Intron-mediated recombinant techniques and reagents

The present invention makes available methods and reagents for novel manipulation of nucleic acids. As described herein, the present invention makes use of the ability of intronic sequences, such as derived from group I, group II, or nuclear pre-mRNA introns, to mediate specific cleavage and ligation of discontinuous nucleic acid molecules. For example, novel genes and gene products can be generated by admixing nucleic acid constructs which comprise exon nucleic acid sequences flanked by intron sequences that can direct trans-splicing of the exon sequences to each other. The flanking intronic sequences can, by intermolecular complementation, form a reactive complex which promotes the transesterification reactions necessary to cause the ligation of discontinuous nucleic acid sequences to one another, and thereby generate a recombinant gene comprising the ligated exons.

BACKGROUND OF THE INVENTION 
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. 
Group II introns, which are classed together on the basis of a conserved 
secondary structure, have been identified in certain organellar genes of 
lower eukaryotes and plants. The group II introns also undergo 
self-splicing reactions in vitro, but in this instance, a residue within 
the intron, rather than added guanosine, initiates the reaction. Another 
key difference between group II and group I introns is in the structure of 
the excised introns. Rather than the linear products formed during 
splicing of group I introns, spliced group II introns typically occur as 
lariats, structures in which the 5'-phosphoryl end of the intron RNA is 
linked through a phoshodiester bond to the 2'-hydroxyl group of an 
internal nucleotide. As with group I introns, the splicing of group II 
introns occurs via two transesterification steps, one involving cleavage 
of the 5' splice site and the second resulting in cleavage of the 3' 
splice site and ligation of the two exons. For example, 5' splice site 
cleavage results from nucleophilic attack by the 2'-hydroxyl of an 
internal nucleotide (typically an adenosine) located upstream of the 3' 
splice site, causing the release of the 5' exon and the formation of a 
lariat intermediate (so called because of the branch structure of the 
2',5' phosphodiester bond thus produced). In the second step, the 3'-end 
hydroxyl of the upstream exon makes a nucleophilic attack on the 3' splice 
site. This displaces the intron and joins the two exons together. 
Eukaryotic nuclear pre-mRNA introns and group II introns splice by the same 
mechanism; the intron is excised as a lariat structure, and the two 
flanking exons are joined. Moreover, the chemistry of the two processes is 
similar. In both, a 2 hydroxyl group within the intron serves as the 
nucleophile to promote cleavage at the 5' splice site, and the 3' hydroxyl 
group of the upstream exon is the nucleophile that cleaves the 3' splice 
site by forming the exon-exon bond. However, in contrast to the conserved 
structural elements that reside within group I and II introns, the only 
conserved features of nuclear pre-mRNA introns are restricted to short 
regions at or near the splice junctions. In yeast, these motifs are (i) a 
conserved hexanucleotide at the 5' splice, (ii) an invariant 
heptanucleotide, the UACUAAC Box, surrounding the branch point A, (iii) a 
generally conserved enrichment for pyrimidine residues adjacent to the 
invariant AG dinucleotide at the 3' splice site. Further characteristics 
of nuclear pre-mRNA splicing in vitro that distinguish it from 
autocatalytic splicing are the dependence on added cell-free extracts, and 
the requirement for adenosine triphosphate (ATP). Another key difference 
is that nuclear pre-mRNA splicing generally requires multiple small 
nuclear ribonucleoproteins (snRNPs) and other accessory proteins, which 
can make-up a larger multi-subunit complex (splicesome) that facilitates 
splicing. 
SUMMARY OF THE INVENTION 
The present invention makes available methods and reagents for novel 
manipulation of nucleic acids. As described herein, the present invention 
makes use of the ability of intronic sequences, such as derived from group 
I, group II, or nuclear pre-mRNA introns, to mediate specific cleavage and 
ligation of discontinuous nucleic acid molecules. For example, novel genes 
and gene products can be generated by admixing nucleic acid constructs 
comprising "exon" nucleic acid sequences flanked by intron sequences that 
can direct trans-splicing of the exon sequences to each other. The 
flanking intronic sequences, by intermolecular complementation between the 
flanking intron sequences of two different constructs, form a functional 
intron which mediates the transesterification reactions necessary to cause 
the ligation of the discontinuous nucleic acid sequences to one another, 
and thereby generate a recombinant gene comprising the ligated exons. As 
used herein, the term exon denotes nucleic acid sequences, or exon 
"modules", that can, for instance, encode portions of proteins or 
polypeptide chains, such as corresponding to naturally occurring exon 
sequences or naturally occurring exon sequences which have been mutated 
(e.g. point mutations, truncations, fusions), as well as nucleic acid 
sequences from "synthetic exons" including sequences of purely random 
construction. However, the term "exon", as used in the present invention, 
is not limited to protein-encoding sequences, and may comprises nucleic 
acid sequences of other function, including nucleic acids of "intronic 
origin" which give rise to, for example, ribozymes or other nucleic acid 
structure having some defined chemical function. 
As described herein, novel genes and gene products can be generated, in one 
embodiment of the present method, by admixing nucleic acid constructs 
which comprise a variegated population of exon sequences. As used herein, 
"variegated" refers to the fact that the population includes nucleic acids 
of different nucleotide compositions. When the interactions of the 
flanking introns are random, the order and composition of the internal 
exons of the combinatorial gene library generated is also random. For 
instance, where the variegated population of exons used to generate the 
combinatorial genes comprises N different internal exons, random 
trans-splicing of the internal exons can result in N.sup.y different genes 
having y internal exons. However, the present trans-splicing method can 
also be utilized for ordered gene assembly such that nucleic acid 
sequences are spliced together in a predetermined order, and can be 
carried out in much the same fashion as automated oligonucleotide or 
polypeptide synthesis. In similar fashion, an ordered combinatorial 
ligation can be carried out in which particular types of exons are added 
to one and other in an ordered fashion, but, at certain exon positions, 
more than one type of exon may be added to generate a library of 
combinatorial genes. 
Furthermore, the present invention makes available methods and reagents for 
producing circular RNA molecules. In particular, exon constructs flanked 
by either group II or nuclear pre-mRNA fragments can, under conditions 
which facilitate exon ligation by splicing of the flanking intron 
sequences, drive the manufacture of circularly premuted exonic sequences 
in which the 5' and 3' ends of the same exon are covalently linked via a 
phosphodiester bond. Circular RNA moieties generated in the present 
invention can have several advantages over the equivalent "linear" 
constructs. For example, the lack of a free 5' or 3' end may render the 
molecule less susceptible to degradation by cellular nucleases. Such a 
characteristic can be especially beneficial, for instance, in the use of 
ribozymes in vitro, as might be involved in a particular gene therapy. The 
circularization of mature messenger-RNA transcripts can also be 
beneficial, by conferring increased stability as described above, as well 
as potentially increasing the level of protein translation from the 
transcript.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION 
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. 
As used herein, the term exon denotes nucleic acid sequences, or exon 
"modules", that can, for instance, encode portions of proteins or 
polypeptide chains. The exons can correspond to discrete domains or 
motifs, as for example, functional domains, folding regions, or structural 
elements of a protein; or to short polypeptide sequences, such as reverse 
turns, loops, glycosylation signals and other signal sequences, or 
unstructured polypeptide linker regions. The exons modules of the present 
combinatorial method can comprise nucleic acid sequences corresponding to 
naturally occurring exon sequences or naturally occurring exon sequences 
which have been mutated (e.g. point mutations, truncation, fusions), as 
well as nucleic acid sequences from "synthetic exons" including sequences 
of purely random construction, that is, nucleic acid sequences not 
substantially similar to naturally occurring exon sequences. In some 
instances, the exon module can correspond to a functional domain and the 
module may comprise a number of naturally occurring exon sequences spliced 
together, with the intron sequences flanking only the exon sequences 
disposed at the extremity of the module. 
Moreover, the term "exon", as used in the present invention, is not limited 
to protein-encoding sequences, and may comprises nucleic acid sequences of 
other function, including nucleic acids of "intronic origin" which give 
rise to, for example, ribozymes or other nucleic acid structure having 
some defined chemical function. As illustrated below, group II intron 
domains (e.g. domains 1-6) and group I intron domains (e.g. paired regions 
P1-P10) can themselves be utilized as "exons", each having flanking 
intronic sequences that can mediate combinatorial splicing between 
different group I or group II domains to produce novel catalytic intron 
structures. In another illustrative embodiment, the exon can comprise a 
cloning or expression vector into which other nucleic acids are ligated by 
an intron-mediated trans-splicing reaction. 
With respect to generating the protein-encoding exon constructs of the 
present invention, coding sequences can be isolated from either cDNA or 
genomic sources. In the instance of cDNA-derived sequences, the addition 
of flanking intronic fragments to particular portions of the transcript 
can be carried out to devise combinatorial units having exonic sequences 
that correspond closely to the actual exon boundaries in the pre-mRNA. 
Alternatively, the choice of coding sequences from the cDNA clone can be 
carried out to create combinatorial units having "exon" portions chosen by 
some other criteria. For example, as described below with regard to the 
construction of combinatorial units from either antibody or plasminogen 
activator cDNA sequences, the criteria for selecting the exon portions of 
each splicing construct can be based on domain structure or function of a 
particular portion of the protein. 
Several strategies exist for identifying coding sequences in mammalian 
genomic DNA which can subsequently be used to generate the present 
combinatorial units. For example, one strategy frequently used involves 
the screening of short genomic DNA segments for sequences that are 
evolutionarily conserved, such as the 5' splice site and branch acceptor 
site consensus sequences (Monaco et al. (1986) Nature 323:646-650; Rommens 
et al. (1989) Science 245:1059-1065; and Call et al. (1990) Cell 
60:509-520). Alternative strategies involve sequencing and analyzing large 
segments of genomic DNA for the presence of open reading frames (Fearson 
et al. (1990) Science 247:49-50), and cloning hypo-methylated CpG islands 
indicative of 5' transcriptional promoter sequences (Bird et al. (1986) 
Nature 321:209-213). Yet another technique comprises the cloning of 
isolated genomic fragments into an intron which is in turn disposed 
between two known exons. The genomic fragments are identified by virtue of 
the ability of the inserted genomic sequences to direct alternate splicing 
which results in the insertion into a mature transcript of at least one 
genomic-derived exon between the two know exons (Buckler et al. (1991) 
PNAS 88:4005-4009). 
Exons identified from genomic DNA can be utilized directly as combinatorial 
units by isolating the identified exon and appropriate fragments of the 
flanking intron sequences normally associated with it. Alternatively, as 
with the cDNA derived exons, the genomic-derived exon can be manipulated 
by standard cloning techniques (Molecular Biology: A Laboratory Manual, 
eds. Sambrook, Fritsch and Maniatis (New York: CSH Press, 1989); and 
Current Protocols in Molecular Biology, Eds. Ausebel et al. (New York: 
John Wiley & Sons, 1989)) into vectors in which appropriate flanking 
intronic sequences are added to the exon upon transcription. In yet 
another embodiment, the reversal of splicing reactions, described below 
for the various intron groups, can be used to specifically add flanking 
intron fragments to one or both ends of the exonic sequences, and thereby 
generate the combinatorial units of the present invention. 
Furthermore, generating the splicing units useful in the present 
combinatorial methods, one skilled in the art will recognize that in the 
instance of protein-encoding exons, particular attention should be payed 
to the phase of the intronic fragments. Introns that interrupt the reading 
frame between codons are known as "Phase 0" introns; those which interrupt 
the codons between the first and second nucleotides are known as "Phase 1" 
introns; and those interrupting the codons between the second and third 
nucleotides are known as "Phase 2" introns. In order to prevent a shift in 
reading frame upon ligation of two exons, the phase at both the 5' splice 
site and 3' splice site must be the same. The phase of the flanking 
intronic fragments can be easily controlled during manipulation, 
especially when reverse splicing is utilized to add the intronic 
fragments, as the each insertion site is known. However, as described 
below, when the variegated population of combinatorial units comprises 
flanking intronic fragments of mixed phase, particular nucleotides in the 
intronic sequences can be changed in such a manner as to lower the 
accuracy of splice site choice. In addition, the splicing reaction 
conditions can also be manipulated to lower the accuracy of splice site 
choice. 
I. Intronic Sequences 
The present invention makes use of the ability of introns to mediate 
ligation of exons to one and other in order to generate a combinatorial 
library of genes from a set of discontinuous exonic sequences. This method 
is not limited to any particular intron or class of introns. By way of 
example, the intronic sequences utilized can be selected from group I, 
group II, or nuclear pre-mRNA introns. Furthermore, in light of 
advancement made in delineating the critical and dispensable elements in 
each of the classes of introns, the present invention can be practiced 
with portions of introns which represent as little as the minimal set of 
intronic sequences necessary to drive exon ligation. 
Group I introns, as exemplified by the Tetrahymena ribosomal RNA (rRNA) 
intron, splice via two successive phosphate transfer, transesterification 
reactions. As illustrated in FIG. 1, the first transesterfication is 
initiated by nucleophilic attack at the 5' junction by the 3' OH of a free 
guanosine nucleotide, which adds to the 5' end of the intron and liberates 
the 5' exon with a 3' OH. The second transesterification reaction is 
initiated by nucleophilic attack at the 3' splice junction by the 3' OH of 
the 5' exon, which results in exon ligation and liberates the intron. 
Group II introns also splice by way of two successive phosphate transfer, 
transesterification reactions (see FIG. 2). There is, however, one 
prominent difference between the reaction mechanisms proposed for group I 
and group II introns. While cleavage at the 5' junction in group I 
splicing is due to nucleophilic attack by a free guanosine nucleotide, 
cleavage at the 5' junction in group II splicing is typically due to 
nucleophilic attack by a 2'-OH from within the intron. This creates a 
lariat intermediate with the 5' end of the intron attached through a 2', 
5'-phosphodiester bond to a residue near the 3' end of the intron. 
Subsequent cleavage at the 3' junction results in exon ligation and 
liberates the "free" intron in the form of a lariat. The nature of the 
initiating nucleophile notwithstanding, the two self-splicing mechanism 
appear quite similar as both undergo 5' junction cleavage first, and 
subsequently 3' junction cleavage and exon ligation as a consequence of 
nucleophilic attack by the 5' exon. Furthermore, nuclear pre-mRNA, in 
similar fashion to group II--intron splicing, also proceed through a 
lariat intermediate in a two-step reaction. 
All three intron groups share the feature that functionally active introns 
able to mediate splicing can be reconstituted from intron fragments by 
non-covalent interactions between the fragments (and in some instances 
other trans-acting factors). Such "trans-splicing" by fragmented introns, 
as described herein, can be utilized to ligate discontinuous exon 
sequences to one and other and create novel combinatorial genes. Moreover, 
autocatalytic RNA (i.e. group I and group II introns) are not only useful 
in the self-splicing reactions used generate combinatorial libraries, but 
can also catalyze reactions on exogenous RNA. 
The following description of each of the group I, group II, and nuclear 
pre-mRNA intronic sequences is intended to illustrate the variation that 
exists in each group of introns. Moreover, the descriptions provide 
further insight to one skilled in the art to devise exon constructs useful 
in the present splicing methods, using as little as a minimal set of 
intronic-fragments. 
A. Group II Introns 
Group II introns, which are classed together on the basis of a conserved 
secondary structure, are found in organellar genes of lower eukayotes and 
plants. Like introns in nuclear pre-mRNA, group II introns are excised by 
a two-step splicing reaction to generate branched circular RNAs, the 
so-called intron lariats. A remarkable feature of group II introns is 
their self-splicing activity in vitro. In the absence of protein or 
nucleotide cofactors, the intronic RNA catalyzes two successive 
transesterfication reactions which lead to autocatalytic excision of the 
intron-lariat from the pre-mRNA and concomitantly to exon ligation (see 
FIG. 2). 
More than 100 group II intron sequences from fungal and plant mitochondria 
and plant chloroplasts have been analyzed for conservation of primary 
sequence, secondary structure and three-dimensional base pairings. Group 
II introns show considerable sequence homology at their 3' ends (an AY 
sequence), and have a common G.sub.1 W.sub.2 G.sub.3 Y.sub.4 G.sub.5 motif 
at their 5' ends, but do not show any other apparent conserved sequences 
in their interior parts. However, group II introns are generally capable 
of folding into a distinctive and complex secondary structure typically 
portrayed as six helical segments or domains (designated herein as domains 
1-6) extending from a central hub (see FIG. 3). This core structure is 
believed to create a reactive center that promotes the transesterification 
reactions. 
However, mutational analysis and phylogenetic comparison indicate that 
certain elements of the group II intron are dispensable to self-splicing. 
For example, several group II introns from plants have undergone some 
rather extensive pruning of peripheral and variable stem structures. 
Moreover, while the group II intron can be used to join two exons via 
cis-splicing, a discontinuous group II intron form of trans-splicing can 
be used which involves the joining of independently transcribed coding 
sequences through interactions between intronic RNA pieces. In vitro 
studies have shown that breaks, for example within the loop region of 
domain IV, can be introduced without disrupting self-splicing. The ability 
of group II intron domains to reassociate specifically in vivo is 
evidenced by trans-spliced group II introns, which have been found, for 
example, in the rps-12 gene of higher plant ctDNA, the psaA gene in 
Chlamydomonas reinhardtii ctDNA, and the nad1 and nad5 genes in higher 
plant mtDNA (Michel et al. (1989) Gene 82:5-30; and Sharp et al. (1991) 
Science 254:663). These genes consist of widely separated exons flanked by 
5'- or 3'-segments of group II introns split in either domains 3 or 4. The 
exons at different loci are transcribed into separate precursor RNAs, 
which are trans-spliced, presumably after the association of the two 
segments of the group II intron. Moreover, genetic analysis of 
trans-splicing of the Chlamydomonas reinhardtii psaA gene has demonstrated 
that the first intron of this gene is split into three segments. The 5' 
exon is flanked by parts of domain 1 and the 3' exon by parts of domains 4 
to 6, respectively. The middle segment of the intron is encoded at a 
remote locus, tscA, and consists of the remainder of domains 1 to 4. This 
tscA segment can apparently associate with the other two intron segments 
to reconstitute an intron capable of splicing the two exons (Goldschmidt 
et al. (1991) Cell 65:135-143). 
The functional significance to self-splicing of certain control structural 
elements have been further deduced by analysis of minimal trans-splicing 
sets, and found to generally comprise an exon-binding site and 
intron-binding site, a structural domain 5, and (though to lesser extent) 
a "branch-site" nucleotide involved in lariat formation. Domain 1 contains 
the exon-binding sequences. Domain 6 is a helix containing the branch 
site, usually a bulged A residue. Domain 5, the most highly conserved 
substructure, is required for catalytic activity and binds to at least a 
portion of domain 1 to form the catalytic core. 
The 5' splice sites of group II introns are defined by at least three 
separate tertiary base pairing contacts between nucleotides flanking the 
5' splice site and nucleotides in substructures of domain I. The first 
interaction involves a loop sequence in the D sub-domain of domain 1 (axon 
binding site 1 or EBS 1) that base pairs with the extreme 3' end of the 5' 
exon (intron binding site 1 or IBS 1). The second interaction involves the 
conserved dinucleotide-G.sub.3 Y.sub.4 -(designated .epsilon.) that base 
pairs with a dinucleotide in the C1 subdomain of domain I (designated 
.epsilon.'). The third interaction involves base pairing between intron 
binding site (IBS 2), a sequence located on the 5' side of IBS 1, with 
exon binding site 2 (EBS 2), a loop sequence of the D subdomain of domain 
1 near EBS 1. Of the two exon-binding sites identified in group II 
introns, only EBS 1 is common to all group II members. The EBS 1 element 
comprises a stretch of 3 to 8 consecutive residues, preferably 6, located 
within domain 1, which are complementary to the last 3 to 8 nucleotides of 
the 3' exon end of the 5' exon. The EBS 2-IBS 2 pairing also typically 
consists of two 4-8 nucleotide stretches. Its exonic component (IBS 2) 
lies from 0 to 3 nucleotides upstream from the IBS 1 element, and the 
intronic component (EBS 2) also lies within domain 1. However, while IBS 
2-EBS 2 pairing can improve the efficiency of 5' splice site use, 
particularly in trans, it is subject to many more variations from the IBS 
1-EBS 1 interaction, such as reduced length, presence of bulging 
nucleotides or a mismatch pair. Disrupting the IBS 2-EBS 2 pairing, in the 
Sc.aS group II intron for example, is essentially without effect on the 
normal splicing reaction, and in at least twelve group II introns 
analyzed, the IBS 2-EBS 2 interaction seems to be missing altogether and 
is apparently less important than the IBS 1-EBS 2 interaction. As already 
noted, only that pairing is absolutely constant in (typical) group II 
introns, and always potentially formed at cryptic 5' splice sites. 
Further studies, while confirming that the EBS 1-IBS 1 base pairing is 
necessary for activation of the 5' junction, indicate that this 
interaction alone is not always sufficient for unequivocal definition of 
the cleavage site. Is have been established that altering the first 
nucleotide of the group II intron (e.g., G.sub.1 of G.sub.1 W.sub.2 
G.sub.3 Y.sub.4 G.sub.5) can reduce the self-splicing rate in vitro. 
Characterization of the products of self-splicing from G.sub.1 .fwdarw.N 
mutant transcripts have demonstrated that the relative order of function 
is G&gt;U&gt;A&gt;C. It is also suggested that the 5' G of the intron helps to 
position the cleavage site precisely (Wallasch et al. (1991) Nuc. Acids 
Res. 19:3307-3314). For example, the presence of an additional adenosine 
following IBS 1 can lead to ambiguous hydrolytic cleavages at the 5' 
intron/exon boundary. As described herein, such ambiguity can be used to 
address exon phasing. 
Another well conserved feature of group II introns is the bulging A located 
7 to 8 nt upstream from the 3' intron-exon junction on the 3' side of 
helix VI. This is the nucleotide which participates in the long range, 
2'-5' lariat bond (Van der Veen et al. (1986) Cell 44:225-234; Schmelzer 
et al. (1986) Cell 46:557-565; Jacquier et al. (1987) Cell 50:17-29; 
Schmelzer et al. (1987) Cell 51:753-762). Evidence from electron 
microscopy, attempts at reverse transcription of circular introns, and 
treatment with the 2',5'-phosphodiesterase of HeLa cells indicate that 
group II introns are excised as lariats (Van der Veen et al. (1986) Cell 
44:225-234; Schmitt et al. (1987) Curr. Genet. 12:291-295; Kroller et al. 
(1985) Embo J. 4:2445-2450). However, lariat formation is not absolutely 
essential for correct exon ligation to occur. Cleavage at the 5' splice 
site, presumably mediated by free hydroxide ions rather than a 2'-OH 
group, followed by normal exon ligation, has been observed both in 
trans-splicing reactions (Jacquier and Rosbash (1986) Science 
234:1099-1104; and Koch et al. (1992) Mol. Cell Biol. 12:1950-1958) and, 
at high ionic strength, in cis-splicing reactions with molecules mutated 
in domain 6 (Van der Veen et al. (1987) Embo. J. 12:3827-3821). Also, 
several group II introns lack a bulging A on the 3' side of helix VI. For 
instance, all four CP tRNA-VAL introns of known sequence have a fully 
paired helix VI, and their 7th nucleotide upstream from the 3' intron-exon 
junction is a G, not an A. Furthermore, correct lariat formation has been 
observed with a mutant of intron Sc.bl whose helix VI should be fully 
paired, due to the insertion of an additional nucleotide (a U) at the site 
facing the normally bulging A (Schmelzer and Muller (1987) Cell 
51:753-762). 
Perhaps one of the best conserved structural elements of group II introns 
is domain 5. The typical domain 5 structure contains 32-34 nucleotides and 
is predicted to fold as a hairpin. The hairpin is typically an extended 14 
base pair helix, capped by a four base loop involving 15-18, and 
punctuated by a 2 base bulge at positions 25 and 26. Comparative sequence 
analysis (Michel et al. (1989) Gene 82:5-30) has shown that group II 
introns can generally be classified into one of two classes (e.g. group 
IIA and IIB). FIG. 4 shows the consensus sequences of domain 5 for each of 
the IIA and IIB introns. Base pairs that are highly conserved are 
indicated by solid lines. Dashed lines indicate less well conserved base 
pair interactions. The unpaired loop at the apex of the hairpin is 
typically an NAAA sequence, where N is most often a G for IIA introns. 
Nucleotides which are highly conserved are circled, while less conserved 
nucleotides are uncircled. A black dot indicates a lack of discernible 
sequence consensus. 
Degenerate group II introns can be functional despite lacking some domains. 
Euglena ctDNA, for example, contains a large number of relatively short 
group II introns which sometimes lack recognizable cognates of domain 2,3, 
or 4 The view that the only group II structures required for splicing 
activities are domains 1 and 5 is supported by a detailed mutational 
analysis of a yeast mitochondrial group II intron in which various domains 
were deleted, either singly or in combinations (Koch et al. (1992) Mol. 
Cell. Biol. 12:1950-1958). For example, the removal or disruption of the 
domain 6 helix simply reduces 3' splice site fidelity and reaction 
efficiency. This analysis has led to the belief that domain 5 probably 
interacts with domain 1 to activate the 5' splice site, since a transcript 
lacking domains 2-4, and 6, but retaining domain 1 and domain 5 was 
capable of specific hydrolysis of the 5' splice junction. 
With regard to 3' splice-site selection, two weak contacts are believed to 
play a role in defining the 3' splice-site but are, however, not essential 
to splicing. The first of these contacts is a lone base pair, termed 
.gamma./.gamma.', between the 3' terminal nucleotide of the introns and a 
single base between domains 2 and 3. (Jacquier et al. (1990) J. Mol. Biol. 
13:437-447). A second single base pair interaction, termed the internal 
guide, has been defined between the first base of the 3' exon and the 
nucleotide adjacent to the 5' end of EBS 1 (Jacquier et al. (1990) J. Mol. 
Biol. 219:415-428). 
In addition to the ability of autocatalytic RNAs such as group I and group 
II introns to excise themselves from RNA and ligate the remaining exon 
fragments, ample evidence has accumulated demonstrating that the 
autocatalytic RNAs can also catalyze their integration into exogenous 
RNAs. For example, both group I and group II introns can integrate into 
foreign RNAs by reversal of the self-splicing reactions. The mechanism of 
the group II intron reverse-splicing reaction is shown in FIG. 2. In the 
first step of the reverse reaction, the attack of the 3'-OH group of the 
intron 3' terminus at the junction site of the ligated exons yields a 
splicing intermediate, the intron-3' exon lariat, and the free 5' exon. In 
the second step, the 5' exon, which is still bound to the lariat via the 
EBS 1/EBS 1 base pairing, can attack the 2'-5' phosphodiester bond of the 
branch. This transesterification step leads to reconstitution of the 
original precursor. The analogous reaction of the intron with a foreign 
RNA harboring an IBS 1 motif results in site-specific integration 
downstream of the IBS 1 sequence. 
The exon constructs of the present invention, whether comprising the group 
II intronic sequences described above or the group I or nuclear pre-mRNA 
intronics described below, can be generated as RNA transcripts by 
synthesis in an in vitro transcription system using well known protocols. 
For example, RNA can be transcribed from a DNA template containing the 
exon construct using a T3 or T7 RNA polymerase, in a buffer solution 
comprising 40 mm Tris-HCI (pH 7.5), 6 mM MgCl.sub.2, 10 mM dithiothreitol, 
4 mM spermridine and 500 mM each ribonucleoside triphosphate. In some 
instances, it will be desirable to omit the spermidine from the 
transcription cocktail in order to inhibit splicing of the transcribed 
combinatorial units. 
Several reaction conditions for facilitating group II-mediated splicing are 
known. For example, the reaction can be carried out in "Buffer C" which 
comprises 40 mM Tris-HCl (pH 7.0), 60 mM MgCl.sub.2, 2 mM spermidine, and 
500 mM KC1 (Wallasch et al. (1991) Nuc. Acid Res. 19:3307-3314; and Suchy 
et al. (1991) J. Mol. Biol. 222:179-187); or "Buffer S" which comprises 70 
mM Tris-SO.sub.4 (pH 7.5) 60 mM MgSO.sub.4, 2 mM spermidine, and 500 mM 
(NH.sub.4)2 SO.sub.4 (Morl et al. (1990) Nuc. Acid Res. 18:6545-6551; and 
Morl et al. (1990) Cell 60:629-636). The group II ligation reactions can 
be carried out, for instance, at 45.degree. C., and the reaction stopped 
by EtOH precipitation or by phenol:chloroforrn (1:1) extraction. Suitable 
reaction conditions are also disclosed in, for example, Jacquier et al. 
(1986) Science 234:1099-1104; Franzer et al. (1993) Nuc. Acid Res. 
21:627-634; Schmelzer et al. (1986) Cell 46:557-565; Peebles et al. (1993) 
J. Biol. Chem. 268:11929-11938; Jarrell et al. (1988) J. Biol. Chem. 
263:3432-3439; and Jarrell et al. (1982) Mol. Cell Biol. 8:2361-2366. 
Moreover, manipulation of the reaction conditions can be used to favor 
certain reaction pathways, such as reverse-splicing reaction (e.g., by 
increasing the MgSO.sub.4, concentration to 240 mM in Buffer S); bypassing 
the need for a branch nucleotide acceptor (e.g. high salt); and decreasing 
the accuracy of splice-site choice (Peebles et al. (1987) CSH Symp. Quant. 
Biol. 52:223-232). 
B. Group I Introns 
Group I introns are present in rRNA, tRNA, and protein-coding genes. They 
are particularly abundant in fungal and plant mitochondrial DNAs (mtDNAs), 
but have also been found in nuclear rRNA genes of Tetraphymena and other 
lower eukaryotes, in chloroplast DNAs (ctDNAs), in bacteriophage, and 
recently in several tRNA genes in eubacteria. 
As first shown for the Tetrahymena large rRNA intron, group I introns 
splice by a mechanism involving two transesterification reactions 
initiated by nucleophilic attack of guanosine at the 5' splice site (see 
FIG. 1). The remarkable finding for the Tetrahymena intron was that 
splicing requires only guanosine and Mg.sup.2+. Because bond formation and 
cleavage are coupled, splicing requires no external energy source and is 
completely reversible. After excision, some group I introns circularize 
via an additional transesterfication, which may contribute to shifting the 
equilibrium in favor of spliced products. 
The ability of group I introns to catalyze their own splicing is related to 
their highly conserved secondary and tertiary structures. The folding of 
the intron results in the formation of an active site juxtaposing key 
residues that are widely separated in primary sequence. This RNA structure 
catalyzes splicing by bring the 5' and 3' splice sites and guanosine into 
proximity and by activating the phosphodiester bonds at the splice sites. 
Different group I introns have relatively little sequence similarity, but 
all share a series of the short, conserved sequence elements P, Q, R, and 
S. These sequence elements always occur in the same order and basepair 
with one another in the folded structure of the intron (see FIG. 5). 
Element R [consensus sequence (C/G)YUCA(GA/AC)GACUANANG; SEQ ID NO: 1] and 
S [consensus AAGAUAGUCY; SEQ ID NO: 2] are the most highly conserved 
sequences within group I introns, and typically serve as convenient 
"landmarks" for the identification of group I introns. The boundaries of 
group I introns are marked simply by a U residue at the 3' end of the 5' 
exon and a G residue at the 3' end of the intron. (see, for example, 
Michel et al. (1990) J Mol Biol 216:585-610; Cech, T R (1990) Annu Rev 
Biochem 59:543-568; Cech, T R (1988) Gene 73:259-271; Burke (1989) Methods 
in Enzymology 190:533-545; and Burke et al. (1988) Gene 73:273-294). 
The conserved group I intron secondary structure was deduced from 
phylogenetic comparisons, and specific features have been confirmed by 
analysis of in vivo and in vitro mutations and by structure mapping. The 
structure, shown in FIG. 5, consists of a series of paired regions, 
denoted P1-P10, separated by single-stranded regions (denoted J) or capped 
by loops (denoted L), from the core of the structure. The fundamental 
correctness of the model is supported by the observation that a vast 
number of group I intron sequences can be folded into this basis 
structure. 
P1 and P10, which contain the 5' and 3' splice sites, respectively, are 
formed by base pairing between an internal guide sequence (IGS), generally 
located just downstream of the 5' splice site, and exon sequences flanking 
the splice sites. Group I introns have been classified into four major 
subgroups, designated IA to ID, based on distinctive structural and 
sequence features. Group IA introns, for example, contain two extra 
pairings, P7.1/P7.1a or P7.1/P7.2, between P3 and P7, whereas many group 
IB and IC introns may contain additional sequences, including open reading 
frames (ORFs), in positions that do not disrupt the conserved core 
structure. Indeed, many of the peripheral stem-loops can be completely 
deleted without major loss of splicing function. For example, the phage I4 
sunY intron has been re-engineered to contain as few as 184 nucleotides 
while still retaining greater than 10-percent activity. Presumably, if the 
criterion for activity were lowered, the minimal size one could achieve 
would be decreased. 
The region of the Tetrahymena intron required for enzymatic activity, the 
catalytic core, consists of P3, P4, P6, P7, P8, and P9.0. Mutation of a 
nucleotide involved in one of these core structural elements typically 
decreases the maximum velocity of splicing, increase K.sub.m for 
guanosine, or both. In those instances where the primary importance of the 
nucleotide is its contribution to the formation of a duplex region, a 
second-site mutations that restores base-pairing also restores splicing 
function. Studies using Fe(II)-EDTA, a reagent that cleaves the 
sugar-phosphate backbone, have shown that parts of the core are buried in 
the structure inaccessible to the solvent, that Mg.sup.2+ is necessary 
for folding of the intron, and that individual RNA domains fold in a 
specific order as Mg.sup.2+ is increased. All group I introns have 
fundamentally similar core structures, but subgroup-specific structures 
such as P7.1, P7.2, and P5abc appear to participate in additional 
interactions that stabilize the core structure in different ways (Michel 
et al. (1990) J. Mol. Biol. 216:585-610; and Michel et al. (1992) Genes 
Dev. 6:1373-1385). 
A three dimensional model of the group I intron catalytic core has been 
developed by Michel and Westhof (Michel et al. (1990) J. Mol. Biol. 
216:585-610) through comparative sequence analysis. In the Michel-Westhof 
model, the relative orientation of the two helices is constrained by a 
previously proposed triple helix involving parts of J3/4-P4-P6-J6/7 and by 
potential tertiary interactions identified by co-variation of nucleotides 
that are not accounted for by secondary structure. A number of these 
binding sites accounts for the known splicing mechanism, which requires 
appropriate alignments of guanosine and the 5' and 3' exons in the first 
and second steps of splicing. Deoxynucleotide and phosphorothioate 
substitution experiments suggest that finctionally important Mg.sup.2+ 
ions are coordinated at specific positions around the active site (e.g., 
P1 and J8/7) where they may function directly in phosphodiester bond 
cleavage (Michel et al. (1990) J. Mol. Biol. 216:585-610; and Yarus, M 
(1993) FASEB J. 7:31-9). Basic features of the predicted three-dimensional 
structure have been supported by mutant analysis in vitro and by the use 
of specifically positioned photochemical cross-linking and affinity 
cleavage reagents. 
The 5' and 3' splice sites of group I introns are substrates that are acted 
on by the catalytic core, and they can be recognized and cleaved by the 
core when added on separate RNA molecules (Cech (1990) Annu. Rev. Biochem. 
59:543-568). In group I introns the last 3-7 nucleotides of the 5' exon 
are paired to a sequence within the intron to form the short duplex region 
designated P1. The intron-internal portion of P1 is also known as the 5' 
exon-binding site and as a portion of the internal guide sequences, IGS. 
The P1s of different group I introns vary widely in sequence. Neither the 
sequence nor length of P1 is fixed, but the conserved U at the 3' end of 
the 5' exon always forms a wobble base pair with a G residue in the IGS 
(FIG. 5). The conserved U:G is one important recognition element that 
defines the exact site of guanosine attack. In general, other base 
combinations do not substitute well. One exception is C:G, which maintains 
the accuracy of splicing but decreases the K.sub.cat /K.sub.m by a factor 
of 100. Another exception is C:A; the ability of this pair to substitute 
well for U:G has been interpreted as an indication that disruption of P1 
by a wobble base pair is a key element in recognition of the splice site. 
Position within the P1 helix is another determinant of 5' splice site. 
Analysis of in vitro mutants has shown that the distance of the U:G pair 
from the bottom of the P1 helix is critical for efficient cleavage in the 
Tetrahymena intron and that J1/2 and P2 also play a role in the 
positioning of P1 relative to the core (Michel et al. (1990) J. Mol. Biol. 
216:585-610; Young et al. (1991) Cell 67:1007-1019; and Salvo et al. 
(1992) J. Biol. Chem. 267:2845-2848). The U:G pair is most efficiently 
used when located 4-7 base pairs from the base of the P1. 
The positioning of the 3' splice site in group I introns depends on at 
least three interactions, whose relative importance differs in different 
introns. These are the P10 pairing between the IGS and the 3' exon, 
binding of the conserved G residue at the 3' end of the intron to the 
G-binding site in the second step of splicing, and an additional 
interaction, P9.0, which involves base paring between the two nucleotides 
preceding the terminal G of the intron and two nucleotides in J7/9 (Cech 
(1990) Annu. Rev. Biochem. 59:543-568). 
Group I introns have K.sub.m values for guanosine that are as low as 1 
.mu.M and readily discriminate between guanosine and other nucleosides. 
The major component of the guanosine-binding site corresponds to a 
universally conserved CG pair in P7. Guanosine was initially proposed to 
interact with this base pair via formation of a base triple, but the 
contribution of neighboring nucleotides and the binding of analogs are 
also consistent with a model in which guanosine binds axially to the 
conserved G and flanking nucleotides. The guanosine-binding site of group 
I introns can also be occupied by the guanidino groups of arginine or 
antibiotics, such as streptomycin, which act as competitive inhibitors of 
splicing (von Ahsen et al. (1991) Nuc. Acids Res. 19:2261-2265). 
Group I introns can also be utilized in both trans-splicing and 
reverse-splicing reactions. For example, the ribozyme core of a group I 
intron can be split in L6, and through intermolecular complementation, a 
functional catalytic core can be reassembled from intronic fragments (i.e. 
P1-6.5 and P6.5-10) on separately transcribed molecules (Galloway et al. 
(1990) J. Mol. Biol. 211:537-549). 
Furthermore, as described for group II intron constructs, combinatorial 
units comprising group I introns can be transcribed from DNA templates by 
standard protocols. The group I self-splicing reaction has an obligatory 
divalent cation requirement, which is commonly met by Mg.sup.2+. The 
reaction can be in fact be stopped using a chelating agent such as EDTA. 
The group I-mediated splicing of exonic sequences can be carried out, for 
example, in a buffer comprising 100 mM (NH.sub.4).sub.2 SO.sub.4, 50 mM 
HEPES (pH 7.5), 10 mM MgCl.sub.2, and 25 .mu.M GTP, at a temperature of 
42.degree. C. (Woodson et al. (1989) Cell 57:335-345). In another 
embodiment, the reaction buffer comprises 50 mM Tris-HCI (pH 7.5), 50 mM 
NH.sub.4C 1, 3 mM MgCl.sub.2, 1 mM spermidine, and 100 mM GTP, and the 
reaction proceeds at 55.degree. C. (Salvo et al. (1990) J. Mol. Biol. 
211:537-549). To form the reverse-splicing reaction, the Mg.sup.2+ 
concentration can be increased (e.g., to 25 mM) and the GTP omitted. 
Typically, the reversal of splicing reaction is favored by high RNA 
concentrations, high magnesium and temperature, and the absence of 
guanosine. Other examples of useful reaction conditions for group I intron 
splicing can be found, for example, in Mohr et al. (1991) Nature 
354:164-167; Guo et al. (1991) J. Biol.Chem. 266:1809-1819; Kittle et al. 
(1991) Genes Dev. 5:1009-1021; Dounda et al. (1989) PNAS 86:7402-7406; and 
Pattanju et al. (1992) Nuc. Acid Res. 20:5357-5364. 
The efficiency of splicing of group II and group I introns can often be 
improved by, and in some instances may require, the addition of protein 
and/or RNA co-factors, such as maturases. (Michel et al. (1990) J. Mol. 
Biol. 216:585-610; Burke et al. (1988) Gene 71:259-271; and Lambowitz et 
al. (1990) TIBS 15:440-444). This can be especially true when more 
truncated versions of these introns are used to drive ligation by 
trans-splicing, with the maturase or other co-factor compensating for 
structural defects in the intron structure formed by intermolecular 
complementation by the flanking intron fragments. Genetic analysis of 
mitochondrial RNA splicing in Neurospora and yeast has shown, for example, 
that some proteins involved in splicing group I and group II introns are 
encoded by host chromosomal genes, whereas others are encoded by the 
introns themselves. Several group I and group II introns in yeast mtDNA, 
for instance, encode maturases that function in splicing the intron that 
encodes them. These include group I introns Cob-I2, -I3, and -I4, and 
group II introns cox1-I1 and -I2. Thus, the conditions for splicing of 
group I and group II introns can further comprise maturases and other 
co-factors as necessary to form a functional intron by the flanking intron 
sequences. 
C. Nuclear pre-mRNA introns 
Nuclear pre-mRNA splicing, like group II intron-mediated splicing, also 
proceeds through a lariat intermediate in a two-step reaction. In contrast 
to the highly conserved structural elements that reside within group II 
introns, however, the only conserved features of nuclear pre-mRNA introns 
are restricted to short regions at or near the splice junctions. For 
instance, in yeast motifs are (i) a conserved hexanucleotide at the 5' 
splice, (ii) an invariant heptanucleotide, the UACUAAC box, surrounding 
the branch point A (underlined), and (iii) a generally conserved 
enrichment for pyrimidine residues adjacent to an invariant AG 
dinucleotide at the 3' splice site. 
Two other characteristics of nuclear pre-mRNA splicing in vitro that 
distinguish it from autocatalytic splicing are the dependence on added 
cell-free extracts and the requirement for adenosine triphosphate (ATP). 
Once in vitro systems had been established for mammalian and yeast 
pre-mRNA splicing, it was found that a group of trans-acting factors, 
predominately made up of small nuclear ribonucleoprotein particles 
(snRNP's) containing U1, U2, U4, U5 and U6 RNA's was essential to the 
splicing process. Together with the discovery of autocatalytic introns, 
the demonstration that snRNAs were essential, trans-acting components of 
the spliceosome argued strongly that group II self-splicing and nuclear 
pre-mRNA splicing occurring by fundamentally equivalent mechanisms. 
According to this view, the snRNAs compensate for the low information 
content of nuclear introns and, by the formation of intermolecular 
RNA--RNA interactions, achieve the catalytic capability inherent in the 
intramolecular structure of autocatalytic introns. 
As illustrated in FIG. 6A, consensus sequences of the 5' splice site and at 
the branchpoint are recognized by base pairing with the U1 and U2 snRNP's, 
respectively. The original proposal that the U1 RNA interacted with the 5' 
splice site was based solely on the observed nine-base-pair 
complementarity between the two mammalian sequences (Rogers et al. (1980) 
Nature 283:220). This model has since been extensively verified 
experimentally (reviewed in Steitz et al., in Structure and Function of 
Major and Minor snRNP Particles, M. L. Bimstiel, Ed. (Springer-Verlag, New 
York, 1988)). Demonstration of the Watson-Crick interactions between these 
RNAs was provided by the construction of compensatory base pair changes in 
mammalian cells (Zhuang et al. (1986) Cell 46:827). Subsequently, 
suppressor mutations were used to prove the interaction between U1 and 5' 
splice site in yeast (Seraphin et al. (1988) EMBO J. 7:2533). 
The base pairing interaction between U2 and sequences surrounding the 
branchpoint was first tested in yeast (Parker et al. (1987) Cell 49:229), 
where the strict conservation of the branchpoint sequence readily revealed 
the potential for complementarity. The branchpoint nucleotide, which 
carries out nucleophilic attack on the 5' splice site, is thought to be 
unpaired (FIG. 6A), and is analogous to the residue that bulges out of an 
intramolecular helix in domain 6 of group II introns. The base pairing 
interaction between U2 and the intron has also been demonstrated 
genetically in mammalian systems (Zhaung et al. (1989) Genes Dev. 3:1545). 
In fact, although mammalian branchpoint sequences are notable for their 
deviation from a strict consensus, it has been demonstrated that a 
sequence identical to the invariant core of the yeast consensus, CUAAC is 
the most preferred (Reed et al. (1989) PNAS 86:2752). 
Genetic evidence in yeast suggests that the intron base pairing region at 
the 5' end of U1 RNA per se is not sufficient to specify the site of 5' 
cleavage. Mutation of the invariant G at position 5 of the 5' splice site 
not only depresses cleavage efficiency at the normal GU site but activates 
cleavage nearby; the precise location of the aberrant site varies 
depending on the surrounding context (Jacquier et al. (1985) Cell 43:423; 
Parker et al. (1985) Cell 41:107; and Fouser et al. (1986) Cell 45:81). 
Introduction of a U1 RNA, the sequence of which has been changed to 
restore base pairing capability at position 5, does not depress the 
abnormal cleavage event; it enhances the cleavage at both wild-type and 
aberrant sites. These results indicate that the complementarity between U1 
and the intron is important for recognition of the splice-site region but 
does not determine the specific site of bond cleavage (Seraphin et al. 
(1988) Genes Dev. 2:125; and Seraphin et al. (1990) Cell 63:619). 
With regard to snRNPs, genetic experiments in yeast have revealed that the 
U5 snRNP is an excellent candidate for a trans-acting factor that 
functions in collaboration with U1 to bring the splice sites together in 
the spliceosome. U5 is involved in the fidelity of the first and the 
second cleavage-ligation reactions. For example, a number of U5 mutants 
exhibit a distinct spectrum of 5' splice-site usage; point mutations with 
the invariant nine-nucleotide loop sequence (GCCUUUUAC) in U5 RNA allows 
use of novel 5' splice sites when the normal 5' splice site was mutated. 
For instance, splicing of detective introns was restored when positions 5 
or 6 of the invariant U5 loop were mutated so that they were complementary 
to the nucleotides at positions 2 and 3 upstream of the novel 5' splice 
site when the normal 5' splice site was mutated. For instance, splicing of 
defective introns was restored when positions 5 or 6 of the invariant U5 
loop were mutated so that they were complementary to the nucleotides at 
positions 2 and 3 upstream of the novel 5' splice site. Likewise, 
mutational analysis has demonstrated the role of the U5 loop sequence in 
3' splice site activation. For example, transcripts which are defective in 
splicing due to nucleotide changes in either one of the first two 
nucleotides of the 3' exon were subsequently rendered functional by 
mutations in positions 3 or 4 of the U5 loop sequence which permitted 
pairing with the mutant 3' exon. (See Newman et al. (1992) Cell 68:1; and 
Newman et al. (1991) Cell 65:115). It is suggested that first U1 base 
pairs with intron nucleotides at the 5' splice site during assembly of an 
early complex (also including U2). This complex is joined by a tri-snRNP 
complex comprising U4, U5 and U6 to form a Holliday-like structure which 
serves to juxtaposition the 5' and 3' splice sites, wherein U1 base pairs 
with intronic sequences at both splice site. (Steitz et al. (1992) Science 
257:888-889). 
While each of the U1, U2 and U5 snRNPs appear to be able to recognize 
consensus signals within the intron, no specific binding sites for the 
U4-U6 snRNP has been identified. U4 and U6 are well conserved in length 
between yeast and mammals and are found base paired to one another in a 
simple snRNP (Siliciano et al. (1987) Cell 50:585). The interaction 
between U4 and U6 is markedly destablized specifically at a late stage in 
spliceosome assembly, before the first nucleolytic step of the reaction 
(Pikienly et al. (1986) Nature 324:341; and Cheng et al. (1987) Genes Dev. 
1:1014). This temporal correlation, together with an unusual size and 
sequence conservation of U6, has lead to the understanding that the 
unwinding of U4 and U6 activates U6 for participation in catalysis. In 
this view, U4 would function as an antisense negative regulator, 
sequestering U6 in an inert conformation until it is appropriate to act 
(Guthrie et al. (1988) Annu Rev. Genet. 22:387). Recent mutational studies 
demonstrate a functional role for U6 residues in the U4-U6 interaction 
domain in addition to base pairing (Vanken et al. (1990) EMBO J 9:3397; 
and Madhani et al. (1990) Genes Dev. 4:2264). 
Mutational analysis of the splicesomal RNAs has revealed a tolerance of 
substitutions or, in some cases, deletion, even of phylogentically 
conserved residues (Shuster et al. (1988) Cell 55:41; Pan et al. (1989) 
Genes Dev. 3:1887; Liao et al. (1990) Genes Dev. 4:1766; and Jones et al. 
(1990) EMBO J 9:2555). For example, extensive mutagenesis of yeast U6 has 
been carried out, including assaying the function of a mutated RNA with an 
in vitro reconstitution system (Fabrizo et al. (1990) Science 250:404), 
and transforming a mutagenized U6 gene into yeast and identifying mutants 
by their in vivo phenotype (Madhani et al. (1990) Genes Dev. 4:2264). 
Whereas most mutations in U6 have little or no functional consequence 
(even when conserved residues were altered), two regions that are 
particularly sensitive to nucleotide changes were identified: a short 
sequence in stem I (CAGC) that is interrupted by the S. prombe intron, and 
a second, six-nucleotide region (ACAGAG) upstream of stem I. 
As described above for both group I and group II introns, exonic sequences 
derived from separate RNA transcripts can be joined in a trans-splicing 
process utilizing nuclear pre-mRNA intron fragments (Konarska et al. 
(1985) Cell 42:165-171; and Solnick (1985) Cell 42:157-164). In the 
trans-splicing reactions, an RNA molecule, comprising an exon and a 3' 
flanking intron sequences which includes a 5' splice site, is mixed with 
an RNA molecule comprising an exon and 5' flanking intronic sequences, 
including a 3' splice site, and a branch acceptor site. As illustrated in 
FIGS. 6B and 6C, upon incubation of the two types of transcripts (e.g. in 
a cell-free splicing system), the exonic sequences can be accurately 
ligated. In a preferred embodiment the two transcripts contain 
complementary sequences which allow basepairing of the discontinuous 
intron fragments. Such a construct, as FIG. 6B depicts, can result in a 
greater splicing efficiency relative to the scheme shown in FIG. 6C in 
which no complementary sequences are provided to potentiate 
complementation of the discontinuous intron fragments. 
The exon ligation reaction mediated by nuclear pre-mRNA intronic sequences 
can be carried out in a cell-free splicing system. For example, 
combinatorial exon constructs can be mixed in a buffer comprising 25 mM 
creatine phosphate, 1 mM ATP, 10 mM MgCl.sub.2, and a nuclear extract 
containing appropriate factors to facilitate ligation of the exons 
(Konarska et al. (1985) Nature 313:552-557; Krainer et al. (1984) Cell 
36:993-1005; and Dignam et al. (1983) Nuc. Acid Res 11:1475-1489). The 
nuclear extract can be substituted with partially purified spliceosomes 
capable of carrying out the two transesterification reactions in the 
presence of complementing extracts. Such spliceosomal complexes have been 
obtained by gradiant sedimentation (Grabowski et al. (1985) Cell 
42:345-353; and Lin et al. (1987) Genes Dev. 1:7-18), gel filtration 
chromatography (Abmayr et al. (1988) PNAS 85:7216-7220; and Reed et al. 
(1988) Cell 53:949-961), and polyvinyl alcohol precipitation (Parent et 
al. (1989) J. Mol. Biol. 209:379-392). In one embodiment, the spliceosomes 
are activated for removal of nuclear pre-mRNA introns by the addition of 
two purified yeast "pre-mRNA processing" proteins, PRP2 and PRP16 (Kim et 
al. (1993) PNAS 90:888-892; Yean et al. (1991) Mol. Cell Biol. 
11:5571-5577; and Schwer et al. (1991) Nature 349:494-499). 
II. Trans-splicing Combination of Exons 
A. Exon Shuffling 
In one embodiment of the present combinatorial method, the intronic 
sequences which flank each of the exon modules are chosen such that gene 
assembly occurs in vitro through ligation of the exons, mediated by a 
trans-splicing mechanism. Conceptually, processing of the exons resembles 
that of a fragmented cis-splicing reaction, though a distinguishing 
feature of trans-splicing versus cis-splicing is that substrates of the 
reaction are unlinked. As described above, breaks in the intron sequence 
can be introduced without abrogating splicing, indicating that coordinated 
interactions between different portions of a functional intron need not 
depend on a covalent linkage between those portions to reconstitute a 
functionally-active splicing structure. Rather, the joining of 
independently transcribed coding sequences results from interactions 
between fragmented intronic RNA pieces, with each of the separate 
precursors contributing to a functional trans-splicing core structure. 
The present trans-splicing system provides an active set of reagents for 
trans-splicing wherein the flanking intronic sequences can interact to 
form a reactive complex which promotes the transesterification reactions 
necessary to cause the ligation of discontinuous exons. In one embodiment, 
the exons are flanked by portions of one of a group I or group II intron, 
such that the interaction of the flanking intronic sequences is sufficient 
to produce an autocatalytic core capable of driving ligation of the exons 
in the absence of any other factors. While the accuracy and/or efficiency 
of these autocatalytic reactions can be improved, in some instances, by 
the addition of trans-acting protein or RNA factors, such additions are 
not necessary. 
In another embodiment, the exon modules are flanked by intronic sequences 
which are unable, in and of themselves, to form functional splicing 
complexes without involvement of at least one trans-acting factor. For 
example, the additional trans-acting factor may compensate for structural 
defects of a complex formed solely by the flanking introns. As described 
above, domain 5 of the group II intron class can be removed from the 
flanking intronic sequences, and added instead as a trans-acting RNA 
element. Similarly, when nuclear pre-mRNA intron fragments are utilized to 
generate the flanking sequences, the ligation of the exons requires the 
addition of snRNPs to form a productive splicing complex. 
In an illustrative embodiment, the present combinatorial approach can make 
use of group II intronic sequences to mediate trans-splicing of exons. For 
example, as depicted in FIG. 7, internal exons can be generated which 
include domains 5 and 6 at their 5' end, and domains 1-3 at their 3' end. 
The nomenclature of such a construct is (IVS5,6) Exon(IVS 1-3), 
representing the intron fragments and their orientation with respect to 
the exon. Terminal exons are likewise constructed to be able to 
participate in trans-splicing, but at only one end of the exon. A 5' 
terminal exon, in the illustrated group II system, is one which is flanked 
by domains 1-3 at its 3' end [Exons(IVS1-3)] and is therefore limited to 
addition of further exonic sequences only at that end; and a 3' terminal 
exon is flanked by intron sequences (domains 5 and 6) at only its 5' end 
[(IVS5,6)Exon]. Under conditions which favor trans-splicing, the flanking 
intron sequences at the 5' end of one exon and the 3' end of another exon 
will associate to form a functionally active complex by intermolecular 
complementation and ligate the two exons together. Such trans-splicing 
reactions can link the 5' terminal exon directly to the 3' terminal exon, 
or alternatively can insert one or more internal exons between the two 
terminal exons. 
In some cases, trans-splicing reactions by intron-flanked internal exons 
may be inhibited by a competing inverse-splicing reaction that such 
internal exons can undergo. As described below and depicted in FIG. 8A, 
intron-flanked internal exons can participate in intramolecular 
"inverse-splicing" reactions in which the 3' end of the exon is spliced to 
its own 5' end, so that the exon is circularized (and the intronic 
sequences are released as a Y-branched ribozyme). Because inverse-splicing 
is an intramolecular reaction, it can sometimes compete effectively with 
any trans-splicing reactions, so that few trans-splicing products are 
produced. In such cases, the inverse-splicing reaction can be inhibited by 
provision of an antisense nucleic acid that binds to one or the other of 
the flanking intronic elements. Of course, the antisense nucleic acid will 
also block one of the trans-splicing reactions that would otherwise be 
available to the internal exon. Accordingly, use of antisense nucleic 
acids to control inverse-splicing also limits trans-splicing experiments 
to a series of sequential reactions. FIG. 9 depicts one embodiment of such 
a controlled, sequential trans-splicing reaction according to the present 
invention. 
In another embodiment of the present trans-splicing combinatorial method, 
the exons, as initially admixed, lack flanking intronic sequences at one 
or both ends, relying instead on a subsequent addition of flanking 
intronic fragments to the exons by a reverse-splicing reaction. Addition 
of the flanking intron sequences, which have been supplemented in the exon 
mixture, consequently activates an exon for trans-splicing. FIG. 10 
illustrates how the reverse-splicing reaction of group II introns can be 
used to add domains 1-3 to the 3' end of an exon as well as domains 5-6 to 
the 5' end of an exon. As shown in FIG. 10, the reversal reaction for 
branch formation can mediate addition of 3' flanking sequences to an exon. 
For example, exon modules having 5' intron fragments (e.g. domains 5-6) 
can be mixed together with little ligation occurring between exons. These 
exons are then mixed with a 2'-5' Y-branched intron resembling the 
lariat-IVS, except that the lariat is discontinuous between domains 3 and 
5. The reverse-splicing is initiated by binding of the IBS 1 of the 5' 
exon to the EBS 1 of the Y-branched intron, followed by nucleophilic 
attack by the 3'-OH of the exon on the 2'-5' phosphodiester bond of the 
branch site. This reaction, as depicted in FIG. 10, results in the 
reconstitution of the 5' splice-site with a flanking intron fragment 
comprising domains 1-3. 
While FIG. 10 depicts both a 5' exon and 3' exon, the reverse splicing 
reaction can be carried out without any 3' exon, the IBS sequence being at 
the extreme 3' end of the transcript to be activated. Alternatively, to 
facilitate addition of 5' flanking sequences, an exon can be constructed 
so as to further include a leader sequence at its 5' end. As shown in FIG. 
10, the leader (e.g. the 5' exon) contains an IBS which defines the splice 
junction between the leader and "mature" exon. The leader sequence can be 
relatively short, such as on the order of 2-3 amino acid residues (e.g. 
the length of the IBS). Through a reverse self-splicing reaction using a 
discontinuous 2'-5' branched intron, the intronic sequences can be 
integrated at the splice junction by reversal of the two transterfication 
steps in forward splicing. The resulting products includes the mature exon 
having a 5' flanking intron fragment comprising domains 5 and 4. 
Addition of intronic fragments by reverse-splicing and the subsequent 
activation of the exons presents a number of control advantages. For 
instance, the IBS:EBS interaction can be manipulated such that a 
variegated population of exons is heterologous with respect to intron 
binding sequences (e.g. one particular species of exon has a different IBS 
relative to other exons in the population). Thus, sequential addition of 
intronic RNA having discrete EBS sequences can reduce the construction of 
a gene to non-random or only semi-random assembly of the exons by 
sequentially activating only particular combinatorial units in the 
mixture. Another advantage derives from being able to store exons as part 
of a library without self-splicing occurring at any significant rate 
during storage. Until the exons are activated for trans-splicing by 
addition of the intronic sequences to one or both ends, the exons can be 
maintained together in an effectively inert state. 
When the interactions of the flanking introns are random, the order and 
composition of the internal exons of the combinatorial gene library 
generated is also random. For instance, where the variegated population of 
exons used to generate the combinatorial genes comprises N different 
internal exons, random trans-splicing of the internal exons can result in 
N.sup.y different genes having y internal exons. Where 5 different 
internal exons are used (N=5) but only constructs having one exon ligated 
between the terminal exons are considered (i.e. y=1) the present 
combinatorial approach can produce 5 different genes. However, where y=6, 
the combinatorial approach can give rise to 15,625 different genes having 
6 internal exons, and 19,530 different genes having from 1 to 6 internal 
exons (e.g. N.sup.1 +N.sup.2 . . . +N.sup.y-1 +N.sup.y. It will be 
appreciated that the frequency of occurrence of a particular exonic 
sequence in the combinatorial library may also be influenced by, for 
example, varying the concentration of that exon relative to other exons 
present, or altering the flanking intronic sequences of that exon to 
either diminish or enhance its trans-splicing ability relative to the 
other exons being admixed. 
However, the present trans-splicing method can be utilized for ordered gene 
assembly, and carried out in much the same fashion as automated 
oligonucleotide or polypeptide synthesis. FIG. 11 describes schematically 
the use of resin-bound combinatorial units in the ordered synthesis of a 
gene. In the illustrated example, mammalian pre-mRNA introns are used to 
flank the exon sequences, and splicing is catalyzed by addition by 
splicing extract isolated from mammalian cells. The steps outlined can be 
carried out manually, but are amenable to automation. The 5' terminal exon 
sequence (shown as exon 1 in FIG. 11) is directly followed by a 5' portion 
of an intron that begins with a 5' splice-site consensus sequence, but 
does not include the branch acceptor sequence. The flanking intron 
fragment further includes an added nucleotide sequence, labeled "A" in the 
diagram, at the 3' end of the downstream flanking intron fragment. The 5' 
end of this terminal combinatorial unit is covalently linked to a solid 
support. 
In the illustrated scheme, exon 2 is covalently joined to exon 1 by 
trans-splicing. The internal shuffling unit that contains exon 2 is 
flanked at both ends by intronic fragments. Downstream of exon 2 are 
intron sequences similar to those downstream of exon 1, with the exception 
that in place of sequence A the intronic fragment of exon 2 has an added 
sequence B that is unique, relative to sequence A. Exon 2 is also preceded 
by a sequence complementary to A (designated A'), followed by the nuclear 
pre-mRNA intron sequences that were not included downstream of exon 1, 
including the branch acceptor sequence and 3' splice-site consensus 
sequence AG. 
To accomplish the trans-splicing reaction, the shuffling units are allowed 
to anneal by hydrogen bonding between the complementary intronic sequences 
(e.g. A and A'). Then, trans-splicing is catalyzed by the addition of a 
splicing extract which contains the appropriate snRNPs and other essential 
splicing factors. The Y-branched intron that is generated, and any other 
by-products of the reaction, are washed away, and a ligated exon 1 and 2 
remain bound to the resin. A second internal shuffling unit is added. As 
shown in FIG. 11, the exon (exon 3) has flanking intronic fragments which 
include a sequence B' in the upstream fragment and a sequence A in the 
downstream fragment. The nucleotide sequence B' is unique relative to 
sequence A', and is complementary to sequence B. As above, the RNA is 
allowed to anneal through the B:B' sequences, splicing of the intervening 
sequences is catalyzed by the addition of extract, and reaction 
by-products other than the resin bound exons are washed away. While FIG. 
11 depicts a non-random assembly of a gene, it is understood that 
semi-random assembly can also be carried out, such as would occur, for 
example, when exon 3 is substituted with a variegated population of exons 
combinatorial units. 
This procedure can be continued with other exons, and may be terminated by 
ligation of a 3' terminal shuffling unit that contains an exon (exon 4 in 
FIG. 11) with upstream intron sequence (and either the A' or B' sequence, 
as appropriate), but lacking any downstream intron sequences. After the 3' 
terminal exon is added, the assembled gene can be cleaved from the solid 
support, reverse transcribed, and the cDNA amplified by PCR and cloned 
into a plasmid by standard methods. 
The domain shuffling experiments described to yield novel protein coding 
genes can also be used to create new ribozymes. FIG. 12 depicts one 
example of how group I intron sequences can be used to shuffle group II 
intron domains. In the illustrative embodiment, the group II intron 
consists of 6 domains and is flanked by exon (E5 and E3); in this 
instance, E5 is shown to include a T7 promoter. The six shuffling 
competent constructs diagramed in FIG. 12 can be made either by standard 
site directed mutagenesis and cloning or by the reversal of splicing. The 
5' terminal exon is followed by sequences from the T4 td intron, beginning 
with the first nucleotide of the intron and including the internal guide 
sequence, and continuing through the 5' half of the P6a stem (i.e. 
including half of L6). The last nucleotide of the exon is a U. The 
internal guide sequence of the intron is changed by site directed 
mutagenesis so that it is complementary to the last 6 nt of the exon. This 
will allow the P1 stem to form. The U at the end of the exon is based 
paired with a G in the internal guide sequence. The 3' terminal "exon", in 
this case, consists of group II intron domain 6 plus E3. The 3' terminal 
exon is preceded by the T4 id intron, beginning with the 3' half of P6a 
and continuing through to the end of the intron. The last nucleotide of 
the intron is followed by the first nucleotide of group II intron domain 
6. The internal exons each consist of a group II intron domain but, in 
contrast to the terminal exons, each internal exon is flanked by group I 
intron sequences on both sides. In each case, the internal guide sequence 
of the group I intron is changed so as to be complementary to the last 6 
nts of the exon and, in each case, the last nucleotide of the exon is a U. 
Constructing a library of group II domains flanked by group I intronic 
sequence allows new group II ribozymes to be assembled from these units by 
random exon shuffling using conditions that allow for efficient 
trans-splicing of "exons" flanked by these group I intron sequences. For 
instance, if only one E5:d1 and d6:E3 are used, but a variegated 
population of d2-d5, the assembled genes will all have the same 5' and 3' 
terminal exons, but will have different arrangements and numbers of 
internal exons. An E3 specific primer plus reverse transcriptase can be 
used to make cDNA of the library of recombined transcript. T7 and E3 
specific primers can be used to amplify the assembled genes by PCR, and 
RNA transcripts of the assembled gene can be generated using T7 
polymerase. The RNA can be incubated under self splicing conditions 
appropriate for group II splicing. Molecules that are capable of self 
splicing will yield intron lariats that migrate anomalously slow on 
denaturing polyacrylamide gels. The lariats can be gel purified and 
represent active ribozyymes. The isolated lariats can be specifically 
debranched with a HeLa debranching activity. Reverse transcription and PCR 
can be used to make and amplify cDNA copies of the ribozymes. The primers 
used for the PCR amplification will include exon sequences so that each 
amplified intron will be flanked by a 5' and a 3' exon. The last 6 nt of 
the 5' exon will be complementary to EBS 1. The amplified DNA can be 
cloned into a plasmid vector and individual interesting variants isolated 
and studied in detail. 
EXAMPLE 1 
Use of Engineered Ribozymes to Catalyze Chimeric Gene Assembly 
Engineered group II introns were utilized to catalyze linkage of human 
exons by trans-splicing (see Mikheeva et al. (1996), PNAS 93:7486-7490, 
incorporated herein by reference). Specifically, group II intron 
derivatives were designed that insert into selected sites in the human 
tissue plasmnogen activator (t-PA) mRNA. The insertion reaction linked 
(t-PA) sequences to the group II intron sequences so that trans-splicing 
reactions catalyzed by the intron sequences shuffled the (t-PA) sequences. 
The three inverse-splicing precursors used in this study were as follows: 
(IVS5, 6)E3,E5(IVS1-3), PY4, and PY7. These precursors were made by 
transcription in vitro, using T7 RNA polymerase, of plasmids pINV1, pY4, 
and pY7, respectively. The three inverse-splicing precursors were 
identical, except for the indicated base substitutions in the EBS and IBS 
sequences of PY4 and PY7 (see FIG. 13). The base substitutions were made 
by site-directed mutagenesis of pINV1 (Jarrell (1993) PNAS 90, 8624-8627) 
DNA using the method of Kunkel, as described (Jarrell et al. (1988) Mol. 
Cell. Biol. 8:2361-2366). Prior to inverse-splicing, full-length splicing 
precursors were purified from an acrylamide gel. Inverse-splicing 
reactions were conducted in buffer containing 40 mM Tris hydrochloride (pH 
7.6), 100 mM MgCl.sub.2, and 1.2 M (NH.sub.4).sub.2 SO.sub.4. 
The substrate RNAs that were attacked by Y-branched ribozymes were made by 
transcription in vitro of plasmids pFn and pProt. Both pFn and pProt are 
subdlones of plasmid pKS.sup.+ t-PA. The pKS.sup.+ t-PA plasmid was 
derived from pETPFR (Pennica et al. (1983) Nature 301:214-221). The cDNA 
region of pETPFR was amplified using PCR primers t- 
(5'-ACGATGCATGCTGGAGAGAAAACCTCTGCG [SEQ ID NO: 3]) and t- 
(5'ACGATGCATTCTGTAGAGAAGCACTGCGCC [SEQ ID NO: 4]). Both primers included 
recognition sites for NsiI. The amplified DNA was cut with NsiI and 
ligated into the KS.sup.+ vector that had been cut with PstI. A 
recombinant plasmid was identified with the insert in the sense 
orientation, relative to the start of T7 transcription. The pFn plasmid 
was generated by deleting the t-PA sequences between the Styl site of t-PA 
and the EcoRI site of the vector. Specifically, the pKS.sup.+ t-PA plasmid 
was cut with StyI and EcoRI, treated with Klenow fragment to blunt the 
EcoRI end and with ligase to join the Styl end to the blunted EcoRI end of 
the vector. The pProt plasmid was generated by subdloning the ScaI-HindIII 
fragment into the pBS.sup.- vector. Specifically, the pKS.sup.+ t-PA 
plasmid was cut with Scal and Hindil. The 922-bp fragment was isolated and 
ligated into pBS.sup.- that had been cut with SmaI and HindIII. Ribozyme 
integration reactions were done in the same buffer that was used for the 
inverse-splicing reactions (see above). 
The reverse transcription (RT)-PCR experiments were done as described 
(Jarrell (1993) PNAS 90:8624-8627). A total of five primers were used in 
RT-PCR experiments. The nucleotide sequences of primers t- and t- 
are given above. Other primers include I654 
(5'-ACGAAGCTTCCTATAGTATAAGTTAGCAGAT [SEQ ID NO: 5]); I5,6 
(5'-GCGAATTCGAGCTCGTGAGCCGTAT [SEQ ID NO: 6]); and t-PA(49) 
(5'-ACGGGTACCGAAAGGGAAGGAGCAAG-CCGTG [SEQ ID NO:7.sub.---- ]). 
The trans-splicing reaction was done in buffer that was identical to that 
used for ribozyme integration and inverse-splicing, except the 1.2 M 
(NH.sub.4).sub.2 SO.sub.4 was replaced by 1.2 M NH.sub.4 Cl. NH.sub.4 Cl 
was included in the trans-splicing buffer because pilot experiments showed 
that the rate of trans-splicing was about 10-fold higher in NH.sub.4 Cl 
was included in the trans-splicing was about 10-fold higher in NH.sub.4 Cl 
buffer than in (NH.sub.4).sub.2 SO.sub.4 buffer (data not shown). However, 
the added NH.sub.4 Cl may have reduced the fidelity of the trans-splicing 
reaction: NH.sub.4 Cl is known to stimulate certain side reactions, such 
as ribozyme catalyzed site specific RNA hydrolysis (Peebles et al. (1987) 
CSH Symp. Quant. Biol. 52:223-232.; Jarrell et al. (1988) J. Biol. Chem. 
263:3432-3439; Koch et al. (1992) Mol. Cell. Biol. 12:1950-1958; Wallasch 
et al. (1991) Nuc. 19:3307-3314; and Jacquier et al. (1991) J. Mol. Biol. 
219:415-428). Trans-splicing reactions conducted in the presence of 
(NH.sub.4).sub.2 SO.sub.4 may occur with higher fidelity. 
The first step in our study involved identification of an efficient means 
of producing Y-branched ribozymes of altered specificity. Y-branched 
products can be produced in forward trans-splicing and inverse-splicing 
reactions. We chose to produce Y-branched ribozymes by inverse-splicing 
because that reaction, being unimolecular, is more efficient than 
trans-splicing. 
Having decided on our strategy, we initiated our experiments to produce 
Y-branched ribozymes that were engineered to target t-PA sequences. Using 
site-directed mutagenesis on DNA plasmids that contained the aI5.gamma. 
group II intron and its natural exons in inverse-splicing orientation, we 
produced two plasmids, pY4 and pY7, from which inverse-splicing substrates 
containing altered EBS1, IBS1, EBS2, and IBS2 sequences could be 
transcribed (FIG. 13). The EMS1 and EBS2 sequences of pY4 and pY7 were 
designed to hybridize to sequences within the t-PA gene, and the IBS1 and 
IBS2 sequences were made complementary to the mutated EBS1 and EBS2 
sequences. Transcripts were produced from pY4 and pY7, and from the 
original inverse-splicing construct (pINV1) and were incubated under 
splicing conditions. 
FIG. 13B shows that all three transcripts spliced to produce an excised 
exon circle [E3,E5(C)] and a Y-branched intron [IVS(Y)]. Although both 
mutated transcripts spliced more slowly than did the control, the 
reactions readily produced useful quantities of Y-branched ribozymes with 
altered EBS1 and EBS2 sequences. For the purpose of exon shuffling, 
efficiency is unimportant; all that is required is that the 
inverse-splicing reactions produce enough Y-branched product for the 
subsequent integration reactions. 
Having produced Y-branched ribozymes with altered EBS1 and EBS2 sequences, 
we tested whether these ribozymes (Y4 and Y7) would insert specifically 
into RNA transcripts corresponding to portions of the t-PA transcript. The 
full-length t-PA transcript encodes five domains (FIG. 14A). We tested the 
ability of Y4 to integrate into a transcript, Fn txt, comprising the 
sequence encoding the fibronectin (Fn) domain followed by part of the 
sequence encoding the EGF domain. Likewise, we tested the ability of the 
Y7 ribozyme to integrate into the Prot txt transcript, which encodes part 
of a kringle domain (K2) and the protease domain (Prot). 
Y4 was designed to insert precisely between the Fn and EGF sequences. Thus, 
for the purposes of this experiment, the last 16 nt of Fn, 
5'-CUCAGUgccUGUCAAA (SEQ ID NO: 8), comprise its IBS2 and IBS1 (uppercase 
letters) sites, respectively. The EBS2 and EBS1 sites of Y4 were made 
complementary to these sequences (see FIG. 13A). Likewise, the Y7 ribozyme 
was engineered to insert precisely between K2 and Prot. Thus, the last 16 
nt of K2, 5'-UGUGCCcucCUGCUCC (SEQ ID NO: 9), comprise its IBS2 and IBS1 
sequences. 
Both the Y4 ribozyme and the Y7 ribozyme were designed to insert at sites 
that, at the genomic level, are occupied by natural introns (Ny et al. 
(1984) PNAS 81:5355-5359). However, unlike the natural introns, which 
disrupt codons, the Y-branched ribozymes were designed to insert between 
codons, so that a subsequent trans-splicing reaction would create an 
in-frame fusion (see below). 
Y4 was incubated with Fn txt under splicing conditions. Precise integration 
of Y4 into Fn txt was expected to yield two products, one corresponding to 
Fn joined to intron domains 1-3[Fn(1-3)], and one corresponding to intron 
domains 5 and 6 joined to the remainder of the Fn transcript [(5,6)E]. The 
Fn(1 -3) product was expected to be 1034 nt; the 803-nt Y4 RNA and the 
RNAs in lane 5 were used in a size standards. 
As shown in FIG. 14B, lane 8, we observed two products (products 1 and 2) 
that migrated more slowly than Y4. Product 1 was the expected size of 
Fn(1-3). Both product 1 and product 2 were isolated for further analysis 
(see below). No attempt was made to identify or characterize the (5,6)E 
product, which, due to its size, was not retained within the gel. 
In a parallel experiment, Y7 was incubated with the Prot txt RNA under 
splicing conditions. As was the case with Y4 and Fn txt, precise 
integration by the Y7 ribozyme into the Prot txt target was expected to 
yield two products: K(1-3) and (5,6)Prot. Our aim was to clone the 
(5,6)Prot product. Unfortunately, as shown in FIG. 14C, lane 8, we were 
not able to separate the 1000-nt (5,6)Prot product from the 955-nt Prot 
txt. Similarly, the 758-nt K(1-3) product was not separable from 
hydrolysis products of Y7. We therefore isolated three regions of the gel 
in order to detect any (5,6)Prot product that had been produced. We 
isolated the large minor triplet of bands, product 3, and also products 4 
and 5 (from the Prot txt region of the gel). We did not expect product 3 
to be (5,6)Prot because it was also observed when Y7 was incubated alone, 
and similar large products were observed when Y4 was incubated alone (see 
FIG. 14B, lane 7). 
We analyzed purified products 1-5 by RT-PCR by using primers that amplify 
only recombinant RNAs. FIG. 15A shows the Fn(1-3) and (5,6)Prot 
recombinant RNAs, and the RT-PCR primers. FIG. 15B shows amplified DNAs 
from products 1-5. As can be seen, RT-PCR on product 1 produced an 
amplified DNA the expected size (1016 bp) of double-stranded Fn(1-3) (FIG. 
15B, lane 1). RT-PCR analysis of product 2 produced three amplified DNAs 
that we did not analyze further, due to their low abundance (lane 2). 
Product 3 did not give rise to an amplification product (lane 3). Products 
4 and 5 both yielded amplified DNAs the expected size (984 bp) of 
double-stranded (5,6)Prot (lanes 4 and 5). 
Both major amplifications products were cloned and analyzed by restriction 
mapping and sequencing. All 18 Fn(1-3) clones analyzed had the expected 
restriction map. Five clones were sequenced and were found to have 
accurately joined Fn and 1-3 sequences (FIG. 15C). Likewise, restriction 
mapping and sequencing of two (5,6)Prot recombinants showed that 5,6 and 
Prot sequencing of two (5,6)Prot recombinants showed that the 5,6 and Prot 
sequences had been accurately joined (FIG. 15D). 
Having demonstrated that engineered Y-branched ribozyrnes insert 
specifically, our objective was to test the hypothesis that chimeric genes 
can be assembled by trans-splicing. The Fn(1-3) and (5,6)Prot RNAs were 
generated by in vitro transcription, mixed, and incubated under splicing 
conditions (FIG. 16). Two trans-splicing products (products 1 and 2) were 
characterized by RT-PCR (data not shown), using recombinant-specific 
primers (FIG. 16A). Product 1 did not yield amplified DNA; product 2 gave 
rise to amplified DNA the expected size (1204 bp) of Fn-Prot DNA. The 
amplified DNA was cloned, restriction mapped, and sequenced. Of 24 
analyzed recombinants, 21 had the expected sequence; the remaining three 
had a deletion of a single adenosine residue (FIG. 16C). Although the 
mechanism of adenosine deletion is unknown, at least three mechanisms can 
be proposed based on known aspects of the group II intron splicing 
mechanism (see above). 
EXAMPLE 2 
Anti-sense Nucleic Acid Inhibits Inverse-splicing by a Flanked Internal 
Exon 
Two different constructs were produced that encode flanked internal exons 
capable of both inverse- and trans-splicing. These constructs were 
prepared by isolating the Kringle-1 (K1) domain from the human t-PA gene 
and inserting this domain into appropriate inverse-splicing vectors. 
Specifically, the cDNA clone of the human tissue plasminogen activator 
(t-PA) gene (pETPFR) was obtained from the ATCC collection (ATCC 40403; 
and U.S. Pat. No. 4,766,075). The entire cDNA clone was amplified by PCR 
using primers 5'-ACGATGCATGCTGGAGAGAAAACCTCTGCG (SEQ ID NO:3) and 
5'-ACGATGCATTCTGTAGAGAAGCACTGCGCC (SEQ ID NO:4). TPA sequences from 70 
base pairs (bp) upstream of the translation initiation site (AUG) to 88 bp 
downstream of the translation termination site (TGA) were amplified (SEQ 
ID NO:10). In addition, the primers added Nsi I sites to both ends of the 
amplified DNA. The amplified DNA was cut with Nsi I and ligated into the 
KS.sup.+ vector that had been cut with Pst I. A clone TPA-KS.sup.+, was 
isolated with the insert oriented such that in vitro transcription with 
T.sub.7 RNA polymerase yields an RNA that is the same polarity as the tPA 
mRNA. 
Separately, two unique restriction sites were added to the pINV1 plasmid 
(SEQ ID NO:11) by site directed mutagenesis, to facilitate insertion of 
portions of the tPA clone. A Kpn I site (GGTACC) was inserted at precisely 
the boundary between the end of the intron and the beginning of E3. An Xho 
I site was added to E5 by changing the sequence GTGGGA to a Xho I site 
(CTCGAG). Thus, the last seven bp of the exon were unchanged, but the six 
preceding base pairs were changed to create a Xho I site. The resulting 
plasmid is termed here INV-KX. 
The region of the TPA cDNA clone that encodes the kringle-1 (Kp1) domain 
was amplified by PCR. The primers added a Kpn I site at the upstream end 
of the domain and a Xho I site to the downstream end. The amplified DNA 
was cut with Kpn I and Xho I and ligated into INV-KX such that the K1 
sequences replaced the E3,E5 sequences. 
Oligonucleotide splints were used in a site-directed mutagenesis experiment 
to change the sequences at the boundaries of the INV-KX derived introns 
and the K1 exon. The sequences were changed such that the intron sequences 
of domain 6 are directly followed by kringle domain sequences ACC AGG GCC 
and kringle sequences TCT GAG GGA precede the intron sequences of domain 
1. In addition, the EBS 1 sequence in domain 1 was changed to TCCCTCA 
(this sequence is homologous to the last 7 nt of K1 (TGAGGGA)). Thus, the 
resulting transcript, contains complementary IBS1 and EBS1 sequences. The 
plasmid encoding this transcript is known as pY8. A related plasmid, which 
is identical to pY8 except that the KpnI site at the intron/exon boundary 
was not destroyed, was also isolated; This plasmid is known as pKK1. The 
transcript encoded by pKK1 is known as (IVS5,6)KKI(IVS1-3). 
An second site-directed mutagenesis reaction was performed on pY8 to change 
the intron EBS2 site to be complementary to K1 IBS2 region. The plasmid 
produced in this experiment is known as pY9; the encoded transcript is 
(IVS5,6)Kl(IVSS1-3). 
The (IVS 5,6)K1(IVS 1-3) and (IVS 5,6)KK1(IVS 1-3) transcripts were 
produced by transcription of pY9 and pKK1; An inhibitory anti-sense RNA 
that hybridizes to the IVS 1-3 region and also to the 3' end of the K1 
exon was produced by T3 transcription of pY9 digested with Apal I. Each 
transcript was then incubated under splicing conditions with and without 
the anti-sense RNA, and also with and without an Fn(1-3) transcript, 
prepared as described in Example 1, to which the K1 transcripts could 
splice. As is shown in FIG. 17, the antisense RNA completely blocked 
inverse splicing by either transcript, but allowed each transcript to 
splice to the Fn(1-3) RNA. 
EXAMPLE 3 
In Vivo Exon Shuffling 
As depicted in FIG. 18, exon shuffling according to the present invention 
can be made to occur in vivo. All that is required is expression in a cell 
of an inverse-splicing precursor that will produce a Y-branched ribozyme 
of broad specificity. Preferably, the inverse-splicing precursor is 
introduced into the cell in association with an inducible promoter, so 
that its expression may be initiated and terminated under controlled 
circumstances. 
Once the Y-branched ribozyme is produced, it will integrate into other RNAs 
being expressed in the cell and will shuffle the "activated exons" created 
by its own integration. It will be appreciated that a population of 
ribozymes having different specificities may be employed rather than a 
single ribozyme of broad specificity. 
A particularly preferred embodiment of this in vivo exon shuffling reaction 
is described in Example 11, where the shuffling is performed in a cell 
bearing a specialized integration construct, so that shuffling products 
may be integrated into the genome, expressed, and analyzed. 
B. Exon Trapping 
FIG. 19 illustrates an "exon-trap" assay for identifying exons (in the 
traditional use of the term) from genomic DNA, utilizing trans-splicing 
mediated by discontinuous nuclear pre-mRNA intron fragments. One advantage 
of this method is that the DNA does not have to be cloned prior to using 
the method. In contrast to prior techniques, the starting material of the 
exon-trap assay could ultimately be total human genomic DNA. In addition, 
the present method described herein is an in vitro method, and can be 
easily automated. 
In the first step, purified RNA polymerase II is used to transcribe the 
target DNA. In the absence of the basal transcription factors, Pol II will 
randomly transcribe DNA (Lewis et al. (1982) Enzymes 15:109-153). FIG. 19 
shows that some of these transcripts will contain individual exons flanked 
by intron sequences. Since human exons are small, typically less than 300 
nt (Hawkins et al. (1988) Nucleic Acids Res. 16:9893-9908) and introns are 
large (up to 200,000 nt, Maniatis (1991) Science 251:33-34) most 
transcripts will contain either zero or one exon. In the illustrative 
embodiment, a spliced leader RNA of, for instance, trypanosome or nematode 
(Agabian (1990) Cell 61:1157-1160), is covalently linked to a solid 
support by its 5' end. The RNA generated by random transcription of the 
genomic DNA is mixed with the immobilized spliced leader and splicing is 
catalyzed using splicing extract. The resin is then washed to remove 
unwanted reaction products, such as unreacted RNA and the splicing 
extract. 
Furthermore, in a subsequent step, an in vitro polyadenlyation reaction 
(for example, Ryner et al. (1989) Mol. Cell. Biol. 9:4229-4238) can be 
carried out which adds oligo-A (up to a length of 300 nt) to the 3' end of 
the RNA. FIG. 19 shows that an RNA transcript, generated by in vitro 
transcription of a plasmid having an oligo T stretch, followed by the 3' 
portion of an intron (including the branch acceptor site and the AG 
dinucleotide), followed by an exon, can be annealed to the immobilized 
polyadenylated RNA by hydrogen bonding between the poly-A and poly-T 
sequences. In vitro trans-splicing, catalyzed by splicing extract, will 
join the known 3' exon to the "trapped" exon. The RNA can then be stripped 
from the column, copied to DNA by reverse transcriptase and amplified by 
PCR using primers to the 5' leader and known 3' exon. The amplified DNA 
that contains a trapped exon primers to the 5' leader and known 3' exon. 
The amplified DNA that contains a trapped exon will be larger than the 
side product that results from splicing of the spliced leader exon to the 
known 3' exon. Thus, the amplified DNA that contains trapped exons can be 
selected by size. 
Moreover, a "capping" reaction can be done to eliminate products that do 
not contain a trapped exon. After the step of mixing genomically derived 
RNA with the immobilized exon, a "capping RNA", with a 3' splice site and 
a 3' exon, can be added and splicing catalyzed by the addition of splicing 
extract. The 3' exon of the capping RNA is different from the 3' exon of 
the RNA shown with the oligo-T stretch. The capping RNA is one which will 
trans-splice very efficiently to any spliced leader RNA which has not 
already participated in a splicing reaction; but, will splice less 
efficiently to immobilized RNAs that have a trapped exon ligated to them 
as the capping RNA lacks a poly-T sequence to anneal to the trapped exon. 
Therefore, after the capping reaction, the step shown for splicing of the 
oligo-T containing construct will result, primarily, in the generation of 
the desired (leader/trapped exon/known exon) product and not in the 
generation of the unwanted (5' leader/3' known exon) product. 
C. DNA Recombination 
It should be understood that, although much of the discussion herein 
focusses on production of recombinant RNA molecules, the principles and 
techniques taught by the present invention are also applicable to 
manipulation of DNA sequences. Methods are available for linking 
splicing-competent intronic RNA sequences directly to single-stranded or 
double stranded DNA so that, in the language of the present invention, 
activated DNA exons are produced. These activated DNA exons can then be 
linked to one another, much as has been described herein for RNA exons, 
through trans-reactions mediated by the intronic sequences. 
FIG. 20 depicts various DNA "trans-splicing" reactions of the present 
invention. For convenience, the term "trans-splicing" is used herein to 
encompass any intron-mediated trans-reaction--i.e. any reaction between 
two distinct intron-linked exons--that leads to precise ligation of those 
exons, even though the term "splicing" has not traditionally been applied 
to reactions involving DNA (rather than RNA) exons. As shown in FIG. 20, 
intronic RNA sequences are linked to pieces of DNA in manner that allows 
the RNA elements to direct precise ligation of the DNA sequences to one 
another, with concomitant removal of the RNA elements. Specifically, at 
least RNA elements 2 and 3 (or 2' and 3 or 4 and 1') are selected to be 
capable of interacting with one another to form a functional intron that 
directs the trans-splicing reaction of the DNA exons. Preferably, the 
intron elements have been engineered as described herein to target the 
specific relevant exon sequences. If RNA elements 1 and 4 of FIG. 20 are 
also capable of interacting to form a functional intron, chains of ligated 
exons 1 and 2, in random order, can be produced. Also, it should be 
understood that the RNA elements depicted in FIG. 20 need not be 
continuous RNA molecules, so that components of the functional introns may 
themselves be provided in trans as desired (and as discussed above). 
Any available method may be employed to attach intronic RNA sequences to 
DNA exons in order to practice the "trans-splicing" DNA recombination 
methods of the present invention. For example, RNA molecules may be 
ligated to DNA molecules through splint-mediated DNA-RNA ligation (see 
Moore et al. (1992) Science 256:992-997). As shown in FIG. 21A, a DNA 
molecule and an RNA molecule are brought within one bond length of one 
another by hybridization to the same nucleic acid splint molecule. The 
hybridized complex is then incubated with DNA ligase, which covalently 
links the DNA and RNA pieces to one another. In one preferred embodiment 
of DNA-RNA ligation as used herein, the DNA molecule is a double-stranded 
molecule in which the second strand (i.e. the strand to which the RNA 
molecule will not be linked) overhangs the first strand, so that the 
overhang acts as the splint to bring the RNA into register for ligation 
(see FIG. 21B). 
As an alternative to the DNA-RNA ligation methods depicted in FIG. 21, RNA 
molecules may be linked to DNA molecules through intron-mediated 
integration reactions in which the DNA molecule is cleaved and ligated to 
the RNA directly. Certain group II introns have been shown to integrate 
into DNA targets. For example, at least the aIl group II intron can fully 
integrate into double-stranded DNA (Yang et al. (1996) Nature 381:332-335; 
see FIG. 22A); at least the aI2 group II intron can perform a first 
integration step, apparently equivalent to a reversal of the second step 
of splicing, so that a DNA strand is cleaved and the intron becomes linked 
to one of the resultant DNA pieces but not the other (Zimmerly et al. 
(1995) Cell 83:529-538; see FIG. 22B). Several other have been shown to 
perform such partial integrations into single-stranded DNA (Herschlag et 
al. (1990) Nature 344:405-409; Robertson et al. Nature 344:467-468; Morl 
et al (1992) Cell 70:803-810). Also, any intron capable of full insertion 
into a DNA target will perform only the first step of that insertion 
(analogous to a reversal of the second step of splicing) if the intron is 
presented to the target in linear, rather than lariat, form. 
The DNA integration reactions catalyzed by aI1 (complete integration) and 
aI2 (partial integration) rely on an intron-encoded protein that has 
maturase, reverse transcriptase, and endonuclease activity. The 
endonuclease activity appears not to be required for intron integration 
into a DNA strand, but rather cleaves the complementary strand when the 
intron inserts into double-stranded DNA, thereby creating a 3'OH in the 
complementary strand that can serve as a reverse transcription primer so 
that the integrated intron can be copied into DNA (Zimmerly et al. (1995) 
Cell 83:529-538). The reverse transcriptase activity may also be 
dispensable. 
According to the present invention, it will often be desirable to prevent 
reverse transcription of the integrated RNA into DNA, in order that the 
RNA remain available for "splicing" reactions. Accordingly, many preferred 
inventive methods are performed under conditions in which reverse 
transcription of integrated RNA is inhibited. For example, reverse 
transcriptase inhibitors may be employed (particularly in in vitro 
reactions). Alternatively or additionally, mutant proteins may be employed 
that lack the endonuclease and/or reverse transcriptase activities. 
Endonuclease-deficient mutant proteins have been described for at least 
the aI2 protein (the HHVR, .DELTA.ConZn, C-C/1, and C-C/2 derivatives 
described by Zimmerly et al. (1995) Cell 83:529-538); others can be 
readily generated using known mutagenesis techniques. Other approaches to 
avoiding reverse transcription include, for example, use of a reverse 
transcriptase-deficient protein (e.g. the YAHH version of the aI2 protein; 
Zimmerly et al. (1995) Cell 82:545-554), isolation of the target DNA 
strand (before or after linkage to the RNA intron element) away from the 
complementary strand, and use of an integrating ribozyme that does not 
require a protein co-factor. One particularly straightforward approach to 
inhibiting reverse transcriptase activity in in vitro reactions is to 
simply exclude dNTPs from the reaction; such dNTP exclusion does not 
interfere with the endonuclease activity of at least the al2 protein 
(Zimmerly et al. (1995) Cell 83:529-538). 
It will also often be desirable to minimize the effects of cis-splicing 
reactions capable of competing with the trans-splicing reaction(s) in 
issue. Various techniques can be used to prevent such cis-splicing 
reactions. For example, in some cases, it will be possible to utilize 
intron components that are only compatible with other components available 
in trans. Alternatively, cis components that would not participate in the 
desired trans- reaction can be specifically removed by, for example, RNAse 
H digestion, or can be inactivated by provision of a specific anti-sense 
probe (see, for example, Example 2). 
Various embodiments of DNA trans-splicing reactions according to the 
present invention are depicted in FIGS. 23-33 and described below in 
Examples 4-11. Those of ordinary skill in the art will recognize that the 
depicted methods and reagents are merely certain preferred embodiments of 
the invention; various modifications of the presented embodiments can 
readily be made according to the teachings of this specification and the 
knowledge available in the art. 
EXAMPLE 4 
Intron-mediated Cleavage and Ligation of Single-stranded DNA Vectors 
In embodiment of the present invention, depicted in FIG. 23, two Y-branched 
ribozymes (e.g. derived from a group II intron such as aI5.gamma.) are 
produced that are engineered to recognize target sites within a 
single-stranded DNA template. The elements of each Y-branched ribozyme are 
also selected to be compatible with one another for splicing (e.g. the 1-3 
element of ribozyme 1 is compatible with the 5-6 elements of both ribozyme 
1 and ribozyme 2; similarly, the 1-3 element of ribozyme 2 is compatible 
with both 5-6 elements). 
The first single-stranded DNA template depicted in FIG. 23 includes 
sequences encoding a tetracycline resistance gene expressible in E. coli; 
the second single-stranded DNA template includes sequences encoding an 
ampicillin resistance gene. At least one of the single-stranded DNA 
templates also has an m13 origin of replication. Preferably, both 
single-stranded DNA templates have an m13 origin of replication, and are 
produced by infection and lysis of E. coli, as is known in the art. 
The ribozymes shown in FIG. 23 are integrated into their respective target 
sites under "reverse-splicing" conditions, and the resultant DNA-RNA 
hybrid molecules are isolated. Mixed together, and allowed to 
"trans-splice". The single-stranded DNA trans-splicing product is then 
isolated and introduced into E. coli, where it may be converted into a 
double-stranded DNA vector or maintained as a single-stranded phage, as is 
known in the art. It will be appreciated that, as shown in the Figure, the 
trans-splicing reaction also produces "shuffled" ribozymes, in which 
ribozyme 1 element 1-3 is linked to ribozyme 2 element 5-6 and ribozyme 2 
element 1-3 is linked to ribozyme I element 5-6. 
EXAMPLE 5 
Intron-mediated Cleavage and Ligation of Double-stranded DNA Vectors 
FIG. 24 depicts another trans-splicing embodiment of the present invention, 
in which a group II intron, aI1, is utilized to cleave and ligate two 
double-stranded DNA vectors. As shown in FIG. 24A, two aIl-derived 
Y-branched ribozymes are engineered to insert into target sites in 
double-stranded DNA vectors. The integration reactions are performed in 
the presence of the all protein, but under conditions that block reverse 
transcription. Accordingly, the ribozymes insert into one strand of the 
DNA and the other DNA strand is nicked by the endonuclease in a manner 
that produces a 10-nt 5' overhang. The ribozyme is not reverse transcribed 
into DNA. Also, because a Y-branched engineered ribozyme is employed 
instead of a lariat, the DNA target DNA strand is disrupted by the 
integration. 
According to the method shown in FIG. 24A, the DNA-RNA hybrid products of 
the integration reactions are combined with one another in the presence of 
the aI1 protein and DNA ligase, and the integration reaction is allowed to 
reverse itself Because the ribozyme elements are selected to be compatible 
with one another (i.e. so that the 1-3 element of ribozyme 1 forms a 
splicing-competent intron with the 5-6 element of ribozyme 2 and 
vice-versa), trans-interactions will occur between the 1-3 element of 
ribozyme 1 and the 5,6 element of ribozyme 2 (and vice-versa) so that at 
least some of the time, the reversal of integration will produce the 
depicted trans-product. DNA ligase is added to seal the nicks that would 
otherwise be present in the complementary DNA strand (i.e. in the strand 
into which the ribozymes did not integrate). 
The ligation reaction depicted in FIG. 24A is referred to as a 
"trans-reversal of integration" rather than a "tans-splicing" reaction 
because it is performed in the presence of the aI1 protein, and utilizes 
substrates in which the IBS1 (and/or IBS2) sites are double-stranded in 
the exons. It is clear that the aI1 intron can recognize and interact with 
double-stranded sites in the presence of its protein, but it may not be 
able to do so on its own. The aI1 intron does recognize single-stranded 
sites (e.g. in RNA) on its own. In fact, the presence of the al1 protein 
alters the intron's natural preference for single-stranded RNA targets and 
allows it to select double-stranded DNA targets (Zimmerly et al. (1995) 
Cell 83:529-538). Thus, there are at least qualitative differences between 
the reactions catalyzed by the all intron with double-stranded as compared 
with single-stranded targets, and the two types of reactions are 
distinguished herein for purposes of clarity. This distinction is not 
intended to imply a mechanistic difference between the two kinds of 
reactions, but rather is employed to achieve linguistic precision. 
An alternative embodiment of the basic reaction depicted in FIG. 24A, one 
that achieves ligation by "trans-splicing" rather than "trans-ligation", 
is presented in FIG. 24B. As can be seen, the integration reaction is 
performed as in FIG. 24A, but is followed by an exonuclease step that 
removes the complementary DNA strand to the extent that at least IBS1, and 
optionally also IBS2, is uncovered. The exo-treated DNA-RNA hybrids are 
then combined and spliced together, either in the presence or absence of 
the aI1 protein (the aI1 protein may assist in the splicing reaction, but 
it is not necessary, as the aI1 intron is known to be capable of 
autocatalytic splicing). DNA polymerase is added to restore the regions of 
the complementary DNA strand that were removed by exonuclease digestion, 
and DNA ligase seals the final nicks. 
FIG. 24C presents yet a third embodiment of the basic reaction depicted in 
FIG. 24A. In this embodiment, the integration reactions are performed in 
the presence of a mutant aI1 protein that lacks endonuclease activity. 
Under such conditions, separate steps to inhibit reverse transcription 
need not be taken. Also, the integration reactions produce DNA-RNA hybrid 
molecules whose complementary strand is an intact circle. Ligation is then 
performed either by trans-reversal of integration (in the presence of the 
protein) or by trans-splicing (in its absence). As shown in FIG. 24C, 
ligation occurs by trans-splicing. A denaturation step is performed prior 
to the trans-splicing incubation, so that the IBS1 (and/or IBS2) sequence 
can be exposed. The resultant double-stranded DNA product molecule 
resembles a Holiday recombination intermediate and can be resolved into 
one or two DNA circles by passage through E. coli. 
It should be noted that preferred embodiments of the reactions depicted in 
FIG. 24 are performed in vitro. However, such reactions could 
alternatively be performed in vivo if cell lines were produced that 
expressed inverse-splicing constructs capable of generating the Y-branched 
ribozymes and also expressed the all protein. 
EXAMPLE 6 
Intron-catalyzed Shuffling of DNA Exons 
As discussed above, the basic recombinatory principles of the present 
invention are just as applicable to DNA exons as to RNA exons, with the 
caveat that the introns' ability to recognize double-stranded DNA 
templates may be protein-dependent. The present Example demonstrates that 
exon shuffling of the sort described above in Example 1 may also be 
performed at the DNA level. 
As shown in FIG. 25A, Y-branched aI1-type ribozymes can be engineered to 
insert immediately downstream of the t-PA Fn domain and immediately 
upstream of the T-PA Prot domain in DNA. Double-stranded DNAs containing 
these insertion sites can be prepared, for example, by PCR amplification 
of appropriate regions of the t-PA gene. Integration reactions are 
performed in the presence of the aI1 protein, and the resultant products 
are ligated together by trans-reversal of integration in the presence of 
the protein. 
Where it is desirable to improve the efficiency of the trans-reversal of 
integration reaction as compared with the "cis" reversal reaction (which 
would produce the starting materials), it is useful to isolate each of the 
DNA-RNA hybrid products, deprotenate them (to remove any aI1 protein that 
may remain bound to the product after integration), and then mix them back 
together in the presence of more protein. Where it is desirable to favor 
the trans-reaction to the exclusion of the "cis" reaction, the steps 
diagramed in FIG. 25B can be performed. That is, each of the hybrid 
products can be isolated individually and hybridized with an 
oligodeoxyribonucleotide complementary to one element of the engineered 
ribozyme. Removal of one ribozyme element from each DNA-RNA hybrid product 
will prevent any "cis" reaction from occurring when the two products are 
mixed together. 
It should be understood that the ligation reaction depicted in FIG. 25A can 
alternatively be performed as a trans-splicing reaction, in the absence of 
all protein, with an appropriate denaturation step. 
EXAMPLE 7 
Cleavage and Ligation of Double-stranded DNA Exons Using the aI2 Group II 
Intron 
The present Example demonstrates that the sort of double-stranded DNA exon 
manipulation described above in Example 5 can also be performed with 
introns, such as the aI2 group II intron, that do not completely integrate 
into double-stranded DNA. 
As shown in FIG. 26, an aI2-type lariat intron is designed to insert into a 
recognition site that is present in two different double-stranded DNA 
templates. The integration reactions are performed in the presence of the 
aI2 protein under conditions that block reverse transcription. The 
products of the integration reaction are then mixed with one another and 
ligation is performed either by trans-reversal of splicing (shown, no 
exonuclease step) or by trans-splicing (after an exonuclease step). Note 
that use of a lariat intron, rather than a Y-branched intron, requires 
that the insertion sites in the two double-stranded DNA targets be the 
same (or sufficiently similar that productive EBS1/IBS1, and optionally 
also EBS2/IBS2 is formed during the trans-reaction). 
EXAMPLE 8 
Chromosome Recombination 
The trans-splicing DNA manipulation methods of the present invention may be 
performed in vivo to accomplish recombination of chromosomal regions. As 
shown in FIG. 27, a cell is generated that expresses both the aI2 protein 
and a splicing precursor that generates an aI2 lariat having a desired 
specificity. Preferably, one or both of these expressed factors is under 
the control of an inducible promoter, so that expression may be initiated 
and terminated under controlled circumstances. Also, the aI2 protein 
should be a version that has endonuclease activity, though it may lack 
reverse transcriptase activity. 
When both the aI2 lariat and the aI2 protein are being expressed in the 
cell, the lariat will integrate at its recognition site in chromosomal 
DNA. Once it has integrated into sites in two different chromosomes, a 
trans-reversal of integration reaction will swap the relevant regions of 
the chromosomes. If it is desirable to select the chromosomal regions to 
be swapped, an aI2 lariat of high specificity can be engineered, and 
appropriate sites can be inserted into the chromosomes (according to 
standard techniques) prior to expression of the aI2 lariat and/or protein 
in the cell. 
EXAMPLE 9 
Intron-mediated DNA Insertion 
FIG. 28 depicts a trans-splicing reaction of the present invention that can 
be utilized to insert one DNA sequence into another in vivo or in vitro. 
As depicted, an aI2-type intron lariat is designed to insert at a specific 
target site that is present both in a double-stranded DNA target and in a 
single-stranded DNA target. The intron lariat is integrated into both 
targets in the presence of an endonuclease mutant version of the aI2 
protein (although the presence of the protein may not be required for 
integration into the single-stranded DNA target). After integration, the 
DNA-RNA hybrid products interact with one another and undergo a 
tans-reversal of integration reaction so that the single-stranded DNA 
"exon" is inserted into the double-stranded DNA. Addition of DNA 
polymerase (or DNA replication in vivo) generates a fully double-stranded 
DNA molecule in which the sequences of the original single-stranded DNA 
target have been inserted into the double-stranded DNA target at a 
specific site. 
It will be appreciated that these reactions can be performed in vivo if a 
cell is constructed that expresses the aI2 lariat (e.g. via expression of 
a splicing precursor) and the endonuclease mutant aI2 protein. Preferably, 
the expression of one or both of these factors is directed by an inducible 
promoter, so that splicing activity in the cell can be initiated and 
terminated under controlled circumstances. The single-stranded DNA 
template can be introduced into the cell (e.g., by infection) prior to 
expression of the aI2 lariat and/or protein. Alternatively, the reaction 
steps may be performed in vitro. 
It will also be appreciated that use of a Y-branched, rather than a lariat, 
intron would allow insertion and recombination at two different target 
sites. 
EXAMPLE 10 
Intron-mediated DNA Cloning 
FIG. 29 presents another embodiment of the trans-splicing methods of the 
present invention that allows single-or double-stranded DNA targets to be 
cleaved and ligated together. As depicted, two ribozymes are engineered as 
described herein (e.g. by alteration of at least their IBS 1 sites) to 
recognize desired target sites within a double-stranded DNA molecule. The 
ribozymes are depicted as lariats in FIG. 29 but alternatively could be 
Y-branched enzymes The DNA between the target sites, labeled "exon 1" in 
FIG. 29, becomes flanked by ribozyme sequences as a result of the 
integration reactions. An RNAse H cleavage reaction is then optionally 
performed in order to reduce the likelihood that the integrated ribozymes 
will splice themselves back out (i.e. to ensure that the trans-splicing 
reaction does not have to compete with a cis-splicing reaction). 
Ribozyme-flanked exon 1 is then mixed with a second exon, "exon 2" in FIG. 
29, that is also flanked by appropriate ribozyme sequences. That is, the 
ribozyme sequences flanking exon 2 are selected to be competent to 
trans-splice with those flanking exon 1 so that the two exons will be 
linked to one another. 
At certain points in FIG. 29, portions of nucleic acid molecules are 
depicted with dashed rather than solid lines. This dashing represents 
optional, or alternative, forms of the nucleic acids. For example, the 
exact form of the complementary DNA strand (i.e. the strand into which the 
ribozyme did not integrate) after ribozyme integration will depend on 
exactly how the reaction is performed. For example, when a protein 
component that has endonuclease activity is utilized, the complementary 
DNA strand will be nicked. On the other hand, when mutant versions of the 
protein that lack endonuclease activity are utilized, the complementary 
DNA strand is not broken during the integration reaction. 
Also, a portion of ribozyme 1 is dashed after the integration reaction; 
this dashing is intended to indicate that ribozyme 1 may be an aI2-type 
ribozyme, capable of only partial integration into a double-stranded DNA 
template. 
The optional RNAse H cleavage reaction depicted in FIG. 29 is performed by 
preparing DNA oligonucleotides complementary to portions of the integrated 
ribozymes that are not required for trans-splicing, hybridizing these 
oligonucleotides with the integrated ribozymes, and digesting the hybrids 
with RNAse H. Of course, it is particularly important that reverse 
transcription across the integrated ribozyme be prevented in reactions 
where the RNAse H step is to be performed; otherwise, digestion with RNAse 
H will remove the integrated ribozyme to the extent that it is hybridized 
with a reverse-transcribed DNA complement. It is, of course, important to 
leave a sufficient amount of intronic information both upstream and 
downstream of exon 1 that the trans-reaction can successfully. 
Any of a variety of DNA targets may be employed as the second exon depicted 
in FIG. 29. Preferred targets include single- or double-stranded DNA 
cloning vectors such as are known in the art. Exon 2 may be linear or 
circular. Exon 2 may be linked to ribozyme elements by any available 
method. Preferred methods include ribozyme integration and splint-assisted 
DNA-RNA ligation. 
FIG. 29 includes a step labeled "DNA processing". The details of this step 
will depend on the specifics of the constructs utilized. For example, it 
may be necessary to "chew back" and/or "fill in" regions of an exon 1 or 
exon 2 complementary strand. DNA ligation to seal the complementary strand 
may also be desirable. Of course, where the product recombinant DNA 
molecule is intended to be single-stranded (e.g., when exon 2 is a 
filamentous phase vector such as m13), such DNA processing steps are not 
required. 
One particularly useful application of the method depicted in FIG. 29 is in 
isolation and analysis of genomic DNA sequences. For such applications, 
exon 1 is genomic DNA. One feature of the method that makes it desirable 
for this application is that reverse transcription and amplification steps 
are not required. Although such steps may be performed if desired, it will 
often be preferable to omit them as they can introduce errors into the 
sequence of the product DNA. Such errors can be problematic, particularly 
when the goal of a study is to identify and analyze the function of the 
cloned DNA sequence and/or any product it encodes. 
FIG. 30 presents one particular embodiment of the method shown in FIG. 29. 
As depicted in FIG. 30, both ribozymes are aI1-type ribozymes, capable of 
full integration. Under these circumstances, only a single protein factor 
(the aI1 protein) need be used. It will be appreciated that the two 
ribozymes depicted in FIG. 30 are not identical to one another. First of 
all, they will have been engineered to have different integration 
specificities, so at least their EBS 1 sites will differ. Furthermore, in 
order to simplify the RNAse H step, it is desirable to engineer the two 
ribozymes to include different sequences that can be specifically targeted 
for RNAse H digestion. As depicted in FIG. 30, three different 
oligonucleotides are utilized in the RNAse H reaction: one that hybridizes 
with domain 4 in both ribozymes, one that hybridizes with a region at or 
near the 5' end of ribozyme 1, and one that hybridizes with a region at or 
near the 3' end of ribozyme 2. In this version of the inventive method, 
ribozymnes 1 and 2 are engineered to differ from each other at the two 
regions to which the ribozyme-specific oligonucleotides hybridize. 
In the reaction depicted in FIG. 30, exon 2, like exon 1, is flanked by all 
ribozyme sequences. These ribozyme sequences can either splice with each 
other, and thus circularize exon 2, or can splice with the sequences 
flanking exon 1 to produce the desired recombinant product. Because the 
exon 2 circularization reaction is an intramolecular reaction, there is a 
risk that it will be favored over the intermolecular trans-reaction. 
Accordingly, it may be desirable to take steps to shift the competition. 
For example, larger concentrations of the flanked exon 2 reagent will 
increase the efficiency of the trans-reaction without affecting that of 
the cis reaction. Alternatively, the trans-reaction can be performed in 
two steps, one in the presence of an anti-sense oligonucleotide (DNA or 
RNA) that blocks one side of the cis reaction but allows one side of the 
trans reaction, and one in the absence of that oligonucleotide, so that 
the second side of the trans reaction (now a cis-splicing reaction) can 
occur. 
FIG. 31 presents an alternative embodiment of the overall reaction scheme 
shown in FIG. 29. In FIG. 31, ribozyme 1 is an aI2-type ribozyme, whereas 
ribozyme 2 is an aI1-type ribozyme. The advantages of this approach 
include the relative increased simplicity in designing RNAse H 
oligonucleotides specific to each ribozyme and the absence of a competing 
cis-reaction when ribozyme-flanked exon 1 is incubated with 
ribozyme-flanked exon 2. Of course, it will be appreciated that the 
ribozyme designed to integrate downstream of exon 1 (and upstream of exon 
2) must be aI1-type (or there will be no linkage between exon 1 and intron 
domains 1-3). Furthermore, where the ribozyme design to integrate upstream 
of exon 1 (and downstream of exon 2) is of the aI2-type (capable of only 
partial integration), it will not be possible to link exon 2 to intron 
domains 1-3 via an integration reaction. Preferably, this linkage is 
accomplished by splint-assisted DNA-RNA instead. FIG. 32 depicts the 
preferred method for this ligation reaction. As shown in FIG. 32, exon 2 
DNA is cleaved in such a way as to leave both 5' and 3' overhangs on the 
complementary strand (the strand to which the ribozyme components will not 
be attached), which overhangs can act as the splint for DNA-RNA ligation. 
EXAMPLE 11 
Integration and Expression of Novel Genes in the Genome Mediated by 
Trans-splicing 
As has been discussed, the aI1 group II intron is capable of full insertion 
into double-stranded DNA. When steps are not taken to block reverse 
transcription of the inserted construct, the intron is copied into DNA. It 
has been proposed that this feature could provide a useful mode for 
integrating new sequences into an existing DNA molecule. Specifically, it 
has been proposed that desired sequences could be inserted into the domain 
4 loop region of the aI1 intron (or into some other non-conserved region), 
the intron (along with the inserted sequences) could then be integrated 
into a target double-stranded DNA molecule, and the integrated sequences 
could be copied into DNA (see Yang et al. (1996) Nature 381:332-335, 
incorporated herein by reference). One interesting application for this 
integration system is the introduction of new sequences into expressible 
sites, e.g., in a genome. The present invention utilizes this integration 
system, in combination with the above-described RNA-based exon shuffling 
techniques, and provides a system by which novel genes, produced by exon 
shuffling, can be expressed and assayed in cells. This embodiment of the 
invention is depicted in FIG. 33. 
As shown in FIG. 33, a splicing precursor RNA is produced in which an 
incompatible intron elements are nested within the domain 4 loop of an all 
intron. This splicing precursor splices to produce a lariat that includes 
the nested intron elements. The nested intron elements do not splice 
themselves out of the lariat because they are incompatible and therefore 
cannot undergo a splicing reaction. This lariat is then combined with an 
intron-flanked product of exon shuffling. The intron elements flanking the 
shuffled exons are compatible with the intron elements nested in the 
lariat, but not with each other. Under these circumstances, the only 
splicing reaction that the lariat and exon shuffling product can undergo 
is a trans-reaction in which the shuffled exons are specifically inserted 
within the domain 4 loop in the lariat, precisely replacing the 
previously-nested intron sequences. This new lariat is then an all lariat 
with inserted gene sequences, and can be integrated into a specific site 
in double-stranded DNA as described above. It is preferred that the site 
be a genomic insertion site, and that the construct include an inducible 
promoter, so that effects of expressing the novel gene can be studied in 
vivo. 
One preferred embodiment of the method depicted in FIG. 33 occurs entirely 
in vivo. Specifically, an all intron carrying nested intron elements as 
discussed is first integrated into the genome of a host cell in which 
expression of the novel gene is to be analyzed. The cell is also designed 
to express the aIl protein. The exon shuffling product is then also 
introduced into the cell, either because the shuffling itself occurs in 
the cell, or because a construct encoding the shuffled product is 
introduced into and expressed within the cell. Under these circumstances, 
trans-splicing and integration proceed within the cell, and the cell is 
analyzed to detect any effects of expressing the novel gene. 
III. Cis-splicing Combination of Exons 
In yet another embodiment, the combinatorial method can be carried out in a 
manner that utilizes the flanking intronic sequences in a cis-splicing 
reaction to generate a combinatorial gene library. As illustrated 
schematically in FIG. 34, the actual combinatorial event takes place at 
the DNA level through annealing of complementary sequences within the 
intron encoding fragments. Briefly, complementary DNA strands are 
synthesized which correspond to the exonic sequences and flanking intron 
fragments. As used herein, the term (+) strand refers to the 
single-stranded DNA that is of the same polarity as a trans-splicing RNA 
transcript. That is, intronic sequences flanking the 5' end of the exon 
represent a 3' fragment of an intron. Likewise, the term (-) strand refers 
to the single stranded DNA which is complementary to the (+) strand (e.g. 
of opposite polarity). 
The 5' and 3' ends of each of the (+) and (-) strands are complementary and 
can therefore mediate concatenation of single-stranded DNA fragments to 
one and other through basepairing. In the exemplary illustration of FIG. 
34, the exon sequences are flanked by group II domains 4-6 at on end, and 
domains 1-4 at the other. A library of combinatorial units representative 
of a number of different exons is generated, such as by PCR or digestion 
of double-stranded plasmid DNA, to include both (+) and (-) strands. The 
units are combined under denaturing conditions, and then renatured. Upon 
renaturation, the sequences corresponding to domain IV at the 3' end of 
one (+) strand unit can anneal with the complementary domain IV sequences 
at the 3' end of a (-) strand unit, resulting in concatenation of 
combinatorial units (see FIG. 34). 
Double-stranded DNA can be generated from the concatenated single-stranded 
units by incubating with a DNA polymerase, dNTPs, and DNA ligase; and the 
resulting combinatorial genes subsequently cloned into an expression 
vector. In one instance, 5' terminal and 3' terminal combinatorial units 
can be used and the double-stranded genes can be amplified using PCR 
anchors which correspond to sequences in each of the two terminal units. 
The PCR primers can further be used to add restriction endonuclease 
cleavage sites which allow the amplified products to be conveniently 
ligated into the backbone of an expression vector. Upon transcription of 
the combinatorial gene, the intronic RNA sequences will drive ligation of 
the exonic sequences to produce an intron-less transcript. 
While FIG. 34 demonstrates one embodiment which utilizes group II introns, 
the combinatorial process can be carried out in similar fashion using 
either group I intron sequences or nuclear pre-mRNA intron sequences. 
IV. Circular RNA Transcripts 
In addition to generating combinatorial gene libraries, certain splicing 
constructs of the present invention have a number of other significant 
uses. For instance, the present trans-splicing constructs can be used to 
produce circular RNA molecules. In particular, exon constructs flanked by 
either group II or nuclear pre-mRNA fragments can, under conditions which 
facilitate exon ligation by trans-splicing of the flanking intron 
sequences, drive the manufacture of circularly permuted exonic sequences 
in which the 5' and 3' ends of the same exon are covalently linked via a 
phosphodiester bond. 
Circular RNA moieties generated in the present invention can have several 
advantages over the equivalent "linear" constructs. For example, the lack 
of a free 5' or 3' end may render the molecule less susceptible to 
degradation by cellular nucleases. Such a characteristic can be especially 
beneficial, for instance, in the use of ribozymes in vivo, as might be 
involved in a particular gene therapy. In the instance of generating 
ribozymes, the "exonic" sequences circularized are not true exons in the 
sense that they encode proteins, rather, the circularized sequences are 
themselves intronic in origin, and flanked by other trans-acting intron 
fragments. 
However, the circularization of mature messenger-RNA transcripts can also 
be beneficial, by conferring increased stability as described above, as 
well as potentially increasing the level of protein translation from the 
transcript. To illustrate, a ribosome which has completed translation of a 
protein from the present circular transcript may continue to track around 
the transcript without dissociating from it, and hence renew synthesis of 
another protein. Alternatively, the ribosome may dissociate after 
translation is completed but, by design of the circular transcript, will 
disengage the transcript proximate to the start site and thereby provide 
an increased probability that the ribosome will rebind the transcript and 
repeat translation. Either scenario can provide a greater level of protein 
translation from the circular transcript relative to the equivalent linear 
transcript. 
FIGS. 8A-C depicts three examples of intron fragment constructs, designated 
(IVS5,6)-exon-(IVS1-3), and (3'-half-IVS)-exon-(5'-half-IVS), which, in 
addition to being capable of driving trans-splicing between heterologous 
exons as described above, can also be used to generate circular RNA 
transcripts. The (IVS5,6)-exon-(IVSI-3) transcript comprises the group II 
intron domains 5 and 6 at the 5' end of the exon, and domains 1-3 at the 
3' end of the exon. The (3'-half-IVS)-exon-(5'-half-IVS) is a similar 
construct, but replaces the group II domains 5-6 and 1-3 with fragments 
corresponding to the 3'-half and 5'-half of a nuclear pre-mRNA intron. As 
described in Examples 12 and 15 below, each of these transcripts can be 
shown to drive intramolecular ligation of the exon's 5' and 3' end to form 
circular exons. 
Furthermore, as set forth in Example 15, a preferred embodiment of an exon 
construct using mammalian pre-mRNA intron sequences to generate circular 
transcripts provides an added structural element that brings together and 
5' and 3' ends of flanking pre-mRNA intron fragments. The addition of such 
structural elements has been demonstrated to greatly improve the 
efficiency of the intramolecular splicing reaction. For example, the ends 
of the intronic fragments can be non-covalently linked as shown in FIGS. 
8B and C, by hydrogen bonding between complementary sequences. 
Alternatively, the ends of the nuclear pre-mRNA intron fragments can be 
covalently closed. In an illustrative embodiment, FIG. 35 shows how group 
II intronic fragments can be utilized to covalentlyjoin the ends of the 
nuclear pre-mRNA transcripts having flanking nuclear pre-mRNA intron 
fragments, which subsequently drive ligation of the 5' and 3' end of the 
exonic sequences. 
In yet another embodiment, the intronic ends can be brought together by a 
nucleic acid "bridge" which involves hydrogen bonding between the intronic 
fragments flanking the exon and a second discrete nucleic acid moiety. As 
illustrated in FIGS. 36A-C, such nucleic acid bridges can be formed a 
number of ways. Each of the splicing bridges shown differ from each other 
in either the orientation of the bridge oligonucleotide when base-paired 
to the flanking intron fragments, in the size of the bridging 
oligonucleotide, or both. For instance, the bridge oligonucleotide shown 
in FIG. 36A base-pairs in an orientation which can result in a 
stem-structure similar to the (3'IVS-half)-exon-(5'IVS-half) construct 
depicted in FIG. 8B. Moreover, when a bridge similar to one shown in FIG. 
36C is used, and the 5' and 3' ends of the flanking introns base-pair some 
distance apart in the linear sequence of the bridge, the bridge 
oligonucleotide may itself comprise the branch acceptor site. For example, 
the bridge oligonucleotide can be an RNA transcript comprising the yeast 
branch site consensus sequence UACUAAC in a portion of the bridge sequence 
which does not base-pair with the intronic fragments of the exon 
construct. 
Oligonucleotide bridges useful in driving the circularization of exon 
transcripts can also be used to direct alternative splicing by "exon 
skipping", which may be useful, for example, in disrupting expression of a 
particular protein. As shown in FIG. 8D, the splicing of exons 1 and 3 to 
each-other can be the result of an oligonucleotide which loops out exon 2, 
effectively bringing together two complementary halves of the intronic 
sequences flanking exons 1 and 3. As shown in FIG. 8D, exon 2 can, in 
fact, be spliced into a circular RNA. 
Carrying the bridging nucleotide one step further, FIG. 37 illustrates the 
use of an exon construct useful in mediating the alternate splicing of an 
exon through a trans-splicing-like mechanism. For instance, a wild-type 
exon can be trans-spliced into an mRNA transcript so as to replace an exon 
in which a mutation has arisen. The wild-type exon construct comprises 
flanking intronic sequences which include sequences complementary to a 
portion of the continuous introns which connect exons 1, mutant exon 2, 
and exon 3. Thus, through a trans-splicing event as described above, some 
of the resulting mature mRNA transcripts will include the wild-type exon 
2. 
EXAMPLE 12 
Group II Introns can Mediate Circularization of Exonic Sequences 
The (IVS 5,6)-exon-(IVS 1-3) RNA transcript, shown in FIG. 8A, was 
synthesized from plasmid pINV1 (SEQ ID NO:1). The intronic sequences 
correspond to the half molecules generated by interruption of the 5 g 
intron of the yeast mitochondrial oxi3 gene in domain 4; and the exonic 
sequences are the exon sequences E5 and E3 which are naturally disposed at 
the 5' and 3' ends of the 5 g intron, respectively. To construct pINV1, 
the Sac I-Hind III fragment of pJDI5'-75 (Jarrell et al. (1988) Mol. Cell 
Biol. 8:2361-2366) was isolated and the Hind III site was filled in with 
Klenow fragment. This DNA was ligated to pJDI3'-673 (Jarrell et al., 
supra) that had been cleaved with SacI and SmaI. The RNA splicing 
substrates were made by in vitro transcription using T7 RNA polymerase. 
Transcription, RNA purification, and splicing reactions were as described 
(Jarrell et al., supra). The E5-specific oligodexoynucleotide 
(5'-GTAGGATTAGATGCAGATAC-TAGAGC-3' [SEQ ID NO:12]) is identical to 26 
nucleotides of the E5 region of the (IVS 5,6)exon(IVS 1-3) RNA. The 
E3-specific oligonucleotide (5'-GAGGACTTCAATAGTAGTATCCTGC-3' [SEQ ID 
NO:13]) is homologous to 25 nt of the E3 region. 
To purify E3,E5(C), described below, for the reverse transcription 
reaction, a standard 100-.mu.l transcription was done, with pINV1 as a 
template. The (IVS 5,6)E3,E5(IVS 1-3) RNA was concentrated by ethanol 
precipitation and was then incubated under the (NH.sub.4).sub.2 SO.sub.4 
splicing conditions for 1 hr. The E3E5(C) RNA was gel purified and 
dissolved in 30 .mu.I of water. A 9-.mu.l annealing reaction mixture was 
incubated at 65.degree. C. for 3 min and then placed on ice. The annealing 
reacting mixture included 1 .mu.l of the E3,E5(C) RNA plus 100 ng of the 
E3-specific oligonucleotide. As a control, an identical annealing reaction 
was done, except E3,E5(C) was not added. A buffer (4 .mu.l) consisting of 
0.25 M Tris-HCl (pH 8.5), 0.25 M KCl, 0.05 M dithiothreitol, and 0.05M 
MgCl.sub.2 was added to both annealing reaction mixtures. Deoxynucleoside 
triphosphates were each added to a final concentration of 5 mM, followed 
by 40 units of RNasin (Promega) and 22 units of reverse transcriptase 
(Seikagaku America, Rockville, Md.). The final volume was adjusted to 20 
.mu.l with water. The mixture was incubated at 42.degree. C. for 90 min. 
Two polymerase chain reaction (PCR) experiments were done using as 
templates either 1 .mu.l of the reverse transcription mixture that 
included E3,E5(C) or 1 .mu.l of the control reverse transcription mixture, 
which lacked E3,E5(C). The PCRs were performed as described previously and 
were continued for 25 cycles. The E3- and E5-specific oligonucleotides, 
300 ng each, were used as PCR primers. DNA sequencing was done with 
Sequence (United States Biochemical) according to the protocol provided by 
the manufacturer. 
Group II intron excision can occur by transesterification (splicing) or by 
site-specific hydrolysis (cleavage). The former reaction is stimulated by 
(NH.sub.4).sub.2 SO.sub.4, the control RNAs, E5(IVS 1-3) plus (IVS5,6)E3, 
trans-spliced to yield spliced exon S(E5-E3) and a Y branched intron 
[IVS(Y)]. Co-incubation in the presence of KC1 yielded free exons (E5 and 
E3) and a linear intron (IVS 1-3) as major products. 
The (IVS 5,6)exon(IVS 1-3) precursor was also reactive. Most for the 
products could be identified based on their comigration with products of 
the control trans-reaction. In the present of (NH.sub.4).sub.2 SO.sub.4, 
the IVS(Y) and some linear intron were liberated; several novel products 
were also generated. Among these was an RNA (E3,E5) the expected size of 
the linear excised exons (591 nt). A slower migrating RNA [E3,E5(C)] was 
also observed. At short times of incubation (1 min) E3,E5(C) and IVS(Y) 
were the predominant products. In contrast, E3,E5 did not accumulate to 
significant levels before 60 min, indicating that it was not an early 
product of the reaction. Analysis of E3,E5(C) demonstrated that is was 
circular spliced exons. E3,E5(C) accumulated in the presence of 
(NH.sub.4).sub.2 SO.sub.4 but not in the presence of KCl. This was 
significant, given that spliced exons (E3-E5) are not only product of cis 
or trans splicing that accumulates in the presence of(NH.sub.4).sub.2 
SO.sub.4 but not in the presence of KCl. Thus, it was likely that E3,E5(C) 
resulted from splicing rather than hydrolysis. 
E3,E5(C) and E3,E5 were purified and analyzed by denaturing gel 
electrophoresis. During the purification process some E3,E5(C) was 
converted to a faster migrating species that comigrated with E3,E5. The 
extent of conversion of E3,E5(C) to the faster migrating species was 
increased by incubation with the group II intron under conditions that 
promote site-specific hydrolysis of the spliced exons. These observations 
are consistent with E3,E5(C) being a circular RNA that can be broken by 
hydrolysis to yield (linear) E3,E5. 
To demonstrate that E3,E5(C) contains spliced exons, a cDNA copy of 
purified E3,E5(C) RNA was made by reverse transcription. The reverse 
transcription was primed with an oligonucleotide homologous to 25 nt of 
E3. If E3,E5(C) is accurately spliced circular exons, its length is 591 
nt. Reverse transcription of this circular RNA would yield cDNAs of 
variable lengths; in particular, multiple rounds of complete reverse 
transcription of the circular template would generate cDNAs that are &gt;591 
nt long. A sample of the reverse transcription reaction mixture was used 
as a template in a PCT. The E3-specific oligonucleotide and an 
oligonucleotide homologous to 26 nt of the E5 sequence of the expected 
cDNA were used as primers. If E3,E5(C) is the product of a splicing 
reaction, it will contain both E3 and E5 sequences and will yield 
amplification products in this PCR reaction. Analysis of the PCR products 
revealed that the major amplification product is the size expected [313 
base pairs (bp)] for a PCR product derived from spliced exons. This 
product was not seen in a control PCR reaction. Two additional PCR 
products of about 900 bp and 1500 bp were also observed. Amplification of 
longer cDNAs generated by multiple rounds of reverse transcription of the 
circular E3,E5(C) template would yield a set PCR products each an integral 
multiple of 591 bp longer than the 313 bp indicating that the 900 bp and 
1500 bp observed products were likely generated in this manner. 
The 313-bp PCR product was purified and cloned into a plasmid vector. The 
nucleotide sequence of each of four independently isolated clones was 
determined by the dideoxy sequencing method, using the E3-specific 
oligonucleotide as a primer. The sequence showed that the PCR product 
contained both E5 and E3 sequences that were joined by accurate splicing. 
EXAMPLE 13 
Engineered Group II Introns Mediate Circularization of a Human Exon 
A derivative of the yeast aI5.gamma. group II intron was designed to be 
able to catalyze the production of a circular exon encoding the first 
Kringle domain (K1) of the human tissue plasminogen activator protein. The 
circular K1 exon is formed with high fidelity in vitro. Furthermore, the 
system is designed so that the circular exon consists entirely of human 
exon sequence. 
The four plasmid constructs utilized in this study are shown in FIG. 38A. 
pJD20 encodes the full-length aI5.gamma. intron with its flanking 5' and 
3' exons; pY1 is identical to pINV1 described above and encodes an 
inverse-splicing substrate that splices to yield an exon circle and a 
y-branched intron (Jarrell (1993) PNAS 90:8624-8627); pY8 and pY9 are 
plasmids that encode inverse-splicing constructs containing the human K1 
exon. Both pY8 and pY9 were made by site-directed mutagenesis that deleted 
the KpnI site and precisely fused the K1 exon sequence to the last 
nucleotide of the group II intron domain 6. The group II intron sequences 
in pY8 were engineered so that the EBS1 site perfectly matches the 
appropriate region of the K1 exon (see FIG. 38B); there is also a 
two-base-pair interaction between IBS2 and EBS2. The group II intron 
sequences in pY9 were engineered so that there is perfect complementarily 
in both the EBS1-IBS1 and EBS2-IBS2 interactions (see FIG. 38B). 
Since the EBS1-IBS1 interaction is essential and the EBS2-IBS2 interaction 
is important, but not essential, for cis or trans splicing, we compared 
the efficiency and accuracy of inverse splicing by the PY8 and PY9 RNAs 
(encoded by pY8 and pY9, respectively) in order to examine the roles of 
these interactions in inverse splicing. As described above, PY8 lacks the 
EBS2-IBS2 interaction, while PY9 has both interactions. In order that the 
splicing reactions could be compared quantitatively, the 
.DELTA.G37.degree. of the EBS1-IBS1 and EBS2-IBS2 interactions were 
calculated for each splicing precursor used (see FIG. 38B). 
PY8 and PY9 splicing precursors (as well as the WT and PY1 splicing 
precursors) were prepared by in vitro transcription with T7 RNA polymerase 
(Phannacia, Stratagene). The in vitro transcription reactions were 
performed at 40.degree. C. on plasmid templates that had been digested 
with HindIII restriction enzyme. The WT and PY1 transcripts were labeled 
with [.alpha.-.sup.32 P]UTP (given that intron aI5.gamma. and its flanking 
exons are relatively A/T rich, splicing of UTP labeled substrate yields 
readily detectable spliced products) while PY8 and PY9 were labeled with 
[.alpha.-.sup.32 p]GTP (given that the human K1 exon is very G/C rich, 
when PY8 or PY9 were labeled with UTP we observed that products and 
intermediates that contain the K1 exon were barely detectable). The 
splicing precursor RNAs were then purified from an acrylamide gel and were 
incubated under splicing conditions (40 mM Tris-HCl (pH7.6), 100 mM 
MgCl.sub.2, and either 1.5 M (NH.sub.4).sub.2 SO.sub.4, 1.5 M NH.sub.4 Cl, 
or 1.5 M KCI; 45.degree. C.). 
As shown in FIG. 39A, both PY8 and PY9 undergo inverse splicing to yield 
the excised exon circle [K1(C)] when incubated in splicing buffer (i.e., 
buffer that contains a monovalent cation salt). Neither gives the circular 
product when incubated in buffer lacking a monovalent cation (lanes 8 and 
14). Of the four different sets of splicing conditions used, 
(NH.sub.4).sub.2 SO.sub.4 (lanes 9 and 15) stimulates the most abundant 
production of the major products of inverse splicing, IVS(Y) and K1(C). 
The yield of those products is also high inNH.sub.4 Cl buffer (lanes 10 
and 16), but the ratio of branched intron [IVS(Y)] to liner released 
intron (IVS 1-3) is reduced in that buffer. Little IVS(Y) or K1(C) is seen 
when PY8 or PY9 are incubated in the presence of NaCl (lanes 11 and 17) or 
KCl (anes 12 and 18). 
Incubation of PY8 or PY9 under splicing conditions produces other products 
in addition to the inverse-splicing products discussed above. Products of 
trans splicing, labeled TRANS in FIG. 39A, are most abundant when the RNA 
is incubated in (NH.sub.4).sub.2 SO.sub.4 or NH.sub.4 Cl, buffer. 
Schematic representations of the products observed in FIG. 39A are 
presented in FIG. 39B. The expected size of each product is given in 
nucleotides (nt). Two of the products, K1.sub.d (IVS1-3) and 
(IVS5,6)K1.sub.u, are produced by a cryptic cleavage event at a site 
within the K1 exon that resembles the last six nucleotides of that exon. 
As shown in FIG. 39B, the two main products of splicing in both the NaCl or 
the KCl buffer were IVS 1-3 (line 4) and (IVS 5,6)K1 (line 5). These two 
products are generated when the first step of the two step splicing 
reaction occurs by hydrolysis rather than by transesterification . That 
(IVS 5,6)K1 accumulates to significant levels when either PY8 or PY9 is 
incubated in the presence of any of the four monovalent cation salts 
indicates that, for both substrates, there is a general reduction in the 
forward rate of the second step of splicing. There appears to be a 
correlation between the amount of branched intron that accumulates and the 
amount of released exon (both K1 and K1 (C)) that is seen; when the levels 
of IVS(Y) are high (e.g. lane 9) the levels of released K1 and K1(C) are 
also high. This suggests that RNAs that accomplish the first step of 
inverse splicing by branching (rather than by hydrolysis) are more likely 
to complete the second step of splicing than are RNAs that accomplish the 
first step by hydrolysis. That observation is consistent with the known 
observation that branched molecules catalyze the second step of cis 
splicing more efficiently than do liner molecules. 
Time course experiments were performed to establish precursor-product 
relationships and to compare rates of splicing by the different substrates 
(data summarized in Table I). Although the desired products of inverse 
splicing were most abundant when PY9 or PY8 were incubated in 
(NH.sub.4).sub.2 SO.sub.4 buffer, the reaction was slow in the presence of 
that salt. Inverse splicing of PY1, which contains yeast exon sequences, 
proceeds at a relative rate that is less than two-fold slower the rate 
observed for the WT (cis splicing) substrate; in contrast, PY8 and PY9 
each splice at a rate about 20- to 30-fold slower than does WT in the 
(NH.sub.4).sub.2 SO.sub.4 buffer. Interestingly, both PY8 and PY9 undergo 
inverse splicing at approximately the same rate in (NH4).sub.2 SO.sub.4 
buffer even though PY8 lacks a strong EBS2-IBS2 pairing (-2.3 kcal), while 
PY9 has a strong EBS2-IBS2 pairing (-12.2 kcal). 
The inverse splicing reaction was faster in the NH.sub.4 Cl buffer than in 
the (NH.sub.4).sub.2 SO.sub.4 buffer. Although the relative yield to the 
desired products of inverse splicing is somewhat reduced in the NH.sub.4 
Cl buffer, useful quantities of spliced product can be obtained relatively 
quickly upon incubation under these conditions. 
Each of the products depicted in FIG. 39B was characterized individually. 
The K1(C) was gel purified from a 1.5M (NH.sub.4).sub.2 SO.sub.4 splicing 
reaction and was characterized by reverse transcription and PCR 
amplification using K1-specific oligodeoxynucleotides (K1.Cir.1:anti-sense 
5'-GCCAACGCGCTGCTGTTCCAG-3' [SEQ ID NO:14] and K1.Cir.2:sense 
5'-GGCCAGACGCCATCAGGCTG-3' [SEQ ID NO:15]). The 241 bp amplification 
product was cloned into the pCR2.1.RTM. Vector (Invitrogeno.RTM. DNA 
Cloning.RTM. Kit Version D), and was sequenced (Amersham Sequenase.RTM. 
Version 2.0 DNA Sequencing Kit) across the splice junction. 8 independent 
clones of the PY8 K1(C) product were sequenced, as were 10 independent 
clones of the PY9 K1(c) product. 
Y-branched products were characterized by debranching analyses. 
Specifically, IVS Y, K1.sub.d (IVS1-3), and IVS1-3 were gel purified from 
1.5 M (NH.sub.4).sub.2 SO.sub.4 PY8 and PY9 splicing reactions. These 
products were incubated in HeLa cell extract (sl 00) (20 mM HEPES (pH8.0), 
100 mM KCl, 8 mM EDTA, 20% Glycerol), which has debranching activity, for 
one hour a 30.degree. C. for one hour. Proteins were then extracted from 
the reaction mixture and RNA products were precipitated and fractionated 
on a 4% denaturing polyacrylamide gel. 
To further characterize the products of inverse splicing, PY8 (data not 
shown) and PY9 (FIG. 40A) were uniquely labeled at their 5' ends prior to 
splicing. Specifically, non-reactioactive PY9 was dephosphorylated with 
Phosphatase (Boehringer Mannheim) in calf intestinal phosphatase buffer 
(Boehringer Mannheim), and then [.gamma.-.sup.32 P]ATP labeling was 
carried out using Polynucleotide Kinase (Pharmacia) in 1X one-phor-all 
kinase buffer (for 2 hours at 37.degree. C.). The labeled RNA was then 
cleaned and precipitated, spliced in 1.5M (NH.sub.4).sub.2 SO.sub.4, and 
run on a 4% acrylamide gel. Intermediates and products that contained the 
original 5' end of the splicing substrate were thereby identified. Three 
of the intermediates or products were found to harbor the original 5' end 
of the splicing substrate. Note that all three of those RNAs contain the 
IVS 5,6 sequence (FIG. 39B, lines 2, 5 and 6). 
PY8 (data not shown) and PY9 (FIG. 40A, lane 8) were also uniquely labeled 
at their 3' ends prior to splicing. In this experiment, non-radioactive 
PY9 was made in vitro and purified by G25-Sephadex column elution. T4 RNA 
Ligase (Pharmacia) was used to label the 3'-end with [5'-.sup.32 P]pCp. 
The reaction was carried out with 0.25 mM ATP, 1% DMSO (Sigma) water in 
RNA Ligase buffer (0.5 M Tris hydrochloride [pH7.6], 0.15 M MgCl.sub.2, 33 
mM DTT). The reaction was incubated over night at 4.degree. C. Labeled RNA 
was precipitated, spliced and run on a 4% acrylamide gel. Intermediates 
and products that contained the original 3' end of the splicing substrate 
were thus identified. Three of the intermediates or products were found to 
harbor the original 3' end of the splicing substrate. Note that all three 
of those RNAs contain the IVS 1-3 sequence (FIG. 39B, lines 2, 3 and 4). 
The only major products of inverse splicing that were not seen either when 
the substrate was 5' end labeled or when it was 3' end labeled were the 
circular form [K1(C)] and the linear form (K1) of the excised exon. 
The product denoted as IVS(Y) in FIG. 40A contained both the original 5' 
and 3' ends of the splicing substrate. Furthermore, that product migrated 
with IVS(Y) generated by inverse splicing of the control substrate, PY1 
(compare FIG. 38A, lanes 6 and 9). To confirm that product is IVS(Y), it 
was purified from an acrylamide gel and subjected to debranching as 
described above. As a control, IVS(Y) from PY1 was treated with the 
debranching activity. upon debranching a product that migrated with IVS 
1-3 was seen, along with a second product that was the expected size of 
5,6 (lane 2). These same two products were seen when IVS(Y) from PY8 (lane 
4) or from PY9 (lane 6) was treated with the debranching activity, 
confirming that the product was indeed IVS(Y). 
The K1.sub.d (IVS 1-3) product was one of the two unexpected products. As 
part of the characterization of that product, we asked whether it was 
sensitive to debranching. We found that it was not sensitive to 
debranching (compare the treated sample [FIG. 40B, lane 8] to the 
untreated sample [FIG. 40B, lane 7]). Finally, as expected, the IVS 1-3 
RNA was not sensitive to debranching (compare the treated sample [FIG. 
40B, lane 9] to the untreated sample [FIG. 40B, lane 10]). 
RNAase protection was used to analyze the four RNAs shown in lines 5-8 of 
FIG. 39B. The anti-sense probe that was used was complementary to the last 
140 nt of the K1 exon as well as to the first 29nt of group 11 intron 
domain 1. This probe was generated by amplification of a 168nt region of 
the K1 exon of pY9 using anti-sense oligonucleotide I5-29 
(5'-TATTATTTATGATAACTTTCAGACC-3' [SEQ ID NO:16]) and sense oligonucleotide 
K1.Cir.2 (5'-GGCCAGACGCCATCAGGCTG-3' [SEQ ID NO:15]). The product was 
agarose gel purified and cloned into pCR 2.1.RTM.Vector (Invitrogen.RTM.TA 
Cloning Kit Version D). Sequencing was carried out with Sequenase.RTM. 
(Amersham) to positively identify a clone with the PCR product in the 
anti-sense orientation with respect to the pY9 sequence. 
RNAse protection studies were preformed as follows: pK1-ANTI-probe was cut 
with SpeI, transcribed and randomnly labeled with [a-.sup.32 P]UTP, and 
acrylamide gel purified. pY9 RNA was spliced in 1.5 M (NH.sub.4).sub.2 
SO.sub.4 and fractionated on a denaturing 4% acrylamide gel. The 
(IVS5,6)K1, IVS(5,6)K1.sub.u, K1 (C), and linear K1 products were purified 
and hybrizided to the probe over night at 37.degree. C. in hybridization 
buffer: 40 mM pipes (pH 6.4), 1 mM EDTA (pH 8.0), 0.4 M NaCl, 8% 
formamide. The single stranded RNAs were degraded with RNAse A and RNAse 
T.sub.1. The reaction proteins were denatured with 20% SDS and Proteinase 
K followed by phenol/chlorophorm extraction. The double stranded RNA was 
precipitated, dissolved in water and run on a 4% acrylamide gel. 
The probe protected a 140 nt region of: (IVS 5,6)K1, K1(C) and K1, 
indicating that all three RNAs contained the last 140 nt of the K1 exon, 
but lacked the intron domain 1 sequences (see FIG. 40C, lanes 2,4 and 5, 
respectively). The probe protected a 96 nucleotide region of the (IVS 
5,6)K1.sub. RNA, consistent with the hypothesis that RNA results from a 
cryptic cleavage event that occurs within the K1 exon just downstream of a 
sequence (5'-CGGGGA) that is complementary to the EBS I sequence of both 
the PY8 and PY9 intron. 
As part of our characterization of the K1.sub.d (IVS 1-3) and the (IVS 
5,6)K1.sub.u RNAs, we measured the approximate length of both RNAs by 
comparing the electrophoretic mobility of those two RNAs with known RNA 
size standards (data not shown). We calculated that the length of K1.sub.d 
(IVS 1-3) is approximately 760 nt, while the length of (IVS 5,6)K1.sub.u 
is approximately 300 nucleotides. Thus, the combined lengths of those RNAs 
(1060 nt) is approximately the length of the PY9 (or the PY8) precursor 
(1070 nt). Furthermore, our end labeling experiments (above) showed that 
(IVS 5,6)K1.sub.u contains the original 5' end of the precursor RNA while 
K1.sub.d (IVS 1-3) contains the original 3' end of the precursor RNA. 
These data, along with the RNAase protection data (FIG. 40C, lane 2) 
suggested that (IVS 5,6)K1.sub.u was the upstream product of a cryptic 
cleavage event that occurs in the K1 exon, while K1.sub.d (IVS 1-3) is the 
downstream product of that same cleavage event. To test that hypothesis, 
we used primer extension to map the 5' end of K1.sub.d (IVS 1-3). The 
primer was complementary to nucleotides 5 to 29 of the intron. Extension 
of the primer produced a 77 nt extension product, as expected if K1.sub.d 
(IVS 1-3) is the downstream product that is generated by cleavage of the 
K1 exon just downstream of the 5'-CGGGGA sequence. 
The K1(C) RNA was further characterized by reverse transcription and PCR. 
Primers were designed that would amplify the K1 splice point sequence. 
Note that a pair of primers that is designed to amplify the circular K1 
exon [K1(C)] will yield an amplification product when used in RT/PCR with 
K1(C). The two primers face toward each other, and the sequence that lies 
between them is amplified. In contrast, that same pair of primers will not 
amplify linear products that contain a single K1 exon (such as (IVS 
5,6)K1) because the two primers face away from each other on the linear K1 
exon. 
RT/PCR analysis of K1(C) from both PY9 (FIG. 41A, lane 4) and PY8 (FIG. 
41A, lane 5) produced the same 244 base-pair amplification product. That 
product was not observed when either (IVS 5,6)K1 (FIG. 41A, lane 2) or 
(IVS 5,6)K1, (FIG. 41A, lane 3) was used as a template in an RT/PCR 
reaction. The 244 bp product was the size expected of a PCR product 
generated by amplification across the splice point of K1(C). 
For both PY8 and PY9, the amplified product was cloned into a plasmid 
vector and the nucleotide sequence of the splice point was determined. The 
splice point was sequenced in a total of 8% independently isolated PY8 
clones and 10 independently isolated PY9 clones. In every case, the 
expected splice point sequence (5'-CCT/TGG) was observed (FIG. 41B). 
Note that all of the inverse splicing substrates used in this study (PY1, 
PY8 and PY9) lack intron domain 4. Previous work has shown that domain 4 
is not required for cis or trans splicing. Furthermore, the deletion of 
domain 4 (in PY1) does not seem to appreciably reduce the rate of inverse 
splicing (relative to the rate of cis splicing; see Table I). However, it 
was clear in previous studies that the deletion of domain 4 slows the rate 
of the second step of cis and trans splicing. Our current results clearly 
show that, for PY8 and PY9, when the first step of inverse splicing occurs 
by hydrolysis, the second step of splicing is slow (i.e. the products of 
the first step, IVS 1-3 and IVS 5,6)K1, accumulate to high levels). The 
above observations suggest that domain 4 sequences are also important for 
the rate of the second step of inverse splicing, but that the importance 
of the domain 4 sequences was not apparent until the yeast exon sequences 
of PY1 were replaced with human exon sequences (to yield PY8 and PY9). 
Thus, it may be desirable to add domain 4 sequences back to PY8 and PY9 
RNAs in order to increase the rate of inverse splicing by these 
precursors. 
TABLE I 
______________________________________ 
Overall Rates of Reaction from Semi-Log Plots 
Splicing Buffer 
KCI NH.sub.4 CI 
(NR.sub.4).sub.2 SO.sub.4 
Plasmid (min.sup.-1) (min.sup.-1) (min.sup.-1) 
______________________________________ 
Data Summary 
WT (10') 0.0509 0.0408 0.0442 
100% 80% 87% 
INV (10') 0.0425 0.0498 0.0242 
83% 98% 48% 
pY8 (60') 0.01069 0.005735 0.0008356 
21% 11% 2% 
pY9(60') 0.001192 0.005035 0.001745 
23% 10% 3% 
______________________________________ 
Splicing Buffer 
KCI NH.sub.4 CI (NH.sub.4).sub.2 SO.sub.4 
Plasmid Y 1-3 Y 1-3 Y 1-3 
______________________________________ 
Ratio of Y-Branch to IVSI-3 
INV (60') 0.302 1.599 2.233 
0.2818 1.020 0.4454 0.3096 0.7914 0.3583 
pY8 (120') -- 0.9008 1.722 
0.1634 0.1820 0.05785 0.03364 
pY9 (120') -- 0.8879 2.019 
0.1801 0.2070 0.09395 0.04711 
______________________________________ 
EXAMPLE 14 
Sequence Requirements for the Inverse Splicing Reaction 
Inverse splicing provides an efficient means for production of Y-branched 
ribozymes. As shown above in Example 13, the group II intron can catalyze 
inverse splicing even when the EBS1 and/or EBS2 sites in the intron have 
been altered. Such inverse splicing reactions produce engineered ribozymes 
of altered specificity. We analyzed the sequence requirements for the 
inverse splicing reaction by engineering inverse splicing substrates with 
different EBS1, IBS1, EBS2, and IBS2 sequences. 
Nine different inverse splicing precursors, PY1 through PY9, were produced 
(FIG. 42) PY1 through PY7 contained yeast exon sequences; PY8 and PY9 
contained human sequences. PY1, PY8, and PY9 are described above in 
Example 13; the others were produced by site directed mutagenesis of 
encoding plasmids. 
The EBS1-IBS1 and EBS2-IBS2 interactions for each of the nine precursors 
are shown in FIG. 42. 
Yeast exon sequences are shown in red. Standard Watson-Crick base pairs are 
indicated by dashes; G-U pairs are indicated by dots. FIG. 42 summarizes 
the base substitutions that were made using site-directed mutagenesis. 
Base substitutions shown in blue (PY2-PY4) were made to produce Y-branched 
ribozymes targeted to insert in the Fn txt RNA (Mikheeva et al. (1996) 
PNAS 93:7486-7490). At certain positions, the yeast and human exon 
sequences are identical. Thus, it was not necessary to mutate these 
nucleotides and they are shown in red (see for example nucleotide minus 
five of the PY2 exon). Base substitutions in PY5-PY7 are shown in green. 
For PY8 and PY9, the human exon sequences are shown in brown. For each 
precursor, the calculated free energy (Freier et al. (1986) PNAS 
83:9373-9372) of (i) EBS1-IBS1, (ii) EBS2-IBS2, and (iii) the sum of these 
calculated values, are shown. The relative efficiency of splicing is also 
shown, along with an autoradiogram of representative time course data 
(incubation times are in minutes). The time course experiments were 
conducted under transesterification conditions, in buffer containing 1.5 M 
(NH.sub.4).sub.2 SO.sub.4 (Jarrell et al. (1988) J Biol. Chem. 
263:3432-3439); the most abundant product that is seen in the 
autoradiograms is the excised Y-branched intron. Analogous time courses 
were performed in NH.sub.4 Cl and KCl buffers. A summary of the reaction 
rate, amount of Y-branched intron, and amount of linear intron observed 
with each precursor in each salt condition is presented in FIG. 43. 
We tested whether the Y2 ribozyme would in fact integrate into the Fn txt 
target RNA (note that the EBS1 site of Y2 is homologous to the target, but 
the EBS2 site is not). We found that Y2 integrates into the Fn txt less 
efficiently than Y1 integrates into its natural target, E5-E3 (FIG. 44). 
The targets and ribozymes were each present at approximately equimolar 
concentrations (about 1 .mu.M each) (FIG. 44; P, products; Y, Y-branched 
ribozymes; S, substrates). The specific activity of the ribozymes was made 
lower than that of the substrates to ensure that the signal from unreacted 
ribozyme did not obscure the signal from product. Targets and ribozymes 
were mixed and incubated in splicing buffer for 2 hours at either 
4.degree. C. (lanes 1 and 5), 25.degree. C. (lanes 2 and 6), 37.degree. C. 
(lanes 3 and 7), or 45.degree. C. (lanes 4 and 8). The Y2 and Fn txt RNAs 
were fractionated in lanes 1 through 4; the Y1 and E5-E3 RNAs were 
fractionated in lanes 5 through 8. Products the size of Fn(1-3)(1035 nt, 
lane 4) and E5(1-3) (1005 nt, lanes 6-8) were observed. The yield of 
E5(1-3) was clearly much greater than the yield of Fn(1-3). The efficiency 
of integration of Y2 might increase if its EBS2 site were also made 
perfectly homologous to the Fn txt target RNA. 
Since data in the literature indicate that the EBS2-IBS2 interaction is not 
essential for group II intron cis splicing (Jacquier et al. (1987) Cell 
50:17-29), we decided to change the pY2 EBS2 site, to make it homologous 
to the Fn txt target. We did not also change the IBS2 site of pY2. Our 
assumption was that the EBS2-IBS2 interaction would not be critical for 
inverse splicing since this interaction is not critical for cis splicing. 
We used site-directed mutagenesis to change the EBS2 site of pY2. This 
experiment produced the pY3 plasmid. The five nucleotides of the EBS2 site 
that were changed are shown in blue in FIG. 42, line 3. Note that the 
EBS2-IBS2 pairing of PY3 has an estimated free energy of about -1.0 
kcalorie. Also note that the PY3 transcript splices about 30-fold less 
efficiently than does PY1 (see FIG. 42). We hypothesized that the splicing 
defects of PY3 would be rescued if its IBS2 sequence was changed so that 
base-pairing between EBS2 and IBS2 was fully restored. Thus, we generated 
the pY4 plasmid. Consistent with our hypothesis, transcripts from pY4 
spliced in vitro as efficiently as did transcripts from pY2 (FIG. 42). 
In a parallel set of experiments, three plasmids (pY5-pY7) were constructed 
from which Y-branched ribozymes (Y5-Y7), designed to insert into the Prot 
txt target (Mikheeva et al. (1996) PNAS 93:7486-7490, could be produced. 
As can be seen, PY5, PY6 and PY7 all spliced with approximately equal 
efficiency. One interpretation of our results (comparing PY3 with PY6) is 
that when the EBS1-IBS1 pairing is strong (PY6) then the EBS2-IBS2 
interaction is not very important. However, when the EBS1-IBS1 pairing is 
weaker (PY3) the EBS2-IBS2 pairing is important. 
Finally, plasmids pY8 and pY9 contain human exon sequences precisely 
flanked by group II intron sequences (all yeast exon sequences have been 
replaced with human exon sequences). The plasmids were made using standard 
recombinant DNA techniques, including site-directed mutagenesis. PY8 has a 
two base-pair EBS2-IBS2 interaction, while in PY9 the EBS2-IBS2 
interaction has been fully restored. We made the PY8 and PY9 precursors in 
order to determine whether group II introns can be used to catalyze 
accurate production of particular circular human exons by inverse 
splicing. PY8 and PY9 splice more slowly that does PY1 (FIG. 42). This may 
indicate that the human exon sequences slow the rate of group II intron 
splicing. Alternately, the rate may be slowed because the human exon 
sequence is shorter than the yeast exon sequence (267 nt vs. 591 nt). That 
PY9 splices about two-fold more rapidly that does PY8 may result from the 
fact that PY9 has a stronger EBS2-IBS2 pairing. 
Taken together, these results suggest that the wildtype EBS1-IBS 1 pairing 
is the optimal pairing, and that the EBS2-IBS2 interaction can be 
important for inverse splicing (compare PY3 with PY4). However, the 
EBS2-IBS2 pairing is not always critical for inverse splicing (compare PY6 
with PY7). Finally, when the yeast exon sequences are completely replaced 
with human exon sequences, accurate inverse splicing occurs, although the 
rate of splicing is slowed. 
EXAMPLE 15 
Mammalian Nuclear Pre-mRNA Introns can Mediate Circularation of Exonic 
Sequences 
The BGINV plasmid (SEQ ID NO:17) was derived from plasmid HBT7. HBT7 has 
the first intron of the human .beta.-globin gene, flanked by .beta.-globin 
exon 1 and 2 sequences, cloned into the psp73 vector. To construct BGINV, 
HBT7 was cut at the unique BbvI site in the intron and at the unique 
BamnHI site, downstream of Exon 2. The ends were made blunt with klenow 
fragment. The DNA was diluted and ligase was added. A clone was isolated 
(BGUS) that has exon 1 and intron sequence, up to the filled BbvII site. 
In a separate experiment, HBT7 was cut with HindlI and BbvI, the ends were 
filled in, and the DNA was diluted and ligated. A clone was isolated BGDS, 
that had intron sequence, beginning with the filled BbvI site, followed by 
exon 2 sequences. BGDS was cut with XhoI and SmaI and the fragment 
containing the intron and exon 2 sequences was gel purified. This DNA was 
ligated into BGUS that had been cleaved with XhoI and PvuII, to yield 
BGINV. The inverse-.beta.-globin RNA can be transcribed from this plasmid 
in vitro using T7 polymerase. 
BGINV was cut with EcoRI and RNA was transcribed in vitro using T7 
polymerase. In vitro splicing reactions were performed as described in 
Hannon et al. (Hannon et al. (1990) Cell, 61:1247-1255), except that 
mammalian extract was used. The extract was prepared by the method of 
Dignam et al. (Dignam et al. (1983) Nucl. Acids Res. 11:1475-1489). 
Splicing extract is also commercially available (Promega cat.# E3980). 
Spliced products were separated by polyacrylamide gel electrophoresis and 
visualized by autoradiography. 
The transcription reaction that generated the RNA that was used to create 
the circular precursor included GMP (final concentration, 0.8 mM); this 
was to ensure that some of the RNA transcripts initiated with GMP, instead 
of GTP, since a 5' phosphate is a substrate for ligase (while a 5' 
triphosphate is not). The transcript was purified from a polyacrylamide 
gel. Circular precursors were generated using a DNA oligonucleotide 
(5'-CGAGGCCGGTCTCCCAATTCGAGCTCGGTAC [SEQ ID NO:18]) to bring the ends of 
the RNA together, followed by the addition of DNA ligase to covalentlyjoin 
the ends (Moore et al. (1992) Science 256:992-997). The circular precursor 
was purified from a polyacrylamide gel. In vitro splicing reactions were 
done as described above. 
The circular exon product was observed and characterized. This RNA was gel 
purified and a cDNA copy generated using the CIR-1 primer 
(5'-GAGTGGACAGATCCCCAAAGGACTC [SEQ ID NO:19]) which is specific to exon 2 
sequences. The cDNA was amplified by PCR using the CIR-1 and CIR-2 
(5'-GTGATGGCCTGGCTCACCTGGACAA [SEQ ID NO:20]) oligonucleotides as primers. 
A 145 nt product was observed. This amplification product is the expected 
size of a product generated from circular spliced exons. 
The branched intermediate (generated by the first step of the reaction) was 
also observed and characterized. It was gel purified and treated with HeLa 
debranching enzyme (Ruskin et al. (1985) Science 229, 135-140). This 
treatment increased the rate of migration of the RNA through a denaturing 
polyacrylamide gel such that it migrated as a 553 nt RNA, consistent with 
the assignment of the product as the lariat intermediate. 
V. Reagents for Molecular Biology 
Molecular cloning of DNA currently relies heavily on restriction enzymes 
and DNA ligase to specifically cut and join molecules. The 
reverse-splicing introns or "ribozymes" of the present invention can 
fulfill these two functions; they can both cut and join RNA molecules, and 
thus can serve as useful tools for nucleic acid manipulation. In similar 
fashion to the activation of an exon by addition of flanking intronic 
fragments through the reversal of splicing the recombinant RNA technology 
described herein involves attacking a target RNA molecule with an intronic 
molecule and, by the reversal of splicing, cleaving the target into two 
pieces while simultaneouslyjoining specific intron sequences to the 
cleaved ends of the target molecule. The newly formed exon construction 
can be purified, and appropriate exons ligated to each other through 
trans-splicing mediated by the intronic fragments. Alternatively, these 
recombinant RNA molecules can be cloned into a plasmid, and fresh RNA 
transcripts generated from these plasmids, with these second generation 
transcript being used in a trans-splicing reaction (see Example 1 and 
Mikheeva et al. (1996) PNAS 93:7486-7490). Thus, cleavage and ligation 
functions similar to those provided by restriction enzymes and ligase can 
be provided by RNA trans-splicing. 
DNA restriction enzymes and DNA ligase are so routinely used for nucleic 
acid manipulation that the limitations of these reagents are seldom 
considered. Restriction enzymes typically recognize and cleave specific 
DNA sequences that are 4 to 6 basepairs in length. Although there are 
theoretically 4,096 different possible restriction enzymes that recognize 
6 basepair sequences, only 78 such enzymes with distinct specificities are 
commercially available. One reason that most possible specificities are 
unavailable is that it is not feasible to engineer the sequence 
specificity of a restriction enzyme. Instead, micro-organisms must be 
identified that naturally produce enzymes with novel specificities. Often, 
it is difficult to obtain large quantities of pure active enzyme from 
these natural sources, leading to the second limitation, which is that 
restriction enzymes are often impure and the enzyme concentration is low. 
A third limitation is that certain classes of DNA sequences are not 
recognized by any known restriction enzyme. For example, there are no 
known enzymes that recognize sequences comprising only of A and C 
nucleotides, such as 5'-AACCAA. A fourth limitation is that DNA ligase 
only joins DNA molecules with compatible ends, making it often necessary 
to fill in or degrade 5' or 3' overhangs on DNA molecules before they can 
be joined by ligation. Finally, yet another limitation is that DNA 
ligation reactions are often not directional, leading to the generation of 
recombinant clones with inserts in the wrong orientation. 
In contrast, the advantages of this system are that potentially any 3-8 nt 
sequence can be specifically targeted. Accordingly, whereas restriction 
enzymes are much more limited, recognizing only a small subset of, for 
example, the 4,096 possible 6 nt sequences present in DNA, the subject 
ribozymes can be generated for each of the 4,096 different sequences. 
Indeed, under appropriate reaction conditions, the efficiency of the 
reaction can be greatly influenced by the EBS2-IBS2 interation such that 
the specificity of the ribozyme is effectively 12-16 basepairs. 
Consequently, in the instance of the ribozyme which recognizes a specific 
12 nucleotide target sequence, over 16 million different specifities are 
possible and can be assessed by the present invention. Note that because 
G:U basepairs are allowed basepairs are allowed in RNA, the specificity of 
a given inventive engineered ribozyme may be broader (especially under 
appropriate reaction conditions) than the particular sequence precisely 
targeted. 
Moreover, in contrast to restriction enzymes which typically require 
palindromic sequences that may introduce ambiguity into the orientation of 
DNA sequences inserted at a restriction endonuclease cleavage site the 
subject ribozymes can be orientation specific. In addition, once an RNA is 
followed by, or preceded by, the correct intron sequences, any upstream 
molecule can be joined to any downstream molecule (see, for instance, 
Examples 19 and 20). In contrast, when molecular cloning is done with 
restriction enzymes, only molecules with compatible ends can be joined; 
for example, a molecule with EcoRI ends cannot be joined to a molecule 
with HindIII ends without first filling in the ends. Furthermore, 
molecules that are joined by trans-splicing are "seamless". That is, 
recognition sites do not have to be engineered into the target molecules 
in order to cleave and ligate the target molecule. Instead, the ribozyme 
is engineered to match the target. For instance, a library of 
reverse-splicing ribozymes can be generated to comprise every possible 6 
nucleotide combination by manipulating intron sequences which interact 
with the "exon" target (e.g. the IBS1 for group II, and the IGS for group 
I). Thus, sequences can be preciselyjoined without adding, deleting or 
changing any nucleotides. Finally, for the autocatalytic introns, no 
enzymes need be added in order to catalyze the forward or reverse 
reactions. Instead, the RNAs are incubated together in a simple salt 
solution and other appropriate ions and the recombinant molecules are 
generated. 
Accordingly, one aspect of the invention pertains to a preparation of a 
reverse-splicing intron which comprises two or more fragments of 
autocatalytic introns and catalyzes integration of at least a portion of 
the reverse-splicing construct into a substrate ribonucleic acid by a 
reverse-splicing reaction. For example, the autocatalytic intron fragments 
can be derived from one or more group II introns, and preferably are 
derived with exon binding site which have been altered by recombinant 
mutagenesis. In another illustrative embodiment, the autocatalytic intron 
fragments are derived from group I introns. Again, the specificity of the 
intron is preferably altered by recombinant mutagenesis of the internal 
guide sequence of group I intron fragments. 
In one particular embodiment, as is apparent from the description 
throughout the present application, where the inventive reverse-splicing 
intron is derived from a group II intron, it may comprise a first segment 
having a 5' portion of a group II intron, which 5' portion includes an 
exon binding site; and a second segment comprising a 3' portion of a group 
II intron, which 3' portion includes a V motif, a branch site acceptor 
forming a phosphodiester bond with the 5' end of the first segment, and a 
nucleophilic group at the 3' end of the second segment for 
transesterifiing a phosphodiester bond of a ribonucleic acid. By this 
arrangement, the first and second segments together form an autocatalytic 
Y-branched intron which catalyzes integration of at least the first 
segment of the reverse-splicing intron into a substrate ribonucleic acid 
by a reverse-splicing reaction. In an exemplary embodiment, the 5' portion 
of the group II intron includes intron domains 5 and 6, and the 3' portion 
of the group II intron includes intron domains 1-3. It will be understood 
that the reverse-splicing construct can be a Y-branched lariat form of the 
group II intron, e.g., the first and second segments are contiguous via a 
covalent bond other than the phosphodiester bond formed with said branch 
site acceptor, or can be in the form of a Y-branched discontinuous intron, 
e.g., the first and second segments are covalently attached only at the 
branch site acceptor. Alternatively, the reverse-splicing construct can be 
linear. 
The reverse-splicing intron can be represented by the general formula: 
##STR1## 
wherein 
(IVS1-3) represents a 5' portion of a group II intron. 
(IVS5,6) represents a 3' portion of a group II intron, which position 
includes a branch site acceptor. 
2'-5' represents a phosphodiester bond formed between a branch site 
acceptor of (IVS5,6) and the 5' end of (IVS1-3), and 
A, if present, represents a phosphodiester bond between a 3' end of 
(IVS1-3) and a 5' end of (IVS5,6), wherein (IVS1-3) and (IVS5,6) together 
form an autocatalytic Y-branched intron which catalyzes integration of at 
least the (IVS1-3) fragment, if discontinuous with (IVS5,6), into a 
substrate ribonucleic acid by a reverse-splicing reaction. 
In preferred embodiments, the exon binding site, e.g. EBS1 and/or EBS2, is 
altered (or deleted in certain instances) by recombinant mutagenesis. As a 
result, the exon binding site can be chosen to provide specific 
integration into a substrate ribonucleic acid at a selected intron binding 
site, such that the effective exon binding sequence can be from 3-16 
nucleotides in length. Such altered reverse-splicing introns are referred 
to herein as "engineered ribozymes". 
In yet another preferred embodiment, the reverse splicing intron is 
provided as a substantially pure preparation. By "substantially pure" it 
is meant that the construct has been isolated from, or otherwise 
substantially free of other polynucleotides, especially exonic sequences, 
normally associated with the intron. The term "substantially pure" or 
"substantially pure or purified preparations" are defined as encompassing 
preparations of the reversing splicing introns having less than 20% (by 
dry weight) contaminating protein or polynucleotides, and preferably 
having less than 5% contaminating protein or polynucleotides. By 
"purified", it is meant, when referring to a nucleic acid construct of the 
present invention, that the indicated molecule is present in the 
substantial absence of other biological macromolecules, such as other 
proteins or polynucleotides. The term "purified" as used herein preferably 
means at least 80% by dry weight, more preferably in the range of 95-99% 
by weight, and most preferably at least 99.8% by weight, of biological 
macromolecules of the same type present (but water, buffers, and other 
small molecules, especially molecules having a molecular weight of less 
than 3000, can be present). The term "pure" as used herein preferably has 
the same numerical limits as "purified" immediately above. "Isolated" and 
"purified" do not encompass either natural materials in their native state 
or natural materials that have been separated into components (e.g. in an 
acrylarnide gel) but not obtained either as pure (e.g. lacking 
contaminating proteins or polynucleotides, or chromatography reagents such 
as denaturing agents and polymers, e.g. acrylamide or agarose) substances 
or solutions. 
To further illustrate, a group II intron or portion thereof can be used to 
specifically cut and join RNA molecules (see Example 1, above). As 
described above, the group II intron splicing reaction is reversible. If 
an intron lariat, a product of the forward reaction, is incubated with 
spliced exons at high RNA concentration under the reaction conditions used 
for the forward reaction, the intron specifically inserts into the spliced 
exons, thus regenerating the precursor RNA (see FIG. 2). Likewise, as 
illustrated in FIG. 10, a Y-branched form of the intron, generated for 
example by an inverse splicing reaction, can also insert into spliced 
exons. When a Y-branched intron, such as the illustrated (VS5,6).sub.2'-5 
'(IVS 1-3) lariat, is used in a reverse-splicing reaction, the exon target 
is cleaved into two pieces. The upstream piece becomes joined to intron 
domains 1-3 and the downstream piece becomes joined to intron domains 5 
and 6. 
The 3-8 nucleotide EBS1 site on the ribozyne is the primary determinant of 
the specificity of the reverse reaction for group II introns. In the 
reverse reaction, EBS1 selects the site of integration by hydrogen bonding 
to it. The intron is subsequently inserted just downstream of this target 
sequence. By changing the nucleotide sequence of EBS1, the ribozyme can be 
targeted to insert downstream of any specific 3-8 nt sequence. Moreover, 
the manipulation of the EBS2:IBS2 interactions can also influence the 
efficiency of splicing and provide even greater specificity to the 
insertion site (e.g. by expanding the recognition sequence to, for 
example, 10-14 nucleotides; see Example 14, above). Likewise, manipulation 
of the IGS, and other secondary intron exon contacts analogous to EBS2, 
the specificity of a group I reverse splicing ribozyme, such as (IVS 
P1-P6.5-P10) can be controlled. 
FIG. 45 depicts a further embodiment illustrating how an reverse-splicing 
ribozyme, such as the group II lariat IVS, can also be used to cleave and 
ligate target RNA molecules. The site directed mutagenesis is the same as 
described above (the EBS 1 and IBS 1 sequences are changed). The lariat 
ribozyme is generated by the forward reaction. The reverse reaction yields 
a single molecule with the intron specifically inserted in it. A cDNA copy 
is made by reverse transcriptase. Two different sets of PCR primers are 
used to amplify either the upstream portion of the interrupted target 
molecule, plus intron domains 1-3 or to amplify domains 5 and 6 and the 
downstream portion of the target molecule. Each of these amplified DNAs 
can be cloned into a plasmid to generate the same two constructs shown in 
FIG. 46. 
In another illustrative embodiment, FIG. 47 depicts a method by which the 
present trans-splicing constructs can be used to manipulate nucleic acid 
sequences into a plasmid such as a cloning or expression vector. In such a 
scheme, the plasmid sequence is itself an exon being flanked at each end 
by intronic fragments capable of mediating a trans-splicing reaction. For 
example, as shown in FIG. 47, the plasmid can be generated as an RNA 
transcript comprising the backbone sequences of the plasmid, flanked at 
the 5' end with the group II domains 5 and 6, and at the 3' end with the 
group II domains 1-3. To generate such a transcript, a pre-plasmid can be 
utilized in which the 5' and 3' flanking sequences are joined with an 
intervening sequence including a T7 RNA promoter sequences and 
endonuclease cleavage site. The plasmid is linearized by cleavage at the 
endonuclease-sensitive site, and the linearized plasmid transcribed to RNA 
using standard techniques. 
The nucleic acid sequences to be cloned into the plasmid are generated to 
similarly include flanking group II intron fragments. Mixing the two 
transcripts under trans-splicing conditions will therefore result in 
ligation of the nucleic acid of interest into the plasmid, in the 
appropriate orientation and at the correct site. Such a method is 
particularly amenable to the cloning of the above-described combinatorial 
gene libraries into replicable expression vectors. Furthermore, this 
trans-splicing technique of sub-cloning can be used effectively in random 
mutagenesis applications. For instance, the nucleic acid of interest can 
be first treated with actinic acid such that a discrete number of base 
modifications occur, and then ligated into the plasmid. 
In addition to the RNA manipulations described herein, the inventive 
engineered ribozymes can be utilized to cleave and ligate DNA molecules. 
Many group II introns recognize single-stranded DNA, as an alternative to 
RNA, as an integration target (see, for example Herschlag (1990) Nature 
344:405-409; Robertson et al (1990) Nature 344:467-468; Morl et al (1992) 
Cell 70:803-810; Zimmerly et al (1995) Cell 83:529-538); some also 
recognize double-stranded DNA (see, for example Yang et al (1996) Nature 
381:332-335; Ziimmerly et al (1995) Cell 83:529-538). Accordingly, 
engineered ribozymes of the present invention may be integrated into DNA 
targets and linked to DNA "exons" than can then be linked to one another 
by splicing reactions, as described herein for RNA exons. Such inventive 
DNA manipulations as described in more detail above, in the section 
entitled "DNA Recombination". 
Another aspect of the invention pertains to a kit for generating a 
Y-branched ribozyme of a particular specificity. In general, the kit can 
feature an expression vector containing a gene, whose expression product 
will give rise to a Y-branched ribozyme of the present invention. For 
instance, the transcript illustrated by FIG. 46, and described in further 
detail in Example 16 below, can be used to generate the Y-branch ribozymes 
of the present invention (see also above section on "Circular RNA 
Transcript"). For instance, a vector can be constructed containing such a 
gene having unique restriction enzyme sites immediately 5' to the IBS2 
sequence and 3' to the EBS1 sequence, such that the EBS1 and (optionally) 
the EBS2 sequences can be altered by insertion of oligonucleotide 
cassettes. In another embodiment, the restriction sites can be placed 
immediately flanking the EBS1-EBS2 sites and another set of restriction 
sites used to flank the IBS1-IBS2 site such that 2 oligonucleotide are 
used to alter the EBS1 and EBS2 specifities. Where a continuous Y-branched 
lariat structure is desired, a construct as shown in the forward reaction 
of FIG. 2, e.g., exonl -intron-exon2, can be used and appropriately 
manipulated to yield a certain specificity for the EBS1 and EBS2 
recognition sequences. 
Various considerations go into the design of an inventive engineered 
ribozyme having a desired specificity. For example, as mentioned above, 
both EBS1 and EBS2 may be designed to interact specifically with a 
selected target site. Alternatively, the EBS2 may have its wild-type 
sequence, or may be deleted altogether. Engineered ribozymes with targeted 
EBS1 and EBS2 sequences are expected to insert more efficiently (and 
specifically) into their target sites than are those ribozymes having only 
an engineered EBS1 site. Regardless of whether EBS1 alone or both EBS1 and 
EBS2 have been engineered, however, it should be borne in mind that G:U 
base pairs are allowed in RNA, so that ribozymes with G or U residues in 
their EBS1 and/or EBS2 sites may have broadened specificity. 
As an alternative to the above-described splicing methods for generating 
engineered ribozymes of the present invention automated systems can be 
provided for scale-up production of quantities of the subject 
inverse-splicing constructs. An exemplary approach for high throughput 
production in commercial scale synthesis of the subject ribozymes is shown 
in FIG. 48. As can be seen with reference to that Figure, an automated 
system can be developed that relies on the fact that three pieces of the 
IVS(1-3) fragment (which flank EBS1 and EBS2 sequences) can be identical 
in each intron, and can consequently be produced in bulk by RNA synthesis 
procedures. For instance, T7 transcription processes have been scaled up 
to permit purification of milligram or greater quantities of RNA. 
Alternatively, such RNAs can be over produced in a bacterial or fungal 
host. Likewise, the EBS1 and EBS2 sequences can be generated easily by RNA 
solid phase synthesis. Similarly, two DNA oligonucleotides (1 and 2) can 
be synthesized by standard automated approaches. Each of the two DNA 
oligos is homologous to one of the EBS sequences, and to the flanking 
intron sequences. The mixture is annealed to produce DNA/RNA duplex, and 
the nicks in the RNA strand can be sealed using a DNA ligase. The DNA 
oligonucleotides are then removed by treatment with DNase I. Similar 
procedures using DNA/DNA pairs can be carried out in place of the use of 
restriction sites described above. 
Rather than being produced one-at-a-time, as described above, inventive 
engineered ribozymes may be produced in as variegated populations. Because 
the genetic information that encodes the ribozyme and the enzymatic 
activity of the ribozyme reside in the same molecule, in vitro genetics 
can be used to produce a large number of engineered ribozymes, from which 
individual ribozymes may be selected if desired. For example, as shown in 
FIG. 49, a reverse-splicing target can be prepared that is enriched for G 
and U nucleotides. A population of ribozymes is then produced, in which 
the EBS 1 site is comprised of random combinations of A and C nucleotides 
(preferably, the EBS2 site has been deleted). The ribozyme population is 
incubated with the target under splicing conditions and, after the 
reversal reaction has been performed, DNA copies of the products are 
obtained by reverse transcription and PCR. The amplified DNA is cloned 
into a plasmid vector and individual clones are analyzed by DNA sequences 
to determine the sequence of the EBS1 site and of the last six nucleotides 
in the exon. 
Still another embodiment of the present invention pertains to a library of 
reverse-splicing introns comprising a variegated population of Y-branched 
group II introns. In preferred embodiments, the variegated population is 
characterized as including at least 25 different Y-branched group II 
introns of unique specificity, more preferably at least 100 different 
Y-branched group II introns of unique specificity, and even more 
preferably from 10.sup.3 to 10.sup.6 different Y-branched group II introns 
of unique specificity. In one particular embodiment, 64 ribozymes that 
have only A and C residues in their EBS1 sites and that lack EBS2 sites, 
are prepared. Without wishing to be bound by any particular theory, we 
suggest that such ribozymes may have particularly high specificity due to 
the absence of Gs and Us from the EBS1 site. 
In another particular embodiment, a set of 100 ribozymes is constructed 
with specificities representing all possible pairwise combinations of the 
following triplets: ACA, CCC, CCA, AAC, AAA, ACC, CAA, CAC, UUU, and GGG. 
Because of the ability of RNA to form G:U basepairs, this collection of 
ribozymes would consist of 64 that are uniquely targeted to a 6-nt IBS1 
sequence; 32 that have affinity for 8 different IBS1 sequences, and 4 that 
have affinity for 64 different IBS 1 sequences, so that in total the set 
would have affinity for 576 different 6-nt sequences (1/7 of all possible 
6-nt sequences). 
EXAMPLE 16 
Use of Group II Y-branched Lariats as Endonucleases/ligase 
FIG. 46 is an exemplary illustration of the use of these reactions to 
generate recombinant molecules. The last six nucleotides of the 
(IVS5,6)E4,E5(IVS1-3) RNA, which was generated by in vitro transcription 
of pINV1, are ATTTTC. The EBS 1 sequence of the flanking intron fragment 
is GGAAAT. As described in Example 19 below, inverse splicing of RNA 
transcribed from pINV1 yields a Y-branched intron with a wild-type EBS I 
sequence (GGAAA. FIG. 46 shows a 404 nt RNA (TPA S,F) that includes coding 
information for the signal sequence and growth factor domain of the TPA 
cDNA clone. This transcript was generated from plasmid TPA-KS+ that had 
been cut with Sty I. The goal was to attack TPA S,F with a Y-branched 
riboz yme such that the ribozyme inserted downstream of the GTCAAA 
sequence that is present at the end of the growth factor domain. In order 
to use pINV1 to generate a Y-branched ribozyte capable of attacking the 
TPA S,F RNA, the EBS I and IBS I sequences of pINVI were changed by site 
directed mutagenesis. The IBS 1 sequence was changed to GTCAAA (that is, 
to the same sequence present in the PTA transcript that is to be 
attacked), and the EBS 1 sequence was changed to TTTGAC in order that it 
be complementary to the mutated IBS 1 sequence. RNA was transcribed in 
vitro from this altered plasmid (termed here GrII-SIG) and incubated under 
splicing conditions to yield the excised Y-branched molecule (SIG-Y). This 
Y-branched intron is identical to that derived from (IVS5,6)E5,E3,(IVS1-3) 
in Example 19, except the EBS 1 sequence is TTTGAC. This Y-branched 
ribozyme was tested for its ability to insert specifically into TAP S,F 
RNA. As diagramed in FIG. 46, this RNA was incubated with the 404 nt 
target RNA under splicing conditions. Specific reversal generates a 1047 
nt product that consists of the first 332 nt of the TPA-KS+ transcript 
ligated to intron domains 1-3. This 1047 nt product was gel purified and a 
cDNA copy was made by reverse transcription. The cDNA was amplified by PCR 
and cloned into a vector to yield plasmid SIG(IVS1-3). The smaller, 108 
nt, product consists of intron domains 5 and 6 ligated to 72 nt of TPA, 
S,F. A cDNA copy of the smaller product can likewise be made by reverse 
transcription, amplified by PCR, and the amplified product cloned into a 
vector to yield plasmid (IVS5,6)StyI. 
Following the success of the inverse splicing reaction, the role of the 
IBS2-EBS2 interaction was investigated with respect to efficiency of the 
reverse splicing reaction (see Example 14). Starting with the construct 
pINV1, oligonucleotide primer mutagenesis was used to alter the IBS 1 
sequence to CTGCTCC and the EBS1 sequence to GGAGCAG. The EBS2 sequence 
was changed by oligonucleotide directed mutagenesis to the sequence 
GGCACA, while the IBS2 sequence was changed to the corresponding TGTGCC, 
to yield the construct pY7. Surprisingly, the reaction efficiency for the 
Y-branched ribozymes was several orders of magnitude better in the 
reversal of splicing reaction involving both the EBS1 and EBS2 
interactions, relative to the Y-branch lariat having only a matched EBS1 
interaction with the target RNA. Thus, despite the indication in the 
literature that the EBS2-IBS2 interaction is not essential for Group II 
intron splicing, the present data would indicate that this interaction is 
much more important to the efficiency of the reversal of splicing 
reaction. Consequently, as set out above, the subject reverse splicing 
ribozymes can be generated to be solely dependant on the EBS1/IBS1 
interaction, e.g., by deletion or mismatch of the EBS2 with the target 
nucleic acid, or alternatively, can be generated to exploit all or a 
portion of the EBS2/IBS2 interaction by recombinantly engineering the 
sequence of the EBS2 sequence. 
As suggested above, discrete inverse splicing introns can be generated for 
each of the potentially 4096 different 6 base sequences. However, since 
G:U base pairing is permissible in RNA, certain EBS1 sequences can give 
rise to specificity for more than one IBS1 target sequence. For instance, 
and EBS I of GGGGGG or UUUUUU could, in the absence of any contribution 
from EBS2/IBS2 interaction (e.g. EBS2 deleted) have an affinity for 64 
different sequences. Accordingly, a library of 100 ribozymes with 
specificities that are derived from all pairwise combinations of the 
following triplets: ACA, CCC, CCA, AAC, AAA, ACC, CAA, CAC, UUU and GGG 
can be generated which recognize 576 different 6 nucleotide sequences. To 
illustrate, of this set of ribozyvmes, 64 would recognize one unique 
sequence each, 32 of the enzymes would recognize 8 different sequences 
each, and 4 would recognize 64 different sequences each. This library 
therefore represents, in specificity, approximately 1/7th of the total 
possible six nucleotide sequences, and would be expected, on average, have 
a corresponding recognition sequence (IBS1) approximately every 42 
nucleotides. In contrast, the 78 or so restriction enzymes, on average 
have recognition sequences every 320 or so basepairs. 
It is clear from this example that potentially any 4-8 nt RNA sequence can 
be attacked specifically by a Y-branch ribozyme that has been engineered 
to have the appropriate EBS 1 and (optionally) EBS2 sequence. The target 
molecule will be split into two pieces. Intron domains 1-3 will be ligated 
to the upstream piece, while domains 5 and 6 will be ligated to the 
downstream piece. Following reverse transcription and PCR, these 
recombinant molecules can each be cloned into a plasmid vector downstream, 
for example, of the 17 promoter. Synthesis of RNA from the plasmid will 
yield transcripts capable of trans-splicing. Thus, in the above example, 
the original 404 nt target RNA could be regenerated by trans splicing. 
Moreover, it is also true that trans-splicing can be used to join the TPA 
sequences of SIG(IVS1-3) to any other RNA that has intron domains 5 and 6 
upstream of it. The recombinant RNA molecule generated by such a 
trans-splicing reaction could be copied into cDNA, amplified by PCR and 
cloned into a plasmid vector. 
EXAMPLE 17 
Chromosome Disruption 
One application for engineered ribozymes of the present invention that are 
capable of partial integration into double-stranded DNA is as specific 
chromosome disruption agents. For example, FIG. 50 depicts an embodiment 
of the invention is which a ribozyme is engineered to recognize a sequence 
that is present in the genome of an infectious agent (e.g., a bacterium, 
fungus, or virus), but not in the genome of the host cell. The ribozyme is 
exposed to the cell in combination with its protein (which has 
endonuclease activity), but under conditions such that reverse 
transcription is prevented. The ribozyme therefore integrates (fully, as 
depicted, or partially, as would be the case with an aI2-type ribozyme) 
into the genome of the infectious agent, creating double-strand breaks. 
The host genome is unaffected. 
VI. Generating Novel Genes and Gene Products 
A major goal of the present combinatorial method is to increase the number 
of novel genes and gene products that can be created by exon shuffling in 
a reasonable period of time. As described herein, the exon portion of the 
present slicing constructs can encode a polypeptide derived from a 
naturally occurring protein, or can be artificial in sequence. The exon 
portion can also be nucleic acid sequences of other function, such as a 
sequence derived from a ribozyme. By accelerated molecular evolution 
through shuffling of such exons, a far greater population of novel gene 
products can be generated and screened in a meaningful period of time. 
In one embodiment, the field of application of the present combinatorial 
method is in the generation of novel enzymatic activities, such as 
proteolytic enzymes. For example, combinatorial trans-splicing can be used 
to rapidly generate a library of potential thrombolytic agents by randomly 
shuffling the domains of several known blood serum proteins. In another 
embodiment, the trans-splicing technique can be used to generate a library 
of antibodies from which antibodies of particular affinity for a given 
antigen can be isolated. As described below, such an application can also 
be especially useful in grafting CDRs from one variable region to another, 
as required in the "humanization" of non-human antibodies. Similarly, the 
present technology can be extended to the immunoglobulin-super family, 
including the T-cell receptor, etc., to generate novel immunologically 
active proteins. 
In another illustrative embodiment, the present trans-splicing method can 
be used to generate novel signal-transduction proteins which can 
subsequently be used to generate cells which have altered responses to 
certain biological ligands or stimuli. For instance, protein tyrosine 
kinases play an important role in the control of cell growth and 
differentiation. Ligand binding to the extracellular domain of receptor 
tyrosine kinases often provides an important regulatory step which 
determines the selectivity of intracellular signaling pathways. 
Combinatorial exon splicing can be used to shuffle, for example, 
intracellular domains of receptor molecules or signal transduction 
proteins, including SH2 domains, SH3 domains, kinase domains, phosphatase 
domains, and phospholipase domains. In another embodiment, variant of SH2 
and SH3 domains are randomly shuffled with domains engineered as either 
protein kinase or phosphatase inhibitors and the combinatorial polypeptide 
library screened for the ability to block the function of, for example, 
the action of oncogenic proteins such as sic or ras. 
As will be appreciated, the present invention allows shuffling of any 
exonic sequences. Thus, the invention provides methods by which novel 
genes, in which sequences encoding known protein domains can be precisely 
linked together inframe. Many different protein domains have been 
identified in the art (see, for example, Doolittle (1995) Annu. Rev. 
Biochem. 64:287-314, incorporated herein by reference), any of which is 
useful in the practice of the present invention. 
Many techniques are known in the art for screening gene products of 
combinatorial libraries made by point mutations, and for screening cDNA 
libraries for gene products having a certain property. Such techniques 
will be generally applicable to screening the gene libraries generated by 
the present exon-shuffling methodology. The most widely used techniques 
for screening large gene libraries typically comprises cloning the gene 
library into replicable expression vectors, transforming appropriate cells 
with the resulting library of vectors, and expressing the combinatorial 
genes under conditions in which detection of a desired activity 
facilitates relatively easy isolation of the vector encoding the gene 
whose producted was detected. For instance, in the case of shuffling 
intracellular domains, phenotypic changes can be detected and used to 
isolate cells expressing a combinatorially-derived gene product conferring 
the new phenotype. Likewise, interaction trap assays can be used in vivo 
to screen large polypeptide libraries for proteins able to bind a "bait" 
protein, or alternatively, to inhibit binding of two proteins. 
For ribozymes, one illustrative embodiment comprises screening a ribozyme 
library for the ability of molecules to cleave an mRNA molecule and 
disrupt expression of a protein in such a manner as to confer some 
phenotypic change to the cell. Similarly, to assay the ability of novel 
autocatalytic introns to mediate splicing (e.g. see the group II domain 
shuffling described above) the ability of a combinatorial intron to 
mediate splicing between two exons can be detected by the ability to score 
for the protein product of two exons when accurately spliced. 
In yet another screening assay, the gene product, especially if its a 
polypeptide, is displayed on the surface of a cell or viral particle, and 
the ability of particular cells or viral particles to bind another 
molecule via this gene product is detected in a "panning assay". For 
example, the gene library can be cloned into the gene for a surface 
membrane protein of a bacterial cell, and the resulting fusion protein 
detected on the surface of the bacteria (Ladner et al., WO 88/06630; Fuchs 
et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 
18:136-140). In another embodiment, gene library is expressed as fusion 
protein on the surface of a viral particle. For instance, in the 
filamentous phase system, foreign peptide sequences can be expressed on 
the surface of infectious phase, thereby conferring two significant 
benefits. First, since these phage can be applied to affinity matrices at 
very high concentrations, large number of phase can be screened at one 
time. Second, since each infectious phage encodes the exon-shuffled gene 
product on its surface, if a particular phage is recovered from an 
affinity matrix in low yield, the phage can be amplified by another round 
of infection. The group of almost identical E.coli filamentous phages M13, 
fd, and fl are most often used in phase display libraries, as either of 
the phage gIII or gVIII coat proteins can be used to generate fusion 
proteins without disrupting the ultimate packaging of the viral particle 
(Ladner et al. PCT publication WO 90/02909; Garrard et al. PCT publication 
WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffbhs 
et al. (1993) EMBO J. 12:725-734; Clackson et al. (1991) Nature 
352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461). 
A. Antibody Repertoires 
Mouse monoclonal antibodies are readily generated by the fusion of 
antibody-producing B lymphocytes with myeloma cells. However, for 
therapeutic applications, human monoclonal antibodies are preferred. 
Despite extensive efforts, including production of heterohybridomas, 
Epstein-Barr virus immortalization of human B cells, and "humanization" of 
mouse antibodies, no general method comparable to the Kohler-Milstein 
approach has emerged for the generation of human monoclonal antibodies. 
Recently, however, techniques have been developed for the generation of 
antibody libraries in E. coli capable of expressing the antigen binding 
portions of immunoglobulin heavy and light chains. For example, 
recombinant antibodies have been generated in the form of fusion proteins 
containing membrane proteins such as peptidoglycan-associated lipoprotein 
(), as well as fusion proteins with the capsular proteins of viral 
particles, or simply as secreted proteins which are able to cross the 
bacterial membrane after the addition of a bacterial leader sequence at 
their N-termini. (See, for example, Fuchs et al. (1991) Bio/Technology 
9:1370-1372; Bettes et al. (1988) Science 240:1041-1043; Skerra et al. 
(1988) Science 240:1038-1041; Hay et al. (1992) Hum. Antibod Hybridomas 
3:81-85; and Barbas et al. WO 92/18019. 
The display of antibody fragments on the surface of filamentous phage that 
encode the antibody gene, and the selection of phage binding to a 
particular antigen, offer a powerful means of generating specific 
antibodies in vitro. Typically, phage antibodies (phAbs) have been 
generated and expressed in bacteria by cloning repertoires of rearranged 
heavy and light chain V-genes into filamentous bacteriophage. Antibodies 
of a particular specificity can be selected from the phAb library by 
panning with antigen. The present intron-mediated combinatorial approach 
can be applied advantageously to the production of recombinant antibodies 
by providing antibody libraries not readily accessible by any prior 
technique. For instance, in contrast to merely sampling combinations of 
V.sub.H and V.sub.L chains, the present method allows the 
complementarily-determining regions (CDRs) and framework regions (FRs) 
themselves to be randomly shuffled in order to create novel V.sub.H and 
V.sub.L regions which were not represented in the originally cloned 
rearranged V-genes. 
Antibody variable domains consist of a .beta.-sheet framework with three 
loops of hypervariable sequences (e.g. the CDRs) (see FIG. 51A), and the 
antigen binding site is shaped by loops from both heavy (V.sub.H) and 
light (V.sub.L) domains. The loops create antigen binding sites of a 
variety of shapes, ranging from flat surfaces to pockets. For human 
V.sub.H domains, the sequence diversity of the first two CDRs are encoded 
by a repertoire of about 50 germline V.sub.H segments (Tomlinson et al. 
(1992) J. Mol. Biol. 227:). The third CDR is generated from the 
combination of these segments with about 30 D and six J segments (Ichihara 
et al. (1988) EMBO J7:4141-4150). The lengths of the first two CDRs are 
restricted, with the length being 6 amino acid residues for CDR1, 17 
residues, and for CDR2. However, the length of CDR3 can differ 
significantly, with lengths ranging from 4 to 25 residues. 
For human light chain variable domains, the sequence diversity of the first 
two CDRs and part of CDR3 are encoded by a repertoire of about 50 human 
V.sub..kappa. segments (Meindl et al. (1990) Eur. J Immunol. 
20:1855-1863) and &gt;10 V.sub..lambda. segments (Chuchana et al. (1990) 
Eur. J Immunol. 20:1317-1325; and Combriato et al. (1991) Eur. J. Immunol. 
21:1513-1522). The lengths of the CDRs are as follows, CDR1=11-14 
residues; CDR2=8 residues; and CDR3 ranges from 6 to 10 residues for 
V.sub..kappa. genes and 9 to 13 for V.sub..lambda. genes. 
The present invention contemplates combinatorial methods for generating 
diverse antibody libraries, as well as reagents and kits for carrying out 
such methods. In one embodiment, the present combinatorial approach can be 
used to recombine both the framework regions and CDRs to generate a 
library of novel heavy and light chains. In another embodiment, 
trans-splicing can be used to shuffle only the framework regions which 
flank specific CDR sequences. While both schemes can be used to generate 
antibodies directed to a certain antigen, the later strategy is 
particularly amenable to being used for "humanizing" non-human monoclonal 
antibodies. 
The combinatorial units useful for generating diverse antibody repertories 
by the present trans-splicing methods comprise exon constructs 
corresponding to fragments of various immunoglobulin variable regions 
flanked by intronic sequences that can drive their ligation. As 
illustrated in FIG. 51B and 51C, the "exonic" sequences of the 
combinatorial units can be selected to encode essentially just a framework 
region or CDR, or can be generated to correspond to larger fragments which 
may include both CDR and FR sequences. The combinatorial units can be made 
by standard cloning techniques that manipulate DNA sequences into vectors 
which provide appropriate flanking intron fragments upon transcription. 
Alternatively, the combinatorial units can be generated using 
reverse-splicing, as described above, to specifically add intronic 
sequences to fragments of antibody transcripts. 
Methods are generally known for directly obtaining the DNA sequence of the 
variable regions of any immunoglobulin chain by using a mixture of 
oligomer primers and PCR. For instance, mixed oligonucleotide primers 
corresponding to the 5' leader (signal peptide) sequences and/or FRl 
sequences and a conserved 3' constant region primer have been used for PCR 
amplification of the heavy and light chain variable regions from a number 
of human antibodies directed to, for example, epitopes on HIV-I(gp 120, gp 
42), digoxin, tetanus, immunoglobulins (rheumatoid factor), MHC class I 
and II proteins (Larrick et al. (1991) Methods: Companion to Methods in 
Enzymology 2:106-110). A similar strategy has also been used to amplify 
mouse heavy and light chain variable regions from murine antibodies, such 
as antibodies raised against human T cell antigens (CD3, CD6), carcino 
embryonic antigen, and fibrin (Larrick et al. (1991) Bio Techniques 
11:152-156). 
In the present invention, RNA is isolated from mature B cells of, for 
example, peripheral blood cells, bone marrow, or spleen preparations, 
using standard protocols. First-strand cDNA is synthesized using primers 
specific for the constant region of the heavy chain(s) and each of the 
.kappa. and .lambda. light chains. Using variable region PCR primers, such 
as those shown in Table II below, the variable regions of both heavy and 
light chains are amplified (preferably in separate reactions) and ligated 
into appropriate expression vectors. The resulting libraries of vectors 
(e.g. one for each of the heavy and light chains) contain a variegated 
population of variable regions that can be transcribed to generated mRNA 
enriched for V.sub.H and V.sub.L transcripts. Using the reversal of 
splicing reaction, group I or group II introns can be used which are 
designed to insert immediately downstream of specific nucleotide sites 
corresponding to the last (carboxy terminal) 2-3 amino acid residues of 
each framework region. For example, as depicted in FIG. 51B, a set of 
group II Y-branched lariats can be utilized to specifically insert 
flanking group II intron fragments between each CDR sequence and the FR 
sequence immediately upstream. The exon binding sequence (EBS1, and in 
some instances EBS2) of each Y-branched lariat is manipulated to create a 
panel of Y lariats based on sequence analysis of known framework regions 
(FR1-4). The intronic addition can be carried out simultaneously for all 
three FR/CDR boundaries, or at fewer than all three boundaries. For 
instance, the RNA transcripts can be incubated with Y lariats which drive 
insertion at only the FR1/CDR1 and FR2/CDR2 boundaries. The resulting 
intron-containing fragments can be reverse transcribed using a domain VI 
primer, and the cDNA amplified using PCR primers complementary to a 
portion of domain VI, a portion of domain I, and the leader sequence. 
Thus, the Leader,FR1(IVS 1-3) and (IVS 5,6)CDR1,FR2(IVS 1-3) constructs 
will be generated. Likewise, the RNA transcript can instead be incubated 
under reverse-splicing conditions with Y-branched lariats which are 
directed to insertion at the FR2/CDR2 and FR3/CDR3 boundaries, resulting 
in the (IVS 5,6)CDR2,FR3(IVS 1-3) and (IVS 5,6)CDR2,FR4 combinatorial 
units, which can then be isolated by reverse transcription and PCR using 
primers to sequences in domain I, domain VI, and the constant region. 
TABLE II 
__________________________________________________________________________ 
Human Immunoglobulin Variable Region PCR Primers 
__________________________________________________________________________ 
5' End Sense 
Human heavy chains 
Group A 
5'-GGGAATTCATGGACTGGACCTGGAGG(AG)TC(CT)- (SEQ ID NO:21) 
TCT(GT)C-3' 
Group B 
5'-GGGAATTCATGGAG(CT)TTGGGCTGA(CG)CTGG(CG)- (SEQ ID 
NO:22) 
TTTT-3' 
- Group C 
5'-GGGAATTCATG(AG)A(AC)(AC)(AT)ACT(GT)TG(GT)- (SEQ ID 
NO:23) 
(AT)(CG)C(AT)(CT)(CG)CT(CT)CTG-3' 
- Human .kappa. light chain 
5'-GGGAATTCATGGACATG(AG)(AG)(AG)(AGT)(CT)CC- (SEQ ID 
NO:24) 
(ACT)(ACG)G(CT)(GT)CA(CG)CTT-3' 
- Human light chain 
5'-GGGAATTCATG(AG)CCTG(CG)(AT)C(CT)CCTCTC(CT)- (SEQ ID 
NO:25) 
T(CT)CT(CG)(AT)(CT)C-3' 
- 
3' End sense constant region 
Human IgM heavy chain 
5'-CCAAGCTTAGACGAGGGGGAAAAGGGTT-3' (SEQ ID NO:26) 
- Human IgG1 heavy chain 
5'-CCAAGCTTGGAGGAGGGTGCCAGGGGG-3' (SEQ ID NO:27) 
- Human light chain 
5'-CCAAGCTTGAAGCTCCTCAGAGGAGGG-3' (SEQ ID NO:28) 
- Human .kappa. light chain 
5'-CCAAGCTTTCATCAGATGGCGGGAAGAT-3' (SEQ ID NO:29) 
__________________________________________________________________________ 
Murine Immunoglobulin Variable Region PCR Primers 
__________________________________________________________________________ 
5' End Sense 
Leader (signal peptide) region (amino-acids -20 to -13) 
Group A 
5'-GGGGAATTCATG(GA)A(GC)TT(GC)(TG)GG(TC)T(AC)- (SEQ ID 
NO:30) 
A(AG)CT(CT)G(GA)TT-3' 
- Group B 
5'-GGGGAATTCATG(GA)AATG(GC)A(GC)CTGGGT(CT)- (SEQ ID 
NO:31) 
(TA)T(TC)CTCT-3' 
- Framework 1 region (amino acids 1 to 8) 
5'-GGGGAATTC(CG)AGGTG(CA)AGCTC(CG)(AT)(AG)(CG)- (SEQ ID 
NO:32) 
A(AG)(CT)C(CG)GGG-3' 
- 
3' End sense constant region 
Mouse .gamma. constant region 
5'-GGAAGCTTA(TC)CTCCACACACAGG(AG)(AG)CCAGTG- (SEQ ID 
NO:33) 
GATAGAC-3' 
- Mouse .kappa. light chain (amino acids 116 to 122) 
5'-GGAAGCTTACTGGATGGTGGGAAGATGGA-3' (SEQ ID NO:34) 
__________________________________________________________________________ 
Bases in parentheses represent substitutions at a given residue. EcoRI an 
HindIII sites are underlined. 
The Leader, FR1 (IVS 1-3) transcripts can be linked to an insoluble resin 
by standard techniques, and each set of combinatorial units (CDR1/FR2, 
CDR2/FR3, CDR3/FR4) can be sequentially added to the resin-bound nucleic 
acid by incubation under trans-splicing conditions, with unbound reactants 
washed away between each round of addition. After addition of the (IVS 
5,6)CDR3,FR4 units to the resin bound molecules, the resulting 
trans-spliced molecule can be released from the resin, reverse-transcribed 
and PCR amplified using primers for the leader sequence and constant 
region, and subsequently cloned into an appropriate vector for generating 
a screenable population of antibody molecules. 
Taking the dissection of the variable regions one step further, a set of 
exon libraries can be generated for ordered combinatorial ligation much 
the same as above, except that each combinatorial unit is flanked at its 
5' end with an intron fragment that is unable to drive a trans-splicing 
reaction with the intron fragment at its 3' end. As described above 
(section II) with regard to ordered gene assembly, each combinatorial unit 
is effectively protected from addition by another unit having identical 
flanking intron fragments. The 5' and 3' flanking intronic sequences can 
be of the same group, but from divergent enough classes (i.e. group IIA 
versus group IIB) or divided in such a way that intermolecular 
complementation and assembly of an active splicing complex cannot occur; 
or the intron fragments can simply be from different groups (e.g. group I 
versus group II). 
As illustrated in FIG. 51C, the combinatorial units of FIG. 51B can be 
generated with Y lariats derived from group IIA intron fragments (hence 
the designation "IVS-A-5,6"). Each CDR is then split from the downstream 
framework region using a Y-branched lariat derived from a group IIB intron 
having a divergent enough domain V that neither combination of (IVS-A-5,6) 
and (IVS-B-1-3) or (IVS-B-5,6) and (IVS-A-1-3) results in a functional 
splicing complex. In order to avoid the need to determine the sequence of 
each of the cloned CDRs, the exon-binding sites of the IIB intron lariats 
can be constructed to match the much less variable nucleotide sequences 
corresponding to the first (amino terminal) 2-3 a.a. residues of each of 
the framework regions (FR2-4). The resulting constructs include internal 
exon units of the general formula (IVS-A-5,6) CDR (VIS-B-1-3) and 
(VIS-B-5,6) FR (IVS-A-1-3), with each CDR containing an extra 2-3 a.a. 
residues from the FR which previously flanked it. Thus, by sequentially 
adding each pool of combinatorial units to the resin-immobilized FR1, an 
ordered combinatorial ligation of variegated populations of CDRs and FRs 
can be carried out to produce a library of variable region genes in which 
both the CDRs and FRs have been independently randomized. 
Furthermore, CDR combinatorial units can be generated which are completely 
random in sequence, rather than cloned from any antibody source. For 
instance, a plasmid similar to pINV1 (described herein) can be used to 
create a set of random CDR sequences of a given length and which are 
flanked by appropriate intronic fragments. In an illustrative embodiment, 
the plasmid includes restriction endonuclease sites in each of the 5' and 
3' flanking intron sequences such that oligonucleotides having the CDR 
coding sequence can be cloned into the plasmid. For example, a degenerate 
oligonucleotide can be synthesized for CDRI which encodes all possible 
amino acid combinations for the 6 amino acid sequence. The nucleotide 
sequences which flank the CDR-encoding portion of the oligonucleotide 
comprise the flanking intron sequences necessary to allow ligation of the 
degenerate oligonucleotide into the plasmid and reconstitute a construct 
which would produce a spliceable transcript. To avoid creation of stop 
codons which can result when codons are randomly synthesized using 
nucleotide monomers, "dirty bottle" synthesis can instead be carried out 
using a set of nucleotide trimers which encode all 20 amino acids. 
With slight modification, the present ordered combinatorial ligation can be 
used to efficiently humanize monoclonal antibodies of non-human origin. 
The CDRs from the monoclonal antibody can be recombined with human 
framework region libraries (e.g. an FR1 library, an FR2 library, etc.) to 
produce a combinatorial population of variable regions in which the CDR 
sequences are held constant, but each of the framework regions have been 
randomized. The variable regions can be subsequently fused with sequences 
corresponding to the appropriate human constant regions, and the 
antibodies resulting from heavy and light chain association can be 
screened for antigen binding using standard panning assays such as phage 
display. In contrast to contemporary humanization schemes which require 
the practitioner to prejudicially choose a particular human scaffold into 
which the CDRs are grafted, the present technique provides a greater 
flexibility in choosing appropriate human framework regions which do not 
adversely affect antigen binding by the resultant chimeric antibody. 
To illustrate, the variable regions of both the heavy and light chains of a 
mouse monoclonal antibody can be cloned using primers as described above. 
The sequence of each CDR can be obtained by standard techniques. The CDRs 
can be cloned into vectors which provide appropriate flanking intronic 
sequences, or alternatively, isolated by reverse-splicing with Y-branched 
lariats designed to insert precisely at each FR/CDR and CDR/FR boundary. 
As described above, the particular intronic fragments provided with each 
murine CDR and each human FR construct can be selected to disfavor 
multiple ligations at each step of addition to a resin bound nucleic acid. 
The library of human heavy chain leader, FRI(IVS-A-1-3) constructs can be 
immobilized on a resin, and in a first round of ligation, the heavy chain 
murine (IVS-A-5,6) CDR1 (IVS-B-1-3) construct is added under 
trans-splicing conditions. Un-ligated combinatorial units are washed away, 
and the library of human heavy chain (IVS-B-5,6) FR2 (IVS-A-1-3) units are 
admixed and trans-spliced to the resin-bound nucleic acids terminating 
with the murine CDR construct. This process is carried out for the 
remaining murine CDR and human FR units of the heavy chain, and a similar 
process is used to construct combinatorial light chain chimeras as well. 
The resulting chimeric heavy and light chains can be cloned into a phage 
display library, and the phAbs screened in a panning assay to isolate 
humanized antibodies (and their genes) which bind the antigen of interest. 
B. Combinatorial Enzyme Libraries 
Plasminogen activators (PAs) are a class of serine proteases that convert 
the proenzyme plasminogen into plasmin, which then degrades the fibrin 
network of blood clots. The plasminogen activators have been classified 
into two immunologically unrelated groups, the urokinase-type PAs (u-PA) 
and the tissue-type PA (tPA), with the later activator being the 
physiological vascular activator. These proteins, as well as other 
proteases of the fibrinolytic pathway, are composed of multiple structural 
domains which appear to have evolved by genetic assembly of individual 
subunits with specific structural and/or functional properties. For 
instance, the amino terminal region of tPA is composed of multiple 
structural/functional domains found in other plasma proteins, including a 
"finger-like domain" homologous to the finger domains of fibronectin, an 
"epidermal growth factor domain" homologous to human EGF, and two 
disulfide-bonded triple loop structures, commonly referred to as "kringle 
domains," homologous to the kringle regions in plasminogen. The region 
comprising residues 276-527 (the "catalytic domain" is homologous to that 
of other serine proteases and contains the catalytic triad. In addition, 
the gene for tPA encodes a signal secretion peptide which directs 
secretion of the protein into the extracellular environment, as well as a 
prosequence which is cleaved from the inactive form of the protease (the 
"plasminogen") to active tPA during the fibrinolytic cascade. 
These distinct domains in tPA are involved in several functions of the 
enzyme, including its binding to fibrin, stimulation of plasminogen 
activation by fibrin, and rapid in vivo clearance. Approaches used to 
characterize the functional contribution of these structural domains 
include isolation of independent structural domains as well as the 
production of variant proteins which lack one or more domains. For 
example, the fibrin selectivity of tPA is found to be mediated by its 
affinity for fibrin conferred by the finger-like domain and by at least 
one of the kringle domains. 
The present combinatorial method can be used to generate novel plasminogen 
activators having superior thrombolytic properties, by generating a 
library of proteins by RNA-splicing mediated shuffling of the domains of 
plasma proteins (see, for example, Example 1, above, and Examples 18-21, 
below). As described below, one mode of generating the combinatorial 
library comprises the random trans-splicing of a mixture of exons 
corresponding to each of the domains of the mature tPA protein. Briefly, a 
cDNA clone of tPA was obtained and, through the use of specific PCR 
amplimers, each of the 5 protein domains was amplified and isolated. Each 
of these amplified domains was then separately cloned into a plasmid as an 
exon module such that the 5' end of the exon is preceded by group II 
domains 5-6, and the 3' end of the exon is followed by group II domains 
1-3. In addition, the IBS 1 site of each of the exon was mutated in order 
to facilitate base pairing with the EBS 1 sequence of the 3' flanking 
intron fragment. Transcription of the resulting construct thus produces 
RNA transcripts of the general formula (IVS 5,6)-Exon-(IVS 1-3). Mixture 
of these transcripts under trans-splicing conditions can result in random 
ligation of the exons to one and other and assembly of the combinatorial 
gene library which can subsequently be screened for fibrinolytic activity. 
Moreover, combinatorial units can be generated from other proteins, 
including proteins having no catalytic role in blood clotting or 
fibrinolysis. For example, a library of catalytic domains can be generated 
from other thrombolytic proteases, blood clotting factors, and other 
proteases having peptidic activity similar to the typsin-like activity of 
tPA. Likewise, libraries of splicing constructs can be derived from 
EGF-like domains, finger-like domains, kringle domains, and 
calcium-binding domains from a vast array of proteins which contain such 
moieties. 
Preferred combinatorial units include domains of the vampire bat DSPAxl 
protein, a plasminogen activator with high fibrin binding activity (see 
Gulba et al. (1995) Fibrinolysis 9 Supp. 1:91-96, incorporated by 
reference). Shuffling one of these domains with domains of the human t-PA 
protein might produce a novel plasminogen activator with desirable 
characteristics as a human therapeutic. 
EXAMPLE 18 
Construction of Plasmid GrII-Sig 
Two oligonucleotide primers were used to change the IBS 1 sequence of pINV1 
to TGTCAAA and the EBS 1 sequence to TTTGACA. Thus, the last seven 
nucleotides of E5 were changes to the sequence of the last 7 nucleotides 
of TPA fibronectin finger like domain and the EBS 1 sequence was made 
complementary. The resulting plasmid is termed here GrII-Sig. 
EXAMPLE 19 
Construction of Plasmid SIG(IVS1-3) 
The plasmid SIG(IVS1-3) contains the first two protein domains of TPA (the 
signal sequence and the finger domain) followed by group II intron domains 
1-3. It was made by the reversal of splicing. Plasmid Gril-Sig (Example 
18) was linearized with Hind III and RNA made using T.sub.7 polymerase in 
vitro. The RNA was incubated under self splicing conditions for two hours 
and the products fractionated on an acrylamide gel. The Sig(Y) molecule (a 
Y-branched lariat intron comprising domains 5 and 6 joined to domains 1 
through 3 by a 2'-5' phosphodiester bond) was gel purified. This molecule 
was the "enzyme" used for the reverse-splicing reaction. The substrate was 
made by cutting TPA-KS.sup.+ DNA (Example 2) with Sty I, which cuts 17 bp 
downstream of the end of the finger domain. A 404 nt RNA was made using 
T.sub.7 polymerase. The enzyme and substrate were mixed and incubated 
under splicing conditions for two hours. By the reversal of splicing, the 
Sig(Y) RNA attacked the substrate to yield the signal plus finger region 
followed by intron domains 1 through 3. A cDNA copy of the molecule was 
made using reverse transcriptase and amplified by PCR. It was cloned into 
the PBS vector in the T.sub.7 orientation. The clones analyzed each showed 
precise joining of the coding sequence to the group II intron sequence. 
Thus, the nucleotide sequence of the EBS1 was sufficient to direct exact 
integration of the intronic IVS(1-3) fragment. 
EXAMPLE 20 
Construction of Other Shuffling Clones 
Clones with each of the other three protein domains (growth factor (GF) 
domain, kringle 2 (K2) domain and catalytic (cat) domain), flanked by 
group II intron sequences, can also be made by either standard cloning 
methods or by the reversal of splicing method, as described above, to 
yield constructs corresponding to (IVS5,6)FG(IVS1-3), (IVS5,6)K2(IVS1-3), 
and (IVS5,6)cat or (IVS5,6)cat(IVS1-3). 
To further illustrate, the plasmid (IVS5.6)cat was generated by reversal of 
splicing as in Example 19. Briefly, the Y-branched intron of the pY7 
construct (see Example 16) was generated by cutting the pY7 plasmid with 
Hindi, producing RNA with T.sub.7, and incubating the RNA under 
self-splicing conditions for 1.5 hours. The products were fractioned on an 
acylamide gel. The Y7 molecule (a Y-branched intron) was gel purified. 
This molecule was used for the reverse-splicing reaction. The substrate 
for this reaction was generated by cutting a plasmid containing the tPA 
catalytic domain with HindiII and transcribing the linear plasmid with T7. 
The Y-branched enzyme and tPA substrate RNAs were mixed and incubated 
under reverse-splicing conditions for 4 hours. By the reversal of 
splicing, the Y7 RNA attacked the substrate at a site (IBS 1) just 
upstream of the coding sequence for the catalytic domain to yield intron 
domains 5 and 6 followed by the tPA protease domain. A cDNA copy of the 
molecule was made using reverse transcriptase and amplified by PCR. It was 
cloned into a PBS vector in the T7 orientation. Two independent clones 
were characterized by DNA sequence analysis. Both clones had the group II 
intron sequences precisely joined to the tPA sequences. Thus, the 
nucleotide sequence of the fusion protein was 5'-ATCCGGAT/ACCTGCGG (SEQ ID 
NO:.sub.----) (intron/exon, respectively). 
EXAMPLE 21 
Generation of Library 
RNA transcripts are made for each of the tPA combinatorial units, 
SIG(IVS1-3), (IVS5,6)K1(IVS1-3), (VS5,6)K2(IVS1-3), (IVS5,6)GF(IVS-3), and 
(IVS5,6)cat. The transcripts are mixed and incubated under trans-splicing 
conditions. The resulting combinatorial RNA molecules can be 
reverse-transcribed to cDNA using primers complementary to sequences in 
the intron domains I-III, and the cDNA amplified by PCR using a similar 
primer and a primer to the tPA signal sequence. The amplified cDNAs can 
subsequently be cloned into suitable expressions vectors to generate an 
expressions library, and the library screened for fibrinolytic activity by 
standard assays. 
All of the above-cited references and publications are hereby incorporated 
by reference. 
EQUIVALENTS 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, numerous equivalents to the specific 
methods and reagents described herein. Such equivalents are considered to 
be within the scope of this invention and are covered by the following 
claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 46 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "group I intron Element R 
consensus" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - SYUCARMGAC UANANG - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "group I intron Element S 
consensus - #sequence" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - AAGAUAGUCY - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: t- 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - ACGATGCATG CTGGAGAGAA AACCTCTGCG - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: t- 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - ACGATGCATT CTGTAGAGAA GCACTGCGCC - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: I654 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - ACGAAGCTTC CTATAGTATA AGTTAGCAGA T - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: I5,6 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - GCGAATTCGA GCTCGTGAGC CGTAT - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: t-PA(-49) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - ACGGGTACCG AAAGGGAAGG AGCAAGCCGT G - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Ribozyme" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: last 16 n - #t of Fn in Y4 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - CUCAGUGCCU GUCAAA - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Ribozyme" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: last 16 n - #t of K2 in Y7 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - UGUGCCCUCC UGCUCC - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1848 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: amplified t- - #PA clone 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - GCTGGAGAGA AAACCTCTGC GAGGAAAGGG AAGGAGCAAG CCGTGAATTT AA - 
#GGGACGCT 60 
- - GTGAAGCAAT CATGGATGCA ATGAAGAGAG GGCTCTGCTG TGTGCTGCTG CT - 
#GTGTGGAG 120 
- - CAGTCTTCGT TTCGCCCAGC CAGGAAATCC ATGCCCGATT CAGAAGAGGA GC - 
#CAGATCTT 180 
- - ACCAAGTGAT CTGCAGAGAT GAAAAAACGC AGATGATATA CCAGCAACAT CA - 
#GTCATGGC 240 
- - TGCGCCCTGT GCTCAGAAGC AACCGGGTGG AATATTGCTG GTGCAACAGT GG - 
#CAGGGCAC 300 
- - AGTGCCACTC AGTGCCTGTC AAAAGTTGCA GCGAGCCAAG GTGTTTCAAC GG - 
#GGGCACCT 360 
- - GCCAGCAGGC CCTGTACTTC TCAGATTTCG TGTGCCAGTG CCCCGAAGGA TT - 
#TGCTGGGA 420 
- - AGTGCTGTGA AATAGATACC AGGGCCACGT GCTACGAGGA CCAGGGCATC AG - 
#CTACAGGG 480 
- - GCACGTGGAG CACAGCGGAG AGTGGCGCCG AGTGCACCAA CTGGAACAGC AG - 
#CGCGTTGG 540 
- - CCCAGAAGCC CTACAGCGGG CGGAGGCCAG ACGCCATCAG GCTGGGCCTG GG - 
#GAACCACA 600 
- - ACTACTGCAG AAACCCAGAT CGAGACTCAA AGCCCTGGTG CTACGTCTTT AA - 
#GGCGGGGA 660 
- - AGTACAGCTC AGAGTTCTGC AGCACCCCTG CCTGCTCTGA GGGAAACAGT GA - 
#CTGCTACT 720 
- - TTGGGAATGG GTCAGCCTAC CGTGGCACGC ACAGCCTCAC CGAGTCGGGT GC - 
#CTCCTGCC 780 
- - TCCCGTGGAA TTCCATGATC CTGATAGGCA AGGTTTACAC AGCACAGAAC CC - 
#CAGTGCCC 840 
- - AGGCACTGGG CCTGGGCAAA CATAATTACT GCCGGAATCC TGATGGGGAT GC - 
#CAAGCCCT 900 
- - GGTGCCACGT GCTGAAGAAC CGCAGGCTGA CGTGGGAGTA CTGTGATGTG CC - 
#CTCCTGCT 960 
- - CCACCTGCGG CCTGAGACAG TACAGCCAGC CTCAGTTTCG CATCAAAGGA GG - 
#GCTCTTCG 1020 
- - CCGACATCGC CTCCCACCCC TGGCAGGCTG CCATCTTTGC CAAGCACAGG AG - 
#GTCGCCCG 1080 
- - GAGAGCGGTT CCTGTGCGGG GGCATACTCA TCAGCTCCTG CTGGATTCTC TC - 
#TGCCGCCC 1140 
- - ACTGCTTCCA GGAGAGGTTT CCGCCCCACC ACCTGACGGT GATCTTGGGC AG - 
#AACATACC 1200 
- - GGGTGGTCCC TGGCGAGGAG GAGCAGAAAT TTGAAGTCGA AAAATACATT GT - 
#CCATAAGG 1260 
- - AATTCGATGA TGACACTTAC GACAATGACA TTGCGCTGCT GCAGCTGAAA TC - 
#GGATTCGT 1320 
- - CCCGCTGTGC CCAGGAGAGC AGCGTGGTCC GCACTGTGTG CCTTCCCCCG GC - 
#GGACCTGC 1380 
- - AGCTGCCGGA CTGGACGGAG TGTGAGCTCT CCGGCTACGG CAAGCATGAG GC - 
#CTTGTCTC 1440 
- - CTTTCTATTC GGAGCGGCTG AAGGAGGCTC ATGTCAGACT GTACCCATCC AG - 
#CCGCTGCA 1500 
- - CATCACAACA TTTACTTAAC AGAACAGTCA CCGACAACAT GCTGTGTGCT GG - 
#AGACACTC 1560 
- - GGAGCGGCGG GCCCCAGGCA AACTTGCACG ACGCCTGCCA GGGCGATTCG GG - 
#AGGCCCCC 1620 
- - TGGTGTGTCT GAACGATGGC CGCATGACTT TGGTGGGCAT CATCAGCTGG GG - 
#CCTGGGCT 1680 
- - GTGGACAGAA GGATGTCCCG GGTGTGTACA CAAAGGTTAC CAACTACCTA GA - 
#CTGGATTC 1740 
- - GTGACAACAT GCGACCGTGA CCAGGAACAC CCGACTCCTC AAAAGCAAAT GA - 
#GATCCCGC 1800 
- - CTCTTCTTCT TCAGAAGACA CTGCAAAGGC GCAGTGCTTC TCTACAGA - # 
1848 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4542 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "plasmid DNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: pINV1 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GA - 
#GACGGTCA 60 
- - CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TC - 
#AGCGGGTG 120 
- - TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CT - 
#GAGAGTGC 180 
- - ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC AT - 
#CAGGCGAC 240 
- - GCGCCCTGTA GCGGCGCATT AAGCGCGGCG GGTGTGGTGG TTACGCGCAG CG - 
#TGACCGCT 300 
- - ACACTTGCCA GCGCCCTAGC GCCCGCTCCT TTCGCTTTCT TCCCTTCCTT TC - 
#TCGCCACG 360 
- - TTCGCCGGCT TTCCCCGTCA AGCTCTAAAT CGGGGGCTCC CTTTAGGGTT CC - 
#GATTTAGT 420 
- - GCTTTACGGC ACCTCGACCC CAAAAAACTT GATTAGGGTG ATGGTTCACG TA - 
#GTGGGCCA 480 
- - TCGCCCTGAT AGACGGTTTT TCGCCCTTTG ACGTTGGAGT CCACGTTCTT TA - 
#ATAGTGGA 540 
- - CTCTTGTTCC AAACTGGAAC AACACTCAAC CCTATCTCGG TCTATTCTTT TG - 
#ATTTATAA 600 
- - GGGATTTTGC CGATTTCGGC CTATTGGTTA AAAAATGAGC TGATTTAACA AA - 
#AATTTAAC 660 
- - GCGAATTTTA ACAAAATATT AACGCTTTAC AATTTCGCCA TTCGCCATTC AG - 
#GCTGCGCA 720 
- - ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GC - 
#GAAAGGGG 780 
- - GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CG - 
#ACGTTGTA 840 
- - AAACGACGGC CAGTGAATTG TAATACGACT CACTATAGGG CGAATTCGAG CT - 
#CGTGAGCC 900 
- - GTATGCGATG AAAGTCGCAC GTACGGTTCT TACCGGGGGA AAACTTGTAA AG - 
#GTCTACCT 960 
- - ATCGGGATAC TATGTATTAT CAATGGGTGC TATTTTCTCT TTATTTGCAG GA - 
#TACTACTA 1020 
- - TTGAAGTCCT CAAATTTTAG GTTTAAACTA TAATGAAAAA TTAGCTCAAA TT - 
#CAATTCTG 1080 
- - ATTAATTTTC ATTGGGGCTA ATGTTATTTT CTTCCCAATG CATTTCTTAG GT - 
#ATTAATGG 1140 
- - TATGCCTAGA AGAATTCCTG ATTATCCTGA TGCTTTCGCA GGATGAAATT AT - 
#GTCGCTTC 1200 
- - TATTGGTTCA TTCATTGCAC TATTATCATT ATTCTTATTT ATCTATATTT TA - 
#TATGATCC 1260 
- - TCTAGAGTCG ACCTGCAGGC ATGCAAGCTG GGGATCACAT CATATGTATA TT - 
#GTAGGATT 1320 
- - AGATGCAGAT ACTAGAGCAT ATTTCCTATC CGCACTGATG ATTATTGCAA TT - 
#CCAACAGG 1380 
- - AATTAAAATC TTTTCTTGAT TAGCCCTGAT CTACGGTGGT TCAATTAGAT TA - 
#GCACTACC 1440 
- - TATGTTATAT GCAATTGCAT TCTTATTCTT ATTCACAATG GGTGGTTTAA CT - 
#GGTGTTGC 1500 
- - CTTAGCTAAC GCCTCATTAG ATGTGGCATT CCACGATACT TACTACGTGG TG - 
#GGACATTT 1560 
- - TCGAGCGGTC TGAAAGTTAT CATAAATAAT ATTTACCATA TAATAATGGA TA - 
#AATTATAT 1620 
- - TTTTATCAAT ATAAGTCTAA TTACAAGTGT ATTAAAATGG TAACATAAAT AT - 
#GCTAAGCT 1680 
- - GTAATGACAA AAGTATCCAT ATTCTTGACA GTTATTTTAT ATTATAAAAA AA - 
#AGATGAAG 1740 
- - GAACTTTGAC TGATCTAATA TGCTCAACGA AAGTGAATCA AATGTTATAA AA - 
#TTACTTAC 1800 
- - ACCACTAATT GAAAACCTGT CTGATATTCA ATTATTATTT ATTATTATAT AA - 
#TTATATAA 1860 
- - TAATAAATAA AATGGTTGAT GTTATGTATT GGAAATGAGC ATACGATAAA TC - 
#ATATAACC 1920 
- - ATTAGTAATA TAATTTGAGA GCTAAGTTAG ATATTTACGT ATTTATGATA AA - 
#ACAGAATA 1980 
- - AACCCTATAA ATTATTATTA TTAATAATAA AAAATAATAA TAATACCAAT AT - 
#ATATATTA 2040 
- - TTTAATTTAT TATTATTATA TTAATAAAAT TTAATATATA TTATAAATAA TT - 
#ATTGGATT 2100 
- - AAGAAATATA ATATTTTATA GAAATTTTCT TTATATTTAG AGGGTAAAAG AT - 
#TGTATAAA 2160 
- - AAGCTAATGC CATATTGTAA TGATATGGAT AAGAATTATT ATTCTAAAGA TG - 
#AAAATCTG 2220 
- - CTAACTTATA CTATAGGGGG GATCCTCTAG AGTCGACCTG CAGGCATGCA AG - 
#CTTTTGTT 2280 
- - CCCTTTAGTG AGGGTTAATT TCGAGCTTGG CGTAATCATG GTCATAGCTG TT - 
#TCCTGTGT 2340 
- - GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA AA - 
#GTGTAAAG 2400 
- - CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA CT - 
#GCCCGCTT 2460 
- - TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GC - 
#GGGGAGAG 2520 
- - GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CG - 
#CTCGGTCG 2580 
- - TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA TC - 
#CACAGAAT 2640 
- - CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC AG - 
#GAACCGTA 2700 
- - AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CA - 
#TCACAAAA 2760 
- - ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CA - 
#GGCGTTTC 2820 
- - CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC GG - 
#ATACCTGT 2880 
- - CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT AG - 
#GTATCTCA 2940 
- - GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GT - 
#TCAGCCCG 3000 
- - ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CA - 
#CGACTTAT 3060 
- - CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GG - 
#CGGTGCTA 3120 
- - CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA TT - 
#TGGTATCT 3180 
- - GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TC - 
#CGGCAAAC 3240 
- - AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG CG - 
#CAGAAAAA 3300 
- - AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TG - 
#GAACGAAA 3360 
- - ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TA - 
#GATCCTTT 3420 
- - TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT TG - 
#GTCTGACA 3480 
- - GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT CG - 
#TTCATCCA 3540 
- - TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG GGAGGGCTTA CC - 
#ATCTGGCC 3600 
- - CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA TC - 
#AGCAATAA 3660 
- - ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC GC - 
#CTCCATCC 3720 
- - AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AG - 
#TTTGCGCA 3780 
- - ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC GTCGTTTGGT AT - 
#GGCTTCAT 3840 
- - TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC CCCCATGTTG TG - 
#CAAAAAAG 3900 
- - CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GT - 
#GTTATCAC 3960 
- - TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AG - 
#ATGCTTTT 4020 
- - CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG CG - 
#ACCGAGTT 4080 
- - GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA TAGCAGAACT TT - 
#AAAAGTGC 4140 
- - TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG GATCTTACCG CT - 
#GTTGAGAT 4200 
- - CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT AC - 
#TTTCACCA 4260 
- - GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA AT - 
#AAGGGCGA 4320 
- - CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC AT - 
#TTATCAGG 4380 
- - GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA CA - 
#AATAGGGG 4440 
- - TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTCTA AGAAACCATT AT - 
#TATCATGA 4500 
- - CATTAACCTA TAAAAATAGG CGTATCACGA GGCCCTTTCG TC - # 
- #4542 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: E5-specific - #oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - GTAGGATTAG ATGCAGATAC TAGAGC - # - # 
26 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: E3-specific - #oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- - GAGGACTTCA ATAGTATCCT GC - # - # 
22 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: K1.Cir.1:anti-s - #ense 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - GCCAACGCGC TGCTGTTCCA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: K1.Cir.2:sense 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- - GGCCAGACGC CATCAGGCTG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: I5-29 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- - TATTATTTAT GATAACTTTC AGACC - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2939 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "plasmid DNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: BGINV 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- - TATAGTGTCA CCTAAATCGT ATGTGTATGA TACATAAGGT TATGTATTAA TT - 
#GTAGCCGC 60 
- - GTTCTAACGA CAATATGTCC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GA - 
#TGCCGCAT 120 
- - AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GC - 
#TTGTCTGC 180 
- - TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TG - 
#TCAGAGGT 240 
- - TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC CT - 
#ATTTTTAT 300 
- - AGGTTAATGT CATGATAATA ATGGTTTCTT AGACGTCAGG TGGCACTTTT CG - 
#GGGAAATG 360 
- - TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CC - 
#AGAGTATG 420 
- - AGTATTCAAC ATTTCCGTGT CGCCCTTATT CCCTTTTTTG CGAGAGTATG AG - 
#TATTCAAC 480 
- - ATTTCCGTGT CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TT - 
#TGCTCACC 540 
- - CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GT - 
#GGGTTACA 600 
- - TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GA - 
#ACGTTTTC 660 
- - CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT AT - 
#TGACGCCG 720 
- - GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GA - 
#GTACTCAC 780 
- - CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AG - 
#TGCTGCCA 840 
- - TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GG - 
#ACCGAAGG 900 
- - AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CG - 
#TTGGGAAC 960 
- - CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GT - 
#AGCAATGG 1020 
- - CAACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CG - 
#GCAACAAT 1080 
- - TAATAGACTG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GC - 
#CCTTCCGG 1140 
- - CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GG - 
#TATCATTG 1200 
- - CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG AC - 
#GGGGAGTC 1260 
- - AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CT - 
#GATTAAGC 1320 
- - ATTGGTAACT GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AA - 
#ACTTCATT 1380 
- - TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AA - 
#AATCCCTT 1440 
- - AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GG - 
#ATCTTCTT 1500 
- - GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CC - 
#GCTACCAG 1560 
- - CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA AC - 
#TGGCTTCA 1620 
- - GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CA - 
#CCACTTCA 1680 
- - AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GT - 
#GGCTGCTG 1740 
- - CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CC - 
#GGATAAGG 1800 
- - CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CG - 
#AACGACCT 1860 
- - ACACCGAACT GAGATACCTA CAGCGTGAGC ATTGAGAAAG CGCCACGCTT CC - 
#CGAAGGGA 1920 
- - GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC AC - 
#GAGGGAGC 1980 
- - TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CT - 
#CTGACTTG 2040 
- - AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GC - 
#CAGCAACG 2100 
- - CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TT - 
#TCCTGCGT 2160 
- - TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT AC - 
#CGCTCGCC 2220 
- - GCAGCCGAAC GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAGAG CG - 
#CCCAATAC 2280 
- - GCAAACCGCC TCTCCCCGCG CGTTGGCCGA TTCATTAATG CAGGTTAACC TG - 
#GCTTATCG 2340 
- - AAATTAATAC GACTCACTAT AGGGAGACCG GCCTCGAGCA GCTGAAGCTT TG - 
#GGTTTCTG 2400 
- - ATAGGCACTG ACTCTCTCTG CCTATTGGTC TATTTTCCCA CCCTTAGGCT GC - 
#TGGTGGTC 2460 
- - TACCCTTGGA CCCAGAGGTT CTTTGAGTCC TTTGGGGATC TGTCCACTCC TG - 
#ATGCTGTT 2520 
- - ATGGGCAACC CTAAGGTGAA GGCTCATGGC AAGAAAGTGC TCGGTGCCTT TA - 
#GTGATGGC 2580 
- - CTGGCTCACC TGGACAACCT CAAGGGCACC TTTGCCACAC TGAGTGAGCT GC - 
#ACTGTGAC 2640 
- - AAGCTGCACG TGGATCCCCC TGAAGCTTGC TTACATTTGC TTCTGACACA AC - 
#TGTGTTCA 2700 
- - CTAGCAACCT CAAACAGACA CCATGGTGCA CCTGACTCCT GAGGAGAAGT CT - 
#GCCGTTAC 2760 
- - TGCCCTGTGG GGCAAGGTGA ACGTGGATGA AGTTGGTGGT GAGGCCCTGG GC - 
#AGGTTGGT 2820 
- - ATCAAGGTTA CAAGACAGGT TTAAGGAGAC CAATAGAAAC TGGGCATGTG GA - 
#GACAGAGA 2880 
- - AGACTCTTGG GATCCCCGGG TACCGAGCTC GAATTCATCG ATGATATCAG AT - 
#CTGGTTC 2939 
- - - - (2) INFORMATION FOR SEQ ID NO:18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA oligonucleotide" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: DNA oligonuc - #leotide used as splint for 
ligation - #of RNA ends 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- - CGAGGCCGGT CTCCCAATTC GAGCTCGGTA C - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: CIR-1 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- - GAGTGGACAG ATCCCCAAAG GACTC - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: CIR-2 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- - GTGATGCCTG GCTCACCTGG ACAA - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO:21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Group A h - #uman heavy chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- - GGGAATTCAT GGACTGGACC TGGAGGRTCY TCTK - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Group B h - #uman heavy chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- - GGGAATTCAT GGAGYTTGGG CTGASCTGGS TTTT - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Group C h - #uman heavy chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- - GGGAATTCAT GRAMMWACTK TGKWSWYSCT YCTG - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Human kappa - #light chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- - GGGAATTCAT GGACATGRRR DYCCHVYKCA SCTT - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Human lambda - # light chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- - GGGAATTCAT GRCCTGSWCY CCTCTCYTYC TSWYC - # - 
# 35 
- - - - (2) INFORMATION FOR SEQ ID NO:26: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Human IgM - #heavy chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
- - CCAAGCTTAG ACGAGGGGGA AAAGGGTT - # - # 
28 
- - - - (2) INFORMATION FOR SEQ ID NO:27: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Human IgG1 - #heavy chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
- - CCAAGCTTGG AGGAGGGTGC CAGGGGG - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Huam lambda - #light cjain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- - CCAAGCTTGA AGCTCCTCAG AGGAGGG - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Group A m - #urine leader region 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
- - GGGGAATTCA TGRASTTSKG GYTMARCTKG RTT - # - # 
33 
- - - - (2) INFORMATION FOR SEQ ID NO:30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Group B m - #urine leader region 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- - GGGGAATTCA TGRAATGSAS CTGGGTYWTY CTCT - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:31: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Murine frame - #work 1 region 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
- - GGGGAATTCS AGGTGMAGCT CSWRSARYCS GGG - # - 
# 33 
- - - - (2) INFORMATION FOR SEQ ID NO:32: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Murine gamma - # constant region 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
- - GGAAGCTTAY CTCCACACAC AGGRRCCAGT GGATAGAC - # 
- # 38 
- - - - (2) INFORMATION FOR SEQ ID NO:33: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA primer" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Murine kappa - # light chain 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
- - GGAAGCTTAC TGGATGGTGG GAAGATGG - # - # 
28 
- - - - (2) INFORMATION FOR SEQ ID NO:34: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: Not R - #elevant 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: nucleotide s - #equence of fusion point 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
- - ATCCGGATAC CTGCGG - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO:35: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "RNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: ligated rRNA - # exons 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
- - CUCUCUUAAG - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:36: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Ribozyme" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: attack point - # for reverse group I splicing 
reaction 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
- - GGCUCUCUAA AAA - # - # 
- # 13 
- - - - (2) INFORMATION FOR SEQ ID NO:37: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Ribozyme" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Group IIB - #intron domain V 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
- - RAGCURAUGA NNNNAAANUN UCAYGUMUUG UUY - # - # 
33 
- - - - (2) INFORMATION FOR SEQ ID NO:38: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Ribozyme" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Group IIA - #intron domain V 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
- - RAGCNNNRUR CRRNGAAANY YGYANGYNNN GUUY - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:39: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA construct" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: Fn(1-3) liga - #tion point 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
- - TCAAAGAGCG - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:40: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "DNA construct" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: (5,6)Prot li - #gation point 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
- - GGGATACCTG - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:41: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "RNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: PY1 exon - #sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
- - GUGGUGGGAC AUUUUC - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO:42: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "RNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: PY2 and P - #Y3 exon sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
- - GUGGUGGAUG UCAAA - # - # 
- # 15 
- - - - (2) INFORMATION FOR SEQ ID NO:43: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "RNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: PY4 exon - #sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
- - GUGGUGGGAU GUCAAA - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO:44: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "RNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: PY5 and P - #Y6 exon sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
- - CUCGUGGAUG UCAAA - # - # 
- # 15 
- - - - (2) INFORMATION FOR SEQ ID NO:45: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: Not Relev - #ant 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "RNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: PY7 exon - #sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
- - UGUGCCGGAC UGCUCC - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO:46: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "RNA" 
- - (vii) IMMEDIATE SOURCE: 
(B) CLONE: PY8 and P - #Y9 exon sequence 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
- - UGCCUGCUCU GAGGGA - # - # 
- # 16 
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