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During the Metaphase type of cell division in the Bone Marrow, Karyotyping method gives the visual representation of the 46 chromosomes paired and arranged in decreasing order of size. This representation is useful in leukemia purposes. This method is a difficult one because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges. So here, Karyotyping uses new mutual information method which is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. This algorithm is formulated as such a method of combinatorial optimization problem. Where the distances between homologous chromosomes are minimized and the distances between non homologous ones are maximized. It is solved by using an integer programming approach. In this project chromosome dataset Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes was used for this study.
Introduction 1.1 The study of chromosome morphology and its relation with some genetic diseases is the main goal of cytogenetic. Normal human cells have 23 classes of large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The chromosome contains approximately 30 000 genes (genotype) and large tracts of non coding sequences. The analysis of genetic material can involve the examination of specific chromosomal regions using DNA probes, e.g., fluorescent in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic hybridization (CGH) , or the morphological and pattern analysis of entire chromosomes, the conventional cytogenetic, which is the focus of this paper. These cytogenetic studies are very important in the detection of acquired chromosomal abnormalities, such as translocations, duplications, inversions, deletions, monosomies, or trisomies. These techniques are particularly useful in the diagnosis of cancerous diseases and are the preferred ones in the characterization of the different types of leukemia, which is the motivation of this paper . The pairing of chromosomes is one of the main steps in conventional cytogenetic analysis where a correctly ordered karyogram is produced for diagnosis of genetic diseases based on the patient karyotype. The karyogram is an image representation of the stained human chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) , where the chromosomes are arranged in 22 pairs of somatic homologous elements plus two sex-determinative chromosomes (XX for the female or XY for the male), displayed in decreasing order of size. A karyotype is the set of characteristics
extracted from the karyogram that may be used to detect chromosomal abnormalities. The metaphase is the step of the cellular division process where the chromosomes are in their most condensed state. This is the most appropriated moment to its visualization and abnormality recognition because the chromosomes appear well defined and clear. The pairing and karyotyping procedure, usually done manually by visual inspection, is time consuming and technically demanding. The application of the G-banding procedure to the chromosomes generates a distinct transverse banding pattern characteristic for each class, which is the most important feature for chromosome classification and pairing. The International System for Cytogenetic Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as for all the chromosomes of a normal human, and the clinical staff is trained to pair and interpret each specific karyogram according to the ISCN information. Other features, related to the chromosome dimensions and shape, are also used to increase the discriminative power of the manual or automatic classifiers. 1.2 CHROMOSOME A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes,regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Chromosomes vary widely between different organisms. The DNA molecule may be circular or linear, and can be composed of 100,000 to 10,000,000,000 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without
In prokaryotes. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. If these structures are manipulated incorrectly. through processes known as chromosomal instability and translocation. Chromosomes may exist as either duplicated or unduplicated. In practice "chromosome" is a rather loosely defined term. divided. or it may unexpectedly evadeapoptosis leading to the progression of cancer. cells may contain more than one type of chromosome. the term genophore is more appropriate when no chromatin is present. Unduplicated chromosomes are single linear strands. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). which is tightly coiled in on itself. circular DNA molecules called plasmids. DNA is usually arranged as a circle. a large body of work uses the term chromosome regardless of chromatin content. for example. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. In prokaryotes and viruses. This allows the very long DNA molecules to fit into the cell nucleus. In eukaryotes. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. These small circular genomes . However. the cell may undergo mitotic catastrophe and die. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. The structure of chromosomes and chromatin varies through the cell cycle. sometimes accompanied by one or more smaller.defined nuclei) have smaller circular chromosomes. Also. Chromosomes are the essential unit for cellular division and must be replicated. Chromosomal recombination plays a vital role in genetic diversity. although there are many exceptions to this rule.
XX.3 MUTATIONS IN CHROMOSOME NUMBER Normally. 1.g. copies of chromosome 21.+21. the individual carrying the mutation is said to be aneuploid.+21. 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).XY or 47. XX (female) or 46 XY (male).are also found in mitochondria and chloroplasts. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. . Euploid human karyotypes are 46. rather than 2. The individual would have Down Syndrome and his/her karyotype would be written 47. 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. in which an individual has 3. reflecting their bacterial origins. a extra copy of human chromosome 21). Such individuals are called euploid and have the wild-type chromosome complement for the species. An example of aneuploidy is trisomy 21. If the mutation involves only one or a few chromosomes in the genome (e.
the chromatids are uncoiled and DNA can again be transcribed. a pair of sister chromatids attached to each other at the centromere. The shorter arms are called p arms (from the French petit. In spite of their appearance. one of which is present on each sister chromatid. the chromatin strands become more and more condensed. During mitosis. q-g "grande"). A special DNA base sequence in the region of the kinetochores provides. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. The microtubules then pull the chromatids apart toward the centrosomes. small) and the longer arms are called q arms (q follows p in the Latin alphabet. along with special proteins. and they form the classic four arm structure.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). 1. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores.Fig 1. chromosomes are structurally highly condensed. This compact form makes the individual chromosomes visible. so that each daughter cell inherits one set of chromatids.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. This is the only natural context in which individual chromosomes are visible with an optical microscope. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. . longer-lasting attachment in this region. Once the cells have divided. which enables these giant DNA structures to be contained within a cell nucleus.
1. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. bone marrow accounts for approximately 2. which use the bone marrow vasculature as a conduit to the body's systemic circulation. In humans. in two separate nuclei. producing the lymphocytes that support the body's immune system CHAPTER 2 2.6 kg (5. .7 lbs).5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones.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. It is generally followed immediately by cytokinesis. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. bone marrow constitutes 4% of the total body mass of humans. which divides the nuclei. On average. bone marrow in large bones produces new blood cells. in adults weighing 65 kg (143 lbs). cytoplasm.
where the nuclear envelope breaks down before the chromosomes separate. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. cytokinesis and mitosis may occur independently. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. forming single cells with multiple nuclei. This accounts for approximately 10% of the cell cycle. for instance during certain stages of fruit fly embryonic development. metaphase. . Because cytokinesis usually occurs in conjunction with mitosis. 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. to produce two identical daughter cells which are still diploid cells. For example. Even in animals. anaphase and telophase. animals undergo an "open" mitosis. Prokaryotic cells. divide by a process called binary fission. prometaphase. but is found in various different groups. prophase. which lack a nucleus. The process of mitosis is fast and highly complex. there are many cells where mitosis and cytokinesis occur separately.genetically identical to each other and to their parent cell. The cell then divides in cytokinesis. These stages are interphase. This occurs most notably among the fungi and slime moulds. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. However. "mitosis" is often used interchangeably with "mitotic phase". Mitosis occurs only in eukaryotic cells and the process varies in different species.
These two cells are identical and do not differ in any way from the original parent cell. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. Because each resultant daughter cell should be genetically identical to the parent cell.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. the parent cell must make a copy of each chromosome before mitosis. Each chromosome now has an identical copy of itself. and together the two are called sister chromatids. This occurs during the S phase of interphase. .
corresponding sister chromosomes are pulled toward opposite ends. each with a replica of the original genome. so they are renamed to sister chromosomes. each sister chromatid is now considered a chromosome. Eventually. 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). giving rise to two daughter cells.In most eukaryotes. In animal cells. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. As mitosis completes. As the cell elongates. the process of binary fission is very much different from the process of mitosis. In plant cells. the daughter cells will construct a new dividing cell wall between each other. pulling apart the sister chromatids of each chromosome. Prokaryotic cells undergo a process similar to mitosis called binary fission. . The chromosomes align themselves in a line spanning the cell. the parent cell will be split in half. separating the two developing nuclei. A new nuclear envelope forms around the separated sister chromosomes.the cell begins cytokinesis. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. As a matter of convention. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. However.
a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . and finally it divides (M) before restarting the cycle. a cell grows (G1). chromosomes are replicated only during the S phase.2. 2. During all three phases. 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. S (synthesis). prophase is preceded by a pre-prophase stage. Interphase is divided into three phases: G1 (first gap). and G2 (second gap). This is achieved through the formation of a phragmosome. the cell grows by producing proteins and cytoplasmic organelles. where the cell prepares itself for cell division. grows more and prepares for mitosis (G 2).2. However. continues to grow as it duplicates its chromosomes (S).1 Preprophase In plant cells only. All these phases in the interphase are highly regulated. the nucleus has to migrate into the center of the cell before mitosis can begin. mainly via proteins.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. Thus. It alternates with the much longer interphase.
The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. degraded. aligned at the metaphase plate. . In addition to phragmosome formation. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. and microtubules have invaded the nuclear Prophase space. The chromosomes have chromatin has condensed. These microtubules can attach to kinetochores or they can interact with opposing microtubules. after the nuclear membrane breaks down. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Cytokinesis has already begun. This band marks the position where the cell will eventually divide. the pinched area is known as the cleavage furrow. 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. instead. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. The cells of higher plants (such as the flowering plants) lack centrioles. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes.division.
the replicated chromosomes have two sister chromatids. The centrosome is the coordinating center for the cell's microtubules.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. A cell inherits a single centrosome at cell division. 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. bound together at the centromere by the cohesin protein complex. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. giving a pair of centrosomes. they are not essential for the . which are made of a pair of centrioles found in most eukaryotic animal cells. Chromosomes are typically visible at high magnification through a light microscope. At the onset of prophase. 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. Close to the nucleus are structures called centrosomes. Although centrioles help organize microtubule assembly.
2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. Each chromosome forms two kinetochores at the centromere. since they are absent from plants. the motor activates. coupled with polymerisation and depolymerisation of microtubules.formation of the spindle. Although the kinetochore structure and function are not fully understood. This is called open mitosis. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. and it occurs in most multicellular organisms. such as algae or trichomonads. one attached at each chromatid. This motor activity. using energy from ATP to "crawl" up the tube toward the originating centrosome. undergo a variation called closed mitosis where the spindle forms inside the nucleus. kinetochore microtubules begin searching for kinetochores to attach to. provides the pulling force necessary to later separate the chromosome's two chromatids. or its microtubules are able to penetrate an intact nuclear envelope. When the spindle grows to sufficient length. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. When a microtubule connects with the kinetochore.2. on an average 20 ). 2. and centrosomes are not always used in mitosis. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. Fungi and some protists. Prometaphase is sometimes considered part of prophase. it is known that it contains some form of molecular motor. .
only roughly lining up along the midline. analogous to a tug-of-war between people of equal strength. an imaginary line that is equidistant from the two centrosome poles. All chromosomes (blue) but one have arrived at the metaphase plate.3 Metaphase A cell in late metaphase. 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." Microtubules find and attach to kinetochores in prometaphase. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". The centromeres of the chromosomes. Metaphase comes from the Greek meaning "after. As a result. 2. In certain types of cells. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell.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. convene along the metaphase plate or equatorial plane. the kinetochore would be the "hook" that catches a sister chromatid or "fish". in some sense. . the chromosomes come under longitudinal tension from the two ends of the cell.
. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. allowing them to separate. Next. These sister chromatids. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. the nonkinetochore microtubules elongate. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. These two stages are sometimes called early and late anaphase. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. At the end of anaphase. 2.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). The signal creates the mitotic spindle checkpoint.” or “re-”). the proteins that bind sister chromatids together are cleaved. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. which have now become distinct sister chromosomes.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. the cell proceeds to anaphase (from the Greek meaning “up. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned.” “back.” “against. Two events then occur: first. The force that causes the centrosomes to move towards the ends of the cell is still unknown. Early anaphase is usually defined as the separation of the sister chromatids.
but cell division is not yet complete. necessary for completing cell division. elongating the cell even more. Both sets of chromosomes. but rather a separate process. Corresponding sister chromosomes attach at opposite ends of the cell.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. In both animal and plant cells. It "cleans up" the after effects of mitosis.5 Cytokinesis Cilliate undergoing cytokinesis. At telophase. A new nuclear envelope. unfold back into chromatin. cytokinesis is a separate process that begins at the same time as telophase. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. cell . now surrounded by new nuclei. forms around each set of separated sister chromosomes. however. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. Cytokinesis is technically not even a phase of mitosis. the nonkinetochore microtubules continue to lengthen. using fragments of the parent cell's nuclear membrane.2. Mitosis is complete. 2. In animal cells. pinching off the separated nuclei.
New cells are formed by mitosis and so are exact copies of the cells being replaced. Following are the occasions in the lives of organism where mitosis happens: 2. cells are constantly sloughed off and replaced by new ones. e.5. 2. zygote and also the basis of the growth of a multicellular body.1Significance Mitosis is important for the maintenance of the chromosomal set.. Each daughter cell has a complete copy of the genome of its parent cell. 2.5.division is also driven by vesicles derived from the Golgi apparatus. 2. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. skin and digestive tract.5. The phragmoplast is a microtubule structure typical for higher plants. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.2 Development and growth The number of cells within an organism increases by mitosis.3 Cell replacement In some parts of body.4 Regeneration . separating the two nuclei. The end of cytokinesis marks the end of the M-phase. which move along microtubules to the middle of the cell. This is the basis of the development of a multicellular body from a single cell i.e. Similarly.g.5. whereas some green algae use a phycoplast microtubule array during cytokinesis. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.
Mitosis continues in the cells of bud and it grows into a new individual. they fail to complete cell division and retain both nuclei in one cell. For example. These cells are considered aneuploid. The production of new cells is achieved by mitosis.Some organisms can regenerate their parts of bodies. 2. . a chromosome may fail to separate during anaphase. and the latter cell having only one chromosome (the homologous chromosome). Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. a condition often associated with cancer. 2. One daughter cell will receive both sister chromosomes and the other will receive none. the process may go wrong. a condition known as trisomy. a condition known as monosomy. the hydra reproduces asexually by budding.7 Consequences of errors Although errors in mitosis are rare. sea star regenerates its lost arm through mitosis. resulting in binucleated cells. In non-disjunction. For example. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue).5.5. The same division happens during asexual reproduction or vegetative propagation in plants. Occasionally when cells experience nondisjunction. The cells at the surface of hydra undergo mitosis and form a mass called bud. especially during early cellular divisions in the zygote.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction.
causing inversion. Benign tumours are not harmful as soon as they are not moving. non-homologous chromosome. but in reverse orientation. This phenomenon is called metastasis or spreading of disease. Such tumours can send cancer cells to other parts in body where new tumours may form. It results in the synthesis of execessive tissue growths. Now what happens is that cell abnormally continue to divide at a single place. causing chromosomal duplication. . When tissues more than the requirement are synthesized in a single organ.Mitosis is a demanding process for the cell. Occasionally. Errors in the control of mitosis may cause cancer. It results in abnormal cell growth. causing deletion. The fragment may incorrectly reattach to another. sometimes mutuations occur in such genes and cells continue to divide. it results in the formation of Tumors. As soon as they start to move and invade other cells there are said to be malignant tumours. its organelles disintegrate and reform in a matter of hours. Or. All cells have genes that control the timing and number of mitosis. An arm of the chromosome may be broken and the fragment lost. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. and chromosomes are jostled constantly by probing microtubules. causing translocation. It may reattach to the original chromosome. which goes through dramatic changes in ultrastructure. chromosomes may become damaged. it may be treated erroneously as a separate chromosome. As long as these tumours remain in their original location they are called benign tumours. The effect of these genetic abnormalities depends on the specific nature of the error.
align in the middle of the cell before being separated into each of the two daughter cells. Preceded by events in prometaphase and followed by anaphase.2. 2.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. analogous to a tug of war between equally strong people. In certain types of cells. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Early events of metaphase can .7 Metaphase Metaphase. An example of a cell that goes through endomitosis is the megakaryocyte. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. 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. carrying genetic information. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. an imaginary line that is equidistant from the two centrosome poles. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. only roughly lining up along the middleline. This process may also be referred to as endoreduplication and the cells as endoploid. from the ancient Greek(between) and (stage).
coincide with the later events of prometaphase. Only after all chromosomes have become aligned at the metaphase plate. often with Giemsa (G banding) or Quinacrine. when every kinetochore is properly attached to a bundle of microtubules. 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). even if most of the kinetochores have been attached and most of the chromosomes have been aligned. and separase. Such a signal creates the mitotic spindle checkpoint. Chromosomes are condensed(Thickened) and highly coiled in metaphase. securin. 2. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. Normal metaphase spreads are used in . This would be accomplished by regulation of the anaphase-promoting complex. produces a pattern of in total up to several hundred bands. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. does the cell enter anaphase. One of the cell cycle checkpoints occurs during prometaphase and metaphase. For classical cytogenetic analyses.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. Staining of the slides. Metaphase chromosomes make the classical picture of chromosomes (karyotype). which makes them most suitable for visual analysis.
such as bcr-abl in chronic myelogenous leukemia. losses of chromosomal segments or translocations. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. . Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. which may lead to chimeric oncogenes. for example.
Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. and the results may be used in evolutionary biology and medicine. So. and any other physical characteristics. such as. ordered by size and position of centromere for chromosomes of the same size. . be sex chromosomes. or an individual organism. Attention is paid to their length. or may not. and to gather information about past evolutionary events. autosomal chromosomes are present in two copies. in normal diploid organisms. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. and what they look like under a light microscope. to study chromosomal aberrations. The study of karyotypes is important for cell biology and genetics. Karyotypes can be used for many purposes. The term is also used for the complete set of chromosomes in a species. The study of whole sets of chromosomes is sometimes known as karyology. Karyogram of human male using Giemsa staining. Thus. banding pattern. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. the position of the centromeres. any differences between the sex chromosomes.  The preparation and study of karyotypes is part of cytogenetics.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. in humans 2n = 46. Karyotypes describe the number of chromosomes. There may. cellular function. taxonomic relationships. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23).
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 1912.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. 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. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. at first favoring 46. The next stage took place after the development of genetics in the early 20th century. in contrast to their genic contents.3. von Waldeyer in 1888. which swells them and spreads the chromosomes . Pretreating cells in a hypotonic solution. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Considering their techniques. 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. in 1882. the discoverer of mitosis. The name was coined by another German anatomist. and he correctly insisted on humans having an XX/XY system. these results were quite remarkable. concluding an XX/XO sex determination mechanism. Their behavior in animal (salamander) cells was described by Walther Flemming. Using cells in culture 2. The subsequent history of the concept can be followed in the works of Darlington and White.
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.2 Observations on karyotypes 3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 3. reducing the number. Usually.3. the great apes have 48 chromosomes. For humans. is applied after cells have been arrested during cell division by a solution of colchicine.2 Observations Six different characteristics of karyotypes are usually observed and compared: .  Sometimes observations may be made on non-dividing (interphase) cells. 3. Rather interestingly.2. such as Giemsa. Arresting mitosis in metaphase by a solution of colchicines 4. a suitable dye.1 Staining The study of karyotypes is made possible by staining. The sex of an unborn fetus can be determined by observation of interphase cells. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.2. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.
and mainly consists of genetically inactive repetitive DNA sequences. faba chromosomes are many times larger. both have six pairs of chromosomes (n=6) yet V. 3. but the genes have been mostly translocated (added) to other chromosomes. permitting its loss without penalty to the organism (the dislocation hypothesis). A full account of a karyotype may therefore include the number. This feature probably reflects different amounts of DNA duplication. indicating tighter packing. as well as other cytogenetic information. Differences in degree and distribution of heterochromatic regions. Humans have one pair fewer chromosomes than the great apes. which (when they occur) are small bodies attached to a chromosome by a thin thread. 6. 4. 5.1. 2. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. Differences in absolute sizes of chromosomes. This is brought about by translocations. Differences in the position of centromeres. Heterochromatin stains darker than euchromatin. . type. shape and banding of the chromosomes. Differences in number and position of satellites.
XY. males have both an X and a Y chromosome denoted 46. Any variation from the standard karyotype may lead to developmental abnormalities. and in . Between members of a population (chromosome polymorphism) 4. Between the sexes 2. Mosaics or otherwise abnormal individuals. Normal karyotypes for females contain two X chromosomes and are denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. Between the germ-line and soma (between gametes and the rest of the body) 3. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. XX. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. 3. the same cannot be said for their karyotypes.Variation is often found: 1. There is variation between species in chromosome number. which are highly variable. Geographical variation between races 5.
This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. 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. used in conjunction with other phylogenetic data. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species.1 Changes during development Instead of the usual gene repression. 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 some species.. But. 3... .3.detailed organization. portions of the chromosomes are cast away in particular cells. or other kinds of visible adjustment to the karyotype. In some cases there is even significant variation within species. "We have a very poor understanding of the causes of karyotype evolution. In A.. entire chromosomes are eliminated during development. which were previously inexplicable. Chromatin diminution (founding father: Theodor Boveri). found in some copepods and roundworms such as Ascaris suum. as in many sciarid flies. Chromosome elimination. Godfrey and Masters conclude: "In our view. despite their construction from the same macromolecules. This variation provides the basis for a range of studies in evolutionary cytology. it is quite unclear what the general significance might be. the general significance of karyotype evolution is obscure. In a review. Although much is known about karyotypes at the descriptive level. In this process.
"They simply could not believe what they saw. male = 7 chromosomes. was found to be 46.. In human females some 15% of somatic cells escape inactivation. all the somatic cell precursors undergo chromatin diminution. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. 3..suum. the inactivation is random as between the two Xs. The diploid number of the Chinese muntjac. Muntiacus reevesi.. Xinactivation. all telocentric.3. which was investigated by Kurt Benirschke and his colleague Doris Wurster. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. they were astonished to find it had female = 6. thus the mammalian female is a mosaic in respect of her X chromosomes. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. In placental mammals. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). They kept quiet for two or three years because they thought something was wrong with their tissue culture. the high record would be somewhere amongst the ferns. When they looked at the karyotype of the closely related Indian muntjac.. Muntiacus muntjak.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. The existence of supernumerary or B chromosomes . In marsupials it is always the paternal X which is inactivated. where the haploid n = 1.
Endopolyploidy occurs when in adult differentiated . but in grasses the average is much higher. FN. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. and aneuploids are another example. Polyploidy.means that chromosome number can vary even within one interbreeding population. Humans have FN = 82. It has been of major significance in plant evolution according to Stebbins. It is a common arrangement in the Hymenoptera. where there are more than two sets of homologous chromosomes in the cells. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. where one sex is diploid. 14. though in this case they would not be regarded as normal members of the population.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. and in some other groups. FN ≤ 2n. occurs mainly in plants. due to the presence of five acrocentric chromosome pairs (13.3 Fundamental number The fundamental number. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. but it has been significant in some groups. 3.3. Thus. 3. 15. about 70%. Polyploidy in animals is much less common. 21 and 22). horsetails and psilotales) is also common. Haplo-diploidy. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. Polyploidy in lower plants (ferns. and the other haploid.
. 3. the daughter chromosomes separating from each other inside an intact nuclear membrane. 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.tissues the cells have ceased to divide by mitosis. it is diverse and complex. but the nuclei contain more than the original somatic number of chromosomes. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. and serves differentiation and morphogenesis in many ways. Down syndrome and Turner syndrome are examples of this. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. See palaeopolyploidy for the investigation of ancient karyotype duplications. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). In many instances. Abnormalities in chromosome number usually cause a defect in development. The cells do not always contain exact multiples (powers of two).
Aneuploidy may also occur within a group of closely related species. 5. Well-researched examples are the ladybird beetle Chilocorus stigma.6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson. and Crocus. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. In about 6. 3.500 sq mi (17. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. that the two chromosome morphs are adapted to different habitats. When this happens. some mantids of the genus Ameles. 4. 3.  Closer to home.000 km2). the great apes have 24x2 chromosomes whereas humans have 23x2. reducing the number. living from rainforests to . where the gametic (= haploid) numbers form the series x = 3. where every number from x = 3 to x = 15 is represented by at least one species. Human chromosome 2 was formed by a merger of ancestral chromosomes. the European shrew Sorex araneus. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. and 7. Classic examples in plants are the genus Crepis.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. 6. the chromosome number is variable from one individual to another.
which can be dated to 30 mya. There are also cases of colonization back to older islands. . the best-studied group of Hawaiian drosophilids.4 million years ago (mya) (Mauna Kea) to 10mya (Necker).subalpine meadows. Using K-Ar dating. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. Chromosome rearrangements. The results are clear. show a clear "flow" of species from older to newer islands. Although it would be possible for a single gravid female to colonise an island. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. when plotted in tree form (and independent of all other information). and skipping of islands. Drosophila and Scaptomyza. probably 20 million years ago. especially inversions. but these are much less frequent. The inversions. it is more likely to have been a group from the same species. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. make it possible to see which species are closely related. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. in the family Drosophilidae. the present islands date from 0. gene arrangements are visible in the banding patterns of each chromosome. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. at least into the Cretaceous. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. In a sense. The polytene banding of the 'picture wing' group.
It yields a series of lightly and darkly stained bands . R-banding is the reverse of G-banding (the R stands for "reverse"). The pattern of bands is very similar to that seen in G-banding. early-replicating and GC rich.There are other animals and plants on the Hawaiian archipelago which have undergone similar. human genome. • • C-banding: Giemsa binds to constitutive heterochromatin.7. The light regions tend to be euchromatic. • T-banding: visualize telomeres.the dark regions tend to be heterochromatic. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. if less spectacular. . The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). adaptive radiations. 3.1 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. late-replicating and AT rich. This method will normally produce 300-400 bands in a normal.7 Depiction of karyotypes 3. so it stains centromeres.
This yields a dark region where the silver is deposited. Karyotypes are arranged with the short arm of the chromosome on top. denoting the activity of rRNA genes within the NOR.7. is used to stain bands on the chromosomes. In the "classic" (depicted) karyotype. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. Cri du chat syndrome involves a deletion on the short arm of . In addition. both chromosomes in a pair will have the same banding pattern. Each chromosome has a characteristic banding pattern that helps to identify them. Quinacrine binds to the adeninethymine-rich regions. 3. and the long arm on the bottom.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. Giemsa is specific for the phosphate groups of DNA.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. For example. Some karyotypes call the short and long arms p and q. less frequently Quinacrine. a dye. often Giemsa (G-banding). respectively.
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. The critical region for this syndrome is deletion of 15. a combinatorial labeling method is used to generate many different colors.7.2) 3. It is written as 46. Image processing software then assigns a pseudo color to each spectrally different combination. allowing the visualization of the individually colored chromosomes. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally-distinct fluorophores.2. This method is also known as virtual karyotyping.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. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.5p-.XX. .chromosome 5. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. which is written as 46. 3.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX.del(5)(p15.
CHAPTER 4 .
1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. are common numerical abnormalities. is caused by trisomy of chromosome 21. Down syndrome. trisomies. trisomy 9 and trisomy 16. in which three copies of a chromosome are present instead of the usual two. Also documented are trisomy 8. X or 45.4. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. otherwise known as 47. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. large-scale deletions or duplications. as in derivative chromosome. XXY is caused by an extra X chromosome. or structural. often occur as a result of nondisjunction during meiosis in the formation of a gamete. X0). as in the presence of extra or missing chromosomes. Numerical abnormalities. translocations. the most common male chromosomal disease. a common chromosomal disease. including . • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. Structural abnormalities often arise from errors in homologous recombination. Klinefelter syndrome. inversions. Some disorders arise from loss of just a piece of one chromosome. also known as aneuploidy. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. • • Patau syndrome is caused by trisomy of chromosome 13. although they generally do not survive to birth.
Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. A Karyotype refers to a full set of chromosomes from an individual which can be compared to a "normal" Karyotype for the species via genetic testing. a deletion of the paternal genes. one well-documented example is the Philadelphia chromosome. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. The name comes from the babies' distinctive cry. example of imprinting disorder. caused by abnormal formation of the larynx. from the loss of part of the short arm of chromosome 1. from a truncated short arm on chromosome 5. 1p36 Deletion syndrome. They can be organized into two basic groups. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. A chromosome anomaly may be detected or confirmed in this manner. . example of imprinting disorder. a deletion of the maternal genes.• Cri du chat (cry of the cat). A chromosome anomaly. • Prader-Willi 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. There are many types of chromosome anomalies. numerical and structural anomalies. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes.
• • Translocations: When a portion of one chromosome is transferred to another chromosome. resulting in extra genetic material. There are two main types of translocations. Tetrasomy. 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.). etc.3 Structural abnormalities When the chromosome's structure is altered. which is caused by partial deletion of the short arm of chromosome 4. 4. an entire chromosome has .2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. rather than two). segments from two different chromosomes have been exchanged. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.4. and Jacobsen 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. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. an X. Known disorders in humans include Wolf-Hirschhorn syndrome. also called the terminal 11q deletion disorder. In a reciprocal translocation. In a Robertsonian translocation. Duplications: A portion of the chromosome is duplicated. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21.
and are therefore initially not inherited. • Inversions: A portion of the chromosome has broken off.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. resulting in Mosaicism (where some cells have the anomaly and some do not). This is why chromosome studies are often performed on parents when a child is found to have an anomaly. This can happen with or without loss of genetic material. as well .attached to another at the Centromere . the anomaly is present in every cell of the body.in humans these only occur with chromosomes 13. turned upside down and reattached. Chromosome anomalies can be inherited from a parent or be "de novo". • Rings: A portion of a chromosome has broken off and formed a circle or ring. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. however. Therefore. 15. 14. especially the chromosomes. can happen after conception.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. other cytogenetic banding techniques. 4. 4. Some anomalies. therefore the genetic material is inverted. 21 and 22. It includes routine analysis of G-Banded chromosomes.
when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. concluding an XX/XO sex determination mechanism. The name was coined by another German anatomist. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. in contrast to their genic contents. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. The next stage took place after the development of genetics in the early 20th century. 4. New techniques were needed to definitively solve the problem: 1. von Waldeyer in 1888. 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. these results were quite remarkable. the discoverer of mitosis. He revised his opinion later from 46 to 48. Pre-treating cells in a hypotonic solution. in 1882. Their behavior in animal (salamander) cells was described by Walther Flemming. and he correctly insisted on man having an XX/XY system. which swells them and spreads the chromosomes .as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Painter in 1922 was not certain whether the diploid number of man was 46 or 48. Using cells in culture 2. Considering their techniques.
the great apes have 48 chromosomes. reducing the number. Arresting mitosis in metaphase by a solution of colchicine 4. 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Using Painter's technique they studied the polytene . Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Human chromosome 2 was formed by a merger of ancestral chromosomes.6. persimilis from wild populations in California and neighboring states. a find which eventually led to her Nobel Prize in 1983.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. During her cytogenetic work.2 Natural populations of Drosophila In the 1930s.3. Rather interestingly. McClintock discovered transposons. In 1931.6 Applications in biology 4. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). 4.6. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.
This had the benefit of eliminating migration as a possible explanation of the results.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. 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. Down syndrome is also referred to as trisomy 21. Evidence rapidly accumulated to show that natural selection was responsible. In some congenital disorders. such as Down's syndrome. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. but adjust to certain frequencies at which they become stabilised. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. as they would if selectively neutral.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. In 1959. discoveries were quickly made related to aberrant chromosomes or chromosome number. which enabled feeding. 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. It was found that the various chromosome types do not fluctuate at random. . breeding and sampling whilst preventing escape. as with most polymorphisms. 4.
with the development of more advanced techniques. is used today as a diagnostic for CML. . Identification of the Philadelphia chromosome by cytogenetics. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. In 1960. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. resulting in 47 total chromosomes. Thirteen years later.as both scientists were doing their research in Philadelphia. An individual with only one sex chromosome (the X) has Turner syndrome. XYY. has Klinefelter's Syndrome. and XXXX.Other numerical abnormalities discovered include sex chromosome abnormalities. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. This abnormal chromosome was dubbed the Philadelphia chromosome . an additional X chromosome in a male. Many other sex chromosome combinations are compatible with live birth including XXX. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). Pennsylvania. Not all genes on the X Chromosome are inactivated. which is required in normal females to compensate for having two copies of the chromosome. in addition to other tests.
8 Beginnings of molecular cytogenetics . Deletions within one chromosome could also now be more specifically named and understood.FIG Advent of banding techniques In the late 1960s. Deletion syndromes such as DiGeorge syndrome. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. and elongation techniques for all culture types that allow for higher resolution banding. 4. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. 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. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. Caspersson developed banding techniques which differentially stain chromosomes.
cloned and studied in ever greater detail. movement was now made in using fluorescent labeled probes. While radioisotope-labeled probes had been hybridized with DNA since 1969.1 Karyotyping . 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.In the 1980s. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. advances were made in molecular cytogenetics. CHAPTER 5 Techniques 5.
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,
Using MATLAB. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. and FORTRAN. 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. For congenital problems usually 20 metaphase cells are scored. such as C. and numerical computation. CGH and Single nucleotide polymorphism-arrays. data analysis. data visualization. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas.generally between 200 and 1000 cells are counted and scored. such as comparative genomic hybridization arrays. including signal and image processing. C++. test and measurement. and computational biology. you can solve technical computing problems faster than with traditional programming languages. financial modeling and analysis. You can use MATLAB in a wide range of applications. communications. control design. .
The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. These effects must be compensated to improve the results of the pairing algorithm. In many cases. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks.MATLAB provides a number of features for documenting and sharing your work. . one line of MATLAB code can often replace several lines of C or C++ code. You can integrate your MATLAB code with other languages and applications. and allocating memory. such as declaring variables. specifying data types. and distribute your MATLAB algorithms and applications. With the MATLAB language. The image processing step is composed of the following operations. 6. It enables fast development and execution. MATLAB eliminates the need for ‘for’ loops. As a result.
Therefore. geometrical and dimensional differences must be removed.2 Concepts used in this phase 1) Image conversion 2) Denoising . the spatially scaled images are histogram equalized. 6. 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. 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. or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. 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.
MATLAB simply applies the filter to the indices in the indexed image matrix. For example.I. RGB = cat (3. You can perform certain conversions just using MATLAB syntax.2.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. you must first convert it to true color format. MATLAB filters the intensity values in the image.I. as is appropriate. green.3) Edge detection 4) Two dimensional convolutions. For example. so the image displays as shades of gray. In addition to these image type conversion functions.I). The resulting true color image has identical matrices for the red. When you apply the filter to the true color image. and the results might not be meaningful. . For example. 6. If you attempt to filter the indexed image. and blue planes. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. if you want to filter a color image that is stored as an indexed image. listed in the following table. there are other functions that return a different image type as part of the operation they perform.
5 Edge detection Edges contain some of the most useful information in an image.parameters. Usually we know what type of errors to expect. Cleaning an image corrupted by noise is thus an important area of image restoration.3 Two dimensional convolutions C = conv2(A. 6.6.4 Denoising We may define noise to be any degradation in the image signal. The general Matlab command for finding edges is edge(image. or through networked cable. caused by external disturbance. If an image is being sent electronically from one place to another.B) computes the two-dimensional convolution of matrices A and B. 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. to recognize or classify objects. If one of these matrices describes a two-dimensional finite impulse response . and we shall look at some of the more straightforward of them. ) Where the parameters available depend on the method used 6. via satellite or wireless transmission. There is a large number of edge finding algorithms in existence. to isolate particular objects from their background. . hence we can choose the most appropriate method for reducing the effects.'method'.2. and hence the type of noise on the image. . We may use edges to measure the size of objects in an image.2.
A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.. That is.A).'sobel').na] and the size of B is [mb. if the size of then the size of C is [ma+mb-1. rgb2gray im2bw(im. minus one. If hcol is a column vector and hrow is a row vector.hrow. .0.7). nb]+1)/2). this case is the same as C = conv2(hcol*hrow. edge(im1.. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.'shape') subsection of the two-dimensional convolution.nb].[3 3]).bmp'). the other matrix is filtered in two dimensions. C = conv2(. 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.na+nb-1]. The size of matrices. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. imedfilt2(im1..(FIR) filter.
8).imy]=size(BW).[imx. rc = [r c]. mx=max(max(L)). for i=1:m for j=1:n if L(i. MODULE 2 clc [m.n]=size(L). bwlabel(B.j)==L_number(k) flag=1. n1(x1. nzeros(imx.1). Msk conv2(double(BW).j)~=0 for k=1:mx if L(i.double(msk)). [sx sy]=size(rc). flag=0.1). . [r.imy). L_number=zeros(mx. for i=1:sx x1=rc(i. y1=rc(i.y1)=255. Index=1.c] = find(L==22).2).
15. Test_number=[188.8.131.52.42.1).4. end end L_number.14.6.).22.40. [sx sy]=size(rc).9.28.39.c] = find(L==L_number((Test_number(x)))).66].184.108.40.206. rc = [r c].y1)=255.33.29.21.2).31. n1(x1.end end if flag~=1 L_number(Index)=L(i.8.56.55. end. .35.43. end %h=figure.imy).7. n1=zeros(imx.220.127.116.11.41.45.j).18.104.22.168. for x=1:46 [r.20.52.30.imshow(n1. Index=Index+1. for i=1:sx x1=rc(i.60.65.10. end flag=0. 36. y1=rc(i.
1).8).5*graythresh(skel)). skel=im2double(f).'skel'. . for x=1:m for y=1:n if BW1(x.bmp')).1).Inf). BW=im2bw(f).'. f=imcomplement(f).y)==1 Circumference_sum=Circumference_sum+1. Area=zeros(46. for i=1:46 f=imread(strcat(num2str(i). end end end Circumference(i)=Circumference_sum.end Circumference=zeros(46. s1=bwmorph(s. skel=im2bw(skel. Circumference_sum=0.'spur'. BW=double(BW).1.'canny'). s=bwmorph(skel. Arm_length_sum=0.1). BW1=edge(BW. [m n]=size(BW1). Arm_length=zeros(46.
end end end Arm_length(i)=Arm_length_sum. Arm_length. end Circumference. for x=1:m for y=1:n if BW(x. end end end Area(i)=Area_sum. BW=im2bw(f). for x=1:m for y=1:n if s1(x. [m n]=size(BW). . Area_sum=0.[m n]=size(s1).y)==1 Area_sum=Area_sum+1.y)==1 Arm_length_sum=Arm_length_sum+1.
for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)).2)=i.2)=j.2). end end end for i=1:45 if Pair(i. Pair=zeros(46. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)).1)=i. Pair(i.2)==46 Pair(46. .Area. Pair(46. Pair(i.1)=46.2)=i+1.1)=i. end end Pair. Pair(i. Pair(i.
2.figure_flag). flag=0. . end flag=0.bmp')).2)).1)). end f1=imread(strcat(num2str(Pair(i.bmp')).figure_flag). figure_flag=1.'. for i=1:46 for j=1:46 if Pair(i. imshow(f2). figure_flag=figure_flag+1. figure_flag=figure_flag+1. end end if flag~=1 if figure_flag~=47 subplot(23.2).delete=zeros(46. end f2=imread(strcat(num2str(Pair(i. imshow(f1). delete(figure_flag)=Pair(i.1)==delete(j) flag=1.'.2. if figure_flag~=47 subplot(23.1).
2) feature extraction from the processed images . based on the MI. 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. dimensions and banding profiles. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure.end CONCLUTION In this paper. in the scope of karyotyping process used in cytogentic analysis. such as. Copenhagen. and Philadelphia. The proposed algorithm is based on the traditional features extracted from the karyogram. 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. plus a new one.
working within an 8-D feature space.10% mean classification rate. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. and finally. and to normalize their dimensions.characterizing the size. 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 .working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. are processed in order to compensate for geometrical and intensity distortions. achieves a 70. 4) pairing. shape. and band pattern. 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). 3) training of a classifier (performed once) where similarity among chromosomes are characterized. In the image processing step. This normalization is needed to make it possible the band pattern comparison between chromosomes. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. The training process consists in the estimation of each vector of coefficient . Here. 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. Tests using 19 karyograms based on bone marrow cells. extracted from the unordered karyogram. The features extracted from the processed images discriminate each pair with respect to their size. The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram. shape and band pattern. from the chromosomes in the training set. the romosome images.
amean classification rate larger than 93% was obtained in all experiments. This dataset was made publicly available . presenting a uniform level of condensation. a 76. Executing the algorithm on a higher quality dataset.performance of the classifier. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. The results presented in this paper are promising. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. REFERENCES . and from which it is possible to extract additional features. centromere position. called LK1 . In fact. such as Edinburgh.10% classification ratewas obtained. whose images are of significantly higher quality. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. Copenhagen.. Using 27 karyograms andworking with a limited number of classes (≤ 8).g. a new chromosome dataset with 9200 chromosomes from bone marrow cells. e. In addition. or Philadelphia. despite the low quality of this type of chromosomes.
D. Edinburgh. biologie 27. Animal cytology and evolution. ^ Kottler M. The chromosomes. revised and enlarged. Bull. A dictionary of genetics. 11. 9. Anat. 7th ed. 10. ^ a b c White M.D. 4. Applied Bot. 3. The morphology of chromosomes.C. 7. 129.S.P Oxford & NY. p. 23. From 48 to 46: cytological technique. Etudes sur la spermatogenese humaine. 1924. 6th ed. ^ Darlington C.1. ^ Stebbins G. Med. 19-174. Cambridge University Press. State Publication Office of the Ukraine. 1912. 1950. 93. Chapter XII: The Karyotype. 1922. 1939. 1931. Stansfield W. and Mulligan P. The material basis of heredity..L.D. 3rd ed. ^ Levitsky G. ^ Levitsky G. Bull. 2006. ^ Painter T. Oliver & Boyd. [in Russian] 6. 1974.K. Kiev. The spermatogenesis of man.J. Variation and evolution in plants. Res. 28 2. Arch. preconception and the counting of the human chromosomes. Evolution of genetic systems.D. Plant Breed. Hist. Genet. Cambridge University Press.J. ^ Concise Oxford Dictionary London. ^ a b White M. ^ von Winiwarter H. 1958.A. 465-502. p242 5. Chapman & Hall. 8. . 2nd ed. ^ King R.A. 147–49. 27. Oxford U. Columbia University Press NY. 1973. 1973. 48.
Chromosome stains. 1-6. ^ Maynard Smith J. 2nd ed. In The ACT Cytogenetics Laboratory Manual 2nd ed. 1991. Human and mammalian cytogenetics: a historical perspective. Springer-Verlag. 1971. 1979. Oxford. 1996. M. 9821– 9823.nih.pubmedcentral. Chromosome elimination in sciarid flies. Chromatin diminution in nematodes. 17. ed. ^ Müller F. 2001. . Barch. Hereditas 42.C. 13. 1923. London. Evolutionary genetics. Eosin Y and Azure-A. ^ Painter T.C.http://www. ^ Godfrey L. Bioessays23: 242–250. New York.^ a b Gustashaw K. ^ A preparation which includes the dyes Methylene Blue. Studies in mammalian spermatogenesis II.fcgi?artid=34032 19. NY. 1956. 15. The spermatogenesis of humans.J. 2000. Arnold. p218-9 20. The chromosome number of man. Exp. 14.R.^ a b Hsu T. ^ Tjio J.L. 21. Kinetophore reproduction theory may explain rapid chromosome evolution. ^ Goday C. J. p85-6 18. Bernard V. ^ Stebbins G.gov/articlerender. Zoology 37. The Association of Cytogenetic Technologists. Raven Press. & Tobler H. and Masters J.B.M. 1998.R. Bioessays18: 133–138.C 16.H & Levan A.S. 291-336. Chromosomal evolution in higher plants. and Esteban M. PNAS 97.12.
F. Stamford CT. Park. ^ Wyngaard G.F. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. Nam.1007/BF02153623.K.H. J. D. 2001. Ichthyological Research 52 (1): 97. 2006.P Oxford & NY.H. Indian Muntjac. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF).D. Muntiacus muntjak: a deer with a low diploid number. doi:10. ^ King R. (1945-05-15). Botanical Journal of the Linnean Society 102: 205–217. & Gregory T. 7th ed. 28. J. ^ Wurster D. 1364-1366. Science 168. . R.R. Experientia (Basel) 1 (2): 50– 56. and Mulligan P. Zool.. C. Chapter 9 24..22.A. 25. Exp.C.1007/s10228-004-0257-z.S. ^ Kim.. 291: 310–16... Retrieved 2011-03-16. Y. 8th ed. Chromosome evolution in the genus Ophioglossum L. 23. Stansfield W. Noh. 1970.doi:10. Chapman.K. 1990. Oxford U.K. ^ Khandelwal S. and Benirschke K. Developmental biology. 2006. (2005). 26. A dictionary of genetics. Retrieved 2008-03-18.A. ^ Gilbert S. 27. Sinauer Associates. ^ Matthey. "L'evolution de la formule chromosomiale chez les vertébrés".
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