Intron-mediated recombinant techniques and reagents

#Most eukaryotic genes are discontinuous with proteins encoded by them, consisting of coding sequences (exons) interrupted by non-coding sequences (introns). After transcription into RNA, the introns are removed by splicing to generate the mature messenger RNA (mRNA). The splice points between exons are typically determined by consensus sequences that act as signals for the splicing process.

#Structural features of introns and the underlying splicing mechanisms form the basis for classification of different kinds of introns. Since RNA splicing was first described, four major categories of introns have been recognized. Splicing of group I, group II, nuclear pre-mRNA, and tRNA introns can be differentiated mechanistically, with certain group I and group II introns able to be autocatalytically excised from a pre-RNA in vitro in the absence of any other protein or RNA factors. In the instance of the group I, group II and nuclear pre-mRNA introns, splicing proceeds by a two-step transesterification mechanism.

#To illustrate, the nuclear rRNA genes of certain lower eukaryotes (e.g., Tetrahymena thermophila and Physarum polycephalum) contain group I introns. This type of intron also occurs in chloroplast, yeast, and fungal mitochondrial rRNA genes; in certain yeast and fungal mitochondrial mRNA; and in several chloroplast tRNA genes in higher plants. Group I introns are characterized by a linear array of conserved sequences and structural features, and are excised by two successive transesterifications. Splicing of the Tetrahymena pre-rRNA intron, a prototypic group I intron, proceeds by two transesterification reactions during which phosphate esters are exchanged without intermediary hydrolysis. Except for the initiation step, promoted by a free guanosine, all reactive groups involved in the transesterification reactions are contained within the intron sequences. The reaction is initiated by the binding of guanosine to an intron sequence. The unshared pair of electrons of the 3'-hydroxyl group of the bound guanosine can act as a nucleophile, attacking the phosphate group at the 5' exon-intron junction (splice site), resulting in cleavage of the precursor RNA. A free 3'-hydroxyl group is generated at the cleavage site (the end of the 5'exon) and release of the intron occurs in a second step by attack of the 5' exon's 3.sup.1 -hydroxyl group on the 3' splice site phosphate.

#FIG. 1 is a schematic representation of an illustrative group I splicing reaction, as well as a reverse-splicing reaction.

#FIG. 2 is a schematic representation of the group II splicing reaction, as well as the reverse-slicing reaction.

#FIG. 3 illustrates the domain structure of a group II intron.

#Biological selections and screens are powerful tools with which to probe protein and nucleic acid function and to isolate variant molecules having desirable properties. The technology described herein enables the rapid and efficient generation and selection of novel genes and gene products. The present combinatorial approach, for example, provides a means for capturing the vast diversity of exons, and relies on the ability of intron sequences to mediate random splicing between exons.

#As described below, novel genes and gene products can be generated, in one embodiment of the present combinatorial method, by admixing a variegated population of exons which have flanking intron sequences that can direct trans-splicing of the exons of each other. Under conditions in which trans-splicing occurs between the exons, a plurality of genes encoding a combinatorial library are generated by virtue of the ability of the exons to be ligated together in a random fashion. Where the initial variegated exon population are ribonucleotides (i.e. RNA), the resulting combinatorial transcript can be reverse-transcribed to cDNA and cloned into an appropriate expression vector for further manipulation or screening.

#In another embodiment of the present combinatorial method, a variegated population of single-stranded DNA molecules corresponding to exon sequences of both (+) and (-) strand polarity, and which have flanking intron sequences capable of mediating cis-splicing, are provided together such that a portion of the nucleic acid sequence in the flanking intron of an exon of one polarity (e.g. a (+) strand) can base pair with a complementary sequence in the flanking intron of another exon of opposite polarity (e.g. a (-) strand). Using standard techniques, any single-stranded regions of the concatenated exon/intron sequences can be subsequently filled-in with a polymerase, and nicks covalently closed with a ligase, to form a double-stranded chimeric gene comprising multiple exons interrupted by intron sequences. Upon transcription of the chimeric gene to RNA, cis-splicing can occur between the exons of the chimeric gene to produce the mature RNA transcript, which can encode a chimeric protein.