Characterization of a novel reptilian tolloid-like gene in the pond turtle, Pseudemys scripta elegans

Sabirzhanov, Boris; Keifer, Joyce; Clark, Timothy G.

doi:10.1016/j.brainres.2007.04.003

Cited by 5 publications

(10 citation statements)

References 23 publications

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“…The sense sequence was 5′-GGACUGACGAGGAGAGCUUCAUUGT-3′ while the antisense sequence was 5′-ACAAUGAAGCUCUCCUCGUCAGUCCUC-3′. The siRNA for tTLL was designed to bind with both transcripts of tTLL, tTLLs and tTLLc, at sites 575–599 and 686–710 bp, respectively, which are the same for both sequences (Sabirzhanov et al, 2007). Oligonucleotides containing the siRNA sequence were synthesized and purified by Integrated DNA Technologies.…”

Section: Methodsmentioning

confidence: 99%

“…RNA was combined with primer/probe sets and TaqMan Gold RT-PCR Master Mix (Applied Biosystems, Inc., Foster City, CA). Gene-specific primers and probes were created for P. scripta elegans to total tTLL, both splice variants tTLLs and tTLLc, and turtle actin, using the Primer Express Software (Applied Biosystems) and these are shown in Table 1 (Sabirzhanov et al, 2007). Real-time assays were run on an ABI PRISM 7000 analyzer (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we characterized a reptilian orthologue of the tolloid gene family in pond turtles, tTll (turtle tolloid-like gene; Sabirzhanov et al, 2007). Comparison of the amino acid sequence of tTLL revealed it most closely resembles mammalian BMP-1 in its domain architecture and mTLL2 in its sequence.…”

Section: Introductionmentioning

confidence: 99%

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Cleavage of proBDNF to BDNF by a Tolloid-Like Metalloproteinase Is Required for Acquisition ofIn VitroEyeblink Classical Conditioning

Keifer

Sabirzhanov²,

Zheng³

et al. 2009

J. Neurosci.

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The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1-and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cleavage of proBDNF to BDNF by a Tolloid-Like Metalloproteinase Is Required for Acquisition ofIn VitroEyeblink Classical Conditioning

Keifer

Sabirzhanov²,

Zheng³

et al. 2009

J. Neurosci.

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“…We normalized data to actin mRNA levels. For quantitation of gene expression, we used the comparative C T method (DDC T ; Livak and Schmittgen 2001;Sabirzhanov et al 2007). We performed all reactions in duplicate and used average values from the two samples in subsequent analyses.…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

Seasonal Variation of Myostatin Gene Expression in Pectoralis Muscle of House Sparrows (Passer domesticus) Is Consistent with a Role in Regulating Thermogenic Capacity and Cold Tolerance

Swanson

Sabirzhanov²,

VandeZande³

et al. 2009

Physiological and Biochemical Zoology

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Winter acclimatization in small birds overwintering in cold climates, including house sparrows (Passer domesticus), is associated with improved cold tolerance, elevated summit metabolic rates (M(sum) = maximum cold-induced metabolic rate), and increased pectoralis muscle mass compared to summer birds. Myostatin is a potent autocrine/paracrine inhibitor of skeletal muscle growth in mammals and birds and is a potential candidate for regulation of seasonal phenotypic flexibility in birds. As a first step toward examining such a role for myostatin in small birds, we measured summer and winter gene expression of myostatin and its potential metalloproteinase activators TLL-1 and TLL-2 in house sparrows from southeastern South Dakota. Gene expression of myostatin decreased significantly in winter, with summer values exceeding winter values by 1.52-fold. Moreover, gene expression of TLL-1 was also significantly reduced in winter, with summer values exceeding winter values by 1.55-fold. These data are consistent with the hypothesis that the winter increases in pectoralis muscle mass, M(sum), and cold tolerance in house sparrows are mediated by reduced levels of myostatin and its activator TLL-1, and they suggest the possibility that myostatin may be a common mediator of phenotypic flexibility of muscle mass in birds.

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“…Several dsRNAs were previously designed against the pond turtle, P. scripta elegans , tGAPDH mRNA (GenBank accession: FJ514828; Sabirzhanov et al 2007). In the present analysis, six primers specific to these dsRNA sequences and which suit 3′ RACE requirements were synthesized (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

2011

Self Cite

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RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose an approach for primary screening dsRNAs for potential off-target effects by using rapid amplification of cDNA ends. This method can be especially useful for model systems using species that have limited availability of sequence data.

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Characterization of a novel reptilian tolloid-like gene in the pond turtle, Pseudemys scripta elegans

Cited by 5 publications

References 23 publications

Cleavage of proBDNF to BDNF by a Tolloid-Like Metalloproteinase Is Required for Acquisition ofIn VitroEyeblink Classical Conditioning

Cleavage of proBDNF to BDNF by a Tolloid-Like Metalloproteinase Is Required for Acquisition ofIn VitroEyeblink Classical Conditioning

Seasonal Variation of Myostatin Gene Expression in Pectoralis Muscle of House Sparrows (Passer domesticus) Is Consistent with a Role in Regulating Thermogenic Capacity and Cold Tolerance

Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

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