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Results for “R-loop”

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PubMed1989

Human cytomegalovirus RNAs immunoprecipitated by multiple systemic lupus erythematosus antisera.

The association of human cytomegalovirus (HCMV) RNAs with ribonucleoprotein particles that react with antibodies from patients with systemic lupus erythematosus was tested by immunoprecipitation with multiple patients' sera. A major late 2.8 kb RNA and several minor RNAs encoded by the HCMV long repeat region were immunoprecipitated from HCMV-infected cells by La, Ro and, much less abundantly, Sm autoimmune antisera. The exact location of these RNAs was determined by high resolution R-loop mapping and found to be between 0.8093 and 0.8189 map units. The 2.8 kb RNA is polyadenylated and associated with polysomes but does not appear to be spliced. Immunoprecipitation was not seen using normal or other autoimmune antisera. In addition, immunoprecipitation was specific to these RNAs in that other abundant HCMV RNAs were not immunoprecipitated. It was also found that the addition of increasing amounts of purified La antigen to infected cell lysates inhibited immunoprecipitation of the 2.8 kb RNA by La antiserum. The data suggest that specific HCMV RNAs may interact with cellular ribonucleoproteins known to be involved in post-transcriptional regulation of gene expression.

Antigen-Antibody Complex↗

PubMed1985

The genome of Bacillus subtilis phage SPP1: structure of an early promoter.

The strongest of five 'early' promoters of Bacillus subtilis phage SPP1 was localized in a DNA restriction fragment by analysis of RNA polymerase binding and R-loop formation. The nucleotide sequence of the promoter region was established. The signal structures identified were similar to those recognized by the sigma 55 RNA polymerase of B. subtilis. The promoter precedes an open reading frame with 51 codons. A protein with the Mr predicted from the nucleotide sequence was identified in minicells.

Autoradiography↗

PubMed1987

Cloning and expression in Escherichia coli of Proteus vulgaris genes for 16S ribosomal RNA.

In contrast to the established systems of plasmid-coded homologous ribosomal DNA (rDNA) cistrons in Escherichia coli little is known about the fate of heterologous rRNA. In order to study expression of foreign rDNA, rRNA cistrons from Proteus vulgaris were cloned in phage vector Charon 35, subcloned in pBR322 and transformed in E. coli. The inserts of two clones (pPM2 and pPM14) were characterized by restriction analysis and Southern hybridization. Each of them harboured a complete rrn cistron. The location of rRNA genes of clone pPM2 was also verified by R-loop analysis. The 5' flanking region of the 16S rRNA of pPM2 was sequenced and compared to the E. coli counterparts. High-level homologies exist in the functional parts of this region, e.g. promoters, box A and RNAase III recognition site. The copy number of pPM2 and pPM14 was estimated to be 8 and 10, respectively. Clones showed a markedly reduced growth rate (generation time about 57 to 70 min) as compared to the non-transformed cells (generation time 40 min). rDNA cistrons of P. vulgaris were properly expressed and the transcripts are processed as demonstrated by the presence of 16S rRNA from P. vulgaris in both ribosomes and 30S ribosomal subunits isolated from the transformed E. coli cells. The fraction of heterologous rRNA in ribosomes was about 25%.

Amino Acid Sequence↗

PubMed2025

Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells.

Base editors create precise nucleotide changes in DNA, but their off-target activity remains challenging to quantify. Here, we develop and deploy a direct, in cellulo sequencing assay that simultaneously measures both Cas9-mediated unwinding and deaminase editing of genomic DNA (beCasKAS). Our strategy nominates >460-fold more potential off-target sites than other methods by enriching for Cas9-dependent R-loops immediately preceding editing. Using beCasKAS in primary human T-cells, we observe that mRNA-encoded ABE8e and PAMless ABE8e-SpRY base editors have distinct off-target profiles that can be mitigated by optimizing mRNA dose. Finally, we combine beCasKAS with base-resolution deep learning models to risk-stratify off-target edits by their likelihood of epigenetic dysregulation. Collectively, beCasKAS offers a sensitive and facile tool to optimize the balance between base editor on- and off-target activity.

Journal Article↗

PubMed1988

Study on rRNA genes in Mycobacterium smegmatis.

The number of rRNA genes in Mycobacterium smegmatis was examined by hybridization of BamHI and SalI digests of chromosomal DNA with 3'-end-labeled 5S, 16S, 23S rRNA and tRNA. Each RNA probe gave two hybridization bands. The PstI fragments of 6.6 kilobases were cloned to pBR322. The cloned DNA was characterized by restriction endonuclease mapping, DNA-RNA hybridization, and the R-loop technique.

Cloning, Molecular↗

PubMed1979

Enrichment and characterization of the DNA coding for vitellogenin in Xenopus laevis.

Purified vitellogenin mRNA of Xenopus laevis was incubated with mechanically sheared DNA in high concentrations of formamide and the resulting R-loops (i.e. RNA . DNA hybrid fragments) separated from the bulk DNA by caesium chloride buoyant density centrifugation. Hybridization with 125I-labeled vitellogenin mRNA revealed a 15--30-fold enrichment of the DNA coding for vitellogenin. Restriction analysis of the R-loop-enriched DNA demonstrated that all known endonuclease HindIII fragments coding for vitellogenin of unfractionated Xenopus DNA were also present in the enriched material, including the specific fragments for the oligo(A)-containing segment of the RNA. Comparison of these restriction data with the structure found in cloned vitellogenin cDNA, indicates the presence of at least one intervening sequence in the genomic DNA coding for vitellogenin.

Animals↗

PubMed1980

Characterization of cloned complementary DNA covering more than 6000 nucleotides (97%) of avian vitellogenin mRNA.

The messenger RNA coding for chicken vitellogenin, a precursor of the egg-yolk proteins lipovitellin and phosvitin, is synthesized in the liver following estrogen injection. This mRNA is 6600 nucleotides long. We have previously reported the cloning and preliminary characterization of some cDNA fragments representing portions of the vitellogenin mRNA [Biochem. Biophys. Acta, 606, 34--46 (1980)]. In this paper we report the full characterization of a larger series of such clones, representing almost the entire length of the mRNA, by restriction endonuclease mapping, R-loop mapping, RNA-DNA hybridization and by translation in vitro of the RNA which hybridizes to the cloned DNA. From the results we conclude that the chicken vitellogenin mRNA, unlike that of Xenopus laevis, does not vary in sequence over most of its length, although some variations in the cDNA sequences were detected particularly in clones derived from the 3' terminus of the RNA. All sequence variants appear to be present in RNA prepared from single animals. The possible origins of these minor species are discussed. Furthermore, we describe a cDNA clone complementary to an mRNA which is about the same size as vitellogenin mRNA and which codes for an egg yolk protein antigenically related to lipovitellin. This mRNA is synthesized constitutively.

Animals↗

PubMed2026

Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.

CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.

Humans↗

PubMed1987

Organization of rRNA genes in Mycobacterium bovis BCG.

The number of rRNA genes in Mycobacterium bovis BCG was examined by Southern hybridization of end-labeled 5S, 16S, and 23S rRNAs with BamHI, PstI, and SalI digests of M. bovis BCG DNA. Each RNA probe gave only one radioactive band with three kinds of DNA digest. These results suggest that M. bovis BCG chromosomes may carry only a minimum set of rRNA genes. Hybridization of randomly labeled rRNAs with BamHI, PstI, SalI, BglII, and PvuII digests of DNA from the same organism supported these conclusions. The 6.4-kilobase-pair SalI fragment containing the entire structural genes for both 16S and 23S rRNAs was cloned into pBR322. The cloned fragment was characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis, and the R-loop technique. The results indicated that the fragments contained rRNA genes in the following order: 16S, 23S, and 5S rRNA genes. No tRNA gene was detected in the spacer region between the 16S and 23S rRNA genes, but one was found downstream of the 23S rRNA and 5S rRNA genes.

Cloning, Molecular↗

PubMed1979

Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.

Animals↗

PubMed1980

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.

Animals↗

PubMed1980

Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus.

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.

Bacteriophage lambda↗

PubMed1980

Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones.

Unintegrated, circular viral DNA, isolated from Prague A avian sarcoma virus (PrA-ASV)-infected quail cells (QT6), was cloned in the lambda vector lambda gtWES x lambda B. Three independent lambda-ASV recombinants were identified, and each contained a complete copy of the PrA-ASV genome. The arrangement of the ASV sequences within the recombinants was determined by restriction enzyme analysis and hybridization with labeled ASV-specific complementary DNA. One of the recombinants (lambda RPA101) resulted from cloning at the EcoRI site located within the terminally repeated sequence and therefore was virtually co-linear with PrA-ASV virion RNA. The other two recombinants (lambda RPA102 and 103) resulted from cloning at the EcoRI site located within the viral env gene. By restriction enzyme analysis and by measurement of R-loops formed between lambda RPA101 and PrA-ASV virion 35S RNA, the viral genome was estimated to be 9,100 bases in length. Genome length viral DNA purified from clones lambda RPA102 and 103 was biologically active. Transfection of chicken embryo cells with viral DNA, in the form of either circles or linear dimers, produced foci of transformed cells within 8 to 10 days. Linear DNA was much less efficient at inducing transformation. Viral DNA from the clone lambda RPA101 was unable to cause transformation; the basis for this defect is unknown.

Alpharetrovirus↗

PubMed1981

Structures of two spliced herpes simplex virus type 1 immediate-early mRNA's which map at the junctions of the unique and reiterated regions of the virus DNA S component.

We have examined the structures of two herpes simplex virus type 1 immediate-early (IE) RNAs (IE mRNA-4 and IE mRNA-5) which map at the junctions of the unique (Us) and reiterated regions (TRs/IRs) of the virus DNA short component. Hybrids between IE cytoplasmic RNA and herpes simplex virus type 1 DNA restriction fragments were digested with single-strand-specific nucleases S1 and exonuclease VII, and the products were analyzed by agarose gel electrophoresis. Data obtained with the nuclease digestion technique were confirmed by electron microscopy of R-loop structures formed with polyadenylated IE RNA and virus DNA fragments. It was found that both IE mRNA-4 and IE mRNA-5 contained a 260-base 5'-terminal cotranscript which mapped at equivalent loci within TRs/IRs. These 5'-terminal sequences were shown to be spliced to 3'-terminal cotranscripts of 1,450 bases (for IE mRNA-4) and 1,540 bases (for IE mRNA-5). The 3'-terminal cotranscripts contained sequences encoded by both TRs/IRs and opposite ends of Us, indicating that the introns contained by the IE mRNA-4 and IE mRNA-5 genes, found to be approximately 150 base pairs in size, mapped entirely within the reiterated sequences. The data suggest that these genes may contain common and unique components, and the implications of this model are discussed.

Base Sequence↗

PubMed1984

Nucleotide sequence of an immediate-early frog virus 3 gene.

We have used "gene walking" with synthetic oligonucleotides and M13 dideoxynucleotide sequencing techniques to obtain the complete coding and flanking sequences of the gene encoding a major immediate-early RNA (molecular weight, 169,000) of frog virus 3. R-loop mapping of the cloned XbaI K fragment of frog virus 3 DNA with immediate-early RNA from infected cells showed that an RNA of approximately 500 to 600 nucleotides (the right size to code for the immediate-early viral 18-kilodalton protein of unknown function) hybridized to a region within 100 base pairs of one end of the XbaI K fragment; no evidence for splicing was observed in the electron microscope or by single-strand nuclease analysis. Further restriction mapping narrowed the location of the gene to the XbaI end of a 2-kilobase-pair XbaI-Bg/II fragment, which was bidirectionally subcloned into the bacteriophage pair mp10 and mp11 for sequencing. Mung bean nuclease mapping was used to identify both the 5' and the 3' ends of the mRNA. The 5' end mapped within an AT-rich region 19 base pairs upstream from two in-phase AUG start codons that were immediately followed by an open reading frame of 157 amino acids. Another AT-rich sequence was found at -29 base pairs from the 5' end of the mRNA start site; this sequence may function as a TATA box. The 3' end of the message displayed considerable microheterogeneity, but clearly terminated within a third AT-rich region 50 to 60 base pairs from the translation stop codon. The eucaryotic polyadenylic acid addition signal (AATAAA) was not present, a finding to be expected since frog virus 3 mRNA is not polyadenylated. Both the single-stranded mp10 clone of the XbaI-Bg/II fragment and a 15-base oligonucleotide complementary to the region flanking the two AUG translation start codons inhibited translation of the immediate-early 18-kilodalton protein in vitro, confirming the identity of the sequenced gene. As the regulatory sequences of this gene did not resemble those of known eucaryotic genes or of the cytoplasmic vaccinia virus, we conclude that frog virus 3 has evolved unique signals for the initiation and termination of transcription.

Base Sequence↗

PubMed1987

Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures.

Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1. The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region. Species with 5' ends in E7 accounted for over 95% of all transcripts seen. Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region. The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region. This species probably encodes the abundant E4 protein found in plantar warts (F. Breitburd, O. Croissant, and G. Orth, Cancer Cells, vol. 5, in press; J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986). Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF. The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Base Sequence↗

PubMed1987

Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata.

We have identified and mapped a number of RNA species of human papillomavirus types 6 and 11 from condylomata acuminata by the electron microscopic R-loop technique. Each of the early (E)- and late (L)-region open reading frames (ORFs) deduced from the DNA sequences was represented in one or more transcripts. In addition, RNA species that could encode the modulator of DNA replication and the repressor of transcription, functions recently identified in the genetically similar bovine papillomavirus type 1, were also detected. Some ORFs were 5' proximal in one or more transcripts, whereas others were not 5' proximal in any species, suggesting that internal initiation of translation might be required to gain access to these latter ORFs. Virtually all transcripts had their 5' ends located in the E region and were polyadenylated at one of two sites, i.e., at the end of the E region or at the end of the L region. The great majority of the RNAs were derived from the E region of the genome, with one species approximately 50 to 100 times more abundant than the others. For most of the RNAs, the 5' end mapped near nucleotide 700; minor populations had 5' ends near nucleotide 100 or 1200. By correlating our mapping data with the genomic DNA sequences as well as available RNA structures and cDNA sequences of several papillomaviruses, we predict a number of mRNA splice donor and acceptor sites and suggest that the papillomaviruses have sophisticated usage of ORFs through alternative promoters, mRNA splice sites, and polyadenylation sites.

Anus Neoplasms↗

PubMed2025

Developing a disease-specific accessible transcriptional signature as a biomarker for ataxia with oculomotor apraxia type 2.

BACKGROUND: Genetic ataxias are clinically heterogenous neurodegenerative conditions often involving rare or private mutations and it is often difficult to assign pathogenicity to rare gene variants solely based on DNA sequencing. An effective functional assay from an easy-to-obtain biospecimen would aid this assessment and be of high clinical value. SETX encodes a ubiquitous DNA/RNA helicase crucial for resolving R-loops and maintaining genome stability. Loss-of-function mutations cause a recessive disorder, Ataxia with Oculomotor Apraxia Type 2 (AOA2). METHODS: Here we utilize Weighted Gene Co-expression Network Analysis (WGCNA) from patient blood to construct an AOA2-specific transcriptomic signature as a biomarker to evaluate SETX variants in patients clinically suspected of having AOA2. RESULTS: WGCNA from peripheral blood RNA of 11 AOA2 patients from 7 families initially identified a single gene module that was modestly effective in distinguishing individuals with AOA2 from controls (sensitivity 73%, specificity 97%) and was able to robustly differentiate AOA2 patients from those with genetically distinct, yet phenotypically similar, neurological disorders (sensitivity 100%, specificity 100%). An independent derivation of the transcriptional biomarker identified a dual module model that was able to better distinguish individuals with AOA2 from controls (sensitivity 100%, specificity 97%). As validation, we examined a second cohort of 21 patients from 13 families and demonstrate that this dual module transcriptional biomarker could discriminate patients clinically suspected of AOA2 from controls (57%, 95%CI: 34%-78%). Overall, the transcriptional biomarker was able to separate AOA2 subjects (n = 32) from controls (n = 35) with 72% sensitivity and 97% specificity. Notably, this transcriptomic biomarker enabled verification of the first pathogenic SETX mutation found in a non-canonical transcript, expanding the spectrum of mutations that contribute to AOA2. CONCLUSIONS: Our study identified a transcriptional biomarker that was able to differentiate AOA2 from controls and from other related neurological disorders, consequently expanding the spectrum of known pathogenic mutations. This proof-of-concept study illustrates that transcriptional biomarkers may be used to validate variants of uncertain significance in known genetic diseases.

Humans↗