Architecture and Morphology of the Polytene Chromosome Where Polytenized and Underrepresented Regions Meet

A plausible and long-held model for the structure of E-H junctions supposes a branched or nested arrangement of static replication forks, sometimes referred to as an "onion-skin" DNA structure, in which the multiple chromatids of euchromatin are merged with the much fewer chromatids (minimally two) of heterochromatin (see Fig. 2A). Results of recent studies by Glaser and colleagues (188,189) suggest instead that a nonbranched chromosome structure joins regions of high and low polyteny (see Fig. 2B). They propose that underrepresentation of heterochromatin-associated DNA results from the blocking of replication fork progression from euchromatin into heterochroma-tin at certain preferred sites and that this generates truncated euchromatic chro-matids that are amplified in subsequent S-phases by "replication runoff." By

Fig. 2. Models of euchromatin-heterochromatin (E-H) junctions. (A) "Onionskin" model showing branched chromatid structure as if replication forks have stalled. (B) Model of an E-H junction produced by a strong block to replication (filled rectangles) and subsequent "runoff replication" of truncated chromatids (188). In both A and B, the lines represent single chromatids; euchromatin is on the left and underreplicated heterochromatin on the right; for simplicity, only eight chromatids are shown in euchromatin.

Fig. 2. Models of euchromatin-heterochromatin (E-H) junctions. (A) "Onionskin" model showing branched chromatid structure as if replication forks have stalled. (B) Model of an E-H junction produced by a strong block to replication (filled rectangles) and subsequent "runoff replication" of truncated chromatids (188). In both A and B, the lines represent single chromatids; euchromatin is on the left and underreplicated heterochromatin on the right; for simplicity, only eight chromatids are shown in euchromatin.

subjecting genomic DNA extracted from flow-sorted follicle cell nuclei of different ploidies (see Chapter 8) to pulsed-field or two-dimensional gel analysis, Leach et al. (188) found that very large E-H junction-spanning DNA molecules (defined by restriction enzyme digestion) gave rise to truncated progeny molecules during the course of polytenization. For example, a 650-kb restriction fragment across the E-H junction of chromosome 3 gave rise to smaller fragments ranging in size from approx 77 kb to nearly full length. However, molecules of approx 340 kb were the most abundant, suggesting a strong stop site for replication at a position about midway along the 650-kb molecule, possibly involving satellite DNA repeats (188). They showed that the truncation process commences during the first polyp-loid S-phase, but that truncated duplex molecules begin to appear only after the second polyploid S-phase (8C nuclei), after the truncated strand produced in the first S-phase is able to serve as a template for replication.

In addition to E-H junctions, other regions of underrepresentation occur in euchromatic regions of polytene chromosomes, referred to as intercalary het-erochromatin (IH; see Chapter 6). The histone gene cluster at 39DE (190) and the Bithorax Complex locus at 89E1-4 (191) are two prominent examples of underreplicated IH sites. IH sites replicate late compared to bulk euchromatin (192), they are often broken in squash preparations of salivary gland chromosomes (i.e., they are weak points) and they tend to form ectopic associations with nonhomologous sites and pericentric regions of chromosomes. IH sites occur frequently on the paired X chromosomes of females, but they are practically absent from the single X of males, and dosage compensation proteins have been implicated in this sexual dimorphism (see ref. 193). No differences between sexes are observed for autosomal IH sites.

Belyaeva et al. (194) identified a fascinating mutation, SuUR [originally called Su(UR)ES], that suppresses underreplication of DNA in both pericentric and intercalary heterochromatin. Weak spots seen in SuUR+ strains are absent in SuUR homozygotes and are replaced by one or more solid bands (191,194). Much of the P-heterochromatin becomes polytenized and shows reproducible banding. This effect is most striking for chromosome 3 pericentric heterochromatin, where a new banded segment, "Plato Atlantis," is seen that is nearly the size of chromosome 4. Ectopic associations between IH sites are greatly reduced in SuUR mutants, often resulting in perfectly spread polytene chromosomes. SuUR homozygous animals are otherwise normal in terms of morphology, viability, fertility, and meiotic recombination. SuUR/+ heterozygotes have an intermediate suppressor phenotype.

The SuUR gene encodes a predicted protein of 962 amino acids that is not fully homologous to any known protein. The ^-terminal 250 residues of SuUR show moderate similarity to sequence motifs that define the ATPase domain of members of the SWI/SNF family of proteins (195). However, the motifs in SuUR are divergent enough that a putative ATPase function is in doubt. The middle region of the protein contains an AT-hook motif, which suggests that it associates with AT-rich sequences in DNA (see Subheading 3.2.). Antibodies against SuUR stain the chromocenter very strongly, and approx 110 sites in euchromatin are also stained, all but 2 of which correspond to late-replicating IH sites (192). Some specific element shared by IH sites must direct SuUR to bind, but how this is achieved and how the protein interferes with DNA replication locally is not known (195). a-Heterochromatin and some P-heterochro-matin remain underreplicated in SuUR mutants.

Just as the polymerase chain reaction can transform trace amounts of DNA into quantities suitable for experimental manipulation, the amplified and banded heterochromatic domains in SuUR mutants are providing new and valuable chromosomal material for cytology. For example, SuUR polytene chromosomes are being used to determine precise chromosomal locations of unassigned (heterochromatic) DNA sequences produced by the genome project (196), and to study the distribution of proteins in P-heterochromatin (197). Also, libraries of DNA fragments microdissected from SuUR-amplified regions have allowed polytene chromosome E-H junctions to be characterized at the molecular level (196). The SuUR system would also be useful in refining the positions of breakpoints of chromosomal rearrangements involving heterochro-matin (see Chapter 12).

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