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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
which is tightly coiled in on itself. sometimes accompanied by one or more smaller. divided. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. The structure of chromosomes and chromatin varies through the cell cycle. although there are many exceptions to this rule. Chromosomes are the essential unit for cellular division and must be replicated. In prokaryotes. a large body of work uses the term chromosome regardless of chromatin content. In eukaryotes. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. Unduplicated chromosomes are single linear strands. the term genophore is more appropriate when no chromatin is present. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). However. In prokaryotes and viruses. for example. In practice "chromosome" is a rather loosely defined term. cells may contain more than one type of chromosome. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. Also. Chromosomal recombination plays a vital role in genetic diversity. This allows the very long DNA molecules to fit into the cell nucleus. through processes known as chromosomal instability and translocation.defined nuclei) have smaller circular chromosomes. circular DNA molecules called plasmids. Chromosomes may exist as either duplicated or unduplicated. the cell may undergo mitotic catastrophe and die. or it may unexpectedly evadeapoptosis leading to the progression of cancer. These small circular genomes . If these structures are manipulated incorrectly. DNA is usually arranged as a circle. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes.
An example of aneuploidy is trisomy 21. The individual would have Down Syndrome and his/her karyotype would be written 47. in which an individual has 3. reflecting their bacterial origins. . The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.XX.are also found in mitochondria and chloroplasts. rather than 2.3 MUTATIONS IN CHROMOSOME NUMBER Normally. Such individuals are called euploid and have the wild-type chromosome complement for the species. members of the same species have the same numbers of types of chromosomes (with the exception of sex chromosomes in males and females if sex is chromosomally determined). a extra copy of human chromosome 21). the individual carrying the mutation is said to be aneuploid.+21. If the mutation involves only one or a few chromosomes in the genome (e.+21. 1. copies of chromosome 21. 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.g.XY or 47. Euploid human karyotypes are 46. XX (female) or 46 XY (male).
. so that each daughter cell inherits one set of chromatids. 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. longer-lasting attachment in this region. 1. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction.Fig 1. Once the cells have divided. The microtubules then pull the chromatids apart toward the centrosomes. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. and they form the classic four arm structure. which enables these giant DNA structures to be contained within a cell nucleus. During mitosis. along with special proteins. the chromatin strands become more and more condensed. one of which is present on each sister chromatid. The shorter arms are called p arms (from the French petit.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). q-g "grande"). In spite of their appearance. A special DNA base sequence in the region of the kinetochores provides. a pair of sister chromatids attached to each other at the centromere. This compact form makes the individual chromosomes visible. chromosomes are structurally highly condensed. small) and the longer arms are called q arms (q follows p in the Latin alphabet.
On average. bone marrow accounts for approximately 2.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. which divides the nuclei. producing the lymphocytes that support the body's immune system CHAPTER 2 2. in adults weighing 65 kg (143 lbs).1.6 kg (5. In humans.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. in two separate nuclei. bone marrow constitutes 4% of the total body mass of humans. . organelles and cell membrane into two cells containing roughly equal shares of these cellular components. bone marrow in large bones produces new blood cells.7 lbs). cytoplasm. which use the bone marrow vasculature as a conduit to the body's systemic circulation.  Bone marrow is also a key component of the lymphatic system. It is generally followed immediately by cytokinesis. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.
prophase. Mitosis occurs only in eukaryotic cells and the process varies in different species. This accounts for approximately 10% of the cell cycle. However. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. animals undergo an "open" mitosis. These stages are interphase.genetically identical to each other and to their parent cell. cytokinesis and mitosis may occur independently. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. This occurs most notably among the fungi and slime moulds. "mitosis" is often used interchangeably with "mitotic phase". Because cytokinesis usually occurs in conjunction with mitosis. anaphase and telophase. there are many cells where mitosis and cytokinesis occur separately. The process of mitosis is fast and highly complex. where the nuclear envelope breaks down before the chromosomes separate. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. divide by a process called binary fission. where chromosomes divide within an intact cell nucleus. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. . Prokaryotic cells. The cell then divides in cytokinesis. For example. metaphase. for instance during certain stages of fruit fly embryonic development. forming single cells with multiple nuclei. prometaphase. which lack a nucleus. Even in animals. to produce two identical daughter cells which are still diploid cells. but is found in various different groups.
The sister chromatids are held together by a specialized region of the chromosome known as the centromere. This occurs during the S phase of interphase. . Because each resultant daughter cell should be genetically identical to the parent cell. the parent cell must make a copy of each chromosome before mitosis. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. and together the two are called sister chromatids.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. These two cells are identical and do not differ in any way from the original parent cell. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. Each chromosome now has an identical copy of itself.
As a matter of convention. In animal cells. each sister chromatid is now considered a chromosome. separating the two developing nuclei.the cell begins cytokinesis. each with a replica of the original genome. 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). Prokaryotic cells undergo a process similar to mitosis called binary fission.In most eukaryotes. A new nuclear envelope forms around the separated sister chromosomes. Eventually. corresponding sister chromosomes are pulled toward opposite ends. As mitosis completes. As the cell elongates. the process of binary fission is very much different from the process of mitosis. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. the daughter cells will construct a new dividing cell wall between each other. The chromosomes align themselves in a line spanning the cell. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. so they are renamed to sister chromosomes. pulling apart the sister chromatids of each chromosome. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. the parent cell will be split in half. giving rise to two daughter cells. However. . In plant cells.
1 Preprophase In plant cells only. the nucleus has to migrate into the center of the cell before mitosis can begin.2. mainly via proteins. S (synthesis). the cell grows by producing proteins and cytoplasmic organelles. All these phases in the interphase are highly regulated. 2. This is achieved through the formation of a phragmosome. continues to grow as it duplicates its chromosomes (S). Thus. and finally it divides (M) before restarting the cycle. It alternates with the much longer interphase. Interphase is divided into three phases: G1 (first gap). a cell grows (G1). a transverse sheet of cytoplasm that bisects the cell along the future plane of cell .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. and G2 (second gap). However. prophase is preceded by a pre-prophase stage. In highly vacuolated plant cells. During all three phases.2. where the cell prepares itself for cell division. chromosomes are replicated only during the S phase. 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. grows more and prepares for mitosis (G 2).
aligned at the metaphase plate. degraded. the pinched area is known as the cleavage furrow. The chromosomes have chromatin has condensed. Cytokinesis has already begun. . 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. after the nuclear membrane breaks down. This band marks the position where the cell will eventually divide. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. instead. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. and microtubules have invaded the nuclear Prophase space. In addition to phragmosome formation. The cells of higher plants (such as the flowering plants) lack centrioles. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes.division. These microtubules can attach to kinetochores or they can interact with opposing microtubules. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves.
chromatin condenses together into a highly ordered structure called a chromosome. The centrosome is the coordinating center for the cell's microtubules. giving a pair of centrosomes. which is replicated by the cell with the help of the nucleus before a new mitosis begins. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. Close to the nucleus are structures called centrosomes. which are made of a pair of centrioles found in most eukaryotic animal cells. the replicated chromosomes have two sister chromatids. Chromosomes are typically visible at high magnification through a light microscope. Although centrioles help organize microtubule assembly. the genetic material in the nucleus is in a loosely bundled coil called chromatin. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. they are not essential for the . A cell inherits a single centrosome at cell division. Since the genetic material has already been duplicated earlier in S phase.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. bound together at the centromere by the cohesin protein complex. At the onset of prophase.
and it occurs in most multicellular organisms.2. and centrosomes are not always used in mitosis. such as algae or trichomonads. coupled with polymerisation and depolymerisation of microtubules. one attached at each chromatid. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. Fungi and some protists. kinetochore microtubules begin searching for kinetochores to attach to. provides the pulling force necessary to later separate the chromosome's two chromatids. Each chromosome forms two kinetochores at the centromere. This is called open mitosis. on an average 20 ). the motor activates. Although the kinetochore structure and function are not fully understood. When a microtubule connects with the kinetochore. since they are absent from plants.formation of the spindle. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. undergo a variation called closed mitosis where the spindle forms inside the nucleus. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. Prometaphase is sometimes considered part of prophase. 2. or its microtubules are able to penetrate an intact nuclear envelope. using energy from ATP to "crawl" up the tube toward the originating centrosome. This motor activity. it is known that it contains some form of molecular motor. .2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. When the spindle grows to sufficient length.
analogous to a tug-of-war between people of equal strength. only roughly lining up along the midline. In certain types of cells. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. All chromosomes (blue) but one have arrived at the metaphase plate.In the fishing pole analogy." Microtubules find and attach to kinetochores in prometaphase. 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. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". . in some sense. convene along the metaphase plate or equatorial plane. As a result. 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. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. Metaphase comes from the Greek meaning "after. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. The centromeres of the chromosomes. 2.3 Metaphase A cell in late metaphase.
Next.” or “re-”). the cell has succeeded in separating identical copies of the genetic material into two distinct populations. the nonkinetochore microtubules elongate. At the end of anaphase. which have now become distinct sister chromosomes. 2.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres).4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. The signal creates the mitotic spindle checkpoint. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. These sister chromatids.” “against. Early anaphase is usually defined as the separation of the sister chromatids. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. The force that causes the centrosomes to move towards the ends of the cell is still unknown. the cell proceeds to anaphase (from the Greek meaning “up. allowing them to separate. . Two events then occur: first. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. These two stages are sometimes called early and late anaphase. the proteins that bind sister chromatids together are cleaved. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached.” “back.
5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. At telophase. forms around each set of separated sister chromosomes.5 Cytokinesis Cilliate undergoing cytokinesis. 2. unfold back into chromatin. Mitosis is complete.2. now surrounded by new nuclei. elongating the cell even more. the nonkinetochore microtubules continue to lengthen. however. but cell division is not yet complete. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. pinching off the separated nuclei. A new nuclear envelope. Corresponding sister chromosomes attach at opposite ends of the cell. cytokinesis is a separate process that begins at the same time as telophase. In animal cells. It "cleans up" the after effects of mitosis. necessary for completing cell division. In both animal and plant cells. Both sets of chromosomes. 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. cell . but rather a separate process. using fragments of the parent cell's nuclear membrane.
The phragmoplast is a microtubule structure typical for higher plants.5..4 Regeneration .e. which move along microtubules to the middle of the cell. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. 2. e.g.2 Development and growth The number of cells within an organism increases by mitosis. 2.1Significance Mitosis is important for the maintenance of the chromosomal set. The end of cytokinesis marks the end of the M-phase.3 Cell replacement In some parts of body.5. skin and digestive tract. Each daughter cell has a complete copy of the genome of its parent cell.division is also driven by vesicles derived from the Golgi apparatus. zygote and also the basis of the growth of a multicellular body. New cells are formed by mitosis and so are exact copies of the cells being replaced. 2. Similarly. cells are constantly sloughed off and replaced by new ones. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. separating the two nuclei.5.5. whereas some green algae use a phycoplast microtubule array during cytokinesis. This is the basis of the development of a multicellular body from a single cell i. Following are the occasions in the lives of organism where mitosis happens: 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.
The same division happens during asexual reproduction or vegetative propagation in plants. a condition often associated with cancer. a condition known as trisomy. a chromosome may fail to separate during anaphase.Some organisms can regenerate their parts of bodies. These cells are considered aneuploid.7 Consequences of errors Although errors in mitosis are rare.5. In non-disjunction. . especially during early cellular divisions in the zygote. For example. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. and the latter cell having only one chromosome (the homologous chromosome). Mitosis continues in the cells of bud and it grows into a new individual. The production of new cells is achieved by mitosis. 2.5. 2. The cells at the surface of hydra undergo mitosis and form a mass called bud. For example. resulting in binucleated cells. the hydra reproduces asexually by budding. a condition known as monosomy. Occasionally when cells experience nondisjunction.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. the process may go wrong. sea star regenerates its lost arm through mitosis. they fail to complete cell division and retain both nuclei in one cell. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). One daughter cell will receive both sister chromosomes and the other will receive none.
and chromosomes are jostled constantly by probing microtubules. All cells have genes that control the timing and number of mitosis. Or. . causing deletion.Mitosis is a demanding process for the cell. Benign tumours are not harmful as soon as they are not moving. The fragment may incorrectly reattach to another. An arm of the chromosome may be broken and the fragment lost. its organelles disintegrate and reform in a matter of hours. Errors in the control of mitosis may cause cancer. causing chromosomal duplication. Now what happens is that cell abnormally continue to divide at a single place. As soon as they start to move and invade other cells there are said to be malignant tumours. It may reattach to the original chromosome. causing inversion. sometimes mutuations occur in such genes and cells continue to divide. As long as these tumours remain in their original location they are called benign tumours. but in reverse orientation. It results in abnormal cell growth. This phenomenon is called metastasis or spreading of disease. chromosomes may become damaged. it results in the formation of Tumors. it may be treated erroneously as a separate chromosome. Such tumours can send cancer cells to other parts in body where new tumours may form. which goes through dramatic changes in ultrastructure. causing translocation. Occasionally. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. When tissues more than the requirement are synthesized in a single organ. The effect of these genetic abnormalities depends on the specific nature of the error. It results in the synthesis of execessive tissue growths. non-homologous chromosome.
from the ancient Greek(between) and (stage).7 Metaphase Metaphase. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. In certain types of cells. Metaphase accounts for approximately 4% of the cell cycle's duration. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes.2. resulting in cells with many copies of the same chromosome occupying a single nucleus. Preceded by events in prometaphase and followed by anaphase.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. 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. analogous to a tug of war between equally strong people. only roughly lining up along the middleline. An example of a cell that goes through endomitosis is the megakaryocyte. carrying genetic information. 2. This process may also be referred to as endoreduplication and the cells as endoploid. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). Early events of metaphase can . align in the middle of the cell before being separated into each of the two daughter cells.
and separase.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. This would be accomplished by regulation of the anaphase-promoting complex.coincide with the later events of prometaphase. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. 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. For classical cytogenetic analyses. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Such a signal creates the mitotic spindle checkpoint. often with Giemsa (G banding) or Quinacrine. which makes them most suitable for visual analysis. produces a pattern of in total up to several hundred bands. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Staining of the slides. does the cell enter anaphase. 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). Only after all chromosomes have become aligned at the metaphase plate. Normal metaphase spreads are used in . securin. Chromosomes are condensed(Thickened) and highly coiled in metaphase. 2. Metaphase chromosomes make the classical picture of chromosomes (karyotype).
Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. losses of chromosomal segments or translocations. such as bcr-abl in chronic myelogenous leukemia. . Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. for example. which may lead to chimeric oncogenes.
The study of whole sets of chromosomes is sometimes known as karyology. banding pattern. to study chromosomal aberrations. or an individual organism. Attention is paid to their length.  The preparation and study of karyotypes is part of cytogenetics. So. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. autosomal chromosomes are present in two copies. such as.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. Karyogram of human male using Giemsa staining. and any other physical characteristics. . in normal diploid organisms. and what they look like under a light microscope. ordered by size and position of centromere for chromosomes of the same size. Karyotypes can be used for many purposes. and the results may be used in evolutionary biology and medicine. the position of the centromeres. be sex chromosomes. taxonomic relationships. cellular function. and to gather information about past evolutionary events. There may. The study of karyotypes is important for cell biology and genetics. in humans 2n = 46. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. any differences between the sex chromosomes. Karyotypes describe the number of chromosomes. or may not. Thus. The term is also used for the complete set of chromosomes in a species. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies.
in 1882. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. in contrast to their genic contents. The next stage took place after the development of genetics in the early 20th century. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. the discoverer of mitosis. Using cells in culture 2. which swells them and spreads the chromosomes . The name was coined by another German anatomist. 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. Considering their techniques.3. New techniques were needed to definitively solve the problem: 1.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. these results were quite remarkable. The subsequent history of the concept can be followed in the works of Darlington and White. Pretreating cells in a hypotonic solution. von Waldeyer in 1888. and he correctly insisted on humans having an XX/XY system. Their behavior in animal (salamander) cells was described by Walther Flemming. He revised his opinion later from 46 to 48. concluding an XX/XO sex determination mechanism. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. at first favoring 46.
For humans. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Usually. the great apes have 48 chromosomes. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.2.3. reducing the number.1 Staining The study of karyotypes is made possible by staining. a suitable dye. Arresting mitosis in metaphase by a solution of colchicines 4. Human chromosome 2 was formed by a merger of ancestral chromosomes.  Sometimes observations may be made on non-dividing (interphase) cells.2. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. such as Giemsa. 3. is applied after cells have been arrested during cell division by a solution of colchicine. The sex of an unborn fetus can be determined by observation of interphase cells.2 Observations on karyotypes 3. 3. Rather interestingly.2 Observations Six different characteristics of karyotypes are usually observed and compared: .
indicating tighter packing. Differences in the position of centromeres. 3. This is brought about by translocations. both have six pairs of chromosomes (n=6) yet V. Differences in number and position of satellites. This feature probably reflects different amounts of DNA duplication. 5. . permitting its loss without penalty to the organism (the dislocation hypothesis). faba chromosomes are many times larger. Differences in absolute sizes of chromosomes. shape and banding of the chromosomes. Humans have one pair fewer chromosomes than the great apes. 6. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in degree and distribution of heterochromatic regions. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). 4.1. Heterochromatin stains darker than euchromatin. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. type. as well as other cytogenetic information. 2. A full account of a karyotype may therefore include the number. but the genes have been mostly translocated (added) to other chromosomes. and mainly consists of genetically inactive repetitive DNA sequences.
XY. Normal karyotypes for females contain two X chromosomes and are denoted 46. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Any variation from the standard karyotype may lead to developmental abnormalities. Between members of a population (chromosome polymorphism) 4. Between the sexes 2. Between the germ-line and soma (between gametes and the rest of the body) 3. which are highly variable. There is variation between species in chromosome number. XX. the same cannot be said for their karyotypes. 3. and in .Variation is often found: 1. males have both an X and a Y chromosome denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. Mosaics or otherwise abnormal individuals. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Geographical variation between races 5.
. In some species. Godfrey and Masters conclude: "In our view. In a review. it is quite unclear what the general significance might be. In some cases there is even significant variation within species.. Chromatin diminution (founding father: Theodor Boveri). used in conjunction with other phylogenetic data. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. portions of the chromosomes are cast away in particular cells. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. as in many sciarid flies.1 Changes during development Instead of the usual gene repression.. "We have a very poor understanding of the causes of karyotype evolution. But. despite their construction from the same macromolecules. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed.detailed organization. which were previously inexplicable. In this process.. Chromosome elimination. . found in some copepods and roundworms such as Ascaris suum. This variation provides the basis for a range of studies in evolutionary cytology. Although much is known about karyotypes at the descriptive level. 3. despite many careful investigations. In A. or other kinds of visible adjustment to the karyotype. entire chromosomes are eliminated during development. the general significance of karyotype evolution is obscure. some organisms go in for large-scale elimination of heterochromatin.3. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species.
They kept quiet for two or three years because they thought something was wrong with their tissue culture. The low record is held by the nematode Parascaris univalens. was found to be 46. all telocentric... Xinactivation. In human females some 15% of somatic cells escape inactivation. In marsupials it is always the paternal X which is inactivated. In placental mammals. When they looked at the karyotype of the closely related Indian muntjac. Muntiacus reevesi. 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. the inactivation is random as between the two Xs. all the somatic cell precursors undergo chromatin diminution. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. The existence of supernumerary or B chromosomes .suum. male = 7 chromosomes. where the haploid n = 1.3. which was investigated by Kurt Benirschke and his colleague Doris Wurster.. The diploid number of the Chinese muntjac. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. the high record would be somewhere amongst the ferns. Muntiacus muntjak. "They simply could not believe what they saw. thus the mammalian female is a mosaic in respect of her X chromosomes. they were astonished to find it had female = 6. 3..2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation).
FN. Polyploidy in lower plants (ferns. and aneuploids are another example. occurs mainly in plants. Humans have FN = 82. but in grasses the average is much higher. Haplo-diploidy. about 70%. and in some other groups. though in this case they would not be regarded as normal members of the population.means that chromosome number can vary even within one interbreeding population. and the other haploid. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. It has been of major significance in plant evolution according to Stebbins. Thus.3 Fundamental number The fundamental number. It is a common arrangement in the Hymenoptera. 21 and 22). 3. Polyploidy in animals is much less common. where one sex is diploid. horsetails and psilotales) is also common. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. 3.Endopolyploidy occurs when in adult differentiated . where there are more than two sets of homologous chromosomes in the cells. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.3. Polyploidy. 14. 15. but it has been significant in some groups. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. due to the presence of five acrocentric chromosome pairs (13. FN ≤ 2n.
Abnormalities in chromosome number usually cause a defect in development. The cells do not always contain exact multiples (powers of two). . Down syndrome and Turner syndrome are examples of this. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted).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. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. In many instances. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. 3. it is diverse and complex. 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. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost.tissues the cells have ceased to divide by mitosis. the daughter chromosomes separating from each other inside an intact nuclear membrane. See palaeopolyploidy for the investigation of ancient karyotype duplications. and serves differentiation and morphogenesis in many ways. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man.
and Crocus. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 6. Human chromosome 2 was formed by a merger of ancestral chromosomes.000 km2). some mantids of the genus Ameles. the chromosome number is variable from one individual to another. When this happens.  Closer to home. the great apes have 24x2 chromosomes whereas humans have 23x2. and 7. that the two chromosome morphs are adapted to different habitats. In about 6.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations.Aneuploidy may also occur within a group of closely related species. 3. the European shrew Sorex araneus. 5. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups.500 sq mi (17. reducing the number.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. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. Classic examples in plants are the genus Crepis. where every number from x = 3 to x = 15 is represented by at least one species. 3. living from rainforests to . Well-researched examples are the ladybird beetle Chilocorus stigma. 4. where the gametic (= haploid) numbers form the series x = 3.
but these are much less frequent. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. especially inversions. The polytene banding of the 'picture wing' group. at least into the Cretaceous. Using K-Ar dating. The inversions. make it possible to see which species are closely related. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). Drosophila and Scaptomyza. In a sense.subalpine meadows. Chromosome rearrangements. the present islands date from 0. and skipping of islands. it is more likely to have been a group from the same species. The results are clear. the best-studied group of Hawaiian drosophilids. There are also cases of colonization back to older islands. show a clear "flow" of species from older to newer islands. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. . probably 20 million years ago. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. in the family Drosophilidae. Although it would be possible for a single gravid female to colonise an island. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. gene arrangements are visible in the banding patterns of each chromosome. which can be dated to 30 mya. when plotted in tree form (and independent of all other information).
There are other animals and plants on the Hawaiian archipelago which have undergone similar. late-replicating and AT rich. so it stains centromeres.7 Depiction of karyotypes 3. R-banding is the reverse of G-banding (the R stands for "reverse"). . adaptive radiations. The pattern of bands is very similar to that seen in G-banding. The light regions tend to be euchromatic. human genome.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.7. if less spectacular. early-replicating and GC rich. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). • Q-banding is a fluorescent pattern obtained using quinacrine for staining. • • C-banding: Giemsa binds to constitutive heterochromatin. It yields a series of lightly and darkly stained bands . 3.the dark regions tend to be heterochromatic. • T-banding: visualize telomeres. This method will normally produce 300-400 bands in a normal.
Quinacrine binds to the adeninethymine-rich regions. often Giemsa (G-banding). a dye. and the long arm on the bottom. For example. Giemsa is specific for the phosphate groups of DNA. less frequently Quinacrine. In the "classic" (depicted) karyotype.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. both chromosomes in a pair will have the same banding pattern. denoting the activity of rRNA genes within the NOR. Some karyotypes call the short and long arms p and q. This yields a dark region where the silver is deposited. Each chromosome has a characteristic banding pattern that helps to identify them. In addition. Cri du chat syndrome involves a deletion on the short arm of . the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein.7. respectively. is used to stain bands on the chromosomes. 3.
allowing the visualization of the individually colored chromosomes. The critical region for this syndrome is deletion of 15. 3. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination.2) 3. a combinatorial labeling method is used to generate many different colors.XX.XX.del(5)(p15. Because there are a limited number of spectrally-distinct fluorophores. It is written as 46. .chromosome 5. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. which is written as 46.5p-.2. This method is also known as virtual karyotyping. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.7.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. 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.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.
CHAPTER 4 .
as in the presence of extra or missing chromosomes. inversions. large-scale deletions or duplications. X or 45. as in derivative chromosome. in which three copies of a chromosome are present instead of the usual two. trisomies. Also documented are trisomy 8. • • Patau syndrome is caused by trisomy of chromosome 13. a common chromosomal disease. including . Down syndrome. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18.4. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. are common numerical abnormalities. also known as aneuploidy. XXY is caused by an extra X chromosome. X0). Some disorders arise from loss of just a piece of one chromosome. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. although they generally do not survive to birth. trisomy 9 and trisomy 16. often occur as a result of nondisjunction during meiosis in the formation of a gamete. Structural abnormalities often arise from errors in homologous recombination. or structural. translocations. Klinefelter syndrome. otherwise known as 47.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. is caused by trisomy of chromosome 21. the most common male chromosomal disease. Numerical abnormalities. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells.
1p36 Deletion syndrome. a deletion of the paternal genes. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. . one well-documented example is the Philadelphia chromosome. The name comes from the babies' distinctive cry. example of imprinting disorder. A chromosome anomaly.• Cri du chat (cry of the cat). They can be organized into two basic groups. caused by abnormal formation of the larynx. There are many types of chromosome anomalies. a deletion of the maternal genes. numerical and structural anomalies. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. 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. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. A chromosome anomaly may be detected or confirmed in this manner. example of imprinting disorder. from the loss of part of the short arm of chromosome 1. from a truncated short arm on chromosome 5. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual.
Duplications: A portion of the chromosome is duplicated. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. Tetrasomy. In a Robertsonian translocation. 4. segments from two different chromosomes have been exchanged. 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. and Jacobsen syndrome. There are two main types of translocations. also called the terminal 11q deletion disorder.). an entire chromosome has . also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. etc.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. resulting in extra genetic material. Known disorders in humans include Wolf-Hirschhorn syndrome. • • Translocations: When a portion of one chromosome is transferred to another chromosome.3 Structural abnormalities When the chromosome's structure is altered. In a reciprocal translocation. an X. rather than two). Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome.4. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.
15. 4. and are therefore initially not inherited.attached to another at the Centromere . 4. the anomaly is present in every cell of the body.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. Chromosome anomalies can be inherited from a parent or be "de novo". as well . This can happen with or without loss of genetic material. turned upside down and reattached. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 21 and 22. 14. It includes routine analysis of G-Banded chromosomes. They often lead to an increased tendency to develop certain types of malignancies. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. Some anomalies.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. Therefore. other cytogenetic banding techniques. can happen after conception. • Rings: A portion of a chromosome has broken off and formed a circle or ring. especially the chromosomes.in humans these only occur with chromosomes 13. resulting in Mosaicism (where some cells have the anomaly and some do not). therefore the genetic material is inverted. • Inversions: A portion of the chromosome has broken off. however.
Their behavior in animal (salamander) cells was described by Walther Flemming. Using cells in culture 2. in contrast to their genic contents. He revised his opinion later from 46 to 48.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. von Waldeyer in 1888.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). which swells them and spreads the chromosomes . Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. The name was coined by another German anatomist. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. Pre-treating cells in a hypotonic solution. at first favoring 46. concluding an XX/XO sex determination mechanism. 4. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. 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. and he correctly insisted on man having an XX/XY system. Considering their techniques. the discoverer of mitosis. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. New techniques were needed to definitively solve the problem: 1. in 1882. The next stage took place after the development of genetics in the early 20th century. these results were quite remarkable.
Rather interestingly. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. 4. Arresting mitosis in metaphase by a solution of colchicine 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.6. Using Painter's technique they studied the polytene .3. the great apes have 48 chromosomes. a find which eventually led to her Nobel Prize in 1983. McClintock discovered transposons. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). persimilis from wild populations in California and neighboring states.2 Natural populations of Drosophila In the 1930s. During her cytogenetic work. In 1931.6. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. 4.6 Applications in biology 4. Human chromosome 2 was formed by a merger of ancestral chromosomes. reducing the number. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.
cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. which enabled feeding. but adjust to certain frequencies at which they become stabilised. 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. 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. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. as with most polymorphisms. Using a method invented by L'Heretier and Teissier. Down syndrome is also referred to as trisomy 21. Evidence rapidly accumulated to show that natural selection was responsible. such as Down's syndrome. discoveries were quickly made related to aberrant chromosomes or chromosome number. Dobzhansky bred populations in population cages. 4.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. In 1959. breeding and sampling whilst preventing escape. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. as they would if selectively neutral. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. In some congenital disorders. It was found that the various chromosome types do not fluctuate at random. . This had the benefit of eliminating migration as a possible explanation of the results.
. is used today as a diagnostic for CML. An individual with only one sex chromosome (the X) has Turner syndrome. an additional X chromosome in a male. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). which is required in normal females to compensate for having two copies of the chromosome. Identification of the Philadelphia chromosome by cytogenetics. XYY. has Klinefelter's Syndrome. This abnormal chromosome was dubbed the Philadelphia chromosome . Thirteen years later. with the development of more advanced techniques.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. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. resulting in 47 total chromosomes. and XXXX. In 1960. Not all genes on the X Chromosome are inactivated. Pennsylvania. Many other sex chromosome combinations are compatible with live birth including XXX. in addition to other tests. 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.as both scientists were doing their research in Philadelphia.
Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. 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. and elongation techniques for all culture types that allow for higher resolution banding.8 Beginnings of molecular cytogenetics . Deletion syndromes such as DiGeorge syndrome. Caspersson developed banding techniques which differentially stain chromosomes.FIG Advent of banding techniques In the late 1960s. 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. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. 4.
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. advances were made in molecular cytogenetics. CHAPTER 5 Techniques 5. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. movement was now made in using fluorescent labeled probes.1 Karyotyping . While radioisotope-labeled probes had been hybridized with DNA since 1969. cloned and studied in ever greater detail.In the 1980s.
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.
5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •
bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations
5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,
generally between 200 and 1000 cells are counted and scored. data analysis. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. communications. such as comparative genomic hybridization arrays. test and measurement. control design. and FORTRAN. you can solve technical computing problems faster than with traditional programming languages. and computational biology. C++. including signal and image processing. financial modeling and analysis. . You can use MATLAB in a wide range of applications. data visualization. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. such as C. CGH and Single nucleotide polymorphism-arrays. Using MATLAB. For congenital problems usually 20 metaphase cells are scored. and numerical computation. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development.
MATLAB provides a number of features for documenting and sharing your work. and allocating memory. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. 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. The image processing step is composed of the following operations.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. These effects must be compensated to improve the results of the pairing algorithm. With the MATLAB language. such as declaring variables. You can integrate your MATLAB code with other languages and applications. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. MATLAB eliminates the need for ‘for’ loops. In many cases. and distribute your MATLAB algorithms and applications. As a result. one line of MATLAB code can often replace several lines of C or C++ code. 6. . It enables fast development and execution. specifying data types.
4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. the spatially scaled images are histogram equalized. 2) Geometrical compensation—The geometric compensation. or at least attenuated.2 Concepts used in this phase 1) Image conversion 2) Denoising .1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. To compensate for this inhomogeneity. Therefore. geometrical and dimensional differences must be removed. 6. This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding). performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. a dimensional scaling is performed before the pattern features is extracted to make all the chromosome with the same size and aspect ratio by interpolating the original images. To compare chromosomes from a band pattern point of view.
6.I). you must first convert it to true color format. MATLAB filters the intensity values in the image. and the results might not be meaningful. In addition to these image type conversion functions. For example.2. You can perform certain conversions just using MATLAB syntax. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. MATLAB simply applies the filter to the indices in the indexed image matrix. listed in the following table. . if you want to filter a color image that is stored as an indexed image. For example. The resulting true color image has identical matrices for the red. green. For example. When you apply the filter to the true color image. there are other functions that return a different image type as part of the operation they perform.I. as is appropriate.I. If you attempt to filter the indexed image.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. so the image displays as shades of gray.3) Edge detection 4) Two dimensional convolutions. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. and blue planes. RGB = cat (3.
and hence the type of noise on the image.5 Edge detection Edges contain some of the most useful information in an image.B) computes the two-dimensional convolution of matrices A and B.2. caused by external disturbance. or through networked cable. via satellite or wireless transmission. There is a large number of edge finding algorithms in existence.2.3 Two dimensional convolutions C = conv2(A.6.parameters. If an image is being sent electronically from one place to another. Usually we know what type of errors to expect. we may expect errors to occur in the image signal. If one of these matrices describes a two-dimensional finite impulse response . ) Where the parameters available depend on the method used 6. to isolate particular objects from their background. . . These errors will appear on the image output in different ways depending on the type of disturbance in the signal. 6. We may use edges to measure the size of objects in an image. Cleaning an image corrupted by noise is thus an important area of image restoration. hence we can choose the most appropriate method for reducing the effects. to recognize or classify objects. The general Matlab command for finding edges is edge(image.4 Denoising We may define noise to be any degradation in the image signal. and we shall look at some of the more straightforward of them.'method'.
the other matrix is filtered in two dimensions. imedfilt2(im1. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.A). 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.(FIR) filter.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. this case is the same as C = conv2(hcol*hrow. edge(im1.hrow.'sobel').nb]. nb]+1)/2). C = conv2(.na+nb-1]. That is. minus one.'shape') subsection of the two-dimensional convolution. if the size of then the size of C is [ma+mb-1..7).[3 3])..na] and the size of B is [mb. . If hcol is a column vector and hrow is a row vector.bmp').0. rgb2gray im2bw(im. The size of matrices. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns..
[r.imy). bwlabel(B. rc = [r c].8).c] = find(L==22).double(msk)). y1=rc(i. L_number=zeros(mx. n1(x1.[imx. nzeros(imx. mx=max(max(L)).1).2). for i=1:sx x1=rc(i. . flag=0.j)==L_number(k) flag=1.y1)=255.imy]=size(BW).j)~=0 for k=1:mx if L(i.n]=size(L). for i=1:m for j=1:n if L(i. MODULE 2 clc [m. [sx sy]=size(rc). Index=1.1). Msk conv2(double(BW).
8. y1=rc(i.27.39.). Index=Index+1.60.55. n1=zeros(imx.19.41. for i=1:sx x1=rc(i. rc = [r c].45. 36.11. end end L_number.4.y1)=255.29.48.20.126.96.36.199.22.2).26. end %h=figure. end flag=0.43.31.j).c] = find(L==L_number((Test_number(x)))).56.24. end.188.8.131.52.21.7.33. Test_number=[184.108.40.206.30.50.imy).220.127.116.11.1).end end if flag~=1 L_number(Index)=L(i. for x=1:46 [r.9. n1(x1.66].imshow(n1.35. [sx sy]=size(rc).38. .15.
8).5*graythresh(skel)).'. skel=im2double(f). skel=im2bw(skel. Arm_length_sum=0. . BW=im2bw(f). for i=1:46 f=imread(strcat(num2str(i). s1=bwmorph(s.'spur'.'skel'. [m n]=size(BW1).'canny'). Arm_length=zeros(46.end Circumference=zeros(46.1). end end end Circumference(i)=Circumference_sum.y)==1 Circumference_sum=Circumference_sum+1. f=imcomplement(f). for x=1:m for y=1:n if BW1(x.1. BW=double(BW).1). s=bwmorph(skel. BW1=edge(BW. Circumference_sum=0.Inf). Area=zeros(46.bmp')).1).
end end end Area(i)=Area_sum. for x=1:m for y=1:n if s1(x. Area_sum=0. end Circumference. Arm_length. [m n]=size(BW).[m n]=size(s1). BW=im2bw(f). .y)==1 Area_sum=Area_sum+1.y)==1 Arm_length_sum=Arm_length_sum+1. for x=1:m for y=1:n if BW(x. end end end Arm_length(i)=Arm_length_sum.
Pair=zeros(46.1)=i. .2)=i. Pair(i.2). for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)). Pair(i.2)=j.Area.2)=i+1. end end Pair.2)==46 Pair(46. end end end for i=1:45 if Pair(i. Pair(i.1)=i. Pair(46.1)=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)). Pair(i.
'.2. for i=1:46 for j=1:46 if Pair(i.figure_flag).bmp')).'.bmp')). figure_flag=figure_flag+1. end end if flag~=1 if figure_flag~=47 subplot(23.delete=zeros(46.1). end f2=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1. figure_flag=1. end f1=imread(strcat(num2str(Pair(i. flag=0. imshow(f2).figure_flag).2)). end flag=0. if figure_flag~=47 subplot(23.1)). imshow(f1).2.1)==delete(j) flag=1.2). delete(figure_flag)=Pair(i. .
to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. Copenhagen. The main goal of this paper is to provide useful contributions toward the design of a fully automatic chromosomes pairing algorithm of bone marrow cells to be used in the diagnosis of leukemia. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. 2) feature extraction from the processed images . dimensions and banding profiles. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. in the scope of karyotyping process used in cytogentic analysis. plus a new one. and Philadelphia. 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. such as. The proposed algorithm is based on the traditional features extracted from the karyogram. based on the MI.end CONCLUTION In this paper.
and band pattern. 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 . This normalization is needed to make it possible the band pattern comparison between chromosomes.characterizing the size. In the image processing step. Here.10% mean classification rate. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. are processed in order to compensate for geometrical and intensity distortions. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.working within an 8-D feature space. shape and band pattern. The training process consists in the estimation of each vector of coefficient . the romosome images. from the chromosomes in the training set.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. The features extracted from the processed images discriminate each pair with respect to their size. Tests using 19 karyograms based on bone marrow cells. achieves a 70. 4) pairing. shape. extracted from the unordered karyogram. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. 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). 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. 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. and finally. and to normalize their dimensions.
REFERENCES . presenting a uniform level of condensation. such as Edinburgh. In addition. amean classification rate larger than 93% was obtained in all experiments. called LK1 .g. This dataset was made publicly available . or Philadelphia. whose images are of significantly higher quality. The results presented in this paper are promising..performance of the classifier. centromere position. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. Using 27 karyograms andworking with a limited number of classes (≤ 8). Copenhagen. despite the low quality of this type of chromosomes. a 76. In fact. and from which it is possible to extract additional features.10% classification ratewas obtained. a new chromosome dataset with 9200 chromosomes from bone marrow cells. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. Executing the algorithm on a higher quality dataset. e.
1974. [in Russian] 6. Bull. ^ Stebbins G. ^ a b White M. Plant Breed. Columbia University Press NY. ^ von Winiwarter H. 7. State Publication Office of the Ukraine.D. Res. Cambridge University Press.J.S. ^ Levitsky G. and Mulligan P.D. 1912. 1924. ^ Kottler M. 1950. 28 2. 129. 23. 1973. ^ a b c White M. Evolution of genetic systems.. Anat.L. A dictionary of genetics.P Oxford & NY. The material basis of heredity. 19-174. 9.A.D. 1931. 1958.1. The chromosomes. 2nd ed. From 48 to 46: cytological technique. 3rd ed. Animal cytology and evolution. 27. Applied Bot. 10. ^ Painter T. 1922. Stansfield W. 48. Oxford U. p. p242 5. . Hist. 93.C.D.A. ^ Darlington C. Kiev. Chapter XII: The Karyotype. ^ Levitsky G. Edinburgh. Etudes sur la spermatogenese humaine. 2006.K. Genet. ^ King R. 6th ed. Cambridge University Press. 465-502. 7th ed. Chapman & Hall. 147–49. Bull. The spermatogenesis of man. Oliver & Boyd. 1973. revised and enlarged. Med. 4. 1939. preconception and the counting of the human chromosomes. Arch. 8.J. 11. ^ Concise Oxford Dictionary London. Variation and evolution in plants. 3. biologie 27. The morphology of chromosomes.
Zoology 37. and Masters J. ed. Chromosome stains. 21.pubmedcentral.C 16. .H & Levan A. ^ Tjio J. Raven Press.S. Springer-Verlag.C. 14. Oxford. Bioessays18: 133–138. Kinetophore reproduction theory may explain rapid chromosome evolution.C. ^ Maynard Smith J.nih. ^ Painter T. Bernard V.M.gov/articlerender. London. ^ Goday C. 1923. 1-6. & Tobler H. 1998. New York. 1991.J. 2000. ^ Stebbins G.R. Studies in mammalian spermatogenesis II. 13. p85-6 18. Human and mammalian cytogenetics: a historical perspective. 1996. 15.^ a b Gustashaw K. p218-9 20. NY.B. Arnold. and Esteban M. Barch. Bioessays23: 242–250. Chromosome elimination in sciarid flies. Chromatin diminution in nematodes. 1956. The Association of Cytogenetic Technologists. Eosin Y and Azure-A. 2nd ed. Chromosomal evolution in higher plants.R. 17. The spermatogenesis of humans. M. J. Exp. The chromosome number of man. Evolutionary genetics.http://www.12. ^ Müller F. ^ Godfrey L.^ a b Hsu T. PNAS 97. 2001. Hereditas 42. 1979. In The ACT Cytogenetics Laboratory Manual 2nd ed.L.fcgi?artid=34032 19. 1971. 9821– 9823. 291-336. ^ A preparation which includes the dyes Methylene Blue.
Developmental biology. 8th ed. ^ Khandelwal S.. Y. ^ Wurster D. Retrieved 2011-03-16. 27.doi:10.D. Chapter 9 24.F. Oxford U. 1364-1366. Retrieved 2008-03-18. "L'evolution de la formule chromosomiale chez les vertébrés". ^ King R. ^ Gilbert S.. J. 1970. Experientia (Basel) 1 (2): 50– 56. 26. Exp.1007/s10228-004-0257-z. 25. Muntiacus muntjak: a deer with a low diploid number. Ichthyological Research 52 (1): 97. Chromosome evolution in the genus Ophioglossum L. 2006.S.C. Indian Muntjac.A.. doi:10. J.. Botanical Journal of the Linnean Society 102: 205–217. 2006. ^ Matthey. 28. and Mulligan P. Stansfield W. Park.22.H.R. 2001. Stamford CT.K. Zool. . C. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). ^ Wyngaard G.K.A.. (2005). 1990. Nam.K. D. R.H. A dictionary of genetics. & Gregory T. 7th ed. Chapman. Science 168. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods.P Oxford & NY.1007/BF02153623. F. ^ Kim. Noh. 291: 310–16. and Benirschke K. (1945-05-15). Sinauer Associates. 23.
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