RESUMO
Frameshift (+1) proteins such as APP(+1) and UBB(+1) accumulate in sporadic cases of Alzheimer disease (AD) and in older subjects with Down syndrome (DS). We investigated whether these proteins also accumulate at an early stage of neuropathogenesis in young DS individuals without neuropathology and in early-onset familial forms of AD (FAD), as well as in other tauopathies, such as Pick disease (PiD) or progressive supranuclear palsy (PSP). APP(+1) is present in many neurons and beaded neurites in very young cases of DS, which suggests that it is axonally transported. In older DS patients (>37 years), a mixed pattern of APP(+1) immunoreactivity was observed in healthy looking neurons and neurites, dystrophic neurites, in association with neuritic plaques, as well as neurofibrillary tangles. UBB(+1) immunoreactivity was exclusively present in AD type of neuropathology. A similar pattern of APP(+1) and UBB(+1) immunoreactivity was also observed for FAD and much less explicit in nondemented controls after the age of 51 years. Furthermore, we observed accumulation of +1 proteins in other types of tauopathies, such as PiD, frontotemporal dementia, PSP and argyrophylic grain disease. These data suggest that accumulation of +1 proteins contributes to the early stages of dementia and plays a pathogenic role in a number of diseases that involve the accumulation of tau.
Assuntos
Doença de Alzheimer/genética , Mutação da Fase de Leitura , Tauopatias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Substituição de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Córtex Cerebral/química , Córtex Cerebral/ultraestrutura , Síndrome de Down/genética , Síndrome de Down/metabolismo , Feminino , Genes Dominantes , Hipocampo/química , Hipocampo/ultraestrutura , Humanos , Técnicas Imunoenzimáticas , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Degeneração Neural/genética , Degeneração Neural/metabolismo , Linhagem , Presenilina-1 , Tauopatias/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
The molecular bases of late-onset and sporadic Alzheimer's disease (AD) still have to be unraveled. Among putative candidates for molecular variations in AD, we propose LMO4 protein, a transcription regulator, involved in multiple protein complexes. We investigated changes in LMO4 immunoreactivity in vulnerable brain regions of AD cases and controls of comparable age. Immunocytochemical analysis revealed a high level of LMO4 expression in the entorhinal cortex (EC) and in the CA1 hippocampal region of the control brains and a consistent decrease in the AD brains, correlated with the amount of neurofibrillary tangles (NFT) degenerating neurones and the severity of senile plaques deposition. The decrease in LMO4 immunoreactivity resulted both from weaker immunoreactive signals and from a loss of immunoreactive neurones. LMO4 immunocytochemical staining appeared not to be colocalized with NFT in a majority of neurones. Its expression was weak in the dentate gyrus and stronger in CA3-4, two regions with no or low numbers of NFT, but there was no decrease in AD compared to control cases. In the frontal cortex, the ventro-infero-median region (area 12) showed a greater LMO4 expression than the polar one (area 9), but no decrease in AD was observed. As LMO4 has been proposed to inhibit cellular differentiation, it can be hypothesized that a reduced expression is associated in EC and CA1 with attempts of diseased neurones to differentiate (e.g. compensatory neuritogenesis). Taken together, these data indicate that LMO4 protein is involved in the complexity of the disease phenotype, at least as a secondary factor.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Homeodomínio/biossíntese , Fatores de Transcrição/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica , Proteínas com Domínio LIM , Masculino , Pessoa de Meia-Idade , Degeneração Neural/patologia , Emaranhados Neurofibrilares/patologia , Placa Amiloide/patologiaRESUMO
We have investigated how transgenic overexpression of human Bcl-2 (Hu-Bcl-2) modifies cell death proteins activation in the long-term in a model of permanent cerebral ischemia induced by middle cerebral artery occlusion. Hu-Bcl-2, cytochrome c, caspases 9 and 3 expression were examined by immunoblotting and immunohistochemistry. In wild type mice, 1 day after middle cerebral artery occlusion, cytochrome c released from the mitochondria was detected. Middle cerebral artery occlusion induces a lasting activation of caspases in WT mice from day 3 post-injury. Increased level of caspase 3 is accompanied by a decrease in procaspase 3. In contrast, middle cerebral artery occlusion induced a sustained increase of procaspase 9L and a decrease in procaspase 9S concomitant to caspase 9 production. These events were observed in the operated but not in the unoperated hemisphere. Bcl-2 overexpression blocks cytochrome c release and delays caspases activation. Consequently procaspase 3 decrease was no more observed. However, Bcl-2 overexpression did not influence the middle cerebral artery occlusion-induced changes in procaspases 9 L and S. Fourteen days after middle cerebral artery occlusion the apoptotic cascade was no longer blocked in transgenic mice. Caspases 9 and 3 were increased, procaspase 3 was decreased but procaspase 9L and procaspase 9S remained increased and decreased respectively. Hu-Bcl-2 overexpression delays the activation of the cell death molecular machinery but does not control the ischemia-induced change in procaspase 9 L and S. Procaspase 9L increase is a potentially harmful event threatening cells of a rapid destruction when anti-apoptotic treatments by Bcl-2, or caspases inhibitors, are overrun.
Assuntos
Isquemia Encefálica/metabolismo , Caspases/biossíntese , Córtex Cerebral/metabolismo , Precursores Enzimáticos/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Animais , Caspase 9 , Caspases/análise , Córtex Cerebral/química , Indução Enzimática/fisiologia , Precursores Enzimáticos/análise , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
In various animal models of neurodegenerative diseases the long-lasting control of cell death by anti-apoptotic therapies is not successful. We present here our view on the control of procaspase expression in a model of cerebral stroke. We have investigated how Hu-Bcl-2 overexpression modifies cell death protein activation in a model of cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO). In wild type mice MCAO induced release of cytochrome c from the mitochondria, and activation of caspases 9 and 3. In parallel with caspases activation, procaspase 9 and procaspase 3 were, respectively, increased and decreased. In Hu-Bcl-2 transgenic mice cytochrome c release and caspases 9 and 3 activation were blocked. However procaspase 9 increased, like in wt mice, but procaspase 3 remained unchanged. By 2 weeks after MCAO caspases were no longer blocked in Hu-Bcl-2 transgenic mice. Procaspase 9 increase could represent a time bomb in Hu-Bcl-2 mice where caspase 9 activation is blocked. Indeed, cellular accumulation of procaspase 9 is a potentially harmful event able to overcome anti-apoptotic protection by Bcl-2 and threaten cells with rapid destruction. Through understanding of the upstream regulation of procaspase 9, early targets for the pharmacological control of apoptotic cell death may be revealed.
Assuntos
Apoptose/fisiologia , Isquemia Encefálica/enzimologia , Caspases/metabolismo , Precursores Enzimáticos/metabolismo , Doenças Neurodegenerativas/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/fisiopatologia , Caspase 9 , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/fisiopatologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
The etiology of late-onset Alzheimer disease is poorly understood. Predisposing factors such as the apolipoprotein E4 allele, as well as protective factors (e.g., antioxidants) have been proposed to play a role in the disease's process. A search for predisposing factors contributing to sporadic late-onset Alzheimer disease was initiated using the differential display technique. RNA expression profiles of the entorhinal cortex and the cerebellum of Alzheimer-diseased and normal patients were compared. The entorhinal cortex is the first brain region to accumulate neurofibrillary tangles during disease progression, whereas the cerebellum is spared. In the Alzheimer cases of this study, one signal showing preferential expression in the entorhinal cortex corresponded to the apolipoprotein D gene. This preferential expression might be genuine at the RNA level as suggested by the in situ hybridization method used. In addition, immunohistochemical experiments showed higher percentages of Apolipoprotein D reactive pyramidal neurons in the entorhinal cortex and region 1 of Ammon's horn in diseased patients. This increase correlated with the number of neurofibrillary tangles in Alzheimer as well as in normal patients. Colocalization of Apolipoprotein D proteins and neurofibrillary tangles in the same neuron was rare. Thus, these results suggest that in Alzheimer disease and aging, apolipoprotein D gene expression is increased in stressed cortical neurons before they possibly accumulate neurofibrillary tangles.
Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas/metabolismo , Encéfalo/metabolismo , Córtex Entorrinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteínas/genética , Apolipoproteínas D , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Valores de Referência , Distribuição TecidualRESUMO
Frontotemporal dementia (FTD) is the second most common degenerative dementia after Alzheimer's disease and its Lewy body variant. Clinical pathology can be subdivided in three main neuropathological subtypes: frontal lobe dementia, Pick's disease and FTD with motor neuron disease (MND), all characterised by distinct histological features. Until recently the presence of ubiquitin-positive intraneuronal inclusions in the dentate gyrus, and the temporal and frontal cortex was usually associated with the MND type. Such inclusions were also observed in a few sporadic cases of FTD without or with parkinsonism (FTDP) in the absence of MND. We present here clinical, neuropathological and immunohistochemical data about a Swiss FTD family with FTDP-like features but without MND. Spongiosis and mild gliosis were observed in the grey matter. No neurofibrillary tangles, Pick bodies, Lewy bodies, senile plaques or prion-positive signals were present. However, ubiquitin-positive intracytoplasmic inclusions were detected in various structures but predominantly in the dentate gyrus. These observations support the existence of a familial form of FTDP with ubiquitin-positive intracytoplasmic inclusions (Swiss FTDP family).
Assuntos
Demência/patologia , Corpos de Inclusão/química , Neurônios Motores/patologia , Proteínas do Tecido Nervoso/análise , Ubiquitinas/análise , Idoso , Demência/genética , Giro Denteado/patologia , Feminino , Genes Dominantes , Gliose/patologia , Humanos , Masculino , Linhagem , Suíça , SíndromeRESUMO
Frontotemporal dementia (FTD) is considered to have a heterogeneous aetiology. To date the tau gene located on chromosome 17 has been shown to be implicated in the pathogenesis of several FTD families with parkinsonism, the so called FTDP-17 families. The mutations reported so far are located within exons 9 to 13, a region coding for the microtubule-binding sites. They are causing various cytoskeletal disturbances. We are describing here the main clinical and neuropathological features of a Swiss FTD family with members presenting a FTDP-like clinical phenotype. However, if we except two silent polymorphic sites at position 227 and 255 in exon 9, neither a known FTDP-17 mutation nor a novel one was detected in this region of the tau gene. Thus, the existence of a yet unknown mechanism of neurodegeneration, other than via mutations near or within the microtubule-binding sites, or the exon 10 splice sites of the tau gene, has to be considered to explain dementia in this family. A mutation in another gene is still possible.
Assuntos
Cromossomos Humanos Par 17 , Demência/genética , Saúde da Família , Mutação , Proteínas tau/genética , Sítios de Ligação/genética , Análise Mutacional de DNA , Primers do DNA , Demência/patologia , Éxons , Feminino , Heterogeneidade Genética , Humanos , Masculino , Microtúbulos/metabolismo , Linhagem , Splicing de RNA , Suíça , Proteínas tau/metabolismoAssuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Príons/metabolismo , Idade de Início , Idoso , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Pessoa de Meia-Idade , Príons/genéticaRESUMO
Mutations have been found in more than a hundred early-onset families with Alzheimer's disease (AD) in the genes for the amyloid precursor protein, presenilin 1 and presenilin 2. The object of our investigation was to identify if these mutations or novel ones were operating in a Swiss early-onset AD family (mean age of onset: 53.3 years) with 7 members available, all neuropathologically confirmed. No known or new mutations were detected. Thus, our data support the existence of a yet unknown mutation, or other genes, contributing to familial early-onset AD. CopyrightCopyright 1999S.KargerAG,Basel
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Genes Dominantes/genética , Proteínas de Membrana/genética , Adulto , Idade de Início , Idoso , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , DNA/genética , Primers do DNA , Éxons/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Linhagem , Presenilina-1 , Presenilina-2 , Escalas de Graduação Psiquiátrica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuíçaRESUMO
We have previously reported that presenilin-1 (PS-1)-immunoreactive neurons survive in late-onset sporadic Alzheimer's disease (AD). To examine if this is also the case in other dementing conditions, and if it is associated with changes in the expression of the main apoptosis-related proteins, a quantitative immunocytochemical study of presenilin-1, Bax, and Bcl-X(L) in the cerebral cortex of non-demented and AD patients, and patients with frontotemporal dementia (FTD) was performed. In non-demented cases, the frequency of neurons showing PS-1 immunoreactivity was 25-60%, Bax immunoreactivity 36-54%, and Bcl-X(L) immunoreactivity 26-63% depending on the cortical area. The frequency of NFT-free neurons which contained PS-1 or Bax was consistently increased in all of the areas in AD. In FTD cases, the percentage of PS-I-, but not Bax-immunoreactive neurons was increased only in areas displaying a substantial neuronal loss. Conversely, there was no difference in the densities of Bcl-X(L)-containing neurons among the three diagnosis groups. These data suggest that surviving neurons in affected cortical areas in AD show a high expression of PS-1 and Bax, indicating that these proteins play a key role in the mechanisms of cell death in this disorder. In FTD, neurons containing PS-1 are preserved, further supporting a neuroprotective role for this protein in other neurodegenerative disorders.
Assuntos
Doença de Alzheimer/metabolismo , Demência/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Lobo Frontal/fisiopatologia , Humanos , Imuno-Histoquímica , Masculino , Presenilina-1 , Valores de Referência , Lobo Temporal/fisiopatologia , Distribuição Tecidual/fisiologia , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Primers specific to the long isoform of leptin receptor (OB-Rb) mRNA were used in reverse transcriptase-linked polymerase chain reactions (RT-PCR) to investigate the expression of this receptor in the hypothalamus and cerebellum of human and rat. For both regions, we observed RT-PCR cDNAs as well as restriction enzyme cleavage fragments of expected sizes. Additionally, in situ hybridization of human cerebellum using two independent [35S]oligonucleotide probes complementary to the OB-Rb mRNA sequence revealed a prominent hybridization signal within the granular layer. Overall, our findings demonstrate the expression of OB-Rb mRNA in the cerebellum and suggest that in such a location, leptin receptors may mediate a function presumably not linked to body weight homeostasis.
Assuntos
Proteínas de Transporte/genética , Cerebelo/metabolismo , Obesidade , Splicing de RNA , RNA Mensageiro/biossíntese , Receptores de Superfície Celular , Receptores de Citocinas/genética , Animais , Humanos , Hibridização In Situ , Ratos , Receptores para LeptinaRESUMO
In this report we describe the developmental expression of the murine (Mobp) gene encoding myelin-associated oligodendrocytic basic protein. We have characterized three Mobp cDNA clones which have been used as probes. Murine Mobp splice variant-1 (mmsv-1), a portion of 3' untranslated region (UTR), is homologous to 3' UTR sequences found in the rat Mobp splice variants rOP1, Mobp81-A and Mobp-99. The mmsv-2 sequence, encoding 81 amino acids, closely resembles the rat Mobp81-A splice variant. The mmsv-3 cDNA, encoding 170 amino acids corresponding closely to the rat rOPRP1 splice variant, detects a single mRNA species present in low levels from E12 onward, suggesting this MOBP may have a function alternative or additional to involvement in myelin formation. The mmsv-1 probe detects an mRNA species abundantly expressed in the postnatal central nervous system (CNS) but barely detectable at E18. This mRNA is located initially in the cell bodies of oligodendrocytes, moving distally into their processes as myelination proceeds. The most abundant mmsv(s) in the adult CNS are present at detectable levels after expression of the myelin basic protein (Mbp) gene and marginally after or coincident with the proteolipid protein (Plp) gene. The level of the abundant, late-expressed mRNA correlates closely with the capacity to form myelin and the maturity of oligodendrocytes, as shown in two hypomyelinated mutants, rumpshaker and jimpy, which represent mildly and severely affected phenotypes, respectively.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glicoproteína Associada a Mielina/genética , Oligodendroglia , Animais , Northern Blotting , Encéfalo/metabolismo , Senescência Celular/genética , Clonagem Molecular , DNA Complementar/isolamento & purificação , Camundongos , Mutação , Proteínas da Mielina , Bainha de Mielina/fisiologia , Glicoproteína Mielina-Oligodendrócito , Reação em Cadeia da Polimerase/métodos , Splicing de RNA , Ratos , Especificidade da Espécie , Transcrição GênicaRESUMO
We report a method providing access to high molecular weight, polymerase chain reaction (PCR)-amplifiable genomic DNA from brains stored in formalin for many years. It consists mainly of an intensive proteinase K treatment of ground tissue previously embedded in agarose plugs, followed by a washing and an elution step. The method was tested on brains fixed and stored in formalin for up to 46 years. All extracted DNA show an identical pattern of degradation ranging from well-preserved (more than 20 kb) to 400-bp-long fragments. This was demonstrated for DNA extracted from the cerebellums of elderly psychiatric and geriatric patients (of more than 60 years of age), male and female, demented or not, with postmortem delays longer than 1 h and shorter than 1 day. In all these cases PCR amplification of a 838-bp-long beta-actin product was successfully performed when proteinase K treatment was sufficiently effective to generate pure DNA. Thus, high molecular weight, PCR-amplifiable genomic DNA can be extracted from brains stored in formalin for almost half a century.
Assuntos
Química Encefálica/genética , DNA/isolamento & purificação , Formaldeído , Fixação de Tecidos/métodos , Idoso , Idoso de 80 Anos ou mais , Cerebelo/química , Endopeptidase K , Feminino , Genoma Humano , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Estudos RetrospectivosRESUMO
The use of subtracted, region-specific cDNA libraries combined with single-pass cDNA sequencing allows the discovery of novel genes and facilitates molecular description of the tissue or region involved. We report the sequence of 497 mouse expressed sequence tags (ESTs) from two subtracted libraries enriched for cDNAs expressed in the substantia nigra, a brain region with important roles in movement control and Parkinson disease. Of these, 238 ESTs give no database matches and therefore derive from novel genes. A further 115 ESTs show sequence similarity to ESTs from other organisms, which themselves do not yield any significant database matches to genes of known function. Fifty-six ESTs show sequence similarity to previously identified genes whose mouse homologues have not been reported. The total number of ESTs reported that are new for the mouse is 407, which, together with the 90 ESTs corresponding to known mouse genes or cDNAs, contributes to the molecular description of the substantia nigra.
Assuntos
Encéfalo/metabolismo , DNA Complementar , Substância Negra/metabolismo , Animais , Embrião de Galinha , DNA Complementar/genética , Camundongos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoAssuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Apolipoproteínas E/genética , Proteínas de Membrana/genética , Distribuição por Idade , Idoso , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 21/genética , Humanos , Presenilina-1 , Fatores de RiscoAssuntos
Mapeamento Cromossômico , Camundongos Endogâmicos C57BL/genética , Muridae/genética , Glicoproteína Associada a Mielina/genética , Animais , Cruzamentos Genéticos , Primers do DNA , Marcadores Genéticos , Camundongos , Proteínas da Mielina , Glicoproteína Mielina-Oligodendrócito , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Recombinação GenéticaAssuntos
Demência , Fatores Etários , Demência/epidemiologia , Demência/genética , Demência/patologia , HumanosRESUMO
Many important phenomena of normal brain physiology and disease are likely to be related to the function of genes expressed in localised regions of the brain. We show that subtracted libraries enriched in clones corresponding to rare mRNAs, which must include genes with very localised and neuron-specific expression, can easily be produced from single-stranded directional cDNA libraries after hybridization to excess photobiotinylated opposite-stranded cDNA (or RNA) from another brain region, followed by the removal of biotinylated molecules. We also demonstrate the use of heterologous probes from anatomically precise small regions of bovine brain to identify cDNA clones that putatively represent mRNAs present at significantly higher levels in a substantia nigra mRNA population enriched for pars compacta mRNA than in the total ventral midbrain or cerebellar mRNA population. Some of these cDNAs may identify genes that play important roles in the specific molecular biology of dopaminergic neurons, including susceptibility to Parkinson's disease.
Assuntos
DNA Complementar/isolamento & purificação , Biblioteca Gênica , Hibridização de Ácido Nucleico , Substância Negra/química , Animais , Bovinos , Cerebelo/química , Clonagem Molecular/métodos , Sondas de DNA , DNA Complementar/biossíntese , Dopamina/genética , Dopamina/metabolismo , Marcação de Genes/métodos , Mesencéfalo/química , Camundongos , Neurônios/fisiologia , Especificidade de Órgãos , Doença de Parkinson/genética , RNA Mensageiro/análise , Reprodutibilidade dos TestesRESUMO
A cDNA encoding the mouse homologue to the rat and human component C5 of proteasome was isolated from a mouse ventral midbrain library. The deduced amino acid sequence shows 93% and 97.5% identity to the human and rat C5 component respectively.
Assuntos
Cisteína Endopeptidases/genética , DNA Complementar , Camundongos/genética , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Biblioteca Gênica , Humanos , Mesencéfalo/metabolismo , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Ratos , Alinhamento de Sequência , Substância Negra/metabolismoRESUMO
To use single-pass cDNA sequencing to characterize low-frequency cDNA clones from a region of the brain that includes the primary site of neurodegeneration in human Parkinson disease, we have developed a prescreening procedure using single brain region first-strand cDNA probes. Selection of cDNA clones giving low hybridization signals allowed the elimination of clones resulting from abundant messages and enrichment for clones corresponding to low-copy messages. Comparative sequencing of standard and prescreened cDNA libraries (191 and 124 clones, respectively) showed that this procedure raised the frequency of novel sequences encountered from 54 to 81%. The increased proportion of novel ESTs justifies the labor of prescreening. Automation of this procedure will accelerate the molecular description of genes expressed in any brain region, or any tissue, and represents a way to maximize access to cDNA sequences for human and mouse genome characterization. In total, the comparative sequencing experiments generated 207 new mouse and 11 new rat brain ESTs.