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Biomedical subjects

R D Wells

Publications and source records attributed to R D Wells.

At least 163 records · Page 9Linked to original sources
PubMed1981

Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments.

Twenty-one DNA restriction fragments ranging in size from 12 to 880 base pairs (bp) were purified to homogeneity in milligram amounts. The developments which facilitated this work were (a) procedures for the rapid preparation of gram quantities of pure recombinant plasmid DNAs, (b) selective poly(ethylene glycol) (PEG) precipitation of DNAs according to broad classes of lengths, and (c) large-scale high-pressure liquid chromatography on RPC-5 for the purification of fragments to homogeneity. The 95- and 301-bp sequences from the lactose control region of Escherichia coli were cloned into the single EcoRI site of pVH51 in up to four copies per plasmid. These tandem inserts are separated by EcoRI sites and have a head to tail orientation in all cases. A total of 50 and 90 mg of th 95- and 301-bp fragments, respectively, were prepared from 300-L fermentations of E. coli cells transformed with these plasmids. A rapid and improved method, which can easily be scaled up, for the purification of plasmids and DNA restriction fragments was developed. Also, the linear pVH51 vector DNA was digested with HaeIII to yield fragments ranging in size from 12 to 880 bp. The five smaller fragments (from 12 to 180 bp) were purified quantitatively by a selective PEG precipitation enrichment step followed by RPC-5 column fractionation. The larger fragments (245-880 bp) were prepared in milligram amounts. Ten subfragments from the 301-bp lac fragment were prepared by HpaII, HinfI, or HaeIII/AluI digestions followed by separation of the reaction products on RPC-5.

Chemical Phenomena↗
PubMed1981

Left-handed DNA in restriction fragments and a recombinant plasmid.

Circular dichroism and 31P-NMR on synthetic oligomers of (dC-dG) inserted within DNA restriction fragments indicate that the right-handed B-structure can exist in close proximity to the left-handed Z-structure. Also, this salt-induced transition to Z-form in a small (dC-dG) segment (1.3%) of a recombinant plasmid markedly influenced the supercoil of the plasmid. These observations have implications for the postulated role of naturally occurring related simple sequences in the regulation of gene activity.

Base Sequence↗
PubMed1981

High resolution experimental and theoretical thermal denaturation studies on small overlapping restriction fragments containing the Escherichia coli lactose genetic control region.

The distribution of thermal stability in the Escherichia coli lac control region is evaluated from the melting behavior of 5 short (80-219 base pairs (bp)) sequenced DNA restriction fragments containing various parts of this sequence. The thermal denaturation of these fragments was measured at 3 salt concentrations. The previous notion that the melting curves for small fragments are sharp and asymmetric in 0.01 M Na+ and broadened and less asymmetric at 0.105 and 0.505 M Na+ is confirmed and the possible explanations are discussed. The existence of two thermodynamic boundaries in this region is also confirmed. The exact location of the boundary upstream of the cyclic AMP receptor protein (CAP) binding site is accurately determined from melting experiments at 260 and 282 nm. The secondary boundary located between the promoter and operator sequence is apparent at the two higher salt concentrations and begins to disappear at the lower salt concentration. The physical interpretation of the melting experiments is compared to the results of theoretical predictions derived from the known sequence of the fragments.

Base Sequence↗
PubMed1981

The leftward promoter of bacteriophage lambda. Isolation on a small restriction fragment and deletion of adjacent regions.

As part of our investigations on the relationship between DNA structure and gene regulation, a 352-base pair Hae III fragment was cloned containing the leftward operator-promoter (PL) region of bacteriophage lambda. This was accomplished without the aid of a phenotypic assay for the cloned fragment. A Hae III digest of a segment of the lambda genome was first fractionated by RPC-5 column chromatography. The partially purified PL fragment was then ligated into the Eco RI site of the pBR322 plasmid vector and cloned into the recBC+ Escherichia coli host C600(R-M-) using a technique that converts the Hae III ends of the fragment into Eco RI sites. Similar cloning attempts into a recBC- host (C600-SF8) were unsuccessful. The cloned fragment has the PL promoter oriented toward the tetracycline resistance genes of the vector, and is isolated from the plasmid (pRW601) by digestion with Eco RI followed by sucrose gradient sedimentation. The fragment was identified as PL by restriction mapping, repressor binding, sequencing, and promoter location. The now complete sequence of this fragment, part of which was known previously, reveals a large A/T-rich region immediately adjacent to the PL promoter. We have generated deletions in this region in order to study the influence of this sequence on promoter function.

Bacteriophage lambda↗
PubMed1981

The leftward promoter of bacteriophage lambda. Structure, biological activity, and influence by adjacent regions.

The effect of regions adjacent to the lambda PL promoter was studied using a sequence deleted in an A/T-rich segment immediately upstream from the promoter. High resolution thermal denaturation analysis showed that the undeleted sequence, as isolated on a 360-bp restriction fragment (360-PL) melted in two distinct steps. Since the deleted sequence (230-PL) melts at a higher temperature than any portion of 360-PL, the deleted promoter is more stable to denaturation. This increased stability was also suggested by a transcription assay in which the strong binding of RNA polymerase is inhibited at low temperature. The deleted promoter fragment required a higher temperature to become functional than did the 360-PL fragment. Furthermore, RNA polymerase bound to the 230-PL fragment is displaced by heparin at a faster rate than from 360-PL, and the deleted fragment initiates transcription at a reduced rate. Thus, by several criteria the deleted promoter is less active than the parent sequence. Evidence is presented arguing that the deletion does not alter the sequence directly recognized by RNA polymerase. Therefore, the altered sequence is reducing promoter activity by changing some other level of overall promoter structure. These results suggest that regions outside of the sequence directly recognized by a regulatory protein can affect genetic expression from the site.

Bacteriophage lambda↗
PubMed1981

Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments.

The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E. coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described. Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment. For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+. Along with the broadening, the melting transitions become more symmetrical. This result is discussed with respect to the irreversibility of melting transitions at low ionic strength. After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+. The dTM/dlog[Na+] values for three fragments fall between 15.7 and 16.7. An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp. The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements. The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed. The possible influences of fragment length and sequence on the thermal stability are discussed. The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described. This transition is about half as wide as the smallest one reported to date.

DNA Restriction Enzymes↗
PubMed1980

A 300 MHz and 600 MHz proton NMR study of a 12 base pair restriction fragment: investigation of structure by relaxation measurements.

The 1H NMR spectrum of a 12 base pair DNA restriction fragment has been measured at 300 and 600 MHz and resonances from over 70 protons are individually resolved. Relaxation rate measurements have been carried out at 300 MHz and compared with the theoretical predictions obtained using an isotropic rigid rotor model with coordinates derived from a Dreiding model of DNA. The model gives results that are in excellent agreement with experiment for most protons when a 7 nsec rotational correlation time is used, although agreement is improved for certain base protons by using a shorter correlation time for the sugar group, or by increasing the sugar-base interproton distances. A comparison of non-selective and selective spin-lattice relaxation rates for carbon bound protons indicates that there is extensive spin diffusion even in this short DNA fragment. Examination of the spin-spin relaxation rates for the same type of proton on different base pairs reveals little sequence effect on conformation.

Base Composition↗
PubMed1980

Circular dichroism studies of the B goes to A conformational transition in seven small DNA restriction fragments containing the Escherichia coli lactose control region.

The B goes to A conformational transition caused by high ethanol concentrations was studied for seven DNA restriction fragments with overlapping and known sequences. Since the DNAs are homogeneous and range in GC content from 44-63%, they permit an evaluation of the influence of DNA sequence and base composition on the B goes to A transition. Moreover, their small size (80-301 bp) minimizes precipitation artifacts. The B- form spectra (in low salt) and the transition toward the C- form (in ethanol concentrations below the B goes to A transition) agree with prior measurements on chromosomal DNAs and are similar for all seven DNAs. At higher ethanol concentrations (80%), all fragments undergo a transition to the A- form as judged by the large increase of the positive CD band at 270 nm. Difference spectra among the fragments reveal minor differences between the A- form spectra. The ethanol concentration necessary to cause this transition is 72 +/- 2% for all fragments, thus excluding a preference of the CAP-, E. coli RNA polymerase-, or lac repressor-binding sequences for the A- form. The kinetics of the B goes to A transition in 80% ethanol are biphasic; the initial rapid transition is an intramolecular B goes to A form shift and the slower transition is an aggregation (but not precipitation) of the DNA

Base Sequence↗