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Chromosome Document

Chromosome Document

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Sections

  • 1.2 CHROMOSOME
  • 1.3 MUTATIONS IN CHROMOSOME NUMBER
  • 1.4 Metaphase chromatin and division
  • 1.5 BONE MARROW
  • 2.2.1 Preprophase
  • 2.2.2 Prometaphase
  • 2.3 Metaphase
  • 2.4 Anaphase
  • 2.5.1Significance
  • 2.5.2 Development and growth
  • 2.5.3 Cell replacement
  • 2.5.4 Regeneration
  • 2.5.6 Asexual reproduction
  • 2.5.7 Consequences of errors
  • 2.6 Endomitosis
  • 2.7 Metaphase
  • 2.8 Metaphase in cytogenetics and cancer studies
  • 3.1 History of karyotype studies
  • 3.2.1 Staining
  • 3.2.2 Observations
  • 3.3.1 Changes during development
  • 3.3.2 Number of chromosomes in a set
  • 3.3.3 Fundamental number
  • 3.4 Ploidy
  • 3.5 Aneuploidy
  • 3.6 Species trees
  • 3.7.1 Types of banding
  • 3.7.2 Classic karyotype cytogenetics
  • 3.7.3 Spectral karyotype (SKY technique)
  • 3.8 Digital karyotyping
  • 4.1 CHROMOSOMAL ABNORMALITIES
  • 4.2 Numerical Disorders
  • 4.3 Inheritance
  • 4.4 Cytogenetics
  • 4.5 Early years
  • 4.6.1 McClintock's work on maize
  • 4.6.2 Natural populations of Drosophila
  • 4.7 Human abnormalities and medical applications
  • 4.8 Beginnings of molecular cytogenetics
  • 5.1 Karyotyping
  • 5.2 Slide preparation
  • 5.3 Analysis
  • 5.4 Fluorescent in situ hybridization
  • 5.5 Slide preparation
  • 5.7 Analysis
  • 6.1 Image Processing
  • 6.2.1 Image conversion
  • 6.2.4 Denoising
  • 6.2.5 Edge detection
  • 6.3 Two dimensional convolutions

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

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

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

The shorter arms are called p arms (from the French petit. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. In spite of their appearance. the chromatids are uncoiled and DNA can again be transcribed. This is the only natural context in which individual chromosomes are visible with an optical microscope. q-g "grande"). During mitosis. and they form the classic four arm structure. The microtubules then pull the chromatids apart toward the centrosomes. chromosomes are structurally highly condensed. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. 1. one of which is present on each sister chromatid. small) and the longer arms are called q arms (q follows p in the Latin alphabet. longer-lasting attachment in this region. .1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. This compact form makes the individual chromosomes visible. A special DNA base sequence in the region of the kinetochores provides. along with special proteins. so that each daughter cell inherits one set of chromatids.Fig 1.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). the chromatin strands become more and more condensed. a pair of sister chromatids attached to each other at the centromere. which enables these giant DNA structures to be contained within a cell nucleus. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. Once the cells have divided.

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

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

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

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

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

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

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

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

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

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

pinching off the separated nuclei. Mitosis is complete. In both animal and plant cells. elongating the cell even more. the nonkinetochore microtubules continue to lengthen. cytokinesis is a separate process that begins at the same time as telophase. using fragments of the parent cell's nuclear membrane. Corresponding sister chromosomes attach at opposite ends of the cell. unfold back into chromatin. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. but rather a separate process. In animal cells. cell .5 Cytokinesis Cilliate undergoing cytokinesis. forms around each set of separated sister chromosomes. It "cleans up" the after effects of mitosis. Both sets of chromosomes. but cell division is not yet complete. Cytokinesis is technically not even a phase of mitosis. necessary for completing cell division. 2. however. At telophase. now surrounded by new nuclei. A new nuclear envelope.2.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be.

2. zygote and also the basis of the growth of a multicellular body.5.5.3 Cell replacement In some parts of body. 2.2 Development and growth The number of cells within an organism increases by mitosis. separating the two nuclei.5. e. skin and digestive tract. which move along microtubules to the middle of the cell. The end of cytokinesis marks the end of the M-phase.e. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.g. The phragmoplast is a microtubule structure typical for higher plants. Each daughter cell has a complete copy of the genome of its parent cell. Similarly.1Significance Mitosis is important for the maintenance of the chromosomal set.4 Regeneration . RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. whereas some green algae use a phycoplast microtubule array during cytokinesis. 2..5. New cells are formed by mitosis and so are exact copies of the cells being replaced.division is also driven by vesicles derived from the Golgi apparatus. Following are the occasions in the lives of organism where mitosis happens: 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. cells are constantly sloughed off and replaced by new ones. This is the basis of the development of a multicellular body from a single cell i.

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

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

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

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

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

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

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

white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. the great apes have 48 chromosomes. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Arresting mitosis in metaphase by a solution of colchicines 4. [16] Sometimes observations may be made on non-dividing (interphase) cells.2. reducing the number.1 Staining The study of karyotypes is made possible by staining.2 Observations on karyotypes 3.3. such as Giemsa. Usually.2. 3. Rather interestingly. The sex of an unborn fetus can be determined by observation of interphase cells. For humans. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. 3. is applied after cells have been arrested during cell division by a solution of colchicine.2 Observations Six different characteristics of karyotypes are usually observed and compared: . a suitable dye. Human chromosome 2 was formed by a merger of ancestral chromosomes.

Heterochromatin stains darker than euchromatin. Differences in number and position of satellites. as well as other cytogenetic information. type. both have six pairs of chromosomes (n=6) yet V. 4.1. 5. faba chromosomes are many times larger. Differences in absolute sizes of chromosomes. 6. shape and banding of the chromosomes. Differences in the position of centromeres. and mainly consists of genetically inactive repetitive DNA sequences. but the genes have been mostly translocated (added) to other chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). This is brought about by translocations. Humans have one pair fewer chromosomes than the great apes. . 3. indicating tighter packing. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Differences in degree and distribution of heterochromatic regions. A full account of a karyotype may therefore include the number. 2. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. This feature probably reflects different amounts of DNA duplication. permitting its loss without penalty to the organism (the dislocation hypothesis). which (when they occur) are small bodies attached to a chromosome by a thin thread.

The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Mosaics or otherwise abnormal individuals. Between members of a population (chromosome polymorphism) 4. Normal karyotypes for females contain two X chromosomes and are denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. the same cannot be said for their karyotypes. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Between the germ-line and soma (between gametes and the rest of the body) 3. Geographical variation between races 5. XX.Variation is often found: 1. which are highly variable. Any variation from the standard karyotype may lead to developmental abnormalities. Between the sexes 2. males have both an X and a Y chromosome denoted 46. XY. There is variation between species in chromosome number. and in . 3.

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

The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). The existence of supernumerary or B chromosomes . The low record is held by the nematode Parascaris univalens. In placental mammals. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. all telocentric. the inactivation is random as between the two Xs. 3..suum. all the somatic cell precursors undergo chromatin diminution. Xinactivation. where the haploid n = 1. The diploid number of the Chinese muntjac. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. In marsupials it is always the paternal X which is inactivated. They kept quiet for two or three years because they thought something was wrong with their tissue culture. they were astonished to find it had female = 6.3. thus the mammalian female is a mosaic in respect of her X chromosomes. the high record would be somewhere amongst the ferns.. Muntiacus reevesi. male = 7 chromosomes. Muntiacus muntjak.. In human females some 15% of somatic cells escape inactivation.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. 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. which was investigated by Kurt Benirschke and his colleague Doris Wurster. was found to be 46. "They simply could not believe what they saw.. When they looked at the karyotype of the closely related Indian muntjac.

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

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

[41] Closer to home. Human chromosome 2 was formed by a merger of ancestral chromosomes. and Crocus.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. the European shrew Sorex araneus. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. that the two chromosome morphs are adapted to different habitats. 3.Aneuploidy may also occur within a group of closely related species. living from rainforests to .000 km2). the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. where the gametic (= haploid) numbers form the series x = 3. the great apes have 24x2 chromosomes whereas humans have 23x2. Classic examples in plants are the genus Crepis. 4.500 sq mi (17. Well-researched examples are the ladybird beetle Chilocorus stigma. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. 5. When this happens. and 7. the chromosome number is variable from one individual to another. In about 6. reducing the number. some mantids of the genus Ameles. 3. where every number from x = 3 to x = 15 is represented by at least one species. 6.

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

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

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

allowing the visualization of the individually colored chromosomes.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX.5p-.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. This method is also known as virtual karyotyping.7. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.chromosome 5.2) 3. 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. . Because there are a limited number of spectrally-distinct fluorophores.del(5)(p15. which is written as 46. Image processing software then assigns a pseudo color to each spectrally different combination. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.2. The critical region for this syndrome is deletion of 15. 3. It is written as 46. a combinatorial labeling method is used to generate many different colors.

CHAPTER 4 .

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

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

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

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

Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. in contrast to their genic contents. Using cells in culture 2. New techniques were needed to definitively solve the problem: 1. He revised his opinion later from 46 to 48. 4. concluding an XX/XO sex determination mechanism. and he correctly insisted on man having an XX/XY system.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). von Waldeyer in 1888. 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. Pre-treating cells in a hypotonic solution. at first favoring 46.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. the discoverer of mitosis. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in 1882. The name was coined by another German anatomist. 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. which swells them and spreads the chromosomes . when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Considering their techniques. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. these results were quite remarkable.

4.6. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.6 Applications in biology 4.3. the great apes have 48 chromosomes.2 Natural populations of Drosophila In the 1930s. In 1931. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. reducing the number. 4. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. During her cytogenetic work. Rather interestingly. persimilis from wild populations in California and neighboring states. Human chromosome 2 was formed by a merger of ancestral chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. McClintock discovered transposons. 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. a find which eventually led to her Nobel Prize in 1983. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.6. Using Painter's technique they studied the polytene .

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

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

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

This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. advances were made in molecular cytogenetics. CHAPTER 5 Techniques 5.1 Karyotyping . While radioisotope-labeled probes had been hybridized with DNA since 1969.In the 1980s. cloned and studied in ever greater detail. 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. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).

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,

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

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

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 compensate for this inhomogeneity. geometrical and dimensional differences must be removed. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. To compare chromosomes from a band pattern point of view. 2) Geometrical compensation—The geometric compensation. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 6.2 Concepts used in this phase 1) Image conversion 2) Denoising . or at least attenuated. Therefore. 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. the spatially scaled images are histogram equalized.

if you want to filter a color image that is stored as an indexed image. MATLAB filters the intensity values in the image. there are other functions that return a different image type as part of the operation they perform.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. so the image displays as shades of gray. and blue planes. In addition to these image type conversion functions. For example.I. RGB = cat (3.2. you must first convert it to true color format.3) Edge detection 4) Two dimensional convolutions. When you apply the filter to the true color image.I. If you attempt to filter the indexed image. For example. MATLAB simply applies the filter to the indices in the indexed image matrix. For example. You can perform certain conversions just using MATLAB syntax. listed in the following table. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. 6. The resulting true color image has identical matrices for the red. and the results might not be meaningful.I). as is appropriate. green. .

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

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

y1)=255. Index=1.j)~=0 for k=1:mx if L(i. MODULE 2 clc [m. mx=max(max(L)). [r.j)==L_number(k) flag=1.8).imy]=size(BW). L_number=zeros(mx. Msk conv2(double(BW).1). for i=1:sx x1=rc(i.2). for i=1:m for j=1:n if L(i.imy). flag=0. bwlabel(B.c] = find(L==22).double(msk)). y1=rc(i. n1(x1. nzeros(imx. .1). [sx sy]=size(rc).[imx. rc = [r c].n]=size(L).

4.21.50. end flag=0.38.y1)=255.35.39.41.6.40.24.19. y1=rc(i.[]).43.31.27.14.45. for x=1:46 [r.j).10.65.7. end.2).imy). end end L_number.c] = find(L==L_number((Test_number(x)))).20.1).48.15.32.66].22.26.54. rc = [r c]. for i=1:sx x1=rc(i.end end if flag~=1 L_number(Index)=L(i. n1(x1.57.59.11. [sx sy]=size(rc). .imshow(n1.9.28. end %h=figure.60.8. n1=zeros(imx.30.56. 36.51.29.42.62.55.33.49.52. Test_number=[3. Index=Index+1.

Circumference_sum=0.'. Arm_length_sum=0.1).end Circumference=zeros(46. Arm_length=zeros(46.1). for x=1:m for y=1:n if BW1(x. Area=zeros(46.1. BW=im2bw(f).8). end end end Circumference(i)=Circumference_sum.Inf). f=imcomplement(f).'canny'). . s1=bwmorph(s.1). [m n]=size(BW1). BW=double(BW).y)==1 Circumference_sum=Circumference_sum+1.'spur'.bmp')). for i=1:46 f=imread(strcat(num2str(i).'skel'. skel=im2bw(skel. s=bwmorph(skel. skel=im2double(f).5*graythresh(skel)). BW1=edge(BW.

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

Pair=zeros(46.2)=j. end end Pair. 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(46. . Pair(i. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)). Pair(i.1)=i. Pair(i.1)=i. Pair(i.2)=i+1.2)=i.2)==46 Pair(46.2). end end end for i=1:45 if Pair(i.1)=46.Area.

figure_flag). for i=1:46 for j=1:46 if Pair(i. flag=0.figure_flag). figure_flag=figure_flag+1.1)==delete(j) flag=1. figure_flag=1.2)). end f1=imread(strcat(num2str(Pair(i.bmp')). end end if flag~=1 if figure_flag~=47 subplot(23.bmp')).'. if figure_flag~=47 subplot(23. imshow(f2).2. end f2=imread(strcat(num2str(Pair(i.1). end flag=0.1)).2).delete=zeros(46.2. figure_flag=figure_flag+1. . delete(figure_flag)=Pair(i. imshow(f1).'.

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

the romosome images.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. In the image processing step. 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 coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. Tests using 19 karyograms based on bone marrow cells. 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. 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. Here. 4) pairing.working within an 8-D feature space. 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 . from the chromosomes in the training set. The features extracted from the processed images discriminate each pair with respect to their size.characterizing the size. 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.10% mean classification rate. achieves a 70. shape and band pattern. This normalization is needed to make it possible the band pattern comparison between chromosomes. extracted from the unordered karyogram. and to normalize their dimensions. and finally. are processed in order to compensate for geometrical and intensity distortions. shape. and band pattern. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.

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

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

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

J. 27. Chapter 9 24.K. Sinauer Associates.. Developmental biology. 26. 1364-1366. ^ Khandelwal S. 2006. D. and Benirschke K. 1970.1007/s10228-004-0257-z.S. Stansfield W..A. 2006. F. (1945-05-15).A. Nam. Science 168. 23.. R.C. Ichthyological Research 52 (1): 97. 25. 1990... Retrieved 2008-03-18.P Oxford & NY. Retrieved 2011-03-16. 291: 310–16. & Gregory T. Y. Chromosome evolution in the genus Ophioglossum L. Muntiacus muntjak: a deer with a low diploid number.1007/BF02153623. ^ Kim.R. C.F.D. A dictionary of genetics.H.doi:10.K. Zool. 8th ed. J. ^ Gilbert S. Stamford CT. . Indian Muntjac. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. Botanical Journal of the Linnean Society 102: 205–217.K. ^ Wyngaard G. 7th ed. "L'evolution de la formule chromosomiale chez les vertébrés". and Mulligan P.H. (2005). Experientia (Basel) 1 (2): 50– 56. ^ Wurster D. Exp. doi:10. Noh. Oxford U. Park. ^ Matthey. 28. 2001. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). ^ King R. Chapman.22.

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