Chromosome Document | Mitosis | Karyotype

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

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

If the mutation involves only one or a few chromosomes in the genome (e. 1. a extra copy of human chromosome 21).+21.XX.are also found in mitochondria and chloroplasts.g.+21. . An example of aneuploidy is trisomy 21. in which an individual has 3. XX (female) or 46 XY (male). reflecting their bacterial origins. copies of 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).XY or 47.3 MUTATIONS IN CHROMOSOME NUMBER Normally. 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. the individual carrying the mutation is said to be aneuploid. Such individuals are called euploid and have the wild-type chromosome complement for the species. Euploid human karyotypes are 46. rather than 2. The individual would have Down Syndrome and his/her karyotype would be written 47. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.

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

cytoplasm. bone marrow in large bones produces new blood cells. [1] Bone marrow is also a key component of the lymphatic system. in adults weighing 65 kg (143 lbs). bone marrow accounts for approximately 2.1. On average.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. which divides the nuclei. It is generally followed immediately by cytokinesis. bone marrow constitutes 4% of the total body mass of humans.6 kg (5. . which use the bone marrow vasculature as a conduit to the body's systemic circulation. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. in two separate nuclei.7 lbs). In humans. 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. producing the lymphocytes that support the body's immune system CHAPTER 2 2.

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

Because each resultant daughter cell should be genetically identical to the parent cell. This occurs during the S phase of interphase. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs.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. Each chromosome now has an identical copy of itself. These two cells are identical and do not differ in any way from the original parent cell. 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.

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

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

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

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

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

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

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

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

4 Regeneration . skin and digestive tract..1Significance Mitosis is important for the maintenance of the chromosomal set. The end of cytokinesis marks the end of the M-phase. New cells are formed by mitosis and so are exact copies of the cells being replaced. e.5. The phragmoplast is a microtubule structure typical for higher plants. zygote and also the basis of the growth of a multicellular body. cells are constantly sloughed off and replaced by new ones.2 Development and growth The number of cells within an organism increases by mitosis. separating the two nuclei.e. Each daughter cell has a complete copy of the genome of its parent cell. Similarly. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. 2. which move along microtubules to the middle of the cell.5.g. whereas some green algae use a phycoplast microtubule array during cytokinesis. 2. Following are the occasions in the lives of organism where mitosis happens: 2.5.3 Cell replacement In some parts of body. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. 2.5. This is the basis of the development of a multicellular body from a single cell i.division is also driven by vesicles derived from the Golgi apparatus.

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

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

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

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

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

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

these results were quite remarkable. He revised his opinion later from 46 to 48. New techniques were needed to definitively solve the problem: 1. which swells them and spreads the chromosomes . the discoverer of mitosis.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. 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. Their behavior in animal (salamander) cells was described by Walther Flemming. Pretreating cells in a hypotonic solution. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. von Waldeyer in 1888. The next stage took place after the development of genetics in the early 20th century. at first favoring 46. and he correctly insisted on humans having an XX/XY system. in 1882. Using cells in culture 2. in contrast to their genic contents. Considering their techniques. The subsequent history of the concept can be followed in the works of Darlington and White.3. The name was coined by another German anatomist. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. concluding an XX/XO sex determination mechanism. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.

Rather interestingly.2 Observations Six different characteristics of karyotypes are usually observed and compared: . [16] Sometimes observations may be made on non-dividing (interphase) cells. a suitable dye. the great apes have 48 chromosomes. Usually. 3.2 Observations on karyotypes 3.2. reducing the number.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. For humans. 3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. such as Giemsa. is applied after cells have been arrested during cell division by a solution of colchicine.2. Arresting mitosis in metaphase by a solution of colchicines 4.3. 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. The sex of an unborn fetus can be determined by observation of interphase cells.

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

and in . Between members of a population (chromosome polymorphism) 4.3 The human karyotype Most (but not all) species have a standard karyotype. Between the sexes 2. Between the germ-line and soma (between gametes and the rest of the body) 3. the same cannot be said for their karyotypes. Mosaics or otherwise abnormal individuals. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes.Variation is often found: 1. Any variation from the standard karyotype may lead to developmental abnormalities. males have both an X and a Y chromosome denoted 46. There is variation between species in chromosome number. which are highly variable. 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. 3. XX. XY. Geographical variation between races 5.

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

The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). the inactivation is random as between the two Xs. When they looked at the karyotype of the closely related Indian muntjac. 3. The low record is held by the nematode Parascaris univalens. all telocentric. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.suum. In placental mammals.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. which was investigated by Kurt Benirschke and his colleague Doris Wurster. Muntiacus muntjak. thus the mammalian female is a mosaic in respect of her X chromosomes. Xinactivation. They kept quiet for two or three years because they thought something was wrong with their tissue culture. "They simply could not believe what they saw. was found to be 46. all the somatic cell precursors undergo chromatin diminution.3. Muntiacus reevesi. In human females some 15% of somatic cells escape inactivation.. 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. the high record would be somewhere amongst the ferns. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes.. In marsupials it is always the paternal X which is inactivated.. male = 7 chromosomes. The diploid number of the Chinese muntjac. where the haploid n = 1. The existence of supernumerary or B chromosomes ..

about 70%.Endopolyploidy occurs when in adult differentiated . Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. It is a common arrangement in the Hymenoptera. occurs mainly in plants. where one sex is diploid.means that chromosome number can vary even within one interbreeding population. horsetails and psilotales) is also common. 14.3 Fundamental number The fundamental number. and aneuploids are another example.3. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Polyploidy. FN. FN ≤ 2n. Haplo-diploidy. Polyploidy in lower plants (ferns. 3. but it has been significant in some groups. due to the presence of five acrocentric chromosome pairs (13. It has been of major significance in plant evolution according to Stebbins. but in grasses the average is much higher. Polyploidy in animals is much less common. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. Humans have FN = 82. 15.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. 3. Thus. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. and in some other groups. though in this case they would not be regarded as normal members of the population. and the other haploid. 21 and 22). where there are more than two sets of homologous chromosomes in the cells.

The cells do not always contain exact multiples (powers of two). 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. 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. . 3. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. In many instances. See palaeopolyploidy for the investigation of ancient karyotype duplications.tissues the cells have ceased to divide by mitosis. and serves differentiation and morphogenesis in many ways. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. Abnormalities in chromosome number usually cause a defect in development. 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. it is diverse and complex.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species.

Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. In about 6. 4. 5.6 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. that the two chromosome morphs are adapted to different habitats. 3. where every number from x = 3 to x = 15 is represented by at least one species. [41] Closer to home. living from rainforests to . There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.000 km2).Aneuploidy may also occur within a group of closely related species. Well-researched examples are the ladybird beetle Chilocorus stigma.500 sq mi (17. Human chromosome 2 was formed by a merger of ancestral chromosomes. some mantids of the genus Ameles. When this happens. 3.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. Classic examples in plants are the genus Crepis. the chromosome number is variable from one individual to another. reducing the number. and 7. where the gametic (= haploid) numbers form the series x = 3. the European shrew Sorex araneus. the great apes have 24x2 chromosomes whereas humans have 23x2. and Crocus. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 6.

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

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

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

Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Image processing software then assigns a pseudo color to each spectrally different combination.7. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. This method is also known as virtual karyotyping. .2) 3.chromosome 5. 3. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.XX.del(5)(p15.XX.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.5p-. The critical region for this syndrome is deletion of 15.2. which is written as 46. a combinatorial labeling method is used to generate many different colors. allowing the visualization of the individually colored chromosomes. 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.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. It is written as 46.

CHAPTER 4 .

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

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

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

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

Pre-treating cells in a hypotonic solution. New techniques were needed to definitively solve the problem: 1.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). the discoverer of mitosis. in 1882. von Waldeyer in 1888. 4. and he correctly insisted on man having an XX/XY system. Their behavior in animal (salamander) cells was described by Walther Flemming. The next stage took place after the development of genetics in the early 20th century. He revised his opinion later from 46 to 48.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. concluding an XX/XO sex determination mechanism. 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. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. which swells them and spreads the chromosomes . 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. The name was coined by another German anatomist. in contrast to their genic contents. Considering their techniques. at first favoring 46.

During her cytogenetic work. persimilis from wild populations in California and neighboring states. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. 4. Human chromosome 2 was formed by a merger of ancestral chromosomes.6 Applications in biology 4.6. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. the great apes have 48 chromosomes. In 1931. Using Painter's technique they studied the polytene .2 Natural populations of Drosophila In the 1930s. 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). Arresting mitosis in metaphase by a solution of colchicine 4. reducing the number. Rather interestingly. 4. McClintock discovered transposons.3. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.6. a find which eventually led to her Nobel Prize in 1983.

In some congenital disorders. Down syndrome is also referred to as trisomy 21. such as Down's syndrome. Dobzhansky bred populations in population cages. as they would if selectively neutral. discoveries were quickly made related to aberrant chromosomes or chromosome number. Abnormalities arising from nondisjunction events can cause cells with aneuploidy (additions or deletions of entire chromosomes) in one of the parents or in the fetus. as with most polymorphisms. 4. Using a method invented by L'Heretier and Teissier.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. Evidence rapidly accumulated to show that natural selection was responsible. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. which enabled feeding. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. It was found that the various chromosome types do not fluctuate at random. . In 1959.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. This had the benefit of eliminating migration as a possible explanation of the results. breeding and sampling whilst preventing escape. but adjust to certain frequencies at which they become stabilised.

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

Caspersson developed banding techniques which differentially stain chromosomes. Deletion syndromes such as DiGeorge syndrome. Deletions within one chromosome could also now be more specifically named and understood. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. and elongation techniques for all culture types that allow for higher resolution banding. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.8 Beginnings of molecular cytogenetics . 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. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. 4.FIG Advent of banding techniques In the late 1960s.

advances were made in molecular cytogenetics. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).In the 1980s. movement was now made in using fluorescent labeled probes. cloned and studied in ever greater detail. While radioisotope-labeled probes had been hybridized with DNA since 1969.1 Karyotyping . This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. 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,

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

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

2) Geometrical compensation—The geometric compensation. 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. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. Therefore. To compensate for this inhomogeneity. 6.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. the spatially scaled images are histogram equalized.2 Concepts used in this phase 1) Image conversion 2) Denoising . performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. or at least attenuated. 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). To compare chromosomes from a band pattern point of view.

as is appropriate. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension.I. The resulting true color image has identical matrices for the red. MATLAB simply applies the filter to the indices in the indexed image matrix. so the image displays as shades of gray. For example. MATLAB filters the intensity values in the image. green. You can perform certain conversions just using MATLAB syntax. For example. and the results might not be meaningful. 6. there are other functions that return a different image type as part of the operation they perform. RGB = cat (3.3) Edge detection 4) Two dimensional convolutions. When you apply the filter to the true color image. In addition to these image type conversion functions. For example.2. if you want to filter a color image that is stored as an indexed image. . you must first convert it to true color format. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. If you attempt to filter the indexed image.I).I.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. listed in the following table. and blue planes.

and hence the type of noise on the image. There is a large number of edge finding algorithms in existence. or through networked cable.6.3 Two dimensional convolutions C = conv2(A.B) computes the two-dimensional convolution of matrices A and B. We may use edges to measure the size of objects in an image. to isolate particular objects from their background. . Usually we know what type of errors to expect. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. to recognize or classify objects. .2. Cleaning an image corrupted by noise is thus an important area of image restoration. If an image is being sent electronically from one place to another. we may expect errors to occur in the image signal.'method'. ) Where the parameters available depend on the method used 6.parameters.2.4 Denoising We may define noise to be any degradation in the image signal. caused by external disturbance. If one of these matrices describes a two-dimensional finite impulse response . 6. via satellite or wireless transmission. The general Matlab command for finding edges is edge(image. and we shall look at some of the more straightforward of them. hence we can choose the most appropriate method for reducing the effects.5 Edge detection Edges contain some of the most useful information in an image.

. 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. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. the other matrix is filtered in two dimensions..(FIR) filter.'sobel').'shape') subsection of the two-dimensional convolution. edge(im1. minus one. If hcol is a column vector and hrow is a row vector. The size of matrices. nb]+1)/2).0.7).hrow. this case is the same as C = conv2(hcol*hrow.. C = conv2(.bmp').A). . That is.na+nb-1]. imedfilt2(im1. if the size of then the size of C is [ma+mb-1.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.nb]. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.na] and the size of B is [mb. rgb2gray im2bw(im.[3 3]).

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

15.42.55.y1)=255.48. [sx sy]=size(rc).27. n1=zeros(imx. end flag=0. end end L_number.38.60. y1=rc(i.end end if flag~=1 L_number(Index)=L(i. Test_number=[3.45.8.c] = find(L==L_number((Test_number(x)))).40.imshow(n1. . for i=1:sx x1=rc(i.41.10.39.22.66]. for x=1:46 [r.19. end %h=figure.28.6.57.[]).14. rc = [r c].21.65. Index=Index+1.9.30.29.32.imy).49.54.50.26.51.7.52.35.24.2).59. n1(x1.31.56.20.1).62.33. end.4.j). 36.43.11.

. skel=im2double(f).5*graythresh(skel)). Arm_length=zeros(46. for x=1:m for y=1:n if BW1(x.'spur'. s1=bwmorph(s.'.1). BW=double(BW). Arm_length_sum=0. BW=im2bw(f).'skel'. BW1=edge(BW. end end end Circumference(i)=Circumference_sum.'canny'). Circumference_sum=0.1).1.Inf). skel=im2bw(skel. for i=1:46 f=imread(strcat(num2str(i). f=imcomplement(f). Area=zeros(46. [m n]=size(BW1).end Circumference=zeros(46.bmp')). s=bwmorph(skel.8).y)==1 Circumference_sum=Circumference_sum+1.1).

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

end end end for i=1:45 if Pair(i. Pair(i.Area. Pair=zeros(46. Pair(i. Pair(i. Pair(i.2)=i. end end Pair.1)=i. 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)). for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)). .1)=46.2)=j.2)==46 Pair(46.2)=i+1.1)=i.2).

figure_flag=1. if figure_flag~=47 subplot(23.delete=zeros(46.bmp')).2.1)).'.bmp')).figure_flag).'.2. end flag=0.1). for i=1:46 for j=1:46 if Pair(i.figure_flag). figure_flag=figure_flag+1.2). end end if flag~=1 if figure_flag~=47 subplot(23. delete(figure_flag)=Pair(i. imshow(f2). . flag=0. imshow(f1).1)==delete(j) flag=1. figure_flag=figure_flag+1.2)). end f2=imread(strcat(num2str(Pair(i. end f1=imread(strcat(num2str(Pair(i.

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

In the image processing step. The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram. The addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the . The training process consists in the estimation of each vector of coefficient . 3) training of a classifier (performed once) where similarity among chromosomes are characterized. by minimizing the overall distances between chromosomes of the same class (intraclass) and by maximizing the distances between chromosomes when at least one of them does not belong to that class (interclass). shape.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. Tests using 19 karyograms based on bone marrow cells. 4) pairing. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. Here. achieves a 70.10% mean classification rate.working within an 8-D feature space. extracted from the unordered karyogram. are processed in order to compensate for geometrical and intensity distortions. This normalization is needed to make it possible the band pattern comparison between chromosomes. 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. 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 and band pattern. The features extracted from the processed images discriminate each pair with respect to their size. and finally. from the chromosomes in the training set. and to normalize their dimensions. the romosome images.characterizing the size.

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

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

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