Are natural hybrids fit or unfit relative to their parents?

The process of natural hybridization may produce genotypes that establish new evolutionary lineages. However, many authors have concluded that natural hybridization is of little evolutionary importance because hybrids, in general, are unfit relative to their progenitors. Deciding between these alternative conclusions requires that fitness be measured for hybrid classes and parental species. Recent analyses have found that hybrids are not uniformly unfit, but rather are genotypic classes that possess lower, equivalent or higher levels of fitness relative to their parental taxa.     

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

Heat-inducible expression of FLP gene in maize cells

The soybean heat-shock gene promoter ( Gmhsp 17.5-E ) has been used to direct expression of gusA and FLP genes in maize cells. At inducible temperatures, in transient expression assays, gusA gene expression controlled by the heat-shock promoter is about 10-fold higher than the expression directed by the CaMV 35S promoter. The Gmhsp 17.5-E promoter preserves its regulatory functions in heterologous maize cells after random integration into genomic DNA.
Heat-shock inducible expression of the FLP gene was investigated by co-transformation of the FLP expression vector (pHsFLP) and a recombination test vector (pUFNeo-FmG) into maize protoplasts. Co-transformed protoplasts were incubated at 42°C for 2 h. This treatment induced recombination of 20–25% of the available FRT sites in transient assays. As a result of heat-shock treatment of stably co-transformed maize cells, activation of gusA gene expression and an associated decrease or elimination of NPT-II activity in transgenic maize lines was observed. Molecular evidence was obtained of the expected DNA excision process catalyzed by the FLP protein in maize transgenic cells. Thus, the experiments presented in this paper indicate that the FLP protein can recognize and subsequently recombine the FRT target sites that had integrated into plant genomic DNA, and that regulated expression of the FLP gene is possible in maize cells using the soybean heat-shock promoter.     

Ethylene-Induced Chlorosis in the Pathogenesis of Bipolaris sorokiniana Leaf Spot of Poa pratensis

Endogenous ethylene of Poa pratensis leaves infected by Bipolaris sorokiniana was evaluated as a factor in leaf chlorosis during pathogenesis. Detectable increases in endogenous ethylene of leaves of intact plants under normal ambient pressure occurred 12 hours after inoculation and was maximum at 48 hours; from 48 to 96 hours the ethylene progressively decreased. Necrotic lesions surrounded by chlorotic halos occurred on infected leaves between 24 and 48 hours. Midvein chlorosis interconnecting individual lesions and complete chlorosis of all tissues not directly affected by the lesions occurred between 72 and 96 hours, after maximum production of ethylene at 48 hours. The chlorophyll loss in infected leaves by 96 hours was 44% compared with controls.

Subjecting inoculated leaves of intact plants to a controlled atmospheric-environmental system with an atmospheric pressure of 233 millibars and O₂ and CO₂ partial pressures adjusted to approximately that of normal ambient pressure during infection and disease development prevented most midvein chlorosis and complete chlorosis, but did not prevent necrotic lesion or chlorotic halo development. Under the hypobaric conditions, chlorophyll loss during disease development was reduced to 22% compared with controls at 96 hours. The observations suggest that ethylene may function late in pathogenesis of this host-pathogen interaction and is responsible for much of the chlorophyll loss after its maximum production at 48 hours.

     

I. Photoaffinity probes provide a general method to prepare synthetic peptide-conjugates

A general synthetic strategy is described for the preparation of peptide-conjugates where the peptides contain the NH₂ terminal, COOH terminal, or internal regions of the protein sequence. Glycoprotein D of herpes simplex virus type 1 is used as a representative protein. Ten-residue peptide fragments of the native sequence were synthesized using standard solid-phase methodology. Photoprobes stable to conditions of synthesis and HF cleavage were coupled directly to the protected-peptide resin during synthesis. This one-step procedure eliminates the potential modification of functional groups in the sequence of interest that can occur when using chemically labile bifunctional reagents. Since the photoprobe is inert until photolysis, the synthetic peptide-probe can be readily purified by high-performance liquid chromatography before cross-linking to the carrier molecule. The following photoprobe derivatives were investigated: thep-azidobenzoyl,p-nitrophenylalanyl, andp-benzoylbenzoyl groups. The benzophenone photoprobes were shown to give the highest incorporation of peptide-probe with the protein carrier over a wide range ofpH and solvent conditions. For solid-phase synthesis three benzophenone photoprobes can be used: benzoylbenzoic acid, benzoylbenzoylglycine, andN ^e-(4-benzoylbenzoyl)-N -t-butyloxycarbonyl-lysine.     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

Essential fatty acid deficiency and adrenal cortical function in vitro.

Adrenocortical cells were prepared from rats maintained on essential fatty acid-deficient diets and control litter mates. Cells from control rats had high concentrations of essential fatty acids in the cholesteryl ester fraction of which approximately 22% was arachidonate. In contrast, cells from EFA-deficient rats had only 2.5% arachidonate in the cholesteryl esters, even though the total esterified cholesterol level was comparable to that of controls. In place of the essential fatty acids, the cholesteryl esters of these cells were rich in 20:3(n--9) and 22:3(n--9). When cells from EFA-deficient rats were incubated with ACTH or dibutyryl cyclic AMP, the output of corticosterone was the same as in controls. Also sterol esters were hydrolyzed to the same extent as in controls despite the unusual composition of the fatty acid esters. The phospholipids in both control and EFA-deficient cells contained high levels of arachidonate but were not hydrolyzed in either type of cell during incubation with ACTH or dibutyryl cyclic AMP. The results indicate that high levels of the prostaglandin precursors, namely linoleate and arachidonate, are not a sine qua non for the steroidogenic action of ACTH or cyclic AMP.     

Effect of diethylstilbestrol on ion fluxes in oat roots

Effects of diethylstilbestrol (DES) on ion fluxes in oat roots (Avena sativa L.) were investigated by measuring K⁺ and Cl⁻ absorption and K⁺ efflux. DES rapidly decreased the absorption of K⁺ (⁸⁶Rb) and ³⁶Cl⁻ by excised roots; 10⁻⁴ molar DES inhibited Cl⁻ absorption in 1 minute and K⁺ absorption in 1 to 2 minutes. With a 10-minute incubation period, K⁺ and Cl⁻ absorption were inhibited 50% by 1.1×10⁻⁵ molar and 8.4×10⁻⁶ molar DES, respectively. Treatment for 3 minutes with 10⁻⁴ molar DES caused irreversible inhibition of K⁺ absorption. Increasing concentrations of KCl in the absorption media decreased the DES inhibition. Experiments with the DES analogs, DES dipropionate, dienestrol and hexestrol, showed that the steric configuration and the hydroxyl group of the DES molecule are important in determining the inhibitory capacity of the compound.