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

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

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

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

cytoplasm. bone marrow in large bones produces new blood cells. [1] Bone marrow is also a key component of the lymphatic system.6 kg (5. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. . in adults weighing 65 kg (143 lbs).1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. On average. which divides the nuclei.7 lbs).5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. which use the bone marrow vasculature as a conduit to the body's systemic circulation.1. It is generally followed immediately by cytokinesis. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. bone marrow accounts for approximately 2. in two separate nuclei. In humans. producing the lymphocytes that support the body's immune system CHAPTER 2 2. bone marrow constitutes 4% of the total body mass of humans.

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

The sister chromatids are held together by a specialized region of the chromosome known as the centromere. 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 parent cell must make a copy of each chromosome before mitosis. and together the two are called sister chromatids. This occurs during the S phase of interphase. . The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. Because each resultant daughter cell should be genetically identical to the parent cell.

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

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

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

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

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

. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". convene along the metaphase plate or equatorial plane. As a result. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.In the fishing pole analogy. In certain types of cells. analogous to a tug-of-war between people of equal strength. 2. the chromosomes come under longitudinal tension from the two ends of the cell. the kinetochore would be the "hook" that catches a sister chromatid or "fish". Metaphase comes from the Greek meaning "after." Microtubules find and attach to kinetochores in prometaphase. only roughly lining up along the midline. The centromeres of the chromosomes. an imaginary line that is equidistant from the two centrosome poles. All chromosomes (blue) but one have arrived at the metaphase plate. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell.3 Metaphase A cell in late metaphase. in some sense.

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

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

Following are the occasions in the lives of organism where mitosis happens: 2. 2.3 Cell replacement In some parts of body.g. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. The end of cytokinesis marks the end of the M-phase.2 Development and growth The number of cells within an organism increases by mitosis. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. Each daughter cell has a complete copy of the genome of its parent cell.5. zygote and also the basis of the growth of a multicellular body. separating the two nuclei.5. New cells are formed by mitosis and so are exact copies of the cells being replaced. e. 2.5. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. which move along microtubules to the middle of the cell. This is the basis of the development of a multicellular body from a single cell i..1Significance Mitosis is important for the maintenance of the chromosomal set. 2. whereas some green algae use a phycoplast microtubule array during cytokinesis.e. skin and digestive tract. cells are constantly sloughed off and replaced by new ones. Similarly. The phragmoplast is a microtubule structure typical for higher plants.division is also driven by vesicles derived from the Golgi apparatus.5.4 Regeneration .

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

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

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

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

.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. for example. 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. such as bcr-abl in chronic myelogenous leukemia. losses of chromosomal segments or translocations.

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

the discoverer of mitosis. in 1882. at first favoring 46. The name was coined by another German anatomist. The subsequent history of the concept can be followed in the works of Darlington and White. and he correctly insisted on humans having an XX/XY system. Using cells in culture 2. concluding an XX/XO sex determination mechanism. which swells them and spreads the chromosomes . Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912.3. Their behavior in animal (salamander) cells was described by Walther Flemming. von Waldeyer in 1888. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. in contrast to their genic contents. New techniques were needed to definitively solve the problem: 1. Pretreating cells in a hypotonic solution. these results were quite remarkable. He revised his opinion later from 46 to 48. The next stage took place after the development of genetics in the early 20th century. Considering their techniques.1 History of karyotype studies 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.

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

and mainly consists of genetically inactive repetitive DNA sequences. 5. type. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. This feature probably reflects different amounts of DNA duplication. Differences in number and position of satellites.1. as well as other cytogenetic information. Humans have one pair fewer chromosomes than the great apes. This is brought about by translocations. 4. Heterochromatin stains darker than euchromatin. . Differences in degree and distribution of heterochromatic regions. 6. 3. permitting its loss without penalty to the organism (the dislocation hypothesis). A full account of a karyotype may therefore include the number. but the genes have been mostly translocated (added) to other chromosomes. shape and banding of the chromosomes. both have six pairs of chromosomes (n=6) yet V. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). 2. faba chromosomes are many times larger. which (when they occur) are small bodies attached to a chromosome by a thin thread. indicating tighter packing. Differences in the position of centromeres. Differences in absolute sizes of chromosomes. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths.

which are highly variable. the same cannot be said for their karyotypes. There is variation between species in chromosome number. XY. XX. Mosaics or otherwise abnormal individuals. males have both an X and a Y chromosome denoted 46. Geographical variation between races 5.3 The human karyotype Most (but not all) species have a standard karyotype. Between the germ-line and soma (between gametes and the rest of the body) 3. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. and in . 3. Between the sexes 2.Variation is often found: 1. Any variation from the standard karyotype may lead to developmental abnormalities. Between members of a population (chromosome polymorphism) 4. Normal karyotypes for females contain two X chromosomes and are denoted 46. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes.

In A. which were previously inexplicable. "We have a very poor understanding of the causes of karyotype evolution. .3. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. entire chromosomes are eliminated during development. In some species. despite many careful investigations. Although much is known about karyotypes at the descriptive level. some organisms go in for large-scale elimination of heterochromatin. despite their construction from the same macromolecules. In a review. But. it is quite unclear what the general significance might be.1 Changes during development Instead of the usual gene repression. the general significance of karyotype evolution is obscure. In some cases there is even significant variation within species. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. portions of the chromosomes are cast away in particular cells. This variation provides the basis for a range of studies in evolutionary cytology.. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. used in conjunction with other phylogenetic data. Chromosome elimination. 3... as in many sciarid flies.. In this process. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. found in some copepods and roundworms such as Ascaris suum. Godfrey and Masters conclude: "In our view. or other kinds of visible adjustment to the karyotype.detailed organization. Chromatin diminution (founding father: Theodor Boveri).

. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). male = 7 chromosomes. In human females some 15% of somatic cells escape inactivation. Muntiacus reevesi. The diploid number of the Chinese muntjac.suum. When they looked at the karyotype of the closely related Indian muntjac. In marsupials it is always the paternal X which is inactivated. all the somatic cell precursors undergo chromatin diminution. The existence of supernumerary or B chromosomes . Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. the high record would be somewhere amongst the ferns.. Xinactivation.3. Muntiacus muntjak. "They simply could not believe what they saw. They kept quiet for two or three years because they thought something was wrong with their tissue culture. where the haploid n = 1. which was investigated by Kurt Benirschke and his colleague Doris Wurster. was found to be 46.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac... all telocentric. thus the mammalian female is a mosaic in respect of her X chromosomes. they were astonished to find it had female = 6. 3. In placental mammals. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. the inactivation is random as between the two Xs. The low record is held by the nematode Parascaris univalens.

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

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

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

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

• • C-banding: Giemsa binds to constitutive heterochromatin.7 Depiction of karyotypes 3.1 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. It yields a series of lightly and darkly stained bands . • Q-banding is a fluorescent pattern obtained using quinacrine for staining. so it stains centromeres. • T-banding: visualize telomeres. if less spectacular. human genome. R-banding is the reverse of G-banding (the R stands for "reverse"). The pattern of bands is very similar to that seen in G-banding. early-replicating and GC rich. adaptive radiations.7. late-replicating and AT rich. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). The light regions tend to be euchromatic. . This method will normally produce 300-400 bands in a normal.the dark regions tend to be heterochromatic. 3.There are other animals and plants on the Hawaiian archipelago which have undergone similar.

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

7.2. which is written as 46. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough. allowing the visualization of the individually colored chromosomes.XX. .del(5)(p15.chromosome 5. The critical region for this syndrome is deletion of 15. Image processing software then assigns a pseudo color to each spectrally different combination.2) 3. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.5p-. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. This method is also known as virtual karyotyping. It 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. Because there are a limited number of spectrally-distinct fluorophores. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. a combinatorial labeling method is used to generate many different colors.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX. 3.

CHAPTER 4 .

1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. as in derivative chromosome. the most common male chromosomal disease.4. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. also known as aneuploidy. Also documented are trisomy 8. Structural abnormalities often arise from errors in homologous recombination. Down syndrome. or structural. XXY is caused by an extra X chromosome. X or 45. X0). Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. in which three copies of a chromosome are present instead of the usual two. Klinefelter syndrome. as in the presence of extra or missing chromosomes. Numerical abnormalities. trisomies. a common chromosomal disease. often occur as a result of nondisjunction during meiosis in the formation of a gamete. although they generally do not survive to birth. inversions. Some disorders arise from loss of just a piece of one chromosome. large-scale deletions or duplications. translocations. are common numerical abnormalities. including . • • Patau syndrome is caused by trisomy of chromosome 13. is caused by trisomy of chromosome 21. trisomy 9 and trisomy 16. otherwise known as 47. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18.

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

In humans an example of a condition caused by a numerical anomaly is Down Syndrome. rather than two).). • • Translocations: When a portion of one chromosome is transferred to another chromosome. an X. In a Robertsonian translocation. etc. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. which is caused by partial deletion of the short arm of chromosome 4.4. In a reciprocal translocation. also called the terminal 11q deletion disorder. 4. Known disorders in humans include Wolf-Hirschhorn syndrome. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. There are two main types of translocations. resulting in extra genetic material. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). Tetrasomy. 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. Duplications: A portion of the chromosome is duplicated. and Jacobsen syndrome.3 Structural abnormalities When the chromosome's structure is altered. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. an entire chromosome has . segments from two different chromosomes have been exchanged.

4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. can happen after conception. the anomaly is present in every cell of the body. Therefore. however. 4. as well .attached to another at the Centromere . especially the chromosomes. 14.in humans these only occur with chromosomes 13. This can happen with or without loss of genetic material. It includes routine analysis of G-Banded chromosomes. • Rings: A portion of a chromosome has broken off and formed a circle or ring. therefore the genetic material is inverted. other cytogenetic banding techniques. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. Some anomalies. Chromosome anomalies can be inherited from a parent or be "de novo". turned upside down and reattached. 4. 21 and 22. • Inversions: A portion of the chromosome has broken off. 15.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. They often lead to an increased tendency to develop certain types of malignancies. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. resulting in Mosaicism (where some cells have the anomaly and some do not). and are therefore initially not inherited.

Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. The name was coined by another German anatomist. New techniques were needed to definitively solve the problem: 1.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. The next stage took place after the development of genetics in the early 20th century. at first favoring 46. which swells them and spreads the chromosomes . von Waldeyer in 1888. these results were quite remarkable. concluding an XX/XO sex determination mechanism. Using cells in culture 2. He revised his opinion later from 46 to 48.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). and he correctly insisted on man having an XX/XY system. Their behavior in animal (salamander) cells was described by Walther Flemming. Pre-treating cells in a hypotonic solution. Considering their techniques. in 1882. in contrast to their genic contents. 4. 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. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.

McClintock discovered transposons. the great apes have 48 chromosomes. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).3.2 Natural populations of Drosophila In the 1930s. Arresting mitosis in metaphase by a solution of colchicine 4. reducing the number.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. 4. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Rather interestingly. 4. Using Painter's technique they studied the polytene . Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. a find which eventually led to her Nobel Prize in 1983. Human chromosome 2 was formed by a merger of ancestral chromosomes.6 Applications in biology 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. In 1931. persimilis from wild populations in California and neighboring states. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. During her cytogenetic work.6.6.

It was found that the various chromosome types do not fluctuate at random. This had the benefit of eliminating migration as a possible explanation of the results. 4. Dobzhansky bred populations in population cages. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. as they would if selectively neutral. as with most polymorphisms. Down syndrome is also referred to as trisomy 21. but adjust to certain frequencies at which they become stabilised. In 1959. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. 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. such as Down's syndrome. In some congenital disorders. Evidence rapidly accumulated to show that natural selection was responsible. breeding and sampling whilst preventing escape. . 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.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. which enabled feeding. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21.

Many other sex chromosome combinations are compatible with live birth including XXX. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. which is why there is a phenotypic effect seen in individuals with extra X chromosomes.Other numerical abnormalities discovered include sex chromosome abnormalities. in addition to other tests. with the development of more advanced techniques. In 1960. resulting in 47 total chromosomes. an additional X chromosome in a male. Pennsylvania. Peter Nowell and David Hungerford [17] discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. XYY. Not all genes on the X Chromosome are inactivated. has Klinefelter's Syndrome. is used today as a diagnostic for CML. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. Thirteen years later. This abnormal chromosome was dubbed the Philadelphia chromosome . and XXXX. . Identification of the Philadelphia chromosome by cytogenetics. An individual with only one sex chromosome (the X) has Turner syndrome. which is required in normal females to compensate for having two copies of the chromosome.as both scientists were doing their research in Philadelphia.

Deletions within one chromosome could also now be more specifically named and understood. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.8 Beginnings of molecular cytogenetics . and elongation techniques for all culture types that allow for higher resolution banding. Deletion syndromes such as DiGeorge syndrome. Caspersson developed banding techniques which differentially stain chromosomes.FIG Advent of banding techniques In the late 1960s. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. 4.

advances were made in molecular cytogenetics. While radioisotope-labeled probes had been hybridized with DNA since 1969. 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. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).1 Karyotyping . cloned and studied in ever greater detail.In the 1980s. CHAPTER 5 Techniques 5.

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

With the MATLAB language. and allocating memory. specifying data types. 6. 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. In many cases. As a result. You can integrate your MATLAB code with other languages and applications. one line of MATLAB code can often replace several lines of C or C++ code. .1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. The image processing step is composed of the following operations. These effects must be compensated to improve the results of the pairing algorithm. such as declaring variables. MATLAB eliminates the need for ‘for’ loops.MATLAB provides a number of features for documenting and sharing your work. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. It enables fast development and execution. and distribute your MATLAB algorithms and applications. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks.

geometrical and dimensional differences must be removed. 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. 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). or at least attenuated. 2) Geometrical compensation—The geometric compensation. To compare chromosomes from a band pattern point of view.2 Concepts used in this phase 1) Image conversion 2) Denoising . the spatially scaled images are histogram equalized. Therefore.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. To compensate for this inhomogeneity. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 6.

MATLAB filters the intensity values in the image. and the results might not be meaningful. and blue planes.I.I.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. For example. 6. if you want to filter a color image that is stored as an indexed image. The resulting true color image has identical matrices for the red. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. green. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension.3) Edge detection 4) Two dimensional convolutions. so the image displays as shades of gray. If you attempt to filter the indexed image. you must first convert it to true color format. You can perform certain conversions just using MATLAB syntax. listed in the following table. as is appropriate. there are other functions that return a different image type as part of the operation they perform. RGB = cat (3. For example. MATLAB simply applies the filter to the indices in the indexed image matrix. When you apply the filter to the true color image. For example.2. In addition to these image type conversion functions.I). .

'method'. Usually we know what type of errors to expect. . 6. ) Where the parameters available depend on the method used 6. and we shall look at some of the more straightforward of them.4 Denoising We may define noise to be any degradation in the image signal. hence we can choose the most appropriate method for reducing the effects.2. to isolate particular objects from their background. or through networked cable. . There is a large number of edge finding algorithms in existence.6. If an image is being sent electronically from one place to another.B) computes the two-dimensional convolution of matrices A and B.3 Two dimensional convolutions C = conv2(A. we may expect errors to occur in the image signal.2. via satellite or wireless transmission. caused by external disturbance. to recognize or classify objects. The general Matlab command for finding edges is edge(image. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. If one of these matrices describes a two-dimensional finite impulse response . and hence the type of noise on the image. We may use edges to measure the size of objects in an image.5 Edge detection Edges contain some of the most useful information in an image.parameters. Cleaning an image corrupted by noise is thus an important area of image restoration.

the other matrix is filtered in two dimensions.'shape') subsection of the two-dimensional convolution.0. imedfilt2(im1. The size of matrices. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.7). if the size of then the size of C is [ma+mb-1.na] and the size of B is [mb. nb]+1)/2).hrow.A). as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345. C = conv2(. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.. minus one. If hcol is a column vector and hrow is a row vector. .. this case is the same as C = conv2(hcol*hrow.. rgb2gray im2bw(im.bmp').'sobel').(FIR) filter.[3 3]).nb].na+nb-1]. That is. edge(im1.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.

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

65.31.1).6. Index=Index+1.35.27. y1=rc(i. .8. 36.30. end end L_number. n1=zeros(imx.43.15.11.66].7.4. [sx sy]=size(rc).51.j). end flag=0.55.33.y1)=255.45.60.39.imshow(n1.38.59.56.21.[]).26.14.54.24.62.28.imy).10.19.end end if flag~=1 L_number(Index)=L(i.20.32.42.22.2). n1(x1.50. Test_number=[3.41.57. for i=1:sx x1=rc(i. end.52.9. for x=1:46 [r.48.c] = find(L==L_number((Test_number(x)))).49.40. end %h=figure. rc = [r c].29.

for i=1:46 f=imread(strcat(num2str(i).5*graythresh(skel)).1). BW1=edge(BW. for x=1:m for y=1:n if BW1(x.y)==1 Circumference_sum=Circumference_sum+1. Area=zeros(46.end Circumference=zeros(46.8). BW=im2bw(f). [m n]=size(BW1). end end end Circumference(i)=Circumference_sum.bmp')). Arm_length_sum=0. BW=double(BW). . s=bwmorph(skel. f=imcomplement(f).'canny').1).'. skel=im2bw(skel. Arm_length=zeros(46.1. Circumference_sum=0.'spur'. s1=bwmorph(s.Inf).1). skel=im2double(f).'skel'.

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

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

imshow(f2). .figure_flag).2. end end if flag~=1 if figure_flag~=47 subplot(23.1)).2. if figure_flag~=47 subplot(23. end flag=0.1). delete(figure_flag)=Pair(i. for i=1:46 for j=1:46 if Pair(i.'.bmp')).1)==delete(j) flag=1. flag=0. end f1=imread(strcat(num2str(Pair(i.2)). end f2=imread(strcat(num2str(Pair(i.'. figure_flag=1. imshow(f1).bmp')). figure_flag=figure_flag+1.delete=zeros(46.2).figure_flag). figure_flag=figure_flag+1.

and Philadelphia. such as. The proposed algorithm is based on the traditional features extracted from the karyogram. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians.end CONCLUTION In this paper. plus a new one. 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. Copenhagen. 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. dimensions and banding profiles. based on the MI. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. 2) feature extraction from the processed images .

characterizing the size.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. extracted from the unordered karyogram. The features extracted from the processed images discriminate each pair with respect to their size. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. Tests using 19 karyograms based on bone marrow cells. by minimizing the overall distances between chromosomes of the same class (intraclass) and by maximizing the distances between chromosomes when at least one of them does not belong to that class (interclass). The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram. Here. 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 . 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. are processed in order to compensate for geometrical and intensity distortions. and band pattern. achieves a 70. and finally.10% mean classification rate.working within an 8-D feature space. shape. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. The training process consists in the estimation of each vector of coefficient . The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. 4) pairing. from the chromosomes in the training set. This normalization is needed to make it possible the band pattern comparison between chromosomes. the romosome images. shape and band pattern. In the image processing step. and to normalize their dimensions.

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

[in Russian] 6. 1922. Hist. Evolution of genetic systems. ^ a b c White M. ^ Levitsky G. 23.P Oxford & NY. Bull. 6th ed. 2nd ed.J. Variation and evolution in plants. 1924. 9. revised and enlarged. ^ King R. p.D. Animal cytology and evolution. The morphology of chromosomes. biologie 27. 3. 28 2. Stansfield W. Kiev. 8. 11. 129. Edinburgh. Med. 1974.L. The chromosomes.J. 147–49. 1950. 48. ^ Levitsky G. 7th ed. and Mulligan P. Cambridge University Press. 2006. Oxford U.1. Applied Bot. Arch. Bull. 1931. 27. From 48 to 46: cytological technique. A dictionary of genetics. ^ Concise Oxford Dictionary London. Chapman & Hall. ^ Kottler M.D. p242 5.D. Oliver & Boyd. ^ Darlington C. Columbia University Press NY. ^ Stebbins G. 1958. 7.A. 1939. Cambridge University Press. ^ von Winiwarter H.D. ^ a b White M. 1973. 10. Genet. 19-174. The material basis of heredity.K. 93.C. preconception and the counting of the human chromosomes. 1973. State Publication Office of the Ukraine. Anat. Res.A. Plant Breed. 3rd ed. 465-502. 4. Etudes sur la spermatogenese humaine. ^ Painter T. The spermatogenesis of man.S. 1912. .. Chapter XII: The Karyotype.

J. The spermatogenesis of humans. Exp. . & Tobler H. Bioessays23: 242–250.C. 1956. p218-9 20.^ a b Hsu T. Studies in mammalian spermatogenesis II. Kinetophore reproduction theory may explain rapid chromosome evolution. London. and Masters J. 1979. Springer-Verlag.pubmedcentral. Hereditas 42. The Association of Cytogenetic Technologists. Chromosome elimination in sciarid flies. PNAS 97. Chromosome stains. 1996. Zoology 37. Chromosomal evolution in higher plants. ^ Godfrey L. ^ Stebbins G.12.fcgi?artid=34032 19. ^ Tjio J.gov/articlerender.R. 13. 291-336. Raven Press. 2nd ed. 1923. 2000.L. Chromatin diminution in nematodes. ^ A preparation which includes the dyes Methylene Blue. 2001. ed.C 16. J. ^ Goday C. Oxford.S.H & Levan A. Barch. 17. Evolutionary genetics. 9821– 9823. Arnold. 1971.R. p85-6 18. 21. Bioessays18: 133–138. and Esteban M. The chromosome number of man. 1991. Human and mammalian cytogenetics: a historical perspective. ^ Maynard Smith J.B. 1-6.M. 1998. 14.^ a b Gustashaw K.http://www. In The ACT Cytogenetics Laboratory Manual 2nd ed. NY. Eosin Y and Azure-A. New York.C.nih. Bernard V. ^ Müller F. ^ Painter T. M. 15.

Exp. Science 168. ^ Matthey. Noh.. Indian Muntjac. Chapman. Park. 23. Oxford U. ^ Kim. 8th ed.1007/BF02153623. 1364-1366.K. 26. ^ King R. 1990. and Benirschke K. Retrieved 2008-03-18. Developmental biology. C. Chapter 9 24. . J. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). R. 2006. Stamford CT. Stansfield W. ^ Khandelwal S. 2001.. Botanical Journal of the Linnean Society 102: 205–217.. A dictionary of genetics.doi:10. 28. 25.F.C. 2006. Chromosome evolution in the genus Ophioglossum L.R. Retrieved 2011-03-16. 1970.D. ^ Wurster D. Nam. ^ Wyngaard G. 291: 310–16. Zool. doi:10. D. Y. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. "L'evolution de la formule chromosomiale chez les vertébrés". Muntiacus muntjak: a deer with a low diploid number. F.S. 27. J.H. & Gregory T.A. (1945-05-15). 7th ed. Sinauer Associates.A.22. ^ Gilbert S. Experientia (Basel) 1 (2): 50– 56.K.K.1007/s10228-004-0257-z..P Oxford & NY. Ichthyological Research 52 (1): 97.H. (2005).. and Mulligan P.

Sign up to vote on this title
UsefulNot useful