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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
mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. the term genophore is more appropriate when no chromatin is present. the cell may undergo mitotic catastrophe and die. divided. Also. although there are many exceptions to this rule. circular DNA molecules called plasmids. cells may contain more than one type of chromosome. Chromosomes are the essential unit for cellular division and must be replicated. In eukaryotes. In practice "chromosome" is a rather loosely defined term. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). a large body of work uses the term chromosome regardless of chromatin content. However. Unduplicated chromosomes are single linear strands. These small circular genomes . Chromosomes may exist as either duplicated or unduplicated. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. or it may unexpectedly evadeapoptosis leading to the progression of cancer. In prokaryotes and viruses. DNA is usually arranged as a circle. In prokaryotes. sometimes accompanied by one or more smaller. for example. through processes known as chromosomal instability and translocation. If these structures are manipulated incorrectly. which is tightly coiled in on itself. The structure of chromosomes and chromatin varies through the cell cycle. Chromosomal recombination plays a vital role in genetic diversity.defined nuclei) have smaller circular chromosomes. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny.
If the mutation involves only one or a few chromosomes in the genome (e. The individual would have Down Syndrome and his/her karyotype would be written 47. the individual carrying the mutation is said to be aneuploid. Euploid human karyotypes are 46.g.3 MUTATIONS IN CHROMOSOME NUMBER Normally. 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). rather than 2. XX (female) or 46 XY (male).XY or 47. 1. a extra copy of human chromosome 21).XX. in which an individual has 3. copies of chromosome 21. 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.+21. Such individuals are called euploid and have the wild-type chromosome complement for the species. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. .are also found in mitochondria and chloroplasts. An example of aneuploidy is trisomy 21.+21.
so that each daughter cell inherits one set of chromatids. the chromatin strands become more and more condensed. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. A special DNA base sequence in the region of the kinetochores provides. the chromatids are uncoiled and DNA can again be transcribed. small) and the longer arms are called q arms (q follows p in the Latin alphabet. The microtubules then pull the chromatids apart toward the centrosomes. and they form the classic four arm structure. This is the only natural context in which individual chromosomes are visible with an optical microscope. a pair of sister chromatids attached to each other at the centromere. In spite of their appearance. along with special proteins. 1.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. chromosomes are structurally highly condensed. This compact form makes the individual chromosomes visible. which enables these giant DNA structures to be contained within a cell nucleus. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. The shorter arms are called p arms (from the French petit. one of which is present on each sister chromatid. Once the cells have divided. During mitosis. . longer-lasting attachment in this region.Fig 1.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). q-g "grande"). Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction.
It is generally followed immediately by cytokinesis. bone marrow accounts for approximately 2. in two separate nuclei.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.  Bone marrow is also a key component of the lymphatic system.6 kg (5. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. producing the lymphocytes that support the body's immune system CHAPTER 2 2.7 lbs). which divides the nuclei. in adults weighing 65 kg (143 lbs). . bone marrow in large bones produces new blood cells.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. which use the bone marrow vasculature as a conduit to the body's systemic circulation. In humans.1. bone marrow constitutes 4% of the total body mass of humans. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. cytoplasm. On average. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.
while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. metaphase. but is found in various different groups. to produce two identical daughter cells which are still diploid cells. prometaphase. divide by a process called binary fission. This accounts for approximately 10% of the cell cycle. "mitosis" is often used interchangeably with "mitotic phase". forming single cells with multiple nuclei. animals undergo an "open" mitosis. However. Even in animals. These stages are interphase. for instance during certain stages of fruit fly embryonic development. prophase. The process of mitosis is fast and highly complex. Because cytokinesis usually occurs in conjunction with mitosis. Prokaryotic cells. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. where chromosomes divide within an intact cell nucleus. The cell then divides in cytokinesis. For example. there are many cells where mitosis and cytokinesis occur separately. cytokinesis and mitosis may occur independently. . which lack a nucleus. where the nuclear envelope breaks down before the chromosomes separate. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. anaphase and telophase. This occurs most notably among the fungi and slime moulds. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer.genetically identical to each other and to their parent cell. Mitosis occurs only in eukaryotic cells and the process varies in different species.
The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. Because each resultant daughter cell should be genetically identical to the parent cell. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. 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. 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.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. This occurs during the S phase of interphase. Each chromosome now has an identical copy of itself.
In most eukaryotes. each sister chromatid is now considered a chromosome. A new nuclear envelope forms around the separated sister chromosomes. the daughter cells will construct a new dividing cell wall between each other. the parent cell will be split in half. . Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. In plant cells. As a matter of convention. Eventually. the process of binary fission is very much different from the process of mitosis. separating the two developing nuclei. In animal cells. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. giving rise to two daughter cells.the cell begins cytokinesis. each with a replica of the original genome. pulling apart the sister chromatids of each chromosome. Prokaryotic cells undergo a process similar to mitosis called binary fission. However. As mitosis completes. 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). so they are renamed to sister chromosomes. As the cell elongates. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. corresponding sister chromosomes are pulled toward opposite ends. The chromosomes align themselves in a line spanning the cell.
grows more and prepares for mitosis (G 2). Thus. chromosomes are replicated only during the S phase. However. and G2 (second gap). In highly vacuolated plant cells. 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.1 Preprophase In plant cells only. the cell grows by producing proteins and cytoplasmic organelles. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . S (synthesis). Interphase is divided into three phases: G1 (first gap). continues to grow as it duplicates its chromosomes (S). All these phases in the interphase are highly regulated. It alternates with the much longer interphase. This is achieved through the formation of a phragmosome. the nucleus has to migrate into the center of the cell before mitosis can begin. a cell grows (G1).2. prophase is preceded by a pre-prophase stage.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. During all three phases. and finally it divides (M) before restarting the cycle.2. where the cell prepares itself for cell division. mainly via proteins. 2.
instead. The cells of higher plants (such as the flowering plants) lack centrioles. This band marks the position where the cell will eventually divide. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. These microtubules can attach to kinetochores or they can interact with opposing microtubules. The chromosomes have chromatin has condensed. after the nuclear membrane breaks down.division. the pinched area is known as the cleavage furrow. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. 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. In addition to phragmosome formation. degraded. and microtubules have invaded the nuclear Prophase space. . Cytokinesis has already begun. aligned at the metaphase plate. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves.
Close to the nucleus are structures called centrosomes. Since the genetic material has already been duplicated earlier in S phase. which is replicated by the cell with the help of the nucleus before a new mitosis begins. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. A cell inherits a single centrosome at cell division. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. Although centrioles help organize microtubule assembly. they are not essential for the . the genetic material in the nucleus is in a loosely bundled coil called chromatin.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. The centrosome is the coordinating center for the cell's microtubules. chromatin condenses together into a highly ordered structure called a chromosome. bound together at the centromere by the cohesin protein complex. Chromosomes are typically visible at high magnification through a light microscope. giving a pair of centrosomes. the replicated chromosomes have two sister chromatids. At the onset of prophase.
Each chromosome forms two kinetochores at the centromere. This motor activity. on an average 20 ). This is called open mitosis. it is known that it contains some form of molecular motor. provides the pulling force necessary to later separate the chromosome's two chromatids. the motor activates. such as algae or trichomonads.2. Fungi and some protists. 2. . undergo a variation called closed mitosis where the spindle forms inside the nucleus. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. Prometaphase is sometimes considered part of prophase. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Although the kinetochore structure and function are not fully understood. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. coupled with polymerisation and depolymerisation of microtubules.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. When the spindle grows to sufficient length. one attached at each chromatid. using energy from ATP to "crawl" up the tube toward the originating centrosome. and centrosomes are not always used in mitosis. and it occurs in most multicellular organisms. since they are absent from plants. When a microtubule connects with the kinetochore. or its microtubules are able to penetrate an intact nuclear envelope.formation of the spindle. kinetochore microtubules begin searching for kinetochores to attach to.
The centromeres of the chromosomes. analogous to a tug-of-war between people of equal strength. the kinetochore would be the "hook" that catches a sister chromatid or "fish".3 Metaphase A cell in late metaphase. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. in some sense. only roughly lining up along the midline. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur." Microtubules find and attach to kinetochores in prometaphase. an imaginary line that is equidistant from the two centrosome poles. In certain types of cells. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". As a result. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.In the fishing pole analogy. All chromosomes (blue) but one have arrived at the metaphase plate. Metaphase comes from the Greek meaning "after. 2. convene along the metaphase plate or equatorial plane. the chromosomes come under longitudinal tension from the two ends of the cell. .
The signal creates the mitotic spindle checkpoint. 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). 2. These sister chromatids.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate.” “against. the proteins that bind sister chromatids together are cleaved. . pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. Next. The force that causes the centrosomes to move towards the ends of the cell is still unknown. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. which have now become distinct sister chromosomes. Two events then occur: first.” “back.” or “re-”). At the end of anaphase. These two stages are sometimes called early and late anaphase. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. Early anaphase is usually defined as the separation of the sister chromatids. the nonkinetochore microtubules elongate. allowing them to separate. the cell proceeds to anaphase (from the Greek meaning “up.
but rather a separate process. now surrounded by new nuclei. A new nuclear envelope. Corresponding sister chromosomes attach at opposite ends of the cell. pinching off the separated nuclei. however. forms around each set of separated sister chromosomes. In animal cells.5 Cytokinesis Cilliate undergoing cytokinesis. cell . elongating the cell even more. Cytokinesis is technically not even a phase of mitosis. cytokinesis is a separate process that begins at the same time as telophase. At telophase. Both sets of chromosomes.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase.2. the nonkinetochore microtubules continue to lengthen. Mitosis is complete. In both animal and plant cells. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. necessary for completing cell division. It "cleans up" the after effects of mitosis. using fragments of the parent cell's nuclear membrane. 2. but cell division is not yet complete. unfold back into chromatin.
zygote and also the basis of the growth of a multicellular body. skin and digestive tract. e. Similarly. which move along microtubules to the middle of the cell.2 Development and growth The number of cells within an organism increases by mitosis.5. 2. Each daughter cell has a complete copy of the genome of its parent cell. The phragmoplast is a microtubule structure typical for higher plants.4 Regeneration . 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.division is also driven by vesicles derived from the Golgi apparatus. separating the two nuclei.3 Cell replacement In some parts of body. This is the basis of the development of a multicellular body from a single cell i.. whereas some green algae use a phycoplast microtubule array during cytokinesis.g. cells are constantly sloughed off and replaced by new ones.e.5.5. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.1Significance Mitosis is important for the maintenance of the chromosomal set. Following are the occasions in the lives of organism where mitosis happens: 2. The end of cytokinesis marks the end of the M-phase.5. New cells are formed by mitosis and so are exact copies of the cells being replaced.
This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). the hydra reproduces asexually by budding. the process may go wrong.5. especially during early cellular divisions in the zygote. sea star regenerates its lost arm through mitosis. they fail to complete cell division and retain both nuclei in one cell.Some organisms can regenerate their parts of bodies. For example. a chromosome may fail to separate during anaphase. One daughter cell will receive both sister chromosomes and the other will receive none. In non-disjunction. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. 2. For example.5. a condition known as monosomy. and the latter cell having only one chromosome (the homologous chromosome). 2. a condition often associated with cancer. a condition known as trisomy.7 Consequences of errors Although errors in mitosis are rare. Occasionally when cells experience nondisjunction. . The cells at the surface of hydra undergo mitosis and form a mass called bud. The same division happens during asexual reproduction or vegetative propagation in plants. The production of new cells is achieved by mitosis.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. Mitosis continues in the cells of bud and it grows into a new individual. resulting in binucleated cells. These cells are considered aneuploid.
sometimes mutuations occur in such genes and cells continue to divide. It results in the synthesis of execessive tissue growths. its organelles disintegrate and reform in a matter of hours. it may be treated erroneously as a separate chromosome. chromosomes may become damaged. but in reverse orientation. and chromosomes are jostled constantly by probing microtubules. Now what happens is that cell abnormally continue to divide at a single place. Errors in the control of mitosis may cause cancer. As soon as they start to move and invade other cells there are said to be malignant tumours. Or. causing deletion. As long as these tumours remain in their original location they are called benign tumours. causing translocation. non-homologous chromosome. causing chromosomal duplication. The effect of these genetic abnormalities depends on the specific nature of the error. it results in the formation of Tumors. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. It may reattach to the original chromosome. Benign tumours are not harmful as soon as they are not moving. causing inversion. It results in abnormal cell growth. . which goes through dramatic changes in ultrastructure. All cells have genes that control the timing and number of mitosis. This phenomenon is called metastasis or spreading of disease. An arm of the chromosome may be broken and the fragment lost. Such tumours can send cancer cells to other parts in body where new tumours may form. Occasionally. When tissues more than the requirement are synthesized in a single organ. The fragment may incorrectly reattach to another.Mitosis is a demanding process for the cell.
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). chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. An example of a cell that goes through endomitosis is the megakaryocyte. align in the middle of the cell before being separated into each of the two daughter cells. carrying genetic information. 2. This process may also be referred to as endoreduplication and the cells as endoploid. resulting in cells with many copies of the same chromosome occupying a single nucleus. Metaphase accounts for approximately 4% of the cell cycle's duration. In certain types of cells. Early events of metaphase can . only roughly lining up along the middleline. 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.2. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.7 Metaphase Metaphase.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. Preceded by events in prometaphase and followed by anaphase. from the ancient Greek(between) and (stage). analogous to a tug of war between equally strong people.
Metaphase chromosomes make the classical picture of chromosomes (karyotype). This would be accomplished by regulation of the anaphase-promoting complex. Such a signal creates the mitotic spindle checkpoint. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. Chromosomes are condensed(Thickened) and highly coiled in metaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. often with Giemsa (G banding) or Quinacrine. when every kinetochore is properly attached to a bundle of microtubules. Staining of the slides. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). 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. For classical cytogenetic analyses. 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. securin.coincide with the later events of prometaphase. does the cell enter anaphase. and separase. One of the cell cycle checkpoints occurs during prometaphase and metaphase. which makes them most suitable for visual analysis. Normal metaphase spreads are used in . 2.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. Only after all chromosomes have become aligned at the metaphase plate.
. losses of chromosomal segments or translocations. such as bcr-abl in chronic myelogenous leukemia. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. which may lead to chimeric oncogenes.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. for example. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations.
Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. Thus. in humans 2n = 46. and to gather information about past evolutionary events. The study of karyotypes is important for cell biology and genetics. The study of whole sets of chromosomes is sometimes known as karyology. ordered by size and position of centromere for chromosomes of the same size. There may. banding pattern. autosomal chromosomes are present in two copies. Karyogram of human male using Giemsa staining. and the results may be used in evolutionary biology and medicine. So. such as. any differences between the sex chromosomes. and any other physical characteristics. 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. . taxonomic relationships. the position of the centromeres. The term is also used for the complete set of chromosomes in a species. Attention is paid to their length.  The preparation and study of karyotypes is part of cytogenetics. Karyotypes can be used for many purposes. 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). cellular function.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. and what they look like under a light microscope. to study chromosomal aberrations. be sex chromosomes. Karyotypes describe the number of chromosomes. or may not. in normal diploid organisms.
Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. Using cells in culture 2. von Waldeyer in 1888. He revised his opinion later from 46 to 48. these results were quite remarkable. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. The subsequent history of the concept can be followed in the works of Darlington and White. Their behavior in animal (salamander) cells was described by Walther Flemming. at first favoring 46. Considering their techniques.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. 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. New techniques were needed to definitively solve the problem: 1. in contrast to their genic contents. the discoverer of mitosis.3. Pretreating cells in a hypotonic solution. and he correctly insisted on humans having an XX/XY system. The name was coined by another German anatomist. in 1882. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. The next stage took place after the development of genetics in the early 20th century.
Rather interestingly.2 Observations Six different characteristics of karyotypes are usually observed and compared: . 3.2. such as Giemsa. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Arresting mitosis in metaphase by a solution of colchicines 4. is applied after cells have been arrested during cell division by a solution of colchicine. The sex of an unborn fetus can be determined by observation of interphase cells. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. 3. Usually. a suitable dye. the great apes have 48 chromosomes.  Sometimes observations may be made on non-dividing (interphase) cells.1 Staining The study of karyotypes is made possible by staining.2 Observations on karyotypes 3. reducing the number.2. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes.3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. For humans.
2. Differences in absolute sizes of chromosomes. This is brought about by translocations. both have six pairs of chromosomes (n=6) yet V. Differences in the position of centromeres. Differences in number and position of satellites. permitting its loss without penalty to the organism (the dislocation hypothesis). but the genes have been mostly translocated (added) to other chromosomes. Differences in degree and distribution of heterochromatic regions. This feature probably reflects different amounts of DNA duplication. 3.1. indicating tighter packing. and mainly consists of genetically inactive repetitive DNA sequences. Heterochromatin stains darker than euchromatin. 4. shape and banding of the chromosomes. A full account of a karyotype may therefore include the number. Humans have one pair fewer chromosomes than the great apes. type. 5. which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. . faba chromosomes are many times larger. 6. as well as other cytogenetic information. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes).
which are highly variable. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Any variation from the standard karyotype may lead to developmental abnormalities. There is variation between species in chromosome number. Mosaics or otherwise abnormal individuals.Variation is often found: 1. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. and in . Between the sexes 2. Geographical variation between races 5. Between the germ-line and soma (between gametes and the rest of the body) 3. the same cannot be said for their karyotypes. XY.3 The human karyotype Most (but not all) species have a standard karyotype. XX. 3. Normal karyotypes for females contain two X chromosomes and are denoted 46. Between members of a population (chromosome polymorphism) 4. males have both an X and a Y chromosome denoted 46.
despite many careful investigations. This variation provides the basis for a range of studies in evolutionary cytology.detailed organization. some organisms go in for large-scale elimination of heterochromatin. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. In this process. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. But.. which were previously inexplicable. entire chromosomes are eliminated during development. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. Although much is known about karyotypes at the descriptive level. or other kinds of visible adjustment to the karyotype. the general significance of karyotype evolution is obscure. portions of the chromosomes are cast away in particular cells.. In A. Chromatin diminution (founding father: Theodor Boveri).. it is quite unclear what the general significance might be.3. In a review. 3.1 Changes during development Instead of the usual gene repression. Chromosome elimination. Godfrey and Masters conclude: "In our view. In some species. despite their construction from the same macromolecules. as in many sciarid flies. In some cases there is even significant variation within species.. used in conjunction with other phylogenetic data. . "We have a very poor understanding of the causes of karyotype evolution. found in some copepods and roundworms such as Ascaris suum.
male = 7 chromosomes. In human females some 15% of somatic cells escape inactivation.3. they were astonished to find it had female = 6. When they looked at the karyotype of the closely related Indian muntjac. the inactivation is random as between the two Xs. In placental mammals. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). Muntiacus reevesi. was found to be 46. all the somatic cell precursors undergo chromatin diminution. which was investigated by Kurt Benirschke and his colleague Doris Wurster. the high record would be somewhere amongst the ferns. They kept quiet for two or three years because they thought something was wrong with their tissue culture.. 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. all telocentric. The existence of supernumerary or B chromosomes .. where the haploid n = 1. Xinactivation. "They simply could not believe what they saw.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. The low record is held by the nematode Parascaris univalens.. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. 3.suum. The diploid number of the Chinese muntjac. In marsupials it is always the paternal X which is inactivated.. thus the mammalian female is a mosaic in respect of her X chromosomes. Muntiacus muntjak.
horsetails and psilotales) is also common. 14. It has been of major significance in plant evolution according to Stebbins. 3. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. due to the presence of five acrocentric chromosome pairs (13. Haplo-diploidy.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell.means that chromosome number can vary even within one interbreeding population.Endopolyploidy occurs when in adult differentiated . 15.3. Polyploidy. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. about 70%. though in this case they would not be regarded as normal members of the population. and aneuploids are another example. Polyploidy in lower plants (ferns. where there are more than two sets of homologous chromosomes in the cells. Humans have FN = 82. but it has been significant in some groups. Polyploidy in animals is much less common. where one sex is diploid. and in some other groups. Thus. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. FN ≤ 2n. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. but in grasses the average is much higher. FN.3 Fundamental number The fundamental number. and the other haploid. It is a common arrangement in the Hymenoptera. 21 and 22). occurs mainly in plants. 3. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.
endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. Abnormalities in chromosome number usually cause a defect in development. See palaeopolyploidy for the investigation of ancient karyotype duplications. 3.tissues the cells have ceased to divide by mitosis. it is diverse and complex. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. In many instances. 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. . The cells do not always contain exact multiples (powers of two). but the nuclei contain more than the original somatic number of chromosomes. the daughter chromosomes separating from each other inside an intact nuclear membrane. Down syndrome and Turner syndrome are examples of this.
5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations.6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson. Human chromosome 2 was formed by a merger of ancestral chromosomes.Aneuploidy may also occur within a group of closely related species. reducing the number. the chromosome number is variable from one individual to another. 6. and Crocus. Classic examples in plants are the genus Crepis. 5. When this happens. and 7. that the two chromosome morphs are adapted to different habitats. living from rainforests to . Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups.000 km2).  Closer to home. some mantids of the genus Ameles. Well-researched examples are the ladybird beetle Chilocorus stigma. where every number from x = 3 to x = 15 is represented by at least one species. In about 6. where the gametic (= haploid) numbers form the series x = 3. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. the European shrew Sorex araneus. 3. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 4. the great apes have 24x2 chromosomes whereas humans have 23x2.500 sq mi (17. 3.
Drosophila and Scaptomyza. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. There are also cases of colonization back to older islands. show a clear "flow" of species from older to newer islands. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). In a sense. The results are clear. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. and skipping of islands. gene arrangements are visible in the banding patterns of each chromosome. in the family Drosophilidae. the best-studied group of Hawaiian drosophilids. when plotted in tree form (and independent of all other information). it is more likely to have been a group from the same species. which can be dated to 30 mya. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. Chromosome rearrangements. The inversions. but these are much less frequent. especially inversions. make it possible to see which species are closely related. probably 20 million years ago. at least into the Cretaceous. Although it would be possible for a single gravid female to colonise an island. . The polytene banding of the 'picture wing' group. the present islands date from 0.subalpine meadows. Using K-Ar dating. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll.
early-replicating and GC rich. . so it stains centromeres. R-banding is the reverse of G-banding (the R stands for "reverse"). • • C-banding: Giemsa binds to constitutive heterochromatin.the dark regions tend to be heterochromatic. 3. The pattern of bands is very similar to that seen in G-banding. if less spectacular. This method will normally produce 300-400 bands in a normal. The light regions tend to be euchromatic. • T-banding: visualize telomeres.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.There are other animals and plants on the Hawaiian archipelago which have undergone similar.7 Depiction of karyotypes 3.7. adaptive radiations. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). late-replicating and AT rich. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. It yields a series of lightly and darkly stained bands . human genome.
Quinacrine binds to the adeninethymine-rich regions. Each chromosome has a characteristic banding pattern that helps to identify them. Some karyotypes call the short and long arms p and q. For example.7. In addition. less frequently Quinacrine.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. a dye. both chromosomes in a pair will have the same banding pattern. denoting the activity of rRNA genes within the NOR. This yields a dark region where the silver is deposited. often Giemsa (G-banding). and the long arm on the bottom. Karyotypes are arranged with the short arm of the chromosome on top. 3. In the "classic" (depicted) karyotype. is used to stain bands on the chromosomes. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. respectively. Giemsa is specific for the phosphate groups of DNA.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. Cri du chat syndrome involves a deletion on the short arm of .
It is written as 46.2. Image processing software then assigns a pseudo color to each spectrally different combination. This method is also known as virtual karyotyping.chromosome 5.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. .del(5)(p15.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX. Because there are a limited number of spectrally-distinct fluorophores. The critical region for this syndrome is deletion of 15.2) 3. which is written as 46. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. 3.7.XX. a combinatorial labeling method is used to generate many different colors.5p-. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. 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. allowing the visualization of the individually colored chromosomes.
CHAPTER 4 .
Some disorders arise from loss of just a piece of one chromosome. including . or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. X0). Also documented are trisomy 8. X or 45. Structural abnormalities often arise from errors in homologous recombination. or structural. are common numerical abnormalities. often occur as a result of nondisjunction during meiosis in the formation of a gamete. Down syndrome. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. a common chromosomal disease. Klinefelter syndrome. the most common male chromosomal disease. translocations. inversions. XXY is caused by an extra X chromosome. is caused by trisomy of chromosome 21.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. trisomy 9 and trisomy 16.4. otherwise known as 47. also known as aneuploidy. in which three copies of a chromosome are present instead of the usual two. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. large-scale deletions or duplications. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. • • Patau syndrome is caused by trisomy of chromosome 13. Numerical abnormalities. as in derivative chromosome. trisomies. although they generally do not survive to birth. as in the presence of extra or missing chromosomes.
A chromosome anomaly. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. a deletion of the maternal genes. The name comes from the babies' distinctive cry. A chromosome anomaly may be detected or confirmed in this manner. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. . from the loss of part of the short arm of chromosome 1. numerical and structural anomalies. caused by abnormal formation of the larynx. 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.• Cri du chat (cry of the cat). from a truncated short arm on chromosome 5. a deletion of the paternal genes. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. They can be organized into two basic groups. one well-documented example is the Philadelphia chromosome. example of imprinting disorder. 1p36 Deletion syndrome. example of imprinting disorder. There are many types of chromosome anomalies.
segments from two different chromosomes have been exchanged. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. an X. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome.3 Structural abnormalities When the chromosome's structure is altered. Duplications: A portion of the chromosome is duplicated. There are two main types of translocations.). rather than two). This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. 4. resulting in extra genetic material. • • Translocations: When a portion of one chromosome is transferred to another chromosome. Tetrasomy. and Jacobsen syndrome. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. also called the terminal 11q deletion disorder. Known disorders in humans include Wolf-Hirschhorn syndrome. which is caused by partial deletion of the short arm of chromosome 4. In a Robertsonian translocation. an entire chromosome has .2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). etc. In a reciprocal translocation.4. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.
turned upside down and reattached. however. can happen after conception. This can happen with or without loss of genetic material. the anomaly is present in every cell of the body. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. 15. 21 and 22. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. Chromosome anomalies can be inherited from a parent or be "de novo". especially the chromosomes. • Inversions: A portion of the chromosome has broken off.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. Therefore. 14. • Rings: A portion of a chromosome has broken off and formed a circle or ring.attached to another at the Centromere .in humans these only occur with chromosomes 13. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. other cytogenetic banding techniques. as well . and are therefore initially not inherited.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). Some anomalies. They often lead to an increased tendency to develop certain types of malignancies. It includes routine analysis of G-Banded chromosomes. 4. 4. therefore the genetic material is inverted.
5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. and he correctly insisted on man having an XX/XY system. The next stage took place after the development of genetics in the early 20th century. von Waldeyer in 1888. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in contrast to their genic contents. these results were quite remarkable. Pre-treating cells in a hypotonic solution. New techniques were needed to definitively solve the problem: 1. Considering their techniques. concluding an XX/XO sex determination mechanism. at first favoring 46. in 1882. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. He revised his opinion later from 46 to 48. 4. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. the discoverer of mitosis. 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. The name was coined by another German anatomist.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Using cells in culture 2. Their behavior in animal (salamander) cells was described by Walther Flemming. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.
2 Natural populations of Drosophila In the 1930s. a find which eventually led to her Nobel Prize in 1983.3. In 1931. Rather interestingly.6 Applications in biology 4. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).6. 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. Human chromosome 2 was formed by a merger of ancestral chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. persimilis from wild populations in California and neighboring states. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.6. During her cytogenetic work. Arresting mitosis in metaphase by a solution of colchicine 4. 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. 4. the great apes have 48 chromosomes. Using Painter's technique they studied the polytene . reducing the number.
. as they would if selectively neutral. discoveries were quickly made related to aberrant chromosomes or chromosome number. 4. In 1959. as with most polymorphisms. 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. such as Down's syndrome. but adjust to certain frequencies at which they become stabilised. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. It was found that the various chromosome types do not fluctuate at random.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes.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. In some congenital disorders. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. Evidence rapidly accumulated to show that natural selection was responsible. Dobzhansky bred populations in population cages. 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. Down syndrome is also referred to as trisomy 21. which enabled feeding. breeding and sampling whilst preventing escape. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions.
which is why there is a phenotypic effect seen in individuals with extra X chromosomes.as both scientists were doing their research in Philadelphia. is used today as a diagnostic for CML. This abnormal chromosome was dubbed the Philadelphia chromosome . 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. In 1960. an additional X chromosome in a male. which is required in normal females to compensate for having two copies of the chromosome. . Not all genes on the X Chromosome are inactivated. Identification of the Philadelphia chromosome by cytogenetics. An individual with only one sex chromosome (the X) has Turner syndrome. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. Pennsylvania. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). in addition to other tests. resulting in 47 total chromosomes. and XXXX.Other numerical abnormalities discovered include sex chromosome abnormalities. XYY. has Klinefelter's Syndrome. Thirteen years later. with the development of more advanced techniques.
Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.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. 4. Caspersson developed banding techniques which differentially stain chromosomes. and elongation techniques for all culture types that allow for higher resolution banding. Deletion syndromes such as DiGeorge syndrome. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Deletions within one chromosome could also now be more specifically named and understood. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.FIG Advent of banding techniques In the late 1960s.
cloned and studied in ever greater detail. movement was now made in using fluorescent labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. While radioisotope-labeled probes had been hybridized with DNA since 1969. CHAPTER 5 Techniques 5.1 Karyotyping . advances were made in molecular cytogenetics.In the 1980s.
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,
financial modeling and analysis. CGH and Single nucleotide polymorphism-arrays. and FORTRAN. data visualization. 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. communications. and computational biology. control design. and numerical computation. Using MATLAB. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. C++. . including signal and image processing. test and measurement. such as C. such as comparative genomic hybridization arrays.generally between 200 and 1000 cells are counted and scored. you can solve technical computing problems faster than with traditional programming languages. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. data analysis. For congenital problems usually 20 metaphase cells are scored. You can use MATLAB in a wide range of applications.
such as declaring variables. and allocating memory. As a result. . you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. You can integrate your MATLAB code with other languages and applications. specifying data types. and distribute your MATLAB algorithms and applications. These effects must be compensated to improve the results of the pairing algorithm. In many cases. The image processing step is composed of the following operations.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.MATLAB provides a number of features for documenting and sharing your work. With the MATLAB language. 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. one line of MATLAB code can often replace several lines of C or C++ code. MATLAB eliminates the need for ‘for’ loops. It enables fast development and execution. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. 6.
This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding). To compare chromosomes from a band pattern point of view. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. 2) Geometrical compensation—The geometric compensation. geometrical and dimensional differences must be removed. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. the spatially scaled images are histogram equalized.2 Concepts used in this phase 1) Image conversion 2) Denoising . Therefore. 6. To compensate for this inhomogeneity. 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.
. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. so the image displays as shades of gray.3) Edge detection 4) Two dimensional convolutions.I. MATLAB filters the intensity values in the image. green. 6. listed in the following table.I). You can perform certain conversions just using MATLAB syntax.I. MATLAB simply applies the filter to the indices in the indexed image matrix. RGB = cat (3. if you want to filter a color image that is stored as an indexed image. For example. and the results might not be meaningful. and blue planes. In addition to these image type conversion functions. there are other functions that return a different image type as part of the operation they perform. The resulting true color image has identical matrices for the red. as is appropriate.2. When you apply the filter to the true color image. For example.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. For example. you must first convert it to true color format. If you attempt to filter the indexed image. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations.
The general Matlab command for finding edges is edge(image. These errors will appear on the image output in different ways depending on the type of disturbance in the signal.6. ) Where the parameters available depend on the method used 18.104.22.168 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.parameters. we may expect errors to occur in the image signal. caused by external disturbance. Cleaning an image corrupted by noise is thus an important area of image restoration. to recognize or classify objects.3 Two dimensional convolutions C = conv2(A.B) computes the two-dimensional convolution of matrices A and B. We may use edges to measure the size of objects in an image. There is a large number of edge finding algorithms in existence. 6. If an image is being sent electronically from one place to another. via satellite or wireless transmission. hence we can choose the most appropriate method for reducing the effects. . or through networked cable. If one of these matrices describes a two-dimensional finite impulse response .'method'. and we shall look at some of the more straightforward of them. . Usually we know what type of errors to expect. and hence the type of noise on the image. to isolate particular objects from their background.
na] and the size of B is [mb. If hcol is a column vector and hrow is a row vector.nb].A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.(FIR) filter. as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345. if the size of then the size of C is [ma+mb-1. C = conv2(.'shape') subsection of the two-dimensional convolution.0.na+nb-1]. The size of matrices.'sobel'). rgb2gray im2bw(im. imedfilt2(im1. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.A). this case is the same as C = conv2(hcol*hrow. That is.7). nb]+1)/2). edge(im1.. .bmp').. minus one. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.. the other matrix is filtered in two dimensions.hrow.[3 3]).
1).[imx.8).c] = find(L==22).imy).n]=size(L).j)==L_number(k) flag=1.imy]=size(BW).1). MODULE 2 clc [m. flag=0.2). n1(x1.y1)=255. rc = [r c]. . y1=rc(i. [r.double(msk)).j)~=0 for k=1:mx if L(i. for i=1:m for j=1:n if L(i. bwlabel(B. Msk conv2(double(BW). Index=1. [sx sy]=size(rc). mx=max(max(L)). nzeros(imx. L_number=zeros(mx. for i=1:sx x1=rc(i.
22.214.171.124. .33. end.10.6. [sx sy]=size(rc). y1=rc(i. n1(x1.49. end end L_number.j).41.21.end end if flag~=1 L_number(Index)=L(i.7.1).30.51.c] = find(L==L_number((Test_number(x)))).60. n1=zeros(imx.126.96.36.199.52.45.).29.35.31.y1)=255.2). end %h=figure. Index=Index+1. for i=1:sx x1=rc(i.188.8.131.52.62. rc = [r c].22.38.59. for x=1:46 [r.9.imy).184.108.40.206. 36. Test_number=[3.66].54. end flag=0.50.57.imshow(n220.127.116.11.42.
'spur'. end end end Circumference(i)=Circumference_sum. skel=im2double(f). BW=double(BW). s=bwmorph(skel. Arm_length_sum=0.1. Arm_length=zeros(46.1).'.'canny').Inf). s1=bwmorph(s.8).'skel'. f=imcomplement(f). skel=im2bw(skel. Circumference_sum=0. . Area=zeros(46.1).1).y)==1 Circumference_sum=Circumference_sum+1. BW1=edge(BW. for i=1:46 f=imread(strcat(num2str(i).bmp')).end Circumference=zeros(46. [m n]=size(BW1).5*graythresh(skel)). BW=im2bw(f). for x=1:m for y=1:n if BW1(x.
BW=im2bw(f).y)==1 Area_sum=Area_sum+1. [m n]=size(BW). Area_sum=0. for x=1:m for y=1:n if BW(x. end end end Area(i)=Area_sum. . end Circumference. Arm_length. for x=1:m for y=1:n if s1(x. end end end Arm_length(i)=Arm_length_sum.y)==1 Arm_length_sum=Arm_length_sum+1.[m n]=size(s1).
1)=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)).2)=i+1. Pair=zeros(46.2). Pair(i.2)==46 Pair(46. Pair(i. . for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)).2)=i.Area. Pair(i. end end Pair.1)=46. Pair(46.1)=i. Pair(i. end end end for i=1:45 if Pair(i.2)=j.
if figure_flag~=47 subplot(23.2)). . end f2=imread(strcat(num2str(Pair(i. flag=0. end flag=0. figure_flag=1.2.figure_flag).bmp')).'.delete=zeros(46. delete(figure_flag)=Pair(i.bmp')).1). figure_flag=figure_flag+1.2). figure_flag=figure_flag+1.1)).'. imshow(f1).2. imshow(f2). end end if flag~=1 if figure_flag~=47 subplot(23. end f1=imread(strcat(num2str(Pair(i.1)==delete(j) flag=1. for i=1:46 for j=1:46 if Pair(i.figure_flag).
2) feature extraction from the processed images . Copenhagen. 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. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. in the scope of karyotyping process used in cytogentic analysis. dimensions and banding profiles. 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. based on the MI.end CONCLUTION In this paper. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. plus a new one. 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. and Philadelphia.
shape. and finally. 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.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. from the chromosomes in the training set. In the image processing step. and to normalize their dimensions. the romosome images. Here. The features extracted from the processed images discriminate each pair with respect to their size. The training process consists in the estimation of each vector of coefficient . a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. shape and band pattern. extracted from the unordered karyogram. achieves a 70. and band pattern.working within an 8-D feature space. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. Tests using 19 karyograms based on bone marrow cells. 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. 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). are processed in order to compensate for geometrical and intensity distortions.characterizing the size. 4) pairing. 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 .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.
presenting a uniform level of condensation.10% classification ratewas obtained. In addition. called LK1 . In fact. e. such as Edinburgh. The results presented in this paper are promising. a new chromosome dataset with 9200 chromosomes from bone marrow cells. This dataset was made publicly available .performance of the classifier. Using 27 karyograms andworking with a limited number of classes (≤ 8). or Philadelphia. REFERENCES . Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. despite the low quality of this type of chromosomes. and from which it is possible to extract additional features. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. Copenhagen. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset.g. Executing the algorithm on a higher quality dataset. a 76. amean classification rate larger than 93% was obtained in all experiments. centromere position.. whose images are of significantly higher quality.
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