A simple method for molecular cloning of fragments of more than one hundred kilobase pairs of exogenous DNA, by the encapsulation of cell in agarose beads, was reported previously for the construction of a human genomic DNA library in a yeast artificial chromosome (YAC) vector in situ YAC construction [1]. The efficiency of this procedure is impaired by the step in which agarose beads that contain human DNA fragments are melted before transformation. The incomplete solubility of the ligated human DNA fragment. YAC vector often results in lower than desirable frequencies of transformation. To overcome this problem we have developed a new improved method that involves use of an agarose film. The technical manipulations involved in the construction of clones of very large segments of human DNA are discussed.

Genomic clones encoding the Drosophila aldolase gene were isolated and the organization of the gene was determined. The protein-coding region spanning nearly 3.5 kb consists of five coding exons (exon 2, 3, 4 alpha, 4 beta, and 4 gamma). The insect exon 2 corresponds to exons 2 to 7 of vertebrate aldolase genes and thus appears to have been formed by the fusion of these 6 exons into a single exon during evolution. The Drosophila aldolase gene is predicted to generate mRNAs for three isozymes (alpha-, beta-, and gamma-types) from the primary transcripts by alternative usage of the final three exons. The reverse transcriptase-PCR assay revealed the occurrence of mRNAs for the three isozymic forms at different developmental stages, and tissue-specific expression was also found to occur in adult flies. In addition to the usual type mRNA species for the alpha-, beta-, and gamma-isozymes, two novel forms of mRNAs, alpha beta- and beta gamma-type mRNAs, were detected tissue-specifically in adult flies, although their functions are unpredictable. The alpha beta-mRNA is an alpha-type mRNA in which exon 4 beta remains unspliced, while the beta gamma-mRNA is a beta-type mRNA with the exon 4 gamma remaining unspliced. Recombinant enzymes expressed in Escherichia coli were all active and exhibited different enzymatic properties.

SummaryA simple and general method for the molecular cloning of fragments of over one hundred kilobase pairs of exogenous DNA, by the encapsulation of cells in agarose beads, was developed for the construction of a human genomic DNA library in a yeast artificial YAC chromosome vector (in situ YAC construction). The main advantages of this method for use in the construction of a human genome library are as follows. First, linear DNA molecules of up to several hundred kilobase pairs in size can easily be prepared by the partial restriction enzyme digestion of the DNA encapsulated in agarose beads in vitro. Second, less than 2 x l0 G cells scraped from tissue culture plates are sufficient for preparation of the linear DNA molecule for construction of the genome library. The technical manipulations involved in construction of clones of very large segments of DNA, including encapsulation of cells in agarose beads, restriction enzyme digestion, ligation with the YAC vector, transformation into host yeast cells, and stable propagation are discussed.

Three isozymic forms, alpha, beta, and gamma, of Drosophila melanogaster aldolase are produced from a single gene by alternative usage of the triple exons 4 (4 alpha, 4 beta, and 4 gamma) [Shaw-Lee et al. (1992) J. Biol. Chem. 267, 3959-3967; Kim et al. (1992) Mol. Cell. Biol. 12, 773-783; Kai et al. (1992) J. Biochem. 112,677-688]. The expression plasmids for the respective isozymes were transfected into Escherichia coli cells, and the isozymes alpha and beta were purified to homogeneity by a simple procedure, though isozyme gamma was only partially purified. These isozymes are active towards two substrates, fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P), with a preference for Fru-1,6-P2 over Fru-1-P, but they have different kcat/Km values towards these two substrates; isozyme alpha shows the highest value for Fru-1,6-P2. These isozymes show similarity in optimal pHs, thermal stability, and Km values for both Fru-1,6-P2 and Fru-1-P. They are composed of four identical subunits of 40 kDa, forming a tetramer with a molecular weight of approximately 160 kDa. The three isozymes are different in primary structure only at the carboxyl-terminal region encoded by the respective exon 4. Therefore, this region should be primarily responsible for the distinct characteristics of these isozymes.