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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section IIThe Physical Map

The construction of the clonal physical map of the C. elegans genome began in response to the need for an efficient means to recover DNA segments corresponding to genes defined through mutation. Of the various methods available in the early 1980s, the most powerful and broadly applicable approach was one that has become known as positional cloning. Meiotic mapping is used in this method to position a gene between markers which are available as cloned DNA. Recovery of the DNA between these markers assures that a clone for the gene of interest is in hand. DNA transformation or other means can then be used to identify precisely on which segment the gene lies.

To assist in this process and to avoid unnecessary duplication, the community of C. elegans workers joined together at an early stage to generate a clone-based physical map of the entire genome, which would be progressively correlated with the genetic map. Two central laboratories took on the task of establishing the relationship of random clones with respect to one another, and the larger community associated specific genes and markers with the growing map. This has resulted in a map that spans more than 99% of the genes, with only seven gaps at present (Fig. 1).

Figure 1. The C.

Figure 1

The C. elegans genome. The contigs that make up the six chromosomes are represented by blocks, with the remaining seven gaps indicated. The progress in genome sequencing is indicated. The scale to the (more...)

The map consists of mostly overlapping bacterial cosmid clones and yeast artificial chromosome (YAC) clones (Fig. 2). The overlaps of the cosmid clones were established through a fingerprinting technique that relies on shared restriction fragments to determine the extent of overlap between randomly selected clones (Coulson et al. 1986). Analysis of the restriction fragment information and assembly was aided significantly by computer programs, but human validation of each proposed new overlap was critical, especially in the early phases, in preventing misassemblies. Biases in the representation of the genome in the bacterially based cosmid clones, however, severely compromised the level of continuity achieved; after the analysis of more than 17,000 clones, the genome remained in some 700 contigs (80 contigs would be expected from a truly random library).

Figure 2. A detail of the physical map.

Figure 2

A detail of the physical map. This representation taken from an ACeDB screen shows, from the top of the figure, the positions of (1) cDNAs, determined by hybridization against YACs representing the genome (more...)

The representation of the genome in YACs is more complete. This, combined with their larger size (average insert size in the initial sets was ˜250 kb), allowed these clones to bridge many of the gaps between the cosmid contigs (Coulson et al. 1988). Hybridization of the YACs to grids of cosmid clones representing the mapped contigs and singlets, as well as hybridization of selected cosmids to grids of random YAC clones, was used to establish the overlaps between the clone sets, which brought down the contig number to just over 100.

Finally, a more laborious effort was undertaken to establish overlaps directly between YACs in those cases where gaps were not spanned by single YACs (Coulson et al. 1991). For this purpose, sequence was obtained from the insert end and used to develop a polymerase chain reaction (PCR) assay for the site, both to make a probe to hybridize against the YAC set and to verify any potential overlaps. A complementary approach involved the construction of additional YAC libraries, including the use of alternative enzymes in the partial digestion of the genomic DNA and the selection of larger inserts (average insert size >700 kb).

Telomeres in C. elegans consist of the repeated hexamer sequence TTAGGC (Wicky et al. 1996). These sequences are also present internally on the chromosomes, along the chromosome arms, complicating the identification of clones derived from the telomeres. Bal31 digestion experiments show that there are 4–9 kb of the repeat at the ends of chromosomes. Using a combination of Bal31 digestion and restriction digests yielding staggered ends on large DNA, Wicky et al. (1996) have obtained candidate clones for 11 of the 12 telomeres. These clones will be linked to clones on the current map to provide end markers.

Representative YAC clones have been selected across the entire map, arrayed in map order on nylon membranes and distributed to the community as the so-called polytene filters (Coulson et al. 1991). Using this grid, any cloned sequence can be localized on average to a 100-kb interval. Cosmids from the region can then be used to derive finer positioning.

Despite the small size of the C. elegans chromosomes, cytogenetics has played an invaluable part throughout the mapping project to position sequences onto chromosomes and to confirm the physical map order by in situ hybridization (Albertson 1985). The resolution of the method is limited, but the results clearly confirm the overall physical map order (Fig. 3) (Coulson 1994). From comparisons of the clonal map and cytogenetic map distances between markers, it is also clear that the C. elegans chromosomes are not uniformly condensed. This is particularly true near the right (lower) end of chromosome I, where the in situ distance between markers is much greater than the physical map suggests. Unfortunately, this region includes one of the seven remaining gaps in the physical map, and thus a more definitive statement cannot be made. Nevertheless, fluorescent staining of the region does appear under the microscope to be attenuated, as if the region is stretched out (D. Albertson, pers. comm.).

Figure 3. Correlation of markers on the physical and cytogenetic maps.

Figure 3

Correlation of markers on the physical and cytogenetic maps. Small boxes show the physical map location of probes, and bars show the in situ signal localization on the right. The scales at the left represent (more...)

Work from a large number of laboratories has led to an increasingly refined correlation between the genetic and physical maps. Cloned genes provided most of the early markers, and laboratories often shared the clones with the central laboratories at early stages in investigations. In addition, restriction fragment length polymorphisms (RFLPs) between the standard laboratory strain of C. elegans (var. Bristol) and other cross-fertile strains have played an increasingly important part. These RFLPs can often be identified at a frequency of one per cosmid or better, providing very tight mapping resolution where necessary. One of the most useful sources of such polymorphisms is the transposable element Tc1 of the Tc1-mariner class. The Bristol strain has about 30 copies of the element, whereas others have more than 500, distributed widely throughout the entire genome (Plasterk and van Leunen, this volume). Many of these elements have been cloned and positioned on both the meiotic and physical maps. An increasing number have been used to generate sequence tagged sites, which provide mapping tools for PCR-based mapping strategies (Williams et al. 1992; R. Plasterk and A. Coulson, pers. comm.).

The result has been a true genome map. With the substantial amounts of various data available, the need for a convenient database for entering and viewing all the relationships became critical. The need has been met by ACeDB (R. Durbin and J. Thierry-Mieg, pers. comm.), which offers interactive displays of the genetic and physical maps plus the underlying information, including genetic data, genomic and cDNA sequences, and much else besides (for examples, see Figs. 2 and 6).

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK20174
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