Caspersson et al. Since then, researchers have developed a variety of other chromosome banding techniques that have largely supplanted Q-banding in clinical cytogenetics.
Today, most karyotypes are stained with Giemsa dye, which offers better resolution of individual bands, produces a more stable preparation, and can be analyzed with ordinary bright-field microscopy.
The molecular causes for staining differences along the length of a chromosome are complex and include the base composition of the DNA and local differences in chromatin structure. In G-banding , the variant of Giemsa staining most commonly used in North America, metaphase chromosomes are first treated briefly with trypsin, an enzyme that degrades proteins, before the chromosomes are stained with Giemsa.
Trypsin partially digests some of the chromosomal proteins, thereby relaxing the chromatin structure and allowing the Giemsa dye access to the DNA. In general, heterochromatic regions, which tend to be AT-rich DNA and relatively gene-poor, stain more darkly in G-banding. In contrast, less condensed chromatin—which tends to be GC-rich and more transcriptionally active—incorporates less Giemsa stain, and these regions appear as light bands in G-banding.
Most importantly, G-banding produces reproducible patterns for each chromosome, and these patterns are shared between the individuals of a species. An example of Giemsa-stained human chromosomes, as they would appear under a microscope, is shown in Figure 1a. Typically, Giemsa staining produces between and bands distributed among the 23 pairs of human chromosomes. Measured in DNA terms, a G-band represents several million to 10 million base pairs of DNA, a stretch long enough to contain hundreds of genes.
G-banding is not the only technique used to stain chromosomes, however. R-banding, which is used in parts of Europe, also involves Giemsa stain, but the procedure generates the reverse pattern from G-banding.
In R-banding Figure 1c , the chromosomes are heated before Giemsa stain is applied. The heat treatment is thought to preferentially melt the DNA helix in the AT-rich regions that usually bind Giemsa stain most strongly, leaving only the comparatively GC-rich regions to take up the stain. R-banding is often used to provide critical details about gene-rich regions that are located near the telomeres. Yet another method is C-banding Figure 1d , which can be used to specifically stain constitutive heterochromatin , or genetically inactive DNA, but it is rarely used for diagnostic purposes these days.
C-banding is a specialized Giemsa technique that primarily stains chromosomes at the centromeres, which have large amounts of AT-rich satellite DNA. The first method to be used to identify all 46 human chromosomes was Q-banding Figure 1b , which is achieved by staining the chromosomes with quinacrine and examining them under UV light.
This method is most useful for examining chromosomal translocations, especially ones involving the Y chromosome. Taken together, these banding techniques offer clinical cytogeneticists an arsenal of staining methods for diagnosing chromosomal abnormalities in patients. In order to maximize the diagnostic information obtained from a chromosome preparation, images of the individual chromosomes are arranged into a standardized format known as a karyotype, or more precisely, a karyogram Figure 1a-c.
According to international conventions, human autosomes, or non-sex chromosomes, are numbered from 1 to 22, in descending order by size, with the exceptions of chromosomes 21 and 22, the former actually being the smallest autosome. The sex chromosomes are generally placed at the end of a karyogram. Within a karyogram, chromosomes are aligned along a horizontal axis shared by their centromeres.
Individual chromosomes are always depicted with their short p arms—p for "petite," the French word for "small"—at the top, and their long q arms—q for "queue"—at the bottom. Centromere placement can also be used to identify the gross morphology, or shape, of chromosomes.
For example, metacentric chromosomes, such as chromosomes 1, 3, and 16, have p and q arms of nearly equal lengths. Submetacentric chromosomes, such as chromosomes 2, 6, and 10, have centromeres slightly displaced from the center. Acrocentric chromosomes, such as chromosomes 14, 15, and 21, have centromeres located near their ends. Arranging chromosomes into a karyogram can simplify the identification of any abnormalities.
Note that the banding patterns between the two chromosome copies, or homologues, of any autosome are nearly identical. Some subtle differences between the homologues of a given chromosome can be attributed to natural structural variability among individuals. Occasionally, technical artifacts associated with the processing of chromosomes will also generate apparent differences between the two homologues, but these artifacts can be identified by analyzing 15—20 metaphase spreads from one individual.
It is highly unlikely that the same technical artifact would occur repeatedly in a given specimen. Today, G-banded karyograms are routinely used to diagnose a wide range of chromosomal abnormalities in individuals.
Although the resolution of chromosomal changes detectable by karyotyping is typically a few megabases, this can be sufficient to diagnose certain categories of abnormalities.
For example, aneuploidy , which is often caused by the absence or addition of a chromosome, is simple to detect by karyotype analysis. Cytogeneticists can also frequently detect much more subtle deletions or insertions as deviations from normal banding patterns.
Likewise, translocations are often readily apparent on karyotypes. When regional changes in chromosomes are observed on karyotypes, researchers often are interested in identifying candidate genes within the critical interval whose misexpression may cause symptoms in patients. This search process has been greatly facilitated by the completion of the Human Genome Project , which has correlated cytogenetic bands with DNA sequence information.
Consequently, investigators are now able to apply a range of molecular cytogenetic techniques to achieve even higher resolution of genomic changes. Fluorescence in situ hybridization FISH and comparative genomic hybridization CGH are examples of two approaches that can potentially identify abnormalities at the level of individual genes.
Molecular cytogenetics is a dynamic discipline, and new diagnostic methods continue to be developed. As these new technologies are implemented in the clinic, we can expect that cytogeneticists will be able to make the leap from karyotype to gene with increasing efficiency. Caspersson, T.
Differential banding of alkylating fluorochromes in human chromosomes. Experimental Cell Research 60 , — doi Gartler, S. The chromosome number in humans: A brief history. Nature Reviews Genetics 7 , — doi Speicher, M. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nature Genetics 12 , — link to article. Strachan, T. Human Molecular Genetics , 2nd ed.
The karyotype is used to look for abnormal numbers or structures of chromosomes. When I hear the word "karyotype", I think about a picture of chromosomes. When somebody has their blood studied to look at how many chromosomes they have and whether the chromosomes are complete, we come up with a picture in which we can line up all the chromosomes and count them.
That way we can tell whether or not somebody has all the proper number of chromosomes, which is 46, and that way we can look at the X and the Y chromosomes and determine if it's a female or male. Autosomes are those chromosomes present in the same number in males and females while sex chromosomes are those that are not. When sex chromosomes were first discovered their function was unknown and the name X was used to indicate this mystery.
The next ones were named Y, then Z, and then W. The combination of sex chromosomes within a species is associated with either male or female individuals. In mammals, fruit flies, and some flowering plants embryos, those with two X chromosomes develop into females while those with an X and a Y become males.
In birds, moths, and butterflies males are ZZ and females are ZW. Because sex chromosomes have arisen multiple times during evolution the molecular mechanism s through which they determine sex differs among those organisms. For example, although humans and Drosophila both have X and Y sex chromosomes, they have different mechanisms for determining sex.
In mammals, the sex chromosomes evolved just after the divergence of the monotreme lineage from the lineage that led to placental and marsupial mammals. Thus nearly every mammal species uses the same sex determination system. During embryogenesis the gonads will develop into either ovaries or testes. A gene present only on the Y chromosome called TDF encodes a protein that makes the gonads mature into testes. XX embryos do not have this gene and their gonads mature into ovaries instead default.
Once formed the testes produce sex hormones that direct the rest of the developing embryo to become male, while the ovaries make different sex hormones that promote female development. The testes and ovaries are also the organs where gametes sperm or eggs are produced. How do the sex chromosome behave during meiosis? Well, in those individuals with two of the same chromosome i. In mammals the consequence of this is that all egg cells will carry an X chromosome while the sperm cells will carry either an X or a Y chromosome.
Half of the offspring will receive two X chromosomes and become female while half will receive an X and a Y and become male. The stages shown are anaphase I, anaphase II, and mature sperm. Note how half of the sperm contain Y chromosomes and half contain X chromosomes.
0コメント