The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.     

Pastoral dairy marketing and household wealth interactions and their implications for calves and humans in Ethiopia

Surveys of pastoral households in a semi-nomadic Borana community during 1987–1988 were used to test the hypothesis that poorer families living closest to a market town would be most affected by the enhanced opportunity to sell dairy products, which would intensify competition between people and calves for milk and have negative implications for calf management. These poorer families indeed reported the highest rates of milk offtake per cow, and the milk increment was probably sold to purchase more grain for human consumption at the expense of milk intake for the calf. Consequently, this strategy may increase the susceptibility of malnourished calves to disease, especially those from lower-producing dams. Benefits of improved human energy intake from grain and retention of livestock capital must be weighed against risks of calf death and possible malnutrition of people from milk restriction when assessing dairy marketing trade-offs that are most acute for the poor. Opportunity to sell dairy products at favorable terms of trade helps the poorest people survive, and their risks could be mitigated by policies that facilitate grain marketing in the rangelands and interventions that improve calf feeding management, diversify human diets, and create alternative opportunities for women to generate income. The households postulated to be most at risk were identified from a complex, but logical, interaction among factors of distance to market, household wealth, and the quality of milking cows held. This indicates that targeting such needy groups for development assistance may require a more detailed and interdisciplinary analysis of production systems than is commonly practiced.     

Pharmacology of insect GABA receptors

A GABA-operated Cl^– channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl^– channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABA_A receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl^– channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.Special issue dedicated to Dr. Eugene Roberts     

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Revised enzyme synthesis rate expression in cybernetic models of bacterial growth

A revised enzyme synthesis rate expression for cybernetic models of bacterial growth is presented. The rate expression, which is comprised of inducible and constitutive contributions, provides for a basal enzyme level that is necessary to predict certain types of commonly observed continuous culture transients. The response of a continuous culture to a step change in feed stream composition is simulated using both the old and new formulations, and the ramifications for the "matching-law" formulation are discussed.     

Three new species of Phacelia (Hydrophyllaceae) from Nuevo León,Mexico

Three new species ofPhacelia from Nuevo León, Mexico are described and illustrated. Two of these,P. hintoniorum andP. potosina, belong to theCrenulata group; the former, with included anthers, relates toP. coerulea; the latter, with exserted anthers, relates toP. alba orP. coulteri. The remaining novelty,P. zaragozana, belongs to the subgenusCosmanthus where its closest relationship is withP. laxa.     

Characterization of monoclonal antibodies to histone 2B. Localization of epitopes and analysis of binding to chromatin

Two mouse monoclonal IgM antibodies have been isolated which bind to histone 2B (H2B), as shown by protein blotting and immunostaining and by solid-phase radioimmunoassay (RIA). One of these (HBC-7) was specific for H2B by both techniques whereas the other (2F8) cross-reacted with histone H1 by RIA. Both antibodies failed to recognize H2B limit peptides from trypsin-digested chromatin and did not bind to Drosophila H2B, which differs extensively from vertebrate H2B only in the N-terminal region. These findings indicate that both antibodies recognize epitopes within the trypsin-sensitive, N-terminal region comprising residues 1-20. Binding of antibody HBC-7 was inhibited by in vitro ADP-ribosylation of H2B at glutamic acid residue 2. This strongly suggests that the epitope recognized by HBC-7 is located at the N-terminus of H2B, probably between residues 1 and 8. We have used solid-phase radioimmunoassay to investigate factors which influence the accessibility of this epitope in chromatin. Removal of H1 ('stripping') from high-molecular-mass chromatin had no effect on HBC-7 binding, nor was any difference observed between binding to stripped chromatin and to 146-base-pair (bp) core particles derived from it by nuclease digestion. These results suggest that accessibility of the N-terminal region of H2B is not influenced by H1 itself or by the size or conformation of linker DNA. In contrast, binding of antibody HBC-7 to 146-bp core particles derived from unstripped chromatin was reduced by up to 70%. Binding was restored by exposure of these core particles to the conditions used for stripping. Analysis of the protein content of core particle preparations from stripped and unstripped chromatin suggests that these findings may be attributable to redistribution of non-histone proteins during nuclease digestion. Pre-treatment of high-molecular-mass chromatin or 146-bp core particles with the intercalating dye ethidium bromide resulted in a severalfold increase in binding of HBC-7. The major changes in nucleosome morphology induced by ethidium are therefore accompanied by an increase in accessibility of the N-terminal region of H2B, possibly as a direct result of changes in the spatial relationship between H2B and core DNA.     

Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids

The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers XGCGCp and GCGCYp containing the GCGC core plus a dangling end. Each 5' dangling end increases helix stability at 37 degrees C roughly 0.2 kcal/mol and each 3' end from 0.8 to 1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported for the GGCC core [Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., & Turner, D. H. (1985) Biochemistry 24, 4533-4539], indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G.C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of "stacking" and "pairing". If pairing contributions are due to hydrogen bonding, the results suggest stacking and hydrogen bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment associated with forming a hydrogen bond is estimated to be -1 kcal/mol of hydrogen bond.     

Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability

A solid-phase method using a phosphoramidite approach is described for synthesis of oligoribonucleotides. The method was used to synthesize pairs of oligomers with identical nearest neighbors but different sequences. Comparison of thermodynamic parameters for these pairs provides a test of the nearest-neighbor hypothesis for prediction of helix stability. In general, pairs of sequences with identical nearest neighbors have enthalpy and entropy changes for helix formation that differ by 8% on average, delta Go37 that differ by 6% on average, and melting temperatures within 0-5 degrees C of each other. These limits are typical of the accuracy that should be expected from nearest-neighbor predictions of RNA helix stability. UCAUGA and UGAUCA have the same nearest neighbors but melting temperatures that differ by 7 degrees C. This suggests some sequences will not be approximated well by the nearest-neighbor model.