Scaling relationships for woody tissue respiration in two tropical rain forests

The relationship between gross primary productivity (GPP) and net primary productivity (NPP) is not fully understood. One of the uncertainties relevant to this issue is the magnitude of woody tissue respiration. Although some data exist for temperate and boreal zones, measurements of woody tissue respiration in tropical forests are sparse. We made in situ chamber measurements of woody tissue respiration in two tropical rain forests, one in the Brazilian Amazon (Reserva Jarú) and one in Central Cameroon (Mbalmayo Reserve). We made measurements on a wide range of species at each site and over a range of stem diameters from 0·02 to 1·4 m. The rate of efflux of carbon dioxide (CO₂) from bark at 25 °C, R_t, varied from 0·1 to 5·2 µmol m^?2 s^?1 across the two sites, and the efflux was related to both volume and surface area components of the measured stem sections. The temperature response in R_t was slightly higher at Jarú than at Mbalmayo, with Q₁₀ values of 1·8 (± 0·1 SE) and 1·6 (± 0·1 SE), respectively. A log–log regression showed that R_t was significantly related to stem diameter, D (P < 0·001; r² = 0·58–0·62) and was significantly higher at Mbalmayo than at Jarú (P < 0·001), but that the rate of increase in R_t with stem diameter, D, was similar between sites. At the Mbalmayo site, tree growth measurements made over a 4 month period were used to make two estimates of the maintenance (R_m) and construction (R_c) components of respiration embedded in R_t. The two methods agreed closely, suggesting that R_m was approximately 80% of R_c at this site. R_m could be strongly related to D using a sigmoidal relationship that described both surface area and volume components as sources of respiratory CO₂ (r² = 0·71). This functional model was combined with inventory, growth and climate data for the Mbalmayo site to make a first estimate of annual above‐ground woody tissue respiration, R_A, which was 257 (± 18 SE) g C m^?2 year^?1. This value corresponds to approximately 10% of GPP, slightly lower than that found for another tropical rain forest, but higher than for temperate forests. When combined with data from six other sites in tropical, temperate and boreal settings, a very strong relationship was found between R_A and leaf area index (LAI), and between R_A/GPP and LAI (P < 0·001, r² = 0·98). This indicates that R_A exerts an appreciable constraint on NPP and that this constraint varies closely with LAI across widely differing types of woody vegetation.     

Location of ribosome binding sites on the right end of lambda DNA.

The locations of ribosome binding sites on the right end of bacteriophage lambda DNA were determined by measurement of the positions of ribosomes bound to single-stranded DNA visualized by electron microscopy. A total of ten ribosome binding sites were found between map co-ordinates 0.90 and 1.0. Four of these ribosome binding sites probably correspond to the polypeptide initiation sites for genes Q (0.910), S (0.928), R (0.936) and Rz (0.945). Six other ribosome binding sites were found which presumably indicate the presence of new lambda genes. Four of these ribosome binding sites (0.958, 0.967, 0.975, 0.995) are located to the right of Rz, which is the most rightward known lambda gene. A ribosome binding site is located at 0.923, between genes Q and S, in or near the 6 S RNA sequence. Another is located left of gene Q at 0.900.     

The ribosomal binding site for eukaryotic elongation factor EF-2 contains 5 S ribosomal RNA

The possible location of RNA in the ribosomal attachment site for the eukaryotic elongation factor EF-2 was analysed. Stable EF-2 · ribosome complexes formed in the presence of the non-hydrolysable GTP analogue GuoPP[CH₂]P were cross-linked with the short (4 Å between the reactive groups) bifunctional reagent, diepoxybutane. Non-cross-linked EF-2 was removed and the covalent factor-ribosome complex isolated. No interaction between EF-2 and 18 S or 28 S rRNA could be demonstrated. However, density gradient centrifugation of the cross-linked ribosomal complexes showed an increased density (1.25 g/cm³) of the factor, as expected from a covalent complex between EF-2 and a low-molecular-weight RNA species. Treatment of the covalent ribosome-factor complexes with EDTA released approx 50% of the cross-linked EF-2 from the ribosome together with the 5 S rRNA · protein L5 complex. Furthermore, the complex co-migrated with the 5S rRNA · L5 particle in sucrose gradients. Polyacrylamide gel electrophoresis showed that EF-2 was directly linked to 5 S rRNA in the 5 S rRNA · L5 complex, as well as in the complexes isolated by density gradient centrifugation. No traces of 5.8 S rRNA or tRNA could be demonstrated. The data indicate that the ribosomal binding domain for EF-2 contains the 5 S rRNA · protein L5 particle and that EF-2 is located in close proximity to 5 S rRNA within the EF-2 · GuoPP[CH₂]P · ribosome complex.     

Uptake of homologous single-stranded fragments by superhelical DNA: II. Characterization of the reaction

We have studied the association of superhelical DNA (RFI)³ of phage G4 with defined single-stranded fragments isolated after cleavage of viral (+) strands by endonuclease R · HaeIII. The sedimentation rates of complexes formed by uptake of different single-stranded restriction fragments by G4 RFI were consistent with the view that base-pairing between the two components causes unwinding of superhelical turns, with one negative superhelical turn removed for every ten nucleotide residues of third strand taken up. The combining ratio of superhelical DNA and a single specific fragment was close to unity.At high concentrations of salt, nitrocellulose filters efficiently retained complexes of superhelical DNA and homologous fragments, which provided the basis for a rapid assay, and permitted the estimation of the thermodynamic and kinetic parameters of strand uptake in vitro. The reaction is reversible, with an apparent K_eq of approximately 10⁶m^?1. Apparent rate constants, k₁, for uptake of different fragments (85 to 1100 nucleotides long) varied about fourfold, with no obvious relationship to the length of the fragment. In 10 mm-Tris · HCl (pH 7.5), 200 mm-NaCl, fragments were taken up most rapidly at about 75 °C. Under these conditions, the apparent k₁ for a fragment 250 nucleotides long was approximately 600 m^?1s^?1, which is two or three orders of magnitude slower than the calculated rate of association of complementary strands of that length. At physiological temperatures, appreciable rates of strand uptake were seen only at low concentrations of salt (4 mm-Na⁺ in 10 mm-Tris · HCl), and were one or two orders of magnitude less than the rate at 75 °C in 200 mm-NaCl. At a given concentration of counterion a threshold temperature exists above which the rate of reaction rises sharply from an undetectable level.Thermodynamic calculations indicate that the reaction is entropically driven, and that the rate is limited by a step exhibiting a positive entropy and enthalpy of activation. The data are consistent with a model for strand uptake in which the rate-limiting step is the unstacking of a small number of base-pairs in the superhelical DNA. Stabilization and extension of the nucleus of base-pairs formed with the incoming strand is favored by the decrease in free energy associated with removal of superhelical turns.     

High-level expression of genes under control of cro translation initiation signals in Escherichia coli

The expression of several genes in Escherichia coli under the control of the lambda p_R promoter and translation initiation signals of the lambda cro gene were studied. Fusions were made in frame at the initiation codon and/or with 5′ translated cro fragments. Expression fluctuated strongly when genes were fused directly at the ATG, whereas constructs, which encode hybrid genes that include at least the first nine codons of the cro gene, always directed high-level synthesis. These fusion proteins were mainly intracellularly precipitated. Our results confirm the poor reliability of ATG vectors for the expression of cloned genes. On the other hand, useful levels of expression are obtained when genes are fused to 5′ cro coding sequences, presumably due to an efficient ribosome binding site configuration.     

Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

The precise positions of the origin of replication³ and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.     

Partition of unit-copy miniplasmids to daughter cells: III. The DNA sequence and functional organization of the P1 partition region

The boundaries of the P1 par (plasmid partition) region of the unit-copy plasmid P1 were defined to within 2.7 × 10³ base-pairs of DNA. The DNA sequence of the region revealed two large open reading frames that could encode proteins of M_r 44,000 and M_r 38,000. Both would be read in the same direction. The first open reading frame corresponds to the parA gene, the M_r 44,000 protein product of which was shown to be trans acting and essential for partition. The second open reading frame (parB) follows closely and may be cotranscribed with parA. The codon usage frequency for parB is consistent with its producing a protein product. The ParB protein was identified in cell extracts as a product with an apparent M_r of 45,000, suggesting that it behaves anomolously on gel electrophoresis. Following parB is the incB region, an incompatibility determinant thought to be the cis acting site that constitutes the putative attachment point on the DNA for the cellular partition apparatus. Subcloning of this site showed it to consist of a maximum of 112 base-pairs. The incB sequence is highly A + T-rich and contains a 20 base-pair inverted repeat. Another A + T-rich inverted repeat of similar size but different sequence is found between the putative parA promoter and the ribosome initiation sequence at the start of the parA open reading frame and may be involved in the autoregulation of ParA synthesis.The par region appears to contain a functional analog of the centromere of eukaryotic chromosomes. It is responsible for ensuring that newly replicated plasmids are properly distributed to daughter cells during cell division of its Escherichia coli host.     

Double-helical structures for polyxanthylic acid

Polyxanthylic acid has been found to exist in two different duplex forms, A_I and A_II. a_I, formed at pH5·7, occurs in a compact lattice with nearest neighbor molecules spaced at 2.11 nm. It has an axial translation per residue, h = 0·301 nm, and a rotation per residue, t = 36·0 °. The intensity distribution in its X-ray fiber diffraction pattern is analogous to that of A-RNA (h = 0·281 nm, t = 32·7 °). On the other hand A_II, formed at pH 8·0, has a less compact, statistically disordered crystal packing with nearest neighbors 2·35 nm apart. It has h = 0·252 nm and t = 32·7 ° and gives an X-ray intensity distribution essentially identical to A-DNA (h = 0·256 nm, t = 32·7 °). Similar right-handed helical duplex models, with flexible C-3′-endo sugar rings, have been developed for each molecular structure. Both have purine purine base-pairs, possibly triply hydrogen-bonded, and certainly with the same symmetry as Watson-Crick pairs but with a 0·2 nm greater C-1′ … C-1′ separation.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Acetyl-coenzyme A carboxylase from the developing endosperm of Ricinus communis: II. A two-site kinetic mechanism

The reaction kinetics of acetyl-coenzyme A carboxylase purified from developing castor oil seeds have been examined. On the basis of the substrate interaction and product inhibition results, a hybrid ping-pong mechanism is proposed. This type of mechanism demands that the active site of the enzyme be separated into two functionally distinct catalytic sites. The carboxybiotin intermediate formed at one site by the hydrolysis of ATP swings to the second site where acetyl-CoA is carboxylated to form malonyl-CoA. This hybrid rapid-equilibrium random bi bi uni uni ping-pong mechanism which includes the formation of three abortive complexes, E · HCO₃^? · ADP, E · HCO₃^? · P_i and E · P_i · P_i, is analogous to the hybrid ping-pong mechanism previously described for methylmalonyl-CoA transcarboxylase (D. B. Northrop (1969) J. Biol. Chem., 244, 5808) and pyruvate carboxylase (R. E. Barden, C-H. Fung, M. F. Utter, and M. C. Scrutton (1972) J. Biol. Chem., 247, 1323).     

Competition and interplay between the lithium bonding and hydrogen bonding: R3C···HY···LiY and R3C···LiY···HY triads as a working model (R=H, CH3; Y=CN, NC)

UMP2 calculations with aug-cc-pVDZ basis set were used to analyze intermolecular interactions in R₃C···HY···LiY and R₃C···LiY···HY triads (R=H, CH₃; Y=CN, NC), which are connected via lithium and hydrogen bonds. To better understand the properties of these systems, the corresponding dyads were also studied. Molecular geometries and binding energies of dyads, and triads were investigated at the UMP2/aug-cc-pVDZ computational level. Particular attention was paid to parameters such as cooperative energies, and many-body interaction energies. All studied complexes, with the simultaneous presence of a lithium bond and a hydrogen bond, showed cooperativity with energy values ranging between ?1.71 and ?9.03 kJ mol^?1. The electronic properties of the complexes were analyzed using parameters derived from atoms in molecules (AIM) methodology. Energy decomposition analysis revealed that the electrostatic interactions are the major source of the attraction in the title complexes.     

Charge recombination in photosystem I at low temperatures kinetics of electron tunneling

Absorption changes accompanying light-induced P-700 oxidation and the decay of P-700+ in the dark were measured in the temperature range 294-5 K over a broad time scale (three to four orders of magnitude). Two qualitatively different types of kinetics for the dark decay of P-700⁺ were observed. In the 294-240K region, a usual exponential kinetics is observed with the rate constant κ = 1 · 10¹⁰ · exp(-16 000/RT) s^?1, with R in cal/mol per degree. Below 220 K, a rather unusual logarithmic or near-logarithmic kinetics are observed. These kinetics can be explained quantitatively if one assumes for the various (P-700+ ··· X^-) pairs a broad rectangular or near-rectangular distribution over the values of the rate constant. The following kinetic equation corresponding to this model was obtained: n_t/n_o = [In(κ_max/κ_min)]-1 - [In(1/κ_min)? In t] where no and nt are respectively the initial concentration of P-700⁺ and its concentration at time t, and kmax and kmin the maximum and minimum values of the rate constant, respectively. The decay processes observed can be ascribed to electron tunneling. Distribution over the values of k can be accounted for by different environments or different mutual orientations of P-700⁺ and X^?, or by different distances between them in the various reacting pairs.The corresponding distribution function was reconstructed from the experimentally measured P-700+-decay curves. The rate of tunneling was found to be temperature dependent. In the 160-80-K region, the temperature dependence corresponds to an activation energy of 2.9 kcal/mol. Below 80 K, new modes of P-700+ decay with lower activation energy become operative. The tunneling distance for the majority of the (P-700+ ··· X^?) pairs was estimated from the EPR linewidth of P-700+ to exceed 13.2 A.     

Efficient expression of a promoter-controlled gene: Tandem promoters of lambda PR and PL functional in enteric bacteria

We describe an expression vector that functions in enteric bacteria. The vector contains the coliphage λ promoters P_R and P_L and entire P_R and P_L operators in tandem upstream from the multiple cloning sites containing the kanamycin-resistant gene. The vector also specifies a ribosome binding site and a thermolabile repressor, cI₈₅₇, and the P_RM promoter. These promoters as well as lacUV5 and trp promoters were inserted into the EcoRI site of pKO-1 plasmid so that they drove the expression of a reporter gene, galactokinase (galK). The P_RP_L promoter showed the highest efficiency of galK expression in the Escherichia coli strain K12ΔH1Δtrp; it was strong in Klebsiella aerogenes, and weak in Serratia marcescens and Citrobacter freundii.     

Mixed-bridged bimetallic d8 complexes. Crystal and molecular structure of (η3-C3H5)Pd(μ-pz)(μ-N3)Rh(CO)2, heterobinuclear complex with extended Rh⋯Rh interactions

The preparation and properties of binuclear complexes containing the pyrazolate and azide groups as bridging ligands are reported. Representative formulae are: M₂(μ-pz)(μ-N₃)(CO)₄, M₂(μ-pz)(μ-N₃)- (COD)₂ (M = Rh or Ir), (CO)₂Rh(μ-pz)(μ-N₃)ML₂ (M = Rh, L₂ = COD, M = Ir, L = CO) and (η³-C₃H₅)- Pd(μ-pz)(μ-N₃)Rh(CO)₂. The crystal and molecular structure of the latter complex has been determined by single-crystal X-ray methods. Crystals are monoclinic, space group C₂/c with cell constants a = 18.4750(10), b = 10.0351(3), c = 13.6399(6) Å, α = 90, β = 100.022(4), γ = 90°, and Z = 8. The final R and R_w values were 0.051 and 0.062 for 1417 observed reflexions. This binuclear compound packs in the crystal zig-zag chains of rhodium atoms, along the c axis, wtth intermolecular Rh···Rh contacts of 3.290(1) and 3.604(1) Å. The Rh···Rh···Rh angle is 163.16(4)°.     

Overproduction of E. coli xylose isomerase

Summary A promoterles DNA fragment containing theE. coli xylose isomerase gene and its ribosome binding site was ligated into a plasmid downstream from the strong P_L promoter. The plasmid was then used to transformE. coli strains containing a temperature-sensitive repressor (cI857). The transformants overproduced xylose isomerase when the repressor was thermally inactivated.     

Effects of magnesium ions on two EMCV IRES key activity fragments

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino protons, changes have been monitored in base-pair kinetics induced by Mg^2 + in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus internal ribosome entry site. For r(CACCUGGCGACAGGUG), the addition of Mg^2 + reveals two types of base-pairs: r(U⁵⁴⁵·A) and r(G⁵⁴⁶·C), in the first category, have lifetimes only slightly higher in the presence of Mg^2 +, whereas their dissociation constants are substantially reduced. This behavior has been termed proximal. The base-pairs r(G⁵⁵³·C) and r(G⁵⁵⁴·C), in the second category, have lifetimes substantially higher in the presence of Mg^2 +, whereas their dissociation constants remain almost constant. This behavior has been termed distal. Mg^2 + has a specific effect on r(CACCUGGCGACAGGUG), the magnitude of which is progressively modulated from the proximal region of the 16-mer towards its distal region. For r(GGCCAAAAGCC), an intermediate behavior is found for base-pairs r(G⁵⁶⁵·C) and r(G⁵⁷²·C). Their lifetimes are slightly higher in the presence of Mg^2 + and their dissociation constants are significantly lower, a behavior resembling that of the 16-mer proximal region. These results indicate that Mg^2 + diffusively moves around r(GGCCAAAAGCC).