Search PubMed⌕ Search

Biomedical subjects

J Brosius

Publications and source records attributed to J Brosius.

At least 37 records · Page 2Linked to original sources
PubMed1998

Heterodimer SRP9/14 is an integral part of the neural BC200 RNP in primate brain.

BC200 RNA is a brain-specific, small non-messenger RNA with a somatodendritic localization in primate neurons and a constituent of a ribonucleoprotein (RNP) complex. The primary and secondary structure of the 5' domain of BC200 RNA resembles that of the Alu domain of 7SL RNA, which is an integral part of the signal recognition particle (SRP). This would predict that similar proteins bind to this defined domain of both RNA species in vitro and in vivo. The data presented in this paper reveal that a protein that binds BC200 RNA in vivo is immunoreactive with antibodies against SRP9. This further supports the notion that the 5' domain of the BC200 RNA can fold into structures similar to the SRP Alu domain and, as a result, bind identical or similar proteins in vivo. The SRP9 protein binds only as dimer with SRP14 protein to the Alu domain of 7SL RNA to form a subdomain that, in SRP, is functional in translation arrest. Therefore, our data also indicate that the neuronal BC200 RNP is a candidate for regulating decentralized protein biosynthesis in dendrites, possibly with a mechanism that resembles translation arrest of the SRP.

Animals↗
PubMed1998

The BC200 RNA gene and its neural expression are conserved in Anthropoidea (Primates).

The gene encoding BC200 RNA arose from a monomeric Alu element. Subsequently, the RNA had been recruited or exapted into a function of the nervous system. Here we confirm the presence of the BC200 gene in several primate species among the Anthropoidea. The period following the divergence of New World monkeys and Old World monkeys from their common ancestor is characterized by a significantly higher substitution rate in the examined 5' flanking region than in the BC200 RNA coding region itself. Furthermore, the conservation of CpG dimers in the RNA coding region (200 bp) is drastically increased compared to the 5' flanking region (approximately 400 bp) over all 12 species examined. Finally, the brain-specific expression pattern of BC200 RNA and its presence as a ribonucleoprotein particle (RNP) are conserved in Old World and New World monkeys. Our studies indicate that the gene encoding BC200 RNA was created at least 35-55 million years ago and its presence, mode of expression, and association with protein(s) as an RNP are under selective pressure.

Animals↗
PubMed1998

Identification of human autoantigen La/SS-B as BC1/BC200 RNA-binding protein.

Rodent BC1 RNA and primate BC200 RNA are small cytoplasmic non-messenger RNAs that are phylogenetically unrelated. Nevertheless, the two RNAs exhibit a large degree of parallelism. In addition to some sequence similarities in their 3' domains, they are prevalently expressed in a similar subset of neurons and belong to a small group of transcripts with a somatodendritic location. Both RNAs are complexed with proteins as ribonucleoprotein particles (RNPs). Their similarities may even extend to analogous functional roles, for example, in the regulation of decentralized dendritic translation. To shed further light on the physiological role(s) of the BC1/BC200 RNPs, we began to analyze protein components that specifically bind to these RNAs. Ultraviolet-crosslinking experiments and affinity purification techniques revealed that the human autoantigen La/SS-B is associated with BC1/BC200 RNA in vitro and in vivo. As with other RNA polymerase III transcripts, La protein binds with high affinity to the 3' end of BC200 RNA. Our results suggest that an additional function of La may be control of dendritic translation by providing a link between the 5' Alu domain of BC200 RNP and the ribosome via the La protein dimer. The fact that La binds both BC1 and BC200 RNAs further supports the notion that the RNAs are functional analogs despite the fact that they arose from two separate retroposition events in two different mammalian lineages.

Animals↗
PubMed1998

The 10Sa RNA gene of Thermus thermophilus.

The 10Sa RNA gene of Thermus thermophilus was isolated and sequenced. The tRNA-like structure at the 5' and 3' ends and other secondary structure features of the T. thermophilus 10Sa RNA are similar to E. coli 10Sa RNA. A variant of the sequence motif coding for the tag peptide is located in the centre of T. thermophilus 10Sa RNA.

Amino Acid Sequence↗
PubMed1997

BC1 RNA, the transcript from a master gene for ID element amplification, is able to prime its own reverse transcription.

ID elements are short interspersed elements (SINEs) found in high copy number in many rodent genomes. BC1 RNA, an ID-related transcript, is derived from the single copy BC1 RNA gene. The BC1 RNA gene has been shown to be a master gene for ID element amplification in rodent genomes. ID elements are dispersed through a process termed retroposition. The retroposition process involves a number of potential regulatory steps. These regulatory steps may include transcription in the appropriate tissue, transcript stability, priming of the RNA transcript for reverse transcription and integration. This study focuses on priming of the RNA transcript for reverse transcription. BC1 RNA gene transcripts are shown to be able to prime their own reverse transcription in an efficient intramolecular and site-specific fashion. This self-priming ability is a consequence of the secondary structure of the 3'-unique region. The observation that a gene actively amplified throughout rodent evolution makes a RNA capable of efficient self-primed reverse transcription strongly suggests that self-priming is at least one feature establishing the BC1 RNA gene as a master gene for amplification of ID elements.

Animals↗
PubMed1997

Expression of dendritic BC200 RNA, component of a 11.4S ribonucleoprotein particle, is conserved in humans and simians.

Primate BC200 RNA, a brain-specific small cytoplasmic RNA, is one of the few known cell type specific non-messenger RNAs. It originated from a monomeric Alu short interspersed repetitive element (SINE) in primates. In situ hybridization using rhesus monkey (Macaca mulatta) brain sections reveals a similar cellular and sub-cellular distribution as in human brain. In addition to confirming its dendritic location, the distribution in an old world monkey indicates a discrete regional and subcellular location of BC200 RNA. We also report that BC200 RNA exists as a ribonucleoprotein (RNP) particle in vivo. In sucrose gradients, the BC200 particle has a sedimentation constant of about 11.4 S, significantly more than the corresponding 200 nucleotide long naked RNA (approximately 7.6 S).

Animals↗
PubMed1997

Expression of neural BC200 RNA in human tumours.

BC200 RNA is a 200-nucleotide-long non-messenger RNA that is selectively expressed in the primate nervous system, where it has been identified in somatodendritic domains of a subset of neurons. BC200 RNA is not normally expressed in non-neuronal somatic cells; it has been shown, however, to be expressed in germ cells and in cultured immortal cell lines of various non-neural origins. In order to investigate whether the neuron-specific expression of BC200 RNA is also deregulated during tumourigenesis in non-neural human tissues, 80 different tumour specimens, representing 19 different tumour types, were screened for the presence of the RNA. BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, parotid, and tongue, but not in corresponding normal tissues. BC200 RNA was not detectable in bladder, colon, kidney, or liver carcinoma tissues examined in this study. These results demonstrate that BC200 expression is deregulated under certain neoplastic conditions. The expression of BC200 RNA in non-neural tumours may indicate a functional interrelationship with induction and/or progression of such tumours.

Blotting, Northern↗
PubMed1997

Expression of neural BC1 RNA: induction in murine tumours.

BC1 RNA is a small cytoplasmic RNA polymerase III transcript that is expressed in the rodent nervous system. The RNA is selectively expressed in neurons where it is located in somatodendritic domains. BC1 RNA is not normally detectable in non-neuronal somatic cells; it is however expressed in germ cells and in cultured immortal cell lines of various non-neural origins. We therefore sought to establish whether the neuron-specific regulation of BC1 expression is altered in non-neural tumour cells. Oncogen and chemical carcinogen induced mouse tumours were analysed for the presence of BC1 RNA, using Northern transfer and in situ hybridisation. Here we report that BC1 RNA is selectively expressed in tumour cells, but not in corresponding normal tissues. These results indicate that neural-specific regulation of BC1 expression is lacking in murine tumour cells of non-neural origin.

Animals↗
PubMed1996

Translational machinery in dendrites of hippocampal neurons in culture.

In neurons, several mRNAs are selectively delivered to dendritic domains where they are presumably translated by local protein synthetic machinery. Although electron microscopy has identified polyribosomes in dendrites, in particular in postsynaptic dendritic compartments, the functional composition of the local protein synthetic apparatus and the scope of its translational capacity have not been analyzed. To ascertain the translational competence of dendrites, we have probed hippocampal neurons in primary culture for various integral and associated factors of the translational apparatus. We report here that dendrites of such neurons are equipped with a spectrum of translational machinery components, including ribosomes, tRNAs, initiation and elongation factors, and elements of the cotranslational signal recognition mechanism. These components are differentially and nonuniformly distributed in dendritic arbors. Their dendritic location illustrates the soma-independent potential of dendrites to synthesize selected proteins in local domains.

Animals↗
PubMed1996

Identification and characterization of BC1 RNP particles.

Rodent brain-specific small cytoplasmic BC1 RNA is an unusual RNA in several respects. It is an RNA polymerase III transcript expressed specifically in neurons, with regional and developmental regulation. Moreover, it is one of a few RNAs actively transported into dendrites. Three findings indicate that BC1 RNA exists as a ribonucleoprotein complex in vivo. First, the buoyant density of fractions containing BC1 RNA from brain extract on CsCI and Cs2SO4 gradients is 1.45 g/ml and 1.55 g/ml, respectively; this is consistent with the density of RNA-protein complexes. Second, in sucrose gradients, the BC1 particle has a larger S value (8.7S) than naked RNA (6.1S). Third, BC1 RNA from brain extracts migrates with retarded mobility compared to naked BC1 RNA during agarose gel electrophoresis. Additionally, in comparison to the signal recognition particle (SRP), the BC1 RNP is more heat resistant and less Mg(2+)-dependent. The buoyant density of the BC1 RNP suggests the presence of protein(s) with a total mass of about 138kD.

Animals↗
PubMed1995

Reverse transcriptase: mediator of genomic plasticity.

Reverse transcription has been an important mediator of genomic change. This influence dates back more than three billion years, when the RNA genome was converted into the DNA genome. While the current cellular role(s) of reverse transcriptase are not yet completely understood, it has become clear over the last few years that this enzyme is still responsible for generating significant genomic change and that its activities are one of the driving forces of evolution. Reverse transcriptase generates, for example, extra gene copies (retrogenes), using as a template mature messenger RNAs. Such retrogenes do not always end up as nonfunctional pseudogenes but form, after reinsertion into the genome, new unions with resident promoter elements that may alter the gene's temporal and/or spatial expression levels. More frequently, reverse transcriptase produces copies of nonmessenger RNAs, such as small nuclear or cytoplasmic RNAs. Extremely high copy numbers can be generated by this process. The resulting reinserted DNA copies are therefore referred to as short interspersed repetitive elements (SINEs). SINEs have long been considered selfish DNA, littering the genome via exponential propagation but not contributing to the host's fitness. Many SINEs, however, can give rise to novel genes encoding small RNAs, and are the migrant carriers of numerous control elements and sequence motifs that can equip resident genes with novel regulatory elements [Brosius J. and Gould S.J., Proc Natl Acad Sci USA 89, 10706-10710, 1992]. Retrosequences, such as SINEs and portions of retroelements (e.g., long terminal repeats, LTRs), are capable of donating sequence motifs for nucleosome positioning, DNA methylation, transcriptional enhancers and silencers, poly(A) addition sequences, determinants of RNA stability or transport, splice sites, and even amino acid codons for incorporation into open reading frames as novel protein domains. Retroposition can therefore be considered as a major pacemaker for evolution (including speciation). Retroposons, with their unique properties and actions, form the molecular basis of important evolutionary concepts, such as exaptation [Gould S.J. and Vrba E., Paleobiology 8, 4-15, 1982] and punctuated equilibrium [Elredge N. and Gould S.J. in Schopf T.J.M. (ed). Models in Paleobiology. Freeman, Cooper, San Francisco, 1972, pp. 82-115].

Animals↗
PubMed1995

Molecular cloning and characterization of the mouse dopamine D3 receptor gene: an additional intron and an mRNA variant.

The intron-exon organization for the murine dopamine D3 receptor gene was determined. A novel intron of approximately 1 kb was identified in both rat and mouse D3 receptor genes. This intron (termed intron 4) is situated between coding nucleotides 723 and 724, resulting in a split of former exon 4 (containing nucleotides 527-801) into two separate exons (exon 4 and exon 5). Thus, the coding regions of the D2 and D3 receptor genes contain an identical number of exons (seven exons) and share a very similar gene structure. Reverse transcription-PCR experiments revealed a short form of mouse D3 mRNA (D3Short) that lacks the first 63 nucleotides from exon 6, and results from a splicing event occurring within this exon. However, this mRNA variant was not found in either rat or human brain. No dopamine D3 receptor mRNA variants were found deriving from the alternative splicing of exon 5, although its counterpart, exon 6 in the D2 receptor gene, is spliced out to produce the D2Short mRNA. These data suggest that, although the intron-exon organizations of the D2 and D3 receptor genes are similar, the encoded transcripts may be processed differently.

Alternative Splicing↗
PubMed1995

Expression of the human T-cell receptor V beta 5.3 in Escherichia coli by thermal induction of the trc promoter: nucleotide sequence of the lacIts gene.

We have constructed a vector, pKBi, for the high-level expression of the variable beta chain 5.3 (V beta 5.3) of the human T-cell receptor in Escherichia coli. This vector incorporates the trc promoter, a polylinker, two transcription terminators, and the tetracycline resistance gene. Furthermore, the vector contains the lacIts gene that encodes a temperature-sensitive (ts) lac repressor, thus obviating both the need to use IPTG as a transcriptional inducer, and bacterial strains that harbor either the lacI or lacIq genes. The sequence of the lacIts gene shows an open reading frame of 1,080 nucleotides encoding 360 amino acids, and differs from the lacI gene at nucleotide 559 (with reference to the first nucleotide of the start codon). This nucleotide changes from G to A, causing amino acid residue 187 to change from glycine (GGC) to serine (AGC). This mutation imparts thermal sensitivity to the lac repressor protein. This is the first time that a TCR V beta region has been expressed at high levels (up to 28 mg/liter of culture) without fusion partners. The availability of the lacIts gene for thermal induction of the trc promoter, and the presence of the tetracycline resistance gene should make the expression vector pKBi particularly attractive for the efficient production of human therapeutic proteins in bacteria.

Bacterial Proteins↗
PubMed1995

BC1 RNA: transcriptional analysis of a neural cell-specific RNA polymerase III transcript.

Rodent BC1 RNA represents the first example of a neural cell-specific RNA polymerase III (Pol III) transcription product. By developing a rat brain in vitro system capable of supporting Pol III-directed transcription, we showed that the rat BC1 RNA intragenic promoter elements, comprising an A box element and a variant B box element, as well as its upstream region, containing octamer-binding consensus sequences and functional TATA and proximal sequence element sites, are necessary for transcription. The BC1 B box, lacking the invariant A residue found in the consensus B boxes of tRNAs, represents a functionally related and possibly distinct promoter element. The transcriptional activity of the BC1 B box element is greatly increased, in both a BC1 RNA and a chimeric tRNA(Leu) gene construct, when the BC1 5' flanking region is present and is appropriately spaced. Moreover, a tRNA consensus B-box sequence can efficiently replace the BC1 B box only if the BC1 upstream region is removed. These interactions, identified only in a homologous in vitro system, between upstream Pol II and intragenic Pol III promoters suggest a mechanism by which the tissue-specific BC1 RNA gene and possibly other Pol III-transcribed genes can be regulated.

Animals↗