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1.
Int Immunol ; 13(11): 1415-22, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11675373

ABSTRACT

Somatic hypermutation is a key mechanism in generating Ig with higher affinities to antigen, a process known as affinity maturation. Using Igkappa transgenes, the 3' enhancer (kappaE3') has been shown to play an important role in introducing hypermutations. In order to identify the cis-acting elements that regulate hypermutagenesis, we have generated transgenic substrates containing mutations/deletions in the kappaE3' region. Here, we report that base substitutions in the kappaE3', either in the PU.1 or in the NF-EM5 binding motif, not only reduce the mutation rate but also disrupt the directed mutagenesis in the intrinsic hotspots of the Igkappa transgene.


Subject(s)
B-Lymphocytes/immunology , Genes, Immunoglobulin , Immunoglobulin kappa-Chains/genetics , Somatic Hypermutation, Immunoglobulin , 3' Untranslated Regions/genetics , Animals , Binding Sites , Enhancer Elements, Genetic , Female , Immunoglobulin Variable Region , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic
2.
Neuroreport ; 12(5): 1061-6, 2001 Apr 17.
Article in English | MEDLINE | ID: mdl-11303746

ABSTRACT

We have studied the projection of olfactory sensory neurons (OSNs), during the developmental and regeneration processes, using the transgenic mouse carrying the differently tagged odorant receptor genes, MOR28. We have found that the axon terminals of the two sets of MOR28-positive OSNs, one expressing the lacZ tag and the other expressing the green fluorescent protein gene, are dispersed and intermingled at early developmental or regeneration stages. Projection areas become more distinct and separated at later stages, however, two sets of axon fibers are not typically bundled or segregated during pathfinding. It appears that segregation of axons mainly occurs when they target at the olfactory bulb to form the glomerular structure.


Subject(s)
Axons/physiology , Brain/growth & development , Nerve Regeneration/physiology , Neurons, Afferent/physiology , Animals , Brain/cytology , Fluorescent Antibody Technique , Mice , Mice, Transgenic , Olfactory Pathways/cytology , Olfactory Pathways/growth & development , Olfactory Pathways/physiology , Receptors, Odorant/genetics
3.
Genes Cells ; 6(1): 71-8, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11277098

ABSTRACT

BACKGROUND: We have previously generated transgenic mice carrying the murine odourant receptor gene, MOR28, tagged with lacZ. In this animal, the endogenous MOR28 is differently tagged with GFP. It was found that the transgenic and endogenous MOR28 genes are expressed in a mutually exclusive manner and that the two sets of olfactory sensory neurones (OSNs), each expressing either the transgenic or the endogenous MOR28, project their axons to separate glomeruli. RESULTS: Our fluorescent in situ hybridization (FISH) revealed that the two endogenous alleles of MOR28 are also mutually excluded for their transcriptional activation. Therefore, we studied whether there would be any segregation in the projection of the two subsets of OSNs: one set expressing the paternal and the other expressing the maternal allele. It was found that the OSNs for both alleles shared the same glomerulus for their projection, but the projection targets were segregated within the glomerular structure. CONCLUSION: Two subsets of neurones expressing either the transgenic or the endogenous MOR28 target their axons to two separate glomeruli based on the differences in the genetic backgrounds, nature of tagging, and chromosomal locations. In contrast, neurones expressing a maternal or paternal allele share the same glomeruli, but tend to target to segregated areas within the glomerular structure. The segregation was more prominent with increased differences in the genetic background between the two alleles.


Subject(s)
Alleles , Axons , Neurons/metabolism , Olfactory Pathways/cytology , Receptors, Odorant/genetics , Amino Acid Sequence , Animals , Female , Genomic Imprinting , In Situ Hybridization, Fluorescence , Male , Mice , Mice, Transgenic , Molecular Sequence Data , Neurons/cytology , Receptors, Odorant/chemistry , Sequence Homology, Amino Acid
4.
Nat Neurosci ; 3(7): 687-93, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10862701

ABSTRACT

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.


Subject(s)
Chromosome Mapping , Gene Expression Regulation/physiology , Olfactory Bulb/physiology , Receptors, Odorant/genetics , Animals , Chromosomes, Artificial, Yeast , Green Fluorescent Proteins , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Olfactory Bulb/cytology , Receptors, Odorant/physiology , beta-Galactosidase/genetics
5.
Int Immunol ; 12(5): 721-9, 2000 May.
Article in English | MEDLINE | ID: mdl-10784618

ABSTRACT

DNA-bending proteins are known to facilitate the in vitro V(D)J joining of antigen receptor genes. Here we report that the high-mobility group protein, HMG1, is necessary for the correct nicking of the 23 bp recombination signal sequence (23-RSS) by the recombination [corrected] activating gene (RAG) proteins, RAG1 and RAG2. Without HMG1, the mouse Jkappa1 23-RSS was recognized as if it were the 12-RSS and nicked at a site 12 + 7 nucleotides away from the 9mer signal, even though no 7mer-like sequence was evident at the cryptic nicking site. When increased amounts of HMG1 were added, the 23-RSS substrate was nicked correctly at a site 23 + 7 nucleotides from the 9mer, and nicking at the cryptic site disappeared. Unlike the 23-RSS, the 12-RSS did not require HMG1 for correct nicking, although HMG1 was found to increase the interaction between RSS and RAG proteins. Modification-interference assays demonstrated that HMG1 caused changes in the interaction between the 23-RSS and RAG proteins specifically at the 7mer and the cryptic nicking site.


Subject(s)
High Mobility Group Proteins/pharmacology , Homeodomain Proteins/metabolism , Animals , DNA Topoisomerases, Type I , DNA-Binding Proteins/metabolism , Mice , Proteins
6.
J Neurosci ; 19(19): 8409-18, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10493742

ABSTRACT

We have characterized two separate odorant receptor (OR) gene clusters to examine how olfactory neurons expressing closely linked and homologous OR genes project their axons to the olfactory bulb. Murine OR genes, MOR28, MOR10, and MOR83, share 75-95% similarities in the amino acid sequences and are tightly linked on chromosome 14. In situ hybridization has demonstrated that the three genes are expressed in the same zone, at the most dorsolateral and ventromedial portions of the olfactory epithelium, and are rarely expressed simultaneously in individual neurons. Furthermore, we have found that olfactory neurons expressing MOR28, MOR10, or MOR83 project their axons to very close but distinct subsets of glomeruli on the medial and lateral sides of the olfactory bulb. Similar results have been obtained with another murine OR gene cluster for A16 and MOR18 on chromosome 2, sharing 91% similarity in the amino acid sequences. These results may indicate an intriguing possibility that olfactory neurons expressing homologous OR genes within a cluster tend to converge their axons to proximal but distinct subsets of glomeruli. These lines of study will shed light on the molecular basis of topographical projection of olfactory neurons to the olfactory bulb.


Subject(s)
Axons/physiology , Chromosome Mapping , Gene Expression Regulation , Multigene Family , Olfactory Bulb/physiology , Olfactory Receptor Neurons/physiology , Receptors, Odorant/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Exons , Gene Library , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Olfactory Mucosa/cytology , Olfactory Mucosa/physiology , Olfactory Receptor Neurons/cytology , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
7.
Int Immunol ; 11(9): 1423-9, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10464163

ABSTRACT

During the development of lymphocytes, expression of the Ig genes is strictly regulated in a tissue-specific manner and in a time-ordered fashion. We have previously shown that the PU.1 binding motif in the Igkappa 3' enhancer (kappaE3') and a novel Ets family protein other than PU.1 may be possibly involved in the control of V(kappa)-J(kappa) joining. In the attempt to isolate the novel Ets family protein, we have screened cDNA libraries with the yeast one-hybrid method and identified a new PU.1-related factor, Prf. This novel Ets family protein is shown to interact with the PU.1 binding sequences in various promoters and enhancers, including kappaE3'. It was found that expression of the prf gene is predominant in the B-lineage cells, with the exception of immature B cells. Since Prf does not exhibit functions of transcriptional activity, this novel protein may act as an antagonist against other Ets family proteins, e.g. PU.1 and Spi-B. Possible roles of Prf with respect to the B cell differentiation are discussed.


Subject(s)
B-Lymphocytes/chemistry , Proto-Oncogene Proteins/chemistry , Trans-Activators/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Animals , B-Lymphocytes/cytology , Base Sequence , Bone Marrow Cells/metabolism , Cell Differentiation/drug effects , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic/genetics , Humans , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Proto-Oncogene Proteins c-ets , RNA, Messenger/analysis , Spleen/cytology , Thymus Gland/cytology , Transcription Factors/genetics
9.
Mol Cell Biol ; 18(1): 655-63, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9418911

ABSTRACT

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.


Subject(s)
DNA Footprinting , DNA-Binding Proteins/genetics , Genes, RAG-1 , Homeodomain Proteins , Receptors, Antigen, T-Cell/genetics , Recombination, Genetic , Animals , DNA Nucleotidyltransferases , Humans , Mice , Nuclear Proteins , VDJ Recombinases
10.
J Immunol ; 159(9): 4145-9, 1997 Nov 01.
Article in English | MEDLINE | ID: mdl-9379006

ABSTRACT

We have previously shown that the PU.1 binding motif (GAG GAA) in the 3'-enhancer region in the Ig kappa gene is responsible for the negative regulation of tissue (B/T)-specific Vkappa-Jkappa joining. Here we report that the PU.1 binding site also regulates the stage (pro-B/pre-B) specificity of Vkappa-Jkappa joining. In the substrate with base substitutions in the PU.1 binding motif, recombination took place in both pro-B (B220dull/CD43+) and pre-B (B220dull/CD43-) cells. In the transcriptional regulation, the PU.1 motif acts in a positive manner cooperatively with the nuclear factor-EM5 (or PIP) motif (GAAAAC), which is located 2 bp downstream from the PU.1 motif. Interestingly, base substitutions in the nuclear factor EM5 (PIP) motif did not affect the pro-B/pre-B specificity of Vkappa-Jkappa joining. Thus, the PU.1 motif regulates both temporal and tissue-specific rearrangements, while nuclear factor-EM5 is not involved in the regulation of Ig kappa recombination.


Subject(s)
B-Lymphocytes/immunology , Enhancer Elements, Genetic/immunology , Genes, Immunoglobulin , Proto-Oncogene Proteins/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Trans-Activators/immunology , Animals , Binding Sites/genetics , Gene Rearrangement, B-Lymphocyte , Histones/genetics , Histones/immunology , Mice , Mice, Transgenic , Proto-Oncogene Proteins/genetics , Receptors, Antigen, T-Cell/genetics , Trans-Activators/genetics
12.
Biochem Biophys Res Commun ; 221(2): 240-7, 1996 Apr 16.
Article in English | MEDLINE | ID: mdl-8619840

ABSTRACT

We have characterized genomic structure and transcription of a murine odorant receptor (OR) gene, MOR23. This gene has two distinct transcriptional initiation regions, which are differentially utilized in the olfactory and testicular cells. We have found that the MOR23 gene has an intron within the 5'-untranslated region upstream of the coding sequence. In olfactory cells, transcription of the gene is initiated in the region upstream of this intron, while in testicular cells, transcription starts within the intron. Differential initiation of transcription is discussed in the context of tissue-specific expression of the OR genes.


Subject(s)
Olfactory Mucosa/metabolism , Receptors, Odorant/genetics , Testis/metabolism , Transcription, Genetic , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid
14.
J Immunol ; 153(10): 4520-9, 1994 Nov 15.
Article in English | MEDLINE | ID: mdl-7963524

ABSTRACT

Recombination signal sequences for V(D)J joining consist of a conserved heptamer (CACAGTG) and a nonamer (ACAAAAACC) separated by a spacer of a constant length (12 bp or 23 bp). In the present study, we have analyzed various recombination signal mutations for their effects in V(D)J joining. Using a retroviral vector, we introduced mutant substrates stably into pre-B cells, and assayed recombination using the lacZ gene as a reporter. This method allowed us to study recombination in a single copy within the context of the host cell chromosome. Because this assay did not show any detectable background, it was quite useful in the analysis of low level recombinations. In the heptamer, mutations in the first three residues severely dropped the joining rates. Among them, the first residue adjacent to the recombination site was found to be most essential. Although mutations in the heptamer reduced the joining rate to various extents, they did not lower the site-specificity of recombination. With regard to the nonamer, the presence of three consecutive A residues was necessary for efficient recombination. Furthermore, the nucleotides flanking the A-rich core needed to be other than A residues, probably marking the border of the A-stretch. This may be important when the recombinase measures the distance between the heptamer and the nonamer to satisfy the 12/23-bp spacer rule.


Subject(s)
Antibody Formation/genetics , B-Lymphocytes/immunology , Recombination, Genetic/immunology , Animals , Base Sequence , Blotting, Southern , Cell Line , Galactosides/immunology , Gene Rearrangement, B-Lymphocyte/genetics , Genes, Reporter/genetics , Indoles/immunology , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed/genetics
15.
DNA Cell Biol ; 13(1): 51-8, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8286039

ABSTRACT

The intron of the yeast RP51A gene was cloned with precision using the polymerase chain reaction (PCR) amplification method, and then inserted into several different positions of the yeast URA3 gene as well as the PGK-lacZ fusion gene without introduction of additional exon sequences. Analysis of transcripts of these genes showed that an intron inserted near the transcription start site of the gene was spliced out efficiently, whereas the same intron sequences inserted 200 bp or further downstream of the start site were not, resulting in a reduced level of mRNA. These results explain why intron-containing genes in yeast usually have an intron near the 5' end.


Subject(s)
Gene Expression Regulation, Fungal , Genes, Fungal , Introns , RNA, Fungal/genetics , Saccharomyces cerevisiae/genetics , Base Sequence , DNA Primers/chemistry , Molecular Sequence Data , RNA Splicing , RNA, Messenger/genetics , Ribosomal Proteins/genetics
16.
Science ; 257(5068): 408-10, 1992 Jul 17.
Article in English | MEDLINE | ID: mdl-17832836
17.
Science ; 254(5028): 81-6, 1991 Oct 04.
Article in English | MEDLINE | ID: mdl-1925563

ABSTRACT

A DNA construct containing the bacterial beta-galactosidase gene (lacZ) was used to study somatic DNA recombination in the transgenic mouse brain. Recombination-positive areas of the adult brain were stained blue with X-gal, a substrate of beta-galactosidase. Blue-colored cells appeared soon after birth, and continued to emerge in postnatal tissue. Staining was prominent in sensory as opposed to motor regions of the brain, and was present in more than 70 discrete areas of the nervous system. The possibility of DNA rearrangement is discussed with respect to the development of the central nervous system.


Subject(s)
Brain/physiology , Gene Rearrangement , Recombination, Genetic , Animals , Base Sequence , Blotting, Southern , Brain Mapping , Mice , Mice, Transgenic/genetics , Molecular Sequence Data , Neurons, Afferent/physiology , Promoter Regions, Genetic , beta-Galactosidase/genetics
19.
Science ; 244(4910): 1346-8, 1989 Jun 16.
Article in English | MEDLINE | ID: mdl-2544026

ABSTRACT

Artificial yeast introns that show cold-sensitive splicing have been constructed. These conditional introns can be inserted into a target gene as an "intron cassette" without disrupting the coding information, allowing expression of the gene to be cold sensitive. Insertion of these intron cassettes rendered the yeast URA3 gene cold sensitive in its expression. The advantage of this intron-mediated control system is that any gene can be converted to a controllable gene by simple insertion of an intron.


Subject(s)
Gene Expression Regulation , Genetic Engineering , Introns , Saccharomyces cerevisiae/genetics , Base Sequence , Cold Temperature , DNA Transposable Elements , Molecular Sequence Data
20.
Proc Natl Acad Sci U S A ; 82(24): 8557-61, 1985 Dec.
Article in English | MEDLINE | ID: mdl-3909147

ABSTRACT

Transcription of the HIS4 gene begins at a single site (I) at position -60 from the ATG that begins translation. We have made linker insertions/deletions in the 5' noncoding region to identify the elements required for the specificity of transcription initiation. Although there are four sequences that begin TATA and are near the start of transcription (-170, -132, -123, and -102) only the sequence at -123 (TATA-123) is required for transcription initiation. By inserting synthetic oligonucleotides into a mutant from which TATA-123 had been deleted, we found that just TATA or TATAA does not work but that TATAAA functions almost as well as the wild-type sequence. This hexamer does not work in the opposite orientation (TTTATA). When a synthetic TATA sequence is placed upstream from the normal site, the site of initiation also moves upstream in a roughly cometric way even when TATA-123 is present. Analysis of transcripts in strains where the distance between the TATA sequence and the wild-type site of transcription initiation (I site) has been altered shows that in yeast, unlike higher cells, transcription does not initiate at a strictly defined distance from the TATA sequence. Constructions that alter the distance between the TATA and the I site or remove the I site change the pattern of transcription initiation without affecting the level of HIS4 expression. Deletions that eliminate the I site produce heterogeneous transcripts and deletions that substantially shorten the distance between TATA-123 and the I site initiate at multiple sites downstream from the I site. Thus, both the TATA and the sequences downstream from it determine the pattern of transcription initiation.


Subject(s)
Histidine/genetics , Promoter Regions, Genetic , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Base Sequence , Chromosome Mapping , DNA, Fungal/genetics , Gene Expression Regulation , Repetitive Sequences, Nucleic Acid
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