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Chromosomal Banding

Chromosomal Banding

A chromosome banding pattern is comprised of alternating light and dark stripes, or bands, that appear along its length after being stained with a dye. A unique banding pattern is used to identify each chromosome and to diagnose chromosomal aberrations, including chromosome breakage, loss, duplication or inverted segments. In the 1950s, chromosomes from the cell's nucleus were identified with a uniform (unbanded) stain that allowed for the observation of the overall length and primary constriction (centromere) of each chromosome, as well as a secondary constriction in chromosomes 1, 9, 16 and the acrocentrics (chromosomes whose centromeres are near the tips). The staining techniques used to make the bands visible were developed in the late 1960s and early 1970s.

Chromosome Structure

To understand what chromosomal bands represent, it is helpful to understand the structure of chromosomes. Eukaryotic chromosomes are composed of chromatin, a combination of nuclear DNA and proteins. At metaphase, which is a phase in the cell cycle after the DNA in the nucleus has been replicated, each chromosome contains two identical strands of DNA. (Each strand contains two complementary strands of nucleotides.) The two strands of DNA, or chromatids, are arranged in a double-helix and are held together at a single point, the centromere, or primary constriction point.

During mitosis , each chromatid becomes condensed approximately ten-thousand fold reaching maximal condensation at metaphase . DNA that was roughly 5 centimeters (2 inches) long is compacted to 5 micrometers. The DNA wraps around proteins called histones, forming complexes called nucleosomes. The nucleosomes twist around each other and assume a loop formation projecting out from the chromosome's protein backbone, or scaffold. The loops weave and condense further to package the DNA into a chromosome. Some of the looped segments of DNA remain close together and condense more than others, forming regions known as domains. These domains are the darkly-stained chromosomal bands that appear when specific stains are applied (such as Giemsa staining; see below).

Looped domains are also seen in polytene chromosomes, which are found mainly in insects of the order Diptera, including Drosophila, which are fruit flies. Polytene chromosomes are large chromosomes that are formed after DNA undergoes repeated rounds of replication without cell division. A polytene chromosome in a Drosophila salivary gland cell can contain as many as five thousand alternating dark and light bands. The dark bands correspond to the folded and looped DNA, and the lighter bands are composed of less condensed DNA. The DNA in polytene chromosomes becomes less condensed when genes become active, permitting DNA to be transcribed into messenger RNA. This unraveling is observed as "puffing" of the polytene chromosome. The puffing resolves (the DNA condenses again) as the genes become inactive.

Chromosome Banding Techniques

Quinacrine mustard, an alkylating agent, was the first chemical to be used for chromosome banding. T. Caspersson and his colleagues, who developed the technique, noticed that bright and dull fluorescent bands appeared after chromosomes stained with quinacrine mustard were viewed under a fluorescence microscope. Quinacrine dihydrochloride was subsequently substituted for quinacrine mustard. The alternating bands of bright and dull fluorescence were called Q bands. Quinacrine-bright bands were composed primarily of DNA that was rich in the bases adenine and thymine, and quinacrine-dull bands were composed of DNA that was rich in the bases guanine and cytosine.

Other fluorescent dyes have been used to generate chromosomal banding patterns. The combination of the fluorescent dye, DAPI (4,6-Diamidino-2-Phenylindole) with a non-fluorescent counterstain, such as Distamycin A, will also stain DNA that is rich in adenine and thymine. It will particularly highlight regions that are on the Y chromosome, on chromosomes 9 and 16, and on the proximal short arms of the chromosome 15 homologues , or pair.

Giemsa has become the most commonly used stain in cytogenetic analysis. Staining a metaphase chromosome with a Giemsa stain is referred to as G-banding. Unlike Q-banding, most G-banding techniques require pretreating the chromosomes with either salt or a proteolytic (protein-digesting) enzyme. "GTG banding" refers to the process in which G-banding is preceded by treating chromosomes with trypsin. G-banding preferentially stains the regions of DNA that are rich in adenine and thymine. In general, the bands produced correspond with Q-bright bands. The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light.

Standard G-band staining techniques allow between 400 and 600 bands to be seen on metaphase chromosomes. With high-resolution G-banding techniques, as many as two thousand different bands have been catalogued on the twenty-four human chromosomes. Jorge Yunis introduced a technique to synchronize cells so they are held at the same stage in the cell cycle. Cells are synchronized by making them deficient in folate, thereby inhibiting DNA synthesis. By rescuing the cells with thymidine, DNA synthesis is initiated and the timing of the prophase and prometaphase stages of the cell cycle can be predicted. Yunis's technique allows more bands to be resolved, as chromosomes produced from either prophase or prometaphase are less condensed and are thus longer than metaphase chromosomes.

Other Banding

R-banding is the reverse pattern of G bands so that G-positive bands are light with R-banding methods, and vice versa. R-banding involves pretreating cells with a hot salt solution that denatures DNA that is rich in adenine and thymine. The chromosomes are then stained with Giemsa. R-banding is helpful for analyzing the structure of chromosome ends, since these areas usually stain light with G-banding.

C-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA. NOR-staining, where NOR is an abbreviation for "nucleolar organizing region," refers to a silver staining method that identifies genes for ribosomal RNA that were active in a previous cell cycle.

Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that allows cytogeneticists to analyze chromosome resolution at the DNA or gene level. FISH can be performed on dividing (metaphase) and non-dividing (interphase) cells to identify numerical and structural abnormalities resulting from genetic disorders.

In FISH, cytogeneticists utilize one or more FISH probes that typically fall into one of the following three categories:

  1. Repetitive sequences, including alpha satellite DNA, that bind to the centromere of a chromosome;
  2. DNA segments, representative of the entire chromosome, that will bind to and cover the entire length of a particular chromosome; and
  3. DNA segments from specific genes or regions on a chromosome that have been previously mapped or identified.

A probe is "tagged" either directly, by incorporating fluorescent nucleotides, or indirectly, by incorporating nucleotides with attached small molecules, such as biotin, digoxygenin, or dinitrophenyl, to which fluorescent antibodies can later be bound. The probe and the chromosomes (from either the metaphase or interphase cells) that are being analyzed are denatured and allowed to bind or hybridize to one another. If necessary, antibodies with a fluorescent tag are applied to the cells. The cells are then viewed with a fluorescence microscope. The fluorescent signals represent the probe(s) that is bound to the chromosomes.

see also Cell Cycle; Chromosomal Aberrations; Chromosome, Eukaryotic; Fruit Fly: drosophila ; In Situ Hybridization.

Gail H. Vance

Bibliography

Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Science, 2002.

Craig, J. M., and W. A. Bickmore. "Chromosome BandsFlavours to Savour." Bioassays 15, no. 5 (1993): 349-353.

Earnshaw, W. C. "Meiotic Chromosome Structure." Bioassays 9, no. 5 (1988): 47-150.

Sumner, A. T. "The Nature and Mechanisms of Chromosome Banding." Cancer Genetics and Cytogenetics 6 (1982): 59-87.

Therman E., and M. Susman. Human Chromosomes: Structure, Behavior and Effects, 3rd ed. New York: Springer-Verlag, 1993.

Yunis, J. J., and R. C. Lewandowski. "High-Resolution Cytogenetics." Birth Defects: Original Article Series 19, no. 5 (1983): 11-37.

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Genetic Mapping

Genetic mapping

The aim of genetic mapping is to determine the linear sequence of genes in genetic material. The mapping can be performed at several levels of detail (resolution) that fall into two broad types: traditional genetic or linkage mapping and, more detailed, physical mapping.

Linkage mapping shows the relative rather than absolute positions of genes along a chromosome and is a technique that has been used since the early 1900s. Early geneticists determined that genes were found on chromosomes . They also reasoned that because the various forms of genes, or alleles, could be precisely exchanged during meiosis through crossovers between homologous chromosomes, the genes for specific characteristics must lie at precise points along each chromosome. It followed that the mapping of chromosomes could, therefore, be made from the observation of crossovers. Between 1912 and 1915, the American scientist Thomas Hunt Morgan (18661945) hypothesized that if genes were arranged linearly along chromosomes, then those genes lying closer together would be separated by crossovers less often than those lying further apart. Genes lying closer together would thus have a greater probability of being passed along as a unit. It follows that the percentage of crossovers would be proportional to the distance between two genes on a chromosome. The percentage crossover can be expressed as the number of crossovers between two genes in meiosis. One genetic map unit (m.u.) is defined as the distance between gene pairs for which one product out of 100 is recombinant (a product of crossover). The recombinant frequency (R.F.) of 0.01 (1%) is defined as 1 m.u. and a map unit is sometimes referred to as a centimorgan (cM) in honor of Thomas Hunt Morgan.

As an example of how linkage mapping might work, suppose two characteristics, A and B, show 26% crossover. Assign 26 crossover units to the distance between these two genes. If a characteristic C turns out in breeding experiments to have 9% crossover with B and 17% crossover with A, it would then be located between A and B at a point 9 units from B and 17 units from A. Compiling the information from many such breeding experiments creates a chromosome map that indicates the relative positions of the genes that code for certain characteristics. Accordingly, the further apart any two genes are on the same chromosome, the greater the incidence of crossing over between them.

A linkage map is limited because recombination frequencies can be distorted relative to the physical distance between sites. As a result, the linkage map is not always the best possible representation of genetic material.

While linkage maps only indicate relative positions of genes, physical maps are more accurate and aim to show the actual number of nucleotides between each gene. Restriction maps are constructed by cleaving DNA into fragments with restriction enzymes . These enzymes recognize specific short DNA sequences and cut the duplex. The distances between the sites of cleavage are then measured. The positions of the target restriction sites for these enzymes along the chromosome can be used as DNA markers. Restriction sites generally exist in the same positions on homologous chromosomes so the positions of these target sites can be used rather like milestones along a road and can act as reference points for locating significant features in the chromosome.

A map of the positions of restriction sites can be made for a localized region of a chromosome. It is made by comparing the sizes of single enzyme breakages (digests) of the region of interest with double digests of the same region. This means that two different restriction enzymes are applied, one to each of two separate chromosome extracts of the region of interest, and subsequently the two enzymes are used together in a third digestion with the chromosome extract. The chromosome fragments resulting from the three digestions are then subjected to a biochemical procedure known as gel electrophoresis , which separates them and gives an estimation of their size. Comparison of the sizes of the chromosome fragments resulting from single and double restriction enzyme digestions allows for an approximate location of the target restriction sites. Thus, such maps represent linear sequences of restriction sites. As this procedure determines the sizes of digested chromosome fragments, the distances between sites in terms of the length of DNA can be calculated, because the size of a fragment estimated from an electrophoresis experiment is proportional to the number of base pairs in that fragment.

A restriction map does not intrinsically identify sites if genetic interest. For it to be of practical use, mutations have to be characterized in terms of their effects upon the restriction sites. In the 1980s, it was shown how restriction fragment length polymorphisms (RFLPs) could be used to map human disease genes. RFLPs are inherited by Mendelian segregation and are distributed in populations as classical examples of common genetic polymorphisms. If such a DNA variant is located close to a defective gene (which can not be tested directly), the DNA variant can be used as a marker to detect the presence of the disease-causing gene. The prenatal examination of DNA for particular enzyme sites associated with certain hereditary diseases has proved to be an important method of diagnosis. Clinically useful polymorphic restriction enzyme sites have been detected within the Beta-like globin gene cluster. For example, the absence of a recognition site for the restriction enzyme HpaI is frequently associated with the allele for sickle-cell anemia, and this association has been useful in prenatal diagnosis of this disease.

The ultimate genetic map is the complete nucleotide sequence of the DNA in the whole chromosome complement , or genome, of an organism. Today, several completed genome maps already exist. Simple prokaryotic organisms, e.g., bacteria , with their relatively small (one to two million base pairs) chromosomes of one to two million base pairs were the first to be mapped. Later, eukaryotic organisms such as the yeast , Saccharomyces cerevisiae , and the nematode worm, Caenorhabditis elegans, were mapped. In 2000, the Human Genome Project produced the first draft of the human genome. The project adopted two methods for mapping the 3 billion nucleotides. The earlier approach was a "clone by clone" method. In this, the entire genome was cut into fragments up to several thousand base pairs long, and inserted into synthetic chromosomes known as bacterial artificial chromosomes (BACs) . The subsequent mapping step involved positioning the BACs on the genome's chromosomes by looking for distinctive marker sequences called sequence tagged sites (STSs), whose location had already been pinpointed. Clones of the BACs are then broken into smaller fragments in a process known as shotgun cloning . Each small fragment was then sequenced and computer algorithms, that recognize matching sequence information from overlapping fragments, were used to reconstruct the complete sequence inserted into each BAC. It was later argued that the first mapping step was unnecessary and that the algorithms used to reassemble the shotgunned DNA fragments could be applied to cloned random fragments taken directly from the whole genome. In this whole genome shotgun strategy, fragments were first assembled by algorithms into larger scaffolds and the correct position of these scaffolds on the genome was worked out by STSs. The latter method speeded up the whole procedure considerably and is currently being used to sequence genomes from other organisms.

See also Cloning, application of cloning to biological problems; Fungal genetics; Gene amplification; Gene; Genetic code; Genetic identification of microorganisms; Genetic regulation of eukaryotic cells; Genetic regulation of prokaryotic cells; Genotype and phenotype; Microbial genetics

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chromosome map

chromosome map Any plan that shows the positions of genes, genetic markers, or other landmarks along the length of a chromosome. There are essentially two complementary types of map: linkage maps, which give the relative positions of genetic sites (loci) determined by studies of how frequently recombination occurs between the loci; and physical maps, which show the arrangement of the chromosomal material, whether it be in the form of banding patterns produced by staining (a type of cytological map) or the sequence of bases in the DNA. Maps of either type can be constructed in various ways, depending on such factors as the type of organism, the complexity of its genome, and the amount of pre-existing data. Accumulated data for the chromosomes of many species of organism are now held in databases and available freely via the Internet for geneticists and others worldwide.

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chromosome map

chromosome map A map showing the locations (loci) of genes on a chromosome, deduced from genetic recombination (see RECOMBINANT) experiments. For example, the frequency of cross-overs between pairs of genes indicate their relative positions or linear order, the distances being given in units of cross-over frequency.

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chromosome map

chromosome map A map that shows the locations (loci) of genes on a chromosome, deduced from genetic-recombination experiments. For example, the frequency of cross-overs between pairs of genes indicate their relative positions or linear order, the distances being given in units of cross-over frequency.

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genetic mapping

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