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Results for “Functional characterization”

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PubMed2026

Identification of the BrSK gene family in flowering Chinese cabbage and functional characterization of BrSK2 subfamily involvement in heat stress.

Glycogen synthase kinase 3 (GSK3) kinases are evolutionarily conserved regulators of plant development and stress signaling, yet their contributions to thermotolerance in cool-adapted Brassica crops remain poorly understood. Here, we identified 16 BrSK genes in the Caixin (Brassica rapa ssp. chinensis var. parachinensis) genome, all harboring intact catalytic motifs indicative of functional kinase activity. Spatiotemporal expression profiling revealed preferential accumulation of BrSK transcripts in stem apices and floral organs during reproductive transition, while promoter analysis identified abundant heat- and abiotic stress-responsive cis-elements. Under heat stress, BrSK21, BrSK22, and BrSK23 displayed striking genotype-specific expression dynamics. BrSK21/22/23 transcripts were stably suppressed in the heat-tolerant cultivar '49-19' but transiently declined before rapidly rebounding in the heat-sensitive 'Liuye 50', mirroring RNA-seq profiles. Protein-protein interaction assays (Y2H, BiFC, and LCI) demonstrated specific associations between BrSK kinases and BrHSFA1. Functional validation via VIGS revealed that silencing of BrSK21 significantly enhanced thermotolerance, with triple silencing of BrSK21/22/23 conferring additive protection, indicating functional redundancy within the BrSK2 subfamily. Collectively, these findings establish the BrSK2 subfamily as negative regulators of heat tolerance in Caixin, likely via modulation of BrHSFA1 expression. This work identifies high-priority targets for molecular breeding of climate-resilient Brassica vegetables.

Plant Proteins↗

PubMed2000

DNA sequence and functional characterization of the human and rat epidermal growth factor promoter: regulation by cell growth.

Epidermal growth factor (EGF) regulates cell growth and differentiation through intracellular transduction networks activated by its tyrosine kinase receptor, EGFR. In this report we describe the structure and DNA sequence of transcriptional control regions from both human and Wistar-rat single copy EGF genes and their functional analysis in epithelial cell cultures. By sequence comparison we show these proximal gene regions have remained conserved in evolution to -640 (relative to the rodent mRNA initiation site), where similarity is interrupted by a rodent interspersed-repeat element (SINE). Transcript mapping reveals complexity in EGF initiation site selection: whereas a single rat liver initiation site (+1) appears 30bp 3' to the TTTAA element, an additional upstream site is detected in kidney RNA at -14. In contrast, in human RNA a single initiation is observed, which is displaced 12bp 3' to the rodent RNA terminus. Both promoters were defined in transient expression assays. Our results show the human promoter to be at least 20-fold more active than the equivalent rodent sequence, although both are activated during cell proliferation and negatively regulated in contact inhibited and quiescent cultures. The results indicate EGF gene expression and cell division are temporally linked, suggesting its promoter comprises a growth responsive regulatory domain.

3T3 Cells↗

PubMed1999

Overexpression and functional characterization of kinin receptors reveal subtype-specific phosphorylation.

G protein-coupled receptors such as the receptors for bradykinin are present in low copy numbers in most natural cells. To overcome the problems associated with the analysis of these receptors at the protein level, we used highly efficient expression systems such as the baculovirus/insect cell system. However, the structural and functional statuses of recombinant receptors have often remained elusive. We have expressed the two types of human kinin receptors, B1 and B2, in Sf9 cells. Both receptors are found on the surface of infected cells where they display the same pharmacological profiles as their cognate receptors of native cells. The functional analysis of kinin receptors coupled to the intracellular signaling pathways of Sf9 cells revealed differential patterns of ligand-induced phosphorylation for the two kinin receptors. The B1 receptor failed to undergo ligand-induced phosphorylation. However the B2 receptor showed selective phosphorylation of a minor 38 kDa band and lack of phosphorylation of a dominant 33 kDa band, indicating that only a fraction of the receptor protein is functionally linked to the kinase pathway. A striking discrepancy between the number of binding sites and the amount of receptor protein per cell (molar ratio of 1:20 to 1:1000) indicated that a significant portion of kinin receptors is associated with the intracellular compartments of Sf9 cells. Pulse-chase and immunoprecipitation experiments demonstrated that the heterogeneity of recombinant receptors is not due to proteolytic processing but likely reflects incomplete or lacking N-glycosylation. We conclude that the baculovirus/Sf9 system is suitable for the recombinant expression and functional analysis of kinin receptors though limitations of the system have to be considered.

Animals↗

PubMed2001

Functional characterization of a human receptor for neuropeptide FF and related peptides.

1. Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2. Binding experiments were performed with a new radioiodinated probe, [(125)I]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [(125)I]-EYF with a K(d) of 0.06 nM. Various NPFF analogues and related peptides inhibited [(125)I]-EYF specific binding with the following rank order (K(i)): human NPAF (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF (0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH(2) (3.25 nM), FMRF-NH(2) (10.5 nM) and NPSF (12.1 nM). 3. The stimulatory activity of the same set of peptides was measured by a functional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequorin. The rank order of potency was consistent with the results of the binding assay. 4. Membranes from NPFFR expressing CHO cells bound GTP gamma[(35)S] in the presence of SQA-NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G(i) family members. 5. SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6. RT -- PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

Aequorin↗

PubMed2000

Heterologous expression, functional characterization and localization of two isoforms of the monkey iron transporter Nramp2.

Natural resistance-associated macrophage protein 2 (Nramp2) has been suggested to be involved in transferrin-independent iron uptake. Two isoforms of the Nramp2 gene generated by alternative splicing of the 3' exons were identified in mouse, rat and human, but it is unclear if they perform distinct functions. To rationalize our previous work, which indicated an increase in iron deposition in a Parkinsonian monkey brain, two monkey Nramp2 isoforms were isolated for a comparative study to assess their relative iron-uptake abilities, tissue distribution and subcellular localization. The monkey Nramp2 isoforms, 2a and 2b, exhibit approx. 98% identity at the amino acid level when compared with the human homologues. The Nramp2a transcript contains a canonical iron-responsive element (IRE), whereas that of Nramp2b lacks the IRE motif in the 3' untranslated region. By reverse transcriptase (RT)-PCR, the mRNAs of both isoforms were detected in all tissues examined. The amino acid differences at the C-terminus neither affected the protein expression levels in HEK-293T and COS-7 cells nor altered the subcellular localization and tissue distribution of the isoforms. Similar levels of iron uptake were detected in the HEK-293T cells transfected with either the Nramp2a or 2b gene, and a reduction of iron from the ferric (Fe(3+)) to the ferrous (Fe(2+)) state is necessary before transport can take place. However, this transferrin-independent uptake of iron into the cells is not a Ca(2+)-dependent process.

3' Untranslated Regions↗

PubMed1997

Diversity of the third form of complement, C3, in fish: functional characterization of five forms of C3 in the diploid fish Sparus aurata.

We have recently shown that Sparus aurata, the gilthead sea bream (a diploid species), similarly to rainbow trout (a quasi-tetraploid species), possesses multiple forms of the third form of complement (C3). In the present study we have evaluated the ability of the gilthead sea bream proteins to function as active C3 molecules. All five C3 isoforms could be fixed covalently to sheep erythrocyte ghosts and were able to bind to various complement-activating surfaces in the presence of MgEGTA. In the absence of MgEGTA their binding capacities generally increased, presumably as a result of classical-pathway activation by the natural antibodies present in the serum. The presence of EDTA abrogated the binding of all C3 isoforms to the various surfaces tested. The C3 isoforms differed in the efficiency of their binding to complement-activating surfaces: the two most abundant C3 isoforms (C3-1 and C3-2) bound to zymosan as well as to sheep and rabbit erythrocyte ghosts, whereas C3-3, C3-4 and C3-5 were unable to bind to zymosan. Upon complement activation, all five C3 isoforms were cleaved to 'iC3b' by factor H and I-like proteins, generating fragments similar to those generated from C3 molecules of other species. Furthermore the degradation of methylamine-hydrolysed C3 isoforms to iC3b was significantly inhibited by EDTA. The structural and functional diversity that we have observed in the C3 isoforms of S. aurata would increase the capacity of this fish to recognize a broader spectrum of potential pathogens and reinforce a specific immune response, which in fish is delayed compared with that of higher vertebrates, and is based on a single Ig type (IgM).

Animals↗

PubMed2005

A rat model for functional characterization of pregnancy-induced denervation and postpartum reinnervation in the myometrium and cervix: a superfusion study.

The pregnancy-induced rapid degeneration of the adrenergic nerves innervating the uterus is a well-known but poorly understood phenomenon. Since most of the published investigations were carried out by histological assay, our aim was to describe the loss of the adrenergic function during pregnancy and the re-innervational procedure in the postpartum period. Myometrial and cervical samples from rats were loaded with [3H]noradrenaline and then transferred into a chamber for superfusion. After a wash-out period, fractions were collected. The fifth and fifteenth fraction tissues were stimulated with an electric field. The [3H]noradrenaline contents of the fractions were determined, together with the amount remaining in the tissue. The myometrial [3H]noradrenaline release was substantially decreased in early pregnancy, and absent in the late stage. Differences in release profile were detected between the implantation sites and the interimplantation areas. As a refinement of the results of previous histochemical studies, the noradrenergic functions of the cervix were found to be deeply affected in the early postpartum period. The pregnancy-induced denervational procedure can be followed by means of a superfusional technique after [3H]noradrenaline loading. As our technique is considered to be similar in sensitivity to histological methods, superfusion can be regarded as a model for functional investigations of pregnancy-induced denervation.

Animals↗

PubMed1998

Functional characterization of the human CYP1A1 negative regulatory element: modulation of Ah receptor mediated transcriptional activity.

The mechanisms that underly the regulation of human CYP1A1 have merited considerable attention because of their association both with toxic outcomes and the etiology of several cancers. Previous work conducted in this laboratory has identified a negative regulatory element (NRE) in the 5' region of this gene that appeared to modulate CYP1A1 transcriptional activity. This NRE is present in two functional copies, a high affinity 21-bp palindrome centered at position -784, and an additional element found within a GC-rich region between position -728 and -558. In this report, the regulatory function of the NREs in the context of the CYP1A1 promoter was evaluated. This was accomplished by substituting mutated elements for the corresponding wild-type element in a vector that contained human CYP1A1 sequences positions -1140 to +59 directing the transcription of the chloramphenicol acetyltransferase reporter gene. Expression vectors containing specific mutations in each or both NREs were characterized. We show that eliminating the binding of the CYP1A1 repressor protein to one or both repressor motifs results in a significant 2- to 3-fold increase in the inducibility of CYP1A1 promoter activity. Although mutation of both sites appeared to result in an increase in inducibility over that observed with only one site mutated, the effect was not additive. Such aberrant transcriptional activity correlates with the highly inducible aryl hydrocarbon hydroxylase phenotype that is a reported marker for individuals predisposed to lung cancer. Mutation of the NRE, or more likely, the cognate repressor protein(s), may provide a genetic basis for this phenotype.

Base Sequence↗

PubMed2001

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Amino Acid Motifs↗

PubMed2003

Molecular characterization, functional expression, and developmental profile of an ether à-go-go K+ channel in the tobacco hornworm Manduca sexta.

A very large number of evolutionarily conserved potassium channels have been identified but very little is known about their function or modulation in vivo. Metamorphosis of the tobacco hornworm, Manduca sexta, is a compelling model system for such studies because it permits analysis to be conducted at the level of identified neurons whose roles in simple behaviors and endocrine regulation are known. We present here the characterization of the first ion channel to be cloned from this animal. Partial genomic sequence for Manduca sexta ether à-go-go (Mseag) and a cDNA clone encoding the Mseag open reading frame were obtained. Genomic Southern analysis indicates that Manduca contains a single member of the eag subfamily per haploid genome. When expressed in Xenopus oocytes, MsEag channels conduct a voltage-dependent, K+ selective outward current with an inactivating component that closely resembles the Drosophila eag current. Mseag transcripts were restricted to the nervous system, adult antenna, and one set of larval skeletal muscles. Steroid hormonal regulation of Mseag expression is suggested by the temporal correlation of developmental changes in transcript expression with the changing steroid titers that promote metamorphosis. These results provide the foundation for functional and modulatory studies of the Eag family of K+ channels in Manduca, which will complement the genetic analysis in Drosophila.

Amino Acid Sequence↗

PubMed2004

Functional characterization of SIX3 homeodomain mutations in holoprosencephaly: interaction with the nuclear receptor NR4A3/NOR1.

Holoprosencephaly (HPE) is a relatively common brain malformation resulting in an incomplete separation of the two cerebral hemispheres. A number of mutations in different genes have been linked to this malformation, including three missense mutations in the homeodomain of the transcription factor SIX3. In this study, we investigated the functional consequences of these SIX3 mutations with respect to the ability of the protein to interact with and stimulate the transcriptional activity of the nuclear receptor NOR1 (NR4A3). Using glutathione S-transferase fusion protein pull-down assays and transient cotransfections of Neuro-2a cells with expression and reporter vectors, we found that one mutation, c.676C>G (p.L226V), does not alter the properties of SIX3 toward NOR1. Another mutation, c.749T>C (p.V250A), results in the production of a highly unstable protein in Neuro-2a cells. The third mutation, c.770G>C (p.R257P), results in a mutant SIX3 protein that no longer interacts with NOR1 in vivo. These observations suggest that different SIX3 mutations in HPE2 may affect different signaling pathways, and that one of these pathways may involve the nuclear receptor NOR1.

Cell Line↗

PubMed2001

Structural and functional characterization of the Zn(II) site in dimethylargininase-1 (DDAH-1) from bovine brain. Zn(II) release activates DDAH-1.

L-N(omega),N(omega)-dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-containing enzyme that, through hydrolysis of side-chain methylated l-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concentrations of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenantroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissociation constant of 4.2 nm, determined by 19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid), lies in the range of intracellular free Zn(II) concentrations. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examined by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure observed is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biological implications of these findings are discussed.

Amidohydrolases↗

PubMed2003

Functional characterization of the Arabidopsis thaliana orthologue of Tsc13p, the enoyl reductase of the yeast microsomal fatty acid elongating system.

The protein encoded by the Arabidopsis At3g55360 gene was selected as a candidate for the enoyl reductase of the microsomal elongase system based on its homology to the Tsc13p protein of S. cerevisiae. The studies presented here demonstrate that heterologous expression of At3g55360 functionally complements the temperature-sensitive phenotype of a yeast tsc13 mutant that is deficient in enoyl reductase activity. Furthermore, AtTSC13 is shown to interact physically with the Elo2p and Elo3p components of the yeast elongase complex. At3g55360 apparently encodes the sole enoyl reductase activity associated with microsomal fatty acid elongation in Arabidopsis. Consistent with this conclusion, AtTSC13 is ubiquitously expressed in Arabidopsis.

Arabidopsis↗

PubMed2006

Identification and functional characterization of three novel human melanocortin-4 receptor gene variants in an obese Chinese population.

OBJECTIVE: Mutations in the melanocortin-4 receptor gene (MC4R) are the most common monogenic form of human obesity. However, the contribution of MC4R mutations to obesity in Chinese has not been investigated. We studied the frequency of MC4R mutations in an obese southern Chinese population and the functional consequences of the novel variants identified. METHODS: We screened for MC4R mutations in 227 obese [body mass index (BMI) 35.29 +/- 5.75 kg/m2] and 100 lean (BMI 21.57 +/- 0.29 kg/m2) southern Chinese subjects using PCR-direct sequencing. In vitro functional studies, including cell surface expression, ligand binding, and cyclic adenosine monophosphate (cAMP) accumulation, were performed to examine the functional properties of three novel missense mutations. RESULTS: Apart from two previously reported polymorphisms, V103I and -176 A > C, three novel missense heterozygous variants (Y35C, C40R and M218T) were identified. The polymorphisms -176 A > C and Y35C were detected in both obese and normal subjects with similar frequency. C40R was identified only in an obese subject. Pedigree analysis revealed M218T carriers in both lean and obese subjects. The prevalence of V103I carriers in normal-weight controls was significantly higher than that in obese subjects (5.3%vs. 1.3%, P < 0.05). In vitro functional studies showed that all three novel missense variants have normal functions. CONCLUSIONS: Two known polymorphisms and three novel variants of the MC4R were identified. No overt functional defects were observed for the three novel MC4R variants, suggesting that they might not be the cause of obesity in variant carriers.

Adult↗

PubMed1993

Functional characterization of a 5-HT3 receptor which modulates the release of 5-HT in the guinea-pig brain.

1. The aims of the present study were to confirm the modulation by 5-HT3 receptors of the electrically evoked release of tritium from slices preloaded with [3H]-5-HT of guinea-pig frontal cortex, hippocampus and hypothalamus, and to assess their functional role in 5-HT release. 2. The selective 5-HT3 agonist, 2-methyl-5-HT, introduced 8 min before the electrical stimulation, enhanced in a concentration-dependent manner the evoked release of [3H]-5-HT in the three brain regions studied. The 5-HT3 agonists, phenylbiguanide and m-chlorophenyl-biguanide, did not enhance the release of tritium in frontal cortex and hypothalamus slices. 3. In hypothalamus slices, this response was lost when 2-methyl-5-HT was introduced 20 min before the stimulation, thus indicating that these 5-HT3 receptors desensitize rapidly. When 2-methyl-5-HT was added 20-min before the first stimulation period to desensitize the 5-HT3 receptors, removed for 24 min, and then re-introduced 8 min before the second stimulation period, the enhancing effect of 2-methyl-5-HT was restored, thus indicating that these 5-HT3 receptors can rapidly regain normal sensitivity. 4. The enhancing effect of 2-methyl-5-HT was attenuated by the 5-HT3 receptor antagonists m-chloro-phenylpiperazine = quipazine = ondansetron > or = ICS 205-930 = BRL 24924 > MDL 72222 = zacopride. 5. The 5-HT reuptake blocker, paroxetine, enhanced the electrically evoked release of tritium when introduced 8 min before stimulation; this effect of paroxetine was blocked by ICS 205-930, thus indicating that these 5-HT3 receptors can be activated by endogenous 5-HT. 6. In the absence of electrical stimulation, 2-methyl-5-HT (10 microM) produced a marked enhancement of the basal release of [3H]-5-HT which was calcium-dependent and blocked by S-zacopride but not by paroxetine. 7. The enhancing effect of 2-methyl-5-HT was dependent both on the frequency of stimulation, as indicated by the attenuated effect of 120 stimulations delivered at 1 Hz instead of 5 Hz, and on the duration of the stimulation, as indicated by the more pronounced effect of pulses delivered at 5 Hz for 24 s instead of 72 s or 120 s.

Animals↗

PubMed1989

Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris.

In Pichia pastoris, alcohol oxidase (AOX) is the first enzyme in the methanol utilization pathway and is encoded by two genes, AOX1 and AOX2. The DNA and predicted amino acid sequences of the protein-coding portions of the genes are closely homologous, whereas flanking sequences share no homology. The functional roles of AOX1 and AOX2 in the metabolism of methanol were examined. Studies of strains with disrupted AOX genes revealed that AOX1 was the major source of methanol-oxidizing activity in methanol-grown P. pastoris. The results of two types of experiments each suggested that the difference in AOX activity contributed by the two genes was a consequence of sequences located 5' of the protein-coding portions of the genes. First, the coding portion of AOX2 was able to functionally substitute for that of AOX1 when placed under the control of AOX1 regulatory sequences. Second, when labeled oligonucleotide probes specific for the 5' nontranslated region of each gene were used, it was apparent that the steady-state level of AOX1 mRNA was much higher than that of AOX2. Except for the difference in the amount of mRNA present, the two genes appeared to be regulated in the same manner. A physiological reason for the existence of AOX2 was sought but was not apparent.

Alcohol Oxidoreductases↗

PubMed2002

Alternative splicing yields novel BMAL2 variants: tissue distribution and functional characterization.

The BMAL2 gene encodes a member of the basic helix-loop-helix PER-ARNT-SIM family of transcription factors, which control diverse physiological processes including circadian rhythms. We identified four novel human BMAL2 transcripts that differ by alternative splicing within their NH2-terminal regions. Divergent expression of these and previously reported transcripts was observed among human tissues. The functional consequences of alternative splicing for transcriptional activation by CLOCK:BMAL2 heterodimers were assessed using luciferase reporter gene constructs that contained one of three diurnally regulated promoters, namely, those of the mouse period1, mouse vasopressin, and human plasminogen activator inhibitor-1 genes. These studies revealed that alternative splicing generates BMAL2 isoforms possessing high, medium, low, or no transcriptional activity. Similar results were obtained with each promoter, suggesting that alternative splicing may influence the amplitudes of both central and peripheral oscillators. Indeed, alternative splicing of BMAL2 may provide tissues with a rheostat capable of regulating CLOCK:BMAL2 heterodimer function across a broad continuum of potential transcriptional activities to accommodate varied metabolic demands and physiological roles.

ARNTL Transcription Factors↗

PubMed2026

[Genetic and functional characterization of a novel KIT splicing variant in a Chinese three-generation pedigree with piebaldism].

OBJECTIVES: To investigate the genetic etiology of a three-generation pedigree affected with piebaldism. METHODS: Next-generation sequencing and Sanger sequencing were employed to detect and verify gene variants. Bioinformatics tools were used to predict the effects of candidate variants on splicing and protein function. RT-PCR and Sanger sequencing were further performed to validate the impact of the variant on RNA splicing, and homology modeling was applied to predict its effect on the three-dimensional structure of the KIT protein. The pathogenicity of the variant was then classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the UK Association for Clinical Genomic Science (ACGS). RESULTS: A heterozygous insertion variant near the splice site, c.1990+8_1990+9insTGCACCATTGGAGGTAAA, was identified in the KIT gene in the proband and was found to co-segregate with the phenotype within the family. RT-PCR and cDNA sequencing revealed that this variant led to aberrant splicing during transcription, resulting in a 21 bp in-frame insertion in the mRNA, which encodes an extra 7 amino acids within the tyrosine kinase domain and may thus affect protein function. In silico predictions, together with the experimental findings, supported classification of this variant as likely pathogenic according to relevant variant interpretation guidelines. CONCLUSIONS: The heterozygous splice-site insertion variant KIT:c.1990+8_1990+9insTGCACCATTGGAGGTAAA is the genetic cause of piebaldism in this pedigree.

Genetics diagnosis↗