Simultaneous synthesis and biological applications of DNA fragments: an efficient and complete methodology.
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Base Sequence↗
Biomedical subjects
H Blöcker
Publications and source records attributed to H Blöcker.
Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region.
New expression vectors were constructed for use in strains of Escherichia coli. Their most important feature is a polylinker system that facilitates the insertion of a gene in an optimal relationship to the highly efficient E. coli atpE translational initiation region (from nucleotide -50 to the start codon). Three ATG-containing restriction endonuclease sites can be used for the insertion of the 5' end of a gene at, or near to, its translational initiation codon. These sites may alternatively be used for the creation of a suitable translational start codon. Transcription is started by the bacteriophage lambda major promoters pR and pL in tandem and terminated by the bacteriophage fd terminator. Transcriptional initiation is very effectively repressed at 28-30 degrees C by the product of the bacteriophage lambda cIts857 gene, which is also present on the vectors. Full induction is achieved by shifting the incubation temperature to 42 degrees C. The combination of highly efficient transcriptional and translational signals on these vectors allowed high-level expression of sequences encoding human interferon beta and interleukin 2 and of the E. coli atpA, sucC and sucD genes.
DNA Restriction Enzymes↗
Chemical-enzymatic synthesis, cloning and expression of a synthetic gene coding for an env protein fragment of the human T-cell leukemia virus type 1.
A synthetic gene for a 88 amino acid long env protein fragment of the human T-cell leukemia virus type 1 (HTLV1) has been assembled by ligation of 35 oligodesoxyribonucleotides, which were chemically synthesized by the phosphotriester segmental support method. After cloning into the pEX vector this HTLV1 env-protein fragment was expressed in E. coli.
Cloning, Molecular↗
Conformational analysis of hairpin oligodeoxyribonucleotides by a single-strand-specific nuclease.
Hairpin-forming oligodeoxynucleotides d[ATCCTA(A)NTAGGAT] with n = 3-6 were subjected to nuclease digestion with the nuclease from mung bean. Cleavage occurs only in the loop region of the hairpin molecules. In detail, the number and intensity of cleavage sites were determined in relation to the length of the loop, the temperature and the salt concentration. The restricted accessibility of mung bean nuclease to the loop bases adjacent to the helical region of all hairpins is due to a reduced exposure of these bases in presence of a certain Mg2+ concentration. With increasing temperature the exposure of these bases is increased. It is deduced that the bases adjacent to the helix increase the length of the latter by stacking, the degree of which is dependent on the number of loop bases.
Binding Sites↗
Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases.
We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV.
Base Sequence↗
RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane.
Treatment of E. coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links. In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have already been identified (see ref. 6). Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA-protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629-633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723-724 and positions 1531-1542 (the 3'-end of the 16S RNA).
Cross-Linking Reagents↗
The mom gene of bacteriophage Mu: the mechanism of methylation-dependent expression.
Transcription of the DNA modification gene (mom) of bacteriophage Mu requires methylation of three GATC sites upstream of the mom promoter by the Escherichia coli deoxyadenosine methylation function (Dam). The three sites map within a 40-bp segment termed region I. Small deletions, inversions, duplications and specific point mutations have been introduced in region I. Their effect on mom expression has been studied in dam+ and dam strains. Dam-dependent expression of the mom gene requires a specific arrangement of the three GATC sites and the presence of the methylated base in at least two of the three sites. We show that mom specific modification is regulated by a host protein. The Mom function is expressed in dam strains if they are defective in one component of the methylation-instructed mismatch correction system, mutH. We suggest that the product of mutH functions as a transcriptional repressor by binding to region I.
Base Sequence↗
Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector.
Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species. Analysis of the products in E. coli revealed that in addition to the two primase polypeptides of 118 and 80 kDa the pri region of RP4 encodes two smaller proteins of 16.5 and 8.6 kDa. The transcript for the four RP4-specified products is polycistronic. The vector system used in E. coli is based on the plasmid pKK223-3 (Brosius and Holy, 1984), a ColE1-type replicon which contains a polylinker sequence flanked on one side by the controllable tac promoter and on the other side by two strong transcriptional terminators. The gene for the lac repressor (lacIQ) was inserted to render the use of the plasmid independent from repressor-overproducing strains. The gene cartridge essential for high-level expression and selection was combined with the RSF1010 replicon to generate a vector plasmid functioning in a wide variety of Gram-negative hosts. The versatility of the vector family was extended by constructing derivatives that contain the polylinker in inverted orientation relative to the tac promoter. Therefore, the orientation of the cloned fragment can be chosen by 'forced cloning' into the appropriately selected vector.
Cloning, Molecular↗
Predicting DNA duplex stability from the base sequence.
We report the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions. We obtained the relevant thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. We show how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence. We illustrate our method of calculation by using the nearest-neighbor data to predict transition enthalpies and free energies for a series of DNA oligomers. These predicted values are in excellent agreement with the corresponding values determined experimentally. This agreement demonstrates that a DNA duplex structure thermodynamically can be considered to be the sum of its nearest-neighbor interactions. Armed with this knowledge and the nearest-neighbor thermodynamic data reported here, scientists now will be able to predict the stability (delta G degree) and the melting behavior (delta H degree) of any DNA duplex structure from inspection of its primary sequence. This capability should prove valuable in numerous applications, such as predicting the stability of a probe-gene complex; selecting optimal conditions for a hybridization experiment; deciding on the minimum length of a probe; predicting the influence of a specific transversion or transition on the stability of an affected DNA region; and predicting the relative stabilities of local domains within a DNA duplex.
Base Composition↗
Structure and transforming function of transduced mutant alleles of the chicken c-myc gene.
A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v-myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles.
Alleles↗
Analysis of secondary structures in M13mp8 (+) single-stranded DNA by the pausing of DNA polymerase alpha.
The pausing of DNA replication has been used as a tool for analyzing secondary structures in a single-stranded DNA. M13mp8 (+) single-stranded DNA was replicated in vitro by the DNA polymerase alpha from calf thymus. The positions of pausing were determined from DNA sequencing gels. All experimentally observed pausing sites could be correlated with computer-predicted secondary structures of the M13 single-stranded DNA. In the computer calculations of the secondary structures, long-range base-pairing, G.T mispairs and loop-out of bases were allowed. By using six different primers, the pausing site pattern and the corresponding secondary structure map of a region comprising 1400 nucleotides of the M13 genome has been established. Our experiments indicate that the M13 DNA is highly structured. Most of the stable structures are clustered around the origin of replication. With fragments of the M13 DNA, we show that long-range base-pairing exists in the M13 single-stranded genome and we present evidence for tertiary structure interactions. Furthermore we observe structures that form newly during the course of replication. The Escherichia coli single-stranded DNA-binding protein facilitates replication through the barriers.
Animals↗
Investigation of the tertiary folding of Escherichia coli 16S RNA by in situ intra-RNA cross-linking within 30S ribosomal subunits.
Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, followed in some cases by a second partial digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC). The cross-linked RNA complexes were separated by two-dimensional gel electrophoresis and the sites of cross-linking analysed by our published procedures. Tertiary structural cross-links in the 16S RNA were identified between positions 31 and 48, between oligonucleotides 1090-1094 and 1161-1164, and between oligonucleotides 1125-1127 and 1280-1281. The first of these imposes a rigid constraint on the relative orientations of helices 3 and 4 of the 16S secondary structure. A further tertiary cross-link (which could not be precisely localised) was found between regions 1-72 and 1020-1095, and secondary structural cross-links were identified between positions 497 and 545-548, and positions 1238-1240 and 1298.
Elapid Venoms↗
On the fidelity of DNA polymerase alpha: the influence of alpha-thio dNTPs, Mn2+ and mismatch repair.
The phi X174am16 revertant system has been used to investigate the influence of alpha-thio-dNTPs and of Mn2+ on the fidelity of the 9S DNA polymerase alpha from calf thymus. Upon substituting dGTP by alpha-thio-dGTP during the in vitro replication, a nearly tenfold decrease in the frequency of G:G and G:T mispairs is observed. The formation of all other mispairs is not changed in the presence of the corresponding alpha-thio-dNTP. Mn2+ at concentrations of 0.5 mM does not influence the frequencies of the mispairs. The expression rate of errors formed during in vitro replication in the (-) strand has been determined for all mispairs detectable in the phi Xam16 system. The (-) strand expression of G:T, T:T and C:T mismatches is about 50%, whereas for A:G, G:G and C:A mismatches it is clearly below 50%. We conclude that the different base-base mismatches are repaired with different efficiencies.
Animals↗
Stabilisation of human interferon beta synthesis in Escherichia coli by zinc ions.
Human interferon beta synthesized in Escherichia coli is unstable and toxic for the bacterial cell. Zinc ions are able to stabilise interferon beta in E. coli probably by inhibiting the action of cell internal proteinase(s) which affect the half-life of this foreign protein. As a result up to one order of magnitude more active IFN-beta can be detected in Zn2+-treated E. coli cells.
Cloning, Molecular↗
Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution.
The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.
Amino Acid Sequence↗
The 5'-terminal sequence of U1 RNA complementary to the consensus 5' splice site of hnRNA is single-stranded in intact U1 snRNP particles.
The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing.
Autoantibodies↗
Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro.
Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible lambda pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to 10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(-) orientation is as efficient as the switch from G(-) to G(+).
Journal Article↗
A new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports.
A new approach is described which will allow the simultaneous synthesis of large numbers of pre-defined oligonucleotide chains. No machine aid is needed. The simultaneous syntheses can be performed by one person and do not require much more time than is currently needed for the synthesis of just one oligonucleotide in existing strategies. The general idea is the following: One uses noninterchangeable polymeric entities from each of which enough OD units can be isolated after completion of the syntheses. Whenever growing chains on different entities have to be elongated with the same building block these entities are gathered in the same reaction vessel. After such a common reaction cycle the entities are separated and now combined according to the next common building blocks etc. The practicability of this approach is demonstrated by the synthesis of d(T-A-A-T-A-T-T-A) and d(T-A-G-T-A-C-T-A) on cellulose filter disks following the phosphotriester approach.
Base Sequence↗