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Results for “Blotting, Southern”

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At least 19 recordsLinked to original sources

PubMed1979

Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized.

A HindIII (17.0 kb) and an EcoR1 restriction fragment (6.9 kb) of Klebsiella pneumoniae nif DNA were cloned on two small amplifiable plasmids, pCM1 and pSA30 respectively. These plasmids between them carry 14 of the 15 known Klebsiella nif genes. The operon for the three structural genes for nitrogenase, nifpHDK, is carried on pSA30: four and five of the remaining six operons are on pCRA37 and pCM1 respectively. All of the nif genes were assigned to endonuclease restriction fragments of DNA using the Southern blotting technique (Southern, 1975) with total DNA of nif insertion mutants and radioactive plasmid DNA which contained cloned nif DNA sequences. Their locations were consistent with the genetic map of nif genes. The estimated size of the nif gene cluster was 24 kb.

Base Sequence↗

PubMed2012

Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation.

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Blotting, Southern↗

PubMed1978

A complete immunoglobulin gene is created by somatic recombination.

Using a pCRI plasmid containing an enzymatically synthesized, full-length DNA transcript of immunoglobulin lambda chain mRNA as the hybridization probe in the Southern gel blotting experiments (Southern, 1975), we identified three DNA fragments of 8.6, 4.8 and 3.5 kb in Eco RI-digested total DNA from BALB/c mouse embryos. A fourth fragment of 7.4 kb was found in addition to these three fragments in similarly digested total DNA from a lambda chain-secreting myeloma (HOPC 2020). We have cloned the four DNA fragments in an EK-2 phage vector, lambdaWES, and characterized them with respect to size, type of lambda gene sequences contained and position of these sequences in the fragments, using agarose gel electrophoresis, the gel blotting technique and electron microscopic R loop mapping. The embryonic DNA clones Ig 99 lambda, Ig 25lambda and Ig 13lambda contain one copy each of V lambdaI, C lambdaI and V lambdaII sequences, respectively, while the myeloma DNA clone Ig 303lambda contains one copy each of V lambdaI and C lambdaI sequences that are separated by a 1.2 kb nontranslated DNA segment. Ig 25lambda was also shown to contain a DNA segment of approximately 40 base pairs (bp) (J sequence) that lies 1.2 kb away from the C lambdaI sequence and is homologous to the V-C junction region of a lambdaI mRNA. Heteroduplex analysis of the three lambdaI DNA clones revealed that Ig 303lambda DNA is composed of two parts, one of which is entirely homologous to one end of Ig 99lambda, and the other to one end of Ig 25lambda DNA. The sequence arrangement observed in the cloned DNA is the same as that in the corresponding cellular DNA. This was shown by identifying certain restriction enzyme cleavage sites on the cloned DNAs and demonstrating the presence of these sites in the total cellular DNA by the gel blotting technique. The site of the homology switch is at the boundary of the V sequence and the 1.2 kb nontranslated DNA segment, and corresponds to the position of the J sequence on the Ig 25lambda DNA. We consider the above experimental results the most direct evidence for somatic rearrangement in immunoglobulin genes. We discuss the significance of these findings for the origin of genes in the evolution of higher organisms and in cell differentiation.

Animals↗

PubMed1994

Description of a human papillary thyroid carcinoma cell line. Morphologic study and expression of tumoral markers.

BACKGROUND: The establishment of cell lines from thyroid carcinomas can provide an in vitro model of oncogenesis. B-CPAP is a new cell line that has been obtained from a differentiated papillary thyroid carcinoma. The data presented give a broader characterization and expression of tumoral markers of this cell line and identify the differentiated functions that are preserved. METHODS: An ultrastructural study was performed to confirm the thyroid nature of the new cell line. The cellular markers (thyroglobulin, S100, neuron-specific enolase [NSE]) and the oncogenes (mutated p53, H-ras, c-myc, PTC, trk) were studied by immunohistochemistry, Southern blot, or in situ hybridization. RESULTS: The cells were of a differentiated ultrastructural thyroid type. All of the cells proved immunoreactive with antibodies specific to thyroglobulin, S100 proteins, NSE, and mutant p53 protein. Mutations of H-ras, PTC, and trk were not observed. The c-myc gene was not amplified. CONCLUSIONS: The cell line described in these data provides a suitable model for the study of thyroid carcinogenesis, given that the cells present thyroid characteristics, and metabolic disorders not previously found in such cell lines. In addition, the coexpression of S100 proteins and mutant p53 proteins in the cells should permit the study of the interaction between these two proteins.

Aged↗

PubMed1979

A cleavage map of the TOL plasmid of Pseudomonas putida mt-2.

A cleavage map of the TOL plasmid pWWO has been determined for the restriction endonucleases HindIII and XhoI. A number of techniques were employed including (i) digestion of purified cleavage products with a second enzyme; (ii) hybridisation of purified XhoI fragments to Southern blots of HindIII digest products and (iii) analysis of a number of deletion mutants.

Chromosome Mapping↗

PubMed2026

Rapid Agrobacterium-mediated transformation and high-efficiency regeneration of finger millet (Eleusine coracana) for crop improvement.

Finger millet (Eleusine coracana) is a nutritionally important and climate-resilient cereal cultivated in rainfed regions of India and Eastern Africa, yet its genetic improvement has been limited by the lack of efficient and reproducible transformation systems. In this study, we developed a rapid and efficient Agrobacterium tumefaciens-mediated transformation and regeneration system using shoot apical meristem (SAM) explants, enabling direct, callus-free shoot organogenesis. Optimal regeneration and shoot elongation were achieved on Murashige and Skoog (MS) medium supplemented with 3.5 mg L⁻1 6-benzylaminopurine (BAP), 1.5 mg L⁻1 kinetin, 0.1 mg L⁻1 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg L⁻1 gibberellic acid (GA₃). Genotype-dependent responses were observed, with PR-202 requiring 2 mg L⁻1 AgNO3 to reduce phenolic browning, whereas VL-376 regenerated efficiently without AgNO3. Transformation efficiencies of 30-32% were achieved in PR-202 and VL-376, respectively, by optimising infection and co-cultivation conditions, including reduced MS salt strength and pre-incubation of Agrobacterium. Molecular analyses, including PCR and Southern blot hybridisation, confirmed stable T-DNA integration in independent lines, while segregation analysis of T₁ progenies demonstrated Mendelian inheritance of the transgene. In addition, CRISPR/Cas9 constructs targeting EcCKX2 were successfully introduced via Agrobacterium, demonstrating the suitability of this system for genome engineering applications. Overall, this optimised SAM-based protocol provides a rapid (45-50 days), efficient, and reproducible platform for stable genetic transformation in finger millet and establishes a strong foundation for transgenic research and future genome editing studies in this underutilized crop.

Eleusine↗

PubMed2025

Prevalence of intronic repeat expansions in the RFC1 gene in Polish patients with cerebellar syndrome.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative ataxic disorder, which has been associated with intronic biallelic repeat expansions in the RFC1 gene. Our objective was to assess retrospectively the prevalence of CANVAS in Polish population. We screened 2523 Polish patients in whom other repeat expansions were excluded. To determine the repeat expansions in the RFC1 gene in patients, we performed RFC1-flanking PCR and repeat primed PCR (RP-PCR) and to measure the size of the expansion we used Southern blotting and optical genome mapping to compare the results. We have observed the biallelic pathogenic motif/unit AAGGG expansions in 4.6% and expansions of non-pathogenic motifs AAAAG, AAAGG in 25% patients of our studied population. This is the first large-scale cohort study that confirms the relatively frequent occurrence of the CANVAS in Polish population. To increase the current diagnostics of late-onset ataxias within an unexplained molecular background, we suggest involving the RFC1 repeat expansions analysis to the routine diagnostic workflow.

Humans↗

PubMed1979

The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity.

We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.

Animals↗

PubMed1979

DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man.

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.

Base Sequence↗

PubMed1978

The genes for 18S, 5.8S and 28S ribosomal RNA of Bombyx mori are organized into tandem repeats of uniform length.

The organization of the multiple genes for 18S, 5.8S and 28S rRNA in the genome of the silkworm, Bombyx mori was determined by restriction endonuclease digestion and Southern blot hybridization. The ribosomal genes (rDNA) are tandemly reiterated, with a uniform repeat length of 6.9 . 10(6) daltons. Each rDNA repeat has a single site for EcoRI, HindIII, HpaI and SmaI and each of these sites has been mapped with respect to the others and to the rRNA genes; each repeat consists of a transcribed region (6 . 10(6)daltons) containing the 18S, 5.8S and 28S rRNA genes (5' leads to 3') and also a small non-transcribed spacer (approximately 10(6) daltons). Complete rDNA repeats were cloned using the vector RSF2124 and grown in Escherichia coli. Characterization of the rDNA plasmids confirmed the conclusions from studies of the total rDNA. The organization of B. mori rDNA is similar to that of other eukaryotes, except for the absence of heterogeneity in the rDNA repeat length; thus, there is neither variation in the length of the non-transcribed spacer nor the presence of inserts in a detectable portion of the rDNA. The utility of this map, and particularly of the rDNA plasmids, for detailed studies of rRNA transcription and processing is discussed.

Animals↗

PubMed1979

Cloning of bacteriophage T5 DNA fragments in plasmid pBR322 and bacteriophage lambda gtWES.

Bacteriophage T5 was digested with the restriction endonucleases HindIII and EcoRI and the resulting fragments were inserted into the plasmid pBR322 and the bacteriophage lambda gtWES as vectors. Approx. 15% of the phage genome was recovered in recombinant clones. The recombinants were characterized by restriction analysis, DNA/DNA hybridization employing Southern blots, and ability to complement or recombine with amber mutants of T5. The results obtained allow revisions of the physical map of the T5 genome and partial correlation of the physical map with the genetic map.

Chromosome Mapping↗

PubMed1979

The organization of a nuclear DNA sequence from a higher plant: molecular cloning and characterization of soybean ribosomal DNA.

The recombinant DNA vector, lambda Charon 4A, was used to construct a library of DNA sequences from the genomic DNA of soybean (Glycine max). To define the organization of ribosomal DNA (rDNA) in the soybean genome, clones containing sequences complementary to both 17S and 25S rRNA have been isolated from this library and used in conjunction with Southern blot hybridization. The rRNA genes are tandemly reiterated with a relatively small unit repeat length of 7.8 kb. There is no heterogeneity in the length of the rDNA repeat units although they display limited differences in either base sequence or pattern of methylation. The cloned rDNA sequences are shown to comprise the entire repeat unit and have been used to obtain a detailed restriction map as well as an approximate transcription map of soybean rRNA genes. The cloning of rDNA from soybean suggests that recombinant DNA techniques can be successfully applied to the genomic DNA of higher plants despite the high degree of methylation exhibited by plant DNA.

Bacteriophage lambda↗

PubMed2026

Parallel Analysis of Repeat Expansions: An Updated Clinical Nanopore Cas9-Targeted Sequencing Workflow for Nanopore R10 Flow Cells.

Hereditary ataxias, caused by expansions of short tandem repeats, are difficult to diagnose using traditional PCR and Southern blot methods, which struggle to detect complex repeat expansions and cannot assess repeat interruptions or methylation. An updated Clinical Nanopore Cas9-Targeted Sequencing workflow is presented for analyzing repeat expansions, now compatible with the Oxford Nanopore Technologies R10 flow cell. The workflow incorporates the Oxford Nanopore Technologies wf-human-variation Epi2Me workflow, including the Straglr tool to analyze base-called reads, ensuring compatibility with past, current, and future sequencing chemistries. It expands the number of genes analyzed from 10 to 27 and introduces new gene panels for ataxia, myopathy, neurodegeneration, and amyotrophic lateral sclerosis/motor neuron disease. Validated with Coriell reference and clinical samples, this method improves the analysis of pathogenic repeat expansions, providing deeper insights into repeat structures while addressing the limitations of traditional approaches. In this work, the use of multiplexing, Flongle flow cells, and single-gene targeting were explored as alternatives to panel-based approaches in the Clinical Nanopore Cas9-Targeted Sequencing workflow, finding that only single-gene targeting provides compatibility and reliable performance.

Journal Article↗

PubMed1979

Transposable mating type genes in Saccharomyces cerevisiae.

A functional copy of the alpha mating type gene of Saccharomyces cerevisiae has been cloned by transformation in yeast. Using the Southern Blotting procedure it has been shown that three distinct genetic loci implicated in mating type interconversion (HML, HMR and MAT) contain sequences homologous to the clone fragment. The restriction fragment associated with each locus exhibits a characteristic size which can be correlated with the mating type allele present at that locus. The characteristic size difference between the a and alpha genetic elements made it possible to demonstrate that the homothallic interconversion of mating types in this yeast occurs by DNA rearrangement as proposed in the 'cassette hypothesis'.

Chromosome Mapping↗

PubMed1978

Construction and characterization of a 2.5-kilobase procollagen clone.

Recombinant bacterial plasmids have been constructed by inserting double-stranded chicken procollagen cDNA sequences linked to chemically synthesized decanucleotides containing HindIII sites into the HindIII site of pBR322. After transformation of Escherichia coli chi1776, colonies were selected by ampicillin resistance and recombinants containing procollagen sequences were identified by colony hybridization to 32P-labeled procollagen cDNA. The inserts from three recombinant plasmids, pCg10, pCg13, and pCg45, were 1200, 2200, and 2550 base pairs long respectively. Their sequence homology has been established by restriction mapping and crosshybridization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts, pCg45 has been positively identified as containing the pro alpha2 collagen sequence by partial determination of the DNA sequence of its ends: it has a short thymine-rich sequence at one end and a sequence coding for residues 478--499 in the chicken alpha2 chain at the other end.

Animals↗

PubMed1979

DNA-mediated gene transfer of a circular plasmid into murine cells.

We have used DNA-mediated gene transfer to introduce a recombinant plasmid containing the human beta-globin gene (H beta 1) into cells of a mouse tissue culture line, Ltk-. DNA isolated from independent transfer lines was analyzed by restriction endonuclease digestion, gel electrophoresis, modified Southern blotting, and filter hybridization using H beta 1 as a probe. H beta 1 sequences were present in 80% of the lines at 1-30 copies per cell. Many of the lines gave a hybridization pattern indicative of H beta 1 sequences integrated into high molecular weight DNA. DNA from three cell lines, digested with several restriction enzymes, produced a pattern providing evidence for the presence of circular H beta 1 molecules in the murine recipient cells.

Animals↗

PubMed1979

Analysis of human tonsil and cancer DNAs and RNAs for DNA sequences of group C (serotypes 1, 2, 5, and 6) human adenoviruses.

Group C human adenoviruses (Ads) of serotypes 1, 2, 5, and 6 infect most children and commonly cause latent infections of lymphoid tissues. Ads transform cells into a malignant-like phenotype; the oncogenic genetic information is in the left 8% of the viral genome, in the HindIII-G DNA fragment. We have investigated the molecular basis for group C Ad latent infections in human tonsils as well as whether these viruses are linked to human cancer. Tonsil or cancer DNAs and RNAs were assayed for Ad sequences by liquid-phase saturation-hybridization with in vitro-labeled Ad5 HindIII-G fragment. About 25% of the 52 tonsils analyzed contained DNA or RNA sequences specific to HindIII-G, indicating that Ad transforming sequences are expressed as RNA in tonsils. Southern blotting analysis of four tonsil DNAs revealed multiple copies of the complete Ad genome in a free state and provided evidence for an unusual form of the Ad genome, possibly Ad DNA integrated into cellular DNA. In assays of human cancers, no Ad sequences were detected in DNAs from 26 squamous cell carcinomas (Cas), 3 adenocarcinomas, 4 oat cell Cas, 5 stomach Cas, 5 small intestine Cas, 15 colon Cas, 6 rectum Cas, 5 Hodgkin and 6 non-Hodgkin lymphomas, and 2 breast Cas. Reconstruction experiments indicated that the HindIII-G probe could detect 1 copy per cell of 0.2-0.3% of the viral genome. No HindIII-G-specific sequences were detected in RNAs from 21 squamous cell Cas, 3 oat cell Cas, 2 stomach Cas, or 18 colon Cas. In six other experiments using the complete Ad2 genome as probe, no Ad sequences were found in DNAs from 6 lung Cas, 12 normal lung tissues, 33 gastrointestinal Cas, 19 normal gastrointestinal tissues, 6 Hodgkin lymphomas, 3 breast Cas, or 4 kidney Cas, at a sensitivity of about 1 copy per tumor cell of 5-10% of the Ad2 genome. All Ad-induced cancer cells should contain at least 1 copy of 1-6% of the viral genome, the minimal size of the transforming region, and probably should contain multiple copies of more of the genome. Therefore, our data are definitive evidence against group C Ads being the cause of the cancers tested, which represent about 50% of the cancer incidence in the United States. Of additional interest, we did not detect Ad2 sequences in RNAs from 7 human placentas, 12 normal lungs, or 19 normal gastrointestinal tissues (nor in 44 cancer or 23 tonsil RNAs). Thus, we did not confirm a recent report of the presence of Ad2 RNA in RNAs from human placentas; the possibility that a small population of cells in placenta expresses group C "related" sequences is not ruled out.

Adenoviruses, Human↗

PubMed1979

Immunoglobulin heavy chain gene organization in mice: analysis of a myeloma genomic clone containing variable and alpha constant regions.

We have isolated a myeloma genomic DNA clone containing the variable and constant regions of a mouse alpha chain. Restriction enzyme analyses and electron microscopic R loop mapping have demonstrated that the variable region is separated from the constant region by 6.8 kilobases of intervening DNA. In addition, two intervening DNA sequences of 100--200 bases separate the constant region into three approximately equal units. These intervening sequences may separate each of the segments coding for the three constant region domains of the alpha heavy chain. Southern blot analysis of embryo and myeloma DNA suggests that DNA rearrangement of heavy chain variable and constant regions occurs during the differentiation of antibody-producing cells.

Animals↗