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A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage A vector, X1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in X1059 requires deletion of the X red and gamma genes. The recombinants are therefore spiP and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage Pg. Random fragments suitable for insertion into X1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a. This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because GA-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved X1059. By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed. DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection. Fractionation of genomes was an intractable problem until the introduction of recombinant DNA techniques. These methods eliminate the necessity for physical separations of DNA segments and permit the isolation of structural genes from collections of randomly cloned DNA fragments (1-10).Given a suitable probe, any eukaryotic gene may be isolated from a pool of cloned fragments, provided that it is large enough to give sequence representation of an entire genome. The simple multicellular eukaryote Caenorhabditis elegans has a haploid DNA content of approximately 8 X 107 base pairs (bp) (11). If random DNA cleavage and uniform cloning efficiency are assumed, a collection of 8 X 104 clones with an average length of 104 bp will be sufficient to include any genomic sequence with >99% probability. Similarly, the human genome with 2 X 109 bp will be covered by 106 clones 104 bp long.We have developed a novel bacteriophage X cloning vector, X1059, with properties that simplify the construction of recombinant phage collections representing genomes. The strategy is shown in Fig. 1 The structure of A1059 is shown in Fig. 2. The phage is a BamHI substitution vector composed of three BamHI fragments: a 19.6-kb left arm carrying the genes for the A head and tail proteins, a 17-kb central fragment, and a 9.4-kb right arm carrying the A replication and lysis genes. The two arms of the vector contain all the essential functions required for A replication and maturation in a DNA sequence 58.2% of the wildtype length. Viable phages are produced when these arms are hybridized with internal DNA fragments 12.8% to 49.8% the length of wild-type A (6.3-24.4 kb). The two arms alone do not produce viable phages, because lambdoid phages require genome sizes between 70% and 108% of the wild-type DNA to fill the phage heads properly (37, 38).The central BamHI fragment of the vector balancing the phage arms carried the A red (exo and 3 genes) and gamma functions under the control of the leftward promoter (pL) an...

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