Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.     

S1 nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus

The structures of murine sarcoma virus (MuSV) ts110 viral RNA and intracellular RNA present in MuSV ts110-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV ts110 viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV ts110 RNAs, 4.0 and 3.5 kilobases (kb) in length, containing overlapping central deletions relative to wild-type MuSV 124 viral RNA (Junghans et al., J. Mol. Biol. 161:229-255, 1982). Here we show that the deletion (termed delta 1) in the 4.0-kb RNA has a 5' border located at about nucleotide 2409 (using the numbering system of Van Beveren et al., Cell 27:97-108, 1981), a position 63 bases upstream of the junction of the p30 and p10 coding sequences. The 3' border of the delta 1 deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5-kb MuSV ts110 RNA, the 5' border of the deleted central region (termed delta 2) is located in a splice consensus donor site at approximately nucleotide 2017, 330 bases downstream from the junction of the p12 and p30 coding sequences, and extends about 1,915 bases in the downstream direction to nucleotide 3935, found in a splice consensus acceptor site about 55 nucleotides downstream of the first mos gene initiation codon and 30 bases upstream of the second initiation codon. No alteration of polyadenylate addition sites was observed in either MuSV ts110 RNA species, as compared with MuSV 349 RNA. The observation that the 5' and 3' borders of the deletion in the 3.5-kb RNA are within in-frame splice donor and acceptor sites suggests strongly that the 3.5-kb RNA is derived from the 4.0-kb RNA by a temperature-sensitive splice mechanism. Data presented here show unequivocally that formation of the 3.5-kb MuSV ts110 RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33 degrees C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39 degrees C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33 degrees C permits the reappearance of the 3.5-kb RNA at its original level.     

Nucleotide assignment of alkali-sensitive sites in mouse mitochondrial DNA

Mature, closed circular mouse mitochondrial DNA contains a significant number of ribonucleotides throughout the genome. Previous studies have implicated the two origins of DNA replication as preferred sites of ribonucleotide retention. We have analyzed the site specificity of ribosubstitution by direct sizing of alkali-treated restriction fragments in comparison with the DNA sequence of untreated restriction fragments of cloned mouse mitochondrial DNA. These results have confirmed the observations that ribonucleotides are retained at the two origins of replication and are most likely remnants of RNA priming events associated with DNA replication. The map location of ribonucleotides at the light strand origin of replication has been refined to a triplet nucleotide (5'-CGG-3') in the light strand initiation region. This approach has demonstrated that all four deoxyribonucleotides are subject to ribosubstitution and no single base (or subset of the four bases) predominates. An examination of selected regions of the mitochondrial DNA genome including the putative coding region for cytochrome oxidase subunit III and regions containing the genes for tRNAPhe, tRNAVal, 12 S rRNA, and 16 S rRNA reveals preferred sites for ribosubstitution. These preferred sites do not relate in any obvious way to the functional aspects of these domains. In addition, the data indicate that every position in the DNA sequences examined can be ribosubstituted at a very low frequency.     

The Ayerst Award Lecture 1976. Novel factors in protein biosynthesis.

Comparison of nucleotide sequences surrounding the initiation sites of a number of mRNAs reveals few common features. These may be the presence of in- or out-of-phase nonsense codons and (or) polypurine bases complementary to the 16S RNA of the 30S subunit of ribosomes. Since the bases which precede or follow an initiation site vary in length and composition we have examined whether they play a role as spacers between cistrons or whether they have an active function in the termination and initiation of translation. In vitro we have observed that some sequences 5' terminal to AUG are preferred over others in forming an initiation complex. The same bases have much less effect when present at the 3' terminal end of an AUG codon. When the 5' terminal codon is the termination codon UAA, absolutely no initiation complex can be detected. This suggests that spacing may be needed between a stop and a start codon. Conversely, the hexamer AUGUAA failed to elicit chain termination. This was so in systems that terminated when free UAA was added or when a sense triplet was present between the initiation and termination triplets. These results suggest that ribosomes may recognize the stop triplet. Hence ribosomes may not obey simple A and P site models in the termination reaction.     

Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo

Analysis of the nucleotide sequences at the 5' ends of RNA-primed nascent DNA chains (Okazaki fragments) and of their locations in replicating simian virus 40 (SV40) DNA revealed the precise nature of Okazaki fragment initiation sites in vivo. The primary initiation site for mammalian DNA primase was 3'-purine-dT-5' in the DNA template and the secondary site was 3'-purine-dC-5', with the 5' end of the RNA primer complementary to either the dT or dC. The third position of the initiation site was variable with a preference for dT or dA. About 81% of the available 3'-purine-dT-5' sites and 20% of the 3'-purine-dC-5' sites were used. Purine-rich sites, such as PuPuPu and PyPuPu , were excluded. The 5'-terminal ribonucleotide composition of Okazaki fragments corroborated these conclusions. Furthermore, the length of individual RNA primers was not unique, but varied in size from six to ten bases with some appearing as short as three bases and some as long as 12 bases, depending on the initiation site used. This result was consistent with the average size (9 to 11 bases) of RNA primers isolated from specific regions of the genome. Excision of RNA primers did not appear to stop at the RNA-DNA junction, but removed a variable number of deoxyribonucleotides from the 5' end of the nascent DNA chain. Finally, only one-fourth of the replication forks contained an Okazaki fragment, and the distribution of their initiation sites between the two arms revealed that Okazaki fragments were initiated exclusively (99%) on retrograde DNA templates. The data obtained at two genomic sites about 350 and 1780 bases from ori were essentially the same as that reported for the ori region (Hay & DePamphilis , 1982), suggesting that the mechanism used to synthesize the first DNA chain at ori is the same as that used to synthesize Okazaki fragments throughout the genome.     

Replication of bacteriophage M13. XV. Location of the specific nick in M13 replicative form II accumulated in Escherichia coli polAex1.

M13 replicative form II (RFII) DNA was prepared from Escherichia coli RS5052 (polAex1) cells in the late stage of infection, and the DNA sequence at the discontinuity was examined. The data presented here suggest that the single discontinuity in the late stage of infection RFII maps at the same position as the gene II protein nicking site on fd RFI which was determined in vitro (Meyer et al., Nature (London) 278:365-367, 1979) and has a 5' terminal nucleotide sequence identical to that at the nick produced by gene II protein in vitro. The discontinuity in the in vivo RFII appears to be a single break in the phosphodiester backbone, leaving a 3' OH terminus. RFII molecules containing a gap, i.e., missing nucleotides at the site of discontinuity, were not detected.     

Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides.

The target specificity of DNA strand transfer mediated by human immunodeficiency virus type 1 integrase was examined in vitro with synthetic oligonucleotides. Although insertion occurred at most locations in the target, some sites were preferred over others by at least 15-fold. Changing the nucleotide sequence of the target changed the distribution of preferred sites in complex ways, some of which included changes in target preference distant from the sequence alteration. Alignment of target sequences revealed that adenosine is preferred adjacent to the insertion site. Strand transfer occurred to within 2 nucleotides of the 3' end and to within 3 nucleotides of the 5' end of the target. This suggests that only 2 or 3 nucleotides flanking the target site are required for integration; such restricted contact with target DNA would allow integrase to insert the two ends of viral DNA into two closely spaced sites in host DNA, consistent with the concerted in vivo integration reaction that generates a 5-bp target duplication.     

Intron sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae

Predicted single-stranded structure at the 3' splice site is a conserved feature among intervening sequences (IVSs) in eukaryotic nuclear tRNA precursors. The role of 3' splice site structure in splicing was examined through hexanucleotide insertions at a central intron position in the Saccharomyces cerevisiae tRNA gene. These insertions were designed to alter the structure at the splice site without changing its sequence. Endonuclease cleavage of pre-tRNA substrates was then measured in vitro, and suppressor activity was examined in vivo. A precursor with fully double-stranded structure at the 3' splice site was not cleaved by endonuclease. The introduction of one unpaired nucleotide at the 3' splice site was sufficient to restore cleavage, although at a reduced rate. We have also observed that guanosine at the antepenultimate position provides a second consensus feature among IVSs in tRNA precursors. Point mutations at this position were found to affect splicing although there was no specific requirement for guanosine. These and previous results suggest that elements of secondary and/or tertiary structure at the 3' end of IVSs are primary determinants in pre-tRNA splice site utilization whereas specific sequence requirements are limited.     

Role of the 3' splice site in U12-dependent intron splicing

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.     

Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro.

Three sequences in the vicinity of poly (A) addition sites are conserved among vertebrate mRNAs. We analyze the effects of single base changes in each position of AAUAAA and in the nucleotide to which poly (A) is added on 3' end formation in vitro. All 18 possible single base changes of the AAUAAA sequence greatly reduce addition of poly (A) to RNAs that end at the poly (A) addition site, and prevent cleavage of RNAs that extend beyond. The magnitude of reduction varies greatly with the position changed and the base introduced. For any given mutation, cleavage and polyadenylation are reduced to similar extents, strongly suggesting that the same factor interacts with AAUAAA in both reactions. Mutations at and near the conserved adenosine to which poly (A) is added disturb the accuracy, but not the efficiency, of 3' end formation. For example, point mutations at the conserved adenosine shift the 3' end of the most abundant 5' half-molecule downstream by a single nucleotide. The mechanism by which these mutations might exert their effects on the precision of 3' end formation are discussed.     

Sequences involved in the control of adenovirus L1 alternative RNA splicing.

During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated.     

The role of branchpoint-3'' splice site spacing and interaction between intron terminal nucleotides in 3'' splice site selection in Saccharomyces cerevisiae.

A conserved 3' splice site YAG is essential for the second step of pre-mRNA splicing but no trans-acting factor recognizing this sequence has been found. A direct, non-Watson-Crick interaction between the intron terminal nucleotides was suggested to affect YAG selection. The mechanism of YAG recognition was proposed to involve 5' to 3' scanning originating from the branchpoint or the polypyrimidine tract. We have constructed a yeast intron harbouring two closely spaced 3' splice sites. Preferential selection of a wild-type site over mutant ones indicated that the two sites are competing. For two identical sequences, the proximal site is selected. As previously observed, an A at the first intron nucleotide spliced most efficiently with a 3' splice site UAC. In this context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3' splice site UAC. We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU. Our data indicate that the 3' splice site is not primarily selected through an interaction with the first intron nucleotide. Selection of the 3' splice site depends critically on its distance from the branchpoint but does not occur by a simple leaky scanning mechanism.     

U-rich tracts enhance 3' splice site recognition in plant nuclei

The process of 5' and 3' splice site definition in plant pre-mRNA splicing differs from that in mammals and yeast. In mammals, splice sites are chosen by their complementarity to U1 snRNA surrounding the /GU at the 5' splice site and by the strength of the pyrimidine tract preceding the AG/ at the 3' splice site; in plants, the 3' intron boundary is defined in a position-dependent manner relative to AU-rich elements within the intron. To determine if uridines are utilized to any extent in plant 3' splice site recognition, uridines in the region preceding the normal (−1) 3' splice site of pea rbcS3A intron 1 were replaced with adenosines. This mutant activates two cryptic 3' splice sites (+62, +95) in the downstream exon, indicating that the uridines in the region immediately preceding the normal (−1) site are essential for recognition. Placement of different length uridine tracts upstream from the cryptic +62 site indicated that a cryptic exonic 3' splice site containing 14 or 10 uridine tracts with a G at −4 can effectively outcompete the normal 3' splice site containing an eight uridine tract with a U at −4. Substitutions at the −4 position demonstrated that the identity of the nucleotide at this position greatly affects 3' splice site selection. It has been concluded that several factors affect competition between these 3' splice sites. These factors include the position of the AU transition point, the strength of the uridine tract immediately preceding the 3' terminal CAG/ and the identity of nucleotide −4.     

Nucleotide preferences in sequence-specific recognition of DNA by c-myb protein.

Using a binding site selection procedure, we have found that sequence-specific DNA-binding by the mouse c-myb protein involves recognition of nucleotides outside of the previously identified hexanucleotide motif. Oligonucleotides containing a random nucleotide core were immunoprecipitated in association with c-Myb, amplified by the Polymerase Chain Reaction and cloned in plasmids prior to sequencing. By alignment of sequences it was apparent that additional preferences existed at each of three bases immediately 5' of the hexanucleotide consensus, allowing an extension of the preferred binding site to YGRCVGTTR. The contributions of these 5' nucleotides to binding affinity was established in bandshift analyses with oligonucleotides containing single base substitutions; in particular, it was found that replacement of the preferred guanine at position -2 with any other base greatly reduced c-Myb binding. We found that the protein encoded by the related B-myb gene bound the preferred c-Myb site with similar affinity; however, B-Myb and c-Myb showed distinct preferences for the identity of the nucleotide at position -1 relative to the hexanucleotide consensus. This study demonstrates that the c-Myb DNA-binding site is more extensive than recognised hitherto and points to similar but distinct nucleotide preferences in recognition of DNA by related Myb proteins.     

Sequestering of the 3' splice site in a theophylline-responsive riboswitch allows ligand-dependent control of alternative splicing

Despite the important role of alternative splicing in various aspects of biological processes, our ability to regulate this process at will remains a challenge. In this report, we asked whether a theophylline-responsive riboswitch could be adapted to manipulate alternative splicing. We constructed a pre-mRNA containing a single upstream 5' splice site and two 3' splice sites, of which the proximal 3' splice site is embedded in theophylline-responsive riboswitch. We show that this pre-mRNA spliced with preferential utilization of proximal 3' splice site in vitro. However, addition of theophylline to the splicing reaction promoted splicing at distal 3' splice site thereby changing the ratio of distal-to-proximal 3' splice site usage by more than twofold. Our data suggest that theophylline influenced 3' splice site choice without affecting the kinetics of the splicing reaction. We conclude that an in vitro selected riboswitch can be adapted to control alternative splicing, which may find many applications in basic, biotechnological, and biomedical research.     

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII.

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.     

Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae.

Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.