Showing papers in "Nucleic Acids Research in 1991"

Distribution of repetitive DNA sequences in eubacteria and application to finerpriting of bacterial enomes

Abstract: Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.

A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.

Abstract: The polymerase chain reaction (PCR) has revolutionised the rapid analysis of mammalian genomic DNA (1). However, PCR is less useful in the analysis of plant DNA due to the difficulties in extracting nucleic acids from limited amounts of plant tissue. We have developed a method for the rapid extraction of small amounts of plant genomic DNA suitable for PCR analysis. The method is applicable to a variety of plant species and has the added advantage of not requiring any phenol or chloroform extraction. Thus it is possible to complete an extraction within 15 minutes without handling any hazardous organic solvents. Samples for PCR analysis (usually leaf tissue) are collected using the lid of a sterile Eppendorf tube to pinch out a disc of material into the tube. This ensures uniform sample size and also reduces the possibilities of contamination arising from handling the tissue. DNA is extracted as follows: The tissue is macerated (using disposable grinders from Bel-art Products: Scienceware, Pequannock, NJ, 07440 USA. catalog no 992) in the original Eppendorf tube at room temperature, without buffer, for 15 seconds. 400 yl of extraction buffer (200 mM Tris HC1 pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) is added and the sample vortexed for 5 seconds. This mixture can then be left at room temperature until all the samples have been extracted (> 1 hour). The extracts are centrifuged at 13,000 rpm for 1 minute and 300 y\\ of the supernatant transferred to a fresh Eppendorf tube. This supernatant is mixed with 300 /il isopropanol and left at room temperature for 2 minutes. Following centrifugation at 13,000 rpm for 5 minutes, the pellet is vacuum dried and dissolved in 100 /xl 1XTE. This DNA is stable at 4°C for greater than one year. 2.5 yX of this sample is sufficient for a standard 50 y\\ PCR (Figure 1). When older tissue is used this may be increased to 25 /xl without any deleterious effect on the PCR. Using this protocol we have found it possible to process hundreds of individual samples in a single working day.

A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells.

Abstract: We have developed a simple and rapid method for preparing DNA-binding protein extracts from mammalian cells. The protocol is derived from the large scale procedure of Dignam et al. (1) and utilizes hypotonic lysis followed by high salt extraction of nuclei. The technique described by Dignam has several drawbacks, including the need for large numbers of cells and lengthy incubation and dialysis steps. It is labor-intensive and precludes preparation of multiple samples simultaneously. Our aim in developing this micropreparation procedure was to easily and rapidly extract DNA-binding proteins from small numbers of cells. Frequently, the quantity of cells available for extraction of DNA-binding proteins is limiting, as in analysis of clinical samples, of multiple clones of transfected cells, or of COS cell pools transiently transfected with a cDNA expression library. Ideally, such a technique would allow processing of many samples simultaneously and quickly on the benchtop. The method described in this report accomplishes these goals. In addition, it gives an excellent yield of DNA-binding proteins, comparable to that of the large scale Dignam protocol with minimal proteolysis. We typically start with between 5X10 and 10 cells. All centrifugations of less than 30 seconds are carried out in a room temperature microfuge; between steps, the samples are placed on ice. Adherent cells are scraped into 1.5 ml of cold phosphatebuffered saline (PBS); non-adherent cells are pelleted and resuspended in 1.5 ml cold PBS. The cell suspension is then transferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 yX cold Buffer A (10 mM HEPES-KOH pH 7.9 at 4°C, 1.5 mm MgCl2, 10 mM KC1, 0.5 mM dithiothreitol, 0.2 mM PMSF) by flicking the tube. The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 20—100 y\ (according to starting number of cells) of cold Buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 minutes at 4C and the supernatant fraction (containing DNA binding proteins) is stored at -70°C. The yield is 50 to 75 /tg protein per 10 cells.

A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies

Abstract: In genetic linkage studies using the restriction fragment length polymorphism (RFLP) technique, it is essential to process effectively large numbers of blood samples. One of the problems faced when extracting DNA by standard methods is the requirement of deproteinizing cell digests with hazardous organic solvents like phenol, chloroform and isoamyl alcohol (1-3). The method described in this report avoids the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated sodium chloride solution (4). Most of the procedures also involve prolonged incubation with proteinase K (1, 4, 5). Our procedure eliminates completely the use of proteinase K treatment. The method reported here is the most economical, safe and rapid for preparation of DNA from whole blood.

Simplified mammalian DNA isolation procedure.

Abstract: The reverse genetics technologies that have recently been developed for mice have provided new tools to probe gene function in vivo. Unfortunately these powerful systems often require the analysis of large numbers of DNA samples. The genetargeting technology requires screening of embryonic stem-cell clones and later of the mice themselves, the latter also being the case for standard transgenic technology. It is not always possible or desirable to rely on PCR analyses, necessitating the isolation of large numbers of DNA samples of sufficient quality for Southern blot analysis. We have simplified the standard mammalian DNA isolation procedure with the aim of minimizing the number of manipulations required for each sample. The basic procedure applied to cultured cells does not require any centrifugation steps or organic solvent extractions.

Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains

Abstract: The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that heterodimeric Fab fragments can be assembled on the surface of the phage by linking one chain to the phage coat protein, and secreting the other into the bacterial periplasm. Furthermore by introducing an amber mutation between the antibody chain and the coat protein, we can either display the antibody on phage using supE strains of bacteria, or produce soluble Fab fragment using non-suppressor strains. The use of Fab fragments may offer advantages over single chain Fv fragments for construction of combinatorial libraries.

Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products.

Abstract: Although the polymerase chain reaction (1,2) (PCR) can be used to produce a large amount of a specific DNA from a complex source, cloning the PCR products has not proven to be straightforward. Restriction endonuclease sites are often incorporated into the oligonucleotide primers used for amplification, so that cleavage of the product will create sticky ends that can theoretically be ligated to an equivalently cut vector (3). Unfortunately, many restriction endonucleases fail to cleave when their recognition sequences are located within a few base pairs of the end of a DNA fragment (4, 5). Ligation-independent PCR cloning schemes (6, 7, 8) involve the addition of at least 12 bases to the 5' end of the primer, which can increase the cost of synthesis substantially when done routinely. Attempts to clone PCR products as blunt-ended fragments have been very inefficient, due to the template-independent terminal transferase activity of Taq polymerase, which results in the addition of a single nucleotide at the 3' end of the fragment (9, 10). This nucleotide is almost exclusively an adenosine, due to the strong preference of the polymerase for dATP (9). Thus, cloning the products as blunt-ended fragments requires enzymatic processing to remove of the 3' overhang using an enzyme with 3' to 5' exonuclease activity (11).

PROSITE : a dictionary of sites and patterns in proteins

Abstract: PROSITE is a compilation of sites and patterns found in protein sequences. The use of protein sequence patterns (or motifs) to determine the function of proteins is becoming very rapidly one of the essential tools of sequence analysis. This reality has been recognized by many authors. While there have been a number of recent reports that review published patterns, no attempt had been made until very recently [5,6] to systematically collect biologically significant patterns or to discover new ones. It is for these reasons that we have developed, since 1988, a dictionary of sites and patterns which we call PROSITE. Some of the patterns compiled in PROSITE have been published in the literature, but the majority have been developed,in the last two years, by the author.

Eukaryotic start and stop translation sites

Abstract: Sequences flanking translational initiation and termination sites have been compiled and statistically analyzed for various eukaryotic taxonomic groups. A few key similarities between taxonomic groups support conserved mechanisms of initiation and termination. However, a high degree of sequence variation at these sites within and between various eukaryotic groups suggest that translation may be modulated for many mRNAs. Multipositional analysis of di-, tri-, and quadrinucleotide sequences flanking start/stop sites indicate significant biases. In particular, strong tri-nucleotide biases are observed at the -3, -2, and -1 positions upstream of the start codon. These biases and the interspecific variation in nucleotide preferences at these three positions have lead us to propose a revised model of the interaction of the 18S ribosomal RNA with the mRNA at the site of translation initiation. Unusually strong biases against the CG dinucleotide immediately downstream of termination codons suggest that they may lead to faulty termination and/or failure of the ribosome to disassociate from the mRNA.

The SWISS-PROT protein sequence data bank

Abstract: SWISS-PROT is an annotated protein sequence database established in 1986 and maintained collaboratively, since 1988, by the Department of Medical Biochemistry of the University of Geneva and the EMBL Data Library

Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of chlamydomonas

Abstract: Expression vectors for Chlamydomonas reinhardtii chloroplast transformation have been constructed with transcription and translation signals from chloroplast genes. The bacterial aadA sequence, coding for aminoglycoside 3" adenyl transferase, was inserted in these vectors and introduced into the C. reinhardtii chloroplast by particle gun transformation. The stable transgenic expression of this foreign protein in the chloroplast confers spectinomycin and streptomycin resistance to the transformed cells. This new marker can be used as a reporter of gene expression, and as a portable selectable cassette for chloroplast reverse genetics. Targetted gene disruption mutants of loci required for photosynthesis, tscA and psaC, were thus obtained. A gene disruption of an unidentified open reading frame, ORF472, remained heteroplasmic, suggesting that it has a vital function.

Automated assembly of protein blocks for database searching

Abstract: A system is described for finding and assembling the most highly conserved regions of related proteins for database searching. First, an automated version of Smith's algorithm for finding motifs is used for sensitive detection of multiple local alignments. Next, the local alignments are converted to blocks and the best set of non-overlapping blocks is determined. When the automated system was applied successively to all 437 groups of related proteins in the PROSITE catalog, 1764 blocks resulted; these could be used for very sensitive searches of sequence databases. Each block was calibrated by searching the SWISS-PROT database to obtain a measure of the chance distribution of matches, and the calibrated blocks were concatenated into a database that could itself be searched. Examples are provided in which distant relationships are detected either using a set of blocks to search a sequence database or using sequences to search the database of blocks. The practical use of the blocks database is demonstrated by detecting previously unknown relationships between oxidoreductases and by evaluating a proposed relationship between HIV Vif protein and thiol proteases.

Maximizing sensitivity and specificity of PCR by preamplification heating

Abstract: We have found that assembling the reaction mixture at a temperature greater than the annealing temperature improved both product yield and specificity of PCR. When reactions were maintained at 70°C in a dry heating block during addition of denatured samples to aliquotted reagent master mix, a reproducible increase in product yield was observed compared to duplicates maintained at room temperature (Table 1). Greater specificity was also seen with heating in a gel electrophoretic analysis (not shown). In addition, improved sensitivity and specificity was seen with pre-amplification heating using templates of double stranded plasmid DNA that had not been previously denatured (Figure 1), both with (Figure 1) and without (not shown) the addition of tetramethylammonium chloride (TMAC) (Fisher Scientific) (3) to the reaction master mix. This effect was observed with 3 different primer-template pairs (Table I and Figure 1). It is of note that pre-amplification heating did not improve product yield with primers of the same sequence as those used in the experiment in Table I that had been documented to lack failure sequences by HPLC. We speculate that preamplification heating may promote stringent primer annealing and subsequent extension, thereby increasing effective primer length. Minimization of 'primer-dimer'or primer self-annealing may also contribute. Preamplification heating has allowed the use of primer pairs which were inadequate for amplification of low copy number templates when reactions were assembled at room temperature.

Modified deoxyoligonucleotides stable to exonuclease degradation in serum

Abstract: Unmodified deoxyoligonucleotides are rapidly degraded in serum-containing medium. Utilizing internally labelled deoxyoligonucleotides the deoxyribonuclease profile for fetal calf serum and human serum was determined. It was found that the predominate nuclease activity in both systems was 3' exonuclease. Deoxyoligonucleotides are protected from exonuclease degradation in sera and cell media by simple terminal modifications that maintain high binding activity for the complementary DNA sequence.

Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells

Abstract: A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.

A rapid procedure for extracting genomic DNA from leukocytes

Abstract: 1. Collect 5 ml of blood in a vacutainer tube (Becton Dickinson) containing EDTA and mix. 2. Make volume up to 10 ml with solution 1 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCy. 3. Add 120 /il Nonidet P40 (BDH) to lyse the cells. Mix well by inverting several times. 4. Spin down the nuclear pellet at 2000 rpm for 10 mins. 5. Pour off the supernatant without dislodging the pellet. The pellets can be stored frozen. 6. Gently resuspend pellet well in 800 /il of solution 2 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCl2; 0.5 M NaCl; 0.5% SDS; 2 mM EDTA). Solution 2 will lyse the nuclei so be careful not to shear the DNA. Transfer to a 1.5 ml microcentrifuge tube. 7. Add 400 /il of distilled phenol (saturated with 1 M Tris pH 8.0) and mix well. 8. Microfuge for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. Do not worry about transferring a little of the interface. 9. Add 200 /tl of phenol and 200 /il of chloroform :isoamyl alcohol (24:1). Mix well by inverting. 10. Spin for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. 11. Add 700 /tl of chloroform:isoamyl alcohol and extract as above. 12. Transfer upper aqueous phase to a small clean container. Avoid removing the interface. Add 2 volumes of ice cold ethanol and mix to precipitate the DNA*. 13. With the sealed tip of a pasture pipette, transfer the DNA fibres to a microcentrifuge tube containing 1 ml of 70% ethanol. Mix well to wash the DNA. 14. Spin for 5 mins at full speed. Discard the ethanol and dry pellet in a speed vac. Resuspend DNA in sterile water at 65°C. Do not over-dry genomic DNA or it will be difficult to resuspend. SIZE A / /Kb Hindll l

Isolation of molecular markers from specific chromosomal intervals using DNA pools from existing mapping populations

Abstract: We present a general method for isolating molecular markers specific to any region of a chromosome using existing mapping populations. Two pools of DNA from individuals homozygous for opposing alleles for a targeted chromosomal interval, defined by two or more linked RFLP markers, are constructed from members of an existing mapping population. The DNA pools are then screened for polymorphism using random oligonucleotide primers and PCR (1). Polymorphic DNA bands should represent DNA sequences within or adjacent to the selected interval. We tested this method in tomato using two genomic intervals containing genes responsible for regulating pedicle abscission (jointless) and fruit ripening (non-ripening). DNA pools containing 7 to 14 F2 individuals for each interval were screened with 200 random primers. Three polymorphic markers were thus identified, two of which were subsequently shown to be tightly linked to the selected intervals. The third marker mapped to the same chromosome (11) but 45 cM away from the selected interval. A particularly attractive attribute of this method is that a single mapping population can be used to target any interval in the genome. Although this method has been demonstrated in tomato, it should be applicable to any sexually reproducing organism for which segregating populations are being used to construct genetic linkage maps.

Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping.

Abstract: Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between strains of almost any organism. We applied the technique to the mouse (Mus musculus). The characteristic differences in the AP-PCR genomic fingerprints between strains will be of value in strain identification and verification. Using one primer, we genetically mapped four polymorphisms in a set of C57BL/6J x DBA/2J recombinant inbreds. One of these polymorphisms is a length variant. The method will allow rapid genetic mapping of DNA polymorphisms without Southern blotting.

Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5′ ends of mRNAs and for constructing cDNA libraries by in vitro amplification

Abstract: Cloning full length cDNAs is a difficult task especially if mRNAs are not abundant or if tissue is only available in limited amounts. Current strategies are based on in vitro amplification of cDNAs after adding a homopolymeric tail at the 3' end of the ss-cDNA. Since subsequent amplification steps yield unspecific amplified DNA mostly due to non-specific annealing of the reverse primer containing a homopolymeric tail, we have devised a new strategy based on the ligation of single-stranded oligodeoxyribonucleotide to the 3' end of single-stranded cDNAs. The efficiency of the strategy was assessed by analyzing the 5' ends of the rat pineal gland tryptophan hydroxylase messenger. The 5' end of the least abundant messenger (0.005% of total mRNAs) could be cloned without selection. Sixty percent of the analyzed clones correspond to TPH. This technique revealed a 5-nt stretch not apparent using dG tailing strategy. The potentiality of the method for generating cDNAs libraries was tested with 10(4) PC12 cells. In this library, the abundance of tyrosine hydroxylase clones (0.03%) correlated well with the abundance of the corresponding messenger, showing that no major distortion was introduced into the construction of the library.

A sequence assembly and editing program for efficient management of large projects

Abstract: We describe a sequence assembly and editing program for managing large and small projects. It is being used to sequence complete cosmids and has substantially reduced the time taken to process the data. In addition to handling conventionally derived sequences it can use data obtained from Applied Biosystems,Inc. 373A and Pharmacia A.L.F. fluorescent sequencing machines. Readings are assembled automatically. All editing is performed using a mouse operated contig editor that displays aligned sequences and their traces together on the screen. The editor, which can be used on single contigs or for joining contigs, permits rapid movement along the aligned sequences. Insertions, deletions and replacements can be made in individual aligned readings and global changes can be made by editing the consensus. All changes are recorded. A click on a mouse button will display the traces covering the current cursor position, hence allowing quick resolution of problems. Another function automatically moves the cursor to the next unresolved character. The editor also provides facilities for annotating the sequences. Typical annotations include flagging the positions of primers used for walking, or for marking sites, such as compressions, that have caused problems during sequencing. Graphical displays aid the assessment of progress.

A thermodynamic study of unusually stable RNA and DNA hairpins

Abstract: About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra-loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs.

A simple and rapid method for generating a deletion by PCR

Abstract: Site-directed mutagenesis including deletions, insertions and basesubstitutions is one of the strongest tools for examining the biological or biochemical properties of cloned genes or gene control elements. Application of this technique has been, however, often limited because of the absence of a suitable restriction site or the complexity of procedures for generating a desired mutation. We report here a simple and rapid mutational method by using polymerase chain reaction (PCR) (1), in which oligonucleotide primers are designed in inverted tail-to-tail directions to amplify the cloning vector together with the target sequence (Fig. 1). A deletion is generated by amplification with primers that have a corresponding gap between their 5' ends. After the PCR with these primers, amplified linear DNA is selfligated, and used to transform appropriate competent cells. We use human papillomavirus (HPV) type 16 E7 open reading frame cloned into pGEMEXTM-l (Promega) as a template. A pair of primers, 5'-TTAAATGACAGCTCAGAGGA-3' and 5'-TGTCTCTGGTTGCAAATCTA-3', was prepared for deleting HPV 16 nucleotide position 619-642. PCR of 25 cycles at 94°C, 2 min, 55°C, 2 min and 72°C, 4 min was done in 100 y\\ of reaction mixture containing 20 mM Tris-HCl, pH 8.8 at 25°C, 10 mM KC1, 10 mM (NH^SO,,, 2 mM MgCl2, 0.1% Triton X-100, 100 /tg/ml acetylated BSA, 200 /*M each of dNTPs, 2 ng of the template plasmid, 2 fiM each of the primers and 2 units of VentTM DNA polymerase (New England Biolabs). Use of VentTM DNA polymerase was required for simplifying the protocols because Taq DNA polymerase adds one nontemplate-directed nucleotide to its products and generates 3' overhanging ends (2), whereas VentTM DNA polymerase possesses 3' —5' exonuclease activity to yield blunt ends, which are ready for self-ligation (3). A portion of agarose gel-purified PCR product was self-ligated in 10 /tl of 66 mM Tris-HCl, pH 7.4, 8 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 5 units of T4 polynucleotide kinase and 5 units of T4 DNA ligase at 14°C for 12 h, followed by use for transformation of JM109. Sequencing of about 300 base pairs each of 16 resulting plasmids shows excellent efficiency of 100% with only one mjsincorporation (Figure 2). We note that our inverted amplification method will be available for generating an insertion or a base-substitution.

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus

Abstract: Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.