Molecular cloning and expression of brain-derived neurotrophic factor.
TL;DR: The full primary structure of brain-derived neurotrophic factor is reported and it is established that these two neurotrophic factors are related both functionally and structurally.
Abstract: During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.
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TL;DR: Some of the recent progress in understanding the cellular and molecular mechanisms underlying neurotrophin regulation of neural circuits are summarized in this Review.
Abstract: Brain-derived neurotrophic factor (BDNF)--a member of a small family of secreted proteins that includes nerve growth factor, neurotrophin 3 and neurotrophin 4--has emerged as a key regulator of neural circuit development and function. The expression, secretion and actions of BDNF are directly controlled by neural activity, and secreted BDNF is capable of mediating many activity-dependent processes in the mammalian brain, including neuronal differentiation and growth, synapse formation and plasticity, and higher cognitive functions. This Review summarizes some of the recent progress in understanding the cellular and molecular mechanisms underlying neurotrophin regulation of neural circuits. The focus of the article is on BDNF, as this is the most widely expressed and studied neurotrophin in the mammalian brain.
1,513 citations
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...complex also contributes to the increased presynaptic release (4)....
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TL;DR: It is demonstrated that BDNF and NT-3 are stress-responsive genes and the possibility that alterations in the expression of these or other growth factors might be important in producing some of the physiological and pathophysiological effects of stress in the hippocampus is raised.
Abstract: Chronic stress produces structural changes and neuronal damage especially in the hippocampus. Because neurotrophic factors affect neuron survival, we questioned whether they might be relevant to the heightened vulnerability of hippocampal neurons following stress. To begin investigating this possibility, we examined the effects of immobilization stress (2 hr/d) on the expression of neurotrophic factors in rat brains using in situ hybridization. We found that single or repeated immobilization markedly reduced brain-derived neurotrophic factor (BDNF) mRNA levels in the dentate gyrus and hippocampus. In contrast, NT-3 mRNA levels were increased in the dentate gyrus and hippocampus in response to repeated but not acute stress. Stress did not affect the expression of neurotrophin-4, or tyrosine receptor kinases (trkB or C). Corticosterone negative feedback may have contributed in part to the stress-induced decreases in BDNF mRNA levels, but stress still decreased BDNF in the dentate gyrus in adrenalectomized rats suggesting that additional components of the stress response must also contribute to the observed changes in BDNF. However, corticosterone-mediated increases in NT-3 mRNA expression appeared to be primarily responsible for the effects of stress on NT-3. These findings demonstrate that BDNF and NT-3 are stress-responsive genes and raise the possibility that alterations in the expression of these or other growth factors might be important in producing some of the physiological and pathophysiological effects of stress in the hippocampus.
1,456 citations
Cites background from "Molecular cloning and expression of..."
...In addition to NGF, several related neurotrophic factors have been recently characterized including brain-derived neurotrophic factor (BDNF) (Leibrock et al., 1989), neurotrophin-3 (NT-3) (Hohn et al....
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TL;DR: The results indicate that gp140trk is a functional NGF receptor that mediates at least some of the signal transduction processes initiated by this neurotrophic factor.
1,380 citations
TL;DR: The distribution of NT-3 messenger RNA and its biological activity on a variety of neuronal populations clearly distinguishNT-3 from NGF and BDNF, and provide compelling evidence that NT- 3 is an authentic neurotrophic factor that has its own characteristic role in vivo.
Abstract: The development and maintenance of the nervous system depends on proteins known as neurotrophic factors. Although the prototypical neurotrophic factor, nerve growth factor (NGF), has been intensively studied for decades, the discovery and characterization of additional such factors has been impeded by their low abundance. Sequence homologies between NGF and the recently cloned brain-derived neurotrophic factor (BDNF) were used to design a strategy that has now resulted in the cloning of a gene encoding a novel neurotrophic factor, termed neurotrophin-3 (NT-3). The distribution of NT-3 messenger RNA and its biological activity on a variety of neuronal populations clearly distinguish NT-3 from NGF and BDNF, and provide compelling evidence that NT-3 is an authentic neurotrophic factor that has its own characteristic role in vivo.
1,355 citations
TL;DR: These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood.
Abstract: The integrity of cell membranes is maintained by a balance between the amount of cholesterol and the amounts of unsaturated and saturated fatty acids in phospholipids. This balance is maintained by membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis. To enhance transcription, the active NH2-terminal domains of SREBPs are released from endoplasmic reticulum membranes by two sequential cleavages. The first is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves the hydrophilic loop of SREBP that projects into the endoplasmic reticulum lumen. The second cleavage, at Site-2, requires the action of S2P, a hydrophobic protein that appears to be a zinc metalloprotease. This cleavage is unusual because it occurs within a membrane-spanning domain of SREBP. Sterols block SREBP processing by inhibiting S1P. This response is mediated by SREBP cleavage-activating protein (SCAP), a regulatory protein that activates S1P and also serves as a sterol sensor, losing its activity when sterols overaccumulate in cells. These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood.
1,314 citations
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TL;DR: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia, using primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase of target DNA copies.
Abstract: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
9,107 citations
TL;DR: A simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells are described and linear DNA is almost inactive in mammalian cells.
Abstract: We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
5,481 citations
TL;DR: The efficacy of this cDNA cloning strategy was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases.
Abstract: We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.
4,673 citations
TL;DR: RNA molecular weight measurements were carried out by gel electrophoresis under four different denaturing conditions including 99% formamide, 10 mM methyl mercury, 2.2 M formaldehyde, and 6 M urea at pH 3.8 to demonstrate reliable molecular weight determinations of denatured RNAs, especially those obtained by extrapolation.
Abstract: RNA molecular weight measurements were carried out by gel electrophoresis under four different denaturing conditions including 99% formamide, 10 mM methyl mercury, 2.2 M formaldehyde, and 6 M urea at pH 3.8. Electrophoresis a t a series of gel concentrations and at least two different voltage gradients resulted in some RNA species exhibiting apparent molecular weights that vary with both gel concentration and voltage gradient. Three different deviations from the requirement for hydrodynamically equivalent conformations were observed: (1) deformation of the random coil structure of very large RNAs at moderately high gel concentrations and voltage gradients resulting, in extreme cases, in a molecular weight independent migration of RNA molecules; (2) incomplete denaturation of RNA molecules with very GC rich helical regions; and (3) varying charge/mass ratio due to G e l electrophoresis has become the major analytical procedure for characterizing charged macromolecules both because of the high resolution it provides and the relative simplicity of the technology it requires. The applicability of this procedure to molecular weight determination of nucleic acids and NaDodS04-protein complexes is based both on the fact that these macromolecules are polymers with a constant charge-mass ratio and hydrodynamically equivalent conformations, and on the linearity of the empirical log molecular weight-mobility relation used to determine molecular weights from mobilities. When these assumptions are fulfilled, molecular weights can be measured in a single experiment using standards of known molecular weight. Moreover, since RNA molecules do not have hydrodynamically equivalent conformations in aqueous solutions (Boedtker, 1968; Groot et al., 1970; MacLeod, 1975), several methods were developed in which R N A molecular weights could be determined by gel electrophoresis under denaturing conditions. Denaturing conditions first used included reaction with formaldehyde (Boedtker, 1971), 8 M urea at 60 'C (Rejinders et al., 1973) and 99% formamide at room temperature (Pinder et al., 1974), the latter being by far the most widely used. In addition to the denaturing gel systems first developed, three others have been reported more recently: 6 M urea at pH 3.5 run at 2 "C (Rosen et ai., 1975), 5 mM methyl mercury run at room temperature (Bailey and Davidson, 1976), and 99% formamide run at 45-55 'C (Spohr et al., 1976). This proliferation of denaturing gel systems arose because it was recognized that some of the denaturants first used were not able to denature very GC rich RNA and could not be used in the dilute agarose gels required t From the Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02 138. Receiued March 30, 1977. H. Lehrach was the recipient of a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. John M. Wozney was awarded a Camille and Henry Dreyfus Foundation Summer Research Fellowship. This investigation was supported by National Institutes of Health Grant HD-01229. differential protonation at pH 3.8. Reliable molecular weight measurements of RNA molecules as large as 4.0 X lo6 containing G C rich helical regions could only be made on dilute (0.5-1.0%) agarose gels after reaction with either 2.2 M formaldehyde or 10 mM methyl mercury hydroxide. A theoretical justification for the use of the empirical log molecular weight-mobility relation is presented. It is also demonstrated that the gel electrophoretic behavior of a homologous series of random coils can be approximated by that of a series of spheres with radii proportional to the square root of radius of gyration of a random coil. Consequently, molecular weight determinations of denatured RNAs, especially those obtained by extrapolation, are more reliable if the square root of the molecular weight is plotted vs. log mobility. for molecular weight determinations of very large RNA molecules. We report here a comparison of the behavior of high molecular weight R N A molecules on four different denaturing gels: polyacrylamide in 99% formamide, agarose in 10 mM methyl mercury hydroxide, 2.2 M formaldehyde, and 6 M urea, pH 3.8. In addition, we offer a rationale for the use of the empirical log molecular weight-mobility relation and suggest an alternate method of obtaining RNA molecular weights from mobilities based on the finding that the migration of denatured RNA molecules depends on the square root of the radius of gyration, or the fourth root of the molecular weight. Materials and Methods RNA Samples. TMV and chicken ribosomal RNA were prepared as described previously (Boedtker, 1960; Boedtker et al., 1973). E. coli 23s and 16s rRNA was prepared by phenol-CHC13 extraction (Perry et al., 1972). followed by fractionation on linear 5-20% sucrose gradients. Mouse 28s rRNA was donated by Jesse F. Scott of the Harvard Medical School. Sindbis virus, a gift from Michelene McCarthy of the Department of Biochemistry and Molecular Biology, was extracted with phenol-CHC13 (Perry et al., 1972). Polyacrylamide Gel Electrophoresis in 99% Formamide. To achieve reproducible mobilities, pure reasonably dry deionized formamide is essential. 99% formamide (Eastman Chemical Co.) was deionized following the procedure of Pinder et al. (1 974) by stirring with 40 g /L mixed bed resin (Bio-Rad AG501-X8, 20-50 mesh). After about 5 h, the conductivity should decrease to about 70 pmho. We have found, however, that some batches of formamide are not deionized under these conditions even when more resin is used. Such formamide is unsuitable for gel electrophoresis because irreproducible polymerization of gels occurs. The deionized formamide was filtered through a sintered-glass filter and then distilled under B I O C H E M I S T R Y , V O L . 1 6 , N O . 2 1 , 1 9 7 7 4743 L E H R A C H E T A L electrophoresis buffer (E buffer): 0.05 M boric acid, 0.005 M NazB407.10H~0, 0.01 M sodium sulfate, and 0.001 M Na3EDTA, pH 8.2, without CH3HgOH. The suspension was heated for 5 min in an autoclave and diluted to the appropriate concentration with hot E buffer. CH3HgOH was added to the hot buffered agarose by syringe pipet and stirred rapidly at 60 “C. The gels were poured immediately using 2.5 mL per tube. Before samples were applied, the top of the gel was sliced off with a razor blade to provide a flat surface. RNA samples were prepared by dissolving the RNA in E buffer containing 5 or 10 mM methyl mercury hydroxide as indicated. One-half volume of a 1:l mixture of glycerol-HzO solution containing 0.004% bromophenol blue was added and the sample applied to the gel. Electrophoresis was carried out for various times as indicated at 2.5 or 3 mA per tube, at room temperature inside the closed hood. The latter was clearly designated as being hazardous both because of high voltage and CH3HgOH. All operations involving methyl mercury hydroxide were carried out in the hood, with the operator wearing gloves. The tops of the gels, leftover agarose, used gels, and any material contaminated with CH3HgOH (gloves, paper, disposable items) were placed in a disposable plastic bag and stored in the hood. CH3HgOH waste and contaminated items were disposed of every 3 months by the Department of Chemistry’s hazardous chemical disposal service, the Radiac Corp. The operator was monitored for CH3HgOH accumulation every 6 months by the Harvard Health Services. Agarose Gel Electrophoresis in 2.2 M Formaldehyde. RNA samples were heated in 2.2 M formaldehyde (prepared from 37% Mallinckrodt formaldehyde) in 50% formamide, 0.01 8 M NazHP04-0.002 M NaHzPO4, for 5 min at 60 “C. Agarose gels were prepared by heating a “3%” agarose-water suspension in the autoclave for 5 min and diluting wi th either 1 volume of 4.4 M formaldehyde in 0.036 M NazHP04-0.004 M NaH2P04 to make 1.5% gels or with 2 volumes of the latter plus 1 volume of water to make 0.75% gels. The gels were poured immediately using 2.5 mL per tube. Electrophoresis was carried out at 2 mA per tube at room temperature for the times indicated. The electrophoresis buffer was 2.2 M formaldehyde-0.018 M Na2HP04-0.002 M NaH2P04. Agarose Gel Electrophoresis in 6 M Urea at pH 3.8. Electrophoresis was performed following the procedure described by Rosen et al. ( I 975) with the following modifications. Three times agarose (usually 3 g/lOO mL of HzO) was heated in the autoclave and then diluted with 2 volumes of warm I .5X buffer to give the final concentration of agarose and 6.0 M urea, 0.025 M citric acid (pH 3.8), heated at 65 “ C for 20 s, and fast cooled. One-half volume of the glycerol dye was added as described above. Electrophoresis was carried out for 12 h at 0.38 mA per gel at 4 “C. Staining Gels with Ethidium Bromide. Agarose and polyacrylamide gels were stained overnight in 1 yg/mL ethidium bromide in 0.1 M ammonium acetate (Bailey and Davidson, 1976). After staining, the gels were photographed under short-wave U V light with a Polaroid M P 3 Land camera and high-speed type 57 film (Polaroid Corp.) using a yellow f i l ter.
3,176 citations
TL;DR: A new miniaturized protein and peptide sequenator has been constructed which uses gas phase reagents at the coupling and cleavage steps of the Edman degradation, characterized by a high repetitive yield during the degradation, low reagent consumption, low maintenance requirements, and a degradative cycle time of only 50 min using a complete double cleavage program.
2,096 citations