A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes themale sterile 14 gene and a hotspot for recombination

Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized onems locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that thems14 locus is located in a ～300 kb region. We have identified a YAC clone with an insert size of ～610 kb that contains thems14-linked markers, reflects the organization of the physical map and therefore most probably contains thems14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from ～105–140 kb/cM to less than 24 kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.     

Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) alone does not support hematopoietic colony formation of bone marrow cells from the A/J mouse. To elucidate the molecular lesion in A/J mice, we examined expression of the alpha and beta subunits of the IL-3 receptor (IL-3R). While IL-3R beta was normally expressed, IL-3R alpha was not detectable on the surface of A/J-derived cells by antibody staining. Genetic linkage analysis using recombinant inbred mouse strains between A/J and IL-3-responsive C57BL/6 indicated that the IL-3R alpha gene locus was responsible for the impaired IL-3 response in A/J mice. Molecular cloning and characterization of A/J-derived IL-3R alpha cDNA revealed that it lacked the sequence corresponding to exon 8, which encodes 10 amino acid residues in the extracellular domain. The aberrant splicing was due to a 5 base pair deletion at the branch point in intron 7 and was reproduced in heterologous cells by transfecting with an IL-3R alpha minigene carrying the deleterious intron. The A/J-specific abnormal form of IL-3R alpha was localized inside the cells, but not on the cell surface, providing the molecular basis for the impaired IL-3 response in the A/J mouse.     

Drosophila Immunity: Analysis of Larval Hemocytes by P-Element-Mediated Enhancer Trap

Our aim was to identify new genes involved in the cellular aspects of defense mechanisms of Drosophila, as well as in melanotic tumor formation processes that are linked to blood cell disregulation. We have screened 1341 enhancer detector fly lines for expression of the lacZ reporter gene in larval hemocytes at the end of the third instar. We have selected 21 lines in which we observed a reproducible lacZ expression in blood cells. These lines were classified according to the subsets of hemocytes in which lacZ was expressed, and we identified five lines that can be used as lamellocyte markers. Three lines were selected for further analysis. The first exhibited strong lacZ expression in all lamellocytes. The second expressed lacZ in plasmatocytes and lamellocytes, and exhibited a melanotic tumor phenotype in larvae homozygous for the insertion. A third line showed a striking insertion-linked phenotype of melanized lymph glands (the hematopoietic organ), which resulted in the total absence of circulating hemocytes in the mutant larvae. We anticipate that this mutation, which we named domino, will prove a useful tool in the analysis of the role of hemocytes during the various aspects of immune response and melanotic tumor formation.     

Amphiphysin II (SH3P9; BIN1), a Member of the Amphiphysin/Rvs Family, Is Concentrated in the Cortical Cytomatrix of Axon Initial Segments and Nodes of Ranvier in Brain and around T Tubules in Skeletal Muscle

Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrin_G), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.     

Uterine defense mechanisms in the mare: Serum opsonins affecting phagocytosis of Streptococcus zooepidemicus by equine neutrophils

The addition of serum to uterine secretions was shown to opsonize Streptococcus zooepidemicus and significantly enhance bacterial phagocytosis by equine neutrophils. Treatment of serum by heat inactivation at 56 degrees C, EDTA treatment, and C3 consumption reduced phagocytosis and therefore demonstrated that the process was complement-dependent. The amount of C3 present in uterine secretions was measured in a series of 14 mares infected with Streptococcus zooepidemicus . Ten of the 14 mares had detectable amounts of C3; however, the C3 had been cleaved and rendered nonfunctional. The importance of these findings in relationship to chronic uterine infections in the mare is discussed.     

Leukotriene B4 binding to human neutrophils

[³H] Leukotriene B₄ (LTB₄) binds concentration dependency to intact human polymorophonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4°C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [³H] LTB₄ indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 × 10⁻⁹M and B_max of 1.96 × 10⁴ sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 × 10⁻⁹M and a B_max of 45.6 × 10⁴ sites per neutrophil. Competitive binding experiments with structural analogues of LTB₄ demonstrate that the interaction between LTB₄ and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25°C[³H] LTB₄ is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB₄. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [³H] LTB₄ binding, suggesting that LTB₄ is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB₄ in neutrophils are tightly coupled processes.     

Acetyl glyceryl ether phosphorylcholine (PAF-acether) and leukotriene B4-mediated neutrophil chemotaxis through an intact endothelial cell monolayer

Human endothelial cell monolayers were grown on nucleopore filters, and used to partition the two halves of a modified Boyden chamber. Human neutrophil chemotaxis through the monolayer was studied in response to leukotriene B4 and acetyl glyceryl ether phosphorylcholine (PAF-acether). Both leukotriene B4 and PAF-acether concentration-dependently stimulated neutrophil chemotaxis through intact monolayer. The biologically inactive lyso-PAF, and leukotriene C4 and D4 were inactive as chemotactic agents. Leukotriene A4 was weakly chemotactic. In the absence of chemotaxin, little penetration of the monolayer by neutrophils was observed. Agents that elevate neutrophil cyclic AMP levels inhibit both leukotriene B4 and PAF-acether-stimulated chemotaxis through the endothelial cell monolayer. The specific 5-lipoxygenase inhibitor, 6,8-de-epoxy-6,9-(phenylimino) delta 6,8-prostaglandin I1 (U-60257), inhibits PAF-acether, but not leukotriene B4-mediated chemotaxis. These data suggest that an intact 5-lipoxygenase may be required for normal PAF-acether-mediated chemotaxis, but leukotriene B4-mediated chemotaxis is independent of 5-lipoxygenase activity. This system may prove to be a useful model for the study of neutrophil-endothelial cell interactions.     

Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis.

In a series of experiments, we have determined that Legionella pneumophila will proliferate as an intracellular parasite of the ciliated holotrich Tetrahymena pyriformis in sterile tap water at 35 degrees C. After 7 days of incubation, serpentine chains of approximately 10(3) L. pneumophila cells were observed throughout the cytoplasm of the protozoan infected initially with 1 to 30 L. pneumophila cells. The overall L. pneumophila population increased from ca. 1.0 X 10(2) to ca. 5.0 X 10(4) cells per ml in the coculture within this time frame. The interactions between the protozoan and the bacterium appear to depend upon their concentrations as well as temperature of incubation. L. pneumophila did not multiply in sterile tap water alone, in suspensions of lysed T. pyriformis, or in cell-free filtrates of a T. pyriformis culture. In addition to establishing an ecological model, we found that addition of T. pyriformis to environmental specimens served as an enrichment method that improved isolation of legionella from the specimens.     

An apparatus for simultaneous manual solid-phase synthesis of multiple peptide analogs

A new design of reaction vessel for simultaneous manual solid-phase synthesis of multiple peptide analogs is described. Simultaneous use of four of these vessels attached to a single rotary mixer has been successfully applied to synthesis of two sets of four decapeptide amide analogs. Efficient coupling was indicated by chemical determination at the end of each synthesis cycle and overall final yields of between 78 and 84% were obtained. The products obtained were of a high quality, as assessed by high-performance liquid chromatography and amino acid analysis. This system allows expeditious synthesis of multiple peptide analogs for structure-function studies with economical use of efficiently ventilated laboratory space.     

Effect of supplementall-tyrosine on pigment production in cultures of the Legionnaires' disease bacterium

The etiologic agent of Legionnaires' disease grows on certain agar media. Cultures of this organism on supplemented Mueller-Hinton agar are characterized by the appearance of brown pigment in and around areas of bacterial growth. The major peptone source in Mueller-Hinton agar is an acid hydrolysate of casein. Legionnaires' disease bacterium also grows on a medium in which the peptone source is 0.25% yeast extract, but no pigment is produced. If the yeast extract agar is enriched withl-tyrosine, pigment formation can occur. Pigmentation of cultures of Legionnaires' disease bacterium may be mediated by a phenolo-monooxygenase, or tyrosinase.