Abstract:
:We have developed a low stringency polymerase chain reaction (LSPCR) to isolate the unknown neighboring region around a known DNA sequence, thus allowing efficient targeted gene walking. The method involves the polymerase chain reaction (PCR) with a single primer under conditions of low stringency for primer annealing (40 degrees C) for the first few cycles followed by more cycles at high stringency (55 degrees C). This enables the amplification of a targeted DNA fragment along with other nontargeted fragments. High stringency (55 degrees C) nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). We performed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40 degrees C followed by 27 cycles at 55 degrees C for primer annealing. Subsequently, using higher stringency (55 degrees C) PCR with radiolabeled nested primers for 8 cycles, we have isolated a 0.66-kb fragment of a putative human sodium channel gene. Partial sequence (325 bp) of this fragment revealed a 270-bp region (exon) with homology to the rat brain sodium channel III alpha (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, we putatively assign this sequence as a part of a gene coding the alpha-subunit of a human brain type III sodium channel (SCN3A). Using PCR on two human/rodent somatic cell hybrid panels with primers specific to this putative SCN3A gene, we have localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase chromosomes was used to sublocalize the SCN3A gene to chromosome at 2q24-31. In conclusion, LSPCR is an efficient and sensitive method for targeted gene walking and is also useful for the isolation of homologous genes in related species.
journal_name
Proc Natl Acad Sci U S Aauthors
Malo MS,Srivastava K,Andresen JM,Chen XN,Korenberg JR,Ingram VMdoi
10.1073/pnas.91.8.2975subject
Has Abstractpub_date
1994-04-12 00:00:00pages
2975-9issue
8eissn
0027-8424issn
1091-6490journal_volume
91pub_type
杂志文章abstract::Antisera directed against human dopamine beta-hydroxylase or serotonin were used to characterize the noradrenergic and serotonergic innervation patterns within the primary visual cortex of the squirrel monkey. The noradrenergic and serotonergic projections exhibit a high degree of laminar complementarity: layers V and...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.79.7.2401
更新日期:1982-04-01 00:00:00
abstract::MST1, mammalian STE20-like kinase 1, is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. MST1 is capable of inducing apoptotic morphological changes such as chromatin condensation upon overexpression. In this study, we show that MST1 contains two functional nuclear export signals (...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.181161698
更新日期:2001-08-28 00:00:00
abstract::Brown adipose tissue (BAT) is present throughout life in rodents and plays an important role in energy balance. However, whereas BAT is clearly recognizable in the neonates of larger mammals (including dogs, cats, sheep, cattle, and humans), it is undetectable or present in only small quantities in adults of these spe...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.88.23.10774
更新日期:1991-12-01 00:00:00
abstract::The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression a...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.042684199
更新日期:2002-03-05 00:00:00
abstract::Alphavirus envelope proteins, organized as trimers of E2-E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1414190112
更新日期:2015-02-17 00:00:00
abstract::Aggregation of the receptor for IgE on mast cells, basophils, and a tumor analog, rat basophilic leukemia (RBL) cells, induces a calcium-dependent degranulation of the cells. We have measured the membrane potential (delta psi) of RBL cells during this reaction by using the tetraphenylphosphonium ion (Ph4P+) equilibrat...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.80.18.5744
更新日期:1983-09-01 00:00:00
abstract::Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.93.26.15272
更新日期:1996-12-24 00:00:00
abstract::The self-organization of multicomponent tetrarosette assemblies into ordered nanostructures on graphite surfaces has been studied by atomic force microscopy (AFM). Real-space information on the level of individual molecules allowed us to analyze the underlying structure in unprecedented detail. In highly ordered nanor...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.072685399
更新日期:2002-04-16 00:00:00
abstract::Integrin-dependent adhesions mediate reciprocal exchange of force and information between the cell and the extracellular matrix. These effects are attributed to the "focal adhesion clutch," in which moving actin filaments transmit force to integrins via dynamic protein interactions. To elucidate these processes, we me...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.2010292117
更新日期:2020-12-22 00:00:00
abstract::The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn(2+) and Zn(2+) with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1220341110
更新日期:2013-03-05 00:00:00
abstract::We studied mutants of E. coli originally identified as being deficient in either endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) or exonuclease III [deoxyribonucleate (double-stranded) 5'-nucleotidohydrolase, EC 3.1.4.27] activity. Twelve independently derived mutants were tested, including t...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.72.2.688
更新日期:1975-02-01 00:00:00
abstract::Chemomechanical coupling in muscle contraction may be due to "swinging crossbridges," such that a change in the angle at which the myosin head binds to the actin filament is tightly coupled to release of products of ATP hydrolysis. This model would limit the step size, the unit displacement of actin produced by a sing...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.87.18.7130
更新日期:1990-09-01 00:00:00
abstract::The roots and stems of dicotyledonous plants thicken by the cell proliferation in the cambium. Cambial proliferation changes in response to environmental factors; however, the molecular mechanisms that regulate cambial activity are largely unknown. The quadruple Arabidopsis thaliana mutant atipt1;3;5;7, in which 4 gen...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0805619105
更新日期:2008-12-16 00:00:00
abstract::Childhood exposure to low-level lead can permanently reduce intelligence, but the neurobiologic mechanism for this effect is unknown. We examined the impact of lead exposure on the development of cortical columns, using the rodent barrel field as a model. In all areas of mammalian neocortex, cortical columns constitut...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.97.10.5540
更新日期:2000-05-09 00:00:00
abstract::A major question in plant physiology is how the large amount of sucrose made in leaves is transported to the rest of the plant. Although physiological, biochemical, and anatomical investigations have been performed in this field, to date there have been very few genetic studies. Using a reverse genetic screen, we have...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.250473797
更新日期:2000-12-05 00:00:00
abstract::Our previous work [Levinger, L. & Varshavsky, A. (1982) Cell 28, 375-385] has shown that D1, a 50-kilodalton chromosomal protein of Drosophila melanogaster, is specifically associated with isolated nucleosomes that contain a complex A + T-rich satellite DNA with buoyant density of 1.688 g/ml. We show here that D1 is a...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.79.23.7152
更新日期:1982-12-01 00:00:00
abstract::DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex th...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.2433654100
更新日期:2003-12-09 00:00:00
abstract::Millions of individuals routinely remain awake for more than 18 h daily, which causes performance decrements. It is unknown if these functional impairments are the result of that extended wakefulness or from the associated shortened sleep durations. We therefore examined changes in objective reaction time performance ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1706694115
更新日期:2018-06-05 00:00:00
abstract::Deamidation of asparaginyl and glutaminyl residues causes time-dependent changes in charge and conformation of peptides and proteins. Quantitative and experimentally verified predictive calculations of the deamidation rates of 1,371 asparaginyl residues in a representative collection of 126 human proteins have been pe...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.221463198
更新日期:2001-10-23 00:00:00
abstract::Leptin acts as a potent inhibitory factor against obesity by regulating energy expenditure, food intake, and adiposity. The obese diabetic db/db mouse, which has defects in leptin receptor, displays enhanced neural responses and elevated behavioral preference to sweet stimuli. Here, we show the effects of leptin on th...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.190066697
更新日期:2000-09-26 00:00:00
abstract::The long-lived ethylene, cyclohexene, and norbornenemercurinium ions prepared in superacidic, low-nucleophilic media have been studied by (13)C and (199)Hg NMR spectroscopy. The norbornenemercurinium ion shows temperature-dependent (13)C and (199)Hg NMR spectra, consistent with equilibration via rapid hydride and Wagn...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.77.9.5036
更新日期:1980-09-01 00:00:00
abstract::The human cytokine I-309 is a small glycoprotein secreted by activated T lymphocytes and structurally related to a number of inflammatory cytokines. To investigate the biological activities of I-309 protein, we produced a stable Chinese hamster ovary cell transfectant, CDI.10, which constitutively secretes I-309 prote...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.89.7.2950
更新日期:1992-04-01 00:00:00
abstract::The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with atte...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.76.4.1613
更新日期:1979-04-01 00:00:00
abstract::Ancient societies are often used to illustrate the potential problems stemming from unsustainable land-use practices because the past seems rife with examples of sociopolitical "collapse" associated with the exhaustion of finite resources. Just as frequently, and typically in response to such presentations, archaeolog...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 历史文章,杂志文章
doi:10.1073/pnas.1409646111
更新日期:2014-08-12 00:00:00
abstract::We used a model system of purified components to explore the effects of a downstream target on the signaling properties of a covalent modification cycle, an example of retroactivity. In the experimental system used, a bifunctional enzyme catalyzed the modification and demodification of its substrate protein, with both...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0913815107
更新日期:2010-06-01 00:00:00
abstract::The ability to control the placement of individual protein molecules on surfaces could enable advances in a wide range of areas, from the development of nanoscale biomolecular devices to fundamental studies in cell biology. Such control, however, remains a challenge in nanobiotechnology due to the limitations of curre...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1116099108
更新日期:2011-12-06 00:00:00
abstract::Up to half of all transcription termination events in bacteria rely on the RNA-dependent helicase Rho. However, the nucleic acid sequences that promote Rho-dependent termination remain poorly characterized. Defining the molecular determinants that confer Rho-dependent termination is especially important for understand...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1319193111
更新日期:2014-05-13 00:00:00
abstract::The kinetics of hepatic uptake and degradation of sphingomyelin/cholesterol (2:1, M/M) small unilamellar liposomes were investigated in a BALB/c mouse. The tissue distribution of liposomes was determined by scintillation spectrometry. The percentage of intact liposomes in tissues was estimated by the technique of gamm...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.77.7.4030
更新日期:1980-07-01 00:00:00
abstract::The role of Notch signaling in cartilage differentiation and maturation in vivo was examined. Conditional Notch pathway gain and loss of function was achieved using a Cre/loxP approach to manipulate Notch signaling in cartilage precursors and chondrocytes of the developing mouse embryo. Conditional overexpression of a...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0902306106
更新日期:2009-08-25 00:00:00
abstract::Twice each year, millions of night-migratory songbirds migrate thousands of kilometers. To find their way, they must process and integrate spatiotemporal information from a variety of cues including the Earth's magnetic field and the night-time starry sky. By using sensory-driven gene expression, we discovered that ni...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0409575102
更新日期:2005-06-07 00:00:00