Operator binding by lambda repressor heterodimers with one or two N-terminal arms.

The first 6 amino acids (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6) of bacteriophage lambda cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric lambda repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of lambda repressor to leucine zippers from Fos and Jun. Although only one arm is visible in the cocrystal structure of the N-domain-operator complex, our results indicate that both arms are required for optimal operator binding and normal site discrimination.

One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.

Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.

Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon

The deg-3 gene from the nematode Caenorhabditis elegans encodes an α subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an α subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.

An experimental test for lineage-specific position effects on alcohol dehydrogenase (Adh) genes in Drosophila

Independent transgene insertions differ in expression based on their location in the genome; these position effects are of interest because they reflect the influence of genome organization on gene regulation. Position effects also represent potentially insurmountable obstacles to the rigorous functional comparison of homologous genes from different species because (i) quantitative variation in expression of each gene across genomic positions (generalized position effects, or GPEs) may overwhelm differences between the genes of interest, or (ii) divergent genes may be differentially sensitive to position effects, reflecting unique interactions between each gene and its genomic milieu (lineage-specific position effects, or LSPEs). We have investigated both types of position-effect variation by applying our method of transgene coplacement, which allows comparisons of transgenes in the same position in the genome of Drosophila melanogaster. Here we report an experimental test for LSPE in Drosophila. The alcohol dehydrogenase (Adh) genes of D. melanogaster and Drosophila affinidisjuncta differ in both tissue distribution and amounts of ADH activity. Despite this striking regulatory divergence, we found a very high correlation in overall ADH activity between the genes of the two species when placed in the same genomic position as assayed in otherwise Adh-null adults and larvae. These results argue against the influence of LSPE for these sequences, although the effects of GPE are significant. Our new findings validate the coplacement approach and show that it greatly magnifies the power to detect differences in expression between transgenes. Transgene coplacement thus dramatically extends the range of functional and evolutionary questions that can be addressed by transgenic technology.

Maintenance of Epstein–Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP, enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1–89 and 323–386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379–641), which includes the nuclear localization signal and DNA-binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1–378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1–90 or histone H1–2 could substitute for EBNA-1 amino acids 1–378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60% of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells.

From symmetry to asymmetry: Phylogenetic patterns of asymmetry variation in animals and their evolutionary significance

Phylogenetic analyses of asymmetry variation offer a powerful tool for exploring the interplay between ontogeny and evolution because (i) conspicuous asymmetries exist in many higher metazoans with widely varying modes of development, (ii) patterns of bilateral variation within species may identify genetically and environmentally triggered asymmetries, and (iii) asymmetries arising at different times during development may be more sensitive to internal cytoplasmic inhomogeneities compared to external environmental stimuli. Using four broadly comparable asymmetry states (symmetry, antisymmetry, dextral, and sinistral), and two stages at which asymmetry appears developmentally (larval and postlarval), I evaluated relations between ontogenetic and phylogenetic patterns of asymmetry variation. Among 140 inferred phylogenetic transitions between asymmetry states, recorded from 11 classes in five phyla, directional asymmetry (dextral or sinistral) evolved directly from symmetrical ancestors proportionally more frequently among larval asymmetries. In contrast, antisymmetry, either as an end state or as a transitional stage preceding directional asymmetry, was confined primarily to postlarval asymmetries. The ontogenetic origin of asymmetry thus significantly influences its subsequent evolution. Furthermore, because antisymmetry typically signals an environmentally triggered asymmetry, the phylogenetic transition from antisymmetry to directional asymmetry suggests that many cases of laterally fixed asymmetries evolved via genetic assimilation.

Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system.

Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.

Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1.

The protein encoded by the gamma 134.5 gene of herpes simplex virus precludes premature shutoff of protein synthesis in human cells triggered by stress associated with onset of viral DNA synthesis. The carboxyl terminus of the protein is essential for this function. This report indicates that the shutoff of protein synthesis is not due to mRNA degration because mRNA from wild-type or gamma 134.5- virus-infected cells directs protein synthesis. Analyses of the posttranslational modifications of translation initiation factor eIF-2 showed the following: (i) eIF-2 alpha was selectively phosphorylated by a kinase present in ribosome-enriched fraction of cells infected with gamma 134.5- virus. (ii) Endogenous eIF-2 alpha was totally phosphorylated in cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene but was not phosphorylated in mock-infected or wild-type virus-infected cells. (iii) Immune precipitates of the PKR kinase that is responsible for regulation of protein synthesis of some cells by phosphorylation of eIF-2 alpha yielded several phosphorylated polypeptides. Of particular significance were two observations. First, phosphorylation of PKR kinase was elevated in all infected cells relative to the levels in mock-infected cells. Second, the precipitates from lysates of cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene contained an additional labeled phosphoprotein of M(r) 90,000 (p90). This phosphoprotein was present in only trace amounts in the immunoprecipitate from cells infected with wild-type virus or mutants lacking a portion of the 5' domain of gamma 134.5. We conclude that in the absence of gamma 134.5 protein, PKR kinase complexes with the p90 phosphoprotein and shuts off protein synthesis by phosphorylation of the alpha subunit of translation initiation factor eIF-2.

The theoretical limits of DNA sequence discrimination by linked polyamides

Linked polyamides bind in the minor groove of double-stranded DNA in a partially sequence-specific manner. This report analyzes the theoretical limits of DNA sequence discrimination by linked polyamides composed of two to four different types of heterocyclic rings, determining (i) the optimal choice of base-binding specificity for each ring and (ii) the optimal design for a polyamide composed of these rings to target a given DNA sequence and designed to maximize the fraction of the total polyamide binding to the specified target sequence relative to all other sequences. The results show that, fortuitously, polyamides composed of pyrrole, a naturally occurring G-excluding element, and imidazole, a rationally designed G-favoring element, have features similar to the theoretical optimum design for polyamides composed of two different rings. The results also show that, in polyamides composed of two or three types of heterocyclic rings, choosing a nonspecific “placeholder” ring, which binds equally strongly to each of the four bases, along with one or two base-specific rings will often enhance sequence specificity over a polyamide composed entirely of base-specific rings.

Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two types of subunits: RNR1 contains the active site for reduction and the binding sites for the nucleotide allosteric effectors. RNR2 contains the diiron-tyrosyl radical (Y⋅) cofactor essential for the reduction process. Studies in yeast have recently identified four RNR subunits: Y1 and Y3, Y2 and Y4. These proteins have been expressed in Saccharomyces cerevisiae and in Escherichia coli and purified to ≈90% homogeneity. The specific activity of Y1 isolated from yeast and E. coli is 0.03 μmol⋅min−1⋅mg−1 and of (His)6-Y2 [(His)6-Y2-K387N] from yeast is 0.037 μmol⋅min−1⋅mg−1 (0.125 μmol⋅min−1⋅mg−1). Y2, Y3, and Y4 isolated from E. coli have no measurable activity. Efforts to generate Y⋅ in Y2 or Y4 using Fe2+, O2, and reductant have been unsuccessful. However, preliminary studies show that incubation of Y4 and Fe2+ with inactive E. coli Y2 followed by addition of O2 generates Y2 with a specific activity of 0.069 μmol⋅min−1⋅mg−1 and a Y⋅. A similar experiment with (His)6-Y2-K387N, Y4, O2, and Fe2+ results in an increase in its specific activity to 0.30 μmol⋅min−1⋅mg−1. Studies with antibodies to Y4 and Y2 reveal that they can form a complex in vivo. Y4 appears to play an important role in diiron-Y⋅ assembly of Y2.

Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy

Patch–clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that γ-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (i) the frequency of spontaneous GABAergic currents is similar to that of control and (ii) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded. Furthermore, large network-driven GABAergic inhibitory postsynaptic currents were present in pyramidal cells during interictal discharges. The duration of these interictal discharges was increased by the GABA type A antagonist bicuculline. We conclude that GABAergic inhibition is still present and functional in these experimental models and that the principal defect of inhibition does not lie in a complete disconnection of GABAergic interneurons from their glutamatergic inputs.

Equilibrium distributions of topological states in circular DNA: Interplay of supercoiling and knotting

Two variables define the topological state of closed double-stranded DNA: the knot type, K, and ΔLk, the linking number difference from relaxed DNA. The equilibrium distribution of probabilities of these states, P(ΔLk, K), is related to two conditional distributions: P(ΔLk|K), the distribution of ΔLk for a particular K, and P(K|ΔLk) and also to two simple distributions: P(ΔLk), the distribution of ΔLk irrespective of K, and P(K). We explored the relationships between these distributions. P(ΔLk, K), P(ΔLk), and P(K|ΔLk) were calculated from the simulated distributions of P(ΔLk|K) and of P(K). The calculated distributions agreed with previous experimental and theoretical results and greatly advanced on them. Our major focus was on P(K|ΔLk), the distribution of knot types for a particular value of ΔLk, which had not been evaluated previously. We found that unknotted circular DNA is not the most probable state beyond small values of ΔLk. Highly chiral knotted DNA has a lower free energy because it has less torsional deformation. Surprisingly, even at |ΔLk| > 12, only one or two knot types dominate the P(K|ΔLk) distribution despite the huge number of knots of comparable complexity. A large fraction of the knots found belong to the small family of torus knots. The relationship between supercoiling and knotting in vivo is discussed.

Case study of the effects of atmospheric aerosols and regional haze on agriculture: An opportunity to enhance crop yields in China through emission controls?

The effect of atmospheric aerosols and regional haze from air pollution on the yields of rice and winter wheat grown in China is assessed. The assessment is based on estimates of aerosol optical depths over China, the effect of these optical depths on the solar irradiance reaching the earth’s surface, and the response of rice and winter wheat grown in Nanjing to the change in solar irradiance. Two sets of aerosol optical depths are presented: one based on a coupled, regional climate/air quality model simulation and the other inferred from solar radiation measurements made over a 12-year period at meteorological stations in China. The model-estimated optical depths are significantly smaller than those derived from observations, perhaps because of errors in one or both sets of optical depths or because the data from the meteorological stations has been affected by local pollution. Radiative transfer calculations using the smaller, model-estimated aerosol optical depths indicate that the so-called “direct effect” of regional haze results in an ≈5–30% reduction in the solar irradiance reaching some of China’s most productive agricultural regions. Crop-response model simulations suggest an ≈1:1 relationship between a percentage increase (decrease) in total surface solar irradiance and a percentage increase (decrease) in the yields of rice and wheat. Collectively, these calculations suggest that regional haze in China is currently depressing optimal yields of ≈70% of the crops grown in China by at least 5–30%. Reducing the severity of regional haze in China through air pollution control could potentially result in a significant increase in crop yields and help the nation meet its growing food demands in the coming decades.