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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

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Page 1: Chromosome Document

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.

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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

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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

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defined nuclei) have smaller circular chromosomes, although there are many

exceptions to this rule. Also, cells may contain more than one type of chromosome;

for example, mitochondria in most eukaryotes and chloroplasts in plants have their

own small chromosomes.

In eukaryotes, nuclear chromosomes are packaged by proteins into a condensed

structure called chromatin. This allows the very long DNA molecules to fit into

the cell nucleus. The structure of chromosomes and chromatin varies through

the cell cycle. Chromosomes are the essential unit for cellular division and must be

replicated, divided, and passed successfully to their daughter cells so as to ensure

the genetic diversity and survival of their progeny. Chromosomes may exist as

either duplicated or unduplicated. Unduplicated chromosomes are single linear

strands, whereas duplicated chromosomes contain two identical copies

(called chromatids) joined by a centromere.

Compaction of the duplicated chromosomes

during mitosis and meiosis results in the classic four-arm structure (pictured to the

right). Chromosomal recombination plays a vital role in genetic diversity. If these

structures are manipulated incorrectly, through processes known as chromosomal

instability and translocation, 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. In

prokaryotes and viruses, the term genophore is more appropriate when no

chromatin is present. However, a large body of work uses the term chromosome

regardless of chromatin content. In prokaryotes, DNA is usually arranged as a

circle, which is tightly coiled in on itself, sometimes accompanied by one or more

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smaller, circular DNA molecules called plasmids. These small circular genomes

are also found in mitochondria and chloroplasts, reflecting their bacterial origins.

The simplest gonophores are found in viruses: these DNA or RNA molecules are

short linear or circular gonophores that often lack structural proteins.

1.3 MUTATIONS IN CHROMOSOME NUMBER

Normally, 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). Such individuals are called euploid

and have the wild-type chromosome complement for the species. Euploid human

karyotypes are 46, XX (female) or 46 XY (male).

Chromosomal Mutations are substantial changes in

chromosome structure that are large enough to be visible by karyotyping (see lab

manual) and thus typically affect more than one gene. If the mutation involves

only one or a few chromosomes in the genome (e.g. a extra copy of human

chromosome 21), the individual carrying the mutation is said to be aneuploid. An

example of aneuploidy is trisomy 21, in which an individual has 3, rather than 2,

copies of chromosome 21. The individual would have Down Syndrome and

his/her karyotype would be written 47,+21,XY or 47,+21,XX.

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Fig 1.1 chromosome paired model

Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. Such a

failure of the separation of homologous chromosomes or sister chromatids is called

nondisjunction.

1.4 Metaphase chromatin and division

In the early stages of mitosis or meiosis (cell division), the

chromatin strands become more and more condensed. They cease to function as

accessible genetic material (transcription stops) and become a compact

transportable form. This compact form makes the individual chromosomes visible,

and they form the classic four arm structure, a pair of sister chromatids attached to

each other at the centromere. The shorter arms are called p arms (from

the French petit, small) and the longer arms are called q arms (q follows p in the

Latin alphabet; q-g "grande"). This is the only natural context in which individual

chromosomes are visible with an optical microscope.

During mitosis, microtubules grow from centrosomes located at

opposite ends of the cell and also attach to the centromere at specialized structures

called kinetochores, one of which is present on each sister chromatid. A special

DNA base sequence in the region of the kinetochores provides, along with special

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proteins, longer-lasting attachment in this region. The microtubules then pull the

chromatids apart toward the centrosomes, so that each daughter cell inherits one set

of chromatids. Once the cells have divided, the chromatids are uncoiled and DNA

can again be transcribed. In spite of their appearance, chromosomes are structurally

highly condensed, which enables these giant DNA structures to be contained

within a cell nucleus.

1.5 BONE MARROW

Bone marrow (Latin: medulla ossium) is the flexible tissue found

in the interior of bones. In humans, bone marrow in large bones produces

new blood cells. On average, bone marrow constitutes 4% of the total body mass

of humans; in adults weighing 65 kg (143 lbs), bone marrow accounts for

approximately 2.6 kg (5.7 lbs). The hematopoietic compartment of bone marrow

produces approximately 500 billion blood cells per day, which use the bone

marrow vasculature as a conduit to the body's systemic circulation.[1] Bone marrow

is also a key component of the lymphatic system, producing the lymphocytes that

support the body's immune system

CHAPTER 2

2.1 MITOSIS

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Mitosis is the process by which a eukaryotic cell separates the chromosomes

in its cell nucleus into two identical sets, in two separate nuclei. It is generally

followed immediately by cytokinesis, which divides the nuclei, cytoplasm,

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,

genetically identical to each other and to their parent cell. This accounts for

approximately 10% of the cell cycle.

Mitosis occurs only in eukaryotic cells and the process varies in different

species. For example, animals undergo an "open" mitosis, where the nuclear

envelope breaks down before the chromosomes separate, while fungi such as

Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed"

mitosis, where chromosomes divide within an intact cell nucleus.[1] Prokaryotic

cells, which lack a nucleus, divide by a process called binary fission.

The process of mitosis is fast and highly complex. The sequence of events is

divided into stages corresponding to the completion of one set of activities and the

start of the next. These stages are interphase, prophase, prometaphase, metaphase,

anaphase and telophase. During mitosis the pairs of chromatids condense and

attach to fibers that pull the sister chromatids to opposite sides of the cell. The cell

then divides in cytokinesis, to produce two identical daughter cells which are still

diploid cells.

Because cytokinesis usually occurs in conjunction with mitosis, "mitosis" is

often used interchangeably with "mitotic phase". However, there are many cells

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where mitosis and cytokinesis occur separately, forming single cells with multiple

nuclei. This occurs most notably among the fungi and slime moulds, but is found

in various different groups. Even in animals, cytokinesis and mitosis may occur

independently, for instance during certain stages of fruit fly embryonic

development. Errors in mitosis can either kill a cell through apoptosis or cause

mutations that may lead to cancer.

Fig 3 Mitosis cell division

The primary result of mitosis is the transferring of the parent cell's genome

into two daughter cells. These two cells are identical and do not differ in any way

from the original parent cell. The genome is composed of a number of

chromosomes—complexes of tightly-coiled DNA that contain genetic information

vital for proper cell function. Because each resultant daughter cell should be

genetically identical to the parent cell, the parent cell must make a copy of each

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chromosome before mitosis. This occurs during the S phase of interphase, the

period that precedes the mitotic phase in the cell cycle where preparation for

mitosis occurs.

Each chromosome now has an identical copy of itself, and together the two

are called sister chromatids. The sister chromatids are held together by a

specialized region of the chromosome known as the centromere.

In most eukaryotes, the nuclear envelope which segregates the DNA from

the cytoplasm disassembles. The chromosomes align themselves in a line spanning

the cell. Microtubules — essentially miniature strings— splay out from opposite

ends of the cell and shorten, pulling apart the sister chromatids of each

chromosome. As a matter of convention, each sister chromatid is now considered a

chromosome, so they are renamed to sister chromosomes. As the cell elongates,

corresponding sister chromosomes are pulled toward opposite ends. A new nuclear

envelope forms around the separated sister chromosomes.

As mitosis completes,the cell begins cytokinesis. In animal 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),

separating the two developing nuclei. In plant cells, the daughter cells will

construct a new dividing cell wall between each other. Eventually, the parent cell

will be split in half, giving rise to two daughter cells, each with a replica of the

original genome.

Prokaryotic cells undergo a process similar to mitosis called binary fission.

However, the process of binary fission is very much different from the process of

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mitosis, because of the non-involvement of nuclear dynamics and lack of linear

chromosomes.

2.2 Phases of cell cycle and mitosis Interphase

Fig 4 The cell cycle

The mitotic phase is a relatively short period of the cell cycle. It alternates

with the much longer interphase, where the cell prepares itself for cell division.

Interphase is divided into three phases: G1 (first gap), S (synthesis), and G2 (second

gap). During all three phases, the cell grows by producing proteins and

cytoplasmic organelles. However, chromosomes are replicated only during the S

phase. Thus, a cell grows (G1), continues to grow as it duplicates its chromosomes

(S), grows more and prepares for mitosis (G2), and finally it divides (M) before

restarting the cycle. All these phases in the interphase are highly regulated, mainly

via proteins. 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.

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2.2.1 Preprophase

In plant cells only, prophase is preceded by a pre-prophase stage. In highly

vacuolated plant cells, the nucleus has to migrate into the center of the cell before

mitosis can begin. This is achieved through the formation of a phragmosome, a

transverse sheet of cytoplasm that bisects the cell along the future plane of cell

division. In addition to phragmosome formation, preprophase is characterized by

the formation of a ring of microtubules and actin filaments (called preprophase

band) underneath the plasma membrane around the equatorial plane of the future

mitotic spindle. This band marks the position where the cell will eventually divide.

The cells of higher plants (such as the flowering plants) lack centrioles; instead,

microtubules form a spindle on the surface of the nucleus and are then organized

into a spindle by the chromosomes themselves, after the nuclear membrane breaks

down. The preprophase band disappears during nuclear envelope disassembly and

spindle formation in prometaphase.

Metaphase: The chromosomes have aligned at the metaphase plate.  

Prophase: The two round objects above the nucleus are the centrosomes. The chromatin has condensed.

Prometaphase: The nuclear membrane has degraded, and microtubules have invaded the nuclear space. These microtubules can attach to kinetochores or they can interact with opposing microtubules.

Early anaphase: The kinetochore microtubules shorten.  

Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Cytokinesis has already begun; the pinched area is known as the cleavage furrow.

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Prophase

Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic

spindle in green during prometaphase of mitosis

Normally, the genetic material in the nucleus is in a loosely bundled coil

called chromatin. At the onset of prophase, chromatin condenses together into a

highly ordered structure called a chromosome. Since the genetic material has

already been duplicated earlier in S phase, the replicated chromosomes have two

sister chromatids, bound together at the centromere by the cohesin protein

complex. Chromosomes are typically visible at high magnification through a light

microscope.

Close to the nucleus are structures called centrosomes, which are made of a

pair of centrioles found in most eukaryotic animal cells. The centrosome is the

coordinating center for the cell's microtubules. A cell inherits a single centrosome

at cell division, which is replicated by the cell with the help of the nucleus before a

new mitosis begins, giving a pair of centrosomes. The two centrosomes nucleate

microtubules (which may be thought of as cellular ropes or poles) to form the

spindle by polymerizing soluble tubulin. Molecular motor proteins then push the

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centrosomes along these microtubules to opposite sides of the cell. Although

centrioles help organize microtubule assembly, they are not essential for the

formation of the spindle, since they are absent from plants, and centrosomes are

not always used in mitosis.

2.2.2 Prometaphase

The nuclear envelope disassembles and microtubules invade the nuclear

space. This is called open mitosis, and it occurs in most multicellular organisms.

Fungi and some protists, such as algae or trichomonads, undergo a variation called

closed mitosis where the spindle forms inside the nucleus, or its microtubules are

able to penetrate an intact nuclear envelope.

Each chromosome forms two kinetochores at the centromere, one attached at

each chromatid. A kinetochore is a complex protein structure that is analogous to a

ring for the microtubule hook; it is the point where microtubules attach themselves

to the chromosome ( about 1-40 in number, on an average 20 ). Although the

kinetochore structure and function are not fully understood, it is known that it

contains some form of molecular motor. When a microtubule connects with the

kinetochore, the motor activates, using energy from ATP to "crawl" up the tube

toward the originating centrosome. This motor activity, coupled with

polymerisation and depolymerisation of microtubules, provides the pulling force

necessary to later separate the chromosome's two chromatids.

When the spindle grows to sufficient length, kinetochore microtubules begin

searching for kinetochores to attach to. A number of nonkinetochore microtubules

find and interact with corresponding nonkinetochore microtubules from the

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opposite centrosome to form the mitotic spindle. Prometaphase is sometimes

considered part of prophase.

In the fishing pole analogy, the kinetochore would be the "hook" that catches

a sister chromatid or "fish". The centrosome acts as the "reel" that draws in the

spindle fibers or "fishing line". It is also one of the main phases of mitosis because

without it cytokinesis would not be able to occur.

2.3 Metaphase

A cell in late metaphase. All chromosomes (blue) but one have arrived at the metaphase plate.

Metaphase comes from the Greek meaning "after." Microtubules find and

attach to kinetochores in prometaphase. Then the two centrosomes start pulling the

chromosomes through their attached centromeres towards the two ends of the cell.

As a result, the chromosomes come under longitudinal tension from the two ends

of the cell. The centromeres of the chromosomes, in some sense, convene along the

metaphase plate or equatorial plane, 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, analogous to a tug-of-war

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between people of equal strength. In certain types of cells, chromosomes do not

line up at the metaphase plate and instead move back and forth between the poles

randomly, only roughly lining up along the midline.

Because proper chromosome separation requires that every kinetochore be

attached to a bundle of microtubules (spindle fibres), 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.

2.4 Anaphase

When every kinetochore is attached to a cluster of microtubules and the

chromosomes have lined up along the metaphase plate, the cell proceeds to

anaphase (from the Greek meaning “up,” “against,” “back,” or “re-”).

Two events then occur: first, the proteins that bind sister chromatids together

are cleaved, allowing them to separate. These sister chromatids, which have now

become distinct sister chromosomes, are pulled apart by shortening kinetochore

microtubules and move toward the respective centrosomes to which they are

attached. Next, the nonkinetochore microtubules elongate, pulling the centrosomes

(and the set of chromosomes to which they are attached) apart to opposite ends of

the cell. The force that causes the centrosomes to move towards the ends of the cell

is still unknown, although there is a theory that suggests that the rapid assembly

and breakdown of microtubules may cause this movement.

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These two stages are sometimes called early and late anaphase. Early

anaphase is usually defined as the separation of the sister chromatids, while late

anaphase is the elongation of the microtubules and the chromosomes being pulled

farther apart. At the end of anaphase, the cell has succeeded in separating identical

copies of the genetic material into two distinct populations.

2.5 Telophase

Telophase (from the Greek meaning "end") is a reversal of prophase and

prometaphase events. It "cleans up" the after effects of mitosis. At telophase, the

nonkinetochore microtubules continue to lengthen, elongating the cell even more.

Corresponding sister chromosomes attach at opposite ends of the cell. A new

nuclear envelope, using fragments of the parent cell's nuclear membrane, forms

around each set of separated sister chromosomes. Both sets of chromosomes, now

surrounded by new nuclei, unfold back into chromatin. Mitosis is complete, but

cell division is not yet complete.

2.5 Cytokinesis

Cilliate undergoing cytokinesis, with the cleavage furrow being clearly visible

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Cytokinesis is often mistakenly thought to be the final part of telophase;

however, cytokinesis is a separate process that begins at the same time as

telophase. Cytokinesis is technically not even a phase of mitosis, but rather a

separate process, necessary for completing cell division. In animal cells, a cleavage

furrow (pinch) containing a contractile ring develops where the metaphase plate

used to be, pinching off the separated nuclei. In both animal and plant cells, cell

division is also driven by vesicles derived from the Golgi apparatus, which move

along microtubules to the middle of the cell. In plants this structure coalesces into a

cell plate at the center of the phragmoplast and develops into a cell wall, separating

the two nuclei. The phragmoplast is a microtubule structure typical for higher

plants, whereas some green algae use a phycoplast microtubule array during

cytokinesis. Each daughter cell has a complete copy of the genome of its parent

cell. The end of cytokinesis marks the end of the M-phase.

2.5.1Significance

Mitosis is important for the maintenance of the chromosomal set; each cell

formed receives chromosomes that are alike in composition and equal in number to

the chromosomes of the parent cell.

Following are the occasions in the lives of organism where mitosis happens:

2.5.2 Development and growth

The number of cells within an organism increases by mitosis. This is the

basis of the development of a multicellular body from a single cell i.e.,

zygote and also the basis of the growth of a multicellular body.

2.5.3 Cell replacement

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In some parts of body, e.g. skin and digestive tract, cells are constantly

sloughed off and replaced by new ones. New cells are formed by mitosis and

so are exact copies of the cells being replaced. Similarly, RBCs have short

life span (only about 4 months) and new RBCs are formed by mitosis.

2.5.4 Regeneration

Some organisms can regenerate their parts of bodies. The production of new

cells is achieved by mitosis. For example; sea star regenerates its lost arm

through mitosis.

2.5.6 Asexual reproduction

Some organisms produce genetically similar offspring through asexual

reproduction. For example, the hydra reproduces asexually by budding. The

cells at the surface of hydra undergo mitosis and form a mass called bud.

Mitosis continues in the cells of bud and it grows into a new individual. The

same division happens during asexual reproduction or vegetative

propagation in plants.

2.5.7 Consequences of errors

Although errors in mitosis are rare, the process may go wrong, especially

during early cellular divisions in the zygote. Mitotic errors can be especially

dangerous to the organism because future offspring from this parent cell will carry

the same disorder.

In non-disjunction, a chromosome may fail to separate during anaphase. One

daughter cell will receive both sister chromosomes and the other will receive none.

This results in the former cell having three chromosomes containing the same

genes (two sisters and a homologue), a condition known as trisomy, and the latter

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cell having only one chromosome (the homologous chromosome), a condition

known as monosomy. These cells are considered aneuploid, a condition often

associated with cancer. Occasionally when cells experience nondisjunction, they

fail to complete cell division and retain both nuclei in one cell, resulting in

binucleated cells.

Mitosis is a demanding process for the cell, which goes through dramatic

changes in ultrastructure, its organelles disintegrate and reform in a matter of

hours, and chromosomes are jostled constantly by probing microtubules.

Occasionally, chromosomes may become damaged. An arm of the chromosome

may be broken and the fragment lost, causing deletion. The fragment may

incorrectly reattach to another, non-homologous chromosome, causing

translocation. It may reattach to the original chromosome, but in reverse

orientation, causing inversion. Or, it may be treated erroneously as a separate

chromosome, causing chromosomal duplication. The effect of these genetic

abnormalities depends on the specific nature of the error. Errors in the control of

mitosis may cause cancer. All cells have genes that control the timing and number

of mitosis. sometimes mutuations occur in such genes and cells continue to divide.

It results in abnormal cell growth.

Now what happens is that cell abnormally continue to divide at a single

place. It results in the synthesis of execessive tissue growths. When tissues more

than the requirement are synthesized in a single organ, it results in the formation of

Tumors. As long as these tumours remain in their original location they are called

benign tumours. Benign tumours are not harmful as soon as they are not moving.

As soon as they start to move and invade other cells there are said to be malignant

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tumours. Malignant tumors are also known as cancerous tumours and their cells are

called cancerous tumours. Such tumours can send cancer cells to other parts in

body where new tumours may form. This phenomenon is called metastasis or

spreading of disease.

2.6 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. This process may also be referred to as endoreduplication and the cells as

endoploid. An example of a cell that goes through endomitosis is the

megakaryocyte.

2.7 Metaphase

Metaphase, from the ancient Greek(between) and (stage), is a stage of

mitosis in the eukaryotic cell cycle in which condensed & highly coiled

chromosomes, carrying genetic information, align in the middle of the cell before

being separated into each of the two daughter cells. Metaphase accounts for

approximately 4% of the cell cycle's duration. Preceded by events in prometaphase

and followed by anaphase, microtubules formed in prophase have already found

and attached themselves to kinetochores in metaphase. The centromeres of the

chromosomes convene themselves on the metaphase plate (or equatorial plate), an

imaginary line that is equidistant from the two centrosome poles.

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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. In certain types of cells, chromosomes do not line up at the

metaphase plate and instead move back and forth between the poles randomly,

only roughly lining up along the middleline. Early events of metaphase can

coincide with the later events of prometaphase, 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.

One of the cell cycle checkpoints occurs during prometaphase and

metaphase. Only after all chromosomes have become aligned at the metaphase

plate, when every kinetochore is properly attached to a bundle of microtubules,

does the cell enter anaphase. It is thought that unattached or improperly attached

kinetochores generate a signal to prevent premature progression to anaphase, even

if most of the kinetochores have been attached and most of the chromosomes have

been aligned. Such a signal creates the mitotic spindle checkpoint. This would be

accomplished by regulation of the anaphase-promoting complex, securin, and

separase.

2.8 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, which makes them most suitable for visual analysis.

Metaphase chromosomes make the classical picture of chromosomes (karyotype).

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For classical cytogenetic analyses, cells are grown in short term culture and

arrested in metaphase using mitotic inhibitor. 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). Staining

of the slides, often with Giemsa (G banding) or Quinacrine, produces a pattern of

in total up to several hundred bands. Normal metaphase spreads are used in

methods like FISH and as a hybridization matrix for comparative genomic

hybridization (CGH) experiments.

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, for example, losses of chromosomal segments

or translocations, which may lead to chimeric oncogenes, such as bcr-abl in

chronic myelogenous leukemia.

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CHAPTER 3

KARYOTYPING

A karyotype is the number and appearance of chromosomes in the nucleus

of an eukaryotic cell. The term is also used for the complete set of chromosomes in

a species, or an individual organism. Karyotypes describe the number of

chromosomes, and what they look like under a light microscope. Attention is paid

to their length, the position of the centromeres, banding pattern, any differences

between the sex chromosomes, and any other physical characteristics.[4] The

preparation and study of karyotypes is part of cytogenetics. Karyogram of human

male using Giemsa staining. The study of whole sets of chromosomes is

sometimes known as karyology.

The chromosomes are depicted (by rearranging a microphotograph) in a

standard format known as a karyogram or idiogram: in pairs, ordered by size and

position of centromere for chromosomes of the same size. The basic number of

chromosomes in the somatic cells of an individual or a species is called the somatic

number and is designated 2n. Thus, in humans 2n = 46. In the germ-line (the sex

cells) the chromosome number is n (humans: n = 23). So, in normal diploid

organisms, autosomal chromosomes are present in two copies. There may, or may

not, be sex chromosomes. Polyploid cells have multiple copies of chromosomes

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and haploid cells have single copies. The study of karyotypes is important for cell

biology and genetics, and the results may be used in evolutionary biology and

medicine. Karyotypes can be used for many purposes; such as, to study

chromosomal aberrations, cellular function, taxonomic relationships, and to gather

information about past evolutionary events.

3.1 History of karyotype studies

Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli

in 1842. Their behavior in animal (salamander) cells was described by Walther

Flemming, the discoverer of mitosis, in 1882. The name was coined by another

German anatomist, von Waldeyer in 1888. The next stage took place after the

development of genetics in the early 20th century, when it was appreciated that the

set of chromosomes (the karyotype) was the carrier of the genes. 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.

The subsequent history of the concept can be followed in the works of

Darlington and White. 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, concluding an XX/XO sex determination

mechanism. Painter in 1922 was not certain whether the diploid number of humans

was 46 or 48, at first favoring 46. He revised his opinion later from 46 to 48, and

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he correctly insisted on humans having an XX/XY system. Considering their

techniques, these results were quite remarkable.

New techniques were needed to definitively solve the problem:

1. Using cells in culture

2. Pretreating cells in a hypotonic solution, which swells them and spreads the

chromosomes

3. Arresting mitosis in metaphase by a solution of colchicines

4. Squashing the preparation on the slide forcing the chromosomes into a

single plane

5. Cutting up a photomicrograph and arranging the result into an indisputable

karyogram.

It took until the mid 1950s until it became generally accepted that the karyotype

of humans included only 46 chromosomes. Rather interestingly, the great apes

have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral

chromosomes, reducing the number.

3.2 Observations on karyotypes

3.2.1 Staining

The study of karyotypes is made possible by staining. Usually, a

suitable dye, such as Giemsa, is applied after cells have been arrested during cell

division by a solution of colchicine. For humans, white blood cells are used most

frequently because they are easily induced to divide and grow in tissue culture.

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[16] Sometimes observations may be made on non-dividing (interphase) cells. The

sex of an unborn fetus can be determined by observation of interphase cells.

3.2.2 Observations

Six different characteristics of karyotypes are usually observed and

compared:

1. Differences in absolute sizes of 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), both have six pairs of

chromosomes (n=6) yet V. faba chromosomes are many times larger. This

feature probably reflects different amounts of DNA duplication.

2. Differences in the position of centromeres. This is brought about

by translocations.

3. Differences in relative size of chromosomes can only be caused by

segmental interchange of unequal lengths.

4. Differences in basic number of chromosomes may occur due to successive

unequal translocations which finally remove all the essential genetic

material from a chromosome, permitting its loss without penalty to the

organism (the dislocation hypothesis). Humans have one pair fewer

chromosomes than the great apes, but the genes have been mostly

translocated (added) to other chromosomes.

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5. Differences in number and position of satellites, which (when they occur)

are small bodies attached to a chromosome by a thin thread.

6. Differences in degree and distribution of heterochromatic regions.

Heterochromatin stains darker than euchromatin, indicating tighter packing,

and mainly consists of genetically inactive repetitive DNA sequences.

A full account of a karyotype may therefore include the number, type,

shape and banding of the chromosomes, as well as other cytogenetic

information.

Variation is often found:

1. Between the sexes

2. Between the germ-line and soma (between gametes and the rest of the body)

3. Between members of a population (chromosome polymorphism)

4. Geographical variation  between races

5. Mosaics  or otherwise abnormal individuals.

3.3 The human karyotype

Most (but not all) species have a standard karyotype. The normal human

karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex

chromosomes. Normal karyotypes for females contain two X chromosomes and are

denoted 46, XX; males have both an X and a Y chromosome denoted 46, XY. Any

variation from the standard karyotype may lead to developmental abnormalities.

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Fig Diversity and evolution of karyotype

Although the replication and transcription of DNA is highly standardized

in eukaryotes, the same cannot be said for their karyotypes, which are highly

variable. There is variation between species in chromosome number, and in

detailed organization, despite their construction from the same macromolecules.

This variation provides the basis for a range of studies in evolutionary cytology.

In some cases there is even significant variation within species. In a review,

Godfrey and Masters conclude: "In our view, it is unlikely that one process or the

other can independently account for the wide range of karyotype structures that are

observed... But, used in conjunction with other phylogenetic data, karyotypic

fissioning may help to explain dramatic differences in diploid numbers between

closely related species, which were previously inexplicable.

Although much is known about karyotypes at the descriptive level, and it is

clear that changes in karyotype organization have had effects on the evolutionary

course of many species, it is quite unclear what the general significance might be.

"We have a very poor understanding of the causes of karyotype evolution; despite

many careful investigations... the general significance of karyotype evolution is

obscure.

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3.3.1 Changes during development

Instead of the usual gene repression, some organisms go in for large-scale

elimination of heterochromatin, or other kinds of visible adjustment to the

karyotype. Chromosome elimination. In some species, as in many sciarid flies,

entire chromosomes are eliminated during development. Chromatin diminution

(founding father: Theodor Boveri). In this process, found in some copepods and

roundworms such as Ascaris suum, portions of the chromosomes are cast away in

particular cells. This process is a carefully organised genome rearrangement where

new telomeres are constructed and certain heterochromatin regions are lost. In A.

suum, all the somatic cell precursors undergo chromatin diminution. X-

inactivation.

The inactivation of one X chromosome takes place during the early

development of mammals (see Barr body and dosage compensation). In placental

mammals, the inactivation is random as between the two Xs; thus the mammalian

female is a mosaic in respect of her X chromosomes. In marsupials it is always the

paternal X which is inactivated. In human females some 15% of somatic cells

escape inactivation.

3.3.2 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. The diploid number of the Chinese muntjac, Muntiacus reevesi, was

found to be 46, all telocentric. When they looked at the karyotype of the closely

related Indian muntjac, Muntiacus muntjak, they were astonished to find it had

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female = 6, male = 7 chromosomes. "They simply could not believe what they

saw... They kept quiet for two or three years because they thought something was

wrong with their tissue culture... 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 low record is held by the nematode Parascaris univalens, where the

haploid n = 1; the high record would be somewhere amongst the ferns, with the

Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.

Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a

mere 372 chromosomes. The existence of supernumerary or B chromosomes

means that chromosome number can vary even within one interbreeding

population; and aneuploids are another example, though in this case they would not

be regarded as normal members of the population.

3.3.3 Fundamental number

The fundamental number, FN, of a karyotype is the number of visible major

chromosomal arms per set of chromosomes. Thus, FN ≤ 2n, the difference

depending on the number of chromosomes considered single-armed (acrocentric or

telocentric) present. Humans have FN = 82, due to the presence of five acrocentric

chromosome pairs (13, 14, 15, 21 and 22).

3.4 Ploidy

Ploidy is the number of complete sets of chromosomes in a cell. Polyploidy, where

there are more than two sets of homologous chromosomes in the cells, occurs

mainly in plants. It has been of major significance in plant evolution according to

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Stebbins. The proportion of flowering plants which are polyploid was estimated by

Stebbins to be 30-35%, but in grasses the average is much higher, about 70%.

Polyploidy in lower plants (ferns, horsetails and psilotales) is also common, and

some species of ferns have reached levels of polyploidy far in excess of the highest

levels known in flowering plants.

Polyploidy in animals is much less common, but it has been significant in some

groups. Polyploid series in related species which consist entirely of multiples of a

single basic number are known as euploid. Haplo-diploidy, where one sex is

diploid, and the other haploid. It is a common arrangement in the Hymenoptera,

and in some other groups.Endopolyploidy occurs when in adult differentiated

tissues the cells have ceased to divide by mitosis, 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, the

daughter chromosomes separating from each other inside an intact nuclear

membrane.

In many instances, endopolyploid nuclei contain tens of thousands of

chromosomes (which cannot be exactly counted). The cells do not always contain

exact multiples (powers of two), which is why the simple definition 'an increase in

the number of chromosome sets caused by replication without cell division' is not

quite accurate.

This 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.

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The phenomenon occurs sporadically throughout the eukaryote kingdom from

protozoa to man; it is diverse and complex, and serves differentiation and

morphogenesis in many ways. See palaeopolyploidy for the investigation of

ancient karyotype duplications.

3.5 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.

Abnormalities in chromosome number usually cause a defect in development.

Down syndrome and Turner syndrome are examples of this.

Aneuploidy may also occur within a group of closely related species. Classic

examples in plants are the genus Crepis, where the gametic (= haploid) numbers

form the series x = 3, 4, 5, 6, and 7; and Crocus, where every number from x = 3 to

x = 15 is represented by at least one species. Evidence of various kinds shows that

that trends of evolution have gone in different directions in different groups.[41]

Closer to home, the great apes have 24x2 chromosomes whereas humans have

23x2. Human chromosome 2 was formed by a merger of ancestral chromosomes,

reducing the number.

3.5 Chromosomal polymorphism

Some animal species are polymorphic for chromosome fusions or

dissociations. When this happens, the chromosome number is variable from one

individual to another. Well-researched examples are the ladybird beetle Chilocorus

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stigma, some mantids of the genus Ameles, the European shrew Sorex araneus.

There is some evidence from the case of the mollusc Thais lapillus (the dog whelk)

on the Brittany coast, that the two chromosome morphs are adapted to different

habitats.

3.6 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.

In about 6,500 sq mi (17,000 km2), the Hawaiian Islands have the most

diverse collection of drosophilid flies in the world, living from rainforests to

subalpine meadows. These roughly 800 Hawaiian drosophilid species are usually

assigned to two genera, Drosophila and Scaptomyza, in the family Drosophilidae.

The polytene banding of the 'picture wing' group, the best-studied group of

Hawaiian drosophilids, enabled Carson to work out the evolutionary tree long

before genome analysis was practicable. In a sense, gene arrangements are visible

in the banding patterns of each chromosome. Chromosome rearrangements,

especially inversions, make it possible to see which species are closely related.

The results are clear. The inversions, when plotted in tree form (and

independent of all other information), show a clear "flow" of species from older to

newer islands. There are also cases of colonization back to older islands, and

skipping of islands, but these are much less frequent. Using K-Ar dating, the

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present islands date from 0.4 million years ago (mya) (Mauna Kea) to 10mya

(Necker). The oldest member of the Hawaiian archipelago still above the sea is

Kure Atoll, which can be dated to 30 mya. The archipelago itself (produced by the

Pacific plate moving over a hot spot) has existed for far longer, at least into the

Cretaceous. Previous islands now beneath the sea (guyots) form the Emperor

Seamount Chain.

All of the native Drosophila and Scaptomyza species in Hawaii have

apparently descended from a single ancestral species that colonized the islands,

probably 20 million years ago. The subsequent adaptive radiation was spurred by a

lack of competition and a wide variety of niches. Although it would be possible for

a single gravid female to colonise an island, it is more likely to have been a group

from the same species.

There are other animals and plants on the Hawaiian archipelago which

have undergone similar, if less spectacular, adaptive radiations.

3.7 Depiction of karyotypes

3.7.1 Types of banding

Cytogenetics employs several techniques to visualize different aspects of

chromosomes:

G-banding is obtained with Giemsa stain following digestion of

chromosomes with trypsin. It yields a series of lightly and darkly stained

bands - the dark regions tend to be heterochromatic, late-replicating and AT

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rich. The light regions tend to be euchromatic, early-replicating and GC rich.

This method will normally produce 300-400 bands in a normal, human

genome.

R-banding is the reverse of G-banding (the R stands for "reverse"). The dark

regions are euchromatic (guanine-cytosine rich regions) and the bright

regions are heterochromatic (thymine-adenine rich regions).

C-banding: Giemsa binds to constitutive heterochromatin, so it stains

centromeres.

Q-banding is a fluorescent pattern obtained using quinacrine for staining.

The pattern of bands is very similar to that seen in G-banding.

T-banding: visualize telomeres.

Silver staining: Silver nitrate stains the nucleolar organization region-

associated protein. This yields a dark region where the silver is deposited,

denoting the activity of rRNA genes within the NOR.

3.7.2 Classic karyotype cytogenetics

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Karyogram from a human female lymphocyte probed for the Alu sequence using

FISH. In the "classic" (depicted) karyotype, a dye, often Giemsa (G-banding), less

frequently Quinacrine, is used to stain bands on the chromosomes. Giemsa is

specific for the phosphate groups of DNA. Quinacrine binds to the adenine-

thymine-rich regions. Each chromosome has a characteristic banding pattern that

helps to identify them; both chromosomes in a pair will have the same banding

pattern.

Karyotypes are arranged with the short arm of the chromosome on top, and

the long arm on the bottom. Some karyotypes call the short and long arms p and q,

respectively. In addition, the differently stained regions and sub-regions are given

numerical designations from proximal to distal on the chromosome arms. For

example, Cri du chat syndrome involves a deletion on the short arm of

chromosome 5. It is written as 46,XX,5p-. The critical region for this syndrome is

deletion of 15.2, which is written as 46,XX,del(5)(p15.2)

3.7.3 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. Fluorescently labeled probes for each chromosome are made by labeling

chromosome-specific DNA with different fluorophores. Because there are a

limited number of spectrally-distinct fluorophores, a combinatorial labeling

method is used to generate many different colors. Spectral differences generated by

combinatorial labeling are captured and analyzed by using an interferometer

attached to a fluorescence microscope. Image processing software then assigns a

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pseudo color to each spectrally different combination, 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.

3.8 Digital karyotyping

Digital karyotyping is a technique used to quantify the DNA copy number on

a genomic scale. Short sequences of DNA from specific loci all over the genome

are isolated and enumerated. This method is also known as virtual karyotyping.

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CHAPTER 4

4.1 CHROMOSOMAL ABNORMALITIES

Chromosome abnormalities can be numerical, as in the presence of extra or

missing chromosomes, or structural, as in derivative chromosome, translocations,

inversions, large-scale deletions or duplications. Numerical abnormalities, also

known as aneuploidy, often occur as a result of nondisjunction during meiosis in

the formation of a gamete; trisomies, in which three copies of a chromosome are

present instead of the usual two, are common numerical abnormalities. Structural

abnormalities often arise from errors in homologous recombination. Both types of

abnormalities can occur in gametes and therefore will be present in all cells of an

affected person's body, 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, X or 45, X0).

Klinefelter syndrome , the most common male chromosomal disease,

otherwise known as 47, XXY is caused by an extra X chromosome.

Edwards syndrome is caused by trisomy (three copies) of chromosome 18.

Down syndrome , a common chromosomal disease, is caused by trisomy of

chromosome 21.

Patau syndrome is caused by trisomy of chromosome 13.

Also documented are trisomy 8, trisomy 9 and trisomy 16, although they

generally do not survive to birth.

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Some disorders arise from loss of just a piece of one chromosome, including

Cri du chat (cry of the cat), from a truncated short arm on chromosome 5.

The name comes from the babies' distinctive cry, caused by abnormal

formation of the larynx.

1p36 Deletion syndrome , from the loss of part of the short arm of

chromosome 1.

Angelman syndrome – 50% of cases have a segment of the long arm of

chromosome 15 missing; a deletion of the maternal genes, example of

imprinting disorder.

Prader-Willi syndrome – 50% of cases have a segment of the long arm of

chromosome 15 missing; a deletion of the paternal genes, example of

imprinting disorder.

Chromosomal abnormalities can also occur in cancerous cells of an otherwise

genetically normal individual; one well-documented example is the Philadelphia

chromosome, a translocation mutation commonly associated with chronic

myelogenous leukemia and less often with acute lymphoblastic leukemia.

A chromosome anomaly, abnormality or aberration reflects an atypical

number of chromosomes or a structural abnormality in one or more chromosomes.

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 may be detected or confirmed in this manner.

Chromosome anomalies usually occur when there is an error in cell division

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following meiosis or mitosis. There are many types of chromosome anomalies.

They can be organized into two basic groups, numerical and structural anomalies.

4.2 Numerical Disorders

This is called Aneuploidy (an abnormal number of chromosomes), and

occurs when an individual is missing either a chromosome from a pair

(monosomy) or has more than two chromosomes of a pair (Trisomy, Tetrasomy,

etc.). In humans an example of a condition caused by a numerical anomaly is

Down Syndrome, also known as Trisomy 21 (an individual with Down Syndrome

has three copies of chromosome 21, rather than two). Turner Syndrome is an

example of a monosomy where the individual is born with only one sex

chromosome, an X.

4.3 Structural abnormalities

When the chromosome's structure is altered. This can take several forms:

Deletions : A portion of the chromosome is missing or deleted. Known

disorders in humans include Wolf-Hirschhorn syndrome, which is caused by

partial deletion of the short arm of chromosome 4; and Jacobsen syndrome,

also called the terminal 11q deletion disorder.

Duplications : A portion of the chromosome is duplicated, resulting in extra

genetic material. 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.

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Translocations : When a portion of one chromosome is transferred to

another chromosome. There are two main types of translocations. In a

reciprocal translocation, segments from two different chromosomes have

been exchanged. In a Robertsonian translocation, an entire chromosome has

attached to another at the Centromere - in humans these only occur with

chromosomes 13, 14, 15, 21 and 22.

Inversions : A portion of the chromosome has broken off, turned upside

down and reattached, therefore the genetic material is inverted.

Rings : A portion of a chromosome has broken off and formed a circle or

ring. This can happen with or without loss of genetic material.

Isochromosome : Formed by the mirror image copy of a chromosome

segment including the centromere.

Chromosome instability syndromes are a group of disorders characterized by

chromosomal instability and breakage. They often lead to an increased tendency to

develop certain types of malignancies.

4.3 Inheritance

Most chromosome abnormalities occur as an accident in the egg or sperm,

and are therefore initially not inherited. Therefore, the anomaly is present in every

cell of the body. Some anomalies, however, can happen after conception, resulting

in Mosaicism (where some cells have the anomaly and some do not). Chromosome

anomalies can be inherited from a parent or be "de novo". This is why

chromosome studies are often performed on parents when a child is found to have

an anomaly.

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4.4 Cytogenetics

Cytogenetics is a branch of genetics that is concerned with the study of the

structure and function of the cell, especially the chromosomes. It includes routine

analysis of G-Banded chromosomes, other cytogenetic banding techniques, as well

as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and

comparative genomic hybridization (CGH).

4.5 Early years

Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli

in 1842. Their behavior in animal (salamander) cells was described by Walther

Flemming, the discoverer of mitosis, in 1882. The name was coined by another

German anatomist, von Waldeyer in 1888.

The next stage took place after the development of genetics in the early 20th

century, when it was appreciated that the set of chromosomes (the karyotype) was

the carrier of the genes. 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. 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, concluding an XX/XO sex determination

mechanism. Painter in 1922 was not certain whether the diploid number of man

was 46 or 48, at first favoring 46. He revised his opinion later from 46 to 48, and

he correctly insisted on man having an XX/XY system. Considering their

techniques, these results were quite remarkable.

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New techniques were needed to definitively solve the problem:

1. Using cells in culture

2. Pre-treating cells in a hypotonic solution, which swells them and

spreads the chromosomes

3. Arresting mitosis in metaphase by a solution of colchicine

4. Squashing the preparation on the slide forcing the chromosomes into a

single plane

5. Cutting up a photomicrograph and arranging the result into an

indisputable karyogram.

It took until 1956 until it became generally accepted that the karyotype of man

included only 46 chromosomes. Rather interestingly, the great apes have 48

chromosomes. Human chromosome 2 was formed by a merger of ancestral

chromosomes, reducing the number.

4.6 Applications in biology

4.6.1 McClintock's work on maize

Barbara McClintock began her career as a maize cytogeneticist. In 1931,

McClintock and Harriet Creighton demonstrated that cytological recombination of

marked chromosomes correlated with recombination of genetic traits (genes).

McClintock continued her career in cytogenetics studying the mechanics and

inheritance of broken and ring (circular) chromosomes of maize. During her

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cytogenetic work, McClintock discovered transposons, a find which eventually led

to her Nobel Prize in 1983.

4.6.2 Natural populations of Drosophila

In the 1930s, Dobzhansky and his co-workers collected Drosophila

pseudoobscura and D. persimilis from wild populations in California and

neighboring states. Using Painter's technique they studied the polytene

chromosomes and discovered that the wild populations were polymorphic for

chromosomal inversions. All the flies look alike whatever inversions they carry:

this is an example of a cryptic polymorphism.

Evidence rapidly accumulated to show that natural selection was

responsible. Using a method invented by L'Heretier and Teissier, Dobzhansky bred

populations in population cages, which enabled feeding, breeding and sampling

whilst preventing escape. This had the benefit of eliminating migration as a

possible explanation of the results. Stocks containing inversions at a known initial

frequency can be maintained in controlled conditions. It was found that the various

chromosome types do not fluctuate at random, as they would if selectively neutral,

but adjust to certain frequencies at which they become stabilised. By the time

Dobzhansky published the third edition of his book in 1951 he was persuaded that

the chromosome morphs were being maintained in the population by the selective

advantage of the heterozygotes, as with most polymorphisms.

4.7 Human abnormalities and medical applications

In the event of procedures which allowed easy enumeration of

chromosomes, discoveries were quickly made related to aberrant chromosomes or

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chromosome number. In some congenital disorders, such as Down's syndrome,

cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy.

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.

In 1959, Lejeune discovered patients with Down syndrome had an extra copy of

chromosome 21. Down syndrome is also referred to as trisomy 21.

Other numerical abnormalities discovered include sex chromosome

abnormalities. An individual with only one sex chromosome (the X) has Turner

syndrome, an additional X chromosome in a male, resulting in 47 total

chromosomes, has Klinefelter's Syndrome. Many other sex chromosome

combinations are compatible with live birth including XXX, XYY, and XXXX.

The ability for mammals to tolerate aneuploidies in the sex chromosomes arises

from the ability to inactivate them, which is required in normal females to

compensate for having two copies of the chromosome. Not all genes on the X

Chromosome are inactivated, which is why there is a phenotypic effect seen in

individuals with extra X chromosomes.

Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with

Edward's Syndrome.

In 1960, Peter Nowell and David Hungerford[17] discovered a small

chromosome in the white blood cells of patients with Chronic myelogenous

leukemia (CML). This abnormal chromosome was dubbed the Philadelphia

chromosome - as both scientists were doing their research in Philadelphia,

Pennsylvania. Thirteen years later, with the development of more advanced

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techniques, the abnormal chromosome was shown by Janet Rowley to be the result

of a translocation of chromosomes 9 and 22. Identification of the Philadelphia

chromosome by cytogenetics, in addition to other tests, is used today as a

diagnostic for CML.

FIG Advent of banding techniques

In the late 1960s, Caspersson developed banding techniques which

differentially stain chromosomes. 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. Deletion

syndromes such as DiGeorge syndrome, Prader-Willi syndrome and others were

discovered to be caused by deletions in chromosome material.

Diagrams identifying the chromosomes based on the banding patterns are

known as cytogenetic maps. These maps became the basis for both prenatal and

oncological fields to quickly move cytogenetics into the clinical lab where

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karyotyping allowed scientists to look for chromosomal alterations. Techniques

were expanded to allow for culture of free amniocytes recovered from amniotic

fluid, and elongation techniques for all culture types that allow for higher

resolution banding.

4.8 Beginnings of molecular cytogenetics

In the 1980s, advances were made in molecular cytogenetics. While

radioisotope-labeled probes had been hybridized with DNA since 1969, movement

was now made in using fluorescent labeled probes. Hybridizing them to

chromosomal preparations using existing techniques came to be known as

fluorescent in situ hybridization (FISH). This change significantly increased the

usage of probing techniques as fluorescent labeled probes are safer and can be used

almost indefinitely. Further advances in micromanipulation and examination of

chromosomes led to the technique of chromosome microdissection whereby

aberrations in chromosomal structure could be isolated, cloned and studied in ever

greater detail.

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CHAPTER 5

Techniques

5.1 Karyotyping

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 (R-

banding) 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

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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

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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

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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-2-

phenylindole (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. For congenital

problems usually 20 metaphase cells are scored.

Future of cytogenetics

Advances now focus on molecular cytogenetics including automated

systems for counting the results of standard FISH preparations and techniques for

virtual karyotyping, such as comparative genomic hybridization arrays, CGH and

Single nucleotide polymorphism-arrays.

CHAPTER 6

MATLAB

MATLAB is a high-level technical computing language and interactive

environment for algorithm development, data visualization, data analysis, and

numerical computation. Using MATLAB, you can solve technical computing

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problems faster than with traditional programming languages, such as C, C++, and

FORTRAN.

You can use MATLAB in a wide range of applications, including signal and

image processing, communications, control design, test and measurement,

financial modeling and analysis, and computational biology. Add-on toolboxes

(collections of special-purpose MATLAB functions) extend the MATLAB

environment to solve particular classes of problems in these application areas.

MATLAB provides a number of features for documenting and sharing your

work. You can integrate your MATLAB code with other languages and

applications, and distribute your MATLAB algorithms and applications. The

MATLAB language supports the vector and matrix operations that are fundamental

to engineering and scientific problems. It enables fast development and execution.

With the MATLAB language, you can program and develop algorithms

faster than with traditional languages because you do not need to perform low-

level administrative tasks, such as declaring variables, specifying data types, and

allocating memory. In many cases, MATLAB eliminates the need for ‘for’ loops.

As a result, one line of MATLAB code can often replace several lines of C or C++

code.

6.1 Image Processing

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The image processing step aims at image contrast enhancement and

compensation of geometric distortions observed in each chromosome not related

with its intrinsic shape or size. The image 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. These effects must be compensated to improve the

results of the pairing algorithm.

The image processing step is composed of the following operations.

1) Chromosome extraction—Each chromosome is isolated from the unordered

karyogram.

2) Geometrical compensation—The geometric compensation, performed by

using the algorithm is needed to obtain chromosomes with vertical medial axis,

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, geometrical

and dimensional differences must be removed, or at least attenuated. Therefore, a

dimensional scaling is performed before the pattern features is extracted to make

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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. To compensate

for this inhomogeneity, the spatially scaled images are histogram equalized.

6.2 Concepts used in this phase

1) Image conversion

2) Denoising

3) Edge detection

4) Two dimensional convolutions.

6.2.1 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. For example, if you want to filter

a color image that is stored as an indexed image, you must first convert it to true

color format. When you apply the filter to the true color image, MATLAB filters

the intensity values in the image, as is appropriate. If you attempt to filter the

indexed image, MATLAB simply applies the filter to the indices in the indexed

image matrix, and the results might not be meaningful. You can perform certain

conversions just using MATLAB syntax. For example, you can convert a grayscale

image to true color format by concatenating three copies of the original matrix

along the third dimension.

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RGB = cat (3,I,I,I);

The resulting true color image has identical matrices for the red, green, and blue

planes, so the image displays as shades of gray.

In addition to these image type conversion functions, there are other functions

that return a different image type as part of the operation they perform. For

example, the region of interest functions returns a binary image that you can use to

mask an image for filtering or for other operations.

6.2.4 Denoising

We may define noise to be any degradation in the image signal, caused by

external disturbance. If an image is being sent electronically from one place to

another, via satellite or wireless transmission, or through networked cable, we may

expect errors to occur in the image signal. These errors will appear on the image

output in different ways depending on the type of disturbance in the signal.

Usually we know what type of errors to expect, and hence the type of noise on the

image; hence we can choose the most appropriate method for reducing the effects.

Cleaning an image corrupted by noise is thus an important area of image

restoration.

6.2.5 Edge detection

Edges contain some of the most useful information in an image. We may use

edges to measure the size of objects in an image; to isolate particular objects from

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their background; to recognize or classify objects. There is a large number of edge

finding algorithms in existence, and we shall look at some of the more

straightforward of them. The general Matlab command for finding edges is

edge(image,'method',parameters. . . ) Where the parameters available depend on

the method used

6.3 Two dimensional convolutions

C = conv2(A,B) computes the two-dimensional convolution of matrices A

and B. If one of these matrices describes a two-dimensional finite impulse response

(FIR) filter, the other matrix is filtered in two dimensions. The size of C in each

dimension is equal to the sum of the corresponding dimensions of the input

matrices, minus one. That is, if the size of A is [ma,na] and the size of B is

[mb,nb], then the size of C is [ma+mb-1,na+nb-1].

The indices of the center element of B are defined as floor(([mb nb]+1)/2).

C = conv2(hcol,hrow,A) convolves A first with the vector hcol along the rows and

then with the vector hrow along the columns. If hcol is a column vector and hrow

is a row vector, this case is the same as C = conv2(hcol*hrow,A). C =

conv2(...,'shape') returns a subsection of the two-dimensional convolution, as

specified by the shape parameter

Algorithms description

1) Read the image and convert into gray

2)Remove noise

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3) Background separation

4) Edge detect

5) Separate the pairs

MODULE 1

PSEUDO CODE

iimread('12345.bmp');

rgb2gray

im2bw(im,0.7);

imedfilt2(im1,[3 3]);

edge(im1,'sobel');

[imx,imy]=size(BW);

Msk

conv2(double(BW),double(msk));

bwlabel(B,8);

mx=max(max(L));

[r,c] = find(L==22);

rc = [r c];

[sx sy]=size(rc);

nzeros(imx,imy);

for i=1:sx

x1=rc(i,1);

y1=rc(i,2);

n1(x1,y1)=255;

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MODULE 2

clc

[m,n]=size(L);

L_number=zeros(mx,1);

Index=1;

flag=0;

for i=1:m

for j=1:n

if L(i,j)~=0

for k=1:mx

if L(i,j)==L_number(k)

flag=1;

end

end

if flag~=1

L_number(Index)=L(i,j);

Index=Index+1;

end

flag=0;

end;

end

end

L_number;

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Test_number=[3,4,6,7,8,9,10,11,14,15,19,20,21,22,24,26,27,28,29,30,31,32,33,35,

36,38,39,40,41,42,43,45,48,49,50,51,52,54,55,56,57,59,60,62,65,66];

for x=1:46

[r,c] = find(L==L_number((Test_number(x))));

rc = [r c];

[sx sy]=size(rc);

n1=zeros(imx,imy);

for i=1:sx

x1=rc(i,1);

y1=rc(i,2);

n1(x1,y1)=255;

end

%h=figure;imshow(n1,[]);

end

Circumference=zeros(46,1);

Arm_length=zeros(46,1);

Area=zeros(46,1);

for i=1:46

f=imread(strcat(num2str(i),'.bmp'));

BW=im2bw(f);

BW=double(BW);

BW1=edge(BW,'canny');

[m n]=size(BW1);

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Circumference_sum=0;

for x=1:m

for y=1:n

if BW1(x,y)==1

Circumference_sum=Circumference_sum+1;

end

end

end

Circumference(i)=Circumference_sum;

f=imcomplement(f);

skel=im2double(f);

skel=im2bw(skel,1.5*graythresh(skel));

s=bwmorph(skel,'skel',Inf);

s1=bwmorph(s,'spur',8);

Arm_length_sum=0;

[m n]=size(s1);

for x=1:m

for y=1:n

if s1(x,y)==1

Arm_length_sum=Arm_length_sum+1;

end

end

end

Arm_length(i)=Arm_length_sum;

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Area_sum=0;

BW=im2bw(f);

[m n]=size(BW);

for x=1:m

for y=1:n

if BW(x,y)==1

Area_sum=Area_sum+1;

end

end

end

Area(i)=Area_sum;

end

Circumference;

Arm_length;

Area;

Pair=zeros(46,2);

for i=1:45

min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)-

Arm_length(i+1))+abs(Area(i)-Area(i+1));

Pair(i,1)=i;

Pair(i,2)=i+1;

for j=1:46

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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,2)=j;

end

end

end

for i=1:45

if Pair(i,2)==46

Pair(46,1)=46;

Pair(46,2)=i;

end

end

Pair;

delete=zeros(46,1);

flag=0;

figure_flag=1;

for i=1:46

for j=1:46

if Pair(i,1)==delete(j)

flag=1;

end

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end

if flag~=1

if figure_flag~=47

subplot(23,2,figure_flag);

figure_flag=figure_flag+1;

end

f1=imread(strcat(num2str(Pair(i,1)),'.bmp'));

imshow(f1);

if figure_flag~=47

subplot(23,2,figure_flag);

figure_flag=figure_flag+1;

end

f2=imread(strcat(num2str(Pair(i,2)),'.bmp'));

imshow(f2);

delete(figure_flag)=Pair(i,2);

end

flag=0;

end

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CONCLUTION

In this paper, a newmetric is proposed to measure the distance

between chromosomes to be used in the automatic chromosome pairing procedure,

in the scope of karyotyping process used in cytogentic analysis. The proposed

algorithm is based on the traditional features extracted from the karyogram, such

as, dimensions and banding profiles, plus a new one, based on the MI, to improve

the discriminative power of the pairing algorithm with respect to the the G-banding

pattern. The main goal of this paper is to provide useful contributions toward the

design of a fully automatic chromosomes pairing algorithm of bone marrow cells

to be used in the diagnosis of leukemia. The 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, Copenhagen, and Philadelphia. The

algorithm is composed by four main steps: 1) image processing of the karyograms

provided by the technicians; 2) feature extraction from the processed images

characterizing the size, shape and band pattern; 3) training of a classifier

(performed once) where similarity among chromosomes are characterized; and

finally, 4) pairing. In the image processing step, the romosome images, extracted

from the unordered karyogram, are processed in order to compensate for

geometrical and intensity distortions, and to normalize their dimensions. This

normalization is needed to make it possible the band pattern comparison between

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chromosomes. The features extracted from the processed images discriminate each

pair with respect to their size, shape, and band pattern. Here, a novel metric

distance is proposed to be used in the pairing procedure that linearly combines the

distances associated with each feature. The coefficients of the linear combination

are obtained through a training step using chromosomes paired manually by

experts. 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. The training process

consists in the estimation of each vector of coefficient , from the chromosomes in

the training set, by minimizing the overall distances between chromosomes of the

same class (intraclass) and by maximizing the distances between chromosomes

when at least one of them does not belong to that class (interclass). The pairing

process is performed by efficiently solving a combinatorial problem where a

permutation matrix is obtained from the distance matrix computed with the

extracted features associated with each pair of chromosomes in the karyogram.

Tests using 19 karyograms based on bone marrow cells,working with 22 classes of

chromosomes and a LOOCV strategy allowus to conclude that the proposed

pairing algorithms,working within an 8-D feature space, achieves a 70.10% mean

classification rate. 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

performance of the classifier. Executing the algorithm on a higher quality dataset, a

76.10% classification ratewas obtained. Using 27 karyograms andworking with a

limited number of classes (≤ 8), amean classification rate larger than 93% was

obtained in all experiments. Qualitative comparisons with the results obtained with

the Leica CW 4000 Karyopairing software using the same data were performed

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and have shown relevant improvements. In addition, a new chromosome dataset

with 9200 chromosomes from bone marrow cells, called LK1 , was built to provide

a ground truth to test classification and pairing algorithms for this type of “low”

image quality chromosomes. This dataset was made publicly available [29]. The

results presented in this paper are promising. In fact, despite the low quality of this

type of chromosomes, it was shown that it is possible to achieve comparable

classification rates to the ones obtained with the classical chromosome dataset,

such as Edinburgh, Copenhagen, or Philadelphia, whose images are of significantly

higher quality, presenting a uniform level of condensation, and from which it is

possible to extract additional features, e.g., centromere position.

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