The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA.

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing greater than 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.     

Combining pheromone-baited and food-baited traps for insect pest control: Effects of developmental period

An age-structured population dynamics model is presented that incorporates pheromone-trapping and food-trapping as control methods for an insect pest. The model yields the following results. Low rates of pest survivorship allow lower trapping rates for control. Species with long developmental periods are easier to control than those with shorter developmental periods (other factors being equal) due to lower net survival. The rates of pheromone trapping alone for effective control are usually very high. The combination of pheromone and food trapping allows control with much lower trapping rates than either method alone. Even small amounts of immigration of adult pests into the control area renders pheromone control ineffective, whereas food traps suppress both the immigrants and the resident population. Food- (or odor-) baited traps which attract both males and females are only somewhat more efficient than those which attract females alone. The existence of density-dependent population regulation assists the control program substantially, but this assistance declines as food trapping becomes a more important part of the control program. Larval competition strongly affects the required trapping rates for eradication; species in which all larvae exert strong competition are much easier to control than those in whic the younger larvae contribute little to the total competitive depression.     

Human immunodeficiency virus type 1 envelope gene structure and diversity in vivo and after cocultivation in vitro.

Nested-primer polymerase chain reaction (PCR) has been applied to the molecular cloning of 4.6-kb half-genome fragments of human immunodeficiency virus type 1 (HIV-1) taken directly from the peripheral blood mononuclear cells (PBMC) of an individual with neurological symptoms of HIV-1 infection. In a similar manner, gp120-coding portions of the envelope gene were cloned after PBMC from the same blood sample were cocultivated with uninfected PBMC for 28 days. The complete 1.6-kb nucleotide sequence of the gp120 gene was determined from each of 35 clones examined. Two of 13 (15%) PBMC-derived gp120 genes and 3 of 22 (14%) coculture-derived gp120 genes were defective as a result of frameshifts and an in-frame stop codon(s). Mean diversity between individual gp120-coding sequences in PBMC was fivefold greater (3.24%) than after coculture (0.65%). A predominant sequence of "strain" was found after coculture that was distinct from the diverse viral genotypes detected in vivo and therefore was selectively amplified during in vitro propagation. Multiple distinct third variable (V3) regions encoding the principal neutralizing domain of the envelope protein were detected in PBMC-derived genes, suggesting the presence of immunologic diversity of HIV env genes in vivo not reflected in the cocultured virus sample. The large size of the HIV fragments generated in this study will permit analysis of the diversity of immunologic reactivity, gene function, and pathogenicity of HIV genomes present within infected individuals, including the functional significance of the loss of diversity that occurs upon coculture.     

Maltose chemotaxis involves residues in the N-terminal and C-terminal domains on the same face of maltose-binding protein.

The periplasmic maltose-binding protein (MBP) of Escherichia coli is the recognition component of the maltose chemoreceptor and of the active transport system for maltose. It interacts with the Tar chemotactic signal transducer and the integral cytoplasmic-membrane components (the MalF and MalG proteins) of the maltose transport system. Maltose binds in a cleft between the globular N-terminal and C-terminal domains of MBP, which are connected by a moveable hinge. The two domains undergo a large motion relative to one another as the protein moves from the open, unbound state to the closed, ligand-bound state. We generated, by doped-primer mutagenesis, amino acid substitutions that specifically disrupt the chemotactic function of MBP. These substitutions cluster in two well-defined regions that are nearly contiguous on the surface of MBP in its closed conformation. One region is in the N-terminal domain and one is in the C-terminal domain. The distance between the two regions is expected to change substantially as the protein goes from the open to the closed form. These results support a model in which ligand binding brings two recognition sites on MBP into the proper spatial relationship to interact with complementary sites on Tar. Mutations in MBP that appear to cause defects in interaction with MalF and MalG are distributed differently from mutations that primarily affect maltose taxis. We conclude that the regions of MBP that contact Tar and those that contact MalF and MalG are adjacent on the face of the protein opposite the hinge connecting the two domains and that those regions are largely, although perhaps not entirely, distinct.     

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier     

Ethanol tolerance and carbohydrate metabolism in lactobacilli

Summary This paper describes the ethanol tolerance and metabolism of 31 strains ofLactobacillus on glucose, xylose, lactose, cellobiose and starch. The purpose of this work was to determine the suitability of the 31 strains as potential host for the ethanol producing genes, pyruvate decarboxylase and aldehyde dehydrogenase, fromZymomonas mobilis. The 31 strains were screened for their ability to grow in 0 to 8% v/v ethanol on all five carbohydrates. Those strains that were able to grow to an OD of 1.0 in 8% ethanol were evaluated at ethanol concentrations up to 16%. v/v. The fermentative products from the five carbohydrates were analyzed to determine the ratios of lactic acid, ethanol, and acetic acid.Published as Paper No. 9786, Journal Series Nebraska Agricultural Experiment Station, Lincoln, NE 68583-0704.     

Modelling the effects of population aggregation on the efficiency of insect pest control

A methodology is developed to assess the effects of spatial distribution on the efficiency of insect pest control. This methodology is especially applicable to pest control methods whose efficiency of action depends either positively or negatively on pest density It is applied here to the sterile insect technique and pheromone trapping for male annihilation, which both depend negatively on density. This methodology relies on quantifying clumps of various size and then relating this to efficiency of control and predicting the total pest production given the information on clump sizes and efficiency of control for each clump size. It is found that control is about four times as difficult for a population that is highly clumped (k of the negative binomial distribution=0.25) as for a regularly dispersed population.     

Quinua and Relatives (Chenopodium sect.Chenopodium subsect.Celluloid)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.