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Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 199 records · Page 11Linked to original sources
PubMed1987

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution.

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.

Base Sequence↗
PubMed1987

Long-range interactions of multiple DNA structural transitions within a common topological domain.

Local DNA conformations that are underwound with respect to the right-handed B form are favored in negatively supercoiled DNA. However, when multiple transitional events co-exist within a common topological domain, they must compete with one another for the available free energy of negative supercoiling. Recently we developed a general theoretical model capable of predicting the behavior at equilibrium of defined sequences in a variety of competitive situations. In the present work we have applied this theory to predict the formation of Z-DNA as a function of superhelicity in stretches of d(CG)m and d(CA)n when they are forced to compete with one another in the same plasmid. The observed behavior of these competing sequences is in close accord with theoretical predictions. These results indicate that sequences separated by large distances can effect the transitional behavior of each other in a complex manner which is independent of the relative orientation of the participating segments. The pattern of transitional events is strongly dependent on levels of DNA supercoiling, ambient conditions and on the nature and number of the sequences involved. Although in the present work we apply the model specifically to the Z-DNA conformational transition, the results of this study may have general relevance to a variety of biological processes in which the helical repeat of DNA is reversibly altered, including the initial steps in transcription, replication and recombination.

Computer Simulation↗
PubMed1987

(dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.

Animals↗
PubMed1987

Signal sequence of human preproparathyroid hormone is inactive in yeast.

The biosynthesis of human preproparathyroid hormone (hpreproPTH) and the processing to mature parathyroid hormone (hPTH) was investigated in yeast. Cells were transformed with a plasmid that carried a fusion gene made of the yeast pyruvate kinase promoter, complementary DNA (cDNA) encoding a slightly modified form of hpreproPTH and the transcription termination signal from yeast triosephosphate-isomerase. In transformed yeast cells we identified a protein that was recognized by a PTH antiserum and, on gel electrophoresis, comigrated with hpreproPTH marker. The amino-terminal sequence of the protein was consistent with that of hpreproPTH, indicating that the hormone precursor is not processed. It was localized inside the cell, when analyzed in pulse-chase experiments by trypsin accessibility in intact and lysed spheroplasts. In contrast, when mRNA from these yeast cells and from human parathyroid tissue was translated into preproPTH in a reticulocyte lysate supplemented with canine pancreatic microsomes, the preproPTHs from both mRNAs were transported and cleaved with identical efficiencies. We conclude that hpreproPTH is synthesized in yeast but not recognized and processed like a precursor of a secreted protein by the yeast secretory apparatus.

Animals↗
PubMed1987

A bifurcated hydrogen-bonded conformation in the d(A.T) base pairs of the DNA dodecamer d(CGCAAATTTGCG) and its complex with distamycin.

The crystal structures of d(CGCA3T3GCG) complex to the antitumor drug distamycin and the DNA fragment alone were solved by x-ray diffraction at 2.2 and 2.5 A resolution, respectively. The drug lies in the narrow minor groove near the center of the B-DNA fragment covering 5 of the 6 A.T base pairs. It is bound to the DNA by hydrogen bonding, van der Waals, and electrostatic interactions. In addition, the DNA was found to have an unusual conformation in the (dA)3.(dT)3 regions. These base pairs have a high positive propeller twist so that in the major groove the adenine amino group is located intermediate between the carbonyl O-4 groups of two adjacent thymines of the opposite strand, making bifurcated hydrogen bonds to the two thymine residues. This suggests a model to explain the unusual properties of poly-(dA).poly(dT) in which a modified B conformation is associated with a large propeller twist of the bases and a set of continuous bifurcating hydrogen bonds along the major groove, which may provide incremental stability to these segments. In addition, shorter segments of (dA)3-6.(dT)3-6 may have this conformation in the midst of B-DNA and stabilize bends in the DNA that may be associated with stacking on one of the high propeller-twisted bases at the ends of these segments.

Binding Sites↗
PubMed1987

The crystal structure of d(CCCCGGGG): a new A-form variant with an extended backbone conformation.

The crystal structure of d(CCCCGGGG) has been determined at a resolution of 2.25 A. The oligomers crystallize as A-DNA duplexes occupying crystallographic two-fold axes. The backbone conformation is, in general, similar to that observed in previously reported crystal structures of A-DNA fragments, except for the central linkage, where it adopts an extended structure resulting from all trans conformation at the P-O5'-C5'-C4' bonds. This type of conformation facilitates interstrand stacking between the guanines at the C-G site. The local helix twist at this step is very small (25 degrees) compared to an overall average of 33.5 degrees. The unique structure of the C-G base-pair step, namely the extended backbone and the distinct stacking geometry, may be an important feature in the recognition mechanism between double-stranded DNA molecules and restriction endonucleases such as Msp I, which cuts the sequence CCGG very specifically with a rate unaffected by neighboring base pairs.

DNA↗
PubMed1987

The interactions of ruthenium hexaammine with Z-DNA: crystal structure of a Ru(NH3)6+3 salt of d(CGCGCG) at 1.2 A resolution.

A crystal of d(CGCGCG) in the Z-DNA lattice was soaked with ruthenium(III) hexaammine and its structure refined at 1.2 A resolution. Three unique metal complexes were found absorbed to each hexamer duplex. In addition, two symmetry-related binding sites were located, yielding a total of five ruthenium complexes bound to each d(CGCGCG) duplex. One unique site and its symmetry related site are nearly identical to the binding site of cobalt(III) hexaammine on Z-DNA. At that position, the metal complex bridges the convex surfaces of two adjacent Z-DNA strands by hydrogen bonds to the N7 and O6 functional groups of the guanine bases. The remaining three ruthenium three ruthenium(III) hexaammine binding sites are not present in the cobalt(III) hexaammine Z-DNA structure. Of these, two are related by symmetry and span the gap between the convex outer surface of one Z-DNA strand and the helical groove crevice of a neighboring strand. The third ruthenium site has no symmetry mate and involves interactions with only the deep groove. In this interaction, the metal complex hydrogen bonds to both the phosphate backbone and to a set of primary shell water molecules that extend the hydrogen bonding potential of the deep groove crevice out to the surface of the molecule. Solution studies comparing the circular dichroism spectra of low salt poly(dG-dC).poly(dG-dC) samples in the presence of ruthenium(III) and cobalt(III) hexammine show that the ruthenium complex does stabilize Z-DNA in solution, but not as effectively as the cobalt analogue. This suggests that some of the interactions available for the larger ruthenium complex may not be important for stabilization of the left-handed DNA conformation.

Circular Dichroism↗
PubMed1987

Human preproparathyroid hormone synthesized in Escherichia coli is transported to the surface of the bacterial inner membrane but not processed to the mature hormone.

cDNA encoding human preproPTH (hpreproPTH) was expressed in Escherichia coli to study the processing of the precursor to hPTH and its secretion by the bacterial secretory apparatus. We first constructed hybrid genes that differed randomly in the distance between the E. coli lac promoter's ribosomal binding site and DNA encoding a fusion protein with beta-galactosidase activity and the prepro sequence of hpreproPTH on the aminoterminus. Starting with clones identified as efficient producers of beta-galactosidase on indicator agar plates, the coding sequence for hpreproPTH was reconstituted intact. In a different construction we placed the hpreproPTH coding sequence downstream from the lac promoter at a distance of 12 base pairs from the ribosomal binding site. PTH immunoreactive proteins from multiple clones were identified by protein gel electrophoresis and by protein microsequencing. PTH-related proteins encoded by different plasmids were shown to be hpreproPTH with amino-terminal extensions of either two or four amino acids and as authentic hpreproPTH. Two hPTH fragments, hPTH(3-84) and hPTH(8-84), were also observed. The trypsin accessibility of hpreproPTH and of the two hPTH fragments in pulse-chase, cell-fractionation experiments using intact and lysed spheroplasts lets us conclude that the mammalian signal sequence directs hpreproPTH to the surface of the spheroplast membrane but is not appropriately cleaved by the signal peptidase.

Animals↗
PubMed1986

Rate of B to Z structural transition of supercoiled DNA.

The forward rate of the B to Z transition induced by negative supercoiling of plasmid DNA containing an alternating C-G sequence has been examined using the binding of Z-DNA-specific antibodies to follow the transition. DNA samples of a plasmid containing a d(pCpG)16 X d(pCpG)16 insert were supercoiled to different extents and appropriate amounts of ethidium were bound to the DNAs to relax them and to keep the alternating C-G sequence in the right-hand helical form. Following the rapid removal of ethidium by passage through a column of cation exchange resin, the DNA becomes negatively supercoiled, which induces the flipping of the helical hand of the C-G insert. The rate of the transition is strongly dependent on the degree of supercoiling. The transition is complete in less than 50 seconds for a DNA with a specific linking difference (superhelical density) sigma of -0.09. For the same DNA, the half-time of the transition is about two minutes at sigma = -0.07 and about a factor of 10 slower at sigma = -0.05.

DNA, Superhelical↗
PubMed1986

An assessment of the Z-DNA forming potential of alternating dA-dT stretches in supercoiled plasmids.

The ability of a stretch of alternating dA-dT to adopt the left-handed Z form has been assessed by examining the behavior of the sequence d(CG)6(TA)4(CG)6 contained in the plasmid pBR322. The structural transition occurring within this sequence as a function of negative superhelicity was analyzed by several methods, including (1) the supercoiling-dependent unwinding of the insert as determined by two-dimensional gel electrophoresis, (2) the binding of anti-Z-DNA antibodies to the insert, (3) the sensitivity of the sequence to a single strand specific endonuclease, and (4) the sensitivity of the insert to digestion by a restriction endonuclease that cuts within the d(CG)6 segments when in the right-handed form. These studies have shown that in negatively supercoiled DNA the two d(CG)6 portions of the insert adopt the Z form, while the central d(TA)4 segment forms an underwound structure with a helical repeat that is best approximated as being intermediate between the B form and the Z form. A statistical mechanical treatment of the unwinding of the insert as a function of negative superhelicity provides an estimate of the minimum free energy required to convert an A-T bp from the B form to the Z form, as well as the free energy associated with the conversion of an A-T bp from the B form to the unwound form. These results strongly indicate that Z DNA is an unfavored structural alternative for stretches of d(AT)n in negatively supercoiled DNA.

Base Sequence↗
PubMed1986

Non-Watson-Crick G.C and A.T base pairs in a DNA-antibiotic complex.

The structure of a DNA octamer d(GCGTACGC) cocrystallized with the bisintercalator antibiotic triostin A has been solved. The DNA forms an unwound right-handed double helix. Four base pairs are of the Watson-Crick type while four are Hoogsteen base pairs, including two A.T and two G.C base pairs. This is the first observation in an oligonucleotide of Hoogsteen G.C base pairs where the cystosine is protonated. It is likely that these also occur in solutions of DNA complexed to this antibiotic.

Anti-Bacterial Agents↗
PubMed1986

The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium.

Four different preparations of rabbit and goat anti-Z-DNA antibodies were examined to determine the effects of antibody binding on the B-DNA-Z-DNA equilibrium. One of the four antibodies, a goat IgG, caused a marked lowering in the ionic strength required for the B-DNA to Z-DNA transition in poly(dG-dC) X poly(dG-dC), shifting the midpoint from 2.25 to 2.0 M NaCl. This IgG had a more prominent high affinity antibody population than did the other goat IgG, which caused little change in the midpoint of this transition. The presence of anti-Z-DNA antibodies also reduced the degree of negative supercoiling required for the formation of Z-DNA in (dG-dC)n sequences inserted into closed circular plasmid DNA. The goat IgG with the more marked effect on the salt-induced transition also had a greater effect in favoring Z-DNA formation in negatively supercoiled plasmids. A shift toward Z-DNA formation was observed in circular dichroism measurements upon antibody binding to poly(dG-dC) X poly(dG-dC) in very low ionic strength solution as well. We propose that the stabilization of Z-DNA by antibody binding in poly(dG-dC) X poly(dG-dC) occurs cooperatively, several antibody molecules binding to a single polymer molecule and stabilizing the entire molecule in Z-DNA through their combined binding energies. The stabilization of Z-DNA by antibody binding in a supercoiled plasmid can be significant, and failure to consider this effect and to choose appropriate conditions for measurement can lead to errors in estimating when Z-DNA will form in response to negative supercoiling.

Animals↗
PubMed1986

Local mobility of nucleic acids as determined from crystallographic data. II. Z-form DNA.

Directions and magnitudes of the local mobility of the Z-DNA hexamer duplex CpGpCpGpCpG have been determined by crystallographic refinement of anisotropic displacement parameters using the observed X-ray diffraction data. The cytidine and guanosine residues demonstrate different modes of mobility, implying that a dinucleotide is the smallest repeating unit in terms of flexibility as well as structure. Directions of librational and translational mobility of the cytidine and guanosine residues of Z-DNA are similar to those observed for the same nucleotides in B-DNA. This suggests that the local mobility of DNA is primarily determined by the individual nucleotide type and by the constraints of Watson-Crick base-pairing, rather than by helical form. Differences in the magnitudes of mobility may be responsible for some of the different physical properties of B-DNA and Z-DNA. The B to Z transition is discussed in terms of the observed flexibilities of these two helical forms.

Base Sequence↗
PubMed1986

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC).

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.

Crystallization↗
PubMed1986

Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides.

Five monoclonal anti-Z-DNA antibodies were characterized with respect to their binding of synthetic nucleic acid polymers and of supercoiled circular plasmid DNA. All of the antibodies reacted only with DNA in the Z-conformation; however, they fell into two classes on the basis of sequence specificity. One class, with broad specificity, reacted well with all sequences in the Z-form, including poly(dG-dC), poly(dG-dm5C), and poly (dG-dBr5C) in linear polymers and poly(dG-dC)n and poly[(dC-dA)n.(dT-dG)n] sequences in supercoiled plasmids. The other class bound only Z-DNA formed by poly(dG-dC). Binding of the monoclonal antibodies specifically to inserts of Z-DNA-forming sequences in plasmids was mapped directly by cross-linking of antibody to the DNA, digestion with restriction nuclease, and electrophoretic analysis of both the unbound fragments and the bound fragments recovered from immune complexes. The monoclonal antibodies were used for indirect immunofluorescence staining of Drosophila polytene chromosomes fixed by two procedures. One procedure yielded chromosomes with Z-specific antibody binding in many interbands, a few specific bands, and parts of some puffs. On chromosomes fixed by the second procedure, antibody staining appeared to follow the DNA concentration, staining all bands brightly. For each fixation procedure, chromosomes showed the same staining pattern with each of the broad specificity monoclonal antibodies that had been seen with polyclonal antibodies. The antibodies that reacted only with poly(dG-dC) and poly (dG-dC)n plasmid inserts did not stain chromosomes fixed by either protocol. We conclude that stretches of poly(dG-dC)n sequences do not contribute significantly to the presence of Z-DNA in fixed polytene chromosomes of Drosophila melanogaster.

Animals↗
PubMed1986

A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences.

The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected. We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation. Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date. The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations. The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths. Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2. The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation.

Base Sequence↗
PubMed1986

Expression of biologically active human interleukin 1 subpeptides by transfected simian COS cells.

"Interleukin 1" (IL-1) is a term used to describe the family of macrophage-derived proteins that mediate many immune and inflammatory reactions. We have previously described the molecular cloning and sequencing of the cDNA encoding the predominant (neutral) form of human IL-1, which has been designated IL-1 beta. We report here that transfection of simian COS cells with this cDNA clone results in the transcription of IL-1 mRNA and the synthesis of antibody-neutralizable intracellular IL-1 biological activity. In addition, selective deletion of regions of the IL-1 cDNA judged not to be essential for function, on the basis of conserved sequence homology, resulted in localization of a "core" region responsible for a majority of the biological activity. These results permit mapping the active site of IL-1 to a peptide of 6970 molecular weight located within the carboxyl third (between Met-136 and Gln-197) of the IL-1 precursor.

Animals↗