Reverse Transcription-polymerase Chain Reaction Strategy Research Articles

The Effect of Correlation of Laboratory-Developed Test and Initial Symptoms and False Negatives in RT-PCR Strategies for COVID-19 Patients with Beta Variants

Abstract Background Reverse transcription-polymerase chain reaction (RT-PCR) assays detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The number of viruses in the sample varies between patients; it depends on sample location, nasal or throat, and with time infection spreads. Previous studies showed that the viral load of coronavirus disease 2019 (COVID-19) infection is the peak just before symptoms onset. Furthermore, positive and negative results depend on test site, sampling, and timing method; RT-PCR can be 1 to 30% false-negative result. Methods and Materials Within this study, we took RT-PCR test from COVID-19 positive patients who already had the confirmation of the disease either by lung computed tomography (CT)-scan or the symptoms such as dyspnea. The study was explained to all the patients, and they confirmed to take the RT-PCR test. Negative samples from those patients were retested, and if the result came back negative, we included them as negative in the result. Result A total number of 49 patients (25 females) and (24 males) with a mean age of 53.24 years (ranging from 32 to 77) were enrolled. About 32.3% of patients, despite having COVID-19 disease, had a negative RT-PCR test. There is a positive and significant relationship between weight (r = 0.253) and CT at the time of hospitalization of COVID-19 patients and a negative and significant relationship with O2 saturation without oxygen therapy (r = − 0.296), the model can predict 67.7% of the disease due to the beta value, and the share of O2 saturation without oxygen therapy is more than weight. Conclusion We show that a pragmatic model can be designed to predict which patients have a higher chance of getting false-negative result, and should be retested for COVID-19. Among the variables, weight had a negative and significant relationship, and O2 saturation without respiratory support had a negative and significant relationship with COVID-19 disease.

Evaluation of COVID-19 Testing Strategies for Repopulating College and University Campuses: A Decision Tree Analysis

PurposeThe optimal approach to identify SARS-CoV-2 infection among college students returning to campus is unknown. Recommendations vary from no testing to two tests per student. This research determined the strategy that optimizes the number of true positives and negatives detected and reverse transcription polymerase chain reaction (RT-PCR) tests needed. MethodsA decision tree analysis evaluated five strategies: (1) classifying students with symptoms as having COVID-19, (2) RT-PCR testing for symptomatic students, (3) RT-PCR testing for all students, (4) RT-PCR testing for all students and retesting symptomatic students with a negative first test, and (5) RT-PCR testing for all students and retesting all students with a negative first test. The number of true positives, true negatives, RT-PCR tests, and RT-PCR tests per true positive (TTP) was calculated. ResultsStrategy 5 detected the most true positives but also required the most tests. The percentage of correctly identified infections was 40.6%, 29.0%, 53.7%, 72.5%, and 86.9% for Strategies 1–5, respectively. All RT-PCR strategies detected more true negatives than the symptom-only strategy. Analysis of TTP demonstrated that the repeat RT-PCR strategies weakly dominated the single RT-PCR strategy and that the thresholds for more intensive RT-PCR testing decreased as the prevalence of infection increased. ConclusionBased on TTP, the single RT-PCR strategy is never preferred. If the cost of RT-PCR testing is of concern, a staged approach involving initial testing of all returning students followed by a repeat testing decision based on the measured prevalence of infection might be considered.

Investigation of persistent infection of bovine viral diarrhea virus (BVDV) in Holstein dairy cows.

The aim of this study was to investigate the persistent infection (PI) of bovine viral diarrhea virus (BVDV) along with its coexistence between BVDV antibody titer and BVD virus in blood of Holstein dairy cows. Only large commercial farms (each contained < 1000-3000 unvaccinated cows) were included. There were 11 dairy cattle herds. They included nearly 20,000 dairy cows. Totally, 140 cows, > 3months to almost 10years old, were randomly sampled. Indirect enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect BVDV antibody and virus, respectively. The percent positivity (PP) < 14 and ≥ 14 values are interpreted negative and positive, respectively. Simultaneously, whole blood samples pooled in groups of 10 animals were used for molecular detection of BVDV. The results revealed that 138 (98.56%) out of 140 cows were positive for BVDV antibody, while the BVDV antigen was detected only in 2 (1.42%) cows, which were negative for BVDV antibody and so were considered as persistent infection (PI) cows. They were also retested 3weeks apart. Since the results showed the strong coexistence between seropositivity and BVD virus, in the infected dairy cattle herds, the combination of simple ELISA and pooled whole blood RT-PCR strategy could be an achievable approach to detect PI animals.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

Xenobiotic Metabolism and Disposition in Human Lung Cell Models: Comparison with In Vivo Expression Profiles

Numerous lung cell lines are currently used as in vitro models for pharmacological and toxicological studies. However, no exhaustive report about the metabolic capacities of these models in comparison with those of lung tissues is available. In the present study, we used a high-throughput quantitative real-time reverse transcription-polymerase chain reaction strategy to characterize the expression profiles of 380 genes encoding proteins involved in the metabolism and disposition of xenobiotics in 10 commonly used lung cell lines (A549, H292, H358, H460, H727, Calu-1, 16HBE, 1 HAEO, BEAS-2B, and L-132) and four primary cultures of human bronchial epithelial cells. Expression results were then compared with those previously obtained in human nontumoral and tumoral lung tissues. Our results revealed disparities in gene expression between lung cell lines or when comparing lung cell lines with primary cells or lung tissues. Primary cell cultures displayed the highest similarities with bronchial mucosa in terms of transcript profiling and therefore seem to be the most relevant in vitro model for investigating the metabolism and bioactivation of toxicants and drugs in bronchial epithelium. H292 and BEAS-2B cell lines, which exhibited the highest homology in gene expression pattern with primary cells and the lowest number of dysregulated genes compared with nontumoral lung tissues, could be used as surrogates for toxicological and pharmacological studies. Overall, our study should provide references for researchers to choose the most appropriate in vitro model for analyzing the cellular effects of drugs or airborne toxicants on the airway.

Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR

BackgroundBovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV). One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR) assay based on SYBR Green I dye has not been established for BVDV detection. This study aims to develop a quantitative one-step RT-PCR assay to detect BVDV type-1 in cell culture.ResultsOne-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and using in vitro transcribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation coefficient (R2) of 0.995, and a maximum intra-assay CV of 2.63%. It was 10-fold more sensitive than conventional RT-PCR and can quantitatively detect BVDV RNA levels from 10-fold serial dilutions of titrated viruses containing a titer from 10-1 to 10-5 TCID50, without non-specific amplification. Melting curve analysis showed no primer-dimers and non-specific products.ConclusionsThe one-step SYBR Green I RT-PCR is specific, sensitive and reproducible for the quantification of BVDV in cell culture. This one-step SYBR Green I RT-PCR strategy may be further optimized as a reliable assay for diagnosing and monitoring BVDV infection in animals. It may also be applied to evaluate candidate agents against HCV using BVDV cell culture model.

Molecular Characterization and Expression Pattern of Two General Odorant Binding Proteins from the Diamondback Moth, Plutella xylostella

In the Lepidoptera, odorant signals are thought to be mediated by general odorant binding proteins (GOBPs) in the sensillar lymph surrounding the olfactory receptors. We describe the identification and characterization of two new cDNAs encoding GOBPs from the antennae of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a species for which no GOBPs have been identified to date. We focused our investigation on this olfactory protein family by using reverse transcription-polymerase chain reaction strategies. The deduced amino acid sequences of PxylGOBP1 and PxylGOBP2 revealed open reading frames of 168 and 163 amino acids, respectively, with six cysteine residues in conserved positions relative to other known GOBPs. The alignment of the mature PxylGOBPs with other Lepidoptera GOBPs showed high sequence identity (70-80%) with other full-length sequences from GenBank. Sequence identity between PxylGOBP1and PxylGOBP2 was only 50%, suggesting that the two proteins belong to different classes of lepidopteran GOBPs. The expression patterns of the two PxylGOBP genes, with respect to tissue distribution and sex, were further investigated by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Although the two GOBP genes were expressed only in the antennae of both sexes, reflecting the antennal specificity of GOBPs, the transcription levels of these genes depended on the sex, the age, the mating status, and the genes.

Serological Evaluation of Precolostral Serum Samples to Detect Bovine Viral Diarrhea Virus Infections in Large Commercial Dairy Herds

The authors propose that screening newborn calves for Bovine viral diarrhea virus (BVDV) antibody prior to colostrum feeding is a useful strategy to detect herds with endemic BVDV infection. In the current study, precolostral serum samples of newborn calves in 2 Minnesota and 2 California dairy farms were examined. Precolostral BVDV antibodies were detected by serum neutralization and enzyme-linked immunosorbent assay in 7.4% (33/446) and 6.2% (32/515) of newborn calves in the California and Minnesota herds, respectively. The serum samples were also tested by reverse transcription polymerase chain reaction (RT-PCR), and BVDV was detected in 1.6% (7/446) and 3.5% (18/515) of newborn calves in the California and Minnesota herds, respectively. The primary advantages of precolostral testing are that calves congenitally infected with BVDV and seropositive at birth represent a larger percentage of calves born than BVDV-viremic calves and that fewer animals would need to be tested with an antibody test than a RT-PCR or antigen detection test to detect endemic BVDV infections at the herd level. Testing for BVDV antibody in calves prior to colostrum feeding detects fetal infections in both late-gestating cows and nonlactating heifers. Precolostral serum antibody detection is not confounded by vaccination and may be a more sensitive screening method than bulk milk RT-PCR and nonvaccinated sentinel calf strategies in large dairy herds.

Molecular Cloning and in Situ Expression Patterns of Two New Pheromone-Binding Proteins from the Corn Stemborer Sesamia nonagrioides

We describe the identification and characterization of two new cDNAs encoding pheromone-binding proteins (PBPs) from the male antennae of Sesamia nonagrioides, a species where no PBPs have been identified to date. Because PBPs are thought to participate in the first step of odor detection in a specific manner, we focused our investigation on this olfactory protein family using reverse transcription-polymerase chain reaction strategies. The deduced amino acid sequences of SnonPBP1 and SnonPBP2 revealed mature proteins of 142 and 143 amino acids, respectively, with six cysteine residues in conserved positions relative to other known PBPs. The alignment of the two mature S. nonagrioides PBPs with other noctuid PBPs showed high sequence identity (70-80%) with other full-length sequences from GenBank. Sequence identity between SnonPBP1 and SnonPBP2 was only 46%, suggesting that the two proteins belong to different classes of PBPs already described from the Noctuidae. Furthermore, analyses of expression patterns of SnonPBP1 and SnonPBP2 were performed by in situ hybridization on antennae of both sexes, and these studies revealed the expression of the two PBPs at the bases of olfactory sensilla (basiconica or trichodea) from both sexes. The possible binding properties of these two new PBPs are discussed according to their homologies with other known PBPs and S. nonagrioides pheromone components.

Molecular cloning of several rat ABC transporters including a new ABC transporter, Abcb8, and their expression in rat testis

Several members of the ABC transporter superfamily play an important role in testicular physiology and defence against anticancer drugs. Using a reverse transcription-polymerase chain reaction strategy with degenerate primers and rat testis RNA as template, we have looked for the presence of other members of this superfamily. Of the six partial cDNA found, five corresponded to ABC transporters already known -Mdr1b, Mrp1, Tapl/Abcb9, Umat/Abcb6 and Sur2/Abcc9- and one presented a strong homology with mouse and human ABCB8. Using a 5' and 3' RACE approach, we cloned the full-length cDNA and found that the predicted protein presented 92% and 80% homology with the mouse and human proteins respectively. Strong expression of rat abcb8 was found in heart, brain and testis when compared with liver, lung and spleen. In the testis, rat abcb8 was expressed both in the somatic Sertoli cells and peritubular cells and in the germline (spermatogonia and pachytene spermatocytes). Furthermore, Umat/Abcb6 was very highly expressed in the testis (high amounts in meiotic pachytene spermatocytes and low amount in post-meiotic early spermatids). In conclusion, we confirm the presence of several ABC transporters in the testis and also provide evidence of the presence of Abcb8 in the organ.

Identification and characterization of six new alternatively spliced variants of the human μ opioid receptor gene, Oprm

The μ opioid receptor plays an important role in mediating the actions of morphine and morphine-like drugs. Receptor binding and a wide range of pharmacological studies have proposed several μ receptor subtypes, but only one μ opioid receptor ( Oprm) gene has been isolated. Like the mouse and rat, the human Oprm gene undergoes alternative splicing. In the present studies, we have identified and characterized six new splice variants from the human Oprm gene using a reverse transcription-polymerase chain reaction strategy, yielding a total of 10 human splice variants of the μ opioid receptor MOR-1. All the variants identified contained exons 1, 2 and 3, but differed from MOR-1 itself and each other by splicing downstream from exon 3, resulting in different amino acid sequences. Northern blot analysis demonstrated expression of the variant mRNAs. Receptor binding assays established that these variants belonged to the μ opioid receptor family with limited differences in μ opioid ligand affinities and selectivity. However, adenylyl cyclase and [ 35S]GTPγS binding assays revealed major differences in both potency and efficacy among these variants. The dissociation between binding affinity, potency and efficacy for the opioids among these variants may provide insights into the wide range of opioid responses among these agents observed clinically and opens new avenues in designing selective drugs based upon their efficacy and potency rather simple binding affinity.

Expression of messenger RNA for ADAMTS subtypes changes in the periovulatory follicle after the gonadotropin surge and during luteal development and regression in cattle.

Extensive remodeling of the extracellular matrix occurs in the ovary during the periovulatory period. Matrix metalloproteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases, are believed to play integral roles in this highly regulated series of cellular events, but their specific roles remain unclear. Recent cloning studies have identified a novel family of metalloproteinases, the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family. The regulated expression of distinct ADAMTS subtypes has been shown to be required for tissue morphogenesis during embryonic development and for maintaining the integrity of tissues in the adult. In the present studies, we have determined that multiple ADAMTS subtypes are present in the bovine ovary using a reverse transcription-polymerase chain reaction strategy. In particular, ADAMTS-1, -2, -3, -4, -5 (also known as ADAMTS-11), -7, -8, and -9, but not ADAMTS-6, -10, or -12, mRNA transcripts were detected in granulosa cells of nonatretic ovarian follicles and corpora lutea. The levels of mRNA for these ovarian ADAMTS were up- or down-regulated or remained unchanged in the granulosa and/or theca cells of the dominant follicle following the preovulatory surge of gonadotropins, depending on the subtype and/or the cell compartment, and in the corpus luteum during the luteal phase of the estrous cycle. The complex expression patterns observed for the distinct ADAMTS subtypes in the granulosa and theca cells of the periovulatory follicle and in the luteal tissues of the bovine ovary suggest that these novel proteases mediate, at least in part, the remodeling events underlying folliculogenesis and ovulation and the formation, maintenance, and regression of the corpus luteum.

The α 1A subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing

Calcium influx through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming α 1 subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel α 1A splice variants (α 1A-a, α 1A-b and α 1A-EFe) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have different timings of development throughout the brain: α 1A-b is expressed from embryonic to the adult stage, α 1A-EFe is restricted to the embryonic period whereas α 1A-a is expressed only postnatally. In situ hybridization indicated that α 1A-a and α 1A-b isoforms develop with different regional and cellular patterns. In hippocampus and cerebellum, α 1A-b represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the α 1A calcium channel properties during development.

Diverse chalcone synthase superfamily enzymes from the most primitive vascular plant, Psilotum nudum.

Psilotum nudum Griseb is a pteridophyte and belongs to the single family (Psilotaceae) of the division, Psilophyta. Being the only living species of a once populated division, P. nudum is the most primitive vascular plant. Chalcone synthase (CHS; EC 2.3.1.74) superfamily enzymes are responsible for biosyntheses of diverse secondary metabolites, including flavonoids and stilbenes. Using a reverse transcription-polymerase chain reaction strategy, four CHS-superfamily enzymes (PnJ, PnI, PnL and PnP) were cloned from P. nudum, and heterologously expressed in Escherichia coli. These four enzymes of 396-406 amino acids showed sequence identity of > 50% among themselves and to other higher-plant CHS-superfamily enzymes. PnJ and PnP preferred p-coumaroyl-CoA and isovaleryl-CoA respectively, as starter CoA and catalyzed CHS-type ring formation, indicating that they are CHS and phlorisovalerophenone synthase, respectively. On the other hand, PnI and PnL preferred cinnamoyl-CoA as starter CoA and catalyzed stilbene synthase-type cyclization and thus were determined to be pinosylvin synthases (EC 2.3.1.146). In addition, PnE, which uniquely contains a glutamine in place of otherwise strictly conserved histidine, had no apparent in vitro catalytic activity. Phylogenetic analysis indicated that these P. nudum clones form a separate cluster together with Equisetum arvense CHS. This cluster of pteridophytes is located next to the cluster formed by pine (gymnosperm) enzymes, in agreement with their evolutionary relationships. Psilotum nudum represents a plant with the most diverse CHS-superfamily enzymes and this ability to diverge may have provided a survival edge during evolution.

Structure and Expression of the TREX1 and TREX2 3′→5′ Exonuclease Genes

The TREX1 and TREX2 genes encode mammalian 3'-->5' exonucleases. Expression of the TREX genes in human cells was investigated using a reverse transcription-polymerase chain reaction strategy. Our results show that TREX1 and TREX2 are expressed in all tissues tested, providing direct evidence for the expression of these genes in human cells. Potential transcription start sites are identified for the TREX genes using rapid amplification of cDNA ends to recover the 5'-flanking regions of the TREX transcripts. The 5'-flanking sequences indicate transcription initiation from consensus putative promoters identified -140 and -650 base pairs upstream of the TREX1 open reading frame (ORF) and -623 and -753 base pairs upstream of the TREX2 ORF. Novel TREX1 and TREX2 cDNAs are identified that contain protein-coding sequences generated from exons positioned in genomic DNA up to 18 kilobases 5' to the TREX1 ORF and up to 25 kilobases 5' to the TREX2 ORF. These novel cDNAs and sequences in the GenBank data base indicate that transcripts containing the TREX1 and TREX2 ORFs are produced using a variety of mechanisms that include alternate promoter usage, alternative splicing, and varied sites for 3' cleavage and polyadenylation. These initial studies have revealed previously unrecognized complexities in the structure and expression of the TREX1 and TREX2 genes.

Developmental profile of Sry transcripts in mouse brain.

Transient activation of the gene Sry in the gonadal ridge during a brief period of embryonic development is believed to function as a key signal for sex determination. However, a number of reports suggest that Sry expression is not as restricted in space and time as one would expect if its role was confined to directing male-specific differentiation in the early gonadal anlage. We have previously reported the occurrence of Sry/SRY transcripts in adult murine and human brain. The present communication is concerned with the study of the ontogenetic time course of Sry transcripts in mouse brain as detected by reverse transcription-polymerase chain reaction (RT-PCR). Particular emphasis was placed on the identification of two different forms of Sry mRNA, which can be linear or circular. To this aim, we used specific RT-PCR strategies to distinguish between both. Sry transcripts were found in male brain tissue of all ontogenetic stages investigated. Circular, presumably untranslatable, transcripts were found in embryonic brains of day 11 through 19. In contrast, postnatal Sry transcripts were linear, and thus translatable, and were found in diencephalon, midbrain, and cortex. The change from one transcript form to the other suggests that expression of the Sry gene in mouse brain is developmentally regulated, presumably by a switch in promoter selection. This supports the notion that Sry expression in brain is biologically significant.

Advances in reverse transcription polymerase chain reaction analysis of cellular mRNA levels of transforming growth factor-beta1 by capillary electrophoresis with laser-induced fluorescence detection.

The prosclerotic transforming growth factor beta1 (TGF-beta1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-beta1 mRNA levels, the comparative kinetic reverse transcription-polymerase chain reaction strategy was used. In this method, cellular mRNA levels of the target and a house-keeping gene are reverse transcribed, amplified by the polymerase chain reaction (PCR) and the kinetics of PCR amplification are compared. Since the current determination of the PCR products, using electrophoretic separation in polyacrylamide gel, staining and scanning of the gel, is time-consuming (> or = 5 hours) and inaccurate, we have developed a method using capillary gel electrophoresis (CGE) in combination with laser-induced fluorescence (LIF) detection for quantification of PCR-products. Using the CGE-LIF method, a minute aliquot of the PCR reaction mixture is separated and quantified within 10 min. Comparison of the values with those obtained by polyacrylamide gel electrophoresis demonstrates the improved sensitivity (> 1000 fold) and accuracy of the proposed method. The CGE-LIF procedure offers a convenient way of automated, comparative analysis of low levels of mRNA via reverse transcription PCR in low cell numbers or small amounts of tissue samples.

Heterogeneous Expression of Multiple Putative Patterning Genes by Single Cells from the Chick Hindbrain

The metameric organization of the vertebrate hindbrain into rhombomeres appears to result from the patterned expression of several transcription factors and putative signaling molecules. We have applied a refined single-cell reverse transcription-polymerase chain reaction strategy to examine the molecular logic proposed to pattern the hindbrain at the single-cell level. This technique allows analysis of the concurrent expression of several genes within an individual cell at higher sensitivity than byin situhybridization. Our results demonstrate that cells in rhombomere (r) 4 and r5 are heterogeneous in their expression of Hoxa-3, Hoxb-2, Sek-1, and Krox-20, suggesting that single cells are dynamically regulating their rhombomere-specific gene-expression profiles. Furthermore, the strong correlation between Sek-1 and Krox-20 expression at stage 12 was greatly diminished by stage 16, suggesting that the proposed interdependence of these two genes is present only at early stages of hindbrain development.

The Arabidopsis thaliana FPS1 Gene Generates a Novel mRNA That Encodes a Mitochondrial Farnesyl-diphosphate Synthase Isoform

The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1./EC 2.5.1. 10) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. FPS is considered to play a key role in isoprenoid biosynthesis. We have reported previously that Arabidopsis thaliana contains two differentially expressed genes, FPS1 and FPS2, encoding two highly similar FPS isoforms, FPS1 and FPS2, (Cunillera, N., Arró, M., Delourme, D., Karst, F., Boronat, A., and Ferrer, A. (1996) J. Biol. Chem. 271, 7774-7780). In this paper we report the characterization of a novel Arabidopsis FPS mRNA (FPS1L mRNA) derived from the FPS1 gene. A cDNA corresponding to the FPS1L mRNA was cloned using a reverse transcription-polymerase chain reaction strategy. Northern blot analysis showed that the two FPS1-derived mRNAs are differentially expressed. The FPS1L mRNA accumulates preferentially in inflorescences, whereas the previously reported FPS1 mRNA (FPS1S mRNA) is predominantly expressed in roots and inflorescences. FPS1L mRNA contains an in-frame AUG start codon located 123 nucleotides upstream of the AUG codon used in the translation of the FPS1S isoform. Translation of the FPS1L mRNA from the upstream AUG codon generates a novel FPS1 isoform (FPS1L) with an NH2-terminal extension of 41 amino acid residues, which has all the characteristics of a mitochondrial transit peptide. The functionality of the FPS1L NH2-terminal extension as a mitochondrial transit peptide was demonstrated by its ability to direct a passenger protein to yeast mitochondria in vivo and by in vitro import experiments using purified plant mitochondria. The Arabidopsis FPS1L isoform is the first FPS reported to contain a mitochondrial transit peptide.

Down-regulation of L-type Ca2+ Channel Transcript Levels by 1,25-Dihyroxyvitamin D3: OSTEOBLASTIC CELLS EXPRESS L-TYPE α1C Ca2+ CHANNEL ISOFORMS

Osteoblast Ca2+ channels play a fundamental role in controlling intracellular and systemic Ca2+ homeostasis. A reverse transcription-polymerase chain reaction strategy was used to determine the molecular identity of voltage-sensitive calcium channels present in ROS 17/2.8 osteosarcoma cells. The amino acid sequences encoded by the two resultant PCR products matched the alpha1C-a and the alpha1C-d isoforms. The ability of 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) and structural analogs to modulate expression of voltage-sensitive calcium channel mRNA transcripts was then investigated. ROS 17/2.8 cells were cultured for 48 h in the presence of either 1,25(OH)2D3,1,24-dihydroxy-22-ene-24-cyclopropyl D3 (analog BT) or 25-hydroxy-16-ene-23-yne-D3 (analog AT), and the levels of mRNA encoding alpha1C were quantitated using a competitive reverse transcription-polymerase chain reaction assay. We found that 1, 25(OH)2D3 and analog BT reduced steady state levels of alpha1C mRNA. Conversely, the Ca2+-mobilizing analog AT did not alter steady state levels of voltage-sensitive calcium channel mRNA. Since analog BT, but not analog AT, binds and transcriptionally activates the nuclear receptor for 1,25(OH)2D3, these findings suggest that the down-regulation of voltage-sensitive calcium channel mRNA levels may involve the nuclear receptor.