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111.
Alessandro Negro Irene Martini Emilio Bigon Flavia Cazzola Cristina Minozzi Stephen D. Skaper Lanfranco Callegaro 《Gene》1992,110(2)
The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters pR and pL, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli. 相似文献
112.
113.
G. Giacomo Consalez Carol L. Stayton Nelson B. Freimer Ponmani Goonewardena W. Ted Brown T. Conrad Gilliam Stephen T. Warren 《Genomics》1992,12(4):710-714
Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage. 相似文献
114.
N1E.115 murine neuroblastoma cells differentiating in serum-free medium were used to develop a paradigm for testing neurotoxicity in vitro. The paradigm was designed to test the effects of toxicants on four different aspects of cell function or structure: 1. Viability as shown by the retention of cellular radiolabel (51Cr); 2. Growth and maintenance of neurites as reflected by the incidence and average length of these processes; 3. Gross structure of neurites; and 4. Velocity and flux of rapid anterograde and retrograde axonal transport as judged by video-enhanced differential interference contrast microscopy. To evaluate this paradigm, colchicine and vinblastine were used as neurotoxicants with a well-understood mechanism of action. These agents were only weakly cytotoxic according to the Cr-release assay, but were able to interfere with neurite outgrowth at nanomolar concentrations. Neurites that were elaborated in the presence of vinblastine and colchicine were often disfigured by numerous swellings packed with organelles. In established neurites, micromolar concentrations of vinblastine inhibited organellar motility with great rapidity, blocking all signs of transport within 20 min. The effect of colchicine was slower and less complete, but still impressive. We suggest that this four-part analysis represents a highly sensitive in vitro test for neurotoxicity, and a means of analyzing the relation between abnormalities of transport and structural damage of nerve cells. 相似文献
115.
G Bakkeren B Gibbard A Yee E Froeliger S Leong J Kronstad 《Molecular plant-microbe interactions : MPMI》1992,5(4):347-355
The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae. 相似文献
116.
The behavior of 8 nursery/peer-reared and 16 mother-only reared rhesus macaques was observed between birth and 5 months of
age, with follow-up studies conducted when the animals were 10–21 months old and living in large social groups. Nursery-reared
neonates were more awake, active, and irritable than mother-only reared monkeys. From 1 to 5 months of age the nursery/peer-reared
animals exhibited a greater variety of behaviors than the mother-only reared infants, which spent the majority of the time
in ventral contact with mothers. As juveniles the groups were indistinguishable with the exception of more self-directed behaviors
observed in the nursery/peer-reared monkeys. Both rearing conditions, by virtue of their atypicality, imposed restrictions
on social development. The behavioral similarity of the juveniles while in the large social group may be a function of maturation
or due to the rehabilitative effect of the large social group. 相似文献
117.
Pere Santamaria Michael T. Boyce-Jacino Alan L. Lindstrom Jose J. Barbosa Anthony J. Faras Stephen S. Rich 《Immunogenetics》1991,33(5-6):374-387
Nucleic acid sequences of the second exons of HLA-DRB1, –DRB3/4/5, –DQB1, and –DQA1 genes were determined from 43 homozygous cell lines, representing each of the known class II haplotypes, and from 30 unrelated Caucasian subjects, comprising 60 haplotypes. This systematic sequence analysis was undertaken in order to a) determine the existence of sequence microheterogeneity among cell lines which type as identical by methods other than sequencing; b) determine whether direct sequencing of class II genes will identify the presence of more extensive sequence polymorphism at the population level than that identified with other typing methods; c) accurately determine the molecular composition of the known class II haplotypes; and d) study their evolutionary relatedness by maximum parsimony analysis. The identification of seven previously unidentified haplotypes carrying five new allelic amino acid sequences suggests that sequence microheterogeneity at the population level may be more frequent than previously thought. Maximum parsimony analysis of these haplotypes allowed their evolutionary classification and indicates that the higher mutation rate at DRB1 compared to DQB1 loci in most haplotypic groups is inversed in specific haplotype lineages. Furthermore, the extent and localization of gene conversions and point mutations at class II loci in the evolution of these haplotypes is significantly different at each locus. Identification of additional HLA class II molecular microheterogeneity suggests that direct sequence analysis of class II HLA genes can uncover new allelic sequences in the population and may represent a useful alternative to current typing methodologies to study the effects of sequence allelism in organ transplantation.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M35890 through M35953. 相似文献
118.
D A Leong 《Cell calcium》1991,12(2-3):255-268
A two-state model for the stimulus-induced nongraded response of a single cell is formulated. Individual metestrus gonadotropes stimulated with LHRH operate as a simple switch: either on or off. At a given concentration of stimulus some gonadotropes switch on, while others do not switch on, secretion. The probability of a gonadotrope being in the secretory state is enhanced with each increment of LHRH concentration. Individual gonadotropes in a secretory state are envisioned to decrease their number of LHRH receptors and to switch off LH biosynthesis. On the other hand, individual gonadotropes that are not in a secretory state are thought to increase their number of LHRH receptors and to switch on LH biosynthesis. The group of individuals in the population that have thresholds falling in the range of a given stimulus initiate secretion. And, the group of individuals in the population that have thresholds that fall above the range of a given stimulus do not initiate secretion. More remarkable is evidence that the cells that are protected from hormone secretion nevertheless respond with a set of intracellular signals and this provides a new perspective of how they switch on hormone biosynthesis and up-regulate the LHRH receptors. These changes are envisioned to reduce the threshold of an individual cell and accordingly to enhance the probability that the cell responds in the secretory state with the next stimulus. This scheme would appear to lead to automatic cycles of secretion and biosynthesis since an individual cell can occupy only one of two states at any time and occupancy of either state promotes change to the other. This may provide a solution to the problem of how an endocrine gland might reconcile differences in the time-course of hormone secretion which occurs rapidly and hormone biosynthesis that requires a longer period of time. Parenthetically, the model may also be adapted to the case where the vast majority of individuals in the population are generally subthreshold in relation to the physiological stimulus: such an adaption leads to interesting ways of viewing the mammalian reproductive cycle and the regulation of the preovulatory LH surge. A two-state model of the internal Ca2+ store is outlined here to stimulate thought on how the intracellular signals of each binary state may switch a variety of cellular responses either on or off. The model provides a new perspective on the coordinate regulation of hormone biosynthesis, receptors, and secretion that may be useful in the final reconciliation of population studies with insights about individual cells. 相似文献
119.
Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells 总被引:10,自引:0,他引:10
Yunxia Wang James D. Jones Stephen C. Weller Peter B. Goldsbrough 《Plant molecular biology》1991,17(6):1127-1138
Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes. 相似文献
120.
Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants 总被引:5,自引:0,他引:5
Glyn A. Edwards Andrew Hepher Stephen P. Clerk Donald Boulter 《Plant molecular biology》1991,17(1):89-100
A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons. 相似文献