The rabbit C family of short, interspersed repeats. Nucleotide sequence determination and transcriptional analysis

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.     

Decreased High-Affinity Binding of [3H]Muscimol to Cerebral Synaptic Membranes of Scrapie-Infected Mice

Scrapie is a transmissible disease that results in progressive degeneration of the central nervous system and death. Although scrapie has been studied histopathologically, relatively little is known concerning neurotransmitter alterations. Specific [3H]muscimol binding to whole brain crude synaptic membranes (CSM) from mice clinically affected with scrapie was significantly (p less than 0.01) reduced, to approximately 73% of that of the controls. Of the brain regions examined, binding to only cerebral CSM was significantly (p less than 0.0001) decreased. Scatchard analyses of saturation curves revealed that the high-affinity (KD = 8 +/- 3 nM) site for muscimol was abolished in cerebral CSM from scrapie-infected mice, while the low-affinity site was unaffected. Binding of [3H]flunitrazepam to cerebral CSM was unaffected by scrapie and was stimulated by GABA to the same extent in both scrapie and control mice. These results suggest that scrapie agent 139A in C57BL/6J mice manifests a portion of its CNS pathology via a high-affinity GABA binding site that is unassociated with the benzodiazepine receptor.     

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Application of reaction-diffusion models to cell patterning in Xenopus retina. Initiation of patterns and their biological stability

We have examined the behavior of two reaction-diffusion models, originally proposed by Gierer & Meinhardt (1972) and by Kauffman, Shymko & Trabert (1978), for biological pattern formation. Calculations are presented for pattern formation on a disc (approximating the geometry of a number of embryonic anlagen including the frog eye rudiment), emphasizing the sensitivity of patterns to changes in initial conditions and to perturbations in the geometry of the morphogen-producing space. Analysis of the linearized equations from the models enabled us to select appropriate parameters and disc size for pattern growth. A computer-implemented finite element method was used to solve the non-linear model equations reiteratively. For the Gierer-Meinhardt model, initial activation (varying in size over two orders of magnitude) of one point on the disc's edge was sufficient to generate the primary gradient. Various parts of the disc were removed (remaining only as diffusible space) from the morphogen-producing cycle to investigate the effects of cells dropping out of the cycle due to cell death or malfunction (single point removed) or differentiation (center removed), as occur in the Xenopus eye rudiment. The resulting patterns had the same general shape and amplitude as normal gradients. Nor did a two-fold increase in disc size affect the pattern-generating ability of the model. Disc fragments bearing their primary gradient patterns were fused (with gradients in opposite directions, but each parallel to the fusion line). The resulting patterns generated by the model showed many similarities to results of "compound eye" experiments in Xenopus. Similar patterns were obtained with the model of Kauffman's group (1978), but we found less stability of the pattern subject to simulations of central differentiation. However, removal of a single point from the morphogen cycle (cell death) did not result in any change. The sensitivity of the Kauffman et al. model to shape perturbations is not surprising since the model was originally designed to use shape and increasing size during growth to generate a sequence of transient patterns. However, the Gierer-Meinhardt model is remarkably stable even when subjected to a wide range of perturbations in the diffusible space, thus allowing it to cope with normal biological variability, and offering an exciting range of possibilities for reaction-diffusion models as mechanisms underlying the spatial patterns of tissue structures.     

Spontaneous malignant lymphoma in an African green monkey naturally infected with simian T-lymphotropic virus (STLV)

An African green monkey naturally infected with simian T-lymphotropic virus (STLV) developed spontaneous malignant lymphoma of diffuse pleomorphic type. The clinical, hematological and histopathological characteristics were very similar to those of human adult T-cell leukemia.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

Carbohydrate-binding proteins of tumor lines with different growth properties

Summary A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weights of 80 kDa, 70 kDa, 22 kDa, 18 kDa and 16 kDa and of a fucose-binding protein at apparent molecular weight of 42 kDa. The -glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5–8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5–8#1 and 5–20#20, whereas the metastatic variant 5–20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.     

Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in Charon 4A. 2,800 recombinants containing 10–20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.     

Conformation of antibiotic X-537A (lasalocid-A) and its calcium complex in acetonitrile solvent using circular dichroism spectroscopy

The calcium binding characteristics of antibiotic X-537A (lasalocid-A) in a lipophilic solvent, acetonitrile (CH3CN), have been studied using circular dichroism (CD) spectroscopy. The analysis of the data indicated that in this medium polar solvent, X-537A forms predominantly the charged complexes of stoichiometries 2:1 and 1:1, the relative amounts of the two being dependent on [Ca2+]. The conformations of the complexes, arrived at on the basis of the data, seem to indicate a rigid part encompassing Ca2+, liganded to 3 oxygens of the molecule, viz., the carbonyl, the substituted tetrahydrofuran ring and the substituted pyran ring oxygens (apart from, possibly, the liganding provided by nitrogen atoms of the solvent molecules), and a flexible part consisting of the salicylic acid group of the molecule.