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M H Caruthers

Publications and source records attributed to M H Caruthers.

At least 55 records · Page 3Linked to original sources
PubMed1984

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.

Base Sequence↗
PubMed1984

Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the -35 region.

A set of the lac promoter mutants that have varying lengths of the spacer between the CAP binding site and the -35 region was constructed. The mutants have the spacer length increased by five (I5 mutant), or eleven (I11) residues or decreased by eleven residues (D11). We also present a construction of the hybrid between the gal and lac promoters in which the CAP binding site and the -35 region of the gal promoter are fused to the lac -10 region. The promoter fragments were assembled through ligations of chemically synthesized oligodeoxynucleotides and cloned into a pBR322-derivative vector. The results of the in vivo assays of promoter activity show that the I11 mutation results in an active but weak promoter that can be stimulated by CAP, though to a lesser degree than the wild-type lac promoter. The other mutants exhibit no promoter activity. Since the insertion of 11-bp preserves the location of the CAP binding site on the same side on the DNA helix, the data demonstrate the importance of spatial alignment between the CAP binding site and the promoter. The fact that the gal::lac hybrid is inactive as a promoter indicates also that catabolite activation is a highly complex process in which the -35 and -10 regions cannot be easily exchanged between promoters.

Base Sequence↗
PubMed1984

Isolation and characterization of a cDNA clone for bovine cytochrome c oxidase subunit IV.

We have isolated a cDNA clone for the precursor to subunit IV of bovine cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). A cDNA library was constructed from poly(A)+ RNA of adult beef liver by insertion of cDNA into the plasmid vector pBR322. Transformants were screened by colony hybridization with two mixtures of [32P]-labeled synthetic oligodeoxyribonucleotides. We screened 20,000 transformants with a mixture of heptadecamers complementary to all 16 possible sequences encoding amino acids 98-103 and obtained two cDNA clones encoding subunit IV amino acid sequences. We determined the DNA sequence of the larger (416 base-pair) insert, which contains the coding sequence for amino acids 1-107 of the mature protein and an NH2-terminal extension (presequence). The deduced amino acid sequence of the mature protein is identical with the previously determined protein sequence: the sequence of the NH2-terminal extension contains a potential initiator methionine at amino acid -22 from the NH2-terminus of the processed protein. The presequence is quite basic and contains several arginines, including one at the processing site. No hydrophobic region analogous to that found in bacterial and eukaryotic signal peptides is present, but there are homologies with other mitochondrial protein presequences, which may include a common signal for their destination and processing.

Amino Acid Sequence↗
PubMed1984

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites.

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

Adenoviruses, Human↗
PubMed1983

Point mutations of the adenovirus major late promoter with different transcriptional efficiencies in vitro.

Synthetic oligodeoxyribonucleotides were used to introduce single base changes into the TATAA box of the adenovirus-2 major late promoter. The resultant mutant DNA templates that contained altered TATAA sequences (TATAA leads to TCTAA/TATCA) were tested for their relative transcriptional activity in vitro using HeLa whole cell extracts. The data indicate that the change TATAA leads to TCTAA results in a level of specific transcription reduced to 48.7% of that from the parental template, whereas the change TATAA leads to TATCA reduced transcription to 21%. Neither base change abolished promoter activity.

Adenoviridae↗
PubMed1983

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter.

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.

Bacteriophage lambda↗
PubMed1982

A calorimetric investigation of the interaction of the lac repressor with inducer.

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.

Buffers↗
PubMed1982

Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site.

Early region IA of human adenoviruses encodes a function required for normal induction of early viral genes and virus-induced cell transformation. The region is expressed at early times as two overlapping spliced mRNAs, 12S and 13S, which encode closely related proteins. To distinguish between the functions of these proteins, a single T leads to G transversion was constructed which prevents splicing of the 12S mRNA. This transversion, in the second base of the 12S mRNA intron, does not alter the protein encoded by the 13S mRNA due to degeneracy in the genetic code. Studies with this mutant demonstrated that only the 13S mRNA encodes the regulatory protein required for normal early gene expression.

Adenoviruses, Human↗
PubMed1982

The application of nucleic acid chemistry to studies on the functional organization of gene control regions.

A new, extremely high yield method for synthesizing deoxyoligonucleotides has been developed. Syntheses are completed on a silica gel polymer support using appropriately protected deoxymononucleotide 3'-phosphoramidites. These mononucleotide intermediates are synthesized using simple organo chemical procedures and can be stored indefinitely as dry powders. Activation is achieved by adding a weak acid such as tetrazole to an acetonitrile solution of the mononucleotide. Chemical steps for the addition of one nucleotide to the support are the following: (1) detritylation using ZnBr2 in nitromethane/methanol (5 min); (2) condensation of a 5'-O-dimethoxytrityldeoxynucleoside 3'-methoxy-N, N-dimethylamino phosphine with the support bound nucleoside (5 min); (3) blocking unreacted, support bound nucleoside hydroxyl groups with acetic anhydride (5 min); (4) oxidation of the phosphite to the phosphate with I2 (2 min). Completed deoxyoligonucleotides are isolated by first sequentially treating with thiophenol, t-butylamine, ammonium hydroxide, and 80% acetic acid and then purifying to homogeneity by electrophoresis on polyacrylamide gels. The method is extremely fast (approximately 30 min per cycle), yields in excess of 98% per condensation are obtained, and isolation of the final product is a simple, one step purification. This methodology has been used to synthesize several lac operators containing altered sequences. The results of binding studies indicate that lac repressor does not recognize a unique DNA sequence.

Base Sequence↗
PubMed1979

Studies on gene control regions XII. The functional significance of a lac operator constitutive mutation.

The functional significance of a lac operator constitutive mutation has been determined. The transition adenine-thymine to guanine-cytosine was shown to be a constitutive mutation simply because thymine contains the functionally important 5-methyl group whereas cytosine does not. The remainder of the base pair is of no consequence. The experimental approach was to synthesize various modified operators containing cytosine, 5-methyl-cytosine, and 5-bromocytosine. The synthetic operator containing a guanine-cytosine base pair displays an eightfold reduction in stability with lac repressor whereas the operator containing 5-methylcytosine binds repressor at least as tightly as does the wild type sequence. Results published previously have shown that a similar decrease in stability of the repressor-operator complex can be obtained simply by substituting uracil for thymine or by inverting the base pair to thymine-adenine. All these results taken together implicate the thymine 5-methyl as the only important functional group recognized by the lac repressor at this base pair. Further confirmation of this conclusion was obtained by substitution of 5-bromocytosine and 5-bromouracil at this base pair. Both altered the stability of the repressor-operator complex by about the same percent suggesting that the bromine atom was the important determinant of complex stability for 5-bromopyrimidine analogs.

Base Sequence↗
PubMed1979

Studies on gene control regions X. The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction.

Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group. These operators also contained a poly(dA) . poly(dT) tail 8 to 12 base pairs in length at one end. Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical. For example a seven fold reduction in stability was observed for the transversion. Results also suggest that the operator spans at least 21 base pairs.

Adenine↗