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melanogaster's five chromosomes are the X and Y sex chromosomes, the
two large autosomes (non-sex chromosomes) labeled 2 and 3, and the small
autosome labeled 4.
Of the roughly 215 million bases in the genome of D. melanogaster, about 120 million are in the form of euchromatin, DNA that can unwind and open and that encodes genes. Material known as heterochromatin forms the centers and ends of chromosomes and consists mostly of noncoding sequences; much of its DNA resists sequencing because it occurs in very short sequences of bases that are repeated in long tandem arrays.
DNA is purified from whole flies that are frozen and ground up. Clones are made by cutting all the DNA into pieces with enzymes, then inserting these snippets into various hosts that replicate numerous copies of them.
Each kind of clone has advantages and drawbacks. For example, short viral clones may reproduce sequences of bases very accurately, but these often match more than one location in the genome. At the other extreme, so-called YACs ("yeast artificial chromosomes") are very long stretches of DNA, up to millions of bases. While YACs can reduce the number of steps needed to create a physical map, they are unstable and may incorporate numerous sequence errors.
The STS content map constructed by BDGP's Berkeley Lab researchers relied on BACs, "bacterial artificial chromosomes," DNA clones that are stable at lengths up to hundreds of thousands of bases. The researchers tiled numerous overlapping BAC clones along the length of chromosomes 2 and 3, using sequence-tagged sites at intervals of approximately 50,000 bases.
In a technique pioneered by BDGP workers, over a third of the sequence-tagged sites in the Drosophila mapping project were "end-sequence tags" -- chosen to lie within 500 bases of either end of a BAC clone, greatly aiding the matching of overlapping clones.
The more clones that overlap at any given place along the chromosome, the greater the assurance of high accuracy in sequencing. At most places the BAC-based map of the euchromatin in chromosomes 2 and 3 reached a depth of about 13 overlapping BAC clones.
(Five short gaps in the euchromatin map were not spanned by any clone, however, and these regions were also present as gaps in the whole-genome shotgun sequence produced in collaboration with Celera Genomics.)
The BAC-clone map, made mostly at Berkeley Lab, was augmented and checked against other mapping techniques. At Baylor College of Medicine in Houston, Texas, members of BDGP separated DNA segments by gel electrophoresis to provide distinct visual identification of BAC clones; these were assembled to construct a "fingerprint map" that corroborated Berkeley Lab's map.
Working with BDGP researchers in Gerald Rubin's laboratory on the UC Berkeley campus, Berkeley Lab researchers confirmed the physical BAC map by hybridizing (joining) BAC clones directly to the chromosomes themselves.
"The chromosomes of the Drosophila larval salivary glands are unusual in that their DNA can form multiple, perfectly registered copies," says Susan Celniker. "This can help the fly make lots of a particular protein in a short time. Larvae produce glue copiously in their salivary glands -- to get tons of glue, the fly makes tons of glue genes."
These so-called polytene chromosomes have distinct banding patterns, and when they are stained, variations in the pattern unambiguously identify regions of the chromosomes. When BAC clones were stained with a different color and allowed to hybridize to their matching sites on the polytenes, they demonstrated virtually complete coverage of the chromosomes by corresponding clones.
By integrating the BAC STS content map and the fingerprint map, BDGS researchers achieved both contiguous clone coverage of the genome and assured adequate overlaps, confirming that sequence assemblies reflected the structure of the genome. Then the in situ polytene hybridization data established that their physical map covered more than 97.8 percent of the euchromatic portion of chromosomes 2 and 3.