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During the Metaphase type of cell division in the Bone Marrow, Karyotyping method gives the visual representation of the 46 chromosomes paired and arranged in decreasing order of size. This representation is useful in leukemia purposes. This method is a difficult one because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges. So here, Karyotyping uses new mutual information method which is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. This algorithm is formulated as such a method of combinatorial optimization problem. Where the distances between homologous chromosomes are minimized and the distances between non homologous ones are maximized. It is solved by using an integer programming approach. In this project chromosome dataset Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes was used for this study.
Introduction 1.1 The study of chromosome morphology and its relation with some genetic diseases is the main goal of cytogenetic. Normal human cells have 23 classes of large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The chromosome contains approximately 30 000 genes (genotype) and large tracts of non coding sequences. The analysis of genetic material can involve the examination of specific chromosomal regions using DNA probes, e.g., fluorescent in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic hybridization (CGH) , or the morphological and pattern analysis of entire chromosomes, the conventional cytogenetic, which is the focus of this paper. These cytogenetic studies are very important in the detection of acquired chromosomal abnormalities, such as translocations, duplications, inversions, deletions, monosomies, or trisomies. These techniques are particularly useful in the diagnosis of cancerous diseases and are the preferred ones in the characterization of the different types of leukemia, which is the motivation of this paper . The pairing of chromosomes is one of the main steps in conventional cytogenetic analysis where a correctly ordered karyogram is produced for diagnosis of genetic diseases based on the patient karyotype. The karyogram is an image representation of the stained human chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) , where the chromosomes are arranged in 22 pairs of somatic homologous elements plus two sex-determinative chromosomes (XX for the female or XY for the male), displayed in decreasing order of size. A karyotype is the set of characteristics
extracted from the karyogram that may be used to detect chromosomal abnormalities. The metaphase is the step of the cellular division process where the chromosomes are in their most condensed state. This is the most appropriated moment to its visualization and abnormality recognition because the chromosomes appear well defined and clear. The pairing and karyotyping procedure, usually done manually by visual inspection, is time consuming and technically demanding. The application of the G-banding procedure to the chromosomes generates a distinct transverse banding pattern characteristic for each class, which is the most important feature for chromosome classification and pairing. The International System for Cytogenetic Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as for all the chromosomes of a normal human, and the clinical staff is trained to pair and interpret each specific karyogram according to the ISCN information. Other features, related to the chromosome dimensions and shape, are also used to increase the discriminative power of the manual or automatic classifiers. 1.2 CHROMOSOME A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes,regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Chromosomes vary widely between different organisms. The DNA molecule may be circular or linear, and can be composed of 100,000 to 10,000,000,000 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without
This allows the very long DNA molecules to fit into the cell nucleus. In eukaryotes. sometimes accompanied by one or more smaller. Unduplicated chromosomes are single linear strands.defined nuclei) have smaller circular chromosomes. Chromosomal recombination plays a vital role in genetic diversity. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. Chromosomes are the essential unit for cellular division and must be replicated. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). the cell may undergo mitotic catastrophe and die. These small circular genomes . mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. for example. In prokaryotes and viruses. circular DNA molecules called plasmids. In practice "chromosome" is a rather loosely defined term. although there are many exceptions to this rule. Also. a large body of work uses the term chromosome regardless of chromatin content. divided. However. the term genophore is more appropriate when no chromatin is present. DNA is usually arranged as a circle. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. If these structures are manipulated incorrectly. The structure of chromosomes and chromatin varies through the cell cycle. which is tightly coiled in on itself. through processes known as chromosomal instability and translocation. cells may contain more than one type of chromosome. Chromosomes may exist as either duplicated or unduplicated. In prokaryotes. or it may unexpectedly evadeapoptosis leading to the progression of cancer.
The individual would have Down Syndrome and his/her karyotype would be written 47.are also found in mitochondria and chloroplasts.XX.g.XY or 47.+21.3 MUTATIONS IN CHROMOSOME NUMBER Normally. Euploid human karyotypes are 46. An example of aneuploidy is trisomy 21. copies of chromosome 21. XX (female) or 46 XY (male). . 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). If the mutation involves only one or a few chromosomes in the genome (e. a extra copy of human chromosome 21). rather than 2. reflecting their bacterial origins. the individual carrying the mutation is said to be aneuploid. 1. 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. 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. in which an individual has 3.+21.
Fig 1. . The microtubules then pull the chromatids apart toward the centrosomes. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. A special DNA base sequence in the region of the kinetochores provides. The shorter arms are called p arms (from the French petit.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. along with special proteins. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. This compact form makes the individual chromosomes visible. and they form the classic four arm structure. longer-lasting attachment in this region.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). so that each daughter cell inherits one set of chromatids. During mitosis. 1. one of which is present on each sister chromatid. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. small) and the longer arms are called q arms (q follows p in the Latin alphabet. which enables these giant DNA structures to be contained within a cell nucleus. Once the cells have divided. chromosomes are structurally highly condensed. In spite of their appearance. This is the only natural context in which individual chromosomes are visible with an optical microscope. the chromatids are uncoiled and DNA can again be transcribed. q-g "grande"). a pair of sister chromatids attached to each other at the centromere. the chromatin strands become more and more condensed.
which use the bone marrow vasculature as a conduit to the body's systemic circulation.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.1.6 kg (5. in two separate nuclei. .5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. In humans.7 lbs). which divides the nuclei. bone marrow constitutes 4% of the total body mass of humans. producing the lymphocytes that support the body's immune system CHAPTER 2 2. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. bone marrow accounts for approximately 2. bone marrow in large bones produces new blood cells. On average.  Bone marrow is also a key component of the lymphatic system. It is generally followed immediately by cytokinesis. in adults weighing 65 kg (143 lbs). Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. cytoplasm.
During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. there are many cells where mitosis and cytokinesis occur separately. This accounts for approximately 10% of the cell cycle. prophase. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. metaphase. where the nuclear envelope breaks down before the chromosomes separate. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. which lack a nucleus. . divide by a process called binary fission. forming single cells with multiple nuclei. These stages are interphase. "mitosis" is often used interchangeably with "mitotic phase". anaphase and telophase. animals undergo an "open" mitosis. where chromosomes divide within an intact cell nucleus. Even in animals. Mitosis occurs only in eukaryotic cells and the process varies in different species. For example. for instance during certain stages of fruit fly embryonic development. The cell then divides in cytokinesis. However. Prokaryotic cells. This occurs most notably among the fungi and slime moulds. cytokinesis and mitosis may occur independently. prometaphase. The process of mitosis is fast and highly complex. Because cytokinesis usually occurs in conjunction with mitosis. to produce two identical daughter cells which are still diploid cells. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. but is found in various different groups.genetically identical to each other and to their parent cell.
Because each resultant daughter cell should be genetically identical to the parent cell. These two cells are identical and do not differ in any way from the original parent cell. Each chromosome now has an identical copy of itself. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. This occurs during the S phase of interphase. .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. and together the two are called sister chromatids. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function.
Eventually. so they are renamed to sister chromosomes. . the parent cell will be split in half. corresponding sister chromosomes are pulled toward opposite ends. In animal cells. the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). the daughter cells will construct a new dividing cell wall between each other. Prokaryotic cells undergo a process similar to mitosis called binary fission.In most eukaryotes. As mitosis completes. each with a replica of the original genome. However. As a matter of convention. separating the two developing nuclei. pulling apart the sister chromatids of each chromosome. the process of binary fission is very much different from the process of mitosis. giving rise to two daughter cells. A new nuclear envelope forms around the separated sister chromosomes. The chromosomes align themselves in a line spanning the cell.the cell begins cytokinesis. In plant cells. As the cell elongates. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. each sister chromatid is now considered a chromosome. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten.
2. chromosomes are replicated only during the S phase.2. grows more and prepares for mitosis (G 2). 2. continues to grow as it duplicates its chromosomes (S). the nucleus has to migrate into the center of the cell before mitosis can begin. and G2 (second gap). a cell grows (G1). The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. mainly via proteins.1 Preprophase In plant cells only. where the cell prepares itself for cell division. In highly vacuolated plant cells. Interphase is divided into three phases: G1 (first gap). All these phases in the interphase are highly regulated. It alternates with the much longer interphase. and finally it divides (M) before restarting the cycle. This is achieved through the formation of a phragmosome.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. During all three phases. S (synthesis). Thus. prophase is preceded by a pre-prophase stage. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . the cell grows by producing proteins and cytoplasmic organelles. However.
degraded. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. instead. These microtubules can attach to kinetochores or they can interact with opposing microtubules. In addition to phragmosome formation. This band marks the position where the cell will eventually divide. the pinched area is known as the cleavage furrow. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. after the nuclear membrane breaks down. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. 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. aligned at the metaphase plate. The chromosomes have chromatin has condensed. Cytokinesis has already begun. The cells of higher plants (such as the flowering plants) lack centrioles. and microtubules have invaded the nuclear Prophase space.division. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. . Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes.
the replicated chromosomes have two sister chromatids. Close to the nucleus are structures called centrosomes. bound together at the centromere by the cohesin protein complex. they are not essential for the . Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. The centrosome is the coordinating center for the cell's microtubules. 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. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. Since the genetic material has already been duplicated earlier in S phase. Although centrioles help organize microtubule assembly. At the onset of prophase. Chromosomes are typically visible at high magnification through a light microscope. A cell inherits a single centrosome at cell division. which is replicated by the cell with the help of the nucleus before a new mitosis begins. giving a pair of centrosomes. which are made of a pair of centrioles found in most eukaryotic animal cells.
it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. undergo a variation called closed mitosis where the spindle forms inside the nucleus. This motor activity. since they are absent from plants.formation of the spindle. the motor activates. such as algae or trichomonads. it is known that it contains some form of molecular motor. on an average 20 ). Although the kinetochore structure and function are not fully understood. one attached at each chromatid. coupled with polymerisation and depolymerisation of microtubules. kinetochore microtubules begin searching for kinetochores to attach to. This is called open mitosis. using energy from ATP to "crawl" up the tube toward the originating centrosome. provides the pulling force necessary to later separate the chromosome's two chromatids. When the spindle grows to sufficient length.2. Each chromosome forms two kinetochores at the centromere. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. or its microtubules are able to penetrate an intact nuclear envelope. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. When a microtubule connects with the kinetochore. and centrosomes are not always used in mitosis. 2. and it occurs in most multicellular organisms.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. Fungi and some protists. . Prometaphase is sometimes considered part of prophase.
Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". convene along the metaphase plate or equatorial plane. in some sense. 2. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. In certain types of cells." Microtubules find and attach to kinetochores in prometaphase. The centromeres of the chromosomes. All chromosomes (blue) but one have arrived at the metaphase plate. an imaginary line that is equidistant from the two centrosome poles. the kinetochore would be the "hook" that catches a sister chromatid or "fish". the chromosomes come under longitudinal tension from the two ends of the cell. . As a result. analogous to a tug-of-war between people of equal strength.3 Metaphase A cell in late metaphase. only roughly lining up along the midline. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Metaphase comes from the Greek meaning "after.In the fishing pole analogy.
.” “against. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. Two events then occur: first. 2. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. At the end of anaphase. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. The signal creates the mitotic spindle checkpoint. allowing them to separate. the proteins that bind sister chromatids together are cleaved. Early anaphase is usually defined as the separation of the sister chromatids. Next.” “back. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. These sister chromatids. the nonkinetochore microtubules elongate. The force that causes the centrosomes to move towards the ends of the cell is still unknown. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. which have now become distinct sister chromosomes.” or “re-”). These two stages are sometimes called early and late anaphase. the cell proceeds to anaphase (from the Greek meaning “up.
forms around each set of separated sister chromosomes. Cytokinesis is technically not even a phase of mitosis. unfold back into chromatin. now surrounded by new nuclei. but rather a separate process. It "cleans up" the after effects of mitosis. using fragments of the parent cell's nuclear membrane. necessary for completing cell division. cell . Corresponding sister chromosomes attach at opposite ends of the cell. In both animal and plant cells. the nonkinetochore microtubules continue to lengthen.5 Cytokinesis Cilliate undergoing cytokinesis. but cell division is not yet complete. A new nuclear envelope. 2. Mitosis is complete. In animal cells. At telophase. elongating the cell even more.2. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. however. pinching off the separated nuclei.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. Both sets of chromosomes. cytokinesis is a separate process that begins at the same time as telophase.
The end of cytokinesis marks the end of the M-phase. separating the two nuclei.g. skin and digestive tract. 2.1Significance Mitosis is important for the maintenance of the chromosomal set. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. zygote and also the basis of the growth of a multicellular body. cells are constantly sloughed off and replaced by new ones. 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.2 Development and growth The number of cells within an organism increases by mitosis.e. Each daughter cell has a complete copy of the genome of its parent cell..4 Regeneration . 2. e. Following are the occasions in the lives of organism where mitosis happens: 2.5.5. Similarly. The phragmoplast is a microtubule structure typical for higher plants.5. New cells are formed by mitosis and so are exact copies of the cells being replaced.3 Cell replacement In some parts of body.5. 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. which move along microtubules to the middle of the cell. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. 2.
especially during early cellular divisions in the zygote. Mitosis continues in the cells of bud and it grows into a new individual. The same division happens during asexual reproduction or vegetative propagation in plants. and the latter cell having only one chromosome (the homologous chromosome). sea star regenerates its lost arm through mitosis. 2.7 Consequences of errors Although errors in mitosis are rare.5. The cells at the surface of hydra undergo mitosis and form a mass called bud. The production of new cells is achieved by mitosis.5. 2. a condition often associated with cancer. Occasionally when cells experience nondisjunction. For example. One daughter cell will receive both sister chromosomes and the other will receive none. For example. a chromosome may fail to separate during anaphase. In non-disjunction. These cells are considered aneuploid.Some organisms can regenerate their parts of bodies. . a condition known as monosomy. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). resulting in binucleated cells.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. a condition known as trisomy. they fail to complete cell division and retain both nuclei in one cell. the process may go wrong. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. the hydra reproduces asexually by budding.
Mitosis is a demanding process for the cell. Or. . All cells have genes that control the timing and number of mitosis. Now what happens is that cell abnormally continue to divide at a single place. It may reattach to the original chromosome. It results in abnormal cell growth. It results in the synthesis of execessive tissue growths. but in reverse orientation. and chromosomes are jostled constantly by probing microtubules. Benign tumours are not harmful as soon as they are not moving. The effect of these genetic abnormalities depends on the specific nature of the error. which goes through dramatic changes in ultrastructure. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. The fragment may incorrectly reattach to another. non-homologous chromosome. An arm of the chromosome may be broken and the fragment lost. its organelles disintegrate and reform in a matter of hours. Occasionally. As soon as they start to move and invade other cells there are said to be malignant tumours. it results in the formation of Tumors. chromosomes may become damaged. causing deletion. This phenomenon is called metastasis or spreading of disease. causing chromosomal duplication. Such tumours can send cancer cells to other parts in body where new tumours may form. it may be treated erroneously as a separate chromosome. causing translocation. causing inversion. As long as these tumours remain in their original location they are called benign tumours. sometimes mutuations occur in such genes and cells continue to divide. Errors in the control of mitosis may cause cancer. When tissues more than the requirement are synthesized in a single organ.
resulting in cells with many copies of the same chromosome occupying a single nucleus. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. align in the middle of the cell before being separated into each of the two daughter cells. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.7 Metaphase Metaphase. carrying genetic information. This process may also be referred to as endoreduplication and the cells as endoploid. from the ancient Greek(between) and (stage). an imaginary line that is equidistant from the two centrosome poles. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. Metaphase accounts for approximately 4% of the cell cycle's duration. Early events of metaphase can . analogous to a tug of war between equally strong people. In certain types of cells. only roughly lining up along the middleline. 2.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. Preceded by events in prometaphase and followed by anaphase.2. 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).
as chromosomes with connected kinetochores will start the events of metaphase individually before other chromosomes with unconnected kinetochores that are still lingering in the events of prometaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Only after all chromosomes have become aligned at the metaphase plate. often with Giemsa (G banding) or Quinacrine. One of the cell cycle checkpoints occurs during prometaphase and metaphase. which makes them most suitable for visual analysis. For classical cytogenetic analyses. securin. Staining of the slides. Metaphase chromosomes make the classical picture of chromosomes (karyotype). cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. produces a pattern of in total up to several hundred bands.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. when every kinetochore is properly attached to a bundle of microtubules. and separase.coincide with the later events of prometaphase. Chromosomes are condensed(Thickened) and highly coiled in metaphase. does the cell enter anaphase. Normal metaphase spreads are used in . even if most of the kinetochores have been attached and most of the chromosomes have been aligned. 2. This would be accomplished by regulation of the anaphase-promoting complex. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). Such a signal creates the mitotic spindle checkpoint.
Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. for example. such as bcr-abl in chronic myelogenous leukemia. which may lead to chimeric oncogenes. .methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. losses of chromosomal segments or translocations.
In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). and the results may be used in evolutionary biology and medicine. in humans 2n = 46. and any other physical characteristics. Karyotypes can be used for many purposes. cellular function. and to gather information about past evolutionary events. Thus. The study of karyotypes is important for cell biology and genetics. such as. Karyogram of human male using Giemsa staining.  The preparation and study of karyotypes is part of cytogenetics. So. and what they look like under a light microscope. or may not. Attention is paid to their length. taxonomic relationships. the position of the centromeres. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. Karyotypes describe the number of chromosomes. The study of whole sets of chromosomes is sometimes known as karyology. The term is also used for the complete set of chromosomes in a species. any differences between the sex chromosomes. There may. be sex chromosomes. to study chromosomal aberrations. or an individual organism. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. in normal diploid organisms. autosomal chromosomes are present in two copies. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. banding pattern. ordered by size and position of centromere for chromosomes of the same size. .CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell.
and he correctly insisted on humans having an XX/XY system. Their behavior in animal (salamander) cells was described by Walther Flemming. 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. Considering their techniques. in 1882. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. the discoverer of mitosis. which swells them and spreads the chromosomes . at first favoring 46. in contrast to their genic contents. The subsequent history of the concept can be followed in the works of Darlington and White. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic 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 1912.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. von Waldeyer in 1888. these results were quite remarkable.3. Using cells in culture 2. Pretreating cells in a hypotonic solution. concluding an XX/XO sex determination mechanism. He revised his opinion later from 46 to 48. The next stage took place after the development of genetics in the early 20th century. The name was coined by another German anatomist. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.
For humans. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. 3. reducing the number.3. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. the great apes have 48 chromosomes.  Sometimes observations may be made on non-dividing (interphase) cells.2. Usually. a suitable dye. The sex of an unborn fetus can be determined by observation of interphase cells.1 Staining The study of karyotypes is made possible by staining. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. is applied after cells have been arrested during cell division by a solution of colchicine. 3. such as Giemsa.2.2 Observations on karyotypes 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . Arresting mitosis in metaphase by a solution of colchicines 4. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Human chromosome 2 was formed by a merger of ancestral chromosomes. Rather interestingly.
5.1. permitting its loss without penalty to the organism (the dislocation hypothesis). 3. faba chromosomes are many times larger. 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. 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. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. This feature probably reflects different amounts of DNA duplication. 6. as well as other cytogenetic information. Heterochromatin stains darker than euchromatin. 2. type. shape and banding of the chromosomes. both have six pairs of chromosomes (n=6) yet V. and mainly consists of genetically inactive repetitive DNA sequences. but the genes have been mostly translocated (added) to other chromosomes. Differences in number and position of satellites. Differences in the position of centromeres. A full account of a karyotype may therefore include the number. indicating tighter packing. . 4. Differences in degree and distribution of heterochromatic regions. This is brought about by translocations.
There is variation between species in chromosome number. 3.3 The human karyotype Most (but not all) species have a standard karyotype. Between members of a population (chromosome polymorphism) 4. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Between the germ-line and soma (between gametes and the rest of the body) 3. Between the sexes 2. males have both an X and a Y chromosome denoted 46. XY. Mosaics or otherwise abnormal individuals.Variation is often found: 1. Normal karyotypes for females contain two X chromosomes and are denoted 46. XX. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Geographical variation between races 5. the same cannot be said for their karyotypes. and in . which are highly variable. Any variation from the standard karyotype may lead to developmental abnormalities.
the general significance of karyotype evolution is obscure. Chromosome elimination.. This variation provides the basis for a range of studies in evolutionary cytology. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. Chromatin diminution (founding father: Theodor Boveri). which were previously inexplicable. 3. But. In some species.. despite their construction from the same macromolecules. used in conjunction with other phylogenetic data.1 Changes during development Instead of the usual gene repression. despite many careful investigations.. Godfrey and Masters conclude: "In our view. . entire chromosomes are eliminated during development. found in some copepods and roundworms such as Ascaris suum. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. it is quite unclear what the general significance might be. or other kinds of visible adjustment to the karyotype. In this process. portions of the chromosomes are cast away in particular cells. some organisms go in for large-scale elimination of heterochromatin. Although much is known about karyotypes at the descriptive level. as in many sciarid flies. In some cases there is even significant variation within species. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. "We have a very poor understanding of the causes of karyotype evolution.3. In A.detailed organization.. In a review.
Muntiacus muntjak. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. They kept quiet for two or three years because they thought something was wrong with their tissue culture. all telocentric. 3. In marsupials it is always the paternal X which is inactivated. all the somatic cell precursors undergo chromatin diminution... male = 7 chromosomes. "They simply could not believe what they saw. the inactivation is random as between the two Xs. When they looked at the karyotype of the closely related Indian muntjac. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable.3.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. In human females some 15% of somatic cells escape inactivation. In placental mammals. The diploid number of the Chinese muntjac. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). The existence of supernumerary or B chromosomes . where the haploid n = 1.suum. thus the mammalian female is a mosaic in respect of her X chromosomes. Muntiacus reevesi. which was investigated by Kurt Benirschke and his colleague Doris Wurster. was found to be 46. the high record would be somewhere amongst the ferns. they were astonished to find it had female = 6. Xinactivation. The low record is held by the nematode Parascaris univalens...
15. though in this case they would not be regarded as normal members of the population. occurs mainly in plants. Humans have FN = 82.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. horsetails and psilotales) is also common. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid.means that chromosome number can vary even within one interbreeding population. Polyploidy in animals is much less common. Thus. 21 and 22). but in grasses the average is much higher.3 Fundamental number The fundamental number. Polyploidy. 3. and the other haploid. FN ≤ 2n.3. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. but it has been significant in some groups. 3.Endopolyploidy occurs when in adult differentiated . of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Haplo-diploidy. It has been of major significance in plant evolution according to Stebbins. Polyploidy in lower plants (ferns. It is a common arrangement in the Hymenoptera. 14. FN. about 70%. due to the presence of five acrocentric chromosome pairs (13. where one sex is diploid. and aneuploids are another example. where there are more than two sets of homologous chromosomes in the cells. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. and in some other groups. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.
The cells do not always contain exact multiples (powers of two).tissues the cells have ceased to divide by mitosis. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. Down syndrome and Turner syndrome are examples of this. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. See palaeopolyploidy for the investigation of ancient karyotype duplications. it is diverse and complex. Abnormalities in chromosome number usually cause a defect in development. and serves differentiation and morphogenesis in many ways.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. but the nuclei contain more than the original somatic number of chromosomes. In many instances. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. 3. 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. . endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). the daughter chromosomes separating from each other inside an intact nuclear membrane.
There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.500 sq mi (17. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups.Aneuploidy may also occur within a group of closely related species. When this happens. and 7. 3. Well-researched examples are the ladybird beetle Chilocorus stigma. 4. living from rainforests to . 6. the European shrew Sorex araneus. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world.000 km2). In about 6. where the gametic (= haploid) numbers form the series x = 3. Human chromosome 2 was formed by a merger of ancestral chromosomes.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. 3. some mantids of the genus Ameles. and Crocus. that the two chromosome morphs are adapted to different habitats. reducing the number. where every number from x = 3 to x = 15 is represented by at least one species.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. 5. the great apes have 24x2 chromosomes whereas humans have 23x2.  Closer to home. Classic examples in plants are the genus Crepis. the chromosome number is variable from one individual to another.
The results are clear.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). The inversions. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. Although it would be possible for a single gravid female to colonise an island. when plotted in tree form (and independent of all other information). These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. the present islands date from 0. probably 20 million years ago. Drosophila and Scaptomyza. especially inversions. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. make it possible to see which species are closely related. but these are much less frequent. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. which can be dated to 30 mya. at least into the Cretaceous. in the family Drosophilidae. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. The polytene banding of the 'picture wing' group. . Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. Chromosome rearrangements. and skipping of islands. In a sense. it is more likely to have been a group from the same species. There are also cases of colonization back to older islands. gene arrangements are visible in the banding patterns of each chromosome. Using K-Ar dating. the best-studied group of Hawaiian drosophilids.subalpine meadows. show a clear "flow" of species from older to newer islands.
This method will normally produce 300-400 bands in a normal. The pattern of bands is very similar to that seen in G-banding. late-replicating and AT rich. It yields a series of lightly and darkly stained bands . if less spectacular.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. . • Q-banding is a fluorescent pattern obtained using quinacrine for staining. early-replicating and GC rich. 3. • T-banding: visualize telomeres. adaptive radiations.the dark regions tend to be heterochromatic. • • C-banding: Giemsa binds to constitutive heterochromatin. The light regions tend to be euchromatic. R-banding is the reverse of G-banding (the R stands for "reverse").7 Depiction of karyotypes 3. human genome. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). so it stains centromeres.
Some karyotypes call the short and long arms p and q.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. Karyotypes are arranged with the short arm of the chromosome on top. is used to stain bands on the chromosomes. In addition. often Giemsa (G-banding). denoting the activity of rRNA genes within the NOR. Each chromosome has a characteristic banding pattern that helps to identify them. Cri du chat syndrome involves a deletion on the short arm of . both chromosomes in a pair will have the same banding pattern. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. respectively. 3.7. Quinacrine binds to the adeninethymine-rich regions. a dye.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. and the long arm on the bottom. For example. less frequently Quinacrine. This yields a dark region where the silver is deposited. In the "classic" (depicted) karyotype. Giemsa is specific for the phosphate groups of DNA.
7. which is written as 46. a combinatorial labeling method is used to generate many different colors.5p-. It is written as 46. This method is also known as virtual karyotyping.2.chromosome 5.XX. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.XX. 3.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. Image processing software then assigns a pseudo color to each spectrally different combination. The critical region for this syndrome is deletion of 15.del(5)(p15.3 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. Because there are a limited number of spectrally-distinct fluorophores. allowing the visualization of the individually colored chromosomes. .2) 3.
CHAPTER 4 .
translocations.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. • • Patau syndrome is caused by trisomy of chromosome 13. although they generally do not survive to birth.4. X or 45. a common chromosomal disease. inversions. X0). the most common male chromosomal disease. also known as aneuploidy. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. XXY is caused by an extra X chromosome. as in the presence of extra or missing chromosomes. as in derivative chromosome. including . trisomies. Down syndrome. in which three copies of a chromosome are present instead of the usual two. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. often occur as a result of nondisjunction during meiosis in the formation of a gamete. are common numerical abnormalities. otherwise known as 47. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. or structural. large-scale deletions or duplications. Also documented are trisomy 8. Klinefelter syndrome. Structural abnormalities often arise from errors in homologous recombination. is caused by trisomy of chromosome 21. Numerical abnormalities. Some disorders arise from loss of just a piece of one chromosome. trisomy 9 and trisomy 16.
numerical and structural anomalies. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. example of imprinting disorder. a deletion of the paternal genes. 1p36 Deletion syndrome. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. a deletion of the maternal genes. A chromosome anomaly. A chromosome anomaly may be detected or confirmed in this manner. from the loss of part of the short arm of chromosome 1. They can be organized into two basic groups. There are many types of chromosome anomalies. • • Angelman 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. 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). a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. example of imprinting disorder. one well-documented example is the Philadelphia chromosome. caused by abnormal formation of the larynx. from a truncated short arm on chromosome 5. The name comes from the babies' distinctive cry. .
In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Tetrasomy. segments from two different chromosomes have been exchanged. etc.4.3 Structural abnormalities When the chromosome's structure is altered. In a Robertsonian translocation. and Jacobsen syndrome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). which is caused by partial deletion of the short arm of chromosome 4. rather than two). 4. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. resulting in extra genetic material. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. Duplications: A portion of the chromosome is duplicated.). This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. 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. Known disorders in humans include Wolf-Hirschhorn syndrome. There are two main types of translocations. an X. an entire chromosome has . and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. • • Translocations: When a portion of one chromosome is transferred to another chromosome. In a reciprocal translocation. also called the terminal 11q deletion disorder.
• Rings: A portion of a chromosome has broken off and formed a circle or ring. turned upside down and reattached. 14. and are therefore initially not inherited.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell.in humans these only occur with chromosomes 13. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. especially the chromosomes. Chromosome anomalies can be inherited from a parent or be "de novo". 15. Therefore. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. It includes routine analysis of G-Banded chromosomes. Some anomalies. as well . • Inversions: A portion of the chromosome has broken off. other cytogenetic banding techniques. resulting in Mosaicism (where some cells have the anomaly and some do not). 4. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 21 and 22. therefore the genetic material is inverted. can happen after conception. 4. They often lead to an increased tendency to develop certain types of malignancies.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm.attached to another at the Centromere . however. This can happen with or without loss of genetic material. the anomaly is present in every cell of the body.
Pre-treating cells in a hypotonic solution. and he correctly insisted on man having an XX/XY system. which swells them and spreads the chromosomes . when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Considering their techniques.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). concluding an XX/XO sex determination mechanism. von Waldeyer in 1888. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. New techniques were needed to definitively solve the problem: 1. at first favoring 46. the discoverer of mitosis. in 1882. these results were quite remarkable. 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. Using cells in culture 2. He revised his opinion later from 46 to 48. 4. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Their behavior in animal (salamander) cells was described by Walther Flemming. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. The name was coined by another German anatomist. in contrast to their genic contents.
the great apes have 48 chromosomes. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Using Painter's technique they studied the polytene . 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.6. persimilis from wild populations in California and neighboring states. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).6 Applications in biology 4. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Arresting mitosis in metaphase by a solution of colchicine 4.2 Natural populations of Drosophila In the 1930s. Rather interestingly. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.6. a find which eventually led to her Nobel Prize in 1983. In 1931.3. reducing the number. During her cytogenetic work. McClintock discovered transposons. Human chromosome 2 was formed by a merger of ancestral chromosomes.
cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. Dobzhansky bred populations in population cages. Down syndrome is also referred to as trisomy 21. as with most polymorphisms. Evidence rapidly accumulated to show that natural selection was responsible. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. but adjust to certain frequencies at which they become stabilised. In 1959. 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.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. breeding and sampling whilst preventing escape. Using a method invented by L'Heretier and Teissier. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. 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. It was found that the various chromosome types do not fluctuate at random. such as Down's syndrome. . 4. discoveries were quickly made related to aberrant chromosomes or chromosome number. which enabled feeding. as they would if selectively neutral. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21.
XYY. 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 . Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them.as both scientists were doing their research in Philadelphia. which is required in normal females to compensate for having two copies of the chromosome. is used today as a diagnostic for CML. An individual with only one sex chromosome (the X) has Turner syndrome.Other numerical abnormalities discovered include sex chromosome abnormalities. Not all genes on the X Chromosome are inactivated. in addition to other tests. has Klinefelter's Syndrome. resulting in 47 total chromosomes. In 1960. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. with the development of more advanced techniques. and XXXX. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. Pennsylvania. Many other sex chromosome combinations are compatible with live birth including XXX. Identification of the Philadelphia chromosome by cytogenetics. an additional X chromosome in a male. Thirteen years later. .
4.FIG Advent of banding techniques In the late 1960s. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. Deletion syndromes such as DiGeorge syndrome. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.8 Beginnings of molecular cytogenetics . Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Deletions within one chromosome could also now be more specifically named and understood. and elongation techniques for all culture types that allow for higher resolution banding. Caspersson developed banding techniques which differentially stain chromosomes. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid.
Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). While radioisotope-labeled probes had been hybridized with DNA since 1969. advances were made in molecular cytogenetics. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. cloned and studied in ever greater detail.In the 1980s.1 Karyotyping . Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. CHAPTER 5 Techniques 5. movement was now made in using fluorescent labeled probes.
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.
5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •
bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations
5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,
and numerical computation. You can use MATLAB in a wide range of applications. C++. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. 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. control design. data analysis. such as C.generally between 200 and 1000 cells are counted and scored. CGH and Single nucleotide polymorphism-arrays. communications. data visualization. and FORTRAN. financial modeling and analysis. For congenital problems usually 20 metaphase cells are scored. Using MATLAB. such as comparative genomic hybridization arrays. and computational biology. including signal and image processing. test and measurement. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development.
specifying data types. one line of MATLAB code can often replace several lines of C or C++ code.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. The image processing step is composed of the following operations. As a result. These effects must be compensated to improve the results of the pairing algorithm.MATLAB provides a number of features for documenting and sharing your work. 6. such as declaring variables. MATLAB eliminates the need for ‘for’ loops. 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. and allocating memory. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. You can integrate your MATLAB code with other languages and applications. It enables fast development and execution. In many cases. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. . With the MATLAB language. and distribute your MATLAB algorithms and applications.
To compensate for this inhomogeneity. or at least attenuated. 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.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. 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. Therefore. the spatially scaled images are histogram equalized. 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. 6.2 Concepts used in this phase 1) Image conversion 2) Denoising . 2) Geometrical compensation—The geometric compensation.
MATLAB filters the intensity values in the image. MATLAB simply applies the filter to the indices in the indexed image matrix. 6. For example. If you attempt to filter the indexed image. The resulting true color image has identical matrices for the red.I. there are other functions that return a different image type as part of the operation they perform. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. and blue planes. green. so the image displays as shades of gray.2. and the results might not be meaningful. RGB = cat (3.3) Edge detection 4) Two dimensional convolutions. When you apply the filter to the true color image. you must first convert it to true color format. For example.I). as is appropriate. if you want to filter a color image that is stored as an indexed image.I. . For example. listed in the following table. You can perform certain conversions just using MATLAB syntax. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. In addition to these image type conversion functions.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.
There is a large number of edge finding algorithms in existence. to recognize or classify objects. or through networked cable. . and hence the type of noise on the image. hence we can choose the most appropriate method for reducing the effects. Cleaning an image corrupted by noise is thus an important area of image restoration.4 Denoising We may define noise to be any degradation in the image signal. via satellite or wireless transmission.5 Edge detection Edges contain some of the most useful information in an image. If an image is being sent electronically from one place to another. caused by external disturbance.3 Two dimensional convolutions C = conv2(A.2. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. and we shall look at some of the more straightforward of them.parameters.2. we may expect errors to occur in the image signal. ) Where the parameters available depend on the method used 6. If one of these matrices describes a two-dimensional finite impulse response . Usually we know what type of errors to expect.'method'. We may use edges to measure the size of objects in an image. to isolate particular objects from their background. .B) computes the two-dimensional convolution of matrices A and B. 6.6. The general Matlab command for finding edges is edge(image.
na+nb-1].(FIR) filter. nb]+1)/2). 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. . this case is the same as C = conv2(hcol*hrow. If hcol is a column vector and hrow is a row vector.hrow.'shape') subsection of the two-dimensional convolution.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma..bmp'). imedfilt2(im1. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. C = conv2(.A).7). The indices of the center element of B are defined as floor(([mb C = conv2(hcol. The size of matrices. if the size of then the size of C is [ma+mb-1.0. the other matrix is filtered in two dimensions.. rgb2gray im2bw(im. minus one.[3 3])..'sobel').nb]. That is. edge(im1.na] and the size of B is [mb.
. MODULE 2 clc [m. [sx sy]=size(rc). nzeros(imx. flag=0.y1)=255.j)~=0 for k=1:mx if L(i. L_number=zeros(mx.j)==L_number(k) flag=1. mx=max(max(L)). y1=rc(i.n]=size(L). for i=1:sx x1=rc(i.[imx. Index=1.1).double(msk)).8).1). bwlabel(B.imy]=size(BW). for i=1:m for j=1:n if L(i. [r. n1(x1. Msk conv2(double(BW).c] = find(L==22). rc = [r c].imy).2).
60. for x=1:46 [r. rc = [r c].31. 36.imshow(n18.104.22.168.14.48. for i=1:sx x1=rc(i.66].c] = find(L==L_number((Test_number(x)))).33. n1=zeros(imx.50.1). end end L_number. .22.214.171.124.2).y1)=255.28.32.56.35.imy).126.96.36.199.43.9. end.).188.8.131.52.40.15.24. Test_number=[3.55.j).42.30.57. n1(x1. y1=rc(i.20.27.end end if flag~=1 L_number(Index)=L(i. Index=Index+1. end %h=figure.184.108.40.206.7. [sx sy]=size(rc). end flag=0.41.51.
[m n]=size(BW1). skel=im2bw(skel.1). for i=1:46 f=imread(strcat(num2str(i). s=bwmorph(skel. Arm_length=zeros(46.1). skel=im2double(f).'spur'.'canny'). s1=bwmorph(s. Arm_length_sum=0. Area=zeros(46.1).Inf). BW=double(BW).y)==1 Circumference_sum=Circumference_sum+1.end Circumference=zeros(46. BW=im2bw(f).5*graythresh(skel)). Circumference_sum=0.bmp')).8). end end end Circumference(i)=Circumference_sum. f=imcomplement(f). for x=1:m for y=1:n if BW1(x.'skel'. .'. BW1=edge(BW.1.
for x=1:m for y=1:n if BW(x. end end end Arm_length(i)=Arm_length_sum. .y)==1 Area_sum=Area_sum+1. for x=1:m for y=1:n if s1(x. end Circumference. BW=im2bw(f).y)==1 Arm_length_sum=Arm_length_sum+1. [m n]=size(BW). Area_sum=0. Arm_length. end end end Area(i)=Area_sum.[m n]=size(s1).
Area. Pair(i.1)=i. Pair=zeros(46.1)=i.2)==46 Pair(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)=46. .2). Pair(i. end end end for i=1:45 if Pair(i. Pair(i.2)=j.2)=i+1. Pair(i.2)=i. Pair(46. end end Pair. 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)).
end f1=imread(strcat(num2str(Pair(i. figure_flag=1.1)). end f2=imread(strcat(num2str(Pair(i.1). end end if flag~=1 if figure_flag~=47 subplot(23.figure_flag).'.2).2.1)==delete(j) flag=1. end flag=0.bmp')). . delete(figure_flag)=Pair(i.figure_flag).delete=zeros(46.2)). figure_flag=figure_flag+1. imshow(f2). flag=0. for i=1:46 for j=1:46 if Pair(i. imshow(f1). if figure_flag~=47 subplot(23. figure_flag=figure_flag+1.2.'.bmp')).
Copenhagen. 2) feature extraction from the processed images . plus a new one. 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. in the scope of karyotyping process used in cytogentic analysis. and Philadelphia. such as. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. based on the MI. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern.end CONCLUTION In this paper. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. dimensions and banding profiles. 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.
achieves a 70. 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. shape and band pattern. This normalization is needed to make it possible the band pattern comparison between chromosomes. shape. and band pattern. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.characterizing the size. are processed in order to compensate for geometrical and intensity distortions. In the image processing step. the romosome images. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. 4) pairing.10% mean classification rate. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts.working within an 8-D feature space. 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 . Tests using 19 karyograms based on bone marrow cells. extracted from the unordered karyogram. from the chromosomes in the training set. 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). Here. and finally. and to normalize their dimensions.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram. 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 .
g. The results presented in this paper are promising. despite the low quality of this type of chromosomes. In addition. whose images are of significantly higher quality. Using 27 karyograms andworking with a limited number of classes (≤ 8). REFERENCES .. a 76. centromere position. Executing the algorithm on a higher quality dataset. and from which it is possible to extract additional features. amean classification rate larger than 93% was obtained in all experiments.10% classification ratewas obtained. This dataset was made publicly available . Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. Copenhagen. or Philadelphia. In fact. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. a new chromosome dataset with 9200 chromosomes from bone marrow cells. such as Edinburgh. presenting a uniform level of condensation.performance of the classifier. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. called LK1 . e.
and Mulligan P. ^ Darlington C. 3rd ed. 2nd ed. 7. . Kiev. Chapman & Hall. 1931. 465-502. 2006. Animal cytology and evolution. A dictionary of genetics.D. preconception and the counting of the human chromosomes. Bull.P Oxford & NY.J. [in Russian] 6. Applied Bot. ^ Stebbins G. The material basis of heredity. The spermatogenesis of man. 1973. 4.D. ^ a b c White M. Res. 1924. 147–49. Plant Breed. 11..J. ^ a b White M. p. biologie 27. 1958. Variation and evolution in plants. Oliver & Boyd.1. From 48 to 46: cytological technique. 27. Genet. Anat.K.L. Med. Oxford U. revised and enlarged. 6th ed. ^ Levitsky G. 93. Cambridge University Press. 1974.A. Edinburgh. ^ Painter T.C. 129. The chromosomes. 7th ed. 8. 28 2. 9. ^ Levitsky G. Cambridge University Press.D. ^ Concise Oxford Dictionary London. 1973. Arch. Hist. Columbia University Press NY. 48. Stansfield W. 3. Etudes sur la spermatogenese humaine. ^ von Winiwarter H. Chapter XII: The Karyotype. 23. ^ Kottler M. Bull. Evolution of genetic systems. 1950. 19-174.A. 1922. State Publication Office of the Ukraine.S. 10.D. The morphology of chromosomes. p242 5. ^ King R. 1912. 1939.
New York. ^ Müller F. ^ Painter T. Evolutionary genetics. Oxford. ed.H & Levan A. Chromosome stains. Arnold. 17. 1979. Chromosome elimination in sciarid flies.B. Barch. Raven Press. Human and mammalian cytogenetics: a historical perspective. Bioessays18: 133–138. PNAS 97. The spermatogenesis of humans. & Tobler H. ^ Maynard Smith J. 2001. In The ACT Cytogenetics Laboratory Manual 2nd ed. The chromosome number of man.12.^ a b Hsu T. Springer-Verlag. . ^ Godfrey L. and Esteban M. 1971. ^ Goday C. ^ Stebbins G.http://www. 1-6. 9821– 9823. Bioessays23: 242–250.nih.C.M.C.pubmedcentral. M. 1998. 1991. 2nd ed. ^ Tjio J. 21. Chromatin diminution in nematodes. Hereditas 42. Eosin Y and Azure-A.L.J.^ a b Gustashaw K. Kinetophore reproduction theory may explain rapid chromosome evolution. and Masters J. 2000. NY. Exp. 1956. ^ A preparation which includes the dyes Methylene Blue. 1923. Studies in mammalian spermatogenesis II.S. 291-336. Chromosomal evolution in higher plants.R. The Association of Cytogenetic Technologists. Bernard V.gov/articlerender. London.R. 15. 13. 14. p218-9 20. Zoology 37. J.C 16. 1996.fcgi?artid=34032 19. p85-6 18.
and Mulligan P. Oxford U. J. D. (1945-05-15). Chromosome evolution in the genus Ophioglossum L.K. Stamford CT.S..C. 1990.22. 8th ed. ^ Gilbert S. Park.D. Zool.K. Noh. Science 168.1007/BF02153623.K. A dictionary of genetics.H. Developmental biology. ^ Wurster D. 291: 310–16..H.doi:10.. ^ King R. Botanical Journal of the Linnean Society 102: 205–217. 1364-1366...A.R.1007/s10228-004-0257-z. "L'evolution de la formule chromosomiale chez les vertébrés". Exp. 26. 7th ed. ^ Wyngaard G. Retrieved 2011-03-16. Chapman. (2005). Muntiacus muntjak: a deer with a low diploid number. . Indian Muntjac. Y. Stansfield W. 1970. ^ Khandelwal S. Nam. 27. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. 2006. Experientia (Basel) 1 (2): 50– 56. Sinauer Associates. C. ^ Matthey. 25. doi:10. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF).A. 28. 2001. ^ Kim. Chapter 9 24.F. & Gregory T. Ichthyological Research 52 (1): 97. 2006. and Benirschke K. F. R.P Oxford & NY. Retrieved 2008-03-18. 23. J.
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