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PubMed2003

Integrins regulate neuronal neurotrophin gene expression through effects on voltage-sensitive calcium channels.

Integrin adhesion receptors regulate gene expression during growth and differentiation in various cell types. Recent work, implicating integrins in functional synaptic plasticity, suggest they may have similar activities in adult brain. The present study tested if integrins binding the arginine-glycine-aspartate (RGD) matrix sequence regulate neurotrophin and neurotrophin receptor gene expression in cultured hippocampal slices. The soluble RGD-containing peptide glycine-arginine-glycine-aspartate-serine-proline (GRGDSP) increased neurotrophin mRNA levels in transcript- and subfield-specific fashions. Integrin ligand effects were greatest for brain-derived neurotrophic factor transcripts I and II and barely detectable for transcript III. In accordance with increased nerve growth factor mRNA levels, GRGDSP increased c-fos expression as well. In contrast, growth-associated protein-43, amyloid precursor protein and fibroblast growth factor-1 mRNAs were not elevated. Ligand effects on brain-derived neurotrophic factor transcript II and c-fos mRNA did not depend on the integrity of the actin cytoskeleton, neuronal activity, or various signaling pathways but were blocked by L-type voltage-sensitive calcium-channel blockers. These results indicate that in mature hippocampal neurons integrin engagement regulates expression of a subset of growth-related genes at least in part through effects on calcium influx. Accordingly, these synaptic adhesion receptors may play the same role in maintaining an adult, differentiated state in brain as they do in other tissues and changes in integrin activation and/or engagement may contribute to dynamic changes in neurotrophin expression and to neuronal calcium signaling.

Anesthetics, Local↗
PubMed2005

Dopamine and cognitive control: the influence of spontaneous eyeblink rate and dopamine gene polymorphisms on perseveration and distractibility.

One fundamental problem of intelligent organisms pursuing goal-directed behavior is how to dynamically regulate the balance between maintenance and flexibility. The authors show that central dopaminergic activity, as indicated by spontaneous eyeblink rate and dopamine gene polymorphisms, plays an important role in the modulation of this balance. Seventy-two young adults were examined. Participants with high blink rates showed increased cognitive flexibility but decreased cognitive stability compared with participants with low blink rates. This pattern of results was even more pronounced for carriers of the DRD4 exon III 4/7 genotype, even though no main effects were found for DRD4 and COMT polymorphisms. Results converge with neuropsychological models that suggest a modulatory role of prefrontal dopaminergic activity for processes of cognitive control.

Adolescent↗
PubMed2003

Huntingtin contains a highly conserved nuclear export signal.

Huntington's disease (HD), is a genetic neurodegenerative disease characterized by a DNA CAG triplet repeat expansion in the first exon of the disease gene, HD. CAG DNA expansion results in a polyglutamine tract expansion in mutant huntingtin protein. Wild-type and mutant full-length huntingtin have been detected in the nucleus, but elevated levels of mutant huntingtin and huntingtin amino-terminal proteolytic fragments are seen to accumulate in the nuclei of HD-affected neurons. The presence of huntingtin in both the nucleus and the cytoplasm suggested that huntingtin may be dynamic between these compartments. By live cell time-lapse video microscopy, we have been able to visualize polyglutamine-mediated aggregation and the transient nuclear localization of huntingtin over time in a striatal cell line. A classical nuclear localization signal could not be detected in huntingtin, but we have discovered a nuclear export signal (NES) in the carboxy-terminus of huntingtin. Leptomycin B treatment of clonal striatal cells enhanced the nuclear localization of huntingtin, and a mutant NES huntingtin displayed increased nuclear localization, indicating that huntingtin can shuttle to and from the nucleus. The huntingtin NES is strictly conserved among all huntingtin proteins from diverse species. This export signal may be important in Huntington's disease because this fragment of huntingtin is proteolytically cleaved away during HD. The huntingtin NES therefore defines a potential role for huntingtin as a member of a nucleocytoplasmic dynamic protein complex.

Active Transport, Cell Nucleus↗
PubMed2000

Real-time monitoring of cytomegalovirus infections after stem cell transplantation using the TaqMan polymerase chain reaction assays.

BACKGROUND: Real-time monitoring of cytomegalovirus (CMV) infections in transplant patients demands a rapid and high-throughput CMV DNA quantification method. METHODS: TaqMan polymerase chain reaction assays based on CMV immediate early protein exon 4 and glycoprotein B were developed and were compared with a COBAS AMPLICOR CMV MONITOR (CMM) test for quantifying CMV DNA in peripheral blood leukocytes from seven stem cell transplant patients having received antiviral treatment. RESULTS: There was a good correlation between the TaqMan assays and the CMM test for CMV DNA quantification. The throughput of the TaqMan assays was, however, about 3 times higher than that of the CMM test. The CMV DNA dynamics patterns determined by the TaqMan polymerase chain reaction were well in line with the outcome of the antiviral therapy. CONCLUSIONS: The TaqMan assays may potentially serve as a useful tool for rapid quantification of CMV infections in stem cell transplant patients.

Antiviral Agents↗
PubMed1998

NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein.

The RNA molecules that make up the spliceosome branch-point helix and the binding site for phage GA coat protein share a secondary structure motif in which two consecutive adenine residues occupy the strand opposite a single uridine, creating the potential to form one of two different A.U base pairs while leaving the other adenine unpaired or bulged. During the splicing of introns out of pre-mRNA, the 2'-OH of the bulged adenine participates in the transesterification reaction at the 5'-exon and forms the branch-point residue of the lariat intermediate. Either adenine may act as the branch-point residue in mammals, but the 3'-proximal adenine does so preferentially. When bound to phage GA coat protein, the bulged adenine loops out of the helix and occupies a binding pocket on the surface of the protein, forming a nucleation complex for phage assembly. The coat protein can bind helices with bulged adenines at either position, but the 3'-proximal site binds with greater affinity. We have studied this RNA motif in a 21 nucleotide hairpin containing a GA coat protein-binding site whose four nucleotide loop has been replaced by a more stable loop from the related phage Ms2. Using heteronuclear NMR spectroscopy, we have determined the structure of this hairpin to an overall precision of 2.0 A. Both adenine bases stack into the helix, and while all available NOE and coupling constant data are consistent with both possible A.U base pairs, the base pair involving the 5'-proximal adenine appears to be the major conformation. The 3'-proximal bulged adenine protonates at unusually high pH, and to account for this, we propose a model in which the protonated adenine is stabilized by a hydrogen bond to the uridine O2 of the A.U base pair. The 2'-OH of the bulged adenine adopts a regular A-form helical geometry, suggesting that in order to participate in the splicing reaction, the conformation of the branch-point helix in the active spliceosome may change from the conformation described here. Thus, while the adenine site preferences of the spliceosome and of phage GA may be due to protein factors, the preferred adenine is predisposed in the free RNA to conformational rearrangement involved in formation of the active complexes.

Adenine↗
PubMed1998

Developmental-dependent DNA methylation of the IGF2 and H19 promoters is correlated to the promoter activities in human liver development.

We have previously shown that the four promoters of the IGF2 gene are under a tight but dynamic control during human liver development, whereby P3 and P1 are reciprocally active before and after birth respectively while the P2 and P4 promoters are constitutively active at a relatively lower level. In this study, we investigated the methylation status of the promoters P1 and P3 of IGF2 and the promoter region of the H19 gene in developing human livers ranging from fetal to late adult. A region of about 300 bp immediately upstream of the IGF2 exon 5 was found to be subjected to a developmental-specific methylation and this may correlate to the P3 promoter activity. The P1 domain of IGF2 was also found to be methylated in a developmentally-specific pattern. The promoter region of the H19 gene displayed different methylation patterns in different development stages showing decreased general methylation with increase of age. Therefore, regional- and developmental-specific DNA methylation is displayed in the promoter regions of the IGF2 and H19 genes. This may be an important factor involved in gene regulation in the developing human liver.

Adult↗
PubMed1998

Ionotropic glutamate receptors in the retina: moving from molecules to circuits.

The cloning of the glutamate-gated ion channels of the brain revealed an unexpected level of complexity: there are many different genes that encode distinct subunits of the receptor/channel complex and an even larger number of possible receptor subunit combinations. Many--nearly all--of these gene products are expressed in the retina, and the questions that we face today are: how are they used and why are there so many? Answers to these questions will be found at several levels. At the level of transcription, we have learned that different sets of subunits are expressed by different retinal neurons. Little is known about the transcriptional control of these genes, so it remains to be determined whether these patterns of expression reflect the need for different gene products in different retinal neurons or whether these patterns of expression reflect the functional constraints of gene expression. Another level of complexity is caused by alternative splicing, and here we report that at least four and possibly all eight of the different NMDAR1 transcripts are present in the mouse retina. The consequences of this alternative splicing are poorly understood, but antibodies directed against the two different possible C-termini of NMDAR1 label many of the same cell types. It is possible that these different splice variants are combined to generate the channels. While immunohistochemistry provides us with a glimpse of the subunit proteins, much remains to be learned about their half-life within a retinal cell, their intracellular trafficking, their regulation at the synapse, and the proteins associated with their cytoplasmic domains. An approach we have taken towards studying the dynamic properties of receptor subunits has been to fuse them to the cDNA encoding the jellyfish Green Fluorescent Protein. This makes it possible to follow functional subunits in transfected cells over time and to begin to measure the mobility of the protein.

Animals↗
PubMed1995

Investigation of the oxytocin receptor at the molecular level.

Before the onset of labor, the myometrium becomes extremely sensitive to oxytocin, for which it is a primary target tissue, because of a tremendous increase in the number of oxytocin receptors. To study this dynamic expression and regulation of the receptor, we cloned the human oxytocin receptor cDNA in 1992 and characterized its expression in female reproductive organs and in the rat brain using the receptor cDNA and anti-oxytocin receptor monoclonal antibodies. In the uterus, we revealed that oxytocin receptor expression is mainly regulated at the transcriptional level. For the investigation of this transcriptional regulation, we isolated and characterized the human oxytocin receptor gene. The gene contains 3 introns and 4 exons and is assigned to 3p26.2 of human chromosome 3. In the 5'-upstream region of this gene, we found several known binding sites for transcription regulation factors including the nucleofactor-interleukin 6 binding consensus sequence and an acute phase reactant-responsive element. Our findings on the structure of the oxytocin receptor gene should help to elucidate the mechanism by which expression of the gene is induced dramatically at parturition in the uterus and how the gene is regulated in other organs such as the mammary gland and central nervous system.

Amino Acid Sequence↗
PubMed2006

Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi).

A phylogeny of the fungal phylum Basidiomycota is presented based on a survey of 160 taxa and five nuclear genes. Two genes, rpb2, and tef1, are presented in detail. The rpb2 gene is more variable than tef1 and recovers well-supported clades at shallow and deep taxonomic levels. The tef1 gene recovers some deep and ordinal-level relationships but with greater branch support from nucleotides compared to amino acids. Intron placement is dynamic in tef1, often lineage-specific, and diagnostic for many clades. Introns are fewer in rpb2 and tend to be highly conserved by position. When both protein-coding loci are combined with sequences of nuclear ribosomal RNA genes, 18 inclusive clades of Basidiomycota are strongly supported by Bayesian posterior probabilities and 16 by parsimony bootstrapping. These numbers are greater than produced by single genes and combined ribosomal RNA gene regions. Combination of nrDNA with amino acid sequences, or exons with third codon positions removed, produces strong measures of support, particularly for deep internodes of Basidiomycota, which have been difficult to resolve with confidence using nrDNA data alone. This study produces strong boostrap support and significant posterior probabilities for the first time for the following monophyletic groups: (1) Ustilaginomycetes plus Hymenomycetes, (2) an inclusive cluster of hymenochaetoid, corticioid, polyporoid, Thelephorales, russuloid, athelioid, Boletales, and euagarics clades, (3) Thelephorales plus the polyporoid clade, (4) the polyporoid clade, and (5) the cantharelloid clade. Strong support is also recovered for the basal position of the Dacrymycetales in the Hymenomycetidae and paraphyly of the Exobasidiomycetidae.

Alleles↗
PubMed2006

OPA1 alternate splicing uncouples an evolutionary conserved function in mitochondrial fusion from a vertebrate restricted function in apoptosis.

In most eucaryote cells, release of apoptotic proteins from mitochondria involves fission of the mitochondrial network and drastic remodelling of the cristae structures. The intramitochondrial dynamin OPA1, as a potential central actor of these processes, exists as eight isoforms resulting from the alternate splicing combinations of exons (Ex) 4, 4b and 5b, which functions remain undetermined. Here, we show that Ex4 that is conserved throughout evolution confers functions to OPA1 involved in the maintenance of the DeltaPsi(m) and in the fusion of the mitochondrial network. Conversely, Ex4b and Ex5b, which are vertebrate specific, define a function involved in cytochrome c release, an apoptotic process also restricted to vertebrates. The drastic changes of OPA1 variant abundance in different organs suggest that nuclear splicing can control mitochondrial dynamic fate and susceptibility to apoptosis and pathologies.

Alternative Splicing↗
PubMed2004

Uromodulin storage diseases: clinical aspects and mechanisms.

The recent discovery of mutations in the uromodulin gene ( UMOD ) in patients with medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy (FJHN), and glomerulocystic kidney disease (GCKD) provides the opportunity for a revision of pathogenic aspects and puts forth the basis for a renewed classification. This review focuses on clinical, pathological, and cell biology advances in UMOD -related pathological states, including a review of the associated clinical conditions described to date in the literature. Overall, 31 UMOD mutations associated with MCKD2 and FJHN (205 patients) and 1 mutation associated with GCKD (3 patients) have been described, with a cluster at exons 4 and 5. Most are missense mutations causing a cysteine change in uromodulin sequence. No differences in clinical symptoms between carriers of cysteine versus polar residue changes have been observed; clinical phenotypes invariably are linked to classic MCKD2/FJHN. A common motif among all reports is that many overlapping symptoms between MCKD2 and FJHN are present, and a separation between these 2 entities seems unwarranted or redundant. Cell experiments with mutant variants indicated a delay in intracellular maturation and export dynamics, with consequent uromodulin storage within the endoplasmic reticulum (ER). Patchy uromodulin deposits in tubule cells were found by means of immunohistochemistry, and electron microscopy showed dense fibrillar material in the ER. Mass spectrometry showed only unmodified uromodulin in urine of patients with UMOD mutations. Lack of uromodulin function(s) is associated with impairments in tubular function, particularly the urine-concentrating process, determining water depletion and hyperuricemia. Intracellular uromodulin trapping within the ER probably has a major role in determining tubulointerstitial fibrosis and renal failure. We propose the definition of uromodulin storage diseases for conditions with proven UMOD mutations.

Animals↗
PubMed1986

Regulation of vasopressin gene expression in rat hypothalamic neurons. Response to osmotic stimulation.

Although the steps involved in biosynthesis and secretion of the neuropeptide vasopressin (AVP) have been extensively studied, the factors which regulate AVP gene expression remain unknown. Therefore, we sought to determine the dynamics of AVP mRNA accumulation in response to a strong stimulus for AVP release, i.e. during salt imbibition and the ensuing period of rehydration. AVP mRNA levels were determined in terms of absolute amounts by a novel quantitative densitometric hybridization assay, using in vitro synthesized sense-strand RNA as a quantitative standard and complementary anti-sense RNA as a specific probe. The template used for RNA transcription consisted of a 196-base pair genomic DNA fragment corresponding to exon C of the rat AVP gene. Determination of basal hypothalamic AVP mRNA levels yielded 12.5 +/- 2.7 fmol/hypothalamus. Salt imbibition, which induced a 6% rise in blood osmolality and an 82% loss of pituitary AVP, resulted in a 3-fold increase of AVP mRNA to 35 +/- 5 fmol/hypothalamus. Following rehydration, plasma osmolality returned to control levels by day 2, pituitary AVP by day 6, and hypothalamic AVP by day 14. By contrast, AVP mRNA levels remained significantly elevated throughout the 30-day rehydration period. Furthermore, pituitary AVP reached a level of 177% of control by day 14 of rehydration. These data show that osmotic stimulation results in a long-lasting elevation of hypothalamic AVP mRNA; during rehydration, these elevated mRNA levels direct AVP biosynthesis at a rate which surpasses secretory demands; AVP mRNA accumulation does not appear to be directly regulated by either pituitary or hypothalamic AVP. Therefore, either an unusually long half-life of greater than or equal to 7 days must be assumed for AVP mRNA or, alternatively, a continued stimulation of AVP gene transcription must occur, even in the absence of a secretory stimulus and following complete repletion of cellular AVP stores.

Animals↗
PubMed2005

Alternative splicing suggests extended function of PEX26 in peroxisome biogenesis.

Matsumoto and colleagues recently identified PEX26 as the gene responsible for complementation group 8 of the peroxisome biogenesis disorders and showed that it encodes an integral peroxisomal membrane protein with a single C-terminal transmembrane domain and a cytosolic N-terminus that interacts with the PEX1/PEX6 heterodimer through direct binding to the latter. They proposed that PEX26 functions as the peroxisomal docking factor for the PEX1/PEX6 heterodimer. Here, we identify new PEX26 disease alleles, localize the PEX6-binding domain to the N-terminal half of the protein (aa 29-174), and show that, at the cellular level, PEX26 deficiency impairs peroxisomal import of both PTS1- and PTS2-targeted matrix proteins. Also, we find that PEX26 undergoes alternative splicing to produce several splice forms--including one, PEX26- delta ex5, that maintains frame and encodes an isoform lacking the transmembrane domain of full-length PEX26 (PEX26-FL). Despite its cytosolic location, PEX26- delta ex5 rescues peroxisome biogenesis in PEX26-deficient cells as efficiently as does PEX26-FL. To test our observation that a peroxisomal location is not required for PEX26 function, we made a chimeric protein (PEX26-Mito) with PEX26 as its N-terminus and the targeting segment of a mitochondrial outer membrane protein (OMP25) at its C-terminus. We found PEX26-Mito localized to the mitochondria and directed all detectable PEX6 and a fraction of PEX1 to this extraperoxisomal location; yet PEX26-Mito retains the full ability to rescue peroxisome biogenesis in PEX26-deficient cells. On the basis of these observations, we suggest that a peroxisomal localization of PEX26 and PEX6 is not required for their function and that the interaction of PEX6 with PEX1 is dynamic. This model predicts that, once activated in an extraperoxisomal location, PEX1 moves to the peroxisome and completes the function of the PEX1/6 heterodimer.

Alleles↗
PubMed2001

Heterologous, splicing-dependent RNA editing in chloroplasts: allotetraploidy provides trans-factors.

RNA editing is unique among post-transcriptional processes in plastids, as it exhibits extraordinary phylogenetic dynamics leading to species-specific editing site patterns. The evolutionary loss of a site is considered to entail the loss of the corresponding nuclear-encoded site-specific factor, which prevents the editing of foreign, i.e. heterologous, sites. We investigated the editing of short 'spliced' and 'unspliced' ndhA gene fragments from spinach in Nicotiana tabacum (tobacco) in vivo using biolistic transformation. Surprisingly, it turned out that the spinach site is edited in the heterologous nuclear background. Furthermore, only exon-exon fusions were edited, whereas intron-containing messages remained unprocessed. A homologue of the spinach site was found to be present and edited in Nicotiana tomentosiformis, representing the paternal parent, but absent from Nicotiana sylvestris, representing the maternal parent of tobacco. Our data show that: (i) the cis-determinants for ndhA editing are split by an intron; (ii) the editing capacity cannot be deduced from editing sites; and (iii) allopolyploidization can increase the editing capacity, which implies that it can influence speciation processes in evolution.

Base Sequence↗
PubMed1995

Correcting sequencing errors in DNA coding regions using a dynamic programming approach.

This paper presents an algorithm for detecting and 'correcting' sequencing errors that occur in DNA coding regions. The types of sequencing errors addressed are insertions and deletions (indels) of DNA bases. The goal is to provide a capability which makes single-pass or low-redundancy sequence data more informative, reducing the need for high-redundancy sequencing for gene identification and characterization purposes. This would permit improved sequencing efficiency and reduce genome sequencing costs. The algorithm detects sequencing errors by discovering changes in the statistically preferred reading frame within a putative coding region and then inserts a number of 'neutral' bases at a perceived reading frame transition point to make the putative exon candidate frame consistent. We have implemented the algorithm as a front-end subsystem of the GRAIL DNA sequence analysis system to construct a version which is very error tolerant and also intend to use this as a testbed for further development of sequencing error-correction technology. Preliminary test results have shown the usefulness of this algorithm and also exhibited some of its weakness, providing possible directions for further improvement. On a test set consisting of 68 human DNA sequences with 1% randomly generated indels in coding regions, the algorithm detected and corrected 76% of the indels. The average distance between the position of an indel and the predicted one was 9.4 bases. With this subsystem in place, GRAIL correctly predicted 89% of the coding messages with 10% false message on the 'corrected' sequences, compared to 69% correctly predicted coding messages and 11% falsely predicted messages on the 'corrupted' sequences using standard GRAIL II method (version 1.2).(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1995

Cloning and sequencing a non-ATPase subunit of the regulatory complex of the Drosophila 26S protease.

We have cloned and sequenced a non-ATPase subunit of the regulatory complex of the Drosophila 26S protease. The gene is present in a single copy in the Drosophila genome. By comparing the nucleotide sequence of the genomic and cDNA clones three exons and two introns were localized. Two transcription start sites were identified 9 bp apart. The deduced protein sequence shows no significant similarity to any other protein in the database. In Drosophila embryos where the 26S protease is present in high concentration, the pool of free subunits of the regulatory complex is very low. Among the free subunits of the regulatory complex the cloned subunit is present in very large excess. This observation raises the possibility that this subunit is in a dynamic equilibrium, exchanging between a free and a particle-bound form, which may have important implications concerning its function.

Adenosine Triphosphatases↗
PubMed2001

Inheritance of a stable mutation in a family with early-onset disease.

Autosomal/dominant polycystic kidney disease (ADPKD) exhibits a high inter- and intrafamilial heterogeneity partly explained by the involvement of at least 3 different genes in the disorder transmission. PKD1, the major locus, is located on chromosome 16p. The occurrence of very early-onset cases of ADPKD (sometimes in utero) in a few PKD1 families or the increased severity of the disease in successive generations raise the question of anticipation. This is a subject of controversial discussion. This report deals with the molecular analysis in families with very early-onset ADPKD. The finding of the same stable mutation with such different phenotypes rules out a dynamic mutation. The molecular basis of severe childhood PKD in typical ADPKD families remains unclear; it may include segregation of modifying genes or unidentified factors and the two-hit mechanism.

Adult↗
PubMed2005

Quantitative assay of total dsDNA with PicoGreen reagent and real-time fluorescent detection.

We describe a quantitative assay of dsDNA based on real-time PCR measurement of fluorescence due to the interaction of PicoGreen dye with dsDNA. An aliquot of 1 to 5 ml of the sample is mixed with 45 ml of diluted PicoGreen reagent within an optical PCR tube. This is placed into the real-time apparatus set to read SYBR Green I dye at the end of three cycles of 94 degrees C for 30 s and 65 degrees C for 30 s. The averaged fluorescence value is converted into DNA amount using a calibration curve prepared with lambda-DNA standard. The calibration curve has a dynamic linear range from 0.20 to 50 ng and a standard deviation variability below 5.0%. In conclusion, this method allows reliable determinations on minimal amounts of DNA from biological samples and PCR products in clinical applications of molecular biology.

Bacteriophage lambda↗