Chapter 6. Variation in Chromosome Number and Structure

Chapter 6. Variation in Chromosome Number and Structure 1. Cytogenetics & Cytological Technique 2. Analysis of Mitotic Chromosomes 3. Cytogenetic Variation 1

The cultivation of wheat originated some 10,000 years ago in the Middle East. Today, wheat is the principal food crop for more than a billion people. More than 17,000 varieties have been developed, each adapted to a different locality. 2

Modern cultivated wheat, Triticum aestivum, is a hybrid of at least three different species (AABBDD). Its progenitors were low-yielding grasses that grew in Syria, Iran, Iraq, and Turkey. Although we do not know the exact course of events, two of the grasses apparently interbred, producing a species that excelled as a crop plant. Through human cultivation, this hybrid species was selectively improved, and then it, too, interbred with a third species, yielding a triple-hybrid that was even better suited for agriculture. 3

They had larger grains, they were more easily harvested, and they grew in a wider range of conditions. We now understand the chromosomal basis for these improvements. Triple-hybrid wheat contains the chromosomes of each of its progenitors. 4

1. Cytogenetics & Cytological Techniques The study of chromosome numbers, structure, function, and behavior in relation to gene inheritance, organization and expression 5

Modern cytogenetics attempts to bring together: the organization of chromosomes in the nucleus with the requirements of gene expression, in different developmental and environmental contexts. the behavior of chromosomes with transmission of phenotypes to progeny changes in chromosomal structure and number with speciation the evolution of the genome with the evolution of the species 6

Cytogenetics blossomed during the twentieth century, as microscopes improved and better procedures for preparing and staining chromosomes were developed. The demonstration that genes reside on chromosomes boosted interest in this research and led to important studies on chromosome number and structure. Today, cytogenetics has significant applied aspects, especially in medicine, where it is used to determine whether disease conditions are associated with chromosome abnormalities. 7

2. Analysis of Mitotic Chromosomes Researchers perform most cytological analyses on dividing cells, usually cells in the middle of mitosis. To enrich for cells at this stage, they have traditionally used rapidly growing material such as animal embryos and plant root tips. However, the development of cell-culturing techniques has made it possible to study chromosomes in other types of cells 8

human white blood cells can be collected from peripheral blood, separated from the nondividing red blood cells, and put into culture. The white cells are then stimulated to divide by chemical treatment, and midway through division a sample of the cells is prepared for cytological analysis. The usual procedure is to treat the dividing cells with a chemical that disables the mitotic spindle. 9

Mitotically arrested cells are then swollen by immersion in a hypotonic solution that causes the cells to take up water by osmosis. 10

For many years it was erroneously thought that human cells contained 48 chromosomes. The correct number, 46, was determined only after the swelling technique was used to separate the chromosomes within individual mitotic cells. Until the late 1960s and early 1970s, chromosome spreads were stained with Feulgen's reagent, a purple dye that reacts with the sugar molecules in DNA, or with aceto-carmine, a deep red dye. 11

Chromosomes of the incense cedar (Calocedrus decurrens) stained with aceto-carmine. 12

Quinacrine, a chemical relative of the antimalarial drug quinine. because quinacrine is a fluorescent compound, the bands appear only when the chromosomes are exposed to ultraviolet (UV) light. Parts of the chromosome shine brightly, whereas other parts remain dark. This staining procedure is called Q banding, and the bands that it produces are called Q bands. 13

Metaphase chromosomes of the plant Allium carinatum, stained with quinacrine to reveal Q bands. 14

Excellent nonfluorescent staining techniques have also been developed. The most popular of these uses Giemsa stain, a mixture of dyes named after its inventor, Gustav Giemsa. - Giemsa solution consisting of methylene azure, methylene violet, methylene blue, and eosin. Like quinacrine, Giemsa creates a reproducible pattern of bands on each chromosome. However, the nature of the banding pattern depends on how the chromosomes were prepared prior to staining. 15

One procedure, called G banding, gives dark bands that correspond roughly to the bright bands obtained with quinacrine; (dark regions tend to be heterochromatic, late-replicating and AT rich) another procedure, called R banding (Reverse), gives the reverse pattern dark bands that correspond roughly to light G bands. A third procedure, called C banding, stains the region around the centromere of each chromosome. These different banding techniques provide cytogeneticists with the means to analyze fine details of chromosome structure 16

Metaphase chromosomes of the deerlike Asian muntjak stained to show G banding. 17

Metaphase human chromosomes stained with acridine orange to show R banding. 18

Metaphase chromosomes of the domestic sheep stained to show C banding. 19

Chromatin: is a complex of DNA and protein found inside the nuclei of eukaryotic cells Examination of Feulgen-stained nuclei revealed nuclear substructure distinguished by differing levels of staining, designated heterochromatin, euchromatin and nucleoli in order of decreasing stain intensity It is now known that the intense staining properties of heterochromatin in interphase is a result of an increased concentration of DNA caused by a more extensive condensation of the chromatin than the rest of the chromosome. 20

C-Banding Giemsa stains the satellite-rich centromeric heterochromatin. C-bands reveal the location of constitutive heterochromatin. Constitutive heterochromatin Facultative heterochromatin 21

The regions of DNA packaged in facultative heterochromatin will not be consistent within the cells of a species, and thus a sequence in one cell that is packaged in facultative heterochromatin (and the genes within poorly expressed) may be packaged in euchromatin in another cell (and the genes within no longer silenced). The formation of facultative heterochromatin is regulated, and is often associated with morphogenesis or differentiation. -1, 9, 16, and the Y chromosome contain large regions of constitutive heterochromatin 22

A karyotype is an organized profile of chromosomes. In a karyotype, chromosomes are arranged and numbered by size, from largest to smallest. This arrangement helps scientists quickly identify chromosomal alterations that may result in a genetic disorder. Karyotypes can be based on mitotic or meiotic chromosomes and are enhanced by chromosome banding techniques. 23

Metacentric Submetacentric Acrocentric Telocentric 25 http://www.citruscollege.edu/pic/46/tp11_02.jpg

인간 46 개염색체 A ~ G groups A B A: Metacentric B: Submetacentric C: Submetacentric D: acrocentric E: Submetacentric F: Metacentric G: acrocentric D C E F G 26

3. Cytogenetic Variation The phenotypes of many organisms are affected by changes in the number of chromosomes in their cells; sometimes even changes in part of a chromosome can be significant. These numerical changes are usually described as variations in the ploidy of the organism (from the Greek word meaning fold, as in twofold ). Organisms with complete, or normal, sets of chromosomes are said to be euploid (from the Greek words meaning good and fold ). 27

Organisms that carry extra sets of chromosomes are said to be polyploid (from the Greek words meaning many and fold ), and the level of polyploidy is described by referring to a basic chromosome number, usually denoted n. Organisms in which a particular chromosome, or chromosome segment, is under- or overrepresented are said to be aneuploid (from the Greek words meaning not, good, and fold ). These organisms therefore suffer from a specific genetic imbalance. 28

Cytogeneticists have also catalogued various types of structural changes in the chromosomes of organisms. For example, a piece of one chromosome may be fused to another chromosome, or a segment within a chromosome may be inverted with respect to the rest of that chromosome. These structural changes are called rearrangements. Because rearrangements segregate irregularly during meiosis, they are often associated with aneuploidy. 29

Polyploidy Polyploidy, the presence of extra chromosome sets, is fairly common in plants but very rare in animals. One-half of all known plant genera contain polyploid species, and about two-thirds of all grasses are polyploids. Many of these species reproduce asexually. In animals, where reproduction is primarily by sexual means, polyploidy is rare, probably because it interferes with the sex-determination mechanism. 30

Polyploidy One general effect of polyploidy is that cell size is increased, presumably because there are more chromosomes in the nucleus. Often this increase in size is correlated with an overall increase in the size of the organism. Polyploid species tend to be larger and more robust than their diploid counterparts. 31

Sterile Polyploids Many polyploid species are sterile. Extra sets of chromosomes segregate irregularly in meiosis, leading to grossly unbalanced (that is, aneuploid) gametes. As an example, let us consider a triploid species with three identical sets of n chromosomes. The total number of chromosomes is therefore 3n. When meiosis occurs, each chromosome will try to pair with its homologues. 33

Sterile Polyploids One possibility is that two homologues will pair completely along their length, leaving the third without a partner; this solitary chromosome is called a univalent. Another possibility is that all three homologues will synapse, forming a trivalent in which each member is partially paired with each of the others. 34

Sterile Polyploids It is difficult to predict how the chromosomes will move during anaphase of the first meiotic division. The more likely event is that two of the homologues will move to one pole, and one homologue will move to the other, yielding gametes with one or two copies of the chromosome. However, all three homologues might move to one pole, producing gametes with zero or three copies of the chromosome. Because this segregational uncertainty applies to each trio of chromosomes in the cell, the total number of chromosomes in a gamete can vary from zero to 3n. 37

Sterile Polyploids Zygotes formed by fertilization with such gametes are almost certain to die; thus, most triploids are completely sterile. In agriculture and horticulture, this sterility is circumvented by propagating the species asexually. The many methods of asexual propagation include cultivation from cuttings (bananas), grafts (Winesap, Gravenstein, and Baldwin apples), and bulbs (tulips). In nature, polyploid plants can also reproduce asexually. One mechanism is apomixis, which involves a modified meiosis that produces unreduced eggs; these eggs then form seeds that germinate into new plants. 38

Fertile Polyploids Some tetraploids are able to produce viable progeny. Close examination shows that these species contain two distinct sets of chromosomes and that each set has been duplicated. Thus, fertile tetraploids seem to have arisen by chromosome duplication in a hybrid that was produced by a cross of two different, but related, diploid species; most often these species have the same or very similar chromosome numbers. 39

A plausible mechanism for the origin of such a tetraploid. 40

Fertile Polyploids This scenario of hybridization between different but related species followed by chromosome doubling has evidently occurred many times during plant evolution. In some cases, the process has occurred repeatedly, generating complex polyploids with distinct chromosome sets. One of the best examples is modern bread wheat, Triticum aestivum. 41

Cytogeneticists have identified primitive cereal plants in the Middle East that may have participated in this evolutionary process 42

Because chromosomes from different species are less likely to interfere with each other's segregation during meiosis, polyploids arising from hybridizations between different species have a much greater chance of being fertile than do polyploids arising from the duplication of chromosomes in a single species. Polyploids created by hybridization between different species are called allopolyploids; in these polyploids, the contributing genomes are qualitatively different. Polyploids created by chromosome duplication within a species are called autopolyploids (from the Greek prefix for self ); in these polyploids, a single genome has been multiplied to create extra chromosome sets. 43

Tissue-Specific Polyploidy and Polyteny In some organisms, certain tissues become polyploid during development. This polyploidization is probably a response to the need for multiple copies of each chromosome and the genes it carries. The process that produces such polyploid cells, called endomitosis, involves chromosome duplication, followed by separation of the resulting sister chromatids. However, because there is no accompanying cell division, extra chromosome sets accumulate within a single nucleus. In the human liver and kidney, for example, one round of endomitosis produces tetraploid cells. 44

Tissue-Specific Polyploidy and Polyteny Sometimes polyploidization occurs without the separation of sister chromatids. In these cases, the duplicated chromosomes pile up next to each other, forming a bundle of strands that are aligned in parallel. The resulting chromosomes are said to be polytene, from the Greek words meaning many threads. Polytene chromosomes are found in the salivary glands of Drosophila larvae. Each chromosome undergoes about nine rounds of replication, producing a total of about 500 copies in each cell. 45

Polytene chromosomes of Drosophila. 46

Aneuploidy Aneuploidy describes a numerical change in part of the genome, usually a change in the dosage of a single chromosome. Individuals that have an extra chromosome, are missing a chromosome, or have a combination of these anomalies are said to be aneuploid. 47

Aneuploidy Datura stramonium. This diploid species has 12 pairs of chromosomes. Blakeslee collected peculiar mutants. Those were apparently caused by dominant factors that were transmitted primarily through the female. Belling found that in every case an extra chromosome was present. Detailed analysis established that the extra chromosome was different in each mutant strain. Such triplications are called trisomies. 48

Datura stramonium 49

Aneuploidy Belling also discovered the reason for the preferential transmission of the trisomic phenotypes through the female. During pollen tube growth, aneuploid pollen in particular, pollen with n + 1 chromosomes does not compete well with euploid pollen. Consequently, trisomic plants almost always inherit their extra chromosome from the female parent. 50

Aneuploidy An organism in which a chromosome, or a piece of a chromosome, is underrepresented is referred to as a hypoploid (from the Greek prefix for under ). When a chromosome or chromosome segment is overrepresented, the organism is said to be hyperploid (from the Greek prefix for over ). Each of these terms covers a wide range of abnormalities. 51

trisomics (presence of an extra chromosome of the standard set, 2n + 1), monosomics (loss of a single chromosome, 2n - 1), nullisomics (loss of a chromosome pair, 2n - 2), and tetrasomics (occurrence of an extra chromosome pair, 2n + 2) 52

Trisomy in Human Trisomic 21 is also called Down's Syndrome (1/700 in newborns). People with Down syndrome are typically short in stature and loose-jointed, particularly in the ankles; they have broad skulls, wide nostrils, large tongues with a distinctive furrowing, and stubby hands with a crease on the palm. Impaired mental abilities require that they be given special training and care. 47, XX,+21 54

Trisomy in Human 55

Trisomy in Human the incidence of trisomics 13 (Patau's Syndrome) is about 1/20,000 Trisomic 18 is called Edward's Syndrome (1/8000 in newborns). Trisomic 21 is also called Down's Syndrome (1/700 in newborns). -among mothers younger than 25 years old, the risk of having a child with Down syndrome is about 1 in 1500, whereas among mothers 40 years old, it is 1 in 100. 56

Meiotic nondisjunction and the origin of Down syndrome. Nondisjunction at meiosis I produces no normal gametes. Nondisjunction at meiosis II produces a gamete with two identical sister chromosomes, a gamete lacking 57 chromosome 21, and two normal gametes.

Sex chromosome trisomics (XXX, XXY, XYY) triplo-x karyotype, 47, XXX. These individuals survive because two of the three X chromosomes are inactivated, reducing the dosage of the X chromosome so that it approximates the normal level of one. Triplo-X individuals are female and are phenotypically normal, or nearly so; sometimes they exhibit a slight mental impairment and reduced fertility. 58

The 47, XXY karyotype is also a viable trisomy in human beings. These individuals have three sex chromosomes, two X's and one Y. Phenotypically, they are male, but they can show some female secondary sexual characteristics and are usually sterile. -now called Klinefelter syndrome; these include small testes, enlarged breasts, long limbs, knock-knees, and underdeveloped body hair. 59

The 47, XYY karyotype is another viable trisomy in human beings. These individuals are male, and except for a tendency to be taller than 46, XY men, they do not show a consistent syndrome of characteristics. All the other trisomies in human beings are embryonic lethals, demonstrating the importance of correct gene dosage. Unlike Datura, in which each of the possible trisomies is viable, human beings do not tolerate many types of chromosomal imbalance 60

Monosomy Monosomy occurs when one chromosome is missing in an otherwise diploid individual. In human beings, there is only one viable monosomic, the 45, X karyotype. These individuals have a single X chromosome as well as a diploid complement of autosomes. it is now called Turner syndrome. 62

45, X individuals can originate from eggs or sperm that lack a sex chromosome or from the loss of a sex chromosome in mitosis sometime after fertilization. This latter possibility is supported by the finding that many Turner individuals are somatic mosaics. These people have two types of cells in their bodies; some are 45, X and others are 46, XX. 64

45 X has no Barr body. Why, then, should Turner patients, who have the same number of active X chromosomes as normal XX females, show any phenotypic abnormalities at all? The answer probably involves a small number of genes that remain active on both of the X chromosomes in normal 46, XX females. These noninactivated genes are apparently needed in double dose for proper growth and development. The finding that at least some of these special X-linked genes are also present on the Y chromosome would explain why XY males grow and develop normally. 66

Deletions and Duplications of Chromosome Segments A missing chromosome segment is referred to either as a deletion or as a deficiency. In a diploid organism, the deletion of a chromosome segment makes part of the genome hypoploid. A classic example is the cri-du-chat syndrome (from the French words for cry of the cat ) in human beings This condition is caused by a conspicuous deletion in the short arm of chromosome 5 67

Deletions and Duplications of Chromosome Segments An extra chromosome segment is referred to as a duplication. The extra segment can be attached to one of the chromosomes, or it can exist as a new and separate chromosome, that is, as a free duplication. In either case, the effect is the same: The organism is hyperploid for part of its genome. 70

Effects of duplications for region 16A of the X chromosome on the size of the eyes in Drosophila. 71

The genes that encode the hemoglobin proteins have been tandemly duplicated in mammals. Gene duplications appear to be relatively common and provide a significant source of variation for evolution. 72

Rearrangement of Chromosomes strucrure When a chromosome region flips 180 degrees and rejoins the chromosome, an inversion has taken place. If the inverted segment contains the centromere or not, it is pericentric or paracentric, respectively. -An inversion heterozygote contains a normal chromosome and its homolog bearing an inversion, which form a loop during meiosis by homologous pairing which can be seen cytologically. 73

X-irradiation 74

Pairing between normal and inverted chromosomes. 77

Translocations When segments from 2 nonhomologous chromosomes exchange sites, a reciprocal translocation has occurred and no genetic material is lost. - In translocation heterozygotes, the translocated chromosomes and their nontranslocated homologs form a cruciform during meiosis which is likely to produce 2 different aneuploid gametes via alternate or adjacent disjunction with reduced fertility. Another disjunction event leads to deficiency and duplication gametes. 78

Compound Chromosomes and Robertsonian Translocations -A compound chromosome arises when homologs fuse and is stable so long as it contains a single centromere. -An isochromosome derives from the joining of homologous chromosome regions to a single centromere, creating, for example, a chromosome with a duplicated long arm each attached to the centromere. 84

Compound Chromosomes and Robertsonian Translocations The first compound chromosome was discovered by Lillian Morgan, the wife of T. H. Morgan. This compound was formed by fusing the two X chromosomes in Drosophila, creating double-x or attached-x chromosomes. 85

Compound Chromosomes and Robertsonian Translocations -A Robertsonian translocation arises when two acrocentrics fuse at the centromere creating a single metacentric chromosome. 87

Chromosomes can also fuse end-to-end to form a structure with two centromeres. If one of the centromeres is inactivated, the chromosome fusion will be stable. 89