ABSTRACT

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.

CHAPTER 1

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[1] nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without

cells may contain more than one type of chromosome. for example. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. Chromosomes are the essential unit for cellular division and must be replicated. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). In practice "chromosome" is a rather loosely defined term. through processes known as chromosomal instability and translocation. the term genophore is more appropriate when no chromatin is present. However. If these structures are manipulated incorrectly. DNA is usually arranged as a circle. which is tightly coiled in on itself. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. although there are many exceptions to this rule. circular DNA molecules called plasmids. Chromosomal recombination plays a vital role in genetic diversity.defined nuclei) have smaller circular chromosomes. In prokaryotes. Unduplicated chromosomes are single linear strands. a large body of work uses the term chromosome regardless of chromatin content. In eukaryotes. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. Also. The structure of chromosomes and chromatin varies through the cell cycle. This allows the very long DNA molecules to fit into the cell nucleus. In prokaryotes and viruses. Chromosomes may exist as either duplicated or unduplicated. These small circular genomes . sometimes accompanied by one or more smaller. or it may unexpectedly evadeapoptosis leading to the progression of cancer. the cell may undergo mitotic catastrophe and die. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. divided.

in which an individual has 3. Such individuals are called euploid and have the wild-type chromosome complement for the species. If the mutation involves only one or a few chromosomes in the genome (e.XX. copies of chromosome 21.+21. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. XX (female) or 46 XY (male).+21.g. Euploid human karyotypes are 46. the individual carrying the mutation is said to be aneuploid.XY or 47. An example of aneuploidy is trisomy 21. The individual would have Down Syndrome and his/her karyotype would be written 47. a extra copy of human chromosome 21). members of the same species have the same numbers of types of chromosomes (with the exception of sex chromosomes in males and females if sex is chromosomally determined). 1. rather than 2. . reflecting their bacterial origins. 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.are also found in mitochondria and chloroplasts.

4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). . longer-lasting attachment in this region. q-g "grande"). which enables these giant DNA structures to be contained within a cell nucleus. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. The shorter arms are called p arms (from the French petit. This compact form makes the individual chromosomes visible. the chromatin strands become more and more condensed. Once the cells have divided. and they form the classic four arm structure. In spite of their appearance. The microtubules then pull the chromatids apart toward the centrosomes. the chromatids are uncoiled and DNA can again be transcribed.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. 1. along with special proteins. so that each daughter cell inherits one set of chromatids. chromosomes are structurally highly condensed. a pair of sister chromatids attached to each other at the centromere. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. one of which is present on each sister chromatid. 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. A special DNA base sequence in the region of the kinetochores provides.Fig 1. During mitosis. This is the only natural context in which individual chromosomes are visible with an optical microscope.

bone marrow constitutes 4% of the total body mass of humans. bone marrow accounts for approximately 2. producing the lymphocytes that support the body's immune system CHAPTER 2 2.7 lbs). which divides the nuclei. bone marrow in large bones produces new blood cells. It is generally followed immediately by cytokinesis. In humans. [1] Bone marrow is also a key component of the lymphatic system. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. cytoplasm.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. in two separate nuclei. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. On average.6 kg (5. which use the bone marrow vasculature as a conduit to the body's systemic circulation.1.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. . in adults weighing 65 kg (143 lbs).

Because cytokinesis usually occurs in conjunction with mitosis. The cell then divides in cytokinesis. Even in animals. to produce two identical daughter cells which are still diploid cells. prophase. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. However. for instance during certain stages of fruit fly embryonic development. "mitosis" is often used interchangeably with "mitotic phase".genetically identical to each other and to their parent cell.[1] Prokaryotic cells. anaphase and telophase. metaphase. This occurs most notably among the fungi and slime moulds. animals undergo an "open" mitosis. forming single cells with multiple nuclei. The process of mitosis is fast and highly complex. where chromosomes divide within an intact cell nucleus. cytokinesis and mitosis may occur independently. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. where the nuclear envelope breaks down before the chromosomes separate. but is found in various different groups. which lack a nucleus. These stages are interphase. there are many cells where mitosis and cytokinesis occur separately. . For example. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. This accounts for approximately 10% of the cell cycle. Mitosis occurs only in eukaryotic cells and the process varies in different species. divide by a process called binary fission. prometaphase. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next.

This occurs during the S phase of interphase. These two cells are identical and do not differ in any way from the original parent cell. . Each chromosome now has an identical copy of itself.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. and together the two are called sister chromatids. the parent cell must make a copy of each chromosome before mitosis. Because each resultant daughter cell should be genetically identical to the parent cell.

corresponding sister chromosomes are pulled toward opposite ends. Prokaryotic cells undergo a process similar to mitosis called binary fission. As the cell elongates. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. As a matter of convention. each sister chromatid is now considered a chromosome. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. each with a replica of the original genome. pulling apart the sister chromatids of each chromosome. Eventually. separating the two developing nuclei. the parent cell will be split in half. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. . the process of binary fission is very much different from the process of mitosis. so they are renamed to sister chromosomes. In animal cells.the cell begins cytokinesis. As mitosis completes. However. 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). A new nuclear envelope forms around the separated sister chromosomes. giving rise to two daughter cells. In plant cells. The chromosomes align themselves in a line spanning the cell. the daughter cells will construct a new dividing cell wall between each other.In most eukaryotes.

continues to grow as it duplicates its chromosomes (S). chromosomes are replicated only during the S phase. It alternates with the much longer interphase. where the cell prepares itself for cell division. S (synthesis).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. the cell grows by producing proteins and cytoplasmic organelles. Thus. All these phases in the interphase are highly regulated. grows more and prepares for mitosis (G 2). During all three phases.1 Preprophase In plant cells only. Interphase is divided into three phases: G1 (first gap). a cell grows (G1). The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. However. mainly via proteins.2. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . This is achieved through the formation of a phragmosome. In highly vacuolated plant cells. 2.2. and finally it divides (M) before restarting the cycle. prophase is preceded by a pre-prophase stage. and G2 (second gap). the nucleus has to migrate into the center of the cell before mitosis can begin.

Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten.division. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. The cells of higher plants (such as the flowering plants) lack centrioles. instead. Cytokinesis has already begun. aligned at the metaphase plate. These microtubules can attach to kinetochores or they can interact with opposing microtubules. In addition to phragmosome formation. 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. This band marks the position where the cell will eventually divide. the pinched area is known as the cleavage furrow. after the nuclear membrane breaks down. . Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. The chromosomes have chromatin has condensed. and microtubules have invaded the nuclear Prophase space. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase.

giving a pair of centrosomes. chromatin condenses together into a highly ordered structure called a chromosome. which is replicated by the cell with the help of the nucleus before a new mitosis begins. the genetic material in the nucleus is in a loosely bundled coil called chromatin. the replicated chromosomes have two sister chromatids. which are made of a pair of centrioles found in most eukaryotic animal cells.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. A cell inherits a single centrosome at cell division. At the onset of prophase. Although centrioles help organize microtubule assembly. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. bound together at the centromere by the cohesin protein complex. they are not essential for the . 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. Chromosomes are typically visible at high magnification through a light microscope. The centrosome is the coordinating center for the cell's microtubules. Since the genetic material has already been duplicated earlier in S phase.

undergo a variation called closed mitosis where the spindle forms inside the nucleus.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Fungi and some protists. 2.2. the motor activates. and it occurs in most multicellular organisms. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. using energy from ATP to "crawl" up the tube toward the originating centrosome. one attached at each chromatid.formation of the spindle. Each chromosome forms two kinetochores at the centromere. This motor activity. such as algae or trichomonads. on an average 20 ). . or its microtubules are able to penetrate an intact nuclear envelope. it is known that it contains some form of molecular motor. This is called open mitosis. provides the pulling force necessary to later separate the chromosome's two chromatids. Prometaphase is sometimes considered part of prophase. coupled with polymerisation and depolymerisation of microtubules. When a microtubule connects with the kinetochore. Although the kinetochore structure and function are not fully understood. kinetochore microtubules begin searching for kinetochores to attach to. and centrosomes are not always used in mitosis. since they are absent from plants. When the spindle grows to sufficient length.

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. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. in some sense. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. All chromosomes (blue) but one have arrived at the metaphase plate. only roughly lining up along the midline." Microtubules find and attach to kinetochores in prometaphase. an imaginary line that is equidistant from the two centrosome poles. analogous to a tug-of-war between people of equal strength. 2. As a result. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". Metaphase comes from the Greek meaning "after. the chromosomes come under longitudinal tension from the two ends of the cell. The centromeres of the chromosomes. convene along the metaphase plate or equatorial plane. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. .3 Metaphase A cell in late metaphase. the kinetochore would be the "hook" that catches a sister chromatid or "fish".In the fishing pole analogy.

” “against. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. The force that causes the centrosomes to move towards the ends of the cell is still unknown. These two stages are sometimes called early and late anaphase. allowing them to separate. The signal creates the mitotic spindle checkpoint. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. Early anaphase is usually defined as the separation of the sister chromatids.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres).” or “re-”). it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. Next.” “back. 2. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. which have now become distinct sister chromosomes. the proteins that bind sister chromatids together are cleaved. the cell proceeds to anaphase (from the Greek meaning “up. At the end of anaphase. These sister chromatids. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. the nonkinetochore microtubules elongate. . Two events then occur: first.

however.2. unfold back into chromatin. In animal cells. the nonkinetochore microtubules continue to lengthen. elongating the cell even more.5 Cytokinesis Cilliate undergoing cytokinesis. In both animal and plant cells. cytokinesis is a separate process that begins at the same time as telophase. necessary for completing cell division. Corresponding sister chromosomes attach at opposite ends of the cell. Cytokinesis is technically not even a phase of mitosis. now surrounded by new nuclei. 2. At telophase.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. A new nuclear envelope. cell . a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. It "cleans up" the after effects of mitosis. pinching off the separated nuclei. forms around each set of separated sister 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. but cell division is not yet complete. Mitosis is complete. using fragments of the parent cell's nuclear membrane. Both sets of chromosomes.

. 2. New cells are formed by mitosis and so are exact copies of the cells being replaced. Following are the occasions in the lives of organism where mitosis happens: 2.5. 2. whereas some green algae use a phycoplast microtubule array during cytokinesis.5. Similarly.division is also driven by vesicles derived from the Golgi apparatus. skin and digestive tract.3 Cell replacement In some parts of body.5. This is the basis of the development of a multicellular body from a single cell i. which move along microtubules to the middle of the cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. Each daughter cell has a complete copy of the genome of its parent cell. 2. e. zygote and also the basis of the growth of a multicellular body.1Significance Mitosis is important for the maintenance of the chromosomal set.e. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. cells are constantly sloughed off and replaced by new ones. separating the two nuclei.4 Regeneration . The end of cytokinesis marks the end of the M-phase. The phragmoplast is a microtubule structure typical for higher plants.5.g.2 Development and growth The number of cells within an organism increases by mitosis.

The cells at the surface of hydra undergo mitosis and form a mass called bud. a condition known as monosomy. a condition known as trisomy. a condition often associated with cancer. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue).5. The same division happens during asexual reproduction or vegetative propagation in plants. These cells are considered aneuploid. Occasionally when cells experience nondisjunction.7 Consequences of errors Although errors in mitosis are rare. sea star regenerates its lost arm through mitosis. a chromosome may fail to separate during anaphase. Mitosis continues in the cells of bud and it grows into a new individual. For example. In non-disjunction. For example.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. 2. .5. resulting in binucleated cells. 2. they fail to complete cell division and retain both nuclei in one cell. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. and the latter cell having only one chromosome (the homologous chromosome). especially during early cellular divisions in the zygote. the process may go wrong. One daughter cell will receive both sister chromosomes and the other will receive none. the hydra reproduces asexually by budding.Some organisms can regenerate their parts of bodies. The production of new cells is achieved by mitosis.

It results in the synthesis of execessive tissue growths. but in reverse orientation. . it results in the formation of Tumors. sometimes mutuations occur in such genes and cells continue to divide. it may be treated erroneously as a separate chromosome. which goes through dramatic changes in ultrastructure. Benign tumours are not harmful as soon as they are not moving. Errors in the control of mitosis may cause cancer. chromosomes may become damaged.Mitosis is a demanding process for the cell. When tissues more than the requirement are synthesized in a single organ. causing chromosomal duplication. Such tumours can send cancer cells to other parts in body where new tumours may form. An arm of the chromosome may be broken and the fragment lost. The fragment may incorrectly reattach to another. and chromosomes are jostled constantly by probing microtubules. causing deletion. Or. It may reattach to the original chromosome. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. As soon as they start to move and invade other cells there are said to be malignant tumours. causing translocation. Occasionally. As long as these tumours remain in their original location they are called benign tumours. Now what happens is that cell abnormally continue to divide at a single place. causing inversion. This phenomenon is called metastasis or spreading of disease. non-homologous chromosome. The effect of these genetic abnormalities depends on the specific nature of the error. It results in abnormal cell growth. All cells have genes that control the timing and number of mitosis. its organelles disintegrate and reform in a matter of hours.

This process may also be referred to as endoreduplication and the cells as endoploid. An example of a cell that goes through endomitosis is the megakaryocyte. Preceded by events in prometaphase and followed by anaphase. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). from the ancient Greek(between) and (stage). align in the middle of the cell before being separated into each of the two daughter cells. resulting in cells with many copies of the same chromosome occupying a single nucleus. only roughly lining up along the middleline. carrying genetic information. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. analogous to a tug of war between equally strong people. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. Metaphase accounts for approximately 4% of the cell cycle's duration. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. In certain types of cells.7 Metaphase Metaphase. Early events of metaphase can .2. an imaginary line that is equidistant from the two centrosome poles. 2.

securin. produces a pattern of in total up to several hundred bands. as chromosomes with connected kinetochores will start the events of metaphase individually before other chromosomes with unconnected kinetochores that are still lingering in the events of prometaphase. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). when every kinetochore is properly attached to a bundle of microtubules. which makes them most suitable for visual analysis. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Chromosomes are condensed(Thickened) and highly coiled in metaphase.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. Such a signal creates the mitotic spindle checkpoint. For classical cytogenetic analyses. Only after all chromosomes have become aligned at the metaphase plate. 2. Metaphase chromosomes make the classical picture of chromosomes (karyotype). does the cell enter anaphase.coincide with the later events of prometaphase. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. Staining of the slides. and separase. often with Giemsa (G banding) or Quinacrine. Normal metaphase spreads are used in . This would be accomplished by regulation of the anaphase-promoting complex. One of the cell cycle checkpoints occurs during prometaphase and metaphase.

for example. such as bcr-abl in chronic myelogenous leukemia. losses of chromosomal segments or translocations. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. which may lead to chimeric oncogenes. . Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments.

or may not. and any other physical characteristics. in normal diploid organisms. Attention is paid to their length. autosomal chromosomes are present in two copies. taxonomic relationships. or an individual organism. to study chromosomal aberrations. So. Karyogram of human male using Giemsa staining. The study of karyotypes is important for cell biology and genetics. in humans 2n = 46. and what they look like under a light microscope. and the results may be used in evolutionary biology and medicine. [4] The preparation and study of karyotypes is part of cytogenetics. and to gather information about past evolutionary events. the position of the centromeres. ordered by size and position of centromere for chromosomes of the same size. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. Thus. . The term is also used for the complete set of chromosomes in a species. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. any differences between the sex chromosomes. such as. The study of whole sets of chromosomes is sometimes known as karyology. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). Karyotypes describe the number of chromosomes. banding pattern.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. Karyotypes can be used for many purposes. be sex chromosomes. cellular function. There may.

Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. in 1882. in contrast to their genic contents. Pretreating cells in a hypotonic solution. and he correctly insisted on humans having an XX/XY system. von Waldeyer in 1888. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. New techniques were needed to definitively solve the problem: 1. concluding an XX/XO sex determination mechanism. The next stage took place after the development of genetics in the early 20th century. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. which swells them and spreads the chromosomes . He revised his opinion later from 46 to 48.3. at first favoring 46. Using cells in culture 2. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. these results were quite remarkable. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. The subsequent history of the concept can be followed in the works of Darlington and White. Considering their techniques. the discoverer of mitosis. Their behavior in animal (salamander) cells was described by Walther Flemming. The name was coined by another German anatomist.

reducing the number. [16] Sometimes observations may be made on non-dividing (interphase) cells. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Arresting mitosis in metaphase by a solution of colchicines 4. 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: . such as Giemsa.2.2. For humans. 3.2 Observations on karyotypes 3. Usually. Human chromosome 2 was formed by a merger of ancestral chromosomes. 3. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly. The sex of an unborn fetus can be determined by observation of interphase cells. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. a suitable dye.1 Staining The study of karyotypes is made possible by staining.3. the great apes have 48 chromosomes.

and mainly consists of genetically inactive repetitive DNA sequences. indicating tighter packing. faba chromosomes are many times larger. which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. 4. Differences in absolute sizes of chromosomes. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. Differences in number and position of satellites. 6. 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). .1. type. both have six pairs of chromosomes (n=6) yet V. A full account of a karyotype may therefore include the number. 3. Humans have one pair fewer chromosomes than the great apes. Heterochromatin stains darker than euchromatin. This is brought about by translocations. 2. as well as other cytogenetic information. Differences in the position of centromeres. but the genes have been mostly translocated (added) to other chromosomes. This feature probably reflects different amounts of DNA duplication. Differences in degree and distribution of heterochromatic regions. shape and banding of the chromosomes. 5.

which are highly variable. Normal karyotypes for females contain two X chromosomes and are denoted 46. Mosaics or otherwise abnormal individuals. Between the sexes 2. Between the germ-line and soma (between gametes and the rest of the body) 3. and in . Any variation from the standard karyotype may lead to developmental abnormalities. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Geographical variation between races 5. XX.3 The human karyotype Most (but not all) species have a standard karyotype. Between members of a population (chromosome polymorphism) 4. There is variation between species in chromosome number. XY. the same cannot be said for their karyotypes. 3. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. males have both an X and a Y chromosome denoted 46.Variation is often found: 1.

Godfrey and Masters conclude: "In our view.. In this process. In A. In some cases there is even significant variation within species. In some species. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. which were previously inexplicable. despite their construction from the same macromolecules. despite many careful investigations. In a review. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost.. Chromosome elimination. found in some copepods and roundworms such as Ascaris suum. it is quite unclear what the general significance might be. portions of the chromosomes are cast away in particular cells. Although much is known about karyotypes at the descriptive level. 3. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species.. used in conjunction with other phylogenetic data. But.3.1 Changes during development Instead of the usual gene repression. Chromatin diminution (founding father: Theodor Boveri). some organisms go in for large-scale elimination of heterochromatin. .detailed organization. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. This variation provides the basis for a range of studies in evolutionary cytology. the general significance of karyotype evolution is obscure. as in many sciarid flies.. entire chromosomes are eliminated during development. or other kinds of visible adjustment to the karyotype. "We have a very poor understanding of the causes of karyotype evolution.

all telocentric. In marsupials it is always the paternal X which is inactivated. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. In human females some 15% of somatic cells escape inactivation. Muntiacus reevesi. They kept quiet for two or three years because they thought something was wrong with their tissue culture. they were astonished to find it had female = 6. 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.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac.. Muntiacus muntjak.suum.. The low record is held by the nematode Parascaris univalens. 3. "They simply could not believe what they saw.. male = 7 chromosomes. all the somatic cell precursors undergo chromatin diminution.. the inactivation is random as between the two Xs. the high record would be somewhere amongst the ferns. which was investigated by Kurt Benirschke and his colleague Doris Wurster. was found to be 46.3. The diploid number of the Chinese muntjac. The existence of supernumerary or B chromosomes . In placental mammals. 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). thus the mammalian female is a mosaic in respect of her X chromosomes. where the haploid n = 1. Xinactivation. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes.

where there are more than two sets of homologous chromosomes in the cells.means that chromosome number can vary even within one interbreeding population. 14. Thus. FN. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. Humans have FN = 82. Polyploidy in animals is much less common. but it has been significant in some groups. and aneuploids are another example. occurs mainly in plants. 21 and 22).3 Fundamental number The fundamental number. FN ≤ 2n.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Haplo-diploidy. It has been of major significance in plant evolution according to Stebbins. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. 3. and in some other groups. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. Polyploidy in lower plants (ferns. horsetails and psilotales) is also common. 3.3. where one sex is diploid. Polyploidy. and the other haploid.Endopolyploidy occurs when in adult differentiated . but in grasses the average is much higher. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. though in this case they would not be regarded as normal members of the population. about 70%. 15. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. It is a common arrangement in the Hymenoptera. due to the presence of five acrocentric chromosome pairs (13.

it is diverse and complex. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. 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. Down syndrome and Turner syndrome are examples of this. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. but the nuclei contain more than the original somatic number of chromosomes. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication.tissues the cells have ceased to divide by mitosis. Abnormalities in chromosome number usually cause a defect in development. . and serves differentiation and morphogenesis in many ways. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). the daughter chromosomes separating from each other inside an intact nuclear membrane. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. See palaeopolyploidy for the investigation of ancient karyotype duplications. 3. In many instances. The cells do not always contain exact multiples (powers of two).

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.000 km2).Aneuploidy may also occur within a group of closely related species. reducing the number. 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. Well-researched examples are the ladybird beetle Chilocorus stigma. and 7. When this happens. In about 6.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. 3. living from rainforests to . 4. the European shrew Sorex araneus. Classic examples in plants are the genus Crepis. some mantids of the genus Ameles. Human chromosome 2 was formed by a merger of ancestral chromosomes. [41] Closer to home. where every number from x = 3 to x = 15 is represented by at least one species. where the gametic (= haploid) numbers form the series x = 3. that the two chromosome morphs are adapted to different habitats. 6. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. the great apes have 24x2 chromosomes whereas humans have 23x2. 5. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. and Crocus.500 sq mi (17. 3.

4 million years ago (mya) (Mauna Kea) to 10mya (Necker). in the family Drosophilidae. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. Chromosome rearrangements. it is more likely to have been a group from the same species. probably 20 million years ago. The inversions. and skipping of islands. at least into the Cretaceous. . These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. but these are much less frequent. 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. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. Drosophila and Scaptomyza. gene arrangements are visible in the banding patterns of each chromosome. The results are clear. which can be dated to 30 mya. the present islands date from 0. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. Although it would be possible for a single gravid female to colonise an island. the best-studied group of Hawaiian drosophilids. There are also cases of colonization back to older islands. especially inversions. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer.subalpine meadows. Using K-Ar dating. The polytene banding of the 'picture wing' group. In a sense. when plotted in tree form (and independent of all other information). make it possible to see which species are closely related. enabled Carson to work out the evolutionary tree long before genome analysis was practicable.

The pattern of bands is very similar to that seen in G-banding.7.the dark regions tend to be heterochromatic. This method will normally produce 300-400 bands in a normal. . The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). late-replicating and AT rich. human genome. R-banding is the reverse of G-banding (the R stands for "reverse").There are other animals and plants on the Hawaiian archipelago which have undergone similar. early-replicating and GC rich. It yields a series of lightly and darkly stained bands .7 Depiction of karyotypes 3. • • C-banding: Giemsa binds to constitutive heterochromatin.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. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. if less spectacular. so it stains centromeres. 3. adaptive radiations. • T-banding: visualize telomeres. The light regions tend to be euchromatic.

both chromosomes in a pair will have the same banding pattern. less frequently Quinacrine. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. This yields a dark region where the silver is deposited. In the "classic" (depicted) karyotype. a dye. For example. is used to stain bands on the chromosomes. denoting the activity of rRNA genes within the NOR.7. Karyotypes are arranged with the short arm of the chromosome on top. 3. Cri du chat syndrome involves a deletion on the short arm of . often Giemsa (G-banding). Some karyotypes call the short and long arms p and q. In addition. respectively. Giemsa is specific for the phosphate groups of DNA. Each chromosome has a characteristic banding pattern that helps to identify them.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. Quinacrine binds to the adeninethymine-rich regions. and the long arm on the bottom.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein.

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 method is also known as virtual karyotyping.7.chromosome 5. which is written as 46.8 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. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. allowing the visualization of the individually colored chromosomes. . Image processing software then assigns a pseudo color to each spectrally different combination. a combinatorial labeling method is used to generate many different colors. The critical region for this syndrome is deletion of 15.5p-.2) 3.del(5)(p15. It is written as 46.XX. 3. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.2. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.XX.

CHAPTER 4 .

4. or structural. the most common male chromosomal disease. is caused by trisomy of chromosome 21. Klinefelter syndrome. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. Some disorders arise from loss of just a piece of one chromosome. Down syndrome. trisomies. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. also known as aneuploidy. inversions. Also documented are trisomy 8. a common chromosomal disease. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. as in derivative chromosome. are common numerical abnormalities. translocations. otherwise known as 47. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. X0). large-scale deletions or duplications. Numerical abnormalities. including . Structural abnormalities often arise from errors in homologous recombination. trisomy 9 and trisomy 16.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. XXY is caused by an extra X chromosome. often occur as a result of nondisjunction during meiosis in the formation of a gamete. although they generally do not survive to birth. as in the presence of extra or missing chromosomes. • • Patau syndrome is caused by trisomy of chromosome 13. in which three copies of a chromosome are present instead of the usual two. X or 45.

1p36 Deletion syndrome. numerical and structural anomalies. The name comes from the babies' distinctive cry.• Cri du chat (cry of the cat). a deletion of the maternal genes. example of imprinting disorder. 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. caused by abnormal formation of the larynx. from the loss of part of the short arm of chromosome 1. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. 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. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. They can be organized into two basic groups. . A chromosome anomaly may be detected or confirmed in this manner. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. example of imprinting disorder. A chromosome anomaly. from a truncated short arm on chromosome 5. one well-documented example is the Philadelphia chromosome. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. a deletion of the paternal genes. There are many types of chromosome anomalies.

Known disorders in humans include Wolf-Hirschhorn syndrome.). • • Translocations: When a portion of one chromosome is transferred to another chromosome. In a Robertsonian translocation. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. and Jacobsen syndrome. resulting in extra genetic material. Duplications: A portion of the chromosome is duplicated. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.3 Structural abnormalities When the chromosome's structure is altered. an entire chromosome has . segments from two different chromosomes have been exchanged. There are two main types of translocations. also called the terminal 11q deletion disorder. rather than two). Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. an X. 4. In a reciprocal translocation. Tetrasomy. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. etc.4. which is caused by partial deletion of the short arm of chromosome 4.

in humans these only occur with chromosomes 13. Therefore.attached to another at the Centromere . can happen after conception. other cytogenetic banding techniques. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. This can happen with or without loss of genetic material. 4. resulting in Mosaicism (where some cells have the anomaly and some do not). Some anomalies.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. 15. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 4. Chromosome anomalies can be inherited from a parent or be "de novo". It includes routine analysis of G-Banded chromosomes. • Rings: A portion of a chromosome has broken off and formed a circle or ring. however.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. They often lead to an increased tendency to develop certain types of malignancies. especially the chromosomes. and are therefore initially not inherited. • Inversions: A portion of the chromosome has broken off. the anomaly is present in every cell of the body. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. 14. 21 and 22. turned upside down and reattached. as well . therefore the genetic material is inverted.

Painter in 1922 was not certain whether the diploid number of man was 46 or 48. The next stage took place after the development of genetics in the early 20th century. 4.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). at first favoring 46. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. He revised his opinion later from 46 to 48. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. concluding an XX/XO sex determination mechanism. Their behavior in animal (salamander) cells was described by Walther Flemming. Considering their techniques. Using cells in culture 2. and he correctly insisted on man having an XX/XY system. the discoverer of mitosis. New techniques were needed to definitively solve the problem: 1. these results were quite remarkable. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in 1882. Pre-treating cells in a hypotonic solution.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. in contrast to their genic contents. which swells them and spreads the chromosomes . The name was coined by another German anatomist. von Waldeyer in 1888.

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. reducing the number. Arresting mitosis in metaphase by a solution of colchicine 4. persimilis from wild populations in California and neighboring states. a find which eventually led to her Nobel Prize in 1983. the great apes have 48 chromosomes.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. During her cytogenetic work. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Natural populations of Drosophila In the 1930s. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. In 1931. 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Rather interestingly. 4.6 Applications in biology 4.6.6.3.

but adjust to certain frequencies at which they become stabilised. as with most polymorphisms. It was found that the various chromosome types do not fluctuate at random. In 1959. . Abnormalities arising from nondisjunction events can cause cells with aneuploidy (additions or deletions of entire chromosomes) in one of the parents or in the fetus. such as Down's syndrome. 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. breeding and sampling whilst preventing escape. In some congenital disorders. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. which enabled feeding. Using a method invented by L'Heretier and Teissier. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. Down syndrome is also referred to as trisomy 21. Dobzhansky bred populations in population cages.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. as they would if selectively neutral. Evidence rapidly accumulated to show that natural selection was responsible. 4. This had the benefit of eliminating migration as a possible explanation of the results. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. discoveries were quickly made related to aberrant chromosomes or chromosome number.

which is required in normal females to compensate for having two copies of the chromosome. 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. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. An individual with only one sex chromosome (the X) has Turner syndrome. has Klinefelter's Syndrome. in addition to other tests. is used today as a diagnostic for CML. Thirteen years later. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. with the development of more advanced techniques.as both scientists were doing their research in Philadelphia. Many other sex chromosome combinations are compatible with live birth including XXX. XYY. Not all genes on the X Chromosome are inactivated. Identification of the Philadelphia chromosome by cytogenetics. resulting in 47 total chromosomes.Other numerical abnormalities discovered include sex chromosome abnormalities. Peter Nowell and David Hungerford [17] discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). an additional X chromosome in a male. Pennsylvania. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. In 1960.

Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 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.8 Beginnings of molecular cytogenetics . and elongation techniques for all culture types that allow for higher resolution banding. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. Deletion syndromes such as DiGeorge syndrome. Caspersson developed banding techniques which differentially stain chromosomes. 4.FIG Advent of banding techniques In the late 1960s. 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.

This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.In the 1980s. movement was now made in using fluorescent labeled probes. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. CHAPTER 5 Techniques 5.1 Karyotyping . advances were made in molecular cytogenetics. 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. cloned and studied in ever greater detail.

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,

you can solve technical computing problems faster than with traditional programming languages. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. such as C. C++. data visualization. and computational biology. including signal and image processing. and FORTRAN.generally between 200 and 1000 cells are counted and scored. such as comparative genomic hybridization arrays. financial modeling and analysis. For congenital problems usually 20 metaphase cells are scored. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. data analysis. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. . communications. Using MATLAB. and numerical computation. You can use MATLAB in a wide range of applications. control design. test and measurement. CGH and Single nucleotide polymorphism-arrays.

. With the MATLAB language. and distribute your MATLAB algorithms and applications. 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. specifying data types. and allocating memory. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. In many cases. such as declaring variables. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems.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. You can integrate your MATLAB code with other languages and applications. The image processing step is composed of the following operations. one line of MATLAB code can often replace several lines of C or C++ code. These effects must be compensated to improve the results of the pairing algorithm. MATLAB eliminates the need for ‘for’ loops. As a result. 6.MATLAB provides a number of features for documenting and sharing your work. It enables fast development and execution.

6. 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. or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. Therefore. To compare chromosomes from a band pattern point of view. To compensate for this inhomogeneity. 2) Geometrical compensation—The geometric compensation.2 Concepts used in this phase 1) Image conversion 2) Denoising . the spatially scaled images are histogram equalized. 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. 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).

MATLAB filters the intensity values in the image. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. if you want to filter a color image that is stored as an indexed image. . If you attempt to filter the indexed image. For example. and the results might not be meaningful. green. so the image displays as shades of gray. 6. you must first convert it to true color format. RGB = cat (3. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations.I. You can perform certain conversions just using MATLAB syntax. MATLAB simply applies the filter to the indices in the indexed image matrix. there are other functions that return a different image type as part of the operation they perform. For example. as is appropriate.3) Edge detection 4) Two dimensional convolutions.I). listed in the following table. and blue planes. In addition to these image type conversion functions.2.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. When you apply the filter to the true color image. The resulting true color image has identical matrices for the red.I. For example.

There is a large number of edge finding algorithms in existence. via satellite or wireless transmission. or through networked cable.6. If one of these matrices describes a two-dimensional finite impulse response . we may expect errors to occur in the image signal. Usually we know what type of errors to expect. and we shall look at some of the more straightforward of them. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. hence we can choose the most appropriate method for reducing the effects. to recognize or classify objects. to isolate particular objects from their background.2. caused by external disturbance. The general Matlab command for finding edges is edge(image.4 Denoising We may define noise to be any degradation in the image signal. .B) computes the two-dimensional convolution of matrices A and B.parameters.2. 6. and hence the type of noise on the image.3 Two dimensional convolutions C = conv2(A.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. We may use edges to measure the size of objects in an image. ) Where the parameters available depend on the method used 6. Cleaning an image corrupted by noise is thus an important area of image restoration.'method'.

A). If hcol is a column vector and hrow is a row vector.7).bmp'). minus one. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. That is. nb]+1)/2)..(FIR) filter.0.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. 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.. .na] and the size of B is [mb. imedfilt2(im1.nb].'shape') subsection of the two-dimensional convolution. The size of matrices. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.'sobel').hrow. edge(im1. rgb2gray im2bw(im.[3 3]). the other matrix is filtered in two dimensions.. this case is the same as C = conv2(hcol*hrow. C = conv2(.na+nb-1].

flag=0. bwlabel(B. for i=1:sx x1=rc(i. n1(x1. Msk conv2(double(BW). [r. [sx sy]=size(rc). L_number=zeros(mx. MODULE 2 clc [m. mx=max(max(L)).double(msk)).1). Index=1.imy]=size(BW).1). for i=1:m for j=1:n if L(i. nzeros(imx. .j)==L_number(k) flag=1.[imx.2).y1)=255. rc = [r c].n]=size(L).j)~=0 for k=1:mx if L(i.c] = find(L==22).8). y1=rc(i.imy).

10.66].19.60. end flag=0.35.8.c] = find(L==L_number((Test_number(x)))).imy).21.24. .56. end end L_number.54. n1(x1.65.1).40.48. Test_number=[3.6. for i=1:sx x1=rc(i. for x=1:46 [r.14. 36.52.43.31. rc = [r c].28. end %h=figure. Index=Index+1.59.4.27.7.32.[]).50.20.2).15.22.57. n1=zeros(imx. [sx sy]=size(rc).49.29.j).26.39.11. y1=rc(i.imshow(n1.9.end end if flag~=1 L_number(Index)=L(i.30.38. end.62.33.y1)=255.41.51.45.55.42.

y)==1 Circumference_sum=Circumference_sum+1. s=bwmorph(skel. [m n]=size(BW1).end Circumference=zeros(46. s1=bwmorph(s.'skel'. for i=1:46 f=imread(strcat(num2str(i).1). skel=im2double(f). BW=double(BW).'spur'.'. for x=1:m for y=1:n if BW1(x.8). end end end Circumference(i)=Circumference_sum. BW=im2bw(f). Circumference_sum=0.1).'canny'). skel=im2bw(skel.Inf). Arm_length=zeros(46. Area=zeros(46. BW1=edge(BW.bmp')).1.5*graythresh(skel)). .1). Arm_length_sum=0. f=imcomplement(f).

end end end Arm_length(i)=Arm_length_sum. Arm_length. for x=1:m for y=1:n if BW(x. [m n]=size(BW). BW=im2bw(f). end Circumference.[m n]=size(s1).y)==1 Arm_length_sum=Arm_length_sum+1. end end end Area(i)=Area_sum. for x=1:m for y=1:n if s1(x.y)==1 Area_sum=Area_sum+1. Area_sum=0. .

1)=i.2)=j. Pair(i. Pair(i. Pair(i.1)=i.2). for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)).2)=i. Pair=zeros(46. Pair(46. end end Pair.1)=46.2)==46 Pair(46. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)). Pair(i. .2)=i+1.Area. end end end for i=1:45 if Pair(i.

if figure_flag~=47 subplot(23. imshow(f1). imshow(f2).'. flag=0.2. . figure_flag=figure_flag+1. for i=1:46 for j=1:46 if Pair(i.figure_flag).2)). end f1=imread(strcat(num2str(Pair(i. end flag=0.1).2.delete=zeros(46. figure_flag=figure_flag+1.bmp')).bmp')).figure_flag). delete(figure_flag)=Pair(i.1)).1)==delete(j) flag=1.'.2). figure_flag=1. end f2=imread(strcat(num2str(Pair(i. end end if flag~=1 if figure_flag~=47 subplot(23.

Copenhagen. based on the MI. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. such as. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. The proposed algorithm is based on the traditional features extracted from the karyogram. and Philadelphia. 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. plus a new one. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. dimensions and banding profiles.end CONCLUTION In this paper. 2) feature extraction from the processed images . The images of these chromosomes present less quality and level of detail than the ones usually used in traditional genetic analysis using datasets such as Edinburgh. in the scope of karyotyping process used in cytogentic analysis.

In the image processing step.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. 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).characterizing the size. The features extracted from the processed images discriminate each pair with respect to their size. achieves a 70. and to normalize their dimensions. shape and band pattern. Vectors of coefficients associated with each one of the 22 classes are computed and the distance between two arbitrary chromosomes is the minimum one among all distances obtained with these 22 vectors. shape. The training process consists in the estimation of each vector of coefficient . extracted from the unordered karyogram. are processed in order to compensate for geometrical and intensity distortions. Here. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. from the chromosomes in the training set.10% mean classification rate. and finally. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. 4) pairing. the romosome images. The addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the . Tests using 19 karyograms based on bone marrow cells. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. and band pattern.working within an 8-D feature space. 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. This normalization is needed to make it possible the band pattern comparison between chromosomes.

e. presenting a uniform level of condensation. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. Using 27 karyograms andworking with a limited number of classes (≤ 8). This dataset was made publicly available [29]. amean classification rate larger than 93% was obtained in all experiments. Executing the algorithm on a higher quality dataset. centromere position. REFERENCES . or Philadelphia. a 76. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. despite the low quality of this type of chromosomes. called LK1 . whose images are of significantly higher quality..performance of the classifier. such as Edinburgh. and from which it is possible to extract additional features. In addition. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes.10% classification ratewas obtained.g. a new chromosome dataset with 9200 chromosomes from bone marrow cells. In fact. Copenhagen. The results presented in this paper are promising.

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