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Cyanobacterial mats collected in hypersaline salterns were incubated in a greenhouse under low sulfate concentrations ([SO4]) and examined for their primary productivity and emissions of methane and other major carbon species. Atmospheric greenhouse warming by gases such as carbon dioxide and methane must have been greater during the Archean than today in order to account for a record of moderate to warm paleoclemates, despite a less luminous early sun. It has been suggested that decreased levels of oxygen and sulfate in Archean oceans could have significantly stimulated microbial methanogenesis relative to present marine rates, with a resultant increase in the relative importance of methane in maintaining the early greenhouse. We maintained modern microbial mats, models of ancient coastal marine communities, in artificial brine mixtures containing both modern [SO4=] (ca. 70 mM) and "Archean" [SO4] (less than 0.2 mM). At low [SO4], primary production in the mats was essentially unaffected, while rates of sulfate reduction decreased by a factor of three, and methane fluxes increased by up to ten-fold. However, remineralization by methanogenesis still amounted to less than 0.4 % of the total carbon released by the mats. The relatively low efficiency of conversion of photosynthate to methane is suggested to reflect the particular geometry and chemical microenvironment of hypersaline cyanobacterial mats. Therefore, such mats w-ere probably relatively weak net sources of methane throughout their 3.5 Ga history, even during periods of low- environmental levels oxygen and sulfate.

Summary Mechanisms of dimethyl sulphide (DMS) and methanethiol (MT) production and consumption were determined in moderately hypersaline mats, Guerrero Negro, Mexico. Biological pathways regulated the net flux of DMS and MT as revealed by increases in flux resulting from decreased salinity, increased temperature and the removal of oxygen. Dimethylsulphoniopropionate (DMSP) was not present in these microbial mats and DMS and MT are probably formed by the reaction of photosynthetically produced low-molecular weight organic carbon and biogenic hydrogen sulphide derived from sulphate reduction. These observations provide an alternative to the notion that DMSP or S-containing amino acids are the dominant precursors of DMS in intertidal sediment systems. The major sink for DMS in the microbial mats was biological consumption, whereas photochemical oxidation to dimethylsulphoxide was the major sink for DMS in the overlying water column. Diel flux measurements demonstrated that significantly more DMS is released from the system during the night than during the day. The major consumers of DMS in the presence of oxygen were monooxygenase-utilizing bacteria, whereas under anoxic conditions, DMS was predominantly consumed by sulphate-reducing bacteria and methanethiol was consumed by methanogenic bacteria. Aerobic and anaerobic consumption rates of DMS were nearly identical. Mass balance estimates suggest that the consumption in the water column is likely to be smaller than net the flux from the mats. Volatile organic sulphur compounds are thus indicators of high rates of carbon fixation and sulphate reduction in these laminated sediment ecosystems, and atmospheric sulphur can be generated as a biogenic signature of the microbial mat community.

A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hr. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P1 initiation site is selectively reduced relative to total mRNA, relative to mRNA from the alternative c-myc P2 initiation site, and relative to mRNA derived from the beta-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxyribonucleotide and duplex DNA in the nucleus of treated cells.

Excised cucumber ( Cucumis sativus L. cv 447 Wisconsin SMR 18) cotyledons were sensitive to acifluorfen-methyl (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate) and MC-15608 (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-chlorobenzoate). Injury was detected by monitoring efflux of 3- O -methyl-d-[U- 14 C]glucose from herbicide-treated tissue after exposure to light. Efflux kinetics of 3- O -methyl-[ 14 C]glucose from cotyledons treated with either acifluorfen-methyl (AFM) or MC-15608 were similar. Neither herbicide was active in darkness. Cyclic voltammetry was used to study redox behavior of AFM and MC-15608 in protolytic and aprotic solvents. In protic media reduction of AFM was chemically irreversible. Reduction of MC-15608 in protolytic solvent was not observed. In aprotic solvent, the electrode reaction of AFM was quasireversible. The voltammogram of MC-15608 in aprotic solvent was indicative of a multielectron, completely irreversible electrode reaction. Although the physiological data indicate that AFM and MC-15608 have similar effects in vivo , the redox behavior of the two compounds is quite different. These results suggest that mechanisms involving direct reduction and reoxidation of the diphenyl ether molecule are probably not the basis for the action of these herbicides.

Light-dependent reduction of p -nitro blue tetrazolium chloride (NBT) and 2,6-dichlorophenol-indophenol (DCPIP) was stimulated by nitrofen (2,4-dichlorophenyl p -nitrophenyl ether) in the presence or absence of the photosensitizer riboflavin. Enhancement of NBT reduction occurred at a concentration as low as 17 μ M nitrofen. Nitrofen had no effect on dark reduction of NBT by dithionite, ascorbate, or reduced phenazine methosulfate. NBT reduction proceeded in solution saturated with either air or nitrogen. Stimulation by nitrofen in the presence or absence of riboflavin also occurred independently of oxygen. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1) inhibited light-dependent, aerobic reduction of NBT and DCPIP with and without riboflavin. The nitrogen-stimulated component was also eliminated. Thus, it appears nitrofen can effect reduction of NBT (and DCPIP) anaerobically by transferring electrons directly to NBT (and DCPIP) through an oxygen-independent mechanism or aerobically via superoxide radicals.

Benzo[alpha]pyrene diol epoxide (BPDE) is a well-studied environmental carcinogen that binds covalently to DNA. Here we describe a photochemical technique that allows us to map BPDE-binding sites within cloned gene sequences. The technique is based upon our observation that, when irradiated with laser light at 355 nm, one single-strand DNA cut is produced at each BPDE binding site. In initial experiments we have studied the distribution of such cuts in cloned DNA from the chicken adult beta-globin gene. We find that BPDE binding in this gene sequence is distinctly nonrandom. While several prominent BPDE-binding sites are evident, a 300-base-pair sequence immediately 5' to the RNA cap site is most strongly attacked by the carcinogen. This region is believed to contain important transcriptional control sequences. We discuss the possibility that sequence-specific binding to such regulatory elements may be an important feature of the mechanism of the carcinogen.

We have prepared covalent complexes between defined length DNA fragments and a diol epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene. We have studied the structure of these complexes, using transient electric dichroism, circular dichroism, fluorescence quenching, thermal denaturation, polyacrylamide gel electrophoresis, and nuclease digestion. Our observations suggest that th covalently bound carcinogen is intercalated within the helix, forming a wedge-shaped complex. Binding of the carcinogen distorts the structure of the DNA over a region extending beyond the immediate binding site. The most striking aspect of this distortion is that it produces a bend in the helix.

Redox behavior of nitrofen (2,6-dichlorophenyl-p-nitrophenyl ether) at a glassy carbon working electrode in aprotic and protolytic solvents was examined using cyclic voltammetry. The electrode reaction of 2 mM nitrofen in dimethylformamide (0.1 M tetra-n-butylammonium hexafluorophosphate as supporting electrolyte) was quasireversible. Measured half-wave potential was −1.11 V vs saturated calomel reference electrode (SCE) and peak potential separation was 0.110 V. The reduction product was chemically stable on the time scale of the experiment; cathodic peak current equalled anodic peak current. Linear sweep voltammetry employing a rotating, glassy carbon disk electrode indicated that electron stoichiometry for reduction of nitrofen in dimethylformamide and acetonitrile was one. Reduction of nitrofen under aprotic conditions was most likely a one-electron reduction to the nitro radical anion. Reduction of 1 mM nitrofen in 50% ( v v ) ethanol/water buffered at pH 5.0, 7.0, and 10.2 with 0.25 M sodium acetate, 0.1 M potassium phosphate, and 0.1 M sodium carbonate was chemically irreversible and characteristic of an EC … -type reaction mechanism; heterogeneous electron transfer followed by homogeneous chemical reactions. Cathodic peak potentials at pH 5.0 and 7.0 were −0.760 and −0.820 V vs SCE. At pH 10.2 two cathodic peaks were observed at −0.840 and −1.17 V vs SCE. In protolytic solvent at pH 5.0 cathodic peak current varied linearly as a function of the square root of the scan rate. Thus, the electrode reaction was limited by diffusion. Electron stoichiometry for reduction of nitrofen in protolytic solvent was four. The fate of new redox species generated in protolytic solvent at pH 7.0 was examined upon subsequent multiple scans at 200 mV/s. Initial reduction at −0.820 V vs SCE was most likely centered at the nitro group, yielding the four-electron hydroxylamine product. Oxidation at 0.020 V vs SCE was thought to correspond to the two-electron oxidation of hydroxylamine to the nitroso derivative.

Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these mono-layers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM. The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell-attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 μm, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal. With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer. The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.

A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

New SrH band systems photographed at high dispersion include: (1) an abbreviated $C^{2}\ensuremath{\Sigma}\ensuremath{\rightarrow}N^{2}\ensuremath{\Sigma}$ system with (0,0) band head at 3808A, (2) a many-lined $D^{2}\ensuremath{\Sigma}\ensuremath{\rightarrow}N^{2}\ensuremath{\Sigma}$ system between 5000A and 6700A, (3) an $E^{2}\ensuremath{\Pi}\ensuremath{\rightarrow}N^{2}\ensuremath{\Sigma}$ system consisting of a $\ensuremath{\Delta}v=0$ sequence only with the (0,0) band at 5323A. The (0,0) and (0,1) $C$ bands have three violent perturbations and a sharp cut-off due to predissociation at ${K}^{\ensuremath{'}}=19$. The form and magnitude of the perturbations are computed. Quantum analyses give ${B}_{0}=3.930$ and ${D}_{0}=\ensuremath{-}9.31\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}4}$ for the $C$ state, ${B}_{1}=3.56$ for the normal state. Quantum assignments of the lines of the six principal branches of the (0,0) band of the $E$ system are presented. For the $E$ state $A=117$, ${{B}^{*}}_{0, \ensuremath{-}\frac{1}{2}}=3.6388$, ${{B}^{*}}_{0, +\frac{1}{2}}=3.8687$ and perturbations at small $J$ values distort the $\ensuremath{\Lambda}$-doubling curve for the $^{2}\ensuremath{\Pi}_{\frac{1}{2}}$ state.

The analyses of the $B$ and $C$ band systems of SrD and of a portion of the $D$ system of SrH are presented. $B_{e}^{*}=1.9427$ for SrD and 3.8788 for SrH for the normal $^{2}\ensuremath{\Sigma}$ state. The ratio of these, 0.50635, shows about the expected departure from equality with the ratio ${\ensuremath{\rho}}^{2}=0.50603$ of the reduced masses. The spin doubling of this SrD state is regular, ${\ensuremath{\gamma}}_{0}=+0.0613$ being almost exactly in the ratio ${\ensuremath{\rho}}^{2}$ with the value for SrH. The spin doubling of the $B^{2}\ensuremath{\Sigma}$ state of SrD departs markedly from the usual linear variation with $K+\frac{1}{2}$. Multiple perturbations occur in the (0,0) $C$ band of SrD, and there is a sharp cut-off of the branches at ${K}^{\ensuremath{'}}=29$. For this $C^{2}\ensuremath{\Sigma}$ state ${B}_{0}\ensuremath{\cong}1.95$, but exact analysis is hindered by the lack of any unperturbed lines. Two bands (${v}^{\ensuremath{'}},2$) and (${v}^{\ensuremath{'}},3$), with a common upper state, of the $D^{2}\ensuremath{\Sigma}\ensuremath{\rightarrow}N^{2}\ensuremath{\Sigma}$ "many-lined" SrH system have been analyzed. In this $D$ state, for which ${B}_{v}=1.913$, large irregular perturbations occur. Electronic configurations are discussed.

Fructans synthesized from newly formed assimilates accumulate in wheat stems as nonstructural carbohydrates. Experiments performed tested the hypothesis that the fructose moiety from translocated sucrose is used preferentially in biosynthesis of these fructans. Results indicated: (a) a large percentage of labeled sucrose was translocated and unloaded in an unaltered state; and (b) sucrose contributed its fructose moiety to fructan synthesis in stems.

We have used a photochemical method to map covalent binding sites of the carcinogen benzo[a]pyrenediol epoxide (BPDE) within DNA from the transcriptional control region of the chicken adult beta-globin gene. Our preliminary low-resolution mapping has demonstrated that this region contains highly preferred BPDE binding sites [Boles, T.C., & Hogan, M.E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5623-5627]. Here, we find that BPDE binding at individual G residues in this region is influenced by nearest-neighbor interactions and also by longer range interactions that may be attributable to sequence-specific variation of DNA secondary structure. Our findings suggest that long poly(dG) sequences should be preferred sites for BPDE action in other genes.

As the use of isolated cell cultures increases as an experimental model, there has been a proportional increase in the number of matrices used for cell growth support. These matrices vary in shape, texture and porosity, and as a result, affect the cells grown on them.The ultrastructural characteristics of several of these growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese Hamster Ovary cells and Balb 3T3 Mouse Fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross-linked dextran) matrices. Cells were plated maintaining egual densities and growth surface area. Once the majority of the cells reached confluency (approx. one week), the cell morphology on each matrix was examined using scanning electron microscopy and digital analysis of cell attachment area.Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead), and that of a smooth (glass) surface (Figs. 1 and 2). Even within smooth surfaces, some variation was observed.