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Biomedical subjects

G Zon

Publications and source records attributed to G Zon.

At least 127 records · Page 7Linked to original sources
PubMed1986

Isolation and characterization of Limulus C-reactive protein genes.

Three homologous genes coding for Limulus C-reactive protein (CRP) have been isolated and characterized from a lambda phage EMBL-3 library containing genomic DNA sequences from Limulus amebocytes. The genes have a typical promoter region with a CAAT (nucleotides 50-53) and a TATAA (nucleotides 77-81) box located, respectively, 178 and 149 base pairs 5' upstream from the initiation codon ATG. The polyadenylation site AATAAA is situated within 300 base pairs downstream from the stop codon TAG. Nucleotide sequence analysis reveals a 24-residue signal peptide preceding a coding region of 218 amino acids. Significant differences were found between the genes coding for human and Limulus CRPs. In the human CRP gene there is an intron separating the signal peptide and the coding region. In Limulus this intervening sequence is missing. The Drosophila heat shock consensus sequence CTnGAAnnTTnAG (Simon, J. A., Sutton, C. A., Lobell, R. B., Glaser, R. L., and Lis, J. T. (1985) Cell 40, 805-817), found in the genes of human (Woo, P., Korenberg, J. R., and Whitehead, A. S. (1985) J. Biol. Chem. 260, 13384-13388) and rabbit (Syin, C., Gotschlich, E. C., and Liu, T.-Y. (1986) J. Biol. Chem. 261, 5473-5479) CRP at the 5' end, is not found in the Limulus CRP genes. Whereas a single CRP gene was found in the human, multiple genes were found for the Limulus CRPs. All CRPs exhibit calcium-dependent phosphorylcholine ligand binding properties. The coding regions of the Limulus and human CRP genes share approximately 25% identity and two stretches of highly conserved regions, one of which falls in the region proposed as the phosphorylcholine binding site, while the other site is very similar to the consensus sequence required for calcium binding in calmodulin and related proteins. The nucleotide sequence analysis provides convincing evidence to support the evolutionary relatedness of the human and Limulus CRPs.

Animals↗
PubMed1986

Isolation of an alpha 1 type-IV collagen cDNA clone using a synthetic oligodeoxynucleotide.

We have isolated a cDNA clone (pCIV-1-225) for the alpha 1 subunit of basement membrane (type IV) collagen from a cDNA library made from Engelbreth-Holm-Swarm mouse tumor RNA. The cDNA library was screened with synthetic oligodeoxynucleotides derived from published amino acid (aa) sequences (Schuppan et al., 1982). Nucleotide sequence data established the identity of our cDNA clone to encode an alpha 1 type IV collagen. This clone contains 270 aa of the helical region and has three interruptions in the Gly-X-Y repeat unit.

Amino Acid Sequence↗
PubMed1986

Synthesis and antitumor activity of cyclophosphamide analogues. 4. Preparation, kinetic studies, and anticancer screening of "phenylketophosphamide" and similar compounds related to the cyclophosphamide metabolite aldophosphamide.

Phenyl ketone phosphorodiamidates [C6H5C(O)CH2CH2OP(O)NHR1NR2R3] were synthesized in conjunction with an ongoing investigation into the effects of substituents on the dynamical solution chemistry of the metabolites of cyclophosphamide (1a). In contrast to aldophosphamide (3a), which readily interconverts with its cyclic isomer 4-hydroxycyclophosphamide (2a), phenylketophosphamide (14a: R1 = H, R2 = R3 = CH2CH2Cl) exhibited an apparent "resistance" toward an intramolecular addition reaction such that 4-hydroxy-4-phenylcyclophosphamide (13a) could not be detected either spectroscopically (31P or 13C NMR) or chemically (NaCN trapping experiment). Control studies that compared the relative reactivities of 14a and methylketophosphamide [20: CH3C(O)CH2CH2OP(O)NH2N-(CH2CH2Cl)2] revealed that the factors that modulate the ring closure/opening reactions were not peculiar to the phenyl group; however, differences between phenyl and methyl profoundly influenced the rates of fragmentation of 14a and 20. 31P NMR spectroscopy was used to determine the rates at which each compound generated a cytotoxic alkylating agent. Under a standard set of reaction conditions [1 M lutidine buffer with added Me2SO (8:2), pH 7.4, 37 degrees C], the half-lives of 2a/3a, 14a, phenylketoifosfamide (14b: R1 = R2 = CH2CH2Cl, R3 = H), phenylketotrofosfamide (14c: R1 = R2 = R3 = CH2CH2Cl), and 20 were 72, 66, 63, 56, and 173 min, respectively. Analogues 14a and 14b exhibited good anticancer activity against a variety of test systems.

Animals↗
PubMed1986

A recognition site on synthetic helical oligonucleotides for monoclonal anti-native DNA autoantibody.

The binding site in native DNA for a murine monoclonal anti-DNA autoantibody was investigated by measurements of competitive binding of a series of synthetic helical oligonucleotides. The antibody bound to a (dG-dC)3 or (dG-dC)4 core in the center of a base-paired octadecanucleotide. Reactions of analogues containing modifications or substitutions at specific sites indicated that the antibody bound to portions of cytosine and guanine in the major groove, a limited region of the backbone, and the 2-amino group of one guanine in the minor groove. For these interactions to occur, the antibody combining site would straddle the backbone of one of the helical strands of DNA.

Animals↗
PubMed1986

Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus.

Synthetic oligodeoxyribonucleotide probes were used in the colony hybridization test to examine the association between the Kanagawa phenomenon (KP) and the thermostable direct hemolysin gene (tdh) of Vibrio parahaemolyticus. Representative V. parahaemolyticus strains with a variety of KP reactions and 17 other Vibrio species were examined for homology with four synthetic oligodeoxyribonucleotide probes (19 to 21 bases long) representing different regions of the tdh structural gene. Under stringent conditions, two of the probes were capable of distinguishing KP-positive V. parahaemolyticus from KP-negative or KP weak-positive V. parahaemolyticus which possesses mutated tdh genes. Vibrio hollisae strains hybridized with all four probes under reduced stringency, suggesting that they have tdh-related genes which are homologous but not identical to the tdh gene in all the regions examined. The results suggest that the colony hybridization test with the synthetic oligonucleotide probes is more suitable for the definitive determination of KP-positive strains than the hybridization with the larger gene probe or immunological assays.

DNA, Bacterial↗
PubMed1986

Total assignments of the 1H NMR of decanucleotide duplexes containing the recognition sites of restriction enzymes.

Complementary DNAs, decanucleotides, were synthesized by solid phase synthesis. They contain six base pairs with different sequences in the center, flanked by GC pairs to stabilize the ends of the helix. All the imino and non-exchangeable protons of these decamers in aqueous solution were assigned by NOESY experiment. Sequence dependent exchange rates of imino protons and the chemical shifts of non-exchangeable protons were examined.

Base Composition↗
PubMed1986

Synthesis of reactive metabolite-analogues of cyclophosphamide for comparisons of NMR kinetic parameters and anticancer screening data.

Ozonolysis of compounds with the general structure CH2 = CHR1CHR2CH2OP(O) (NHR3)NR42 gave, in each case, one major product which was an analogue of the cyclophosphamide (CP) metabolites aldophosphamide (AP) or 4-hydroxy-CP. 31P NMR spectra recorded for each of these compounds in aqueous buffered solutions at pH 7.4, 37 degrees C, revealed a cascade of reactions similar to those observed for AP and/or 4-hydroxy-CP, although the individual rate constants for these reactions were substituent-dependent. Aryl ketone analogues of AP did not give rise to detectable amounts of their cyclic hemiaminals, but those which produced phosphoramide mustards at rates similar to that found for AP were active against L1210 lymphoid leukaemia in mice. The results indicated that oncostatic selectivity may not require cell-specific oxidative detoxification.

Animals↗
PubMed1985

Unique pyrimidine 2D-COSY aromatic cross-peaks as monitors of pyrimidine environments and mobility in oligo- and polynucleotides.

Only cytosine contains two adjacent aromatic protons that give rise to cross-peaks in the aromatic region of 2D-COSY spectra of oligodeoxynucleotides. In two GC-containing sequences several such cross-peaks were resolved. The intensity of these cross-peaks is a sensitive monitor of local mobility, and upon the addition of the intercalating drug daunomycin selective intensity losses were observed, indicating binding to specific GC base pairs. We have also monitored the 2D-COSY cross-peaks from mobile pyrimidine bases in tRNA (Phe) as a function of temperature.

Base Composition↗
PubMed1985

Analytical studies of 'mixed sequence' oligodeoxyribonucleotides synthesized by competitive coupling of either methyl- or beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, including 2'-deoxyinosine.

High-performance liquid chromatography (HPLC) and 1H/31P nuclear magnetic resonance (NMR) spectroscopy were used to measure the molar ratio of oligodeoxyribonucleotide products in mixtures obtained with automated DNA synthesizers that employed competitive coupling of either standard methyl- or newer beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, which include deoxyinosine. Mixtures of these reagents when used as freshly prepared solutions afforded ratios of products that indicated negligibly small differences among the rates of the various competitive coupling reactions. However, studies of reagent stability in solution revealed that both types of the N-isobutyryl deoxyguanosine reagent decompose faster than their corresponding dA, dC, and dT phosphoramidites, which led to significantly lower proportions of dG-containing sequences. This problem was attenuated for the beta-cyanoethyl reagents due to their slower rate of decomposition.

Base Sequence↗
PubMed1985

Genomic DNA sequence for human C-reactive protein.

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.

Amino Acid Sequence↗
PubMed1985

Two-dimensional nuclear Overhauser enhancement investigation of the solution structure and dynamics of the DNA octamer [d(GGTATACC)]2.

The resonances of nearly all 70 of the non-exchangeable protons of the duplex [d(GGTATACC)]2 in aqueous solution are assigned by proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra obtained in pure absorption phase at 500 MHz. Experimental and theoretical 2D NOE spectra are compared at each mixing time (100, 175, 250 and 400 ms) using two B-DNA structures: a standard B-form and an energy-minimized form. The GG and CC ends of the octamer duplex are well represented by the regular B-DNA structure. But large discrepancies from these models are observed for the 'TATA' box. All 2D NOE data are consistent with nanosecond correlation times, as indicated by non-selective proton spin-lattice relaxation times, but small variations in the correlation time are observed, suggesting that there are some local differences in mobility within the octamer duplex structure in solution.

Base Sequence↗
PubMed1985

Nuclear magnetic resonance and circular dichroism studies of a duplex--single-stranded hairpin loop equilibrium for the oligodeoxyribonucleotide sequence d(CGCGATTCGCG).

Nuclear magnetic resonance (NMR) and circular dichroism (CD) studies have been carried out with the oligodeoxyribonucleotide mismatch sequence, d(CGCGATTCGCG), 1. It has been found that 1 exists, in solution, as an equilibrium mixture of slowly interconverting, structured conformational isomers, 1a and 1b. On the basis of the concentration dependence of the 1a-1b equilibrium, the 1H NMR spectrum of the imino protons of the nucleotide bases, and the individual CD spectra of 1a and 1b, it is suggested that the two species correspond to a B-type DNA duplex and a single-stranded, hairpin-loop structure; the portion of the single-stranded species not involved in the loop appears to have a B-type DNA structure (on the basis of the CD measurements). To facilitate 1H NMR resonance assignments, the two possible des-methyl thymidine derivatives of 1 were synthesized; the effect of this substitution on the physical chemical properties of 1 was explored. The 1H NMR spectra of 1, as a function of temperature, showed that, under conditions wherein both species were present to a significant extent, the duplex form melted at a lower temperature than the single-stranded, hairpin loop structure.

Circular Dichroism↗
PubMed1985

Investigation of the solution structure of a DNA octamer [d(GGAATTCC)]2 using two-dimensional nuclear Overhauser enhancement spectroscopy.

Proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra in the pure absorption phase were obtained at 500 MHz for [d(GGAATTCC)]2 in aqueous solution at a series of mixing times. The experimental data were analyzed by comparison with theoretical spectra calculated using the complete 70 X 70 relaxation matrix including all proton dipole-dipole interactions and spin diffusion [Keepers, J. W. & James, T. L. (1984) J. Magn. Reson. 57, 404-426]. The theoretical spectra at each mixing time were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on the similarity of the six internal residues of the title octamer with those of the dodecamer [d(CGCGAATTCGCG)]2, for which the crystal structure has been determined. Neither the standard B-form nor the energy-minimized structure will yield theoretical 2D NOE spectra which accurately reproduce all peak intensities in the experimental spectra. However, many features of the experimental spectra can be represented by both the B-form and the energy-minimized structure. Sequence-dependent structural characteristics are manifest in the 2D NOE spectra, in particular at the purine-pyrimidine junction as noted previously in the crystal structure. On the whole, the energy-minimized structure appears to yield theoretical 2D NOE spectra which mimic many, if not all, aspects of the experimental spectra. All 2D NOE data were consistent with nanosecond correction times as implied by proton spin-lattice relaxation time measurements. But better fits of some of the 2D NOE data using small variations in an effective isotropic correlation time suggest that there may be some local variations in mobility within the octamer duplex structure in solution.

Magnetic Resonance Spectroscopy↗
PubMed1985

Reversed-phase high-performance liquid chromatographic separation of diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides and other backbone-modified congeners of DNA.

Diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides, which were synthesized by an automated, solid-phase, phosphoramidite-coupling method, were conveniently separated by reversed-phase high-performance liquid chromatography on a muBondapak C18 column with a gradient of acetonitrile in triethylammonium acetate buffer. These synthetic and chromatographic methods were also used to obtain diastereomerically pure bis-phosphorothioate, alkanephosphonate, and O-alkyl phosphotriester analogues of DNA for exploratory studies of stereochemistry and phosphorolytic enzymes.

Chemical Phenomena↗
PubMed1985

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization.

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.

Bacterial Toxins↗
PubMed1984

The structural gene for tetanus neurotoxin is on a plasmid.

A pool of synthetic oligonucleotides was prepared based on the amino terminal amino acid sequence of tetanus toxin. This probe hybridized to plasmid DNA isolated from three toxigenic strains of Clostridium tetani but not to plasmid DNA from a nontoxigenic strain. These results show that the structural gene for the toxin is on the plasmid. The pCL1 plasmid from one of the toxigenic strains spontaneously deleted 22 kilobase pairs of DNA to form pCL2. Strains harboring this deleted plasmid are nontoxigenic. However, the probe mixture hybridized to pCL2, indicating that the DNA encoding the amino terminus of the toxin had not been deleted. Restriction endonuclease cleavage maps of pCL1 and pCL2 were constructed and indicate the approximate location and orientation of the structural gene for tetanus toxin.

Amino Acid Sequence↗
PubMed1984

Complete assignment of the non-exchangeable proton nmr resonances of [d-(GGAATTCC)]2 using two-dimensional nuclear overhauser effect spectra.

Proton two-dimensional nuclear Overhauser effect (2D NOE) spectra have been obtained for [d-(GGAATTCC)]2 at a series of mixing times. By performing the experiment at 500 MHz and obtaining pure absorption phase spectra, sufficient resolution was achieved to be able to assign all seventy of the non-exchangeable proton resonances in the self-complementary octamer duplex, including the H3' resonances which overlap with the solvent peak.

Indicators and Reagents↗