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2.
Genes Brain Behav ; 7(5): 560-7, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18363856

ABSTRACT

Physiological dependence and associated withdrawal episodes are thought to constitute a motivational force perpetuating continued alcohol use/abuse. Although no animal model duplicates alcoholism, models for specific factors, like the withdrawal syndrome, are useful to identify potential determinants of liability in humans. We previously detected quantitative trait loci (QTLs) with large effects on predisposition to physical dependence and associated withdrawal following chronic or acute alcohol exposure to a large region of chromosome 1 in mice (Alcdp1 and Alcw1, respectively). Here, we provide the first confirmation of Alcw1 in a congenic strain, and, using interval-specific congenic strains, narrow its position to a minimal 1.1 Mb (maximal 1.7 Mb) interval syntenic with human chromosome 1q23.2-23.3. We also report the development of a small donor segment congenic that confirms capture of a gene(s) affecting physical dependence after chronic alcohol exposure within this small interval. This congenic will be invaluable for determining whether this interval harbors a gene(s) involved in additional alcohol responses for which QTLs have been detected on distal chromosome 1, including alcohol consumption, alcohol-conditioned aversion and -induced ataxia. The possibility that this QTL plays an important role in such diverse responses to alcohol makes it an important target. Moreover, human studies have identified markers on chromosome 1q associated with alcoholism, although this association is still suggestive and mapped to a large region. Thus, the fine mapping of this QTL and analyses of the genes within the QTL interval can inform developing models for genetic determinants of alcohol dependence in humans.


Subject(s)
Alcohol Withdrawal Seizures/genetics , Alcoholism/genetics , Chromosome Mapping , Chromosomes, Mammalian , Quantitative Trait Loci , Acute Disease , Animals , GABA Modulators/pharmacology , Genetic Predisposition to Disease/genetics , Genotype , Humans , Mice , Mice, Congenic , Mice, Inbred C57BL , Mice, Inbred DBA , Pentobarbital/pharmacology , Phenotype , Substance Withdrawal Syndrome/genetics
3.
Genes Brain Behav ; 7(2): 214-23, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17696997

ABSTRACT

We report here the confirmation of the quantitative trait locus for haloperidol-induced catalepsy on distal chromosome (Chr) 1. We determined that this quantitative trait locus was captured in the B6.D2-Mtv7a/Ty congenic mouse strain, whose introgressed genomic interval extends from approximately 169.1 to 191.3 Mb. We then constructed a group of overlapping interval-specific congenic strains to further break up the interval and remapped the locus between 177.5 and 183.4 Mb. We next queried single nucleotide polymorphism (SNP) data sets and identified three genes with nonsynonymous coding SNPs in the quantitative trait locus. We also queried two brain gene expression data sets and found five known genes in this 5.9-Mb interval that are differentially expressed in both whole brain and striatum. Three of the candidate quantitative trait genes were differentially expressed using quantitative real-time polymerase chain reaction analyses. Overall, the current study illustrates how multiple approaches, including congenic fine mapping, SNP analysis and microarray gene expression screens, can be integrated both to reduce the quantitative trait locus interval significantly and to detect promising candidate quantitative trait genes.


Subject(s)
Catalepsy/genetics , Chromosome Mapping , Corpus Striatum/pathology , Haloperidol/toxicity , Mice, Inbred Strains/genetics , Quantitative Trait Loci , Animals , Catalepsy/chemically induced , Catalepsy/pathology , Crosses, Genetic , DNA/genetics , DNA/isolation & purification , Female , Male , Mice , Microsatellite Repeats , Oligonucleotide Array Sequence Analysis , Polymorphism, Genetic , Posture , RNA/genetics , RNA/isolation & purification , Species Specificity
4.
J Biol Rhythms ; 18(6): 450-62, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14667146

ABSTRACT

Provisional quantitative trait loci (QTL) for circadian locomotor period and wheel-running period have been identified in recombinant inbred (RI) mouse strains. To confirm those QTL and identify new ones, the genetic component of variance of the circadian period was partitioned among an F2 intercross of RI mouse strains (BXD19 and CXB07). First, a genomic survey using 108 SSLP markers with an average spacing of 15 cM was carried out in a population of 259 (BXD19 x CXB07)F2 animals. The genome-wide survey identified two significant QTL for period of locomotor activity measured by infrared photobeam crossings on mouse chromosomes 1 (lod score 5.66) and 14 (lod score 4.33). The QTL on distal chromosome 1 confirmed a previous report based on congenic B6.D2-Mtv7a/Ty mice. Lod scores greater than 2.0 were found on chromosomes 1, 2, 6, 12, 13, and 14. In a targeted extension study, additional genotyping was performed on these chromosomes in the full sample of 341 F2 progeny. The 6 chromosome-wide surveys identified 3 additional QTL on mouse chromosomes 6, 12, and 13. The QTL on chromosome 12 overlaps with circadian period QTL identified in several prior studies. For wheel-running period, the chromosome-wide surveys identified QTL on chromosomes 2 and 13 and one highly suggestive QTL on proximal chromosome 1. The results are compared to other published studies of QTL of circadian period.


Subject(s)
Circadian Rhythm/genetics , Circadian Rhythm/physiology , Genetic Variation , Motor Activity/physiology , Quantitative Trait Loci , Animals , Epistasis, Genetic , Female , Genome , Genotype , Male , Mice , Mice, Inbred Strains , Phenotype
5.
Cell Death Differ ; 10(6): 698-708, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12761578

ABSTRACT

The aberrant splicing isoform (PS2V), generated by exon 5 skipping of the Presenilin-2 (PS2) gene transcript, is a diagnostic feature of sporadic Alzheimer's disease (AD). We found PS2V is hypoxia-inducible in human neuroblastoma SK-N-SH cells. We purified a responsible trans-acting factor based on its binding to an exon 5 fragment. The factor was identified as the high mobility group A1a protein (HMGA1a; formerly HMG-I). HMGA1a bound to a specific sequence on exon 5, located upstream of the 5' splice site. HMGA1a expression was induced by hypoxia and the protein was accumulated in the nuclear speckles with the endogenous splicing factor SC35. Overexpression of HMGA1a generated PS2V, but PS2V was repressed by cotransfection with the U1 snRNP 70K protein that has a strong affinity to HMGA1a. HMGA1a could interfere with U1 snRNP binding to the 5' splice site and caused exon 5 skipping. HMGA1a levels were significantly increased in the brain tissue from sporadic AD patients. We propose a novel mechanism of sporadic AD that involves HMGA1a-induced aberrant splicing of PS2 pre-mRNA in the absence of any mutations.


Subject(s)
Alternative Splicing/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , HMGA1a Protein/metabolism , Membrane Proteins/genetics , RNA Precursors/genetics , Base Sequence/genetics , Binding Sites/genetics , Exons/genetics , Genes, Regulator/genetics , HMGA1a Protein/genetics , Humans , Hypoxia/genetics , Hypoxia/metabolism , Membrane Proteins/biosynthesis , Mutation/genetics , Presenilin-2 , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA Splice Sites/genetics , RNA, Small Nuclear/genetics
6.
J Biol Chem ; 276(52): 48908-14, 2001 Dec 28.
Article in English | MEDLINE | ID: mdl-11684676

ABSTRACT

We have characterized two RNA-binding proteins, of apparent molecular masses of approximately 40 and 35 kDa, which possess a single N-terminal RNA-recognition motif (RRM) followed by a C-terminal domain rich in serine-arginine dipeptides. Their primary structures resemble the single-RRM serine-arginine (SR) protein, SC35; however their functional effects are quite distinctive. The 40-kDa protein cannot complement SR protein-deficient HeLa cell S100 extract and showed a dominant negative effect in vitro against the authentic SR proteins, SF2/ASF and SC35. Interestingly, the 40- and 35-kDa proteins antagonize SR proteins and activate the most distal alternative 5' splice site of adenovirus E1A pre-mRNA in vivo, an activity that is similar to that characterized previously for the heterogeneous nuclear ribonucleoprotein particles A/B group of proteins. A series of recombinant chimeric proteins consisting of domains from these proteins and SC35 in various combinations showed that the RRM, but not the C-terminal domain rich in serine-arginine dipeptides, has a dominant role in this activity. Because of the similarity to SR proteins we have named these proteins SRrp40 and SRrp35, respectively, for SR-repressor proteins of approximately 40 and approximately 35 kDa. Both factors show tissue- and cell type-specific patterns of expression. We propose that these two proteins are SR protein-like alternative splicing regulators that antagonize authentic SR proteins in the modulation of alternative 5' splice site choice.


Subject(s)
Alternative Splicing/genetics , Neoplasm Proteins , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , 3T3 Cells , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Cell Cycle Proteins , Cell Nucleus/metabolism , Cloning, Molecular , Genes, Reporter , HeLa Cells , Humans , Immunohistochemistry , Mice , Molecular Sequence Data , Molecular Weight , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Sequence Alignment , Serine-Arginine Splicing Factors , Tissue Distribution
7.
J Virol ; 75(18): 8487-97, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11507194

ABSTRACT

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.


Subject(s)
3' Untranslated Regions , Exons , Gene Silencing , HIV-1/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , RNA Splicing , RNA, Viral , Ribonucleoproteins/metabolism , 5' Untranslated Regions , Binding Sites , Consensus Sequence , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Mutagenesis
8.
J Biol Chem ; 276(34): 32300-12, 2001 Aug 24.
Article in English | MEDLINE | ID: mdl-11438536

ABSTRACT

We report here the isolation and characterization of two proteins, NFAR-1 and -2, which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. The NFAR proteins, of 90 and 110 kDa, are derived from a single gene through alternative splicing and are evolutionarily conserved nuclear phosphoproteins that interact with double-stranded RNA. Both NFAR-1 and -2 are phosphorylated by PKR, reciprocally co-immunoprecipitate with PKR, and colocalize with the kinase in a diffuse nuclear pattern within the cell. Transfection studies indicate that the NFARs regulate gene expression at the level of transcription, probably during the processing of pre-mRNAs, an activity that was increased in fibroblasts lacking PKR. Subsequent functional analyses indicated that amino acids important for NFAR's activity were localized to the C terminus of the protein, a region that was found to specifically interact with FUS and SMN, proteins also known as regulators of RNA processing. Accordingly, both NFARs were found to associate with both pre-mRNAs and spliced mRNAs in post-transcriptional studies, similar to the known splicing factor ASF/SF-2. Collectively, our data indicate that the NFARs may facilitate double-stranded RNA-regulated gene expression at the level of post-transcription and possibly contribute to host defense-related mechanisms in the cell.


Subject(s)
Alternative Splicing , Evolution, Molecular , Phosphoproteins , Protein Isoforms/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , eIF-2 Kinase/metabolism , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cell Nucleus/metabolism , DNA Primers , DNA, Complementary , Molecular Sequence Data , Nuclear Factor 90 Proteins , Precipitin Tests , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/physiology , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Substrate Specificity
9.
J Cell Biol ; 154(1): 25-32, 2001 Jul 09.
Article in English | MEDLINE | ID: mdl-11448987

ABSTRACT

The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-beta1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF(35). Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.


Subject(s)
Alternative Splicing , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/physiology , Spliceosomes/physiology , Active Transport, Cell Nucleus , Amino Acid Sequence , Arginine/chemistry , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Cloning, Molecular , Conserved Sequence , Dose-Response Relationship, Drug , Fluorescent Antibody Technique, Indirect , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Models, Genetic , Molecular Sequence Data , Plasmids/metabolism , Precipitin Tests , Protein Structure, Tertiary , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Serine/chemistry , Transfection , Two-Hybrid System Techniques
10.
Rheumatology (Oxford) ; 40(1): 70-3, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11157144

ABSTRACT

OBJECTIVE: To determine ethnic variations of large-joint osteoarthritis (OA) in past populations. METHODS: One thousand two hundred and nine adult skeletons, excavated from archaeological sites in Japan, China and France were assessed for OA as defined by the presence of eburnation. RESULTS: Within Asian skeletal populations, elbow OA and patellofemoral joint OA were more common in hunter-gatherers than in agriculturalists. Compared with Caucasians, the Asian skeletal population had a higher prevalence of tibiofemoral joint OA. CONCLUSION: The relative frequencies of OA within and between ethnic groups at certain joint sites have changed over time from the past to the present.


Subject(s)
Osteoarthritis/history , Adult , Asian People , China/epidemiology , Female , France/epidemiology , History, Ancient , Humans , Japan/epidemiology , Male , Middle Aged , Osteoarthritis/epidemiology , Osteoarthritis/ethnology , Paleopathology , Prevalence , Skeleton , White People
11.
Mol Cell ; 8(6): 1351-61, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11779509

ABSTRACT

SR proteins recognize exonic splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to exonic splicing silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.


Subject(s)
Exons/genetics , Gene Silencing , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , RNA Splicing/genetics , Regulatory Sequences, Nucleic Acid/genetics , Ribonucleoproteins/antagonists & inhibitors , Base Sequence , Binding Sites , Cell Extracts , Gene Products, tat/genetics , Genetic Complementation Test , Glycine/metabolism , HIV-1/genetics , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Kinetics , Models, Genetic , Mutation/genetics , Protein Binding , Protein Structure, Tertiary , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA-Binding Proteins , Ribonucleoproteins/chemistry , Ribonucleoproteins/deficiency , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors , Substrate Specificity , tat Gene Products, Human Immunodeficiency Virus
12.
Mol Cell Biol ; 20(22): 8303-18, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11046128

ABSTRACT

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nuclear Proteins/metabolism , RNA Splice Sites , Ribonucleoprotein, U1 Small Nuclear/metabolism , Ribonucleoproteins/metabolism , Alternative Splicing , Binding Sites , Binding, Competitive , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Models, Biological , Nuclear Proteins/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/genetics , Ribonucleoproteins/genetics , Serine-Arginine Splicing Factors
13.
J Biol Chem ; 275(37): 29170-7, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10880506

ABSTRACT

Splicing of the last intron (intron D) of the bovine growth hormone pre-mRNA requires the presence of a downstream exonic splicing enhancer (ESE). This enhancer is contained within a 115-nucleotide FspI-PvuII (FP) fragment located in the middle of the last exon (exon 5). Previous work showed that the splicing factor SF2/ASF binds to this FP region and stimulates splicing of intron D in vitro. However, the precise sequences recognized by SF2/ASF within the FP region had not been determined. Here we used multiple strategies to map the SF2/ASF binding sites and determine their importance for ESE function. Taking advantage of the fact that SF2/ASF ultraviolet (UV) cross-links specifically to RNA containing the FP sequence, we first mapped a major SF2/ASF binding site by UV cross-linking and reverse transcription. This strategy identified a 29-nucleotide SF2/ASF binding region in the middle of the FP sequence containing the 7-nucleotide purine-rich motif described previously. Interestingly, this binding region is neither sufficient, nor absolutely required for SF2/ASF-mediated splicing, suggesting that additional SF2/ASF binding sites are present. The location of these additional sites was determined by electrophoretic mobility shift analysis of various subfragments of the FP sequence. Antisense 2'-O-methyl oligoribonucleotides complementary to selected SF2/ASF binding sites block bovine growth hormone intron D splicing. Thus, multiple SF2/ASF binding sites within the exonic splicing enhancer contribute to maximal enhancer activity.


Subject(s)
Enhancer Elements, Genetic , Exons , Growth Hormone/genetics , Nuclear Proteins/metabolism , RNA Splicing , Animals , Binding Sites , Cattle , HeLa Cells , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Introns , RNA Precursors/metabolism , RNA-Binding Proteins , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors
14.
Arch Gen Psychiatry ; 57(6): 572-9, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10839335

ABSTRACT

BACKGROUND: Previous studies have suggested that bipolar patients are supersensitive to light suppression of melatonin and that this may be a trait marker for genetic vulnerability. The present study was an attempt to replicate and extend this observation. Propranolol hydrochloride effects were compared with light effects because of the documented influence of beta-adrenergic receptors on melatonin production. Nighttime levels of corticotropin and cortisol were also examined as potential trait vulnerability markers. METHODS: Melatonin levels in euthymic bipolar patients (n= 29) were tested before and after 500-lux light was administered between 2 and 4 AM and on a separate night in the dark. Results were compared with those of a group of patients with unipolar depression (n= 24) and with those of a group of non-psychiatrically ill control subjects (n= 50). Lithium effects and propranolol effects were tested in subgroups. RESULTS: No group differences were seen in light suppression among bipolar patients, unipolar patients, and controls; an analysis of the whole group did not reveal differences in propranolol effect, differences in corticotropin or cortisol levels, or evidence for a lithium effect. However, patients with bipolar I affective disorder showed the following: (1) significantly lower melatonin levels on the light night, at baseline and following light exposure; and (2) a later peak time for melatonin on the dark night. CONCLUSIONS: The general hypothesis of increased light sensitivity in bipolar patients was not supported. However, melatonin secretion abnormalities were confirmed in the subgroup with bipolar I disorder. Further assessments of circadian rhythm disruption as a vulnerability marker in bipolar illness are indicated.


Subject(s)
Bipolar Disorder/blood , Bipolar Disorder/genetics , Circadian Rhythm/physiology , Depressive Disorder/blood , Depressive Disorder/genetics , Light , Melatonin/blood , Adrenocorticotropic Hormone/blood , Adult , Bipolar Disorder/diagnosis , Circadian Rhythm/drug effects , Circadian Rhythm/genetics , Depressive Disorder/diagnosis , Female , Genetic Markers , Genetic Predisposition to Disease , Humans , Hydrocortisone/blood , Lithium/pharmacology , Male , Melatonin/metabolism , Middle Aged , Photic Stimulation , Propranolol/pharmacology , Radioimmunoassay
15.
Behav Genet ; 29(3): 171-6, 1999 May.
Article in English | MEDLINE | ID: mdl-10547923

ABSTRACT

Many genes support the manifestation of the circadian period in mice. In a multiple-gene trait all genes contributing in a minor way to this characteristic are quantitative trait loci (QTL). Screens of both the BXD and the CXB panels of recombinant inbred mice suggested that distal chromosome 1, between 90 and 100 cM, contained a QTL, Cplaq3, for a difference in the circadian period of locomotor activity between the C57BL/6J and the DBA/2J and between the BALB/cBy and the C57BL/6By progenitor strains. The mice studied were a commercially available congenic strain, B6.D2-Mtv7a/Ty, from 50 to 100 days old. This congenic strain contains a small DBA/2J genomic insert that covers the region of the provisional QTL in a 99.9% C57BL/6J background. The congenic mice had a shorter period than C57BL/6J mice, confirming that this region has a QTL for the difference in period between the C57BL/6J and the DBA/2J strains. In addition, these data suggest that this region has a QTL for the mean amount of daily activity and for the pattern of locomotor activity.


Subject(s)
Alleles , Mice, Inbred Strains/genetics , Models, Genetic , Motor Activity/genetics , Phenotype , Animals , Circadian Rhythm/genetics , Female , Genotype , Male , Mice , Recombination, Genetic/genetics
18.
Mol Biochem Parasitol ; 102(1): 103-15, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10477180

ABSTRACT

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.


Subject(s)
Fungal Proteins/chemistry , Fungal Proteins/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Trans-Splicing/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Protozoan/analysis , Fluorescent Antibody Technique , Fungal Proteins/metabolism , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Protozoan/metabolism , RNA, Spliced Leader/genetics , RNA-Binding Proteins/metabolism , Transcription, Genetic
19.
Proc Natl Acad Sci U S A ; 96(19): 10655-60, 1999 Sep 14.
Article in English | MEDLINE | ID: mdl-10485881

ABSTRACT

Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. The in vitro splicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF. These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing.


Subject(s)
Catalysis , Enhancer Elements, Genetic/physiology , RNA Precursors/metabolism , RNA Splicing/physiology , Adenosine Triphosphate/pharmacology , Adenoviridae/genetics , Escherichia coli/genetics , Exons , Immunoglobulin M/metabolism , Kinetics , Models, Genetic , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotides/metabolism , RNA-Binding Proteins , Serine-Arginine Splicing Factors , Time Factors , Trans-Splicing/physiology
20.
EMBO J ; 18(16): 4560-70, 1999 Aug 16.
Article in English | MEDLINE | ID: mdl-10449421

ABSTRACT

Biochemical purification of a pre-mRNA splicing activity from HeLa cells that stimulates distal alternative 3' splice sites in a concentration-dependent manner resulted in the identification of RNPS1, a novel general activator of pre-mRNA splicing. RNPS1 cDNAs, encoding a putative nucleic-acid-binding protein of unknown function, were previously identified in mouse and human. RNPS1 is conserved in metazoans and has an RNA-recognition motif preceded by an extensive serine-rich domain. Recombinant human RNPS1 expressed in baculovirus functionally synergizes with SR proteins and strongly activates splicing of both constitutively and alternatively spliced pre-mRNAs. We conclude that RNPS1 is not only a potential regulator of alternative splicing but may also play a more fundamental role as a general activator of pre-mRNA splicing.


Subject(s)
DNA-Binding Proteins/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Ribonucleoproteins , Alternative Splicing , Amino Acid Sequence , Animals , Cell Line , DNA-Binding Proteins/isolation & purification , HeLa Cells , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Processing, Post-Translational , RNA Precursors , RNA-Binding Proteins/isolation & purification , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Serine-Arginine Splicing Factors , Spodoptera , Subcellular Fractions
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