Starting with the low-density human genetic map, the next task was to build a high- density marker map. That involved obtaining a large number of nonpolymorphic markers and mapping them to quite short, unique chromosomal regions. The key markers here were sequence tagged site (STS) markers, short (<1kb) DNA sequences that occur at a unique location in the genome and that can readily be screened by a PCR assay. They are often nonpolymorphic (but some may contain a polymorphic site). Many STS markers were located outside genes, but some of them were located in genes and this subset of STS markers came to be known as expressed sequence tag (EST) markers.
STS markers were obtained by various routes, including from previously studied DNA clones that had been sequenced, and by randomly sequencing the ends of inserts of genomic DNA clones. They were mapped by different methods too. Sometimes fluorescence in situ hybridization (FISH) mapping was used: a genomic DNA clone containing the STS marker would be fluorescently labeled and hybridized to denatured DNA from a preparation of fixed metaphase or prometaphase chromosomes from human cells. In 1995 a human STS map was published with an average spacing of just less than one STS marker per 200 kb. The chromosomal locations of STS markers had been obtained by either using panels of human–rodent hybrid cells, or by STS content mapping as described below.
Mapping with somatic cell hybrid panels
Somatic cell hybrids are artificially constructed by fusing a human cell to a mouse or hamster cell. Initially unstable, the hybrid cells become stable after selectively jettisoning most of the human chromosomes. The loss of human chromosomes occurs randomly: different human chromosomes are retained in different hybrid cells (Figure 1A).
Fig1. Somatic cell hybrids and the use of radiation hybrids to map human sequence tagged sites (STSs). (A) Principle of somatic cell hybrids. In the presence of certain agents, such as polyethylene glycol (PEG), cultured human and rodent cells can be induced to fuse, generating somatic cell hybrids. The initial fusion cells are heterokaryons, with both a human and a rodent nucleus. At mitosis the two nuclear envelopes dissolve, bringing human and rodent chromosomes together in a single nucleus. For unknown reasons, most human chromosomes fail to replicate in subsequent mitoses, and are lost. Eventually a variety of stable hybrid cell lines arise, each with the full set of rodent chromosomes plus a few types of human chromosome. (B) Mapping with radiation hybrids. Panels of radiation hybrids are produced by exposing cells with human chromosomes to radiation-induced chromosome breakage, prior to fusion to a rodent cell that is deficient in thymidine kinase (TK), followed by selection for TK+ cells. The radiation hybrids retain different sets of small human chromosome fragments; different human DNA markers will be present in some hybrid cells but absent in others. Although the pattern of fragment integration is mostly random, individual markers will give related typing patterns if they originate from closely spaced loci on a particular chromosome. The principle of a radiation hybrid map is therefore reminiscent of meiotic linkage analysis: the nearer together two DNA sequences are on a chromosome, the lower the probability that they will be separated by the chance occurrence of a breakpoint between them. Laboratories can map any unknown STS by assaying for it in a defined panel of radiation hybrids (RH) and comparing the pattern with patterns of previously mapped markers held on a central server.
Monochromosomal hybrids (with a single human chromosome) are particularly useful for mapping. To obtain them, donor human cells are first exposed to colcemid, causing the chromosome set to become partitioned into different discrete subnuclear packets (micronuclei). Subsequent centrifugation can produce microcells that have a single micronucleus containing just a few chromosomes and a thin rim of cytoplasm sur rounded by an intact plasma membrane. When microcells are fused with recipient rodent cells, some hybrid cells retain just a single human chromosome. Monochromosomal hybrid cells were quickly developed to represent each of the human autosomes plus the X chromosome.
Subchromosomal mapping became possible after hybrids were designed to contain human chromosome fragments. The most popular approach involved exposing a cell containing one or more human chromosomes to a lethal dose of radiation (causing multiple chromosome breaks), and then fusing the irradiated cell with a rodent cell. The resulting radiation hybrids contained fragments of human chromosomes that integrated into rodent chromosomes.
Initially, the irradiated cells were monochromosomal hybrid cells, and fragments of a single human chromosome (plus fragments of many rodent chromosomes) randomly integrated into the genome of the rodent cell fusion partner. However, it was more efficient to irradiate a diploid human cell and then fuse it to a rodent cell. The resulting hybrids had multiple fragments from several different human chromosomes. The locations of the breakpoints on any one human chromosome varied from one irradiated cell to the next, and only a minority of the fragments successfully integrated, in a random way, into the rodent chromosomes (Figure 1B).
Constructing clone contigs by STS content mapping
Human genomic DNA libraries containing large insert DNAs were preferred for assembling clone contigs in preparation for DNA sequencing. The first such library was based on yeast artificial chromosomes (YACs) that can be constructed to have DNA inserts over a megabase in size (see Box1 for how YACs are made).
Box1. CLONING OF LARGE DNA FRAGMENTS USING YEAST AND BACTERIAL ARTIFICIAL CHROMOSOMES
To build clone contigs efficiently, a quick and simple way of identifying clones with overlapping inserts was needed, and it was provided by constructing maps based on STS markers. By typing YAC clones with STS markers it was possible to assemble contigs of clones with partially overlapping inserts. But it soon became clear that YAC clones are unstable and YAC inserts were often not faithful representations of the original starting human DNA.
Because of their frequent instability, YAC clones could not be the template for the final genome sequencing effort, and alternative large-insert cloning systems were developed. Bacterial artificial chromosome (BAC) libraries (see Box 1) have smaller inserts than YACs but, crucially, human inserts are comparatively stable in BACs. Eventually, large BAC clone contigs were established for each human chromosome, paving the way for the final genome sequencing phase (see Figure2 for an overview of the different methodological approaches used in the Human Genome Project).
Fig2. Major scientific strategies and approaches used in the Human Genome Project (HGP). The HGP required isolation of human genomic and cDNA clones. The clones were used to construct high-resolution genetic and physical maps that paved the way for genome sequencing. Inevitably, the HGP interacted with research on mapping and identifying human disease genes. The data produced were channeled into mapping and sequence databases permitting rapid electronic access and data analysis. Ancillary projects (not shown here) included studying genetic variation, genome projects for model organisms, and research on ethical, legal, and social implications. CEPH, Centre d’Etude du Polymorphisme Humain; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; lods, logarithm of the odds scores; STS, sequence tagged site.
