Construction of first-generation lactococcal integrative cloning vectors

Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp. lactis LM0230, first-generation lactococcal integrative cloning vectors were developed. Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs. Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L. lactis and L. lactis subsp. cremoris. Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (ery ^r) gene obtained from L. lactis IL1837. Integration of the ery ^r gene into the L. lactis LM0230 chromosome was achieved by a Campbell-like recombination. The nisin (Nis)-resistance (nis ^r) gene from L. lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L. lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nis ^r fragment flanked by the cloned chromosomal DNA. Transformants grown in the absence of either Em or Nis for >200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes. Further studies involving the Nis-resistant (Nis ^r ) transformant suggested that the integrated nis ^r gene may be amplifying within the host chromosome. Correspondence to: S. K. Harlander     

Phylogenetic analysis of the Rhabdocoela (Platyhelminthes) with emphasis on the Neodermata and relatives

Phylogenetic systematic analysis of 24 taxa representing the rhabdocoel platyhelminths, based on a suite of 89 morphological characters, produced two equally parsimonious trees, 181 steps long, with a consistency index (CI) of 0.69 and a rescaled consistency index (RCI) of 0.56, differing only with respect to that portion of the tree containing Umagillidae, Acholadidae, Graffillinae, Pseudograffillinae, Pterastericolidae and Hypoblepharinidae. Our results accommodate all previously proposed sister taxa to the Neodermata in a single clade in which ((Dalyelliidae + Temnocephalida) Typhloplanidae) is the sister group of ((Fecampiidae +  Urastoma ) ( Udonella ((Aspidogastrea + Digenea) (Monogenea (Gyrocotylidea (Amphilinidea + Eucestoda)))))). Bootstrap and jackknife analyses indicate that the groupings of ((Dalyelliidae + Temnocephalida) Typhloplanidae) and of ((Fecampiidae +  Urastoma ) ( Udonella ((Aspidogastrea + Digenea) (Monogenea (Gyrocotylidea (Amphilinidea + Eucestoda)))))) are highly robust, with the latter clade having a CI of 90% and RCI of 82%. Disagreements among previous analyses of these taxa have been due to the influence of missing data for critical characters in key taxa and differences in the taxa analysed, rather than any inherent weakness in the morphological data. Non-phylogenetic systematic approaches to homology assessment and misconceptions regarding phylogenetic systematic methodology are discussed. Recent analyses combining sequence data with a subset of approximately 60% of the morphological characters should be re-assessed using the entire morphological database. Even if Udonella is a monogenean, it is most parsimonious to suggest that the common ancestor of the Neodermata had a vertebrate–arthropod two-host life cycle.     

DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

Genetic analysis of murine strains C57BL/6J and C3H/HeJ to confirm the map position ofAth-1, a gene determining atherosclerosis susceptibility

Previous results suggested that strains C57BL/6J and C3H/HeJ differed in a single gene for atherosclerosis susceptibility, calledAth-1. Based on data from recombinant inbred strainsAth-1 was tentatively assigned to chromosome 1 linked toAlp-2. In this report, a cross between C57BL/6 and C3H/HeJ was carried out in order to test whether the tentative map position was correct. Parental strains and F₁ and F₂ progeny were examined. Susceptible alleles ofAth-1, found in C57BL/6, are associated with relatively low levels of high-density lipoprotein (HDL)-cholesterol in animals fed an atherogenic diet; resistant alleles ofAth-1 are associated with relatively high levels of HDL-cholesterol. F₁ progeny have HDL levels that are intermediate between these of the two parental strains. Among the F₂ progeny,Alp-2 andAth-1 cosegregated, providing confirmatory evidence thatAth-1 is linked toAlp-2 on chromosome 1. Three mice recombinant forAlp-2 andAth-1 were found among the 60 chromosomes tested, giving an estimated map distance between these two genes of 5.0±2.8 (SE) cM. The phenotypic characteristics ofAth-1 resemble a genetic trait in humans, hyperalphalipoproteinemia, which is characterized by elevated levels of HDL-cholesterol, reduced risk of heart disease, and increased longevity.This work was supported by Grant HL-32087 from the Heart, Lung, and Blood Institute, National Institutes of Health, Grant 1858 from the Council for Tobacco Research, Grant 86-1387 from the American Heart Association with funds contributed in part by the Alameda, Orange, and Santa Barbara County Chapters, and Grants 85-N132A and 85-N136A from the California Affiliate of the American Heart Association.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.