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Results for “in situ sequencing”

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At least 19 recordsLinked to original sources
PubMed2024

Diversity of ribosomes at the level of rRNA variation associated with human health and disease.

With hundreds of copies of rDNA, it is unknown whether they possess sequence variations that form different types of ribosomes. Here, we developed an algorithm for long-read variant calling, termed RGA, which revealed that variations in human rDNA loci are predominantly insertion-deletion (indel) variants. We developed full-length rRNA sequencing (RIBO-RT) and in situ sequencing (SWITCH-seq), which showed that translating ribosomes possess variation in rRNA. Over 1,000 variants are lowly expressed. However, tens of variants are abundant and form distinct rRNA subtypes with different structures near indels as revealed by long-read rRNA structure probing coupled to dimethyl sulfate sequencing. rRNA subtypes show differential expression in endoderm/ectoderm-derived tissues, and in cancer, low-abundance rRNA variants can become highly expressed. Together, this study identifies the diversity of ribosomes at the level of rRNA variants, their chromosomal location, and unique structure as well as the association of ribosome variation with tissue-specific biology and cancer.

Humans↗
PubMed1978

Detection of Epstein-Barr virus (EBV) DNA sequences using in situ hybridization.

In situ hybridization was used to detect Epstein-Barr virus (EBV) DNA sequences under conditions where the virus DNA is replicating spontaneously and where it is induced to do so following superinfection. The in situ reaction itself is influenced by several parameters, analogous to conventional nucleic acid hybridization, consideration of which should help to optimize the designing of in situ hybridization reactions in general. Both EBV complementary RNA (cRNA) and EBV DNA synthesized in vitro can efficiently detect the virus DNA sequences in situ. The findings presented here can therefore be utilized in both the study of EBV-cell interactions and, more generally, in studies using in situ hybridization as a general approach.

Antigens, Viral↗
PubMed2019

Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing.

Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a "chromatin-RNA in situ reverse transcription sequencing" (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.

Animals↗
PubMed1975

Detection of virus-specific DNA sequences by quantitative in situ hybridization during infection with SV40.

In situ hybridization has been used as a quantitative method to study the replication of SV40 during lytic infection. Various parameters have been defined such as fixation and denaturation of cytological preparations, concentration of complementary (3H)-cRNA and volume of buffer used for the hybridization reaction. When all these parameters are carefully controlled, a reproducibility of +/-10% can be obtained for the quantitative study of the system SV40-monkey kidney cells (CV-1). In this system, it is evident that the synthesis of viral DNA is not synchronized in confluent cells. Moreover, a proportion of 20 to 30% of cells are not labelled whatever the multiplicity of infection. In SV40-transformed cells, it was not possible to detect significantly the integrated genome due either to the small size of the genome or to the fact that the number of genome-equivalents integrated at the same place is too low to yield a radioactivitity superior to background level for long periods of exposure.

Animals↗
PubMed2024

Diversity of ribosomes at the level of rRNA variation associated with human health and disease.

Ribosomal DNA and RNA (rDNA and rRNA) sequences are usually discarded from sequencing analyses. But with hundreds of copies of rDNA genes it is unknown whether they possess sequence variations that form different types of ribosomes that affect human physiology and disease. Here, we developed an algorithm for variant-calling between paralog genes (termed RGA) and compared rDNA variations found in short- and long-read sequencing data from the 1,000 Genomes Project (1KGP) and Genome In A Bottle (GIAB). We additionally developed a novel protocol for long-read sequencing full-length rRNA (RIBO-RT) from actively translating ribosomes. Our analyses identified hundreds of rDNA variants, most of which, surprisingly, are short insertion-deletions (indels) and dozens of highly abundant rRNA variants that are incorporated into translationally active ribosomes. To visualize variant ribosomes at the single cell level, we developed an in-situ rRNA sequencing method (SWITCH-seq) which revealed that variants are co-expressed within individual cells. Strikingly, by analyzing rDNA, we found that variants assemble into distinct ribosome subtypes. We discovered that these subtypes acquire different rRNA structures by successfully employing dimethyl sulfate (DMS) probing of full length rRNA. With this atlas we investigated rRNA variation changes across human tissues and cancer types. This revealed tissue-specific rRNA subtype expression in endoderm/ectoderm-derived tissues. In cancer, low abundant rRNA variants can become highly expressed, which suggests the presence of cancer-specific ribosomes. Together, this study identifies and comprehensively characterizes the diversity of ribosomes at the level of rRNA variants which is dominated by indel variants, their chromosomal location and unique structure as well as the association of ribosome variation with tissue-specific biology and cancer.

Journal Article↗
PubMed1979

Detection of AKR MuLV-specific RNA in AKR mouse cells by in situ hybridization.

Conditions for the detection of complex RNA sequences by in situ hybridization have been investigated by using a single-stranded 3H-cDNA probe complementary to the AKR MuLV genome and in vitro cultured AKR mouse cells which spontaneously produce AKR MuLV. It is shown that fixation with glutaraldehyde at low concentration allows cellular RNA to be sufficiently well retained during the annealing process and that stringent conditions in situ can be maintained by means of formamide. Some conditions which promote atypical and non-specific binding of the probe have been identified.

Animals↗
PubMed1976

In situ hybridization of repetitive monkey genome sequences isolated from defective simian virus 40 DNA.

The origin of a repetitive monkey DNA sequence that is incorporated into a defective simian virus 40 genome has been studied. A fragment (about 140 base pairs in length) containing essentially all the repetitive monkey DNA present in the defective and few, if any, SV40 sequences can be cleaved from the purified defective DNA by restriction endonucleases Hind(II and III). Radioactive cRNA prepared with the isolated fragment as template was hybridized in situ to African green monkey chromosomes. The results indicate that all or part of the sequence in question occurs at both centromeric and noncentromeric positions in many, but not all, chromosomes. Of the typical 60 chromosomes, between nine and eleven hybridize with the cRNA in noncentromeric regions.

Base Sequence↗
PubMed1978

Detection of viral sequences of low reiteration frequency by in situ hybridization.

The sensitivity of in situ hybridization has been increased at least 10-fold by hybridizing in cDNA excess, by increasing the diffusion of the cDNA through the cells, by hybridizing at optimum temperature, and by stabilizing hybrids during autoradiography. Saturation of intracellular RNA with [3H]cDNA has been achieved. The assay is quantitative. In situ hybridization has been used to detect and quantitate visna virus RNA in infected cells. By using [3H]cDNA with specific activity of 2 X 10(8) dpm/micrograms and conditions that reduce background to negligible levels, 10--20 copies of viral RNA per cell can be detected and quantitated after 2 days of autoradiographic exposure.

Animals↗
PubMed1977

Effect of different denaturing agents on the detectability of specific DNA sequences of various base compositions by in situ hybridisation.

In situ hybridisation of certain AT rich and GC rich satellite DNA complementary RNAs (cRNAs) to their homologous chromosomes at their respective optimal rate temperatures (TOPTS) after denaturation with various reagents (0.2 N HCl, 0.07 N NaOH, 90% formamide and heat) led to the following conclusions. -- Heat denaturation of chromosomal DNA in 0.1 X SSC at 100 degrees C gives significantly higher grain counts regardless of DNA base composition, HCl denaturation discriminates markedly against GC rich DNA. Chromosome morphology is best preserved after HCl and heat denaturation.

Animals↗
PubMed2026

Molecular divergence and genomic composition of B chromosomes in the fish Cyphocharax modestus (Characiformes, Curimatidae).

B chromosomes are supernumerary elements that evolve from standard A chromosomes and are primarily composed of repetitive DNAs, yet their origin, diversification, and molecular composition remain poorly understood in most vertebrates. We investigated two allopatric populations of Cyphocharax modestus (Curimatidae) combining classical cytogenetics, comparative genomic hybridization (CGH), and comparative satellitomics to characterize the repetitive DNA landscape of its B chromosomes. While both populations exhibited a conserved karyotype of 2n=54 biarmed chromosomes, five individuals from the Batalha River (BR) carried supernumerary chromosomes, comprising two distinct variants: a C-positive B1 and an C-negative B2. Comparative satellitome analysis between 3B-carrying and B-lacking individuals identified 116 satellite DNAs (CmoSatDNAs), with the 3B library showing higher abundances of specific sequences. Fluorescence in situ hybridization (FISH) revealed that both B variants share two centromeric satellites (CmoSat01-192 and CmoSat02-108) with the A complement, while CmoSat58-47 was exclusively to B2. Minimum spanning tree analysis of CmoSat58-47 revealed B-exclusive haplotypes alongside haplotypes shared with B-lacking individuals, suggesting a recent origin for these chromosomes. CGH experiments further confirm the sequence sharing between the A and B chromosomes, supporting an intraspecific origin, and revealing substantial genomic differentiation among B variants.

Animals↗
PubMed1976

Clonal derivatives of a herpes type 2 transformed hamster cell line (333-8-9): cytogenetic analysis, tumorigenicity and virus sequence detection.

The characteristics of the Syrian hamster cell line (333-8-9), putatively transformed by herpes virus type 2 (HSV-2), strain 333, have been examined. The line has been cloned and the clones characterized cytogenetically and morphologically. The original line and the clones have been assayed for tumorigenicity and for the presence of herpes-virus-specific nucleic acid sequences using in situ hybridization with 125I HSV-2 DNA. The line 333-8-9 is hyperdiploid with a mode of 49 chromosomes and contains a spectrum of cytogenetic marker chromosomes present in varying frequencies. The clones demonstrate a segregation of these marker chromosomes and other abnormal chromosomes. Cloned lines have distinct stem-cell types with the characteristic abnormal chromosomes present in a high percentage of cells. Some of the marker chromosomes have been identified by Giemsa banding techniques. All the clones have distinctive, persistent in vitro morphologies, ranging from purely epithelial to purely fibroblastic. They differ from the line 333-8-9 and between themselves in tumorigenicity, varying from highly tumorigenic to entirely non-tumorigenic. Herpes virus 2 specific sequences have been detected in the original cell line and in all but two of the clones. There does not appear to be any obvious correlation between the three parameters of: (1) detection of HSV-2 information; (2) marker chromosome occurrence; and (3) tumorigenicity.

Animals↗
PubMed2026

Evolutionary persistence of a highly prevalent multicopy mitochondrial-derived nuclear insertion (Mega-NUMT) in Neotropical Drosophila flies.

Although strict maternal transmission of mitochondria is a general feature of animals for ensuring homogeneity in mitochondrial DNA (mtDNA) across generations, exceptions were reported in the recent past. For example, some extremely rare but spectacular cases of heteroplasmy and paternal transmission in humans have questioned the universal evolutionary principle. Hence, as an alternative, the Mega-NUMT concept was coined to explain this discovery and was thereafter partly proven to exist. This concept expands on the quite common transfer of mtDNA fragments to the nucleus (NUMTs) by considering the existence of multicopy mitochondrial nuclear insertions. Mega-NUMT reports are currently restricted to a few cases in animals, including humans. However, their detailed genomic organization, natural prevalence, and potential biological functions remain unclear. Here, we discovered that up to 60 full-sized mitochondrial genomes are integrated into the nuclear genome of the neotropical Drosophila paulistorum using long-read sequencing and in situ hybridization. The copies are organized in one cluster on chromosome 3, which we designated the "Dpau Mega-NUMT". Contrary to the rarity in humans, this Mega-NUMT is found at high prevalence (40%) in both laboratory lines and natural D. paulistorum populations of different semispecies. Additionally, the Mega-NUMT copies are phylogenetically separated from the current mitotypes of D. paulistorum. Together, these observations suggest long-term maintenance of the Mega-NUMT in nature. Hence, we propose that the Dpau Mega-NUMT may have been transferred to the nuclear genome before the D. paulistorum semispecies radiation and speculate based on these findings that it possibly is maintained at relatively high prevalence in nature by balancing selection or due to a yet undetermined function.

Animals↗
PubMed2026

Application of a Translational Research Platform to Unveil Efficacy Signals and Mechanisms of Resistance of FGFR Inhibitors in Multiple FGFR-Altered Solid Tumors.

PURPOSE: The predictive value of fibroblast growth factor receptor (FGFR) amplifications (amp) and the role of FGFR mutations (mut) beyond known activating variants remain unclear. We aimed to establish a translational research platform to characterize FGFR alterations (alt) and explore their potential as predictive biomarkers for FGFR-targeted agents. EXPERIMENTAL DESIGN: This ambispective study included a retrospective analysis of patients with FGFR-alt tumors treated with selective FGFR inhibitors (FGFRi) and a prospective collection of longitudinal tumor samples. Patient-derived xenografts (PDX) were generated to investigate FGFRi mechanisms of action and resistance. Molecular characterization included genomic, transcriptomic, proteomic, and functional analyses using the Functional Annotation for Cancer Treatment (FACT) assay. RESULTS: Among 36 retrospectively analyzed patients, clinical benefit from FGFRis was observed in cases with FGFR mRNA overexpression or FGFR2/11q co-amp, but no association was found with the amplification levels. In archival tumor samples, exploratory proteomic analysis showed FGFR1-4 protein expression in 78% of FGFR1/2-amp tumors detected by fluorescence in situ hybridization. RNA sequencing identified a higher prevalence of FGFR mRNA overexpression than proteomic analysis. Among patients harboring FGFR-mut, only one bladder cancer with an FGFR3-mut S249C derived benefit. FACT assay supported the functional activity of selected variants, including FGFR3 T689M, and suggested potential resistance mechanisms involving PI3K/PTEN and MAPK pathway co-alterations. A prospective FGFR-alt PDX biorepository enabled exploratory biomarker analyses, supporting the hypothesis that FGFR1-4 mRNA expression may better reflect FGFR dependency than genomic alterations alone. CONCLUSIONS: These findings highlight the complexity of FGFR-driven oncogenesis and support integrative molecular approaches to refine patient selection for FGFR-targeted therapies.

Humans↗
PubMed1977

Sequences associated with intracisternal A particles are reiterated in the mouse genome.

Using a 3H-cDNA for RNA sequences specifically associated with murine intracisternal type A particles, we have found multiple copies of this information in high molecular weight nuclear DNA from tissues of both Mus muscules (BALB/c, NIH Swiss, A/Jax and feral) and Mus cervicolor. Reiteration frequencies varied from 1050-1800 per haploid genome, except that fewer copies (450) were found in BALB/3T3 cells. In the series studied, the reiteration frequencies in the DNA of A particle-rich tumor cells (myeloma and neuroblastoma) were not higher than those in normal tissues (liver and sperm). Multiple copies were retained when cellular DNAs were sedimented through alkaline sucrose gradients, indicating that the sequences are integrated in the mouse genome. In situ hybridization with cDNA showed that the sequences were associated with many chromosomes and were concentrated over certain regions of some chromosomes. Only low levels of homologous sequences were detected in rat, hamster and guinea pig DNA under stringent conditions of hybridization. The presence of reiterated sequence transcripts in poly(A) RNA from a neuroblastoma A particle fraction was confirmed by direct hybridization of the RNA with cellular DNA.

Animals↗
PubMed1977

Extensive in vitro transcription of rous sarcoma virus RNA by avian myeloblastosis virus DNA polymerase and concurrent activation of the associated RNase H.

Conditions are described that promote the efficient reverse transcription of most of Rous sarcoma virus (RSV) RNA sequences by avian myeloblastosis virus DNA polymerase in vitro. A detailed analysis of the reverse transcription reaction was carried out using two procedures: in situ analysis of the RNA sequences transcribed and DNA-RNA annealing studies. Under optimal conditions, after 1 h of reaction, practically all RSV RNA sequences were transcribed with a frequency varying from 30 to 90%. The DNA product was at least 95% single stranded, had a chain length ranging from a few hundred up to 5,000 necleotide residues, half of it being larger than 1,000 residues, and, after hybridization at RNA excess, protected the entire RSV genome from RNase digestion, as monitored by the large T1 oligonucleotides of RSV RNA. Analysis of the product of a very short reaction time (5 min) showed that DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA. This in in agreement with our previous analysis of a much less efficient reverse transcription reaction. Under optimal conditions of reverse transcription, we find now that the RNase H associated with the avian myeloblastosis virus DNA polymerase is active in degrading the RNA moiety of the RNA-DNA hybrids synthesized.

Avian Leukosis Virus↗
PubMed1975

Detection of virus-specific DNA and RNA base-sequences in individual cells transformed or infected by adenovirus type 2.

By means of 3H thymidine-labelled adenovirus 2 DNA, adenovirus-specific DNA sequences have been localized in individual nuclei of adenovirus 2- or 12-infected cells, and adenovirus-specific RNA sequences have been detected in the permissive cells as well as in cells transformed by adenovirus type 2. The ability to detect such specific virus RNA base-sequences in particular, by in situ hybridization, should be useful in studying the transcriptional specificities of tumours and neoplasms.

Adenoviridae↗