Full-Length Single-Gene cDNA Libraries: Applications in Splice Variant Analysis

Regan, Melissa R.; Emerick, Mark C.; Agnew, William S.

doi:10.1006/abio.2000.4819

Cited by 13 publications

(18 citation statements)

References 17 publications

(17 reference statements)

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“…Moreover, because our exon scanning was performed on a cDNA library whose constituent clones need not span complete channel transcripts, it was also worth establishing whether the observed variants were present in full-length ␣ 1A (␣ 1 2.1) cDNAs. To address these issues, we reexamined key transcript-scanning reactions, this time performed on a different library as a template, a full-length, single-gene ␣ 1A (␣ 1 2.1) library, obtained by long PCR amplification (Regan et al, 2000) of the original human cerebellar library. Results of the analysis are summarized in Table 1, inspection of which reveals clear preferences for certain splice variants.…”

Section: Resultsmentioning

confidence: 99%

“…A f ull-length, single-gene ␣ 1 2.1 cDNA library was isolated by heminested PCR amplification of whole-brain and cerebellar cDNA libraries (C lontech; Regan et al, 2000). T wo sets of primers were used: (1) F1, 5Ј-CCG GCA GCC TCA GCA TCA GC -3Ј; and R1, 5Ј-GGA TCA CAG GGG AAT AGG AC -3Ј; and (2) F2, 5Ј-GCG TAA CCC GGA GCC C TT TG-3Ј; and R2, 5Ј-CGG ATC ACA GGG GAA TAG GAC -3Ј.…”

Section: Methodsmentioning

confidence: 99%

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Systematic Identification of Splice Variants in Human P/Q-Type Channel α₁2.1 Subunits: Implications for Current Density and Ca²⁺-Dependent Inactivation

et al. 2002

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P/Q-type (Ca v 2.1) calcium channels support a host of Ca 2ϩ -driven neuronal functions in the mammalian brain. Alternative splicing of the main ␣ 1A (␣ 1 2.1) subunit of these channels may thereby represent a rich strategy for tuning the functional profile of diverse neurobiological processes. Here, we applied a recently developed "transcript-scanning" method for systematic determination of splice variant transcripts of the human ␣ 1 2.1 gene. This screen identified seven loci of variation, which together have never been fully defined in humans. Genomic sequence analysis clarified the splicing mechanisms underlying the observed variation. Electrophysiological characterization and a novel analytical paradigm, termed strength-current analysis, revealed that one focus of variation, involving combinatorial inclusion and exclusion of exons 43 and 44, exerted a primary effect on current amplitude and a corollary effect on Ca 2ϩ -dependent channel inactivation. These findings significantly expand the anticipated scope of functional diversity produced by splice variation of P/Q-type channels.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Systematic Identification of Splice Variants in Human P/Q-Type Channel α₁2.1 Subunits: Implications for Current Density and Ca²⁺-Dependent Inactivation

et al. 2002

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“…The mixture of the plasmids was added to the above affi nity system for saturation hybridization (Reqan et al, 2000), and then eluted with wash buffer (Invitrogen). The eluted plasmids were transformed into the ElectroMAX™ DH10B™ T1 phageresistant cells.…”

Section: Construction Of the Normalized Cdna Librarymentioning

confidence: 99%

Construction of a cDNA Library from the Ephemeral Plant Olimarabidopsis pumila and Preliminary Analysis of Expressed Sequence Tags

Zhao

Wei

Zhao

et al. 2013

Zeitschrift Für Naturforschung C

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Olimarabidopsis pumila is a close relative of the model plant Arabidopsis thaliana but, unlike A. thaliana, it is a salt-tolerant ephemeral plant that is widely distributed in semi-arid and semi-salinized regions of the Xinjiang region of China, thus providing an ideal candidate plant system for salt tolerance gene mining. A good-quality cDNA library was constructed using cap antibody to enrich full-length cDNA with the gateway technology allowing library construction without traditional methods of cloning by use of restriction enzymes. A preliminary analysis of expressed sequence tags (ESTs) was carried out. The titers of the primary and the normalized cDNA library were 1.6 · 10 6 cfu/mL and 6.7 · 10 6 cfu/mL, respectively. A total of 1093 clones were randomly selected from the normalized library for EST sequencing. By sequence analysis, 894 high-quality ESTs were generated and assembled into 736 unique sequences consisting of 72 contigs and 664 singletons. The resulting unigenes were categorized according to the gene ontology (GO) hierarchy. The potential roles of gene products associated with stress-related ESTs are discussed. The 736 unigenes were similar to A. thaliana, A. lyrata, or Thellungiella salsuginea. This research provides an overview of the mRNA expression profi le and fi rst-hand information of gene sequence expressed in young leaves of O. pumila.

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“…Twenty to 30 bacterial colonies (derived from a single neuron) were then PCR amplified with splice-specific primers (bottom right, 220 bp), enabling determination of the ratio of EFa to EFb transcripts in individual neurons (Fig. 4 E) (Welling et al, 1997;Regan et al, 2000;Splawski et al, 2004;Fan et al, 2005). Whereas EFa and EFb were equivalent at 5 d, EFa predominated by 12 d. Although the relative increase of EFa transcript was less pronounced than the age-dependent elevation of CDF (Fig.…”

Section: Developmental Onset Of Neuronal Camentioning

confidence: 99%

Developmental Activation of Calmodulin-Dependent Facilitation of Cerebellar P-Type Ca²⁺Current

Chaudhuri¹,

Alseikhan²,

Sheng³

et al. 2005

J. Neurosci.

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-dependent facilitation (CDF) of channel opening. This facilitation occurs when local Ca 2ϩ influx through individual channels selectively activates the C-terminal lobe of calmodulin. In neurons, however, such calmodulin-mediated processes have yet to be detected, and CDF of native P-type current has thus far appeared different, arguably triggered by other Ca 2ϩ sensing molecules. Here, in cerebellar Purkinje somata abundant with prototypic P-type channels, we find that the C-terminal lobe of calmodulin does produce CDF, and such facilitation augments Ca 2ϩ entry during stimulation by repetitive action-potential and complex-spike waveforms. Beyond recapitulating key features of recombinant channels, these neurons exhibit an additional modulatory dimension: developmental upregulation of CDF during postnatal week 2. This phenomenon reflects increasing somatic expression of Ca V 2.1 splice variants that manifest CDF and progressive dendritic targeting of variants lacking CDF. Calmodulin-triggered facilitation is thus fundamental to native Ca V 2.1 and rapidly enhanced during early development.

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Full-Length Single-Gene cDNA Libraries: Applications in Splice Variant Analysis

Cited by 13 publications

References 17 publications

Systematic Identification of Splice Variants in Human P/Q-Type Channel α₁2.1 Subunits: Implications for Current Density and Ca²⁺-Dependent Inactivation

Systematic Identification of Splice Variants in Human P/Q-Type Channel α₁2.1 Subunits: Implications for Current Density and Ca²⁺-Dependent Inactivation

Construction of a cDNA Library from the Ephemeral Plant Olimarabidopsis pumila and Preliminary Analysis of Expressed Sequence Tags

Developmental Activation of Calmodulin-Dependent Facilitation of Cerebellar P-Type Ca²⁺Current

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Full-Length Single-Gene cDNA Libraries: Applications in Splice Variant Analysis

Cited by 13 publications

References 17 publications

Systematic Identification of Splice Variants in Human P/Q-Type Channel α12.1 Subunits: Implications for Current Density and Ca2+-Dependent Inactivation

Systematic Identification of Splice Variants in Human P/Q-Type Channel α12.1 Subunits: Implications for Current Density and Ca2+-Dependent Inactivation

Construction of a cDNA Library from the Ephemeral Plant Olimarabidopsis pumila and Preliminary Analysis of Expressed Sequence Tags

Developmental Activation of Calmodulin-Dependent Facilitation of Cerebellar P-Type Ca2+Current

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Systematic Identification of Splice Variants in Human P/Q-Type Channel α₁2.1 Subunits: Implications for Current Density and Ca²⁺-Dependent Inactivation

Systematic Identification of Splice Variants in Human P/Q-Type Channel α₁2.1 Subunits: Implications for Current Density and Ca²⁺-Dependent Inactivation

Developmental Activation of Calmodulin-Dependent Facilitation of Cerebellar P-Type Ca²⁺Current