This action might not be possible to undo. Are you sure you want to continue?
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
and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. sometimes accompanied by one or more smaller. This allows the very long DNA molecules to fit into the cell nucleus. If these structures are manipulated incorrectly. cells may contain more than one type of chromosome. Also. However. These small circular genomes . circular DNA molecules called plasmids. In prokaryotes and viruses. Chromosomes may exist as either duplicated or unduplicated. The structure of chromosomes and chromatin varies through the cell cycle. In eukaryotes. the cell may undergo mitotic catastrophe and die. Chromosomes are the essential unit for cellular division and must be replicated. through processes known as chromosomal instability and translocation. divided. or it may unexpectedly evadeapoptosis leading to the progression of cancer. In practice "chromosome" is a rather loosely defined term. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. Unduplicated chromosomes are single linear strands. which is tightly coiled in on itself. a large body of work uses the term chromosome regardless of chromatin content. the term genophore is more appropriate when no chromatin is present.defined nuclei) have smaller circular chromosomes. DNA is usually arranged as a circle. In prokaryotes. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). for example. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. Chromosomal recombination plays a vital role in genetic diversity. although there are many exceptions to this rule.
rather than 2. The individual would have Down Syndrome and his/her karyotype would be written 47.XY or 47. 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). Chromosomal Mutations are substantial changes in chromosome structure that are large enough to be visible by karyotyping (see lab manual) and thus typically affect more than one gene. XX (female) or 46 XY (male). . a extra copy of human chromosome 21). An example of aneuploidy is trisomy 21. copies of chromosome 21. the individual carrying the mutation is said to be aneuploid.are also found in mitochondria and chloroplasts. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. 1. Such individuals are called euploid and have the wild-type chromosome complement for the species.g.+21. If the mutation involves only one or a few chromosomes in the genome (e.+21. reflecting their bacterial origins.XX.3 MUTATIONS IN CHROMOSOME NUMBER Normally. Euploid human karyotypes are 46. in which an individual has 3.
. longer-lasting attachment in this region. This compact form makes the individual chromosomes visible. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. a pair of sister chromatids attached to each other at the centromere. which enables these giant DNA structures to be contained within a cell nucleus. A special DNA base sequence in the region of the kinetochores provides. and they form the classic four arm structure. The microtubules then pull the chromatids apart toward the centrosomes.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. This is the only natural context in which individual chromosomes are visible with an optical microscope. During mitosis. q-g "grande"). They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. Once the cells have divided. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. small) and the longer arms are called q arms (q follows p in the Latin alphabet. chromosomes are structurally highly condensed. The shorter arms are called p arms (from the French petit. In spite of their appearance. so that each daughter cell inherits one set of chromatids.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). the chromatin strands become more and more condensed.Fig 1. the chromatids are uncoiled and DNA can again be transcribed. along with special proteins. one of which is present on each sister chromatid. 1.
5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. It is generally followed immediately by cytokinesis.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. bone marrow constitutes 4% of the total body mass of humans. in adults weighing 65 kg (143 lbs). The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. bone marrow in large bones produces new blood cells. in two separate nuclei.  Bone marrow is also a key component of the lymphatic system. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. which use the bone marrow vasculature as a conduit to the body's systemic circulation. bone marrow accounts for approximately 2. In humans. On average. producing the lymphocytes that support the body's immune system CHAPTER 2 2. cytoplasm.6 kg (5.1.7 lbs). which divides the nuclei. .
"mitosis" is often used interchangeably with "mitotic phase". cytokinesis and mitosis may occur independently. divide by a process called binary fission. This occurs most notably among the fungi and slime moulds. anaphase and telophase. However. where the nuclear envelope breaks down before the chromosomes separate.genetically identical to each other and to their parent cell. For example. The cell then divides in cytokinesis. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. The process of mitosis is fast and highly complex. These stages are interphase. but is found in various different groups. for instance during certain stages of fruit fly embryonic development. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. This accounts for approximately 10% of the cell cycle. to produce two identical daughter cells which are still diploid cells. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. forming single cells with multiple nuclei. Even in animals. Because cytokinesis usually occurs in conjunction with mitosis. which lack a nucleus. where chromosomes divide within an intact cell nucleus. there are many cells where mitosis and cytokinesis occur separately. . metaphase. animals undergo an "open" mitosis. prophase. prometaphase. Mitosis occurs only in eukaryotic cells and the process varies in different species. Prokaryotic cells.
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 parent cell must make a copy of each chromosome before mitosis.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. and together the two are called sister chromatids. This occurs during the S phase of interphase. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. . 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. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs.
the cell begins cytokinesis. Prokaryotic cells undergo a process similar to mitosis called binary fission. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. The chromosomes align themselves in a line spanning the cell. separating the two developing nuclei. the daughter cells will construct a new dividing cell wall between each other. As the cell elongates. each with a replica of the original genome.In most eukaryotes. However. so they are renamed to sister chromosomes. pulling apart the sister chromatids of each chromosome. each sister chromatid is now considered a chromosome. corresponding sister chromosomes are pulled toward opposite ends. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. the process of binary fission is very much different from the process of mitosis. In animal cells. Eventually. A new nuclear envelope forms around the separated sister chromosomes. As a matter of convention. As mitosis completes. . the parent cell will be split in half. giving rise to two daughter cells. In plant 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). because of the non-involvement of nuclear dynamics and lack of linear chromosomes.
S (synthesis). where the cell prepares itself for cell division. It alternates with the much longer interphase.1 Preprophase In plant cells only.2. the cell grows by producing proteins and cytoplasmic organelles. Interphase is divided into three phases: G1 (first gap). a cell grows (G1). Thus. However. During all three phases. and G2 (second gap). prophase is preceded by a pre-prophase stage.2. 2. grows more and prepares for mitosis (G 2). In highly vacuolated plant cells. This is achieved through the formation of a phragmosome. All these phases in the interphase are highly regulated. mainly via proteins. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . and finally it divides (M) before restarting the cycle. 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. chromosomes are replicated only during the S phase. continues to grow as it duplicates its chromosomes (S). the nucleus has to migrate into the center of the cell before mitosis can begin.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.
Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. instead. The chromosomes have chromatin has condensed. In addition to phragmosome formation. the pinched area is known as the cleavage furrow. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase.division. This band marks the position where the cell will eventually divide. These microtubules can attach to kinetochores or they can interact with opposing microtubules. 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. degraded. and microtubules have invaded the nuclear Prophase space. . after the nuclear membrane breaks down. Cytokinesis has already begun. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. aligned at the metaphase plate. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. The cells of higher plants (such as the flowering plants) lack centrioles.
A cell inherits a single centrosome at cell division. giving a pair of centrosomes. which is replicated by the cell with the help of the nucleus before a new mitosis begins. The centrosome is the coordinating center for the cell's microtubules. Close to the nucleus are structures called centrosomes. 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. which are made of a pair of centrioles found in most eukaryotic animal cells. Chromosomes are typically visible at high magnification through a light microscope.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. bound together at the centromere by the cohesin protein complex. chromatin condenses together into a highly ordered structure called a chromosome. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. Although centrioles help organize microtubule assembly. At the onset of prophase. the replicated chromosomes have two sister chromatids. they are not essential for the .
it is known that it contains some form of molecular motor. undergo a variation called closed mitosis where the spindle forms inside the nucleus. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. such as algae or trichomonads. provides the pulling force necessary to later separate the chromosome's two chromatids. on an average 20 ). A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Although the kinetochore structure and function are not fully understood. Each chromosome forms two kinetochores at the centromere. Prometaphase is sometimes considered part of prophase. since they are absent from plants. one attached at each chromatid. the motor activates. Fungi and some protists. using energy from ATP to "crawl" up the tube toward the originating centrosome.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. This motor activity. and centrosomes are not always used in mitosis. When the spindle grows to sufficient length.2. When a microtubule connects with the kinetochore.formation of the spindle. This is called open mitosis. and it occurs in most multicellular organisms. kinetochore microtubules begin searching for kinetochores to attach to. or its microtubules are able to penetrate an intact nuclear envelope. coupled with polymerisation and depolymerisation of microtubules. . 2. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle.
Metaphase comes from the Greek meaning "after. the kinetochore would be the "hook" that catches a sister chromatid or "fish".3 Metaphase A cell in late metaphase. analogous to a tug-of-war between people of equal strength. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. only roughly lining up along the midline. convene along the metaphase plate or equatorial plane. In certain types of cells. As a result. an imaginary line that is equidistant from the two centrosome poles. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line".In the fishing pole analogy. . All chromosomes (blue) but one have arrived at the metaphase plate. the chromosomes come under longitudinal tension from the two ends of the cell. 2." Microtubules find and attach to kinetochores in prometaphase. The centromeres of the chromosomes. in some sense. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell.
The signal creates the mitotic spindle checkpoint.” “back. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. . it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. 2. These two stages are sometimes called early and late anaphase. Next. At the end of anaphase.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). which have now become distinct sister chromosomes. These sister chromatids. Two events then occur: first. the nonkinetochore microtubules elongate. the cell has succeeded in separating identical copies of the genetic material into two distinct populations.” or “re-”). The force that causes the centrosomes to move towards the ends of the cell is still unknown. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. allowing them to separate.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate.” “against. the proteins that bind sister chromatids together are cleaved. 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 cell proceeds to anaphase (from the Greek meaning “up.
Corresponding sister chromosomes attach at opposite ends of the cell. In animal cells.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. pinching off the separated nuclei.2. 2. the nonkinetochore microtubules continue to lengthen. necessary for completing cell division. but rather a separate process. however. At telophase. using fragments of the parent cell's nuclear membrane. It "cleans up" the after effects of mitosis. Cytokinesis is technically not even a phase of mitosis. In both animal and plant cells. Both sets of chromosomes. cytokinesis is a separate process that begins at the same time as telophase. cell . A new nuclear envelope. elongating the cell even more. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. unfold back into chromatin. forms around each set of separated sister chromosomes.5 Cytokinesis Cilliate undergoing cytokinesis. Mitosis is complete. but cell division is not yet complete. now surrounded by new nuclei.
Similarly. Following are the occasions in the lives of organism where mitosis happens: 2. The end of cytokinesis marks the end of the M-phase.5.2 Development and growth The number of cells within an organism increases by mitosis. e. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.1Significance Mitosis is important for the maintenance of the chromosomal set. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. This is the basis of the development of a multicellular body from a single cell i. Each daughter cell has a complete copy of the genome of its parent cell. whereas some green algae use a phycoplast microtubule array during cytokinesis. New cells are formed by mitosis and so are exact copies of the cells being replaced.g. skin and digestive tract. 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. zygote and also the basis of the growth of a multicellular body. 2.e.. 2.5. separating the two nuclei. which move along microtubules to the middle of the cell.5.4 Regeneration .3 Cell replacement In some parts of body.division is also driven by vesicles derived from the Golgi apparatus. The phragmoplast is a microtubule structure typical for higher plants. cells are constantly sloughed off and replaced by new ones.
Mitosis continues in the cells of bud and it grows into a new individual. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. a condition known as trisomy. In non-disjunction. they fail to complete cell division and retain both nuclei in one cell. a chromosome may fail to separate during anaphase.Some organisms can regenerate their parts of bodies. and the latter cell having only one chromosome (the homologous chromosome). a condition known as monosomy. . a condition often associated with cancer. especially during early cellular divisions in the zygote. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). 2. The production of new cells is achieved by mitosis.5.7 Consequences of errors Although errors in mitosis are rare. The cells at the surface of hydra undergo mitosis and form a mass called bud. the process may go wrong. One daughter cell will receive both sister chromosomes and the other will receive none.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. The same division happens during asexual reproduction or vegetative propagation in plants. For example. For example. sea star regenerates its lost arm through mitosis. Occasionally when cells experience nondisjunction. 2. These cells are considered aneuploid. resulting in binucleated cells.5. the hydra reproduces asexually by budding.
. As long as these tumours remain in their original location they are called benign tumours. Benign tumours are not harmful as soon as they are not moving. chromosomes may become damaged. causing deletion. All cells have genes that control the timing and number of mitosis.Mitosis is a demanding process for the cell. It may reattach to the original chromosome. It results in abnormal cell growth. its organelles disintegrate and reform in a matter of hours. non-homologous chromosome. An arm of the chromosome may be broken and the fragment lost. Occasionally. The fragment may incorrectly reattach to another. Such tumours can send cancer cells to other parts in body where new tumours may form. causing inversion. which goes through dramatic changes in ultrastructure. sometimes mutuations occur in such genes and cells continue to divide. but in reverse orientation. Now what happens is that cell abnormally continue to divide at a single place. Or. causing translocation. This phenomenon is called metastasis or spreading of disease. It results in the synthesis of execessive tissue growths. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. it results in the formation of Tumors. and chromosomes are jostled constantly by probing microtubules. 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 may be treated erroneously as a separate chromosome. causing chromosomal duplication. As soon as they start to move and invade other cells there are said to be malignant tumours. Errors in the control of mitosis may cause cancer.
This process may also be referred to as endoreduplication and the cells as endoploid. only roughly lining up along the middleline. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. from the ancient Greek(between) and (stage). resulting in cells with many copies of the same chromosome occupying a single nucleus. Metaphase accounts for approximately 4% of the cell cycle's duration. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Early events of metaphase can .7 Metaphase Metaphase. Preceded by events in prometaphase and followed by anaphase.2. carrying genetic information. align in the middle of the cell before being separated into each of the two daughter cells. analogous to a tug of war between equally strong people.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). In certain types of cells. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. An example of a cell that goes through endomitosis is the megakaryocyte. 2. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. an imaginary line that is equidistant from the two centrosome poles.
cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. does the cell enter anaphase. Chromosomes are condensed(Thickened) and highly coiled in metaphase. securin. Such a signal creates the mitotic spindle checkpoint. Staining of the slides. which makes them most suitable for visual analysis. produces a pattern of in total up to several hundred bands. This would be accomplished by regulation of the anaphase-promoting complex. One of the cell cycle checkpoints occurs during prometaphase and metaphase. and separase. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. Only after all chromosomes have become aligned at the metaphase plate. often with Giemsa (G banding) or Quinacrine. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. when every kinetochore is properly attached to a bundle of microtubules. Normal metaphase spreads are used in . 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). Metaphase chromosomes make the classical picture of chromosomes (karyotype). as chromosomes with connected kinetochores will start the events of metaphase individually before other chromosomes with unconnected kinetochores that are still lingering in the events of prometaphase.coincide with the later events of prometaphase. 2.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. For classical cytogenetic analyses.
. losses of chromosomal segments or translocations.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. 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. which may lead to chimeric oncogenes. such as bcr-abl in chronic myelogenous leukemia.
in humans 2n = 46. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). Thus. autosomal chromosomes are present in two copies. There may. such as.  The preparation and study of karyotypes is part of cytogenetics. 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. or an individual organism. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. taxonomic relationships. Karyotypes can be used for many purposes. and what they look like under a light microscope. Attention is paid to their length. any differences between the sex chromosomes. to study chromosomal aberrations. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. So. and the results may be used in evolutionary biology and medicine. Karyotypes describe the number of chromosomes. the position of the centromeres. or may not. The study of karyotypes is important for cell biology and genetics. cellular function. Karyogram of human male using Giemsa staining. . The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. and to gather information about past evolutionary events.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. and any other physical characteristics. banding pattern. ordered by size and position of centromere for chromosomes of the same size. in normal diploid organisms. be sex chromosomes.
Using cells in culture 2. which swells them and spreads the chromosomes . in contrast to their genic contents. The name was coined by another German anatomist. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. von Waldeyer in 1888. the discoverer of mitosis. He revised his opinion later from 46 to 48. and he correctly insisted on humans having an XX/XY system. Considering their techniques. Their behavior in animal (salamander) cells was described by Walther Flemming. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. concluding an XX/XO sex determination mechanism. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. The next stage took place after the development of genetics in the early 20th century. The subsequent history of the concept can be followed in the works of Darlington and White. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. New techniques were needed to definitively solve the problem: 1. at first favoring 46. Pretreating cells in a hypotonic solution. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. in 1882.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842.3. these results were quite remarkable.
a suitable dye. The sex of an unborn fetus can be determined by observation of interphase cells. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.  Sometimes observations may be made on non-dividing (interphase) cells. Rather interestingly. Arresting mitosis in metaphase by a solution of colchicines 4. For humans. such as Giemsa. Human chromosome 2 was formed by a merger of ancestral chromosomes. 3.2 Observations on karyotypes 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: .2. is applied after cells have been arrested during cell division by a solution of colchicine.1 Staining The study of karyotypes is made possible by staining. reducing the number. 3. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. Usually. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.2. the great apes have 48 chromosomes.
faba chromosomes are many times larger. 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). A full account of a karyotype may therefore include the number. indicating tighter packing. Differences in the position of centromeres. 6.1. . and mainly consists of genetically inactive repetitive DNA sequences. 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. Humans have one pair fewer chromosomes than the great apes. but the genes have been mostly translocated (added) to other chromosomes. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. shape and banding of the chromosomes. type. both have six pairs of chromosomes (n=6) yet V. Heterochromatin stains darker than euchromatin. This is brought about by translocations. 5. This feature probably reflects different amounts of DNA duplication. 3. 2. Differences in absolute sizes of chromosomes. as well as other cytogenetic information. Differences in number and position of satellites. permitting its loss without penalty to the organism (the dislocation hypothesis). 4.
The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. and in . which are highly variable. XX. Normal karyotypes for females contain two X chromosomes and are denoted 46. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Mosaics or otherwise abnormal individuals.3 The human karyotype Most (but not all) species have a standard karyotype. XY. Between the sexes 2. Between the germ-line and soma (between gametes and the rest of the body) 3. 3. Geographical variation between races 5. the same cannot be said for their karyotypes. males have both an X and a Y chromosome denoted 46. Any variation from the standard karyotype may lead to developmental abnormalities.Variation is often found: 1. There is variation between species in chromosome number. Between members of a population (chromosome polymorphism) 4.
found in some copepods and roundworms such as Ascaris suum. In a review. Although much is known about karyotypes at the descriptive level. it is quite unclear what the general significance might be.. Chromosome elimination. despite their construction from the same macromolecules. entire chromosomes are eliminated during development. 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.detailed organization.3. In A. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost.1 Changes during development Instead of the usual gene repression. . portions of the chromosomes are cast away in particular cells.. 3.. Chromatin diminution (founding father: Theodor Boveri). which were previously inexplicable. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. or other kinds of visible adjustment to the karyotype. as in many sciarid flies. Godfrey and Masters conclude: "In our view. used in conjunction with other phylogenetic data. In some species. In this process. despite many careful investigations. But. This variation provides the basis for a range of studies in evolutionary cytology. some organisms go in for large-scale elimination of heterochromatin. 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.. the general significance of karyotype evolution is obscure.
suum. In human females some 15% of somatic cells escape inactivation. all telocentric. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. they were astonished to find it had female = 6. In placental mammals. the inactivation is random as between the two Xs. which was investigated by Kurt Benirschke and his colleague Doris Wurster. The diploid number of the Chinese muntjac. was found to be 46. In marsupials it is always the paternal X which is inactivated. the high record would be somewhere amongst the ferns. The low record is held by the nematode Parascaris univalens. 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.. thus the mammalian female is a mosaic in respect of her X chromosomes. male = 7 chromosomes. 3. When they looked at the karyotype of the closely related Indian muntjac. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). where the haploid n = 1.. all the somatic cell precursors undergo chromatin diminution.. Xinactivation. Muntiacus muntjak. "They simply could not believe what they saw.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. Muntiacus reevesi. They kept quiet for two or three years because they thought something was wrong with their tissue culture. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes.3.. The existence of supernumerary or B chromosomes .
about 70%. 3. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. Polyploidy in lower plants (ferns. and the other haploid. due to the presence of five acrocentric chromosome pairs (13. It has been of major significance in plant evolution according to Stebbins. 21 and 22).Endopolyploidy occurs when in adult differentiated . and in some other groups. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Humans have FN = 82. 14. but it has been significant in some groups. It is a common arrangement in the Hymenoptera. Haplo-diploidy. Polyploidy in animals is much less common. where there are more than two sets of homologous chromosomes in the cells. where one sex is diploid. Polyploidy. Thus. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. horsetails and psilotales) is also common. and aneuploids are another example. 15. though in this case they would not be regarded as normal members of the population. occurs mainly in plants. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. 3.3 Fundamental number The fundamental number.3. FN ≤ 2n. but in grasses the average is much higher.means that chromosome number can vary even within one interbreeding population. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. FN.
5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. but the nuclei contain more than the original somatic number of chromosomes. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. Abnormalities in chromosome number usually cause a defect in development.tissues the cells have ceased to divide by mitosis. Down syndrome and Turner syndrome are examples of this. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). In many instances. and serves differentiation and morphogenesis in many ways. 3. See palaeopolyploidy for the investigation of ancient karyotype duplications. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. . In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. it is diverse and complex. The cells do not always contain exact multiples (powers of two). the daughter chromosomes separating from each other inside an intact nuclear membrane.
the great apes have 24x2 chromosomes whereas humans have 23x2. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 3. 5.Aneuploidy may also occur within a group of closely related species. the chromosome number is variable from one individual to another. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. where every number from x = 3 to x = 15 is represented by at least one species. 6. and Crocus. Classic examples in plants are the genus Crepis.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. reducing the number.000 km2). When this happens. and 7. the European shrew Sorex araneus. 4. In about 6. that the two chromosome morphs are adapted to different habitats. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. where the gametic (= haploid) numbers form the series x = 3. 3. Well-researched examples are the ladybird beetle Chilocorus stigma.6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson.500 sq mi (17. some mantids of the genus Ameles. Human chromosome 2 was formed by a merger of ancestral chromosomes. living from rainforests to .  Closer to home.
The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. but these are much less frequent. the best-studied group of Hawaiian drosophilids. The polytene banding of the 'picture wing' group. probably 20 million years ago. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll.subalpine meadows. especially inversions. it is more likely to have been a group from the same species. Drosophila and Scaptomyza. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. when plotted in tree form (and independent of all other information). . enabled Carson to work out the evolutionary tree long before genome analysis was practicable. 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. and skipping of islands. Chromosome rearrangements. There are also cases of colonization back to older islands. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. In a sense. The inversions. The results are clear. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. Although it would be possible for a single gravid female to colonise an island. gene arrangements are visible in the banding patterns of each chromosome. show a clear "flow" of species from older to newer islands.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). Using K-Ar dating. at least into the Cretaceous. which can be dated to 30 mya. in the family Drosophilidae. the present islands date from 0.
• T-banding: visualize telomeres.7 Depiction of karyotypes 3. 3. 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. early-replicating and GC rich. It yields a series of lightly and darkly stained bands .the dark regions tend to be heterochromatic. human genome.7. This method will normally produce 300-400 bands in a normal. The light regions tend to be euchromatic. . adaptive radiations.There are other animals and plants on the Hawaiian archipelago which have undergone similar. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. • • C-banding: Giemsa binds to constitutive heterochromatin. The pattern of bands is very similar to that seen in G-banding. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). R-banding is the reverse of G-banding (the R stands for "reverse"). so it stains centromeres. late-replicating and AT rich.
7. Giemsa is specific for the phosphate groups of DNA. 3. and the long arm on the bottom. both chromosomes in a pair will have the same banding pattern. This yields a dark region where the silver is deposited. Quinacrine binds to the adeninethymine-rich regions. less frequently Quinacrine. Cri du chat syndrome involves a deletion on the short arm of . often Giemsa (G-banding). Karyotypes are arranged with the short arm of the chromosome on top.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. For example. Each chromosome has a characteristic banding pattern that helps to identify them. respectively. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. denoting the activity of rRNA genes within the NOR.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. a dye. is used to stain bands on the chromosomes. In the "classic" (depicted) karyotype. In addition. Some karyotypes call the short and long arms p and q.
2) 3. which is written as 46. 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.5p-. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.XX. This method is also known as virtual karyotyping.chromosome 5.del(5)(p15.2. Image processing software then assigns a pseudo color to each spectrally different combination. 3. The critical region for this syndrome is deletion of 15. Because there are a limited number of spectrally-distinct fluorophores. It is written as 46.7. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX. . a combinatorial labeling method is used to generate many different colors.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. allowing the visualization of the individually colored chromosomes.
CHAPTER 4 .
Numerical abnormalities. Klinefelter syndrome. including . Also documented are trisomy 8. • • Patau syndrome is caused by trisomy of chromosome 13. as in derivative chromosome. X or 45. in which three copies of a chromosome are present instead of the usual two. as in the presence of extra or missing chromosomes. 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. trisomies. the most common male chromosomal disease. large-scale deletions or duplications. or structural. Down syndrome. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. a common chromosomal disease. often occur as a result of nondisjunction during meiosis in the formation of a gamete. translocations.4. is caused by trisomy of chromosome 21. although they generally do not survive to birth. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. X0). also known as aneuploidy. inversions. XXY is caused by an extra X chromosome. are common numerical abnormalities. Structural abnormalities often arise from errors in homologous recombination.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. trisomy 9 and trisomy 16. otherwise known as 47.
. 1p36 Deletion syndrome. example of imprinting disorder. • • Angelman 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. one well-documented example is the Philadelphia chromosome. They can be organized into two basic groups. There are many types of chromosome anomalies. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. A Karyotype refers to a full set of chromosomes from an individual which can be compared to a "normal" Karyotype for the species via genetic testing. a deletion of the paternal genes. from the loss of part of the short arm of chromosome 1. A chromosome anomaly may be detected or confirmed in this manner. The name comes from the babies' distinctive cry. A chromosome anomaly. 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. example of imprinting disorder. a deletion of the maternal genes. from a truncated short arm on chromosome 5.• Cri du chat (cry of the cat). numerical and structural anomalies. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. caused by abnormal formation of the larynx.
segments from two different chromosomes have been exchanged. • • Translocations: When a portion of one chromosome is transferred to another chromosome. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). also called the terminal 11q deletion disorder. etc. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. an X. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. which is caused by partial deletion of the short arm of chromosome 4. There are two main types of translocations.). resulting in extra genetic material.3 Structural abnormalities When the chromosome's structure is altered. Known disorders in humans include Wolf-Hirschhorn syndrome. an entire chromosome has . Tetrasomy. and Jacobsen syndrome. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. rather than two). In a reciprocal translocation.4. Duplications: A portion of the chromosome is duplicated. In a Robertsonian translocation. 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 occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. 4.
the anomaly is present in every cell of the body. and are therefore initially not inherited. Chromosome anomalies can be inherited from a parent or be "de novo". 4. • Rings: A portion of a chromosome has broken off and formed a circle or ring. Some anomalies. other cytogenetic banding techniques. however. 14. 4.in humans these only occur with chromosomes 13.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. This can happen with or without loss of genetic material. turned upside down and reattached. It includes routine analysis of G-Banded chromosomes. 15. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.attached to another at the Centromere . 21 and 22.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. Therefore. resulting in Mosaicism (where some cells have the anomaly and some do not). as well . especially the chromosomes. They often lead to an increased tendency to develop certain types of malignancies. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. • Inversions: A portion of the chromosome has broken off. therefore the genetic material is inverted. can happen after conception. This is why chromosome studies are often performed on parents when a child is found to have an anomaly.
4. Pre-treating cells in a hypotonic solution. at first favoring 46. in 1882. He revised his opinion later from 46 to 48. New techniques were needed to definitively solve the problem: 1. which swells them and spreads the chromosomes . von Waldeyer in 1888.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Using cells in culture 2. 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. The name was coined by another German anatomist. Their behavior in animal (salamander) cells was described by Walther Flemming. in contrast to their genic contents. these results were quite remarkable. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. Considering their techniques. and he correctly insisted on man having an XX/XY system. concluding an XX/XO sex determination mechanism. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. The next stage took place after the development of genetics in the early 20th century. the discoverer of mitosis.
2 Natural populations of Drosophila In the 1930s.3. Using Painter's technique they studied the polytene . It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. McClintock discovered transposons. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.6 Applications in biology 4. 4. Arresting mitosis in metaphase by a solution of colchicine 4. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). the great apes have 48 chromosomes.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Human chromosome 2 was formed by a merger of ancestral chromosomes. During her cytogenetic work. 4. persimilis from wild populations in California and neighboring states.6. reducing the number. In 1931. Rather interestingly. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. a find which eventually led to her Nobel Prize in 1983.6.
In 1959. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. breeding and sampling whilst preventing escape. 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. as with most polymorphisms. Down syndrome is also referred to as trisomy 21. Evidence rapidly accumulated to show that natural selection was responsible. 4. 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. as they would if selectively neutral. Dobzhansky bred populations in population cages. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. It was found that the various chromosome types do not fluctuate at random.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. Using a method invented by L'Heretier and Teissier.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. . In some congenital disorders. This had the benefit of eliminating migration as a possible explanation of the results. discoveries were quickly made related to aberrant chromosomes or chromosome number. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. but adjust to certain frequencies at which they become stabilised. such as Down's syndrome. which enabled feeding.
XYY. with the development of more advanced techniques. Thirteen years later.Other numerical abnormalities discovered include sex chromosome abnormalities. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). has Klinefelter's Syndrome. Identification of the Philadelphia chromosome by cytogenetics. In 1960. An individual with only one sex chromosome (the X) has Turner syndrome. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. is used today as a diagnostic for CML. resulting in 47 total chromosomes. Pennsylvania.as both scientists were doing their research in Philadelphia. in addition to other tests. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. . Many other sex chromosome combinations are compatible with live birth including XXX. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. Not all genes on the X Chromosome are inactivated. which is required in normal females to compensate for having two copies of the chromosome. an additional X chromosome in a male. and XXXX. This abnormal chromosome was dubbed the Philadelphia chromosome . Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome.
Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material.8 Beginnings of molecular cytogenetics . Caspersson developed banding techniques which differentially stain chromosomes. Deletions within one chromosome could also now be more specifically named and understood. 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. and elongation techniques for all culture types that allow for higher resolution banding. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid.FIG Advent of banding techniques In the late 1960s. 4. 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.
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.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. advances were made in molecular cytogenetics. cloned and studied in ever greater detail.In the 1980s. 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. 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,
such as C. communications. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. control design. For congenital problems usually 20 metaphase cells are scored. including signal and image processing.generally between 200 and 1000 cells are counted and scored. . financial modeling and analysis. CGH and Single nucleotide polymorphism-arrays. data analysis. and numerical computation. and FORTRAN. such as comparative genomic hybridization arrays. and computational biology. data visualization. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. test and measurement. You can use MATLAB in a wide range of applications. C++. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. you can solve technical computing problems faster than with traditional programming languages. Using MATLAB.
You can integrate your MATLAB code with other languages and applications. specifying data types. and distribute your MATLAB algorithms and applications. With the MATLAB language.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 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. As a result. and allocating memory. . It enables fast development and execution. 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. one line of MATLAB code can often replace several lines of C or C++ code. The image processing step is composed of the following operations. 6. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. 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. such as declaring variables.
2) Geometrical compensation—The geometric compensation. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. geometrical and dimensional differences must be removed. To compensate for this inhomogeneity. the spatially scaled images are histogram equalized. 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. 6. or at least attenuated. To compare chromosomes from a band pattern point of view.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram.2 Concepts used in this phase 1) Image conversion 2) Denoising . Therefore. 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).
1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. For example. listed in the following table. RGB = cat (3. For example. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. you must first convert it to true color format. so the image displays as shades of gray. green. 6. and the results might not be meaningful. When you apply the filter to the true color image. MATLAB simply applies the filter to the indices in the indexed image matrix. You can perform certain conversions just using MATLAB syntax. MATLAB filters the intensity values in the image.I.2.3) Edge detection 4) Two dimensional convolutions. there are other functions that return a different image type as part of the operation they perform. For example. The resulting true color image has identical matrices for the red. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. if you want to filter a color image that is stored as an indexed image. If you attempt to filter the indexed image. .I).I. as is appropriate. and blue planes. In addition to these image type conversion functions.
6.2.'method'. via satellite or wireless transmission. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. . to isolate particular objects from their background. to recognize or classify objects.3 Two dimensional convolutions C = conv2(A.B) computes the two-dimensional convolution of matrices A and B.5 Edge detection Edges contain some of the most useful information in an image. 6. Usually we know what type of errors to expect. Cleaning an image corrupted by noise is thus an important area of image restoration.parameters. There is a large number of edge finding algorithms in existence.4 Denoising We may define noise to be any degradation in the image signal. and hence the type of noise on the image. caused by external disturbance. . If an image is being sent electronically from one place to another. or through networked cable. The general Matlab command for finding edges is edge(image. ) Where the parameters available depend on the method used 6. and we shall look at some of the more straightforward of them. If one of these matrices describes a two-dimensional finite impulse response .2. We may use edges to measure the size of objects in an image. hence we can choose the most appropriate method for reducing the effects. we may expect errors to occur in the image signal.
'sobel'). if the size of then the size of C is [ma+mb-1. 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. the other matrix is filtered in two dimensions. this case is the same as C = conv2(hcol*hrow.bmp'). edge(im1.na] and the size of B is [mb..'shape') subsection of the two-dimensional convolution. rgb2gray im2bw(im.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. That is.na+nb-1].[3 3]).hrow. C = conv2(.nb]. minus one. If hcol is a column vector and hrow is a row vector.. . convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.(FIR) filter. The size of matrices.A). nb]+1)/2).. imedfilt2(im1. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.7).0.
rc = [r c].double(msk)).n]=size(L).[imx.y1)=255.8). for i=1:m for j=1:n if L(i.j)~=0 for k=1:mx if L(i. nzeros(imx.2). Msk conv2(double(BW).1). n1(x1.imy]=size(BW).imy).j)==L_number(k) flag=1.1). MODULE 2 clc [m. . flag=0. Index=1. y1=rc(i. for i=1:sx x1=rc(i. L_number=zeros(mx. [sx sy]=size(rc). mx=max(max(L)). [r. bwlabel(B.c] = find(L==22).
45.66].18.104.22.168.22.29. end. for i=1:sx x1=rc(i. end %h=figure.22.214.171.124.24.4.55. n1(x1.20.60. y1=rc(i.c] = find(L==L_number((Test_number(x)))).126.96.36.199. . Index=Index+1.imshow(n1.33.imy).51.y1)=255.62.31.48.65.1). Test_number=[3.30. [sx sy]=size(rc).32.9.56. end flag=0. end end L_number.end end if flag~=1 L_number(Index)=L(i. 188.8.131.52.j).15.39.26. for x=1:46 [r.2).184.108.40.206. rc = [r c].).19.10. n1=zeros(imx.43.
s=bwmorph(skel.1).1).Inf). BW1=edge(BW. s1=bwmorph(s. Area=zeros(46. [m n]=size(BW1). BW=im2bw(f). for i=1:46 f=imread(strcat(num2str(i).'canny').1). for x=1:m for y=1:n if BW1(x. skel=im2double(f). .end Circumference=zeros(46. Arm_length_sum=0.'.8). Circumference_sum=0.bmp')).5*graythresh(skel)).1.'skel'. skel=im2bw(skel. end end end Circumference(i)=Circumference_sum. Arm_length=zeros(46.'spur'. BW=double(BW).y)==1 Circumference_sum=Circumference_sum+1. f=imcomplement(f).
y)==1 Area_sum=Area_sum+1. end Circumference. end end end Area(i)=Area_sum.y)==1 Arm_length_sum=Arm_length_sum+1. for x=1:m for y=1:n if BW(x.[m n]=size(s1). BW=im2bw(f). Arm_length. Area_sum=0. end end end Arm_length(i)=Arm_length_sum. . for x=1:m for y=1:n if s1(x. [m n]=size(BW).
Pair(46. Pair(i.2)==46 Pair(46.1)=i.2)=j. Pair=zeros(46.1)=46. 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. .Area.2)=i+1. end end end for i=1:45 if Pair(i. Pair(i. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)). end end Pair.1)=i.2)=i. Pair(i.2).
1)). imshow(f2).1)==delete(j) flag=1.1). . figure_flag=figure_flag+1.2.bmp')). figure_flag=figure_flag+1.figure_flag).delete=zeros(46. if figure_flag~=47 subplot(23.2)). end f2=imread(strcat(num2str(Pair(i. imshow(f1). flag=0.'. end flag=0. end end if flag~=1 if figure_flag~=47 subplot(23.bmp')). end f1=imread(strcat(num2str(Pair(i. figure_flag=1.'.2.figure_flag).2). delete(figure_flag)=Pair(i. for i=1:46 for j=1:46 if Pair(i.
2) feature extraction from the processed images . and Philadelphia. such as. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. 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 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. Copenhagen. based on the MI. The proposed algorithm is based on the traditional features extracted from the karyogram. in the scope of karyotyping process used in cytogentic analysis. dimensions and banding profiles. plus a new one. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure.end CONCLUTION In this paper.
shape. This normalization is needed to make it possible the band pattern comparison between chromosomes. 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. 4) pairing.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. and to normalize their dimensions. 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). and finally.10% mean classification rate. 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 addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the . achieves a 70. Tests using 19 karyograms based on bone marrow cells.working within an 8-D feature space. The training process consists in the estimation of each vector of coefficient . the romosome images. shape and band pattern. Here. In the image processing step. are processed in order to compensate for geometrical and intensity distortions. The features extracted from the processed images discriminate each pair with respect to their size. 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. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. and band pattern. extracted from the unordered karyogram. from the chromosomes in the training set.
REFERENCES . and from which it is possible to extract additional features. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. amean classification rate larger than 93% was obtained in all experiments. Executing the algorithm on a higher quality dataset. centromere position. despite the low quality of this type of chromosomes. Copenhagen. In addition. e. or Philadelphia. This dataset was made publicly available . a 76. 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.10% classification ratewas obtained. presenting a uniform level of condensation. called LK1 . In fact. such as Edinburgh.performance of the classifier. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes.g.. whose images are of significantly higher quality. The results presented in this paper are promising. Using 27 karyograms andworking with a limited number of classes (≤ 8).
10. p242 5.D. Bull. Oxford U. . Edinburgh. The chromosomes. 1924.K. Oliver & Boyd. Cambridge University Press.1. 147–49. From 48 to 46: cytological technique. 28 2. Genet. Kiev. 1931. ^ a b c White M. 1939.A. Applied Bot. 3rd ed.S. Chapter XII: The Karyotype. Evolution of genetic systems. Animal cytology and evolution. Plant Breed. 2006. The morphology of chromosomes. 1950. Hist. preconception and the counting of the human chromosomes. Anat. A dictionary of genetics. and Mulligan P. 48. The spermatogenesis of man. 1958.L. ^ von Winiwarter H. 3. 93. [in Russian] 6. biologie 27. 2nd ed. 1922. ^ a b White M. Med. ^ Levitsky G. State Publication Office of the Ukraine. 465-502. Etudes sur la spermatogenese humaine. 1974..D.J.J. 1973. revised and enlarged. Bull.C.P Oxford & NY. 23. 4. ^ Levitsky G. Columbia University Press NY. ^ Darlington C. 11. Stansfield W. 7th ed. 19-174.D. ^ Kottler M. 1912. 1973.A. p. ^ Painter T. 9. 129. Chapman & Hall. Cambridge University Press. 8. Arch. 7. 6th ed. Res. The material basis of heredity. 27. ^ Concise Oxford Dictionary London. ^ Stebbins G.D. ^ King R. Variation and evolution in plants.
PNAS 97. 1996. Exp.fcgi?artid=34032 19. 2000.C. 1991. p85-6 18. Raven Press. Springer-Verlag. 14.J. Eosin Y and Azure-A. ed. ^ Stebbins G. 1923. 1998. In The ACT Cytogenetics Laboratory Manual 2nd ed. 9821– 9823. ^ Godfrey L. and Masters J. Chromosome stains. 1971. NY. p218-9 20. 21. ^ Goday C. 1-6. 2nd ed. Bioessays18: 133–138. J.C 16. 13.L. 291-336.C. Evolutionary genetics.12.R. M.R. Chromosome elimination in sciarid flies. Human and mammalian cytogenetics: a historical perspective. Arnold. The Association of Cytogenetic Technologists. ^ A preparation which includes the dyes Methylene Blue. The chromosome number of man.^ a b Gustashaw K. 1979. Zoology 37. Oxford. ^ Müller F. Bernard V. ^ Maynard Smith J.H & Levan A. and Esteban M. Chromosomal evolution in higher plants.http://www. New York.gov/articlerender.M. ^ Tjio J. Barch.pubmedcentral. Hereditas 42. 1956. 17.nih. The spermatogenesis of humans. London. Chromatin diminution in nematodes.B. Bioessays23: 242–250.^ a b Hsu T. . 15. ^ Painter T. & Tobler H.S. 2001. Studies in mammalian spermatogenesis II. Kinetophore reproduction theory may explain rapid chromosome evolution.
Park. Noh. Oxford U.doi:10. 2006.1007/BF02153623.S. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). Chromosome evolution in the genus Ophioglossum L. Stansfield W. doi:10. R.D. Developmental biology. (2005). 291: 310–16. Nam. Ichthyological Research 52 (1): 97.. C. ^ Wyngaard G. 7th ed. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. Botanical Journal of the Linnean Society 102: 205–217. J.. and Mulligan P. Exp. 1970. Retrieved 2008-03-18. D. ^ Matthey. Indian Muntjac.F. 26. Chapman. ^ Kim. 23. Experientia (Basel) 1 (2): 50– 56. ^ Khandelwal S. & Gregory T. A dictionary of genetics. (1945-05-15). Sinauer Associates. 2006. ^ King R.. F. and Benirschke K. Muntiacus muntjak: a deer with a low diploid number.H.. "L'evolution de la formule chromosomiale chez les vertébrés". 27.P Oxford & NY.R. ^ Wurster D. Zool. Science 168.K. Chapter 9 24. Stamford CT. 25.A. 1364-1366. J.H.K.22. 8th ed.C. Retrieved 2011-03-16. 28.K. Y. 2001.1007/s10228-004-0257-z. .A.. ^ Gilbert S. 1990.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue reading from where you left off, or restart the preview.