Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrogen↗

PubMed1984

Deoxyadenosine-deoxycytidine pairing in the d(C-G-C-G-A-A-T-T-C-A-C-G) duplex: conformation and dynamics at and adjacent to the dA X dC mismatch site.

Deoxyadenosine-deoxycytidine pairing at symmetrically related dA X dC mismatch sites in the d(C1-G2-C3-G4-A5-A6-T6-T5-C4-A3-C2-G1) self-complementary duplex (henceforth called 12-mer AC) has been investigated by proton and phosphorus NMR studies in aqueous solution. We demonstrate that base pairing is maintained on either side of the mismatch site in the 12-mer AC duplex at low temperature. The proton chemical shifts and NOE measurements rule out models in which the H-2 proton of adenosine at the mismatch site is stacked over adjacent dG X dC base pairs. A comparison of the hydrogen-exchange kinetics in the d(C-G-C-G-A-A-T-T-C-G-C-G) duplex (henceforth called 12-mer) with standard dG X dC base pairs at position 3 from either end with the 12-mer AC duplex, which contains dA X dC mismatches at these positions, demonstrates kinetic destabilization at dG X dC base pair 4 adjacent to the mismatch site and at dA X dT base pairs 5 and 6 far from this site. This contrasts with previous hydrogen-exchange studies on the 12-mer GT and 12-mer GA duplexes where the kinetic destabilization was localized to base pair 4, which is adjacent to the mispairing site. The melting temperature of the 12-mer AC duplex in 0.1 M phosphate is approximately 30 degrees C lower than the corresponding value for the 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)

Deoxyadenosines↗

PubMed1984

Prenatal diagnosis of alpha 1-antitrypsin deficiency by direct analysis of the mutation site in the gene.

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Chromosome Mapping↗

PubMed1984

Quantitation of apolipoprotein A-I in pooled human serum by single radial immunodiffusion and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Apolipoprotein A-I was released from human HDL particles by treatment with 8 M urea, and the free apolipoprotein exhibited identical antigenicity and the same low mobility as purified apolipoprotein A-I in electrophoresis. Treatment of serum with 8 M urea enabled enabled quantitation of apolipoprotein A-I by single radial immunodiffusion assay, as judged by comparison with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Apolipoprotein A-I↗

PubMed1984

Two-fold symmetry of crystalline DNA-EcoRI endonuclease recognition complexes.

Recognition complexes between EcoRI endonuclease and either of two synthetic oligonucleotides (sequences CGCGAATTCGCG and TCGCGAATTCGCG) crystallize in Space Group P321 with unit cell parameters a = 128 and c = 47 A and a = 118.4 and c = 49.7 A, respectively. Native diffraction data to 3 A resolution have been collected from the form containing the tridecameric sequence. Electrophoretic analyses of dissolved crystals demonstrate that this form contains DNA and protein in a ratio of one double helix per enzyme dimer. The most likely asymmetric unit contents are one 31,000 dalton enzyme subunit and one strand of DNA, yielding VM values of 3.1 A3/dal and 2.8 A3/dal for the forms containing dodecameric and tridecameric DNA, respectively. This implies that the DNA-protein complex possesses two-fold rotational symmetry, which has been incorporated in the crystalline lattice.

Base Sequence↗

PubMed1984

Synthesis and use of synthetic oligonucleotides.

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Base Sequence↗

PubMed1984

[Studies on oocyte maturation and mammals and human fertilization].

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3-Hydroxysteroid Dehydrogenases↗

PubMed1984

Dynamics of DNA duplexes containing internal G.T, G.A, A.C, and T.C pairs: hydrogen exchange at and adjacent to mismatch sites.

We have undertaken high-resolution NMR studies to elucidate the conformation and dynamics of the d(C1-G2-C3-G4-A5-A6-T6-T5-C4-G3-C2-G1) 12-mer duplex and its analogs where the symmetrically related dG.dC base pairs in position 3 are replaced by dG.dT, dG.dA, dA.dC, and dT.dC mismatch pairs. The pairing schemes for the dG.dA and dA.dC mismatch interactions have been elucidated from intra- and inter-base pair nuclear Overhauser effect measurements. The replacement of two dG.dC pairs by two dG.dA or two dG.dT pairs results in an approximately 20 C destabilization of the 12-mer; replacement by two dA.dC or two dT.dC pairs results in an approximately 30 C destabilization of the 12-mer. The hydrogen exchange kinetics of the resolved and assigned imino protons in the 12-mer duplex and its mismatch analogs in 0.1 M phosphate have been monitored by saturation recovery measurements. We observe kinetic destabilization at dG.dC base pair 4 adjacent to the mismatch site, which increases in the order 12-mer less than 12-mer GT less than 12-mer GA less than 12-mer AC less than 12-mer TC. By contrast, the kinetic destabilization at dA.dT base pairs 5 and 6 increases in the order 12-mer approximately 12-mer GT approximately 12-mer GA less than 12-mer AC less than 12-mer TC. These results demonstrate that the introduction of dG.dT and dG.dA mismatches results in dynamic perturbations that are localized at the adjacent base pairs, whereas introduction of dA.dC and dT.dC mismatches results in perturbations that extend several base pairs into the duplex. Both base pair opening and preequilibrium pathways contribute to the imino proton hydrogen exchange rates in the 12-mer GA, 12-mer AC, and 12-mer TC duplexes, which is in contrast to earlier studies that demonstrated that the imino proton hydrogen exchange rates were a direct measure of the base pair opening rates in the 12-mer and 12-mer GT duplexes.

Base Composition↗

PubMed1983

Requirement for signal peptide cleavage of Escherichia coli prolipoprotein.

Oligonucleotide-directed site-specific mutagenesis was applied to alter the cleavage site in the signal peptide of the major outer membrane lipoprotein of Escherichia coli. Replacing the glycine residue at the cleavage site with an alanine residue did not affect the processing of the signal peptide. However, when the same cleavage site was constructed by the deletion of the glycine residue, the signal peptide was no longer cleaved. These results indicate that stringent structural integrity at the cleavage site in the lipoprotein signal sequence is required for correct processing of prolipoprotein.

Amino Acid Sequence↗

PubMed1983

Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli.

We have examined the importance of the positively charged NH2 terminus of the major outer membrane lipoprotein precursor, prolipoprotein, in the early steps of secretion in Escherichia coli. For this purpose, we have generated three mutants using oligonucleotide-directed mutagenesis in which the charge at the NH2-terminal region was changed from +2 to +1, 0, and -2. The results indicate that the synthesis of prolipoprotein is facilitated by the presence of a positively charged NH2 terminus. In addition, the translocation of prolipoprotein across the cytoplasmic membrane does not absolutely require any basic amino acids at its NH2 terminus. However, the presence of a net negatively charged NH2 terminus causes an initial cytoplasmic accumulation of prolipoprotein which is slowly, post-translationally translocated across the cytoplasmic membrane at a rate which is dependent on the number of positive charges present in this region. The analysis of these mutants clearly demonstrates the importance of the NH2 terminus of the lipoprotein signal peptide in initiating the secretion of this protein in E. coli.

Amino Acid Sequence↗

PubMed1983

Purification of insulin receptor with full binding activity.

Insulin receptor was purified 2400-fold with an overall yield of 40% from human placental membranes by affinity chromatography on wheat germ agglutinin-Sepharose and insulin-Sepharose. The receptor was eluted from insulin-Sepharose using mild conditions, eliminating urea, so that it was stable and retained full insulin-binding activity. Chromatofocusing and gel filtration analysis indicated that the receptor preparation was apparently pure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three high molecular weight protein bands with Mr = 320,000, 300,000, and 270,000 under nonreducing conditions and two major protein bands with Mr = 135,000 and 90,000 under reducing conditions. The purified receptor showed a curvilinear Scatchard plot with maximum insulin binding of 28.5 micrograms per mg of protein. In comparison, the receptor eluted from insulin-Sepharose with previously used conditions in the presence of urea resulted in maximum insulin binding of only 6 micrograms per mg of protein. This indicates that a 4-to 5-fold increase in specific activity can be obtained by using the new elution conditions.

Chromatography, Affinity↗

PubMed1983

Solid phase synthesis of polynucleotides. VIII. Synthesis of mixed oligodeoxyribonucleotides by the phosphotriester solid phase method.

A solid phase method for the simultaneous synthesis of mixed oligonucleotides using a phosphotriester approach has been developed. For this synthesis, a mixture of mono or dimeric coupling units is used, and a slight difference in the reactivity of those units is found. However, this difference does not hamper the simultaneous, mixed oligonucleotide synthesis, and the sequence analysis of a product demonstrates the existence of all desired sequences in the final mixture.

Base Sequence↗

PubMed1983

Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site.

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.

Bacterial Outer Membrane Proteins↗

PubMed1983

Saccharomyces cerevisiae actin--Escherichia coli lacZ gene fusions: synthetic-oligonucleotide-mediated deletion of the 309 base pair intervening sequence in the actin gene.

Plasmids carrying gene fusions between the yeast (Saccharomyces cerevisiae) actin gene and an initiation-defective Escherichia coli lacZ (beta-galactosidase) gene have been constructed. Expression of beta-galactosidase in such fusion plasmids depends on transcription of the actin gene, and is possible only after the RNA-splicing machinery has removed from the primary RNA transcript the 309-bp intervening sequence (IVS) interrupting the actin coding region. Mutants deleting the actin IVS were constructed via synthetic oligonucleotide-mediated in vitro mutagenesis of the actin-beta-galactosidase fusion plasmid. A 17-base synthetic oligonucleotide was used to generate a 309-bp deletion which precisely removed the actin IVS. A partial deletion mutant was also constructed in which 272-bp, starting at the 5' end of the actin IVS, and including the 5' splice junction signal, were deleted. Both the complete and partial IVS-deletion mutants were transformed into yeast hosts. However, the partial deletion resulted in a greater than 98% reduction in beta-galactosidase activity. The precise deletion of the actin IVS did not reduce the levels of beta-galactosidase activity as compared with the parental fusion plasmid containing the intact IVS.

Actins↗