Human 60-kDa SS-A/Ro ribonucleoprotein autoantigen gene (SSA2) localized to 1q31 by fluorescence in situ hybridization.
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Autoantigens↗
Biomedical subjects
A G Matera
Publications and source records attributed to A G Matera.
The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes.
Epstein-Barr virus (EBV), an oncogenic herpesvirus, encodes two small RNAs (EBERs) that are expressed at high levels during latent transformation of human B lymphocytes. Here we report that a 15-kDa cellular protein called EAP (for EBER associated protein), previously shown to bind EBER1, is in fact the ribosomal protein L22. Approximately half of the L22 in EBV-positive cells is contained within the EBER1 ribonucleoprotein (RNP) particle, whereas the other half residues in monoribosomes and polysomes. Immunofluorescence with anti-L22 antibodies demonstrates that L22 is localized in the cytoplasm and the nucleoli of uninfected human cells, as expected, whereas EBV-positive lymphocytes also show strong nucleoplasmic staining. In situ hybridization indicates that the EBER RNPs are predominantly nucleoplasmic, suggesting that L22 relocalization correlates with binding to EBER1 in vivo. Since incubation of uninfected cell extracts with excess EBER1 RNA does not remove L22 from preexisting ribosomes, in vivo binding of L22 by EBER1 may precede ribosome assembly. The gene encoding L22 has recently been identified as the target of a chromosomal translocation in certain patients with leukemia, suggesting that L22 levels may be a determinant in cell transformation.
B-Lymphocytes↗
Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry.
The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.
Base Sequence↗
Localization of the human Sp1 transcription factor gene to 12q13 by fluorescence in situ hybridization.
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Chromosome Banding↗
An oligonucleotide probe specific to the centromeric region of human chromosome 5.
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Base Sequence↗
Nucleoplasmic organization of small nuclear ribonucleoproteins in cultured human cells.
The organization of eight small nuclear ribonucleoproteins (the U1, U2, U4, U5, and U6 RNAs previously studied by others and three additional snRNAs, U11, U12, and 7SK) has been investigated in cultured human cells by fluorescence in situ hybridization with antisense DNA and 2'-O-Me RNA oligonucleotides. Using highly sensitive digital imaging microscopy we demonstrate that all of these snRNAs are widespread throughout the nucleoplasm, but they are excluded from the nucleoli. In addition, the U2, U4, U5, U6, and U12 snRNAs are concentrated in discrete nuclear foci, known as coiled bodies, but U1 and 7SK are not. In addition to coiled bodies, a classic speckled pattern was observed in the nucleoplasm of monolayer-grown HeLa cells, whereas suspension-grown HeLa cells revealed a more diffuse nucleoplasmic labeling. Immunofluorescence staining using various snRNP-specific antisera shows complete agreement with that of their antisense snRNA oligonucleotide counterparts. Although U2 RNA is concentrated in coiled bodies, quantitation of the fluorescence signals from the U2 antisense probe reveals that the bulk of the U2 snRNP is located in the nucleoplasm. Furthermore, simultaneous visualization of the U2 snRNAs and the tandemly repeated U2 genes demonstrates that coiled bodies are not the sites of U2 transcription.
Base Sequence↗
Oligonucleotide probes for the analysis of specific repetitive DNA sequences by fluorescence in situ hybridization.
Five non-isotopically labeled oligonucleotides have been designed and synthesized to facilitate the analysis of specific human and murine repetitive DNA sequences by fluorescence in situ hybridization (FISH). Three of the oligonucleotides contain alphoid DNA sequences; one hybridizes to the centromeres of all human and mouse chromosomes except the Y, while the other two are specific for human chromosomes 2 and 12, respectively. The fourth oligomer, containing sequences from the spacer region of a human 5S rDNA repeat, was used to confirm the map location of a approximately 100 copy 5S rDNA tandem repeat locus. The fifth oligomer, specific to the Alu family of repeats, generates a sharp R-banding pattern on human metaphase chromosomes, suitable for FISH karyotyping. These probes permit highly specific chromosome enumeration and aneuploidy detection with hybridization times as short as 30 minutes.
Animals↗
mTFE3, an X-linked transcriptional activator containing basic helix-loop-helix and zipper domains, utilizes the zipper to stabilize both DNA binding and multimerization.
Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending.
Amino Acid Sequence↗
Recently transposed Alu repeats result from multiple source genes.
A human Alu repeat subfamily (the PV subfamily) whose members include insertional polymorphisms is found, as predicted, to differ by five tightly linked mutations relative to another subfamily of recently inserted Alu repeats. Based on these sequence differences some of the small number of polymorphic Alus can be selected from the background of nearly one million member sequences which are fixed in the human genome. Shared patterns of mutations suggest that PV subfamily members are the progeny of several different founder sequences. The additional observation that all members of the PV subfamily end in a stretch of uninterrupted polyadenine residues rather than merely A-rich sequences is evidence for post-transcriptional polyadenylation of the presumptive RNA intermediate. The drift of polyadenine sequences toward tandemly repeated A-rich motifs suggests a biological function that may select for the fixation of dispersed Alu repeats.
Base Sequence↗
A transpositionally and transcriptionally competent Alu subfamily.
DNA base sequence comparisons indicate that a subfamily of recently transposed human Alu repeats are distinguished from most Alu repeats by diagnostic sequence differences. Using an oligonucleotide hybridization probe that incorporates these sequence features, we found that there was an expansion of this Alu subfamily following the divergence of humans and African apes. This oligonucleotide was used to select human genomic clones containing representatives of this subfamily. One representative member of this subfamily was evidently absent from the corresponding chimpanzee locus and was associated with a restriction fragment length polymorphism in the human genome. This apparently polymorphic member had all the diagnostic sequence features that initially predicted the existence of a newly expanding Alu subfamily. A transpositionally active sequence variant should also be transcriptionally active in at least some cell types or tissues. Northern (RNA) blot hybridization, primer extension, and RNA sequence analysis demonstrated the existence of different-length polyadenylated and nonpolyadenylated transcripts corresponding to this subfamily. Evidence for 3' processing and subcellular localization of these transcripts is discussed. Most of the nearly one million human Alu repeats are pseudogenes with respect to coding for either an RNA product or new family members; a select and identifiable subset of Alu repeats serve as transcriptionally and transpositionally competent source genes.
Base Sequence↗
Structure and evolution of the U2 small nuclear RNA multigene family in primates: gene amplification under natural selection?
The organization of U2 genes was compared in apes, Old World monkeys, and the prosimian galago. In humans and all apes (gibbon, orangutan, gorilla, and chimpanzee), the U2 genes were organized as a tandem repeat of a 6-kb element; however, the restriction maps of the 6-kb elements in these divergent species differed slightly, demonstrating that mechanisms must exist for maintaining sequence homogeneity within this tandem array. In Old World monkeys, the U2 genes were organized as a tandem repeat of an 11-kb element; the restriction maps of the 11-kb elements in baboon and two closely related macaques, bonnet and rhesus monkeys, also differed slightly, confirming that efficient sequence homogenization is an intrinsic property of the U2 tandem array. Interestingly, the 11-kb monkey repeat unit differed from the 6-kb hominid repeat unit by a 5-kb block of monkey-specific sequence. Finally, we found that the U2 genes of the prosimian galago were dispersed rather than tandemly repeated, suggesting that the hominid and Old World monkey U2 tandem arrays resulted from independent amplifications of a common ancestral U2 gene. Alternatively, the 5-kb monkey-specific sequence could have been inserted into the 6-kb array or deleted from the 11-kb array soon after divergence of the hominid and Old World monkey lineages.
Animals↗
Recombination mediates production of an extrachromosomal circular DNA containing a transposon-like human element, THE-1.
An abundant class of HeLa extrachromosomal circular DNA containing the transposon-like element, THE-1, is shown to arise via site specific recombination. The chromosomal locus from which these circles are derived, however, is single-copy. Northern blot analysis detects homology to two polyadenylated RNAs in HeLa cells. The possible presence of an origin of replication and its role in generating these small polydisperse circles is discussed.
Base Sequence↗
Repetitive nucleotide sequence insertions into a novel calmodulin-related gene and its processed pseudogene.
A gene containing a transposon-like human repeat element, called THE 1, has been isolated and characterized. The gene, termed T+, encodes a polypeptide resembling known calcium-binding proteins. The THE 1 element is present in the 3'-untranslated region of its message. The cDNA clone corresponding to the gene's mRNA product led to the identification of this gene. A processed RNA pseudogene related to the authentic gene has also been isolated. In addition to intron processing, this pseudogene differs from the gene in that it contains an interspersed Alu repeat instead of a THE 1 element in the 3'-untranslated region. Thus, we compare a site containing a THE 1 element to an ancestrally related transposon-less target site. The comparison suggests a retroviral-related mechanism of THE 1 insertion. This system is unusual in that the parent gene is associated with three distinct retrotransposition events: the parent gene was converted to a processed RNA pseudogene, an Alu repeat inserted into the pseudogene, and a THE 1 element inserted into the parent gene.
Amino Acid Sequence↗
Transcription of a human transposon-like sequence is usually directed by other promoters.
The transcriptional activity of a human transposon-like family of repeats, called the THE-1 family, has been studied in cell culture and in human tissue. Both strands of THE-1 are present in several discrete length poly A plus RNAs. Primer extension studies and the structures of cDNA clones show that these THE-1 transcripts are usually the product of other transcription units. The THE-1 LTR provides the polyadenylation processing site for two transcripts, which result from upstream non THE-1 promoters. Yet another transcript, containing an internal THE-1 element in the probable sense orientation, is greatly enriched in a polysomal size fraction.
Base Sequence↗
The U22 host gene (UHG): chromosomal localization of UHG and distribution of U22 small nucleolar RNA.
The U22 host gene (UHG) is very unusual because it encodes a spliced, polyadenylated RNA that has little apparent coding capacity and is rapidly degraded. The stable RNA products from this locus are actually encoded within eight different introns of the UHG pre-RNA. These small nucleolar RNAs (snoRNAs) assemble into ribonucleoproteins, some of which have been shown to function in rRNA processing and modification. In order to more fully characterize the locus, we have mapped UHG to chromosome 11q13 by fluorescence in situ hybridization (FISH). Radiation hybrid mapping placed this sequence-tagged site with very high probability (lod >19) to chromosome 11, approximately 10.1 cR distal to framework marker WI-8652. We also investigated the possibility that the expression of UHG was subject to genomic imprinting. Several laboratories have shown that non-protein-coding mRNAs are frequently associated with imprinted domains in mammalian cells. We used a novel somatic cell hybrid method to assay parent-of-origin effects in the expression of UHG alleles and found that, unlike XIST, IPW, and H19, this RNA is expressed biparentally. Additional FISH experiments using anti-U22 oligonucleotides revealed that, as with U3, this sno-RNA is localized throughout the nucleolus.
Cell Nucleolus↗