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Interphase Polytene Chromosomes Arise by DNA Amplification

The larval salivary glands of Drosophila species and other dipteran insects contain enlarged interphase chromosomes that are visible in the light microscope. When fixed and stained, these polytene chromosomes are characterized by a large number of reproducible, well-demarcated bands that have been assigned standardized numbers (Figure 10-30a). The highly reproducible banding pattern seen in Drosophila salivary gland chromosomes provides an extremely powerful method for locating specific DNA sequences along the lengths of the chromosomes in this species. For example, the chromosomal location of a cloned DNA sequence can be accurately determined by hybridizing a labeled sample of the cloned DNA to polytene chromosomes prepared from larval salivary glands (Figure 10-30b).

A generalized amplification of DNA gives rise to the polytene chromosomes found in the salivary glands of Drosophila. This process, termed polytenization, occurs when the DNA repeatedly replicates, but the daughter chromosomes do not separate. The result is an enlarged chro-

▲ FIGURE 10-31 Amplification of DNA along the polytene fourth chromosome of Drosophila melanogaster.

(a) Morphology of the stained polytene fourth chromosome as it appears in the light microscope at high magnification. The fourth chromosome is by far the smallest (see Figure 10-30). The homologous chromatids (paternal and maternal) are paired. The banding pattern results from reproducible packing of DNA and protein within each amplified site along the chromosome. Dark bands are regions of more highly compacted chromatin.

(b) The pattern of amplification of one of the two homologous chromatids during five replications. Double-stranded DNA is represented by a single line. Telomere and centromere DNA are not amplified. In salivary gland polytene chromosomes, each parental chromosome undergoes «10 replications (210 = 1024 strands). [Part (a) from C. Bridges, 1935, J. Hered. 26:60; part (b) adapted from C. D. Laird et al., 1973, Cold Spring Harbor Symp. Quant. Biol. 38:311.]

mosome composed of many parallel copies of itself (Figure 10-31). The amplification of chromosomal DNA greatly increases gene copy number, presumably to supply sufficient mRNA for protein synthesis in the massive salivary gland cells. Although the bands seen in human metaphase chromosomes probably represent very long folded or compacted stretches of DNA containing about 107 base pairs, the bands in Drosophila polytene chromosomes represent much shorter stretches of only 50,000-100,000 base pairs.

Heterochromatin Consists of Chromosome Regions That Do Not Uncoil

As cells exit from mitosis and the condensed chromosomes uncoil, certain sections of the chromosomes remain dark-staining. The dark-staining areas, termed heterochromatin, are regions of condensed chromatin. The light-staining, less condensed portions of chromatin are called euchromatin. Heterochromatin appears most frequently—but not exclusively—at the centromere and telomeres of chromosomes and is mostly simple-sequence DNA.

In mammalian cells, heterochromatin appears as darkly staining regions of the nucleus, often associated with the nuclear envelope. Pulse labeling with 3H-uridine and autoradi-ography have shown that most transcription occurs in regions of euchromatin and the nucleolus. Because of this and because heterochromatic regions apparently remain condensed throughout the life cycle of the cell, they have been regarded as sites of inactive genes. However, some transcribed genes have been located in regions of heterochro-matin. Also, not all inactive genes and nontranscribed regions of DNA are visible as heterochromatin.

Three Functional Elements Are Required for Replication and Stable Inheritance of Chromosomes

Although chromosomes differ in length and number between species, cytogenetic studies have shown that they all behave similarly at the time of cell division. Moreover, any eukary-otic chromosome must contain three functional elements in order to replicate and segregate correctly: (1) replication origins at which DNA polymerases and other proteins initiate synthesis of DNA (see Figures 4-34 and 4-36), (2) the centromere, and (3) the two ends, or telomeres. The yeast transformation studies depicted in Figure 10-32 demonstrated the functions of these three chromosomal elements and established their importance for chromosome function.

As discussed in Chapter 4, replication of DNA begins from sites that are scattered throughout eukaryotic chromosomes. The yeast genome contains many «100-bp sequences, called autonomously replicating sequences (ARSs), that act as replication origins. The observation that insertion of an ARS into a circular plasmid allows the plasmid to replicate in yeast cells provided the first functional identification of origin sequences in eukaryotic DNA (see Figure 10-32a).

Plasmid with sequence from normal yeast

Transfected leu~ cell

Progeny of transfected cell Conclusion

Growth without leucine

Mitotic segregation

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