Hormonal modulation of alpha2u globulin mRNA: sequence measurements using a specific cDNA probe.
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Aging↗
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Results for “cDNA sequencing”
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Nucleotide sequences in mouse DNA and RNA specific for Moloney sarcoma virus.
Complementary DNA (cDNA) synthesized by Moloney murine sarcoma virus (M-MSV) was separated into two parts, the first, termed MSV-specific cDNA, composed of nucleotide sequences found only in M-MSV viral RNA, and the second, termed MSV-MuLV common cDNA, composed of nucleotide sequences that were found in both M-MSV and murine leukemia virus (MuLV) VIRAL RNAs. RNA complementary to the MSV-specific cDNA was not found in several other MSV isolates, nor in ecotropic MuLV, mouse mammary tumor virus, or several murine xenotropic oncoviruses. Cellular DNA of several species was examined for the presence of nucleotide sequences complementary to MSV-specific cDNA. Cells transformed by M-MSV did contain MSV-specific cDNA in their DNA. Normal mouse cell DNA apparently contained the majority of MSV-specific nucleotide sequences. Cellular DNA of related species contained proportionally less MSV-specific cDNA. Hybrids of MSV-spedivic cDNA and cellular DNA of related species melted at lower temperatures than hybrids of MSV-specific cDNA and mouse cellular DNA. RNA from normal mouse adult or embryonic cells did not contain detectable nucleotide sequences complementary to MSV-specific cDNA. Transformation of cells with M-msv resulted in transcription of RNA hybridizing with MSV-specific cDNA. Methylcholanthrene-induced mouse sarcomas and cell lines derived from them did not contain RNA complementary to MSV-specific cDNA. Mouse cell lines transformed with avian sarcoma virus or Kirsten MSV-specific cDNA. RNA homologous to MSV-specific nucleotide sequences is measurably present only in cells transformed by M-MSV and not in cells transformed by other biological or chemical agents that also cause sarcomas.
Animals↗
Dimensions of homo- and heteropolymeric sections of DNA synthesized by reverse transcription from globin mRNA matrices.
The lengths of globin messenger RNAs, isolated from rabbit and pigeon reticulocytes, and single-stranded complementary DNAs, synthesized from mRNA in presence of RNA-directed DNA-polymerase and oligo(dt) have been investigated by polyacrylamide gel electrophoresis under denaturing conditions. The cDNA preparations contained a fraction which corresponded in length to the alpha and beta chains of the mRNA used as a matrix. The lengthwise distribution of poly(A) sequences, isolated from globin mRNAs, corresponded to a statistical distribution, with a maximum at 75-80 nucleotides and with a maximum length of approximately 150 nucleotides. The lengthwise distribution of poly(dt) sequences, isolated from cDNA, corresponded to the poly(A) sequence distribution. It was concluded that: 1) cDNA is heterogeneous with respect to the length of poly(dt) sequences located at the 5' end of the molecule; 2) there was no selective use as primer of only that oligo(dt) which was located within the complex formed with the 5' end of the mRNA poly(A) sequence; 3) the heterogeneity of poly(DT) corresponded qualitatively to two models of template-primer interaction: selective use as a true primer of the oligonucleotide located in the complex formed with the 3' end of mRNA, and a statistical distribution of primer along the poly(A) sequence; and 4) the heterogeneity of the homopolymeric section of the cDNA may affect the kinetics of hybridization with mRNA, which possibly explains the previously observed [1] anomalous dependence of the hybridization rate on time.
Animals↗
Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing.
Three synthetic oligonucleotides were prepared to be complementary to known regions of the mouse immunoglublin light chain mRNA, and their ability to prime the transcription of complementary DNA (cDNA) was studied. The sequence of the cDNA was determined by adapting for mRNA the DNA sequencing method of Sanger, Nicklen and Coulson (1977) which uses 2'3' dideoxy ribonucleotides. A continuous sequence of 532 nucleotides was obtained, 321 corresponding to the whole of the constant region of the mRNA and the remaining 211 being the complete 3' noncoding region of the mRNA. The termination codon U-A-G occurs at the expected position in the mRNA corresponding to the triplet following the C terminal cystine. The nucleotide sequence is partially corroborated by the sequence of fragments obtained previously from 32P-mRNA fingerprints and endonuclease IV digests of 32P-cDNA, and is in agreement with the amino acid sequence of the constant region, except for a rearrangement of four amino acids (between amino acid positions 163 and 166). A revision of the amino acid sequence confirms the nucleic acid sequence.
Amino Acid Sequence↗
Nucleotide sequence of a mouse kappa light chain cDNA cloned in a bacterial plasmid.
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Animals↗
Human globin gene analysis for a patient with beta-o/delta beta-thalassemia.
Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene.
Adolescent↗
Detection and partial sequence analysis of gastrin mRNA by using an oligodeoxynucleotide probe.
We have used a specific deoxyoligonucleotide probe to detect gastrin mRNA in poly(A)-enriched RNA preparations from hog antrum. The nucleotide sequence of the oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C-A), was deduced from the unique amino acid sequence Trp-Met-Glu-Glu of gastrin. When used with hog antral RNA, the dodecanucleotide is an effective primer for the synthesis of gastrin-specific cDNA as judged by nucleotide sequence analysis of cDNA isolated by polyacrylamide gel electrophoresis. We have determined an 81-nucleotide sequence corresponding to the region of the gastrin mRNA that codes for the known amino acid sequence of the G34 progastrin intermediate species, and we have demonstrated the presence of two consecutive basic residues preceding the G34 sequence in the prohormone. Hybridization of gastrin cDNA or synthetic dodecanucleotide to hog antral RNA separated by gel electrophoresis on agarose gels in the presence of methylmercuric hydroxide indicates that the mRNA coding for gastrin is about 620 nucleotides long. These results suggest that the gastrin precursor peptide contains 110-140 amino acids. This method should be of general application for detection and characterization of mRNAs corresponding to proteins of known amino acid sequence.
Animals↗
IMAGE cDNA clones, UniGene clustering, and ACeDB: an integrated resource for expressed sequence information.
In this study we describe a new information resource that provides integrated access to information on IMAGE (integrated molecular analysis of genomes and their expression) cDNA library clones and derived expressed sequence tags (ESTs). We have developed an automated procedure that collates data from various public sources into a single ACeDB database. This database is a valuable tool for electronic cloning experiments and gene expression studies. It allows researchers to find information about cDNA libraries, plate addresses, insert sizes, and sequence data for IMAGE clones, the assignment of ESTs to UniGene clusters, and the chromosomal location of those genes in an efficient, graphically oriented manner.
Cloning, Molecular↗
Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.
We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.
Animals↗
Deciphering mixed infections by plant RNA virus and reconstructing complete genomes simultaneously present within-host.
Local co-circulation of multiple phylogenetic lineages is particularly likely for rapidly evolving pathogens in the current context of globalisation. When different phylogenetic lineages co-occur in the same fields, they may be simultaneously present in the same host plant (i.e. mixed infection), with potentially important consequences for disease outcome. This is the case in Burkina Faso for the rice yellow mottle virus (RYMV), which is endemic to Africa and a major constraint on rice production. We aimed to decipher the distinct RYMV isolates that simultaneously infect a single rice plant and to sequence their genomes. To this end, we tested different sequencing strategies, and we finally combined direct cDNA ONT (Oxford Nanopore Technology) sequencing with the bioinformatics tool RVhaplo. This method was validated by the successful reconstruction of two viral genomes that were less than a hundred nucleotides apart (out of a genome of 4450nt length, i.e. 2-3%), and present in artificial mixes at a ratio of up to a 99/1. We then used this method to subsequently analyze mixed infections from field samples, revealing up to three RYMV isolates within one single rice plant sample from Burkina Faso. In most cases, the complete genome sequences were obtained, which is particularly important for a better estimation of viral diversity and the detection of recombination events. The method described thus allows to identify various haplotypes of RYMV simultaneously infecting a single rice plant, obtaining their full-length sequences, as well as a rough estimate of relative frequencies within the sample. It is efficient, cost-effective, as well as portable, so that it could further be implemented where RYMV is endemic. Prospects include unravelling mixed infections with other RNA viruses that threaten crop production worldwide.
Genome, Viral↗
Human pegivirus 1 in healthy blood donors and in acute febrile patients from Mato Grosso, Midwestern Brazil.
The association between Human Pegivirus 1 (HPgV-1, Pegivirus hominis, family Hepaciviridae) with disease etiology remains unclear, yet this virus is widely distributed among people exposed to contaminated blood. This study aimed to identify the prevalence and characterize HPgV-1 genomes obtained from acute febrile patients (n = 92 from 2019) and from blood donors (n = 633 from 2022) sampled in Mato Grosso, Brazil. The serum samples were tested by RT-qPCR for a conserved 5' UTR region of HPgV-1. Nucleic acid of 28 positive samples was converted to ds-cDNA, purified and sequenced with an Illumina NextSeq platform. The prevalence of HPgV-1 among acute febrile patients was 4.35% (4/92), who were mostly female (3/4; 75.00%) mean 28.86 (12-67) years-old; whereas among blood donors it was 3.79% (24/633), which were predominantly male (16/24; 66.67%), aged 30-45 years-old (13/24; 54.17%). Eight HPgV-1 genomic sequences (7,708-9,248) were recovered; two belong to subgenotype 2a and clustered with strains from United States, Brazil, France and Japan, while the remaining six genomes from subgenotype 2b grouped with strains from Pará, Brazil and abroad. HPgV-1 detection in acute febrile patients and in healthy blood donors highlights the necessity to investigate the epidemiological aspects of this infection, as well as the possible impact to public health in Midwestern Brazil.
Cross-Sectional Studies↗
Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts.
Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.
Age Factors↗
Reverse transcription of thyroglobulin 33-S mRNA.
Bovine thyroglobulin 33-S mRNA has been used as a template for the synthesis of a complementary DNA, using RNA-directed DNA polymerase from the avian myeloblastosis virus. The yield of the reaction was relatively poor and the size of the cDNA did not exceed 10 S. Nevertheless, a copy of high specific radioactivity (approximately 10(7) counts. min-1 microgram-1) could be obtained which hybridized specifically back to its template with an rot1/2 value about 5 times higher than that observed in hybridizations between hemoglobin mRNA (alpha + beta chain) and hemoglobin cDNA. This suggests that thyroglobulin mRNA does not contain extensive internal repetitive sequences. Quantification of thyroglobulin mRNA sequences among various RNA preparations from the beef thyroid was performed using cDNA/RNA hybridizations in RNA excess. The results confirmed that thyroglobulin mRNA represents the large majority of mRNA in membrane-bound polysomes and indicated the virtual absence of thyroglobulin sequences on free polyosomes. The cDNA transcribed from mRNA of bovine origin hybridized efficiently with thyroid RNA from goats, dogs and humans. Although the heterologuous hybrids exhibited the expected decrease in thermal stability, the bovine cDNA provides an appropriate probe for studies dealing with the expression of the thyroglobulin gene in various mammals including man.
Animals↗
Nanopore sequencing to detect A-to-I editing sites.
Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.
Inosine↗
Complete nucleotide sequence of the 5' noncoding region of rabbit beta-globin mRNA.
The 5' noncoding region sequence of rabbit beta-globin mRNA has been determined. This region is 53 nucleotides long, not including the A-U-G initiator sequence or m7Gppp "cap" structure. 32P-labeled DNA complementary to the 5' noncoding region was synthesized using reverse transcriptase with the synthetic deoxyoctanucleotide d(G-C-A-C-C-A-T-T) as a "primer". The cDNA produced was then sequenced using a modification of the gel-sequencing technique previously developed for DNA sequencing (G.G. Brownlee and E.M. Cartwright, manuscript in preparation). The sequence obtained was checked by depurination and nearest-neighbor analysis. The known sequence at the 3' end of rabbit 18S ribosomal RNA cannot base-pair extensively with the 5' noncoding region of beta-globin mRNA; however, it does form 6 base pairs around the initiation codon.
Animals↗
Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.
Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.
RNA Editing↗
EnsMart: a generic system for fast and flexible access to biological data.
The EnsMart system (www.ensembl.org/EnsMart) provides a generic data warehousing solution for fast and flexible querying of large biological data sets and integration with third-party data and tools. The system consists of a query-optimized database and interactive, user-friendly interfaces. EnsMart has been applied to Ensembl, where it extends its genomic browser capabilities, facilitating rapid retrieval of customized data sets. A wide variety of complex queries, on various types of annotations, for numerous species are supported. These can be applied to many research problems, ranging from SNP selection for candidate gene screening, through cross-species evolutionary comparisons, to microarray annotation. Users can group and refine biological data according to many criteria, including cross-species analyses, disease links, sequence variations, and expression patterns. Both tabulated list data and biological sequence output can be generated dynamically, in HTML, text, Microsoft Excel, and compressed formats. A wide range of sequence types, such as cDNA, peptides, coding regions, UTRs, and exons, with additional upstream and downstream regions, can be retrieved. The EnsMart database can be accessed via a public Web site, or through a Java application suite. Both implementations and the database are freely available for local installation, and can be extended or adapted to 'non-Ensembl' data sets.
Animals↗
Full-length single-cell spatial transcriptomics reveals spatial and cell-type-specific transcript isoforms in the primate brain.
The primate brain exhibits complex RNA alternative splicing heterogeneity crucial for functional complexity, yet systematic spatial isoform characterization has been lacking. We developed Fullscope-seq, a full-length single-molecule large field-of-view spatial transcriptomics sequencing method at single-cell resolution, based on programmed concatenation cDNA for multiple long-read sequencing platforms. Applying Fullscope-seq to the macaque brain, we uncovered thousands of genes exhibiting differential transcript usage (DTU) across cortical layers, cell types and brain regions. Fullscope-seq resolved hundreds of major isoform switches across distinct brain regions and identified DTUs between superficial and deep cortical layers. Cortical layer-specific DTUs showed cell-composition dependence, whereas regional DTUs were regulated according to both cellular composition and spatial contexts. These isoform variations showed substantial enrichment for neuropsychiatric disorder-associated genes and were conserved across platforms and species. Our study establishes a scalable framework for spatial isoform analysis and provides a resource for understanding transcriptomic diversity in complex tissues.
Animals↗