Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003

Cosmids from the 1A3–1A10 region of the complete miniset were individually subcloned by using the vector M13 mp18. Sequences of each cosmid were assembled from about 400 DNA fragments generated from the ends of these phage subclones and merged into one 189-kb contig. About 160 ORFs identified by the CodonUse program were subjected to similarity searches. The biological functions of 80 ORFs could be assigned reliably by using the WIT and Magpie genome investigation tools. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Most of the ORFs lacking significant similarity with reference databases also were grouped. There are two large clusters of these ORFs, one located between 45 and 67 kb of the map, and the other between 150 and 183 kb. Nine of the loosely identified ORFs (of 15) of the first of these clusters match ORFs from phages or transposons. The other cluster also has four ORFs of possible phage origin.

Behavioral alterations associated with apoptosis and down-regulation of presenilin 1 in the brains of p53-deficient mice

Presenilin 1 (PS1) expression is repressed by the p53 tumor suppressor. As shown herein, wild-type PS1 is an effective antiapoptotic molecule capable of significantly inhibiting p53-dependent and p53-independent cell death. We analyzed, at the functional and molecular levels, the brains of p53 knockout mice. Surprisingly, we found that lack of p53 expression induces apoptotic brain lesions, accompanied by learning deficiency and behavioral alterations. p53-deficient mice show an unexpected overexpression of p21waf1 with subsequent down-regulation of PS1 in their brains. This process is progressive and age-dependent. These data indicate that the p53 pathway, besides affecting tumor suppression, may play a major role in regulating neurobehavioral function and cell survival in the brain.

Correlation between Binding Affinity and Necrosis-Inducing Activity of Mutant AVR9 Peptide Elicitors1

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.

Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression.

Developmentally regulated genes in Drosophila, which are conserved through evolution, are potential candidates for key functions in biological processes such as cell cycle, programmed cell death, and cancer. We report cloning and characterization of the human homologue of the Drosophila seven in absentia gene (HUMSIAH), which codes for a 282 amino acids putative zinc finger protein. HUMSIAH is localized on human chromosome 16q12-q13. This gene is activated during the physiological program of cell death in the intestinal epithelium. Moreover, human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA. A similar pattern of expression is also displayed by the p21waf1. These results suggest that mammalian seven in absentia gene, which is a target for activation by p53, may play a role in apoptosis and tumor suppression.

Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.

This report demonstrates that the investigational prostatic carcinoma marker known as the prostate-specific membrane antigen (PSM) possesses hydrolytic activity with the substrate and pharmacologic properties of the N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is a membrane hydrolase that has been characterized in the mammalian nervous system on the basis of its catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) to yield glutamate and N-acetylaspartate and that has been hypothesized to influence glutamatergic signaling processes. The immunoscreening of a rat brain cDNA expression library with anti-NAALADase antisera identified a 1428-base partial cDNA that shares 86% sequence identity with 1428 bases of the human PSM cDNA [Israeli, R. S., Powell, C. T., Fair, W. R. & Heston, W.D.W. (1993) Cancer Res. 53, 227-230]. A cDNA containing the entire PSM open reading frame was subsequently isolated by reverse transcription-PCR from the PSM-positive prostate carcinoma cell line LNCaP. Transient transfection of this cDNA into two NAALADase-negative cell lines conferred NAAG-hydrolyzing activity that was inhibited by the NAALADase inhibitors quisqualic acid and beta-NAAG. Thus we demonstrate a PSM-encoded function and identify a NAALADase-encoding cDNA. Northern analyses identify at least six transcripts that are variably expressed in NAALADase-positive but not in NAALADase-negative rat tissues and human cell lines; therefore, PSM and/or related molecular species appear to account for NAAG hydrolysis in the nervous system. These results also raise questions about the role of PSM in both normal and pathologic prostate epithelial-cell function.

Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina.

Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.