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Rapid accumulation of measles virus leader RNA in the nucleus of infected HeLa cells and human lymphoid cells.

The 3' terminus of the single-stranded, negative-sense genome of the measles virus comprises a 55-nucleotide-long sequence, which is transcribed into a short, positive-sense RNA called the leader sequence. In other viral systems, this RNA has been shown to modulate host cell transcription. Here, we report the presence of measles virus leader RNA in both cytoplasmic and nuclear fractions of infected HeLa cells as well as T- and B-lymphoid cells. A sharp and rapid increase in the concentration of leader RNA in the nucleus of infected HeLa cells was also observed. The presence and accumulation of leader RNA in the nucleus of infected cells supports the hypothesis that the leader RNA plays a role in the down regulation of host cell transcription and may be responsible for the suppression of immunoglobulin synthesis by measles virus-infected B cells. Such alterations in immune responsiveness could aid in the establishment of a persistent infection by measles virus.

Base Sequence↗

Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability.

Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40 + IL-4. To address this issue, single-cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse-transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing mu, gamma 1, gamma 3, or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase.

Antibodies, Anti-Idiotypic↗

The Chinese hamster cell emetine resistance gene. Analysis of cDNA and genomic sequences encoding ribosomal protein S14.

We used an intersecting pool strategy to recognize chimeric plasmids containing Chinese hamster ribosomal protein cDNAs. The screening procedure involved hybridization-selection of messenger RNAs, cell-free translation of selected mRNAs, and electrophoresis of polypeptide products on one- and two-dimensional polyacrylamide gels. The protocol was designed to recognize ribosomal protein S14 cDNAs specifically. Of 500 chimeric plasmids screened, two possessed cDNAs complementary to S14 mRNA and 18 contained sequences complementary to other ribosomal protein messages. Previously we demonstrated that mutations affecting Chinese hamster ovary cell ribosomal protein S14 are responsible for genetic resistance to the translational inhibitor emetine (emt b). Because emetine-resistant mutant and wild type Chinese hamster ovary cells elaborate mRNAs that encode electrophoretically distinguishable forms of S14 protein, we were able to identify S14 cDNA clones unambiguously. The data described here indicate that: 1) clone pCS14-1 contains most, if not all, of the S14 coding sequence as a cDNA; 2) S14 mRNA is approximately 0.01% of a Chinese hamster cell's polyadenylated messenger RNA; and 3) genomic DNA-encoding ribosomal protein S14 is a low, perhaps single, copy sequence with a complex structure, including several, long intervening sequences.

Animals↗

mRNA localisation during development.

Although there are many differences, mRNA localisations in the Xenopus oocyte show some tantalizing similarities to those occurring in Drosophila development. As in Drosophila, transcripts localise to opposite poles of the oocyte, this localisation is hierarchical and occurs in a multistep process in which localisation is followed by anchoring at the cortex. This distinction between initial transport and long-term maintenance reflects the dynamic nature of the cytoskeleton: the microtubule tracks form and reform according to the needs of the cell so that stable localisation must be mediated by a more constant structure--the cortex. A possible exception is the localisation of gurken mRNA where it is unknown whether there are separate mechanisms for transport to and maintenance at the oocyte nucleus. However, gurken is responsible for the transmission of a transitory signal; once this has been received, and the fate of the recipient follicle cells determined, there is no further need for localisation. It is possible that the time scale over which the localisation machinery is stable is sufficient for transmission of this signal without the need for a separate maintenance phase. The existence of a nanos homologue, Xcat-2 (Mosquera et al., 1993), associated with the Xenopus germ plasm is particularly interesting because of the morphological and functional similarities between Drosophila polar granules, Caenorhabditis P-granules, and Xenopus germ plasm. These electron-dense protein-RNA complexes are maternally supplied and in each case segregate with the germ line. These granules may represent a fundamental conserved pathway to germ-cell specification and it is now at least a possibility that they are also involved in establishing the embryonic axis through translational repression. In the case of Drosophila, this occurs through localised nanos acting (via Pumilio) on nanos response elements in hunchback mRNA. No such regulatory pair has yet been demonstrated in C. elegans or X. laevis, but each contains a candidate for one half of the interaction: glp-1 could be a target for an unidentified nanos-like protein; Xcat-2 may control translation of an unknown NRE-containing mRNA. Another common feature of mRNA localisation is that in every case where the targeting signal has been determined, it has been mapped to a region of the 3' UTR capable of forming an extensive secondary structure (e.g., David and Ish-Horowicz, 1991; Dalby and Glover, 1992; Gavis and Lehmann, 1992; Kim-Ha et al., 1993; Kislauskis et al., 1993, 1994; Lantz and Schedl, 1994). In several cases, translational control and transcript stability signals have also been mapped to these regions (Jackson and Standart, 1990; Standart et al., 1990; Standart and Hunt, 1990; Davis and Ish-Horowicz, 1991; Wharton and Struhl, 1991; Dalby and Glover, 1993; Evans et al., 1994; Kim-Ha et al., 1995). The large secondary structures may provide a means for stably exposing sequence-specific regions of RNA to proteins. Due to the ease with which RNA forms base pairs, it is likely that rather than remaining single-stranded, RNA within the cell forms some sort of secondary structure. The geometry of purely double-stranded RNA does not permit sequence specific interactions between proteins and the bases because the major groove is inaccessible to amino acid side chains (Weeks and Crothers, 1993). However, the distortions to the dsRNA helix provided by bulges, pseudoknots, and the single-strand loop regions in stem-loop structures do present sequence information that can be "read" by proteins. The extensive 3'UTRs may produce a stable secondary structure which ensures that regulatory elements remain exposed in such regions rather than hidden in double-stranded stems. (ABSTRACT TRUNCATED)

Animals↗

Extensive homology of nuclear ribonucleic acid and polysomal poly(adenylic acid) messenger ribonucleic acid between normal and neoplastically transformed cells.

A cell line, designated BP6T, derived from Syrian hamster embryo (SHE) cells following treatment with benzo[a]pyrene is capable of producing tumors in newborn hamsters following the injection of as few as 1-10 cells. Polysomal poly(A) mRNA and total nuclear RNA obtained from this highly tumorigenic cell line were compared to RNAs obtained from the nonneoplastic parental embryo cells by a variety of techniques. RNA excess hybridizations to normal cell radiolabeled single-copy DNA or to a single-copy DNA tracer enriched for sequences transcribed in neoplastically transformed cells were unable to detect any significant differences in RNA sequence complexity between normal SHE cells and neoplastic BP6T cells. This finding of extensive homology of polysomal poly(A) mRNA and total nuclear RNA between normal and neoplastic cells, together with our previous finding of extensive homology of the major 35S-labeled nuclear or cytoplasmic polypeptides observable on two-dimensional gels [Leavitt, J. C., & Moyzis, R. K. (1978) J. Biol. Chem. 253, 2497-2500], demonstrates that the phenotypic changes associated with neoplastic transformation by chemical carcinogens are accompanied by relatively few changes in the qualitative pattern of gene expression in cells cultured in vitro.

Animals↗

The nucleotide sequence of spinach cytoplasmic 5 S ribosomal RNA.

The nucleotide sequence of the cytoplasmic 5 S ribosomal RNA from Spinacia oleracea has been determined. A secondary structural model possessing four base-paired regions can be constructed from the primary structure. This RNA shows 90 to 93% nucleotide sequence homology with other higher plant cytoplasmic 5 S RNAs and 73% homology with that of the lower eukaryote Chlorella. The spinach 5 S RNA has the nucleotide sequence identical with that of Chlorella in two important single-stranded regions, the sequence C10 AUACC and the dodecanucleotide sequence at positions 33 to 44. A nucleotide sequence similar or identical with C10 AUACC is found in most other eukaryotic 5 S RNAs, including the 5 S RNA from human KB cells. In addition, a single-stranded loop of 12 residues corresponding to positions 33 to 44 in the spinach 5 S RNA sequence may be a general feature of eukaryotic cytoplasmic 5 S RNAs, while prokaryotic 5 S RNAs have a 13-member loop for the corresponding residues. Several other important homologies in primary and secondary structure have also been observed in comparing spinach 5 S RNA to other 5 S RNAs.

Base Sequence↗

Transcriptional regulation of two genes specifically induced by glucose starvation in a hamster mutant fibroblast cell line.

This report concerns the characterization of the RNA transcripts encoded by two cDNA sequences p4A3 and p3C5, derived from a hamster temperature-sensitive mutant cell line K12. Using the two cDNA sequences as hybridization probes, we show that they occur as single copy genes in the hamster genome and encode for RNA transcripts which are highly inducible in K12 cells at 40.5 degrees C. After incubation at 40.5 degrees C for 16 h, there is a 10-fold increase in the p4A3 and p3C5 mRNA levels, reaching a final concentration of about 1% of the cytoplasmic polyadenylated RNA. We demonstrate that the kinetics of transcription of p4A3 and p3C5 directly parallel the accumulation of the mRNA levels at 40.5 degrees C. Thus, our data indicate that the expression of these two genes are primarily regulated at the transcriptional level. In addition, there is a 3- to 4-fold increase in the p4A3 and p3C5 mRNA levels when the cells are specifically starved of glucose. This implies that the expression of these two genes are stringently regulated by the availability of glucose in the culture medium. The relationship between the cDNA clones and two glucose-regulated proteins which are overproduced in K12 cells at 40.5 degrees C is discussed.

Animals↗

RD 114 virus-specific sequences in feline cellular RNA: detection and characterization.

RNA extracted from cat cells contains sequences homologous to RD-114 viral RNA. The sequences are measured by molecular hybridization with a single-stranded DNA probe synthesized by the virion polymerase using the endogenous viral RNA as template. Viral-specific RNA has been detected in all cells of cat origin tested thus far, but not in cells of other animals, except for the virus-producing human rhabdomyosarcoma cell, RD-114. The extent of hybridization of the DNA probe to cellular RNA was equivalent to that obtained with viral 70S RNA indicating that an equal extent of viral specific sequences is present in all cat cells as well as in RD-114 cells. The amounts of this viral RNA reach approximately 100 copies per cell in cat cells, while virus-producing RD-114 cells contain about 1,000 copies per cell. The viral RNA is present in cat cells in two distinct sizes of about 35S and 18S, whereas in RD-114 cells virus-specific RNA is quite heterogeneous in size.

Animals↗

Intron 4 mutation in APC gene results in splice defect and attenuated FAP phenotype.

The adenomatous polyposis coli (APC) protein is a tumor suppressor frequently involved in the development of inherited and sporadic colon cancers. Somatic mutations of the APC gene are found in 80% of all colon cancers. Inherited mutations result in familial adenomatous polyposis (FAP) as well as an attenuated form of this syndrome. FAP is characterized by the early age onset of hundreds to thousands of colonic adenomatous polyps and a virtual certainty of colon cancer unless the colon is removed. The attenuated form of FAP (AFAP) is characterized by fewer adenomas, later onset of adenomas and cancer, and a decreased lifetime cancer risk. We report a 37-year-old man with a history of more than 50 colonic adenomatous polyps, located predominately in the right colon. An insertion of a single thymidine between the second and third base pairs of intron 4 of the APC gene was identified (c.531+2_531+3insT). Monoallelic hybrid cells harboring a single copy of human chromosome 5 were generated from patient lymphoblasts. Sequencing of the APC cDNA product from these cells revealed a single RNA transcript with aberrant splicing in the mutant mRNA whereby exon 4 is deleted. The translational reading frame is shifted after codon 140 and a translational stop is generated predicting a truncated protein of 147 amino acids, thus indicating that the intronic mutation is disease causing. The lack of a secondary transcript from the mutant allele suggests that incomplete exon skipping is not the molecular mechanism behind the attenuated phenotype.

Adenomatous Polyposis Coli↗

Direct sequence analysis of amplified dengue virus genomic RNA from cultured cells, mosquitoes and mouse brain.

A method is described for direct sequence analysis of selected regions of dengue virus genomic RNA in infected tissues. Using specific primers, total high-molecular-weight infected-cell RNA is reverse transcribed to single-stranded (ss) complementary DNA, amplified using the polymerase chain reaction (PCR) and sequenced using ssDNA obtained after lambda exonuclease digestion of one strand of the PCR product (R.G. Higuchi and H. Ochman, Nucleic Acids Research, 17, 5865, 1989). Sequence data for the envelope protein gene of two dengue-3 virus isolates were obtained using RNA from small numbers (10(5)) of cultured mosquito or monkey kidney cells, from one mg of infected mouse brain and from 1/300th of an infected Toxorhynchites amboinensis mosquito. Independent determinations showed that errors occurring during reverse transcription or PCR were not represented to a significant degree in the sequence of the amplified DNA. The method does not depend on extensive passaging of virus or large-scale growth to generate material for sequencing and therefore provides a means of obtaining sequence data for unadapted dengue virus isolates.

Aedes↗

Decoding the landscape of cell-type-specific co-expressed transcription factors in soybean.

Soybean (Glycine max) is an essential source of protein and oil with high nutritional value for human and animal consumption. To enhance our understanding of soybean biology, it is essential to have accurate information regarding the expression of each of its protein-coding genes. Here, we present Tabula Glycine max, a soybean single-cell resolution transcriptome atlas. This atlas comprises single-nucleus RNA-sequencing data from ten different G. max organs and morphological structures constituting the entire soybean plant. These nuclei are grouped into 156 different clusters based on their transcriptomic profiles. The breadth of various organs, tissues and cell types represented in Tabula Glycine max reveals that the pattern of co-expressed transcription factor genes is sufficient to define most cell types based on their function and organ of origin. Defining cell-type-specific co-expressed transcription factor genes offers a new perspective to engineer cell-type-specific programmes and enhance the biology of unique soybean cell types. This cellular resolution and breadth make the Tabula Glycine max an exceptional resource for the plant and soybean communities.

Journal Article↗

Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.

A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan. Significant homology was found between the deduced amino acid sequence of the proteoglycan and two regions of a chicken hepatic lectin. Immunoprecipitation of the products of cell-free translation yielded a component of about 340 kDa, and transfer blot hybridization of sternal cartilage RNA showed a single mRNA of about 8.1 kilobases. Hybridizable mRNA sequences were readily detectable by dot-blot analyses of the cytoplasm of cartilaginous tissues of the chicken embryo, whereas similar analyses of prechondrogenic limb mesenchymal cells did not demonstrate such hybridizable mRNA signals.

Amino Acid Sequence↗

"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.

A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes.

Animals↗

Putative glioblastoma origin-like cells in the subventricular zone: isolation and characterization.

Glioblastoma (GBM) remains lethal despite maximal therapy. The adult subventricular zone (SVZ), a neural stem-cell niche, has been implicated as a potential site of origin, yet the identity and functional properties of putative GBM origin-like cells (GBM-OCs) within the SVZ remain unclear. An SVZ-restricted somatic mutation mouse model (Cre-induced EGFRvIII expression with Trp53 and Pten disruption) was established and mouse SVZ-derived cells were prospectively isolated for functional and molecular profiling. Self-renewal, multipotency, invasive potential and tumour-initiating capacity were assessed relative to control SVZ cells and matched tumour-derived tumourspheres. Whole-genome and RNA sequencing defined genomic and transcriptional alterations during early progression. Mouse GBM-OCs exhibited self-renewal and multilineage differentiation and initiated tumours only after re-implantation into the SVZ (11/29, 38%), whereas direct striatal implantation failed (0/25, 0%), indicating context-dependent tumorigenic potential associated with the SVZ microenvironment. In contrast, tumour-derived tumourspheres retained tumorigenic capacity upon implantation into both the SVZ and the striatum. During progression from mouse GBM-OCs to tumours, whole-chromosome and arm-level aneuploidies accumulated. In patients with GBM, multi-region single-nucleus RNA sequencing of tumour-free SVZ, matched tumours and tumour-free cortex identified rare neural stem cell-like, astrocyte-like and oligodendrocyte precursor-like SVZ populations transcriptionally aligned with GBM programmes. These cells showed single-nucleus RNA-inferred chromosome 7 gain and/or chromosome 10 loss signals, with concordant low-frequency copy-number alterations in the SVZ detected by exome sequencing and enriched in matched tumours. Together, these findings support the presence of SVZ-resident stem or progenitor-like populations with early GBM-associated features, consistent with putative GBM-OCs, and highlight the SVZ niche as a potential target for early detection and niche-informed therapeutic strategies.

Animals↗

Identification of malignant cells in multiple myeloma bone marrow with immunoglobulin VH gene probes by fluorescent in situ hybridization and flow cytometry.

Because it has been difficult to identify and separate malignant cells in human lymphoid malignancies, we have developed a flow cytometry-based fluorescent in situ hybridization (FISH) technique using immunoglobulin (Ig) heavy chain variable region (VH) gene probes. After obtaining the specific VH gene sequence expressed by the multiple myeloma IM-9 cell line and the malignant cells in five multiple myeloma patients, sense and antisense biotinylated single-stranded RNA probes were prepared by transcription from the malignant clone's VH DNA sequences. The cells from the IM-9 cell line and from the mononuclear bone marrow cells of multiple myeloma patients were fixed, hybridized with the above biotinylated RNA probes, incubated with streptavidin-phycoerythrin, and analyzed by FACS analysis. The myeloma cells stained positive with their own specific antisense VH biotinylated RNa probes, whereas sense and irrelevant antisense biotinylated probes demonstrated only background staining. Dilutional concentrations of the IM-9 cell line with normal bone marrow cells were also accurately quantitated by this procedure. The application of this technique will allow a more accurate assessment of tumor burden in patients with multiple myeloma and should permit an accurate method of tumor cell purification for clinical as well as biological studies. Furthermore, this technological advance should be equally effective at identifying specific VH gene-expressing cells in other lymphoid malignancies, as well as in nonmalignant B cell disorders.

Bacterial Proteins↗

Glycine loops in proteins: their occurrence in certain intermediate filament chains, loricrins and single-stranded RNA binding proteins.

Quasi-repetitive, glycine-rich peptide sequences are widespread in at least three distinct families of proteins: the keratins and other intermediate filament proteins, including nuclear lamins; loricrins, which are major envelope components of terminally differentiated epithelial cells; and single-stranded RNA binding proteins. We propose that such sequences comprise a new structural motif termed the 'glycine loop'. The defining characteristics of glycine loop sequences are: (1) they have the form x(y)n, where x is usually an aromatic or occasionally a long-chain aliphatic residue; y is usually glycine but may include polar residues such as serine, asparagine, arginine, cysteine, and rarely other residues; and the value of n is highly variable, ranging from 1 to 35 in examples identified to date. (2) Glycine-loop-containing domains are thought to form when at least two and to date, as many as 18, such quasi-repeats are configured in tandem, so that the entire domain in a protein may be 50-150 residues long. (3) The average value of n, the pattern of residues found in the x position and the non-glycine substitutions in the y position appear to be characteristic of a given glycine loop containing domain, whereas the actual number of repeats is less constrained. (4) Glycine loop sequences display a high degree of evolutionary sequence variability and even allelic variations among different individuals of the same vertebrate species. (5) Glycine loop sequences are expected to be highly flexible, but possess little other regular secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Single-cell transcriptomic atlas of Alzheimer's disease middle temporal gyrus reveals region, cell type, and sex specificity of gene expression with novel genetic risk for MERTK in female.

BackgroundAlzheimer's disease (AD), the most common age-related neurodegenerative disease, is closely associated with both amyloid-β plaque and neuroinflammation. Two thirds of AD patients are female, and they have a higher disease risk; women with AD have more extensive brain histological changes than men along with more severe cognitive symptoms and neurodegeneration.ObjectiveThis study aimed to determine how sex difference induces structural brain changes and molecular cell vulnerabilities in AD, with a focus on identifying sex-specific transcriptional alterations and genetic risk factors.MethodsWe performed single nucleus RNA sequencing on postmortem brains from individuals with AD and age- and sex-matched controls, focusing on the middle temporal gyrus, a cortical brain region strongly affected by the disease, and integrated single nucleus RNA sequencing results with genome-wide association study (GWAS) data using cell type-specific enrichment and generalized gene-set analysis approaches. The analysis pipeline is provided with threshold information.ResultsWe identified a selectively vulnerable subpopulation of layer 2/3 excitatory neurons that were RORB-negative and CDH9-expressing in both males and females. Disease-associated, but sex-independent, reactive astrocyte signatures were also present. In clear contrast, the microglia signatures of AD brains differed between males and females. Integrating single cell transcriptomic data with results from GWAS, we identified MERTK genetic variation as a candidate novel risk factor for AD selectively in females.ConclusionsTaken together, our single cell atlas of middle temporal gyrus revealed a unique cellular-level view of sex-specific transcriptional changes in AD, illuminating GWAS identification of sex-specific AD genes. These data serve as a rich resource for interrogation of the molecular and cellular basis of AD.

Alzheimer's disease↗

From wild to domestic: Single-cell transcriptomic perspectives on hippocampal regulation and evolution.

How domestication shapes brain evolution remains an open question. In this study, we integrated single-nucleus RNA sequencing (snRNA-seq), population genomics, and machine learning to investigate the hippocampal evolution under domestication. Across-species comparisons revealed that hippocampal cell type profiles are largely conserved across vertebrate species, while supporting the presence of adult hippocampal neurogenesis in birds. We further found that domestication and selective breeding likely influence the cellular composition and molecular regulation of the hippocampus. Our findings provide cellular evidence supporting the hypothesis that domestication affects adult hippocampal neurogenesis. Additionally, we showed that genes associated with neural progenitor cells (NPC) states and cell-marker programs are enriched for signatures of selection. Many of these genes function as regulators of neurogenesis and pathways mediating stress and fear reduction. Specifically, we identified selection at the FKBP5 promoter that may influence its expression in the NPC lineage, potentially contributing to stress-response regulation during domestication. Collectively, these results suggest that domestication is associated with hippocampal remodeling as part of an adaptive response to human-managed environments. This study provides a cellular and genetic perspective on how domestication reshapes the brain and offers a basis for further investigation into the mechanisms of neural evolution within the context of microevolution.

Animals↗