The mammalian transcriptome and the cellular complexity of the brain.
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Animals↗
Biomedical subjects
S Gustincich
Publications and source records attributed to S Gustincich.
The transcriptional landscape of the mammalian genome.
This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.
3' Untranslated Regions↗
Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.
Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.
Alternative Splicing↗
Functional annotation of a full-length mouse cDNA collection.
The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.
Animals↗
Extrasynaptic release of dopamine in a retinal neuron: activity dependence and transmitter modulation.
Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of approximately 40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the neurons generate in a rhythmic fashion upon removal of all synaptic influences and therefore depends upon the electrical events at the neuronal surface. Furthermore, it is stimulated by kainate and abolished by GABA and quinpirole, an agonist at the D(2) dopamine receptor. Since the somatic receptors for these ligands are extrasynaptic, we suggest that the composition of the extracellular fluid directly modulates extrasynaptic release.
Action Potentials↗
Real-time amperometric measurements of zeptomole quantities of dopamine released from neurons.
Amperometry with carbon-fiber microelectrodes provides a unique way to measure very small chemical concentration changes at the surface of biological cells. In this work, an investigation of dopamine release from individual neurons isolated from the mouse retina is described. The mice were genetically modified so that, in cells that expressed the protein responsible for catecholamine synthesis, tyrosine hydroxylase, the marker protein, placental alkaline phosphatase, was also expressed. This modification allowed for identification of the dopamine-containing cells among the many present in the freshly dissociated retina. Release of dopamine was evoked by chemical secretagogues delivered from micropipets that were calibrated with respect to response time and concentration delivered. Amperometric measurements were recorded with low-noise patch clamp amplifiers, and the primary noise source was found to be the electrode capacitance. Dopamine release occurred in the form of transient concentration spikes, consistent with release from small intracellular vesicles. With optimized filtering of the data, the quantity secreted during each release event could be determined. The average quantity determined at one cell was 52 zmol. However, the spikes were quite variable in size and the amount released per event ranged from 8 to 170 zmol. These measurements allow an estimation of the concentration of released transmitter in a synapse.
Animals↗
Pharmacology of GABA(A) receptors of retinal dopaminergic neurons.
When the vertebrate retina is stimulated by light, a class of amacrine or interplexiform cells release dopamine, a modulator responsible for neural adaptation to light. In the intact retina, dopamine release can be pharmacologically manipulated with agonists and antagonists at GABA(A) receptors, and dopaminergic (DA) cells receive input from GABAergic amacrines. Because there are only 450 DA cells in each mouse retina and they cannot be distinguished in the living state from other cells on the basis of their morphology, we used transgenic technology to label DA cells with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. We could therefore identify DA cells in vitro after dissociation of the retina and investigate their activity with whole cell voltage clamp. We describe here the pharmacological properties of the GABA(A) receptors of solitary DA cells. GABA application induces a large inward current carried by chloride ions. The receptors are of the GABA(A) type because the GABA-evoked current is blocked by bicuculline. Their affinity for GABA is very high with an EC(50) value of 7.4 microM. Co-application of benzodiazepine receptor ligands causes a strong increase in the peak current induced by GABA (maximal enhancement: CL-218872 220%; flunitrazepam 214%; zolpidem 348%) proving that DA cells express a type I benzodiazepine-receptor (BZ1). GABA-evoked currents are inhibited by Zn(2+) with an IC(50) of 58.9 +/- 8.9 microM. Furthermore, these receptors are strongly potentiated by the modulator alphaxalone with an EC(50) of 340 +/- 4 nM. The allosteric modulator loreclezole increases GABA receptor currents by 43% (1 microM) and by 107% (10 microM). Using outside-out patches, we measured in single-channel recordings a main conductance (29 pS) and two subconductance (20 and 9 pS) states. We have previously shown by single-cell RT-PCR and immunocytochemistry that DA cells express seven different GABA(A) receptor subunits (alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L)) and by immunocytochemistry that all subunits are expressed in the intact retina. We show here that at least alpha1, beta3 and gamma2 subunits are assembled into functional receptors.
Alkaline Phosphatase↗
Composition of the GABA(A) receptors of retinal dopaminergic neurons.
Transgenic technology, single-cell RT-PCR, and immunocytochemistry were combined to investigate the composition of the GABA(A) receptors of dopaminergic (interplexiform) amacrine (DA) cells. A mouse line was used in which these neurons were labeled with human placental alkaline phosphatase and could therefore be identified in vitro after dissociation of the retina. We performed single-cell RT-PCR on the isolated cells and showed that (1) DA cells contained the messages for alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L) subunits; (2) this transcript repertory did not change on dissociation of the retina and throughout the time required for cell harvesting; and (3) all DA cells contained the entire transcript repertory. Immunocytochemistry with subunit-specific antibodies showed that all subunits were expressed and appeared homogeneously distributed throughout the cell membrane at a low concentration. In addition, with the exception of alpha4, the subunits formed clusters at the surface of the dendrites and on the inner pole of the cell body. Because of their size, shape, and topographic coincidence with GABAergic endings, the clusters were interpreted as postsynaptic active zones containing GABA(A) receptors. The composition of the synaptic receptors was not uniform: clusters distributed throughout the dendritic tree contained alpha3, beta3, and, less frequently, beta1 subunits, whereas clusters containing the alpha1 subunit were confined to large dendrites. Therefore, DA cells possess at least two types of GABA(A) receptors localized in different synapses. Furthermore, they exhibit multiple extrasynaptic GABA(A) receptors.
Alkaline Phosphatase↗
The human serum deprivation response gene (SDPR) maps to 2q32-q33 and codes for a phosphatidylserine-binding protein.
The serum deprivation response gene (SDPR, alias sdr) has been previously isolated for its high mRNA expression in serum-starved cells compared to contact-inhibited NIH3T3 cells; such regulation is not observed in single-oncogene transformed NIH3T3 cells after serum starvation. More recently Sdpr has been identified as a substrate of protein kinase C (PKC): this interaction determines the compartimentalization of PKC to caveolae, a plasma membrane invagination of which Sdpr is a major component. Lack of Sdpr-PKC interaction in transformed cells has been proposed to be involved in the alteration of PKC subcellular localization and substrate specificity. Here we report the cloning of the human SDPR homologue (HGMW-approved symbol SDPR) and its mapping to 2q32-q33 in the human genome. In analogy with the murine system, SDPR mRNA expression is increased when human fibroblasts are serum starved, it becomes down-regulated during synchronous cell-cycle reentry, but it is not induced in cells arrested by contact inhibition. Analysis of SDPR expression in human tissues reveals a near ubiquitous expression, with highest levels found in heart and lung. We show that human SDPR encodes PS-p68, a previously characterized phosphatidylserine-binding protein purified from human platelets. Accordingly, recombinant Sdpr is able to specifically bind phosphatidylserine in the absence of Ca2+. SDPR is homologous to two genes in the databank, one of which, srbc, is similarly regulated during growth arrest and encodes a phosphatidylserine-binding protein that is a substrate of PKC.
Amino Acid Sequence↗
Spontaneous activity of solitary dopaminergic cells of the retina.
Dopaminergic interplexiform amacrine cells were labeled in transgenic mice with human placental alkaline phosphatase and could therefore be identified after dissociation of the retina and used for whole-cell current and voltage clamp. In absence of synaptic inputs, dopaminergic amacrines spontaneously fired action potentials in a rhythmic pattern. This activity was remarkably robust in the face of inhibition of various voltage-dependent ion channels. It was minimally affected by external cesium or cobalt, suggesting no involvement of either the hyperpolarization-activated cation current Ih or voltage-dependent calcium channels. Inhibiting calcium-activated potassium channels by charybdotoxin or tetraethylammonium slowed the repolarizing phase of the action potentials and eliminated a slow afterhyperpolarization but had a scarce effect on the frequency of spontaneous firing. Voltage-clamp experiments showed that the interspike depolarization leading to threshold results from tetrodotoxin-sensitive sodium channels active at the interspike voltages of -60 to -40 mV. Because dopamine acts on distant targets in the retina, the pacemaker activity of dopaminergic amacrines may be necessary to ensure a tonic release of the modulator from their dendritic tree. Pacemaking is a property that this type of retinal amacrine cell shares with the dopaminergic mesencephalic neurons, but the ionic mechanisms responsible for the spontaneous firing are apparently different.
Animals↗
Control of dopamine release in the retina: a transgenic approach to neural networks.
Dopaminergic, interplexiform amacrines (DA cells) were labeled in transgenic mice with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. It was therefore possible to investigate their activity in vitro after dissociation of the retina with whole-cell current and voltage clamp, as well as their connections in the intact retina with the electron microscope. DA cells generate action potentials even in the absence of synaptic inputs. This activity is abolished by the amacrine cell transmitters GABA and glycine, which induce an inward current carried by chloride ions, and is stimulated by kainate, an agonist at the receptor for the bipolar cell transmitter glutamate, which opens nonselective cation channels. Since DA cells are postsynaptic to amacrine and bipolar cells, we suggest that the spontaneous discharge of DA cells is inhibited in the dark by GABAergic amacrines that receive their input from off-bipolars. Upon illumination, the GABA-inhibition is removed, DA cells generate action potentials, and their firing is modulated by the excitation received from on-bipolars.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
New lambda and plasmid vectors for expression cloning in mammalian cells.
This report describes the construction of a new family of lambda phage and plasmid cloning vectors. lambda GDST3/T7 allows cDNA insertion up to 14 kb; it is derived from lambda NM1151 by the insertion of a multiple cloning site containing eight unique restriction sites. The two asymmetrical SfiI sites are flanked by the T3 and T7 promoters for direct sequencing and in vitro transcription/translation. The same multiple cloning site is also present in both orientations in the eukaryotic expression plasmids, pGDSV3 and pGDSV7. By exploiting the superior discrimination of the signal-to-noise ratio of the lambda vectors for primary screening (by either nucleic acids or antibody probes), relevant cDNAs can thus be efficiently transferred through SfiI sites into the plasmids pGDSV3/7 for functional secondary screening by expression in mammalian cells.
Animals↗
Dissection of c-myc domains involved in S phase induction of NIH3T3 fibroblasts.
The product of the c-myc proto-oncogene is an important regulator of cell proliferation and apoptosis in murine fibroblasts. Addition of the tumor promoter, phorbol myristate acetate (PMA), prevents apoptotic cell death induced by low serum concentrations in NIH3T3 cells that constitutively express and are transformed by v-myc. The protective effect of PMA allowed us to analyse the ability of normal c-Myc and Myc deletion mutants to induce serum starved, untransformed NIH3T3 cells to enter S phase. By microinjecting these quiescent cells with wild type and mutant human c-myc plasmids, we showed that full length c-myc is able to induce S phase entry in presence of PMA, but that c-Myc mutants that delete amino acids delta 7/91, delta 41/53, delta 56/103, delta 106/143, delta 265/317 and delta 414/433 are totally inactive. c-Myc did not shorten the period before entry into S phase, since Myc overexpressing cells entered S phase with the same kinetics as control cells when both were stimulated with 20% fetal calf serum (FCS). However, c-Myc overexpression did increase the percentage of cells entering S phase when these cells were stimulated with 2% fetal calf serum. Interestingly, this ability to enhance stimulation by a suboptimal concentration of FCS was retained to a significant degree by Myc mutants that delete amino acids delta 41/53, delta 56/103 or delta 265/317. Finally, Myc mutants that delete delta 106/143 or delta 414/433 exerted a dominant negative effect on S phase entry both in quiescent cells stimulated with 2% FCS and in unsynchronized, cycling cells.
3T3 Cells↗
Serum deprivation response gene is induced by serum starvation but not by contact inhibition.
The relationship between quiescence induced by serum deprivation and that induced by high cell density (contact inhibition) is still unclear. Here, we describe a gene, named sdr (serum deprivation response), whose mRNA level is highly induced in serum starved but not in density dependent growth arrested NIH3T3 cells. sdr induction seems to be coupled to growth arrest since it does not become apparent when transformed NIH3T3 cells are cultured in low serum. The expression of sdr is down-regulated within 6 h after the addition of serum or epidermal growth factor to serum starved cells. Although a transient reinduction is noticed at later times after fetal calf serum stimulation, this is not the case for epidermal growth factor, for which the sdr mRNA level remains down-regulated. The data presented here point to the possibility that the density dependent growth arrest state cannot be simply explained by local growth factor depletion, as occurs in overcrowded cultures, but it could be the consequence of a more complex pathway mediated by cellular interactions.
3T3 Cells↗
[The molecular genetics of cystic fibrosis: the delta F508 allele in Mexican families].
We have initiated the screening of the delta F508 cystic fibrosis (CF) mutation in our country basing our study on the polymerase chain reaction technique. In a sample of 10 western and a northeastern Mexican families that have at least one CF affected child, we found a delta F508 frequency of 59.1% among CF chromosomes. This frequency is similar to those reported for Spain. Furthermore, we found a correlation between the delta F508 allele and pancreatic insufficiency. This paper points out the advantage of the polymerase chain reaction technology for the analysis of Mendelian disorders.
Alleles↗
Sex determination and species exclusion in forensic samples with probe cY97.
A total of 120 human samples of blood, saliva and semen stains, hair roots, bone and skin fragments, obtained from 30 males and 16 females were analyzed in Southern blots with probe cY97. Only the male samples gave a specific band of 5.7 kb. In dot blot, under high stringency conditions, male DNA gave signals equivalent to a quantity of female DNA eight times higher. Probe cY97 did not react with 9 different vertebrate species but gave a signal for monkey DNA when used at low stringency. The advantage of using a probe specific for the centromeric region for sex determination and species exclusion is discussed.
Animals↗
The complexity of cell proliferation control in mammalian cells.
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Animals↗
A fast method for high-quality genomic DNA extraction from whole human blood.
A simple and fast protocol is described for the purification of genomic DNA from 0.3 ml of whole human blood. The recovery of DNA is quantitative and reproducible; the quality is such that it can be used for all relevant molecular biology techniques.
Base Sequence↗