A Comparison of Internal Standards for Plant Cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

The effect of nectar guides on pollinator preference: experimental studies with a montane herb

Summary Rare albino morphs of the montane larkspur Delphinium nelsonii differ from common blue-flowered morphs in overall flower color, and in the strength of a contrasting color pattern at the center of the flower that presumably guides pollinators to concealed nectar. Previous studies showed that bumblebees and hummingbirds discriminate against albinos when presented with mixtures of the 2 morphs, and that it takes these pollinators longer to fly between successive flowers on albino than on blue-flowered inflorescences. To explore the link between these observations, we measured pollinator preferences and flower-to-flower flight times (handling times) before and after painting flowers in 2 alternative ways that enhanced albino nectar guides. In all of 16 experimental replicates discrimination against albinos was reduced or eliminated after painting, and albino handling times declined toward values for blue-flowered inflorescences. This consistent result indicates that an inferior nectar guide increases the energetic cost of foraging at albinos. Increased cost in turn explains discrimination, under the reasonable assumption that hummingbirds and bumblebees are sensitive to foraging economics.     

Expression of cytochrome o in hydrogen uptake constitutive mutants of Rhizobium japonicum.

Mutant strains of Rhizobium japonicum constitutive for H2 uptake activity (Hupc) contained significantly more membrane-bound b-type cytochrome than did the wild type when grown heterotrophically. The Hupc strains contained approximately three times more dithionite- and NADH-reducible CO-reactive b-type cytochrome than did the wild type; the absorption features of the CO spectra were characteristic of cytochrome o. This component, designated cytochrome b', was not reduced by NADH in the presence of cyanide. Cytochrome o from the wild type (SR) and cytochrome b' from mutants SR476 and SR481 bound to CO with similar dissociation constants of 5.4, 7.4, and 5.6 microM, respectively. NADH-dependent reduction of cytochrome b' from SR476 and SR481 and the cytochrome o from SR followed pseudo-first-order kinetics with similar rate constants. Based on these spectral, ligand-binding, and kinetic measurements, it was concluded that cytochrome b' expressed by the Hupc mutants is equivalent to cytochrome o found in the wild type. H2, NADH, and succinate each reduced the same amount of total b-type cytochrome in membranes from SR481, and the rate of H2-dependent cytochrome o reduction was significantly less than with succinate or NADH as the reductants. It was concluded that neither cytochrome o nor any b-type cytochrome expressed by the Hupc mutants was unique to the H2 oxidation system. At low O2 concentrations, the inhibition of H2 and NADH oxidase activities by CO closely paralleled the binding of CO to cytochrome o rather than cytochromes a3 or c'. This suggested that NADH and H2 oxidation involved primarily cytochrome o as the terminal oxidase at low O2 tensions.     

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.     

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

Summary A cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Kinetics of microbial growth on pentachlorophenol

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

Interactions betweenAcyrthosiphon kondoi [Homoptera: Aphidoidea] andAphelinus asychis [Hymenoptera: Chalcidoidea] and other parasites and hosts

In laboratory trials to investigate the parasite/host spectra of certain aphid pests and hymenopterous parasites, the aphidAcyrthosiphon kondoi Shinji encapsulated the egg of the aphelinid parasiteAphelinus asychis Walker. The resultant brown, sclerotic capsule was formed within 24 h of exposure of the aphid to parasitization and as far as is known prevented the development of the parasite to the larval stage. The capsule remained throughout the life of the aphid, whose longevity and fecundity were apparently not seriously impaired. A small number ofAphelinus escaped encapsulation, especially in aphids already containing capsule(s), and developed into normal, reproductive adults.A. kondoi did not encapsulate, andA. asychis was not encapsulated by any other species. However, thoughA. asychis readily parasitizedAphis citricola van der Goot,A. nerii Boyer de Fonscolombe andToxoptera citricidus (Kirkaldy), most of its progeny ceased development in these aphids before reaching the mummification stage, and died within the dead or dying, non-mummified aphid host.     

Comparison of patterns of hybrid resistance to the BALB/c plasmacytomas LPC-1 and MPC-11

Summary Patterns of genetic control of hybrid resistance to the BALB/c plasmacytoma LPC-1 were studied for comparison with those to MPC-11, a plasmacytoma investigated previously. The overall patterns of hybrid resistance to the two tumors were similar, i.e., hybrids between BALB/c and BALB congenic resistant (CR) strains, A and A CR strains, SJL and DBA/2 were as susceptible to LPC-1 as BALB/c mice themselves, whereas hybrids between BALB/c and AKR, C57BL/Ks, DBA/1, C57BL/6 (B6), C57BL/10 (B10) and B10 CR strains were resistant to LPC-1 as previously shown with MPC-11. Heterozygosity within the H-2 complex alone was insufficient for resistance to either tumor. Among hybrids between BALB/c and the B10 CR strains, however, the presence of certain H-2 haplotypes influenced the degree of resistance seen and this H-2 effect was different for the two tumors. A sex effect on resistance to LPC-1, but not to MPC-11, was seen among F₁ hybrids between BALB/c and DBA/1 although not in any other F₁ hybrids. Among ((B10×BALB/c)F₁×BALB/c) and (BALB/c×(B10×BALB/c)F₁) and ((BALB/c×B10)F₁×BALB/c) and ((BALB/c×B10)F₁×BALB/c) backcross mice, however, significantly more males than females were resistant to LPC-1 and the results of this study are compatible with the idea that in F₁ hybrids between BALB/c and B10, resistance to LPC-1 is controlled by two dominant autosomal genes, one of which is sex-limited and neither of which is linked to H-2. In contrast, hybrid resistance to MPC-11 in this cross is controlled by a single gene. Cross-protection experiments indicated that the two tumors share at least one tumor-associated transplantation antigen.