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Table 15.1 The Constituency of Calf
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Thymus Chromatin
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Constituent DNA Histone proteins Nonhistone proteins RNA
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* Weight relative to 100 units of DNA.
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Relative Weight* 100 114 33 1
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Tamarin: Principles of Genetics, Seventh Edition
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III. Molecular Genetics
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15. The Eukaryotic Chromosome
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The McGraw Hill Companies, 2001
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Fifteen
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The Eukaryotic Chromosome
BOX 15.2
o facilitate the creation of recombinant genomic libraries, for mapping purposes, and for other reasons, it is useful to be able to isolate individual human chromosomes. To these ends, several methods have been developed to isolate chromosomes. Here we discuss a highspeed sorting method based on uorescent staining and ow cytometry. DNA can be treated with several uorescent dyes. Chromosomes can then be recognized individually by their relative uorescent intensities. The dyes Hoechst 33258 and chro-
Experimental Methods
High-Speed Chromosomal Sorting
momycin A3 are a valuable combination because they respond to different wavelengths of light and they bind DNA differently. Hoechst binds preferentially to DNA rich in adenine and thymine, whereas chromomycin
Flow karyotype of human chromosomes at very high resolution, measured under low-speed sorting ( fteen to thirty- ve chromosomes per second). The ordinate is Hoechst 33258 uorescence intensity, and the abscissa is chromomycin A3 uorescence intensity. All chromosomes are resolved except numbers 9 12. (Reprinted with permission from J. W. Gray, et al.,
High-Speed Chromosome Sorting, Science, 238:323 329, 1987. Copyright 1987 American Association for the Advancement of Science.)
binds preferentially to DNA rich in guanine and cytosine. Thus, since every human chromosome has a unique ratio of bases, the relative intensity of each chromosome is different when uoresced. Chromomycin uoresces in the presence of a laser tuned to 458 nm, and Hoechst uoresces in the presence of a UV laser. The chromosomes can be identi ed when their relative uorescence in the two lasers is plotted, producing a ow karyotype ( g. 1). Modern ow cytometry techniques then allow the isolation of these identi ed chromosomes. In practice, chromosomes are isolated in large numbers from cells that have been arrested in metaphase by treatment with colcemid, which inhibits spindle formation. These chromosomes are then puri ed in buffer and treated with the two dyes. The chromosomes are separated at high speed (two hundred chromosomes per second) in a ow cytometry device ( g. 2). As the chromosomecontaining buffer passes through the laser beams, identi cation is made. The liquid is then forced to form minute droplets (215,000 per second) by passing through a vibrator. Speci c droplets carrying the identi ed chromosomes are then charged, either positively or negatively, and passed between de ection plates. Positively charged droplets pass one way, and negatively charged droplets pass the other way, thus allowing the simultaneous isolation of two different chromosomes. At a rate of two hundred chromosomes per second, it
of histones from the cellular pool with the help of proteins called chromatin assembly factors; at least three of these factors are known. For example, in fruit ies, a protein complex called the replication-coupling assembly factor assembles new nucleosomes. In addition, a protein complex called condensin is needed for the condensation of interphase
chromosomes to mitotic chromosomes. This complex includes two SMC proteins (for structural maintenance of chromosomes) and two non-SMC proteins. SMC proteins also aid other chromosomal activities, such as mitotic segregation, sister-chromatid adhesion, dosage compensation, and recombination. Thus, a diverse array of proteins is involved in creating nucleosomes and chromato-
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
15. The Eukaryotic Chromosome
The McGraw Hill Companies, 2001
The Eukaryotic Chromosome
is possible to isolate 0.1 g of DNA in less than an hour; 0.1 g of DNA is adequate for library construction and represents about 5 105 average chromosomes. The technique is not perfect. During isolation, debris and clumps of chromosomes are produced that
cause contamination problems.Then, some chromosomes are so similar in their uorescence that they are hard to separate. This is true, for example, for chromosomes 9 to 12. Also, chromosome 21 is hard to separate because its uorescence tends to fall into the debris area.
Some of these problems, however, can be overcome by using hybrid cell lines of hamsters, for example, containing only one human chromosome. It is much easier to isolate the human chromosome from the hybrid line. Purity values of 90% are not unreasonable, with some in excess of 95%.
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