Insertion Of DNA Sequences Research Articles

Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli

We have constructed a set of expression vectors which contain synthetic DNA sequences comprising a computer-generated model ribosomal binding site located downstream from the tightly regulated phage λ p L promoter. These vectors have been used in several laboratories to produce significant amounts of eukaryotic and prokaryotic gene products in Escherichia coli, either as fusion proteins (with two to nine extra N-terminal amino acids) or as proteins containing the naturally occurring amino terminus. For inserting DNA sequences downstream of an initiation codon, we used synthetic oligonucleotides to introduce multiple-use restriction sites recognized by EcoRI, Bam HI and ClaI which generate termini complementary to those of a variety of enzymes (e.g., Eco RI ∗, MboI, TaqI, and HpaII), in addition to their own. A set of three of these vectors was made to accommodate all three translational reading frames. In combination, the features of these vectors afford useful advantages over expression vectors previously described, especially for the application of shot-gun cloning of genomic DNA to generate expression libraries.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

4359535 Autonomously replicating DNA containing inserted DNA sequences: George Pieczenik

4359535 Autonomously replicating DNA containing inserted DNA sequences: George Pieczenik

Intronless human dihydrofolate reductase genes are derived from processed RNA molecules.

Three groups of recombinant bacteriophage containing coding sequences for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) were isolated from two human DNA clone libraries. One recombinant (lambda hDHFR-1) contains three exons that encode the COOH-terminal portion of human DHFR. The other two human DHFR genes (hDHFR-psi 1 and hDHRF-psi 2) lack introns. hDHFR-psi 2 contains several in-phase termination codons and is only 93% homologous to the normal human DHFR coding sequences, whereas hDHFR-psi 1 has an open reading frame and is virtually identical to the coding sequence of the normal DHFR gene. The region of DNA sequence homology between each intronless gene and the normal DHFR gene extends 2.9 kilobases beyond the end of the coding sequences. At the 3' end of this homologous sequence, each intronless gene has an A-rich tract. The lack of introns and the presence of the 3' A-rich tract suggest that hDHFR-psi 1 and hDHFR-psi 2 were derived from processed RNA molecules. A short DNA sequence, 60 nucleotides 5' to the ATG start codon in lambda hDHFR-psi 2, is directly repeated immediately after the 3' A-rich tract; such terminal direct repeats also flank integrated proretroviruses and transposable DNA elements and are thought to be the hallmark of inserted DNA sequences.

Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7.

Double-stranded cDNA sequences for rat alpha 1-acid glycoprotein and rat glutathione S-transferase mRNAs were inserted into the Pst I site of bacteriophage M13mp7 and used to develop a new method for preparing specific cDNA hybridization probes directly from cloned template DNA. A palindrome sequence surrounding the Pst I site in the vector DNA permitted single-stranded DNA isolated from the recombinant phage to fold back, thus forming a stable hybrid bounded on the ends by a large loop of M13mp7 single-stranded DNA and a small loop of inserted foreign DNA. A primer corresponding to an internal sequence of the foreign DNA was hybridized, then Escherichia coli DNA polymerase I was used to synthesize a 32P-labeled complementary DNA copy of the cloned inserted DNA. The single-stranded cDNA reaction product was easily isolated by subsequent sedimentation through alkaline sucrose gradients. Gel electrophoresis of the labeled cDNA product, after denaturation with glyoxal, indicated a single discrete band with an electrophoretic mobility corresponding to the length of the inserted DNA sequence. About 95% of the cDNA product formed S1 nuclease-resistant hybrids in hybridization reactions with excess RNA in solution. DNA sequences complementary to the M13mp7 vector DNA were not detected in the cDNA product. Thus, these M13mp7-derived probes are the functional equivalent of cDNA copies to mRNAs and can be employed for quantitative measurements of mRNA concentration. This simple, rapid method probably can be used for most cloned DNA sequences to yield single-stranded radioactively labeled DNA, without contaminating vector DNA sequences, for virtually any hybridization requirement.

Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA.

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.

Construction and identification of a hybrid plasmid containing DNA sequence complementary to phenobarbital-inducible cytochrome P-450 messenger RNA from rat liver.

Cytochrome P-450 mRNA has been partially purified from membrane-bound polysomes of the livers of phenobarbital-treated rats by SDS-phenol-chloroform extraction, followed by poly(U)-Sepharose chromatography and by centrifugation through a sucrose density gradient. Cytochrome P-450 mRNA activity was detected near 18S in the sucrose density gradient, accounting for approximately 5% of total mRNA activity on the basis of [3H]leucine incorporation in an in vitro translation system of wheat germ. Complementary DNA (cDNA) which had been synthesized on the partially purified mRNA by AMV reverse transcriptase was inserted into the Pst I site of pBR 322. After bacterial transformation, and in situ colony hybridization using [32P]cDNA as a probe, a colony carrying cytochrome P-50 cDNA sequence was identified by a hybridization-arrested translation assay. Sequence complementarity of the inserted DNA sequence to cytochrome P-450 mRNA was further confirmed by a positive hybridization-translation assay. The mRNA isolated from the partially purified mRNA preparation by hybridizing it with the recombinant DNA (III-8-10) showed enriched synthesis of a protein product whose apparent molecular weight was consistent with that of cytochrome P-450, and which was immunoprecipitable with anti-cytochrome P-450 antibody.

Transformation of mouse fibroblasts to methotrexate resistance by a recombinant plasmid expressing a prokaryotic dihydrofolate reductase.

A recombinant plasmid has been constructed for the expression of inserted DNA sequences coding for polypeptide chains using the simian virus 40 early promoter and splicing and polyadenylylation signals from the rabbit beta-globin gene. The coding regions for two prokaryotic methotrexate-resistant dihydrofolate reductases were introduced into the expression vector. When mouse fibroblasts were exposed to these recombinant plasmids, it was possible to select methotrexate-resistant clones that had integrated the plasmids and produced a chimeric RNA coding for the prokaryotic enzyme.

Construction and characterization of recombinant plasmid DNAs containing sequences of the origin of bacteriophage phi X174 DNA replication.

The synthetic DNA fragment (formula, see text) (corresponding to nucleotides 4299-4314 of the phi X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177. The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 FnudII restriction DNA fragment N6 (345 b.p.). Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined. Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head). In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the phi X origin region was 14 (No. 4300-4313), 16 (No. 4299-4314) and 20 nucleotides (No. 4299-4318), respectively. None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with phi X gene A protein. Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for phi X RFI DNA in the in vitro (+) strand synthesizing system. It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the phi X gene A protein. The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the phi X origin region of more than 20 nucleotides is required. These results suggest a model for initiation of phi X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the phi X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the phi X gene A protein. This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by phi X gene A protein.

80] Annealing of Oligo(dC)-tailed ds cDNA with oligo(dG)-tailed plasmid and transformation of Escherichia coli χ 1776 with recombinant DNA

This chapter focuses on preparing recombinants, DNA fragments can be joined to plasmid or virus vectors in a number of ways. Vectors linearized with restriction enzymes can be annealed to DNA fragments terminating in homologous sequences. Alternatively, vectors and DNA fragments can be terminated in the complementary oligodeoxynucleotides oligo(dA) and oligo(dT) or oligo(dG) and oligo(dC). With the use of the plasmid pBR322 linearized with the restriction enzyme PstI, the addition of oligo(dG) restores the PstI site. This conveniently permits the excision of the inserted DNA sequence from the recombinants. The PstI site is within the β-lactamase gene, so that these pBR322 recombinants with inserts are penicillinsensitive, but retain their tetracycline resistance. The transformation efficiency of χ1776 by intact plasmid pBR322 by the above procedure yields approximately 1 to 3×10 6 transformants per microgram of pBR322 DNA.

Number and evolutionary conservation of α- and β-tubulin and cytoplasmic β- and γ-actin genes using specific cloned cDNA probes

Bacterial clones containing inserted DNA sequences specific for α-tubulin, β-tubulin, β-actin and γ-actin have been constructed from mRNA of embryonic chick brain. Plasmids containing approximately 75, 90 and >90%, respectively, of the sequences present in α-tubulin, β-tubulin and β-actin mRNAs have been isolated as well as clones containing parts of the extensive 3′ untranslated regions of the β- and γ-actin mRNAs. The sequences for the two tubulins do not cross hybridize. Hybridization of labeled, cloned probes for each of the tubulins with chicken DNA digested with several restriction endonucleases reveals about four fragments for α- and four for β-tubulin. This seems to be the number of genes, since both the 5′ and 3′ ends of either cloned tubulin cDNAs hybridize to at least four common fragments in genomic DNA which has been digested with restriction endonucleases. The tubulin probes are able to hybridize under stringent conditions to DNA of all vertebrate genomes tested, as well as to sea urchin DNA, but not to yeast DNA. In digested sea urchin sperm DNA there are more than 20 different fragments which hybridize to both the 5′ and 3′ ends of the tubulin cDNAs. A full-length β-actin cDNA clone hybridizes to 4–7 bands in restricted chicken DNA and cross hybridizes to DNA from every other species tested, including sea urchin and yeast. Hybridization to chicken DNA of cloned probes specific for the 3′ untranslated regions of β- and γ-actin mRNA indicates that the β sequence is present only once in the genome and the γ is present in at most three copies. Neither 3′ untranslated sequence is conserved evolutionarily.

Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.

Isolation of bacteriophage λ containing yeast ribosomal RNA genes: Screening by in situ RNA hybridization to plaques

We have developed an in situ hybridization technique which can be used to screen large numbers of hybrid bacteriophage for the presence of a particular inserted DNA sequence. Plaques of hybrid phage are formed on E. coli lawns on nitrocellulose filters, and their DNA is released, denatured, and fixed directly on the filters for hybridization to radioactive RNA probes. We have used this technique to isolate a number of hybrid bacteriophage λ which contain EcoRI restriction fragments of the ribosomal RNA genes from yeast, and have examined the DNA from several of these phage.