Random-clone strategy for genomic restriction mapping in yeast.

Abstract

An approach to global restriction mapping is described that is applicable to any complex source DNA. By analyzing a single restriction digest for each member of a redundant set of A clones, a data base is constructed that contains fragment-size lists for all the clones. The clones are then grouped into subsets, each member of which is related to at least one other member by a significant overlap. Finally, a tree-searching algorithm seeks restriction maps that are consistent with the fragment-size lists for all the clones in each subset. The feasibility of the approach has been demonstrated by collecting data on 5000 A clones containing random 15-kilobase inserts of yeast DNA. It is shown that these data can be analyzed to produce regional maps of the yeast genome, extending in some cases for over 100 kilobases. In combination with hybridization probes to previously cloned genes, these local maps are already useful for defining the physical arrangement of closely linked genes. They may in the future serve as building blocks for the construction of a continuous global map.Fifteen years after the discovery of site-specific restriction endonucleases, the restriction map has become the standard medium for displaying the functional organization of short segments of DNA. For regions spanning up to 50 kilobases (kb), restriction maps are routinely constructed and have become indispensable tools both for organizing existing data and for planning experiments. In a few favorable cases, maps as long as 600 kb have been constructed at a resolution of a few kb (1-3), but it has proven difficult to extend existing mapping methods beyond this range. Given that the average human chromosome contains 105 kb of DNA, there is a gross disparity between the present limits of restriction-mapping techniques and the sequence complexity of cellular genomes.In this paper, we describe a project that is aimed at constructing a restriction map of total nuclear DNA from the yeast Saccharomyces, whose haploid genome size has been estimated to be 1.5 x 104 kb (4). The data collection has involved picking X clones at random and measuring the sizes of the restriction fragments generated by a single digest of each clone. Because enough clones have been analyzed to provide redundant sampling of most regions of the genome, it is possible to extract mapping information from the unordered fragment-size lists by an algorithm that systematically imposes the requirement that the set of restriction fragments generated from a single clone must be contiguous.At the present stage of the yeast project, we have analyzed 5000 X clones with average insert sizes of 15 kb, thereby achieving a sampling redundancy on the order of five. Using the data, it is now possible to construct maps of from 20 to >100 kb around most arbitrary starting points in the yeast genome. These local maps have already defined the physical arrangement of clusters of genes that previously had only been linked genetically, and they point the way toward the development of a global phy...

The Saccharomyces cerevisiae mutation elm4-1 facilitates pseudohyphal differentiation and interacts with a deficiency in phosphoribosylpyrophosphate synthase activity to cause constitutive pseudohyphal growth.

“…The known location of FASI (18), therefore, implies that PPS1 is located on the left arm of chromosome XI. Physical mapping confirmed this genetic location; the 3.1-kb Pstl fragment containing PPSJ hybridized to clone 71161 of the ordered clone bank of Olson et al (19,20) (data not shown), known to be located on the left arm of chromosome XI. PPSI specifies PRPP synthase activity. PPS1 codes for a deduced amino acid sequence of 427 residues, with a predicted molecular mass of 47,017 Da.…”

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

“…Nucieotide and Predicted Amino Acid Sequence of MDJl The mitochondrial DnaJ (A&IJI) gene was identified during the DNA sequencing of a S. cerevisiae genomic library (a gift of M. V. Olson et al, 1986;Riles et al, 1993). MDJl lies head to head with HSP12IGLP1, and between the SUF9 and CDC4 genes, on chromosome VI.…”

Integrated Mapping Package—A Physical Mapping Software Tool Kit

“…For example, physical mapping may consist of restriction fingerprinting (Coulson et al, 1986;Olson et al, 1986;Kohara et al, 1987;Carrano et al, 1989;Stallings et al, 1990;Trask et al, 1992), PCR-based STS content mapping (Green and Olson, 1990;Foote et al, 1992;Chumakov et al, 1992), and clone-to-clone hybridization and often incorporates maps and mapping reagents from external sources.…”