Biomedical subjects

A Watakabe

Publications and source records attributed to A Watakabe.

9 recordsLinked to original sources

PubMed2001

Similarity and variation in gene expression among human cerebral cortical subregions revealed by DNA macroarrays: technical consideration of RNA expression profiling from postmortem samples.

The functional regionality of the human cerebral cortex suggests that a set of genes might be activated in each subregion of the neocortex to support its specific functions. To test this hypothesis, we employed the DNA array technique to compare the mRNA expression profiles of three neocortical subregions of the human brain: prefrontal cortex (Area 46), motor cortex (Area 4) and visual cortex (Area 17). The macroarray analysis on high quality mRNA from postmortem brains revealed that the expression profiles of the different cortical areas are almost similar: only six out of 1088 known genes exhibited significant differences (>2-fold) in their expression. RT-PCR studies with an increased number of samples confirmed that expression of only two genes, annexin II and early growth response protein 1, varied by 2-fold among the regions, whereas expression of the others showed large inter-individual difference. These results suggest that the whole neocortex of humans is more homogeneous than we expected at the level of gross gene expression profiles. In parallel, sensitivity and accuracy of radioisotope-based DNA macroarrays and fluorescence-based DNA microarrays were tested.

Aged↗

PubMed2001

The occ1 gene is preferentially expressed in the primary visual cortex in an activity-dependent manner: a pattern of gene expression related to the cytoarchitectonic area in adult macaque neocortex.

Marker molecules to visualize specific subsets of neurons are useful for studying the functional organization of the neocortex. One approach to identify such molecular markers is to examine the differences in molecular properties among morphologically and physiologically distinct neuronal cell types. We used differential display to compare mRNA expression in the anatomically and functionally distinct areas of the adult macaque neocortex. We found that a gene, designated occ1, was preferentially transcribed in the posterior region of the neocortex, especially in area 17. Complete sequence analysis revealed that occ1 encodes a macaque homolog of a secretable protein, TSC-36/follistatin-related protein (FRP). In situ hybridization histochemistry confirmed the characteristic neocortical expression pattern of occ1 and showed that occ1 transcription is high in layers II, III, IVA and IVC of area 17. In addition, occ1 transcription was observed selectively in cells of the magnocellular layers in the lateral geniculate nucleus (LGN). Dual labeling immunohistochemistry showed that the occ1-positive neurons in area 17 include both gamma-aminobutyric acid (GABA)-positive aspiny inhibitory cells and the alpha-subunit of type II calcium/calmodulin-dependent protein kinase (CaMKII alpha)-positive spiny excitatory cells. With brief periods of monocular deprivation, the occ1 mRNA level decreased markedly in deprived ocular dominance columns of area 17. From this we conclude that the expression of occ1 mRNA is present in a subset of neurons that are preferentially localized in particular laminae of area 17 and consist of various morphological and physiological neuronal types, and, furthermore, occ1 transcription is subject to visually driven activity-dependent regulation.

Age Factors↗

PubMed2001

Growth/differentiation factor 7 is preferentially expressed in the primary motor area of the monkey neocortex.

We applied a differential display PCR technique to isolate molecules that are area-specific in expression in the primate neocortex, and found that growth/differentiation factor 7 (GDF7), a member of the bone morphogenetic protein (BMP)/transforming growth factor (TGF) beta super-family, is preferentially expressed in the primary motor area of African green monkeys (Cercopithecus aethiops). We proved that GDF7 is 10 times more abundant in the motor cortex than in the visual cortex by northern blotting and quantitative RT-PCR. When we examined the neocortex of closely related rhesus monkeys (Macaca mulatta), GDF7 was also most abundant in the motor cortex, although the regional difference was reduced to 3-fold. This differential expression pattern was observed in both newborn and infant rhesus monkeys. We found that several type I/II receptors of BMP, candidates of the receptors for GDF7, are uniformly expressed in the mature neocortex. The unique expression pattern of GDF7 suggests that it may play an active role in the motor area of the primate neocortex.

Aging↗

PubMed1996

N-tropomodulin: a novel isoform of tropomodulin identified as the major binding protein to brain tropomyosin.

We have identified and characterized two proteins in rat brain that bind to the neuron-specific tropomyosin isoform, TMBr3. The two proteins were identified by blot overlay assay, in which the proteins immobilized on the membrane were probed by epitope-tagged TMBr3, followed by detection with anti-epitope antibody. We have purified these proteins using a TMBr3 affinity column. Peptide sequencing as well as immunoblotting showed that one of the two proteins is identical to tropomodulin, a tropomyosin-binding protein originally identified in erythrocytes. The cDNA for the other protein was cloned from an adult rat brain cDNA library using degenerate oligonucleotides that we designed based on the peptide sequences. Sequence analysis of the cDNA clone revealed this protein to be a novel isoform of tropomodulin which is the product of a distinct gene, and is herein referred to as N-tropomodulin. Recombinant N-tropomodulin bound to TMBr3 as well as to other low molecular mass tropomyosins (TM5a or TM5), but not to high molecular mass tropomyosins (TM2 or TMBr1). Northern blotting and RNase protection assays as well as immunoblotting showed that N-tropomodulin is expressed predominantly in brain. Furthermore, RNase protection assays revealed no alternatively spliced regions within the coding sequence. Developmentally, N-tropomodulin was detected in rat brain as early as embryonic day 14 and reaches the adult level before birth. Immunofluorescence of primary frontal cortex cell cultures showed that N-tropomodulin is specifically expressed in neurons. The neuron-specific expression of N-tropomodulin strongly suggests specialized roles of this TM-binding protein in neurons.

Amino Acid Sequence↗

PubMed1995

Specificity of dimer formation in tropomyosins: influence of alternatively spliced exons on homodimer and heterodimer assembly.

Tropomyosins consist of nearly 100% alpha-helix and assemble into parallel and in-register coiled-coil dimers. In vitro it has been established that nonmuscle as well as native muscle tropomyosins can form homodimers. However, a mixture of muscle alpha and beta tropomyosin subunits results in the formation of the thermodynamically more stable alpha/beta heterodimer. Although the assembly preference of the muscle tropomyosin heterodimer can be understood thermodynamically, the presence of multiple tropomyosin isoforms expressed in nonmuscle cells points toward a more complex principle for determining dimer formation. We have investigated the dimerization of rat tropomyosins in living cells by the use of epitope tagging with a 16-aa sequence of the influenza hemagglutinin. Employing transfection and immunoprecipitation techniques, we have analyzed the dimers formed by muscle and nonmuscle tropomyosins in rat fibroblasts. We demonstrate that the information for homo- versus heterodimerization is contained within the tropomyosin molecule itself and that the information for the selectivity is conferred by the alternatively spliced exons. These results have important implications for models of the regulation of cytoskeletal dynamics.

Alternative Splicing↗

PubMed1994

Polypurine sequences within a downstream exon function as a splicing enhancer.

We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.

Animals↗

PubMed1993

The role of exon sequences in splice site selection.

Using mouse immunoglobulin mu (IgM) pre-mRNA as the model substrate for in vitro splicing, we have explored the role of exon sequences in splicing. We have found that deletion of the 5' portion of exon M2 of the IgM gene abolishes the splicing of its immediately upstream intron. Splicing was restored when a purine-rich sequence found within the deleted region was reinserted into the deletion construct. This M2 exon sequence was able to stimulate the splicing of a heterologous intron of the Drosophila doublesex pre-mRNA that contains a suboptimal 3' splice site sequence. These results show that the IgM M2 exon sequence functions as a splicing enhancer. We found that the assembly of the early splicing complex is stimulated by the M2 exon sequence. In vitro competition experiments show that this stimulatory effect is mediated by the interaction of some trans-acting factors. Our results suggest that the U1 snRNP is one such factor. We propose that recognition of an enhancer exon sequence by the components of splicing machinery plays a vital role in the selection of splice sites, not only for the IgM pre-mRNA but for other pre-mRNAs. We designate such a sequence as exon recognition sequence (ERS).

Animals↗

PubMed1991

Repositioning of an alternative exon sequence of mouse IgM pre-mRNA activates splicing of the preceding intron.

Using a transient expression system of mouse IgM mini-gene constructs in mouse B-cell lines and in fibroblast L cell, we investigated splicing of the IgM transcript. We observed that the efficiency of splicing between exons C4 and M1 (C4-to-M1 splicing), the splicing reaction leading to the production of membrane-bound form (microns) mRNA, was drastically affected by mutations in a specific portion of the downstream exon (M2). The results show that the specific exon M2 sequence activates the C4-to-M1 splicing. This activation was not observed when splicing between exons M1 and M2 was abolished by base substitutions at the splice sites. These results indicate that positioning of the downstream exon is crucial for efficient splicing of the preceding intron.

Animals↗

PubMed1989

A secondary structure at the 3' splice site affects the in vitro splicing reaction of mouse immunoglobulin mu chain pre-mRNAs.

The expression of the IgM (immunoglobulin mu) heavy chain gene is known to be regulated at the post-transcriptional level. The two isoforms, the membrane-bound and secreted forms, are generated from the same gene by alternative processing at the 3' end of the primary transcript. The processing reactions involved are polyadenylation at the upstream poly(A) site (for the secreted form) and polyadenylation at the downstream poly(A) site coupled with splicing between exon C4 and exon M1 (for the membrane-bound form). The regulatory mechanism underlying these differential processing reactions is still not well understood. We investigated the splicing reaction between exon C4 and exon M1 in a HeLa nuclear extract using model transcripts containing the 5' and 3' splice sites of the C4-M1 intron. We found that the 3' splice site of the C4-M1 intron is sequestered in a stem-loop structure, which inhibits the splicing reaction in vitro. The inhibition by the stem-loop structure was also observed with a mouse lymphoma extract.

Animals↗