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
the term genophore is more appropriate when no chromatin is present. the cell may undergo mitotic catastrophe and die. Chromosomes are the essential unit for cellular division and must be replicated. Unduplicated chromosomes are single linear strands. In practice "chromosome" is a rather loosely defined term. This allows the very long DNA molecules to fit into the cell nucleus. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. The structure of chromosomes and chromatin varies through the cell cycle. In prokaryotes. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). which is tightly coiled in on itself. although there are many exceptions to this rule. divided. cells may contain more than one type of chromosome. through processes known as chromosomal instability and translocation. a large body of work uses the term chromosome regardless of chromatin content. DNA is usually arranged as a circle. circular DNA molecules called plasmids. These small circular genomes . or it may unexpectedly evadeapoptosis leading to the progression of cancer. In prokaryotes and viruses. Also. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny.defined nuclei) have smaller circular chromosomes. Chromosomes may exist as either duplicated or unduplicated. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. sometimes accompanied by one or more smaller. However. If these structures are manipulated incorrectly. Chromosomal recombination plays a vital role in genetic diversity. for example. In eukaryotes.
An example of aneuploidy is trisomy 21. If the mutation involves only one or a few chromosomes in the genome (e. . in which an individual has 3. 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). 1. The individual would have Down Syndrome and his/her karyotype would be written 47. a extra copy of human chromosome 21). XX (female) or 46 XY (male). 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.3 MUTATIONS IN CHROMOSOME NUMBER Normally. rather than 2. Such individuals are called euploid and have the wild-type chromosome complement for the species.g. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.XX. Euploid human karyotypes are 46.+21. copies of chromosome 21. the individual carrying the mutation is said to be aneuploid.+21.XY or 47. reflecting their bacterial origins.are also found in mitochondria and chloroplasts.
. a pair of sister chromatids attached to each other at the centromere. longer-lasting attachment in this region. the chromatin strands become more and more condensed. 1. chromosomes are structurally highly condensed. The microtubules then pull the chromatids apart toward the centrosomes. one of which is present on each sister chromatid. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. along with special proteins. Once the cells have divided. This compact form makes the individual chromosomes visible. which enables these giant DNA structures to be contained within a cell nucleus. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. During mitosis.Fig 1. In spite of their appearance. A special DNA base sequence in the region of the kinetochores provides.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). The shorter arms are called p arms (from the French petit. small) and the longer arms are called q arms (q follows p in the Latin alphabet. and they form the classic four arm structure. the chromatids are uncoiled and DNA can again be transcribed. q-g "grande"). so that each daughter cell inherits one set of chromatids.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. This is the only natural context in which individual chromosomes are visible with an optical microscope.
in adults weighing 65 kg (143 lbs). producing the lymphocytes that support the body's immune system CHAPTER 2 2.  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. bone marrow constitutes 4% of the total body mass of humans. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. which use the bone marrow vasculature as a conduit to the body's systemic circulation. in two separate nuclei. .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. On average. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. cytoplasm.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. bone marrow in large bones produces new blood cells. bone marrow accounts for approximately 2.1.7 lbs). which divides the nuclei.
which lack a nucleus. forming single cells with multiple nuclei. The cell then divides in cytokinesis. animals undergo an "open" mitosis. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. where chromosomes divide within an intact cell nucleus. The process of mitosis is fast and highly complex. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. metaphase. These stages are interphase. anaphase and telophase. For example. "mitosis" is often used interchangeably with "mitotic phase". prometaphase. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. Prokaryotic cells. Because cytokinesis usually occurs in conjunction with mitosis.genetically identical to each other and to their parent cell. cytokinesis and mitosis may occur independently. Mitosis occurs only in eukaryotic cells and the process varies in different species. . where the nuclear envelope breaks down before the chromosomes separate. for instance during certain stages of fruit fly embryonic development. However. This accounts for approximately 10% of the cell cycle. This occurs most notably among the fungi and slime moulds. to produce two identical daughter cells which are still diploid cells. Even in animals. but is found in various different groups. prophase. there are many cells where mitosis and cytokinesis occur separately. divide by a process called binary fission. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer.
This occurs during the S phase of interphase. . The sister chromatids are held together by a specialized region of the chromosome known as the centromere. Each chromosome now has an identical copy of itself.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. the parent cell must make a copy of each chromosome before mitosis. and together the two are called sister chromatids. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. 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.
pulling apart the sister chromatids of each chromosome. giving rise to two daughter cells. Eventually.the cell begins cytokinesis. As the cell elongates. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. In animal cells. the parent cell will be split in half. As a matter of convention. the process of binary fission is very much different from the process of mitosis. so they are renamed to sister chromosomes. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. the daughter cells will construct a new dividing cell wall between each other. A new nuclear envelope forms around the separated sister chromosomes. . Prokaryotic cells undergo a process similar to mitosis called binary fission. 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). corresponding sister chromosomes are pulled toward opposite ends. each with a replica of the original genome. However.In most eukaryotes. each sister chromatid is now considered a chromosome. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. In plant cells. separating the two developing nuclei. As mitosis completes. The chromosomes align themselves in a line spanning the cell.
chromosomes are replicated only during the S phase. During all three phases. grows more and prepares for mitosis (G 2). This is achieved through the formation of a phragmosome. and G2 (second gap). the nucleus has to migrate into the center of the cell before mitosis can begin. and finally it divides (M) before restarting the cycle. where the cell prepares itself for cell division. Thus. It alternates with the much longer interphase. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . a cell grows (G1). 2. S (synthesis). All these phases in the interphase are highly regulated. 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.2. prophase is preceded by a pre-prophase stage. mainly via proteins. However. In highly vacuolated plant cells. continues to grow as it duplicates its chromosomes (S). the cell grows by producing proteins and cytoplasmic organelles.1 Preprophase In plant cells only. Interphase is divided into three phases: G1 (first gap).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.2.
Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. aligned at the metaphase plate. In addition to phragmosome formation. This band marks the position where the cell will eventually divide. 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. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. the pinched area is known as the cleavage furrow. and microtubules have invaded the nuclear Prophase space. The cells of higher plants (such as the flowering plants) lack centrioles. These microtubules can attach to kinetochores or they can interact with opposing microtubules.division. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. . The chromosomes have chromatin has condensed. instead. after the nuclear membrane breaks down. degraded. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. Cytokinesis has already begun.
the replicated chromosomes have two sister chromatids. they are not essential for the . giving a pair of centrosomes. At the onset of prophase. chromatin condenses together into a highly ordered structure called a chromosome.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. Chromosomes are typically visible at high magnification through a light microscope. Although centrioles help organize microtubule assembly. bound together at the centromere by the cohesin protein complex. Since the genetic material has already been duplicated earlier in S phase. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. which are made of a pair of centrioles found in most eukaryotic animal cells. which is replicated by the cell with the help of the nucleus before a new mitosis begins. A cell inherits a single centrosome at cell division. Close to the nucleus are structures called centrosomes. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. the genetic material in the nucleus is in a loosely bundled coil called chromatin. The centrosome is the coordinating center for the cell's microtubules.
undergo a variation called closed mitosis where the spindle forms inside the nucleus. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. such as algae or trichomonads. using energy from ATP to "crawl" up the tube toward the originating centrosome. . Each chromosome forms two kinetochores at the centromere. provides the pulling force necessary to later separate the chromosome's two chromatids. and it occurs in most multicellular organisms.2. since they are absent from plants. the motor activates. kinetochore microtubules begin searching for kinetochores to attach to. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. it is known that it contains some form of molecular motor. Prometaphase is sometimes considered part of prophase. on an average 20 ). and centrosomes are not always used in mitosis. coupled with polymerisation and depolymerisation of microtubules. or its microtubules are able to penetrate an intact nuclear envelope. When the spindle grows to sufficient length. Although the kinetochore structure and function are not fully understood. one attached at each chromatid. When a microtubule connects with the kinetochore. This motor activity.formation of the spindle. 2. Fungi and some protists. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. This is called open mitosis.
Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. only roughly lining up along the midline. The centromeres of the chromosomes. As a result. an imaginary line that is equidistant from the two centrosome poles. All chromosomes (blue) but one have arrived at the metaphase plate. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line".In the fishing pole analogy. analogous to a tug-of-war between people of equal strength. the chromosomes come under longitudinal tension from the two ends of the cell.3 Metaphase A cell in late metaphase. 2. in some sense. . the kinetochore would be the "hook" that catches a sister chromatid or "fish". Metaphase comes from the Greek meaning "after. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. In certain types of cells." Microtubules find and attach to kinetochores in prometaphase. convene along the metaphase plate or equatorial plane.
Two events then occur: first.” “back.” or “re-”). These sister chromatids. the proteins that bind sister chromatids together are cleaved. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. which have now become distinct sister chromosomes. 2. the nonkinetochore microtubules elongate. .Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). The force that causes the centrosomes to move towards the ends of the cell is still unknown. At the end of anaphase. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart.” “against. Next. the cell proceeds to anaphase (from the Greek meaning “up. allowing them to separate. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. Early anaphase is usually defined as the separation of the sister chromatids.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. The signal creates the mitotic spindle checkpoint. These two stages are sometimes called early and late anaphase.
Corresponding sister chromosomes attach at opposite ends of the cell. necessary for completing cell division. now surrounded by new nuclei. forms around each set of separated sister chromosomes. the nonkinetochore microtubules continue to lengthen. Mitosis is complete. using fragments of the parent cell's nuclear membrane. unfold back into chromatin. pinching off the separated nuclei. cell . cytokinesis is a separate process that begins at the same time as telophase.5 Cytokinesis Cilliate undergoing cytokinesis. A new nuclear envelope.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events.2. In both animal and plant cells. At telophase. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. Both sets of chromosomes. but rather a separate process. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. It "cleans up" the after effects of mitosis. but cell division is not yet complete. elongating the cell even more. 2. Cytokinesis is technically not even a phase of mitosis. In animal cells. however.
The end of cytokinesis marks the end of the M-phase. e.division is also driven by vesicles derived from the Golgi apparatus.e.4 Regeneration .5. whereas some green algae use a phycoplast microtubule array during cytokinesis. The phragmoplast is a microtubule structure typical for higher plants.5. Each daughter cell has a complete copy of the genome of its parent cell. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.2 Development and growth The number of cells within an organism increases by mitosis.3 Cell replacement In some parts of body. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. New cells are formed by mitosis and so are exact copies of the cells being replaced. 2. cells are constantly sloughed off and replaced by new ones. which move along microtubules to the middle of the cell.5. Similarly.. This is the basis of the development of a multicellular body from a single cell i.5.1Significance Mitosis is important for the maintenance of the chromosomal set. skin and digestive tract. separating the two nuclei.g. Following are the occasions in the lives of organism where mitosis happens: 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. zygote and also the basis of the growth of a multicellular body. 2. 2.
These cells are considered aneuploid.5. Mitosis continues in the cells of bud and it grows into a new individual.5. 2. the process may go wrong. a chromosome may fail to separate during anaphase.7 Consequences of errors Although errors in mitosis are rare. For example.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. The same division happens during asexual reproduction or vegetative propagation in plants. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). a condition often associated with cancer. For example. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder.Some organisms can regenerate their parts of bodies. The cells at the surface of hydra undergo mitosis and form a mass called bud. especially during early cellular divisions in the zygote. In non-disjunction. 2. The production of new cells is achieved by mitosis. Occasionally when cells experience nondisjunction. the hydra reproduces asexually by budding. One daughter cell will receive both sister chromosomes and the other will receive none. a condition known as trisomy. a condition known as monosomy. they fail to complete cell division and retain both nuclei in one cell. resulting in binucleated cells. sea star regenerates its lost arm through mitosis. and the latter cell having only one chromosome (the homologous chromosome). .
Benign tumours are not harmful as soon as they are not moving. causing chromosomal duplication. The fragment may incorrectly reattach to another. but in reverse orientation. causing translocation. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. The effect of these genetic abnormalities depends on the specific nature of the error. causing inversion. Errors in the control of mitosis may cause cancer. which goes through dramatic changes in ultrastructure. and chromosomes are jostled constantly by probing microtubules. it may be treated erroneously as a separate chromosome. It may reattach to the original chromosome. As soon as they start to move and invade other cells there are said to be malignant tumours.Mitosis is a demanding process for the cell. Or. Occasionally. chromosomes may become damaged. This phenomenon is called metastasis or spreading of disease. non-homologous chromosome. As long as these tumours remain in their original location they are called benign tumours. causing deletion. An arm of the chromosome may be broken and the fragment lost. When tissues more than the requirement are synthesized in a single organ. it results in the formation of Tumors. Such tumours can send cancer cells to other parts in body where new tumours may form. sometimes mutuations occur in such genes and cells continue to divide. It results in abnormal cell growth. It results in the synthesis of execessive tissue growths. Now what happens is that cell abnormally continue to divide at a single place. . All cells have genes that control the timing and number of mitosis. its organelles disintegrate and reform in a matter of hours.
Early events of metaphase can . an imaginary line that is equidistant from the two centrosome poles. Metaphase accounts for approximately 4% of the cell cycle's duration. 2. from the ancient Greek(between) and (stage). In certain types of cells. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Preceded by events in prometaphase and followed by anaphase. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.2. resulting in cells with many copies of the same chromosome occupying a single nucleus. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. This process may also be referred to as endoreduplication and the cells as endoploid. carrying genetic information. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). only roughly lining up along the middleline. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. align in the middle of the cell before being separated into each of the two daughter cells. An example of a cell that goes through endomitosis is the megakaryocyte. analogous to a tug of war between equally strong people.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division.7 Metaphase Metaphase.
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). This would be accomplished by regulation of the anaphase-promoting complex. when every kinetochore is properly attached to a bundle of microtubules. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. securin. and separase. 2. 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. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. often with Giemsa (G banding) or Quinacrine. One of the cell cycle checkpoints occurs during prometaphase and metaphase. which makes them most suitable for visual analysis. Metaphase chromosomes make the classical picture of chromosomes (karyotype).8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. produces a pattern of in total up to several hundred bands. does the cell enter anaphase. Chromosomes are condensed(Thickened) and highly coiled in metaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Staining of the slides. Only after all chromosomes have become aligned at the metaphase plate. For classical cytogenetic analyses. Such a signal creates the mitotic spindle checkpoint.
methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. for example. such as bcr-abl in chronic myelogenous leukemia. which may lead to chimeric oncogenes. . Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. losses of chromosomal segments or translocations.
and any other physical characteristics. or may not. Karyogram of human male using Giemsa staining. be sex chromosomes. Thus. the position of the centromeres. The term is also used for the complete set of chromosomes in a species. So. and to gather information about past evolutionary events. and what they look like under a light microscope. Karyotypes describe the number of chromosomes. banding pattern. taxonomic relationships. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. in humans 2n = 46. in normal diploid organisms. to study chromosomal aberrations. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). Karyotypes can be used for many purposes. . autosomal chromosomes are present in two copies. any differences between the sex chromosomes. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. such as. and the results may be used in evolutionary biology and medicine.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. or an individual organism. There may.  The preparation and study of karyotypes is part of cytogenetics. The study of karyotypes is important for cell biology and genetics. cellular function. The study of whole sets of chromosomes is sometimes known as karyology. ordered by size and position of centromere for chromosomes of the same size. Attention is paid to their length.
Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Considering their techniques. 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. concluding an XX/XO sex determination mechanism. in 1882. which swells them and spreads the chromosomes . The next stage took place after the development of genetics in the early 20th century. at first favoring 46. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48.3. the discoverer of mitosis. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. and he correctly insisted on humans having an XX/XY system. The subsequent history of the concept can be followed in the works of Darlington and White. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Using cells in culture 2. these results were quite remarkable. He revised his opinion later from 46 to 48. in contrast to their genic contents. New techniques were needed to definitively solve the problem: 1. The name was coined by another German anatomist. Their behavior in animal (salamander) cells was described by Walther Flemming. Pretreating cells in a hypotonic solution. von Waldeyer in 1888.
2. 3.3. is applied after cells have been arrested during cell division by a solution of colchicine.2 Observations Six different characteristics of karyotypes are usually observed and compared: . Arresting mitosis in metaphase by a solution of colchicines 4. Usually. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. reducing the number.  Sometimes observations may be made on non-dividing (interphase) cells. Rather interestingly.2 Observations on karyotypes 3.2. such as Giemsa. the great apes have 48 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Human chromosome 2 was formed by a merger of ancestral chromosomes. For humans. a suitable dye. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. The sex of an unborn fetus can be determined by observation of interphase cells.1 Staining The study of karyotypes is made possible by staining. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. 3.
6. shape and banding of the chromosomes. type. 3. Differences in absolute sizes of chromosomes. Differences in number and position of satellites. 2. as well as other cytogenetic information. . This feature probably reflects different amounts of DNA duplication. permitting its loss without penalty to the organism (the dislocation hypothesis). Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. 4. 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. but the genes have been mostly translocated (added) to other chromosomes. 5. Differences in the position of centromeres. Humans have one pair fewer chromosomes than the great apes. Heterochromatin stains darker than euchromatin. faba chromosomes are many times larger. indicating tighter packing. and mainly consists of genetically inactive repetitive DNA sequences. This is brought about by translocations. A full account of a karyotype may therefore include the number. both have six pairs of chromosomes (n=6) yet V.1.
Between the germ-line and soma (between gametes and the rest of the body) 3. XY. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. XX. Between the sexes 2.3 The human karyotype Most (but not all) species have a standard karyotype. which are highly variable. and in . Geographical variation between races 5.Variation is often found: 1. 3. Normal karyotypes for females contain two X chromosomes and are denoted 46. Mosaics or otherwise abnormal individuals. There is variation between species in chromosome number. Between members of a population (chromosome polymorphism) 4. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. 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.
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. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species.1 Changes during development Instead of the usual gene repression. despite many careful investigations. which were previously inexplicable. Chromosome elimination. Although much is known about karyotypes at the descriptive level. the general significance of karyotype evolution is obscure.. In some cases there is even significant variation within species. it is quite unclear what the general significance might be. In some species. "We have a very poor understanding of the causes of karyotype evolution. entire chromosomes are eliminated during development. 3. portions of the chromosomes are cast away in particular cells. But. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. Chromatin diminution (founding father: Theodor Boveri). Godfrey and Masters conclude: "In our view. found in some copepods and roundworms such as Ascaris suum.. In this process. . used in conjunction with other phylogenetic data. In a review. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species.. some organisms go in for large-scale elimination of heterochromatin.detailed organization. or other kinds of visible adjustment to the karyotype. This variation provides the basis for a range of studies in evolutionary cytology. as in many sciarid flies.. In A.3.
3. male = 7 chromosomes. the inactivation is random as between the two Xs. In human females some 15% of somatic cells escape inactivation. Muntiacus reevesi. In marsupials it is always the paternal X which is inactivated. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. In placental mammals. The existence of supernumerary or B chromosomes .3.. which was investigated by Kurt Benirschke and his colleague Doris Wurster.. 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. Muntiacus muntjak. all the somatic cell precursors undergo chromatin diminution. was found to be 46. 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. The low record is held by the nematode Parascaris univalens. When they looked at the karyotype of the closely related Indian muntjac.suum.. Xinactivation. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. "They simply could not believe what they saw. the high record would be somewhere amongst the ferns. all telocentric.. they were astonished to find it had female = 6. They kept quiet for two or three years because they thought something was wrong with their tissue culture. The diploid number of the Chinese muntjac.
Polyploidy. where there are more than two sets of homologous chromosomes in the cells. Polyploidy in lower plants (ferns. and in some other groups. and aneuploids are another example. but it has been significant in some groups. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. 3. 3. 14. Polyploidy in animals is much less common. FN ≤ 2n. It is a common arrangement in the Hymenoptera. though in this case they would not be regarded as normal members of the population. about 70%. FN. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Haplo-diploidy. It has been of major significance in plant evolution according to Stebbins.3 Fundamental number The fundamental number. Thus. and the other haploid. occurs mainly in plants.Endopolyploidy occurs when in adult differentiated . horsetails and psilotales) is also common. 15. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. but in grasses the average is much higher. 21 and 22). Humans have FN = 82.3.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. due to the presence of five acrocentric chromosome pairs (13. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.means that chromosome number can vary even within one interbreeding population. where one sex is diploid. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants.
tissues the cells have ceased to divide by mitosis. This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. the daughter chromosomes separating from each other inside an intact nuclear membrane. 3. it is diverse and complex. See palaeopolyploidy for the investigation of ancient karyotype duplications.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. Abnormalities in chromosome number usually cause a defect in development. Down syndrome and Turner syndrome are examples of this. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. The cells do not always contain exact multiples (powers of two). In many instances. and serves differentiation and morphogenesis in many ways. but the nuclei contain more than the original somatic number of chromosomes. .
Human chromosome 2 was formed by a merger of ancestral chromosomes. where the gametic (= haploid) numbers form the series x = 3. reducing the number. the great apes have 24x2 chromosomes whereas humans have 23x2. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. In about 6. the chromosome number is variable from one individual to another. the European shrew Sorex araneus. Well-researched examples are the ladybird beetle Chilocorus stigma. 6. that the two chromosome morphs are adapted to different habitats. 5.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. and 7.6 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. living from rainforests to . and Crocus.500 sq mi (17. 3. 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.Aneuploidy may also occur within a group of closely related species. where every number from x = 3 to x = 15 is represented by at least one species. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. some mantids of the genus Ameles. 3. 4.  Closer to home. When this happens.000 km2).
the present islands date from 0.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). The results are clear. There are also cases of colonization back to older islands. Although it would be possible for a single gravid female to colonise an island. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. at least into the Cretaceous. The inversions. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. Chromosome rearrangements. In a sense. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. Drosophila and Scaptomyza. Using K-Ar dating. make it possible to see which species are closely related. it is more likely to have been a group from the same species. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. when plotted in tree form (and independent of all other information). Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. especially inversions. in the family Drosophilidae. the best-studied group of Hawaiian drosophilids. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. which can be dated to 30 mya. The polytene banding of the 'picture wing' group. gene arrangements are visible in the banding patterns of each chromosome. probably 20 million years ago. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. . show a clear "flow" of species from older to newer islands. but these are much less frequent. and skipping of islands.subalpine meadows.
• Q-banding is a fluorescent pattern obtained using quinacrine for staining. • • C-banding: Giemsa binds to constitutive heterochromatin. adaptive radiations. if less spectacular. R-banding is the reverse of G-banding (the R stands for "reverse"). so it stains centromeres. 3.7. The pattern of bands is very similar to that seen in G-banding. The light regions tend to be euchromatic.7 Depiction of karyotypes 3. early-replicating and GC rich.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. human genome. late-replicating and AT rich. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).There are other animals and plants on the Hawaiian archipelago which have undergone similar. It yields a series of lightly and darkly stained bands . This method will normally produce 300-400 bands in a normal.the dark regions tend to be heterochromatic. • T-banding: visualize telomeres. .
a dye. Quinacrine binds to the adeninethymine-rich regions. is used to stain bands on the chromosomes. In addition. This yields a dark region where the silver is deposited. Karyotypes are arranged with the short arm of the chromosome on top. 3. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. For example. often Giemsa (G-banding). and the long arm on the bottom. Giemsa is specific for the phosphate groups of DNA. both chromosomes in a pair will have the same banding pattern.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein.7. respectively. In the "classic" (depicted) karyotype. Each chromosome has a characteristic banding pattern that helps to identify them. Some karyotypes call the short and long arms p and q. less frequently Quinacrine. denoting the activity of rRNA genes within the NOR. Cri du chat syndrome involves a deletion on the short arm of .
XX. which is written as 46.3 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. 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. Image processing software then assigns a pseudo color to each spectrally different combination. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. 3.XX.2.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Because there are a limited number of spectrally-distinct fluorophores. The critical region for this syndrome is deletion of 15.7. .chromosome 5. This method is also known as virtual karyotyping. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.2) 3. a combinatorial labeling method is used to generate many different colors. It is written as 46. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. allowing the visualization of the individually colored chromosomes.del(5)(p15.5p-.
CHAPTER 4 .
1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. the most common male chromosomal disease. X or 45. translocations. XXY is caused by an extra X chromosome. is caused by trisomy of chromosome 21. Klinefelter syndrome. in which three copies of a chromosome are present instead of the usual two. are common numerical abnormalities. including . Some disorders arise from loss of just a piece of one chromosome. a common chromosomal disease. or structural. • • Patau syndrome is caused by trisomy of chromosome 13. although they generally do not survive to birth. as in the presence of extra or missing chromosomes. trisomies. Numerical abnormalities. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. Down syndrome. large-scale deletions or duplications. Structural abnormalities often arise from errors in homologous recombination. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body.4. inversions. otherwise known as 47. also known as aneuploidy. often occur as a result of nondisjunction during meiosis in the formation of a gamete. as in derivative chromosome. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. Also documented are trisomy 8. trisomy 9 and trisomy 16. X0). or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells.
• Cri du chat (cry of the cat). example of imprinting disorder. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. 1p36 Deletion syndrome. The name comes from the babies' distinctive cry. 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. There are many types of chromosome anomalies. A chromosome anomaly. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. a deletion of the maternal genes. . a deletion of the paternal genes. A chromosome anomaly may be detected or confirmed in this manner. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. one well-documented example is the Philadelphia chromosome. numerical and structural anomalies. They can be organized into two basic groups. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. from the loss of part of the short arm of chromosome 1. from a truncated short arm on chromosome 5. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. caused by abnormal formation of the larynx. example of imprinting disorder. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia.
4. In a Robertsonian translocation. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21.3 Structural abnormalities When the chromosome's structure is altered. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. segments from two different chromosomes have been exchanged. 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. In a reciprocal translocation. etc. Tetrasomy. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). 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. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. and Jacobsen syndrome. an entire chromosome has . Known disorders in humans include Wolf-Hirschhorn syndrome. rather than two). which is caused by partial deletion of the short arm of chromosome 4. There are two main types of translocations. an X. • • Translocations: When a portion of one chromosome is transferred to another chromosome. resulting in extra genetic material. Duplications: A portion of the chromosome is duplicated. also called the terminal 11q deletion disorder.
15. however. This can happen with or without loss of genetic material. and are therefore initially not inherited. turned upside down and reattached. 21 and 22. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. • Rings: A portion of a chromosome has broken off and formed a circle or ring. Therefore. 14. especially the chromosomes.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. It includes routine analysis of G-Banded chromosomes. Some anomalies. resulting in Mosaicism (where some cells have the anomaly and some do not). the anomaly is present in every cell of the body. They often lead to an increased tendency to develop certain types of malignancies. Chromosome anomalies can be inherited from a parent or be "de novo".in humans these only occur with chromosomes 13. as well .attached to another at the Centromere . 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. other cytogenetic banding techniques.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. 4. 4. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. • Inversions: A portion of the chromosome has broken off.
which swells them and spreads the chromosomes . 4. The next stage took place after the development of genetics in the early 20th century. 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 discoverer of mitosis. The name was coined by another German anatomist. He revised his opinion later from 46 to 48. in contrast to their genic contents. von Waldeyer in 1888. in 1882. concluding an XX/XO sex determination mechanism. Using cells in culture 2. at first favoring 46. these results were quite remarkable.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Their behavior in animal (salamander) cells was described by Walther Flemming. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. New techniques were needed to definitively solve the problem: 1. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. and he correctly insisted on man having an XX/XY system.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Considering their techniques. Pre-treating cells in a hypotonic solution. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.
reducing the number.6. Human chromosome 2 was formed by a merger of ancestral chromosomes. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). a find which eventually led to her Nobel Prize in 1983. Arresting mitosis in metaphase by a solution of colchicine 4.3. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. 4. 4. Rather interestingly.2 Natural populations of Drosophila In the 1930s. In 1931. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.6 Applications in biology 4. During her cytogenetic work.6. Using Painter's technique they studied the polytene . persimilis from wild populations in California and neighboring states. McClintock discovered transposons. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. the great apes have 48 chromosomes.
cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. Down syndrome is also referred to as trisomy 21. 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. but adjust to certain frequencies at which they become stabilised. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. This had the benefit of eliminating migration as a possible explanation of the results. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. breeding and sampling whilst preventing escape. Evidence rapidly accumulated to show that natural selection was responsible. as with most polymorphisms. It was found that the various chromosome types do not fluctuate at random. 4. Dobzhansky bred populations in population cages.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. which enabled feeding. 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. discoveries were quickly made related to aberrant chromosomes or chromosome number. In some congenital disorders. In 1959. such as Down's syndrome. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. . Using a method invented by L'Heretier and Teissier. as they would if selectively neutral.
which is why there is a phenotypic effect seen in individuals with extra X chromosomes. in addition to other tests. In 1960. Identification of the Philadelphia chromosome by cytogenetics. is used today as a diagnostic for CML. has Klinefelter's Syndrome.Other numerical abnormalities discovered include sex chromosome abnormalities.as both scientists were doing their research in Philadelphia. XYY. Thirteen years later. Not all genes on the X Chromosome are inactivated. . Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. This abnormal chromosome was dubbed the Philadelphia chromosome . The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. which is required in normal females to compensate for having two copies of the chromosome. with the development of more advanced techniques. resulting in 47 total chromosomes. An individual with only one sex chromosome (the X) has Turner syndrome. Pennsylvania. Many other sex chromosome combinations are compatible with live birth including XXX. and XXXX. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). an additional X chromosome in a male.
Caspersson developed banding techniques which differentially stain chromosomes. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 4.FIG Advent of banding techniques In the late 1960s. Deletion syndromes such as DiGeorge syndrome. and elongation techniques for all culture types that allow for higher resolution banding.8 Beginnings of molecular cytogenetics . Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Deletions within one chromosome could also now be more specifically named and understood. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations.
cloned and studied in ever greater detail. 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.In the 1980s. While radioisotope-labeled probes had been hybridized with DNA since 1969. advances were made in molecular cytogenetics. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). CHAPTER 5 Techniques 5. movement was now made in using fluorescent labeled probes.
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.
5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •
bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations
5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,
Using MATLAB.generally between 200 and 1000 cells are counted and scored. test and measurement. C++. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. including signal and image processing. 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. data visualization. you can solve technical computing problems faster than with traditional programming languages. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. CGH and Single nucleotide polymorphism-arrays. such as comparative genomic hybridization arrays. communications. You can use MATLAB in a wide range of applications. control design. and FORTRAN. data analysis. financial modeling and analysis. and numerical computation. For congenital problems usually 20 metaphase cells are scored. . and computational biology. such as C.
In many cases.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. MATLAB eliminates the need for ‘for’ loops. The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted. The image processing step is composed of the following operations. and allocating memory.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. such as declaring variables. . The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. It enables fast development and execution. specifying data types. and distribute your MATLAB algorithms and applications. You can integrate your MATLAB code with other languages and applications. These effects must be compensated to improve the results of the pairing algorithm. As a result. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. 6. With the MATLAB language.
geometrical and dimensional differences must be removed. 6. Therefore. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 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 compensate for this inhomogeneity. or at least attenuated. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. To compare chromosomes from a band pattern point of view.2 Concepts used in this phase 1) Image conversion 2) Denoising . 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). 2) Geometrical compensation—The geometric compensation. the spatially scaled images are histogram equalized.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram.
RGB = cat (3. MATLAB simply applies the filter to the indices in the indexed image matrix. For example.2. so the image displays as shades of gray. as is appropriate. and the results might not be meaningful. if you want to filter a color image that is stored as an indexed image. When you apply the filter to the true color image. For example. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. . listed in the following table. If you attempt to filter the indexed image. you must first convert it to true color format. For example. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. there are other functions that return a different image type as part of the operation they perform. 6. You can perform certain conversions just using MATLAB syntax. and blue planes. green. The resulting true color image has identical matrices for the red. In addition to these image type conversion functions.3) Edge detection 4) Two dimensional convolutions. MATLAB filters the intensity values in the image.I.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.I).I.
4 Denoising We may define noise to be any degradation in the image signal. ) Where the parameters available depend on the method used 6. There is a large number of edge finding algorithms in existence. we may expect errors to occur in the image signal. and hence the type of noise on the image.'method'. .B) computes the two-dimensional convolution of matrices A and B. We may use edges to measure the size of objects in an image.parameters.3 Two dimensional convolutions C = conv2(A. and we shall look at some of the more straightforward of them. to recognize or classify objects. . hence we can choose the most appropriate method for reducing the effects. via satellite or wireless transmission. If one of these matrices describes a two-dimensional finite impulse response . These errors will appear on the image output in different ways depending on the type of disturbance in the signal. Usually we know what type of errors to expect. Cleaning an image corrupted by noise is thus an important area of image restoration.2. or through networked cable. The general Matlab command for finding edges is edge(image. 6.6. caused by external disturbance. to isolate particular objects from their background.2.5 Edge detection Edges contain some of the most useful information in an image. If an image is being sent electronically from one place to another.
rgb2gray im2bw(im.0.nb].bmp').'shape') subsection of the two-dimensional convolution..'sobel').(FIR) filter. If hcol is a column vector and hrow is a row vector. That is. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.hrow. C = conv2(...[3 3]). The size of matrices. .na] and the size of B is [mb. this case is the same as C = conv2(hcol*hrow. edge(im1. the other matrix is filtered in two dimensions. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.A).7). minus one. 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.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. imedfilt2(im1. nb]+1)/2).na+nb-1]. if the size of then the size of C is [ma+mb-1.
8).2). flag=0.c] = find(L==22).j)==L_number(k) flag=1.y1)=255. mx=max(max(L)). MODULE 2 clc [m. Index=1.n]=size(L).1).imy). bwlabel(B. nzeros(imx. n1(x1. for i=1:m for j=1:n if L(i. [r. for i=1:sx x1=rc(i.j)~=0 for k=1:mx if L(i.imy]=size(BW). [sx sy]=size(rc). rc = [r c]. L_number=zeros(mx.1). .double(msk)). Msk conv2(double(BW). y1=rc(i.[imx.
40.29.26.imy). Test_number=[3.22.38. end end L_number.65.end end if flag~=1 L_number(Index)=L(i. n1=zeros(imx. [sx sy]=size(rc).32.27.48. rc = [r c].10. for i=1:sx x1=rc(i.41.6. for x=1:46 [r.y1)=255.35.30. .4.8. end flag=0.11.1). 184.108.40.206.220.127.116.11.18.104.22.168.24.66].22.214.171.124. n1(x1.j). y1=rc(i.62.52.c] = find(L==L_number((Test_number(x)))).2). Index=Index+1.21. end %h=figure.).imshow(n126.96.36.199. end.188.8.131.52.
[m n]=size(BW1).'canny').'spur'.'skel'. end end end Circumference(i)=Circumference_sum.y)==1 Circumference_sum=Circumference_sum+1.'. s=bwmorph(skel. s1=bwmorph(s. f=imcomplement(f). Arm_length=zeros(46. .1). Arm_length_sum=0. for x=1:m for y=1:n if BW1(x.5*graythresh(skel)). skel=im2bw(skel.1). skel=im2double(f). BW1=edge(BW. for i=1:46 f=imread(strcat(num2str(i).bmp')).8). BW=im2bw(f). BW=double(BW).1.1). Area=zeros(46.end Circumference=zeros(46. Circumference_sum=0.Inf).
y)==1 Arm_length_sum=Arm_length_sum+1. BW=im2bw(f). Area_sum=0. [m n]=size(BW). for x=1:m for y=1:n if s1(x.[m n]=size(s1). Arm_length. . end end end Arm_length(i)=Arm_length_sum. end end end Area(i)=Area_sum. end Circumference.y)==1 Area_sum=Area_sum+1. for x=1:m for y=1:n if BW(x.
2)=i. Pair(i. Pair(i.2)=j. end end end for i=1:45 if Pair(i. end end Pair. Pair(i. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)).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.1)=i.Area.2)==46 Pair(46.2).1)=i. . Pair=zeros(46. Pair(46.2)=i+1.
figure_flag). . end f1=imread(strcat(num2str(Pair(i. for i=1:46 for j=1:46 if Pair(i. flag=0.2.bmp')). figure_flag=figure_flag+1.1)). delete(figure_flag)=Pair(i.1)==delete(j) flag=1.delete=zeros(46.2)). figure_flag=figure_flag+1.2).'.'. imshow(f1). end f2=imread(strcat(num2str(Pair(i. if figure_flag~=47 subplot(23. end end if flag~=1 if figure_flag~=47 subplot(23. figure_flag=1. imshow(f2).bmp')).2.1).figure_flag). end flag=0.
such as. plus a new one.end CONCLUTION In this paper. The proposed algorithm is based on the traditional features extracted from the karyogram. 2) feature extraction from the processed images . based on the MI. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. The images of these chromosomes present less quality and level of detail than the ones usually used in traditional genetic analysis using datasets such as Edinburgh. and Philadelphia. dimensions and banding profiles. in the scope of karyotyping process used in cytogentic analysis. Copenhagen. 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 addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the . and to normalize their dimensions. 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 romosome images. The training process consists in the estimation of each vector of coefficient . achieves a 70. are processed in order to compensate for geometrical and intensity distortions. In the image processing step. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts.working within an 8-D feature space. The features extracted from the processed images discriminate each pair with respect to their size.characterizing the size.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. Here. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. Tests using 19 karyograms based on bone marrow cells. from the chromosomes in the training set. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. shape and band pattern. This normalization is needed to make it possible the band pattern comparison between chromosomes. extracted from the unordered karyogram. 4) pairing. 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 band pattern. and finally.10% mean classification rate. shape. 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.
In addition.performance of the classifier. a 76.10% classification ratewas obtained. e. a new chromosome dataset with 9200 chromosomes from bone marrow cells. called LK1 . whose images are of significantly higher quality. centromere position. such as Edinburgh.. This dataset was made publicly available . In fact. despite the low quality of this type of chromosomes. amean classification rate larger than 93% was obtained in all experiments. presenting a uniform level of condensation. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. Copenhagen. Using 27 karyograms andworking with a limited number of classes (≤ 8).g. or Philadelphia. The results presented in this paper are promising. REFERENCES . Executing the algorithm on a higher quality dataset. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. and from which it is possible to extract additional features.
The spermatogenesis of man. 1912. ^ Concise Oxford Dictionary London.J. Applied Bot.1. 1939. Anat. A dictionary of genetics. ^ Painter T. 147–49. Stansfield W. Bull. ^ a b White M.J. 28 2. 93. 7. Cambridge University Press. p. 1931. From 48 to 46: cytological technique. Oliver & Boyd. Evolution of genetic systems. [in Russian] 6.D. and Mulligan P.D. 1922. The chromosomes. 8. Arch. The morphology of chromosomes. 7th ed. Chapman & Hall.L. Cambridge University Press. Plant Breed. 3rd ed. Genet. . 1973. Edinburgh. 1950. ^ King R. Etudes sur la spermatogenese humaine. Columbia University Press NY. Kiev. 1973.D. revised and enlarged. Oxford U.P Oxford & NY. 129.D. ^ Levitsky G. Animal cytology and evolution.S.K. ^ Darlington C. Variation and evolution in plants. Hist. 1924. 10. Res. 2006. Med. biologie 27.C. 11. 9. 6th ed. Bull. ^ Kottler M. 4. 1958. The material basis of heredity. 23.A.A. 3. ^ Stebbins G. ^ von Winiwarter H. 2nd ed. Chapter XII: The Karyotype. ^ Levitsky G. p242 5. State Publication Office of the Ukraine. 27. 1974. ^ a b c White M. 19-174.. preconception and the counting of the human chromosomes. 48. 465-502.
2nd ed. 1996.C. 1956.B. London. NY. The chromosome number of man. Studies in mammalian spermatogenesis II. ^ A preparation which includes the dyes Methylene Blue. ^ Müller F.fcgi?artid=34032 19.gov/articlerender.C 16. and Esteban M.^ a b Hsu T. 21. Hereditas 42. 14. p85-6 18. Human and mammalian cytogenetics: a historical perspective. J.pubmedcentral. Bernard V. The spermatogenesis of humans. Barch.12. Bioessays23: 242–250. Springer-Verlag.R. & Tobler H. Arnold. 13. Zoology 37.M. ^ Maynard Smith J. Bioessays18: 133–138.nih. 1979. M. 1971.H & Levan A. In The ACT Cytogenetics Laboratory Manual 2nd ed. 2000. ^ Painter T. 17.http://www. ed. p218-9 20.C. PNAS 97. ^ Godfrey L. Chromosome elimination in sciarid flies. 291-336. Eosin Y and Azure-A. 1-6.R. 1998.J. Evolutionary genetics.L.^ a b Gustashaw K. Chromosomal evolution in higher plants. Chromosome stains. 15. 1991. Chromatin diminution in nematodes.S. Kinetophore reproduction theory may explain rapid chromosome evolution. and Masters J. Raven Press. ^ Tjio J. The Association of Cytogenetic Technologists. New York. ^ Stebbins G. 1923. 2001. Oxford. Exp. . ^ Goday C. 9821– 9823.
2006. 28. Indian Muntjac. 26. Oxford U..C.K.R. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). 1990. ^ Wurster D. Sinauer Associates.P Oxford & NY. F. Stamford CT. Retrieved 2008-03-18. ^ Wyngaard G.doi:10. Botanical Journal of the Linnean Society 102: 205–217. Noh.. A dictionary of genetics. Muntiacus muntjak: a deer with a low diploid number. Chapman.A. Retrieved 2011-03-16. (1945-05-15).1007/s10228-004-0257-z. 25.F. R. 1364-1366. Nam. J.1007/BF02153623. J. ^ Gilbert S. Experientia (Basel) 1 (2): 50– 56. 23. "L'evolution de la formule chromosomiale chez les vertébrés". ^ King R. 2001.D. Chapter 9 24.S.K. Park. and Mulligan P. D.A. Developmental biology. ^ Khandelwal S. Stansfield W. ^ Matthey. 7th ed. Ichthyological Research 52 (1): 97. (2005). and Benirschke K. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods.22. Exp.K. Zool. . Science 168.H..H. Chromosome evolution in the genus Ophioglossum L. ^ Kim. 8th ed. C.. 27.. & Gregory T. 291: 310–16. Y. 2006. 1970. doi:10.
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.