island. especially inversions. the best-studied group of Hawaiian drosophilids. which can be dated to 30 mya. The results are clear. Drosophila and Scaptomyza. gene arrangements are visible in the banding patterns of each chromosome. but these are much less frequent. probably 20 million years ago.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). .subalpine meadows. The polytene banding of the 'picture wing' group.
early-replicating and GC rich. . if less spectacular. It yields a series of lightly and darkly stained bands .the dark regions tend to be heterochromatic. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).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. • T-banding: visualize telomeres. The pattern of bands is very similar to that seen in G-banding.There are other animals and plants on the Hawaiian archipelago which have undergone similar. The light regions tend to be euchromatic. 3. R-banding is the reverse of G-banding (the R stands for "reverse").7.7 Depiction of karyotypes 3. • • C-banding: Giemsa binds to constitutive heterochromatin. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. human genome. adaptive radiations. so it stains centromeres. late-replicating and AT rich.
a dye. Quinacrine binds to the adeninethymine-rich regions. is used to stain bands on the chromosomes. This yields a dark region where the silver is deposited. Giemsa is specific for the phosphate groups of DNA. Each chromosome has a characteristic banding pattern that helps to identify them. In addition. respectively. Karyotypes are arranged with the short arm of the chromosome on top. 3.7.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. less frequently Quinacrine.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. In the "classic" (depicted) karyotype. For example. often Giemsa (G-banding). denoting the activity of rRNA genes within the NOR. Some karyotypes call the short and long arms p and q. and the long arm on the bottom. both chromosomes in a pair will have the same banding pattern. Cri du chat syndrome involves a deletion on the short arm of .
which is written as 46. Image processing software then assigns a pseudo color to each spectrally different combination. a combinatorial labeling method is used to generate many different colors.chromosome 5.5p-.del(5)(p15. The critical region for this syndrome is deletion of 15. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.2.XX. allowing the visualization of the individually colored chromosomes.2) 3. This method is also known as virtual karyotyping. . Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors.XX. 3. 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. It is written as 46. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.7. Because there are a limited number of spectrally-distinct fluorophores.
CHAPTER 4 .
as in the presence of extra or missing chromosomes. often occur as a result of nondisjunction during meiosis in the formation of a gamete. including . are common numerical abnormalities. Klinefelter syndrome. Also documented are trisomy 8. a common chromosomal disease. or structural.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. trisomy 9 and trisomy 16. large-scale deletions or duplications. X or 45. in which three copies of a chromosome are present instead of the usual two. Down syndrome. Some disorders arise from loss of just a piece of one chromosome. although they generally do not survive to birth. the most common male chromosomal disease. otherwise known as 47. trisomies. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. Numerical abnormalities. • • Patau syndrome is caused by trisomy of chromosome 13. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. as in derivative chromosome. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. is caused by trisomy of chromosome 21. inversions. XXY is caused by an extra X chromosome. X0).4. Structural abnormalities often arise from errors in homologous recombination. translocations. also known as aneuploidy.
from a truncated short arm on chromosome 5. . A chromosome anomaly may be detected or confirmed in this manner. 1p36 Deletion syndrome. There are many types of chromosome anomalies. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. A chromosome anomaly. caused by abnormal formation of the larynx. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. a deletion of the maternal genes. They can be organized into two basic groups. a deletion of the paternal genes. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. 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. one well-documented example is the Philadelphia chromosome. The name comes from the babies' distinctive cry. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. from the loss of part of the short arm of chromosome 1. example of imprinting disorder. example of imprinting disorder. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. numerical and structural anomalies.• Cri du chat (cry of the cat).
also called the terminal 11q deletion disorder. Known disorders in humans include Wolf-Hirschhorn syndrome. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. resulting in extra genetic material. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. and Jacobsen syndrome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). Tetrasomy.). • • Translocations: When a portion of one chromosome is transferred to another chromosome. etc. In a reciprocal translocation. rather than two). 4. In a Robertsonian translocation. segments from two different chromosomes have been exchanged. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. 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. an entire chromosome has . which is caused by partial deletion of the short arm of chromosome 4.3 Structural abnormalities When the chromosome's structure is altered. an X.4. There are two main types of translocations. Duplications: A portion of the chromosome is duplicated.
and are therefore initially not inherited. however.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. They often lead to an increased tendency to develop certain types of malignancies. It includes routine analysis of G-Banded chromosomes. • Rings: A portion of a chromosome has broken off and formed a circle or ring. 15.attached to another at the Centromere . Therefore.in humans these only occur with chromosomes 13. as well .3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. resulting in Mosaicism (where some cells have the anomaly and some do not). the anomaly is present in every cell of the body. 14. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. 21 and 22. especially the chromosomes. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. • Inversions: A portion of the chromosome has broken off. Chromosome anomalies can be inherited from a parent or be "de novo". 4. can happen after conception. other cytogenetic banding techniques. This can happen with or without loss of genetic material. 4. therefore the genetic material is inverted. Some anomalies. turned upside down and reattached.
in contrast to their genic contents. He revised his opinion later from 46 to 48. 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 1912.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. concluding an XX/XO sex determination mechanism. these results were quite remarkable. the discoverer of mitosis.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Using cells in culture 2. Considering their techniques. which swells them and spreads the chromosomes . in 1882. Pre-treating cells in a hypotonic solution. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. von Waldeyer in 1888. at first favoring 46. The next stage took place after the development of genetics in the early 20th century. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Their behavior in animal (salamander) cells was described by Walther Flemming. New techniques were needed to definitively solve the problem: 1. The name was coined by another German anatomist. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. and he correctly insisted on man having an XX/XY system. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. 4.
McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.2 Natural populations of Drosophila In the 1930s. 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.3. Arresting mitosis in metaphase by a solution of colchicine 4. In 1931. Rather interestingly. 4.6. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. persimilis from wild populations in California and neighboring states. a find which eventually led to her Nobel Prize in 1983. McClintock discovered transposons. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D.6. Human chromosome 2 was formed by a merger of ancestral chromosomes. During her cytogenetic work. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Using Painter's technique they studied the polytene . McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). the great apes have 48 chromosomes.6 Applications in biology 4. reducing the number.
Using a method invented by L'Heretier and Teissier. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. but adjust to certain frequencies at which they become stabilised.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. It was found that the various chromosome types do not fluctuate at random. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. breeding and sampling whilst preventing escape. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. Dobzhansky bred populations in population cages. In some congenital disorders. as with most polymorphisms. discoveries were quickly made related to aberrant chromosomes or chromosome number. as they would if selectively neutral. 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. Evidence rapidly accumulated to show that natural selection was responsible. . Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. 4. which enabled feeding. such as Down's syndrome. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. In 1959.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. This had the benefit of eliminating migration as a possible explanation of the results. Down syndrome is also referred to as trisomy 21.
Not all genes on the X Chromosome are inactivated. Many other sex chromosome combinations are compatible with live birth including XXX. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. Thirteen years later. and XXXX. which is required in normal females to compensate for having two copies of the chromosome. In 1960.as both scientists were doing their research in Philadelphia. resulting in 47 total chromosomes. Pennsylvania. in addition to other tests. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. Identification of the Philadelphia chromosome by cytogenetics. XYY. An individual with only one sex chromosome (the X) has Turner syndrome. 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). This abnormal chromosome was dubbed the Philadelphia chromosome . . which is why there is a phenotypic effect seen in individuals with extra X chromosomes. has Klinefelter's Syndrome. with the development of more advanced techniques.Other numerical abnormalities discovered include sex chromosome abnormalities. an additional X chromosome in a male. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them.
Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 4. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Caspersson developed banding techniques which differentially stain chromosomes.FIG Advent of banding techniques In the late 1960s. Deletions within one chromosome could also now be more specifically named and understood. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. and elongation techniques for all culture types that allow for higher resolution banding.8 Beginnings of molecular cytogenetics . 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. Deletion syndromes such as DiGeorge syndrome. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid.
CHAPTER 5 Techniques 5. advances were made in molecular cytogenetics.1 Karyotyping . This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. movement was now made in using fluorescent labeled probes.In the 1980s. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). cloned and studied in ever greater detail. While radioisotope-labeled probes had been hybridized with DNA since 1969.
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,
CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. such as comparative genomic hybridization arrays. you can solve technical computing problems faster than with traditional programming languages. C++. For congenital problems usually 20 metaphase cells are scored. and computational biology. control design. data visualization. financial modeling and analysis. You can use MATLAB in a wide range of applications. . and FORTRAN. test and measurement. such as C. communications. 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.generally between 200 and 1000 cells are counted and scored. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. Using MATLAB. CGH and Single nucleotide polymorphism-arrays. data analysis. and numerical computation. including signal and image processing.
The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems.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. With the MATLAB language. As a result. . You can integrate your MATLAB code with other languages and applications. specifying data types. and distribute your MATLAB algorithms and applications. The image processing step is composed of the following operations. 6. such as declaring variables. It enables fast development and execution. These effects must be compensated to improve the results of the pairing algorithm. The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted.MATLAB provides a number of features for documenting and sharing your work. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. In many cases. and allocating memory. one line of MATLAB code can often replace several lines of C or C++ code. MATLAB eliminates the need for ‘for’ loops.
To compare chromosomes from a band pattern point of view. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 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. Therefore.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. 6. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. the spatially scaled images are histogram equalized. geometrical and dimensional differences must be removed.2 Concepts used in this phase 1) Image conversion 2) Denoising . or at least attenuated. 2) Geometrical compensation—The geometric compensation. This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding). To compensate for this inhomogeneity.
3) Edge detection 4) Two dimensional convolutions. you must first convert it to true color format. and the results might not be meaningful. The resulting true color image has identical matrices for the red. as is appropriate.I. If you attempt to filter the indexed image. You can perform certain conversions just using MATLAB syntax. and blue planes. there are other functions that return a different image type as part of the operation they perform.2. so the image displays as shades of gray. RGB = cat (3. MATLAB simply applies the filter to the indices in the indexed image matrix. . For example. green. MATLAB filters the intensity values in the image. For example. For example. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. 6. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.I). When you apply the filter to the true color image. In addition to these image type conversion functions. listed in the following table.I. if you want to filter a color image that is stored as an indexed image.
2. to isolate particular objects from their background. If an image is being sent electronically from one place to another.B) computes the two-dimensional convolution of matrices A and B.6. and we shall look at some of the more straightforward of them. The general Matlab command for finding edges is edge(image. or through networked cable. There is a large number of edge finding algorithms in existence. . via satellite or wireless transmission. to recognize or classify objects. hence we can choose the most appropriate method for reducing the effects.'method'.3 Two dimensional convolutions C = conv2(A. If one of these matrices describes a two-dimensional finite impulse response .5 Edge detection Edges contain some of the most useful information in an image.4 Denoising We may define noise to be any degradation in the image signal. Usually we know what type of errors to expect. . We may use edges to measure the size of objects in an image.2. Cleaning an image corrupted by noise is thus an important area of image restoration. and hence the type of noise on the image.parameters. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. we may expect errors to occur in the image signal. caused by external disturbance. 6. ) Where the parameters available depend on the method used 6.
convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. minus one. The size of matrices.A). That is.na] and the size of B is [mb. nb]+1)/2). The indices of the center element of B are defined as floor(([mb C = conv2(hcol. 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. imedfilt2(im1.bmp').na+nb-1].0..'shape') subsection of the two-dimensional convolution.. edge(im1.. the other matrix is filtered in two dimensions. rgb2gray im2bw(im.7). this case is the same as C = conv2(hcol*hrow. If hcol is a column vector and hrow is a row vector.hrow.nb]. C = conv2(.(FIR) filter.'sobel').[3 3]).A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. if the size of then the size of C is [ma+mb-1. .
8). Index=1.[imx.imy). mx=max(max(L)).2).double(msk)).y1)=255. [r. Msk conv2(double(BW).1). nzeros(imx. rc = [r c].j)~=0 for k=1:mx if L(i. flag=0.j)==L_number(k) flag=1. for i=1:sx x1=rc(i.imy]=size(BW). n1(x1. y1=rc(i.1). [sx sy]=size(rc).n]=size(L). MODULE 2 clc [m. for i=1:m for j=1:n if L(i. . bwlabel(B. L_number=zeros(mx.c] = find(L==22).
54.31.50. . Test_number=[3.10.48. for i=1:sx x1=rc(i.end end if flag~=1 L_number(Index)=L(i.45.19.35. end %h=figure.59. 36. for x=1:46 [r.22.214.171.124.55.56. [sx sy]=size(rc).24. end flag=0.imshow(n126.96.36.199.21.6.62. rc = [r c].1).188.8.131.52.22.y1)=255. Index=Index+1.60.65. end end L_number.26.41.27.).29.2).184.108.40.206]. end.14.7. n1=zeros(imx.c] = find(L==L_number((Test_number(x)))).220.127.116.11.28. y1=rc(i.j).imy). n1(x1.
8). for i=1:46 f=imread(strcat(num2str(i). Area=zeros(46.'skel'. s1=bwmorph(s.y)==1 Circumference_sum=Circumference_sum+1. Arm_length_sum=0.1. Arm_length=zeros(46. .5*graythresh(skel)).1).end Circumference=zeros(46. end end end Circumference(i)=Circumference_sum. BW=double(BW).'canny'). Circumference_sum=0.'spur'.1). skel=im2bw(skel. [m n]=size(BW1). BW1=edge(BW.1). s=bwmorph(skel. BW=im2bw(f).bmp')). f=imcomplement(f). skel=im2double(f).'. for x=1:m for y=1:n if BW1(x.Inf).
Area_sum=0. [m n]=size(BW). for x=1:m for y=1:n if BW(x. . end Circumference. end end end Area(i)=Area_sum.y)==1 Arm_length_sum=Arm_length_sum+1. Arm_length. for x=1:m for y=1:n if s1(x.y)==1 Area_sum=Area_sum+1. end end end Arm_length(i)=Arm_length_sum.[m n]=size(s1). BW=im2bw(f).
. 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)).2)=i. Pair(i.2).2)=i+1. end end end for i=1:45 if Pair(i. Pair=zeros(46.1)=i.1)=i. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)). Pair(46. end end Pair.Area.1)=46. Pair(i. Pair(i.2)==46 Pair(46.2)=j. Pair(i.
delete(figure_flag)=Pair(i. for i=1:46 for j=1:46 if Pair(i. figure_flag=1.'.2. end f1=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1. end flag=0.delete=zeros(46.2)).1)==delete(j) flag=1. end end if flag~=1 if figure_flag~=47 subplot(23. flag=0. imshow(f1). imshow(f2). . figure_flag=figure_flag+1.'.figure_flag). if figure_flag~=47 subplot(23.figure_flag).1).bmp')).bmp')).2). end f2=imread(strcat(num2str(Pair(i.2.1)).
plus a new one. 2) feature extraction from the processed images . 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. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. 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. in the scope of karyotyping process used in cytogentic analysis. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. The proposed algorithm is based on the traditional features extracted from the karyogram. and Philadelphia.end CONCLUTION In this paper. based on the MI. dimensions and banding profiles. such as. Copenhagen.
working within an 8-D feature space. and band pattern. In the image processing step. The training process consists in the estimation of each vector of coefficient . are processed in order to compensate for geometrical and intensity distortions. extracted from the unordered karyogram.characterizing the size. and to normalize their dimensions. shape and band pattern. the romosome images. 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). from the chromosomes in the training set. 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. This normalization is needed to make it possible the band pattern comparison between chromosomes. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. The addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the . The features extracted from the processed images discriminate each pair with respect to their size. 4) pairing. and finally. Here. The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. shape. achieves a 70. Tests using 19 karyograms based on bone marrow cells.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.
e. centromere position.10% classification ratewas obtained. whose images are of significantly higher quality. REFERENCES . This dataset was made publicly available . and from which it is possible to extract additional features. called LK1 . In addition. Executing the algorithm on a higher quality dataset. a new chromosome dataset with 9200 chromosomes from bone marrow cells.performance of the classifier. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes.g. Using 27 karyograms andworking with a limited number of classes (≤ 8). The results presented in this paper are promising. or Philadelphia. such as Edinburgh. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. a 76. Copenhagen. despite the low quality of this type of chromosomes.. amean classification rate larger than 93% was obtained in all experiments. presenting a uniform level of condensation. In fact.
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