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PubMedDate not supplied

[Structure of integrated oncogens E1A and E1B in a malignant line of rat SH2 fibroblasts, transformed by monkey adenovirus SA7 (C8) DNA].

In this investigation the primary structure of E1A and E1B regions of SA7 (C8) simian adenovirus integrated in malignant SH2 rat cell line was studied. Southern blotting revealed at least two copies of the SA7 oncogene integrated in the SH2 genome. PCR analysis of E1A and E1B regions showed heteroduplex structures, proving the different structure of the integrated copies. The heteroduplex molecules with different electrophoretic mobility were separated, and chains corresponding to different copies were sequenced according to the modified Sanger method. We found that two copies differ mainly in microsatellite regions, in E1A between positions 894-902 (GCG)3/(GCG)4, in E1B between positions 2037-2048 (GCA)3/(GCA)4. It is necessary to stress that all deviations found belong to the coding regions of the SA7 oncogene.

Adenovirus E1A Proteins↗
PubMed1995

Molecular cloning and sequencing of cytochrome c' from the phototrophic purple sulfur bacterium Chromatium vinosum.

The gene for cytochrome c' from Chromatium vinosum was cloned from a HindIII-SalI digest of genomic DNA. A 1.4 kbp fragment containing the gene was sequenced in both directions using the Sanger dideoxy method. The cytochrome c' gene codes for a 154-residue peptide, of which the last 131 amino acids match the previously determined sequence of the protein. The remaining 23 residues represent a signal sequence that is cleaved from the polypeptide upon translocation to the periplasmic space. An additional open reading frame on the other strand of the fragment codes for a peptide that contains four regions that are homologous to corresponding regions of the cytochrome b-type subunit of several Ni-Fe hydrogenases.

Amino Acid Sequence↗
PubMed1999

[The feature of influenza virus activity and genetic characterization of HA1 gene of influenza A H3N2 viruses isolated from 1994-1997 in Shenzhen].

OBJECTIVE: To understand the feature of influenza virus activity and genetic characterization of HA1 gene of influenza A H3N2 viruses isolated in recently years in Shenzhen. METHODS: The specimens were inoculated into amniotic and allantoic cavities of the embryonated chicken eggs for viral isolation. The isolates were typed with HI test. Virion RNA extracted from allantoic fluid by Rneasy RNA extraction kit was used for RT-PCR amplification. Afterward, the product of PCR was purified with purification kit. RNA sequence analysis was performed by Sanger dideoxy-mediated nucleotide chain termination method. RESULTS: Influenza A H3N2, H1N1 and B viruses were cocirculating in men in Shenzhen in recent years. However, the extent of their activities was not the same in a single year. The influenza B virus was the predominant strain in 1994 and 1997, but the predominant strain was H3N2 virus in 1996. The genetic analysis showed that the substitution of amino acids occured year by year and was mainly located at antigenic determinants or receptor binding site on HA1 domain of H3N2 viruses. CONCLUSION: Influenza A H3N2, H1N1 and B viruses were still co-circulating in men in Shenzhen in recent years, the predominant strain of influenza virus was different in different years. An increase of influenza A H3N2 virus activity in 1996 was due to the substitution of amino acids sequences on viral HA1 domain.

Amino Acid Sequence↗
PubMed2002

Analysis of the human hephaestin gene and protein: comparative modelling of the N-terminus ecto-domain based upon ceruloplasmin.

Hephaestin was implicated in mammalian iron homeostasis following its identification as the defective gene in murine sex-linked anaemia. It is a member of the family of copper oxidases that includes mammalian ceruloplasmin, factors V and VIII, yeast fet3 and fet5 and bacterial ascorbate oxidase. Hephaestin is different from ceruloplasmin, a soluble ferroxidase, in having a membrane-spanning region towards the C-terminus. Here we report the gene structure, spanning approximately 100 kb, of the human homologue of mouse hephaestin. The sequence was assembled from the cDNA clones and the chromosome X genomic sequence data available at the Sanger Centre. It has an open reading frame that encodes a protein of 1158 residues, 85% identical with the murine homologue. A model of the N-terminal ecto-domain has been built based on the known three-dimensional structure of human ceruloplasmin. The overall tertiary structure for the hephaestin and the putative residues involved in binding copper and iron appear to be highly conserved between these proteins, which suggests they share the same fold and a conserved function.

Base Sequence↗
PubMed1998

Toward a complete human genome sequence.

We have begun a joint program as part of a coordinated international effort to determine a complete human genome sequence. Our strategy is to map large-insert bacterial clones and to sequence each clone by a random shotgun approach followed by directed finishing. As of September 1998, we have identified the map positions of bacterial clones covering approximately 860 Mb for sequencing and completed >98 Mb ( approximately 3.3%) of the human genome sequence. Our progress and sequencing data can be accessed via the World Wide Web (http://webace.sanger.ac.uk/HGP/ or http://genome.wustl.edu/gsc/).

Contig Mapping↗
PubMed2003

Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing.

A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.

Base Sequence↗
PubMed1997

Detection of a R173W mutation in the porphobilinogen deaminase gene in the Nova Scotian "foreign Protestant" population with acute intermittent porphyria: a founder effect.

OBJECTIVES: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG-->TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP. DESIGN AND METHODS: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger's dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an Ncil (also Mspl) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion. RESULTS: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the "Foreign Protestants," who were recruited to Nova Scotia in the 1750s. CONCLUSION: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.

Adult↗
PubMed2026

Clinical, genetic and bioinformatic analysis of Saudi families with Joubert syndrome and related disorders.

BACKGROUND: Joubert syndrome and related disorders (JSRD) are clinically and genetically heterogeneous ciliopathies caused by pathogenic variants in over 40 genes, mainly encoding ciliary proteins. However, data on JSRD in the Saudi population remain limited. This study aimed to identify novel and reported JSRD-causing variants in Saudi families and analyze their potential functional impact on protein structure using advanced computational tools. METHODS: Patients with genetically or clinically confirmed/suspected JSRD were recruited according to ethical protocols. Clinical data were collected, and exome sequencing was performed, followed by Sanger validation of identified variant. Pathogenicity was assessed using bioinformatics tools, while conservation, expression and RNA structure analyses were conducted to evaluate the potential functional consequences. RESULTS: Homozygous variants were identified in three consanguineous Saudi families, including a novel stop-gain variant in KIF7 (c.2992 C > T; p.(Gln998Ter)), and novel splice-site variant in CEP104 (c.489 + 1G > A) and a previously reported splice-site variant in TMEM237 (c.869 + 1G > A). All variants were predicted to be pathogenic as per ACMG criteria and located in highly conserved regions, suggesting potential functional impact. RNA analysis suggested possible alterations in folding. CONCLUSION: This study identified three pathogenic variants in JSRD-related genes in Saudi families, including two novel variants. These findings expand the variant spectrum for JSRD, particularly in the Saudi population. Establishing a comprehensive regional variant database is essential for improving molecular diagnosis and genetic counseling in population with high consanguinity. Finally, these results highlight the need for further functional studies to validate their pathogenicity and elucidate their role in JSRD pathogenesis.

Humans↗
PubMed2026

XXYLT1 and Mendelian Retinal Dystrophy.

IMPORTANCE: Substantial unexplained heritability remains for pathogenic inherited retinal disease (IRD) variants. Application of genome-wide association studies (GWAS) could help identify causal genes in rare diseases. OBJECTIVE: To leverage a GWAS for the discovery of IRD-associated genes. DESIGN, SETTING, AND PARTICIPANTS: This GWAS analysis was combined with replication of findings in 2 independent IRD cohorts. The study was conducted from January 2024 to December 2025 in a multicenter setting through FinnGen, 100&#x202f;000 Genomes Project, and the National Health Service Genomic Medicine Service combined with clinical cohort from the Oulu University Hospital. Using IRD criteria from the International Classification of Diseases, 9th and 10th Revisions, 540 individuals with IRD and 473&#x202f;945 control individuals were identified in the FinnGen study. For validation of FinnGen results, 49 patients were recruited from Oulu University Hospital. Results were further validated in 2 individuals identified from the UK cohort. MAIN OUTCOMES AND MEASURES: The GWAS and proteomics analysis were performed in the FinnGen cohort. Sanger and whole-genome sequencing and RNA approaches were used in a clinical IRD cohort to validate pathogenicity of the identified XXYLT1 variant. RESULTS: This GWAS identified 13 recessive loci reaching genome-wide significance (defined as P&#x2009;<&#x2009;5&#x2009;&#xd7;&#x2009;10-8). Of these, 4 (near or within XXYLT1, ANKRD10, DYM, and CBLN4) had not been associated with IRD, including the XXYLT1 c.505-1G>C founder variant. This variant was further genotyped in the clinical replication cohort, leading to identification of 5 more homozygous individuals from 4 families. The phenotype was consistent with a cone-rod or macular dystrophy, with visual deterioration, cystoid macular edema and/or schisislike macular abnormalities. The effect of the XXYLT1 c.505-1G>C variant was further investigated using RNA sequencing and complementary DNA amplicon sequencing, demonstrating exon 2 skipping and a loss-of-function effect. These findings were replicated in an independent population identifying 2 patients from the UK harboring a homozygous XXYLT1 c.766G>A, p.(Glu256Lys) missense variant. CONCLUSIONS AND RELEVANCE: This GWAS identified an association between XXYLT1 and IRD. These results affirm that GWAS in a founder population can be used as a potential tool for the discovery of rare mendelian disease genes and that XXYLT1 should be considered in clinical IRD gene panels.

Humans↗
PubMed2025

Novel Insights into the Clinical Features, Genetic Spectrum and Clonal Evolution of Patients Carrying NLRP3 Mosaicism.

NLRP3 mosaicism is a well-established mechanism causing the monogenic autoinflammatory disease named cryopyrin-associated periodic syndromes (CAPS). The number of reported patients with NLRP3 mosaicism is small, and the knowledge about the long-term disease behavior is limited. Herein we assembled the largest cohort of individuals with NLRP3 mosaicism reported to date to obtain additional evidence that strengthens the understanding of this disease. The novel genetic data were obtained by using Sanger and next-generation sequencing methods, whereas in vitro analyses determined the functional consequences of detected variants. A total of seventeen individuals with NLRP3 mosaicism were enrolled, with 16/17 experiencing different CAPS phenotypes. An overrepresentation of late-onset forms was detected (37.5%). Overall, clinical manifestations, analytical results, and outcomes of treatments were markedly similar to those detected in patients with germline variants. A large mutational diversity was identified, with 16 different variants among 17 individuals. Two main patterns of mosaicism (extended vs. myeloid-restricted) were detected, with the last one overrepresented in the late-onset group. The evaluation of mosaicism over time identified three different patterns, being the group with stable mosaicism the largest one. Collected evidence supports the marked similarities among patients carrying somatic or germline NLRP3 variants. The overrepresentation of NLRP3 mosaicism in late-onset forms should be considered in patients with inflammatory manifestations starting in adulthood. Analysis of mosaicism at the biological level confirms the two known patterns of corporal distribution and reveals that mosaicism remains stable over time in most patients, but it may also vary during the course of the disease.

Humans↗
PubMed2025

Clinical and genetic variant re-analysis among pediatric probands undergoing genetic testing for arrhythmia syndromes.

BACKGROUND: Despite increases in genetic testing, longitudinal data regarding changes in diagnostic yield and variant reclassification for inherited arrhythmia syndromes are limited. OBJECTIVE: Determine longitudinal changes in diagnostic yield and variant classification. METHODS: Single-center retrospective study of probands <18 years undergoing genetic testing for suspected inherited cardiac conditions associated with arrhythmias, 2007 to 2018. Variants were classified as diagnostic (pathogenic/likely pathogenic), non-diagnostic (benign/likely benign [B/LB]), or variants of uncertain significance (VUS). Variant reclassification was performed in October 2023 using VarSome and American College of Medical Genetics criteria. We evaluated results by era (early 2007-2013 vs. later 2014-2018, coinciding with Sanger and next-generation sequencing, respectively) and by likelihood of disease based on clinical evaluation. RESULTS: Of 306 probands, initial testing was 23.2% diagnostic, 55.6% non-diagnostic (33.7% no variant, 21.9% B/LB), and 21.2% VUS. When comparing eras, diagnostic yield decreased (34.1%-15.3%), VUS increased (9.3%-29.9%), and non-diagnostic remained similar (55% to 57%). Variants for 22.7% (46/203) of probands with &#x2265;1 variant changed: 9.9% of diagnostic variants (7/71) downgraded to VUS or non-diagnostic, and 60.0% of VUS changed (23.1% upgraded, 36.9% downgraded). B/LB variants did not change. Probands with higher disease likelihood had 6-times the odds of diagnostic results compared to lower disease likelihood, regardless of era (odds ratio 6.3, 95% confidence interval 3.2-12.4, P < .0001). CONCLUSION: Variant reclassification led to changes in 23% of probands, both downgrading and upgrading status, even among probands initially thought to be pathogenic. When comparing later to earlier eras, VUS variants increased while diagnostic yield decreased. Findings support the need for variant re-interpretation and periodic reclassification over time.

Humans↗
PubMed1994

[Molecular heterogeneity of beta thalassemia].

PURPOSE: To check out the incidence of beta-thalassaemia in different regions of Spain and to demonstrate its heterogeneity. PATIENTS AND METHODS: The study was performed on 60 peripheral blood samples from patients diagnosed of beta-thalassaemia by conventional methods, namely, microcytosis, HbA2 over 3.5%, increased HbF. DNA was isolated with phenol-chloroform and amplified by ARMS technique, and in one case its sequence was established according to Sanger's method. RESULTS: The mutations found in the 67 alleles studied showed similar incidence, although NT1 (IVS 1) mutation was increased and NT110 (IVS 1) mutation was decreased when compared with the findings in other Mediterranean populations. CONCLUSION: Due to historical reasons, the racial characteristics of Spanish people are the product of different ethnic groups, so hereditary disorders are here usually heterogeneous. This study appears to confirm such fact with respect to beta-thalassaemia.

Genetic Heterogeneity↗
PubMed2003

400000 nematode ESTs on the Net.

The parasitic nematode expressed sequence tag (EST) project, a collaboration between University of Edinburgh and the Wellcome Trust Sanger Institute in the UK and the Genome Sequencing Center, St Louis, MO, USA, is currently generating sequence information from >30 different species of nematode. Over 400000 nematode ESTs are now available and at least another 130000 are planned. Here, an update is provided on the status of the project and describes the database tools being developed to disseminate these data.

Animals↗
PubMedDate not supplied

[A simple system of cloning in phage M13 and DNA sequencing with terminators].

A compilation of techniques for DNA cloning in filamentous phage M13 based vectors for a novice in cloning is presented. It does not require either specialized microbiological facilities, or any specific knowledge in Escherichia coli genetics. The cloning strategy uses only blunt-end ligation into a vector that has been prepared once for several hundred experiments. The first part describes the isolation, preparation and checking of a blunt-ended M13 vector (with M13 mp series vectors as an example), and also the isolation of clonable fragments, transformation of competent cells and preliminary analysis of recombinants. The second part describes procedures and equipment, which enable to sequence recombinant M13 clones by the chain termination procedure of Sanger et al. It includes simplified procedures for the preparation of sequencing gels, and the rules of interpretation of the sequencing ladders. Reference material is added, which includes trouble-shooting guide, E. coli K12 strain list and polylinker sequences for use of mp-series vectors as well as a fully documented cloning and sequencing experiment.

Base Sequence↗
PubMed1993

The cloning and sequencing of coat protein gene from beet necrotic yellow vein virus.

The beet necrotic yellow vein virus (BNYVV) isolate NM was isolated from sugarbeet infected with rhizomania in Inner Mongolia of China. The cDNA of BNYVV coat protein (CP) gene was amplified from the extracted RNA of BNYVV isolate NM by using the polymerase chain reaction (PCR) and cloned into pGEM-7zf(+). Its complete nucleotide sequence was determined by means of Sanger's dideoxy-mediated chain-termination method. The result shows that CP gene of BNYVV isolate has 567 nucleotides. It shares 98.8% and 96.7% identity with the CP gene of isolate F13 (report in reference [1]) in terms of amino acid and nucleotide sequence, respectively.

Amino Acid Sequence↗
PubMed2002

[Cloning and expression in Escherichia coli of oligo-1,6-glucosidase gene from Bacillus subtilis HB002].

The gene coding for oligo-1,6-glucosidase of Bacillus subtilis HB002 was cloned by the shotgun-cloning method and sequenced by the chain-termination method of Sanger et al. It consists of an open reading frame of 1683 bp. The amino acid sequence of oligo-1,6-glucosidase deduced from its nuecleotide sequence predicts a protein of 561 amino acid residues with a Mr of 65.985 kD, which is 81% and 67% identical to those of oligo-1,6-glucosidase from Bacillus sp. and Bacillus coagulans, respectively, 89% and 79% similar to those of oligo-1,6-glucosidase from Bacillus sp. and Bacillus coagulans, respectively. The oligo-1,6-glucosidase gene of Bacillus subtilis HB002 was cloned into Escherichia coli expression plasmid pBV220, the result of SDS-PAGE showed that the oligo-1,6-glucosidase gene had been expressed in Escherichia coli DH5 alpha, the expressed oligo-1, 6-glucosidase has enzymatic activity.

Amino Acid Sequence↗
PubMed1998

[Cloning and sequencing of E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus].

OBJECTIVE: To clone E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus into mammalian expression vector and analyze its primary nucleotide structure. METHODS: Reverse transcription nested-PCR methods were employed for amplification E2/NS1 gene which was cloned into mammalian expression vector pcDNA3 by directed clone. Sequence of inserted gene was analyzed by Sanger's method. RESULTS: E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus was cloned into eukaroytic expression vector effectively. Sequence of E2/NS1 showed 88.37% identity in nucleotide and 89.29% in putative amino acid to that of a Japanese genotype III isolate of hepatitis C virus. Nevertheless it showed 70.69% and 75.55% identity to that of a Chinese genotype II isolate of hepatitis C virus. CONCLUSION: E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus was successfully cloned into mammalian expression vector by technique of DNA recombination. Sequence analysis showed that E2/NS1 gene has good homology intra-genotype and poor homology intergenotype of hepatitis C virus. Research of primary nucleotide structure of different genotype hepatitis C virus might be helpful for the development of vaccine against the virus.

Amino Acid Sequence↗