RESUMO
Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.
Assuntos
Linfócitos B , Aprendizado de Máquina , Análise de Sequência de RNA , Análise de Célula Única , Linfócitos T , Transcriptoma , Células Cultivadas , Humanos , Especificidade de ÓrgãosRESUMO
The DNA sequence of cDNA clones prepared from transcripts of the mouse met proto-oncogene reveals that the mouse met gene encodes a 1380 amino acid protein with the characteristics of a growth factor receptor. This protein can be divided into several putative domains, including an intracellular protein tyrosine kinase domain, a transmembrane domain and a 929 amino acid extracellular domain, possessing a potential proteolytic cleavage site with the sequence Lys-Arg-Arg-Lys-Arg-Ser. To gain additional insights into the function of the met protein we have examined the level of met transcripts in tissues of the late-gestation mouse conceptus. Transcription of met was observed in most of the tissues analysed, but the highest levels of met mRNA were detected in the yolk sac, amnion and kidney; no transcripts were detectable in the calvaria. Chromosomal localization using a series of mouse-hamster hybrid cell lines has demonstrated that met is located on mouse chromosome 6.
Assuntos
Camundongos/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Substâncias de Crescimento , Dados de Sequência MolecularRESUMO
We have previously described the primary structure of the entire met domain and part of the tpr domain present in the human tpr-met oncogene. The isolation and sequencing of an additional cDNA clone now enables us to present the complete primary sequence of the tpr domain. A computer search has unearthed a remarkable identity between tpr and a rat sequence found at the 5-prime end of the activated raf oncogene. The occurrence of tpr-like sequences in combination with two oncogenes suggests that tpr contributes a domain(s) relevant to the observed activation of met and raf.
Assuntos
DNA de Neoplasias/genética , Oncogenes , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Humanos , Dados de Sequência Molecular , RatosRESUMO
The primary structure of the protein tyrosine kinase domain of the human met gene has been determined from cDNA clones prepared from transcripts of the activated human met gene. These analyses reveal that the met kinase domain (located on human chromosome 7) possesses unique features that distinguish met from other members of the src family of protein tyrosine kinases. The results also demonstrate that the product of the activated met gene is a fusion protein and that the amino terminal end of this fusion protein, which is encoded by human chromosome 1, exhibits homology to laminin B1.
Assuntos
Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 7 , Clonagem Molecular , DNA/genética , Humanos , Laminina/genética , Dados de Sequência Molecular , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
V79 Chinese hamster cells were cultured in the presence of 3-methylcholanthrene-diolepoxide (10r,9t-dihydroxy-7,8t-epoxy-tetrahydro-3-methylcholanthrene, MCDE) and mutants were selected in medium containing 6-thioguanine (TG). Of 22 TG-resistant mutants examined, 18 were devoid of HPRT (hypoxanthine-guanine phosphoribosyltransferase, EC 2.4.2.8) activity. Two mutants had suffered a total and one a partial gene deletion. The 1.6-kb HPRT mRNA was not detected in these three mutants nor in two others. The remaining mutants did not, however, have a readily demonstrable lesion.
Assuntos
Metilcolantreno/análogos & derivados , Mutação/efeitos dos fármacos , Animais , Carcinógenos , Células Cultivadas , Cricetinae , Cricetulus , DNA/genética , Enzimas de Restrição do DNA , Resistência a Medicamentos , Genes , Hipoxantina Fosforribosiltransferase/genética , Ponto Isoelétrico , Metilcolantreno/farmacologia , RNA Mensageiro/genética , Tioguanina/farmacologiaAssuntos
Carcinógenos , Transformação Celular Neoplásica/efeitos dos fármacos , Compostos Policíclicos , Animais , Benzo(a)pireno , Biotransformação , Células Cultivadas , Fenômenos Químicos , Química , DNA , Compostos de Epóxi , Regulação da Expressão Gênica , Humanos , Hipoxantina Fosforribosiltransferase/genética , Metilcolantreno , Mutação , Oncogenes , Relação Estrutura-AtividadeRESUMO
We have previously described the induction by r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) of 8-azaguanine resistant (AGr) Chinese hamster V79 cell mutants, 40% of which were found to contain material which cross-reacted (CRM) with antiserum to hypoxanthine-guanine phosphoribosyltransferase (HPRT) and whose AGr phenotype we ascribed to missense mutation (Brookes et al., 1982). We now report that we have been unable to demonstrate by Southern blotting any change in the HPRT gene in 11 CRM-negative mutants. We have, moreover, found HPRT mRNA of normal size and amount in most of these mutants. Examination of the revertants of one mutant indicates the probable occurrence of changes within an amino acid codon in the genesis of mutant and revertant. Our results suggest that BPDE functions primarily as a point mutagen.
Assuntos
Benzopirenos/toxicidade , Carcinógenos , Mutação , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido , Animais , Cricetinae , Cricetulus , Enzimas de Restrição do DNA/farmacologia , Hipoxantina Fosforribosiltransferase/genética , RNA Mensageiro/análiseRESUMO
A series of 8-azaguanine resistant mutants was induced by treatment of V79 Chinese hamster cells with either r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (antiBPDE) or methylnitrosourea (MNU). Hypoxanthine phosphoribosyltransferase (HPRT) activity in the mutants was determined for both hypoxanthine and azaguanine as substrates. With antiserum to purified brain HPRT, cross-reacting material was also determined and analysed by two dimensional polyacrylamide gel electrophoresis. By these criteria mutants induced by anti-BPDE or MNU did not differ appreciably and the data obtained was consistent with the induction of point mutations by both carcinogens. The relevance of these results to the correlation of carcinogenicity with mutagenicity in V79 cells, but not in bacteria, is discussed.
Assuntos
Benzopirenos/toxicidade , Mutagênicos , Mutação , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido , Animais , Cricetinae , Cricetulus , Reações Cruzadas , DNA/metabolismo , Hipoxantina Fosforribosiltransferase/análise , Metilnitrosoureia/toxicidadeRESUMO
The previously reported reaction at N2- and N7- of guanine following addition of 7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to an aqueous solution of DNA has been studied in more detail. The extent of reaction and the relative yields of N2- and N7-products was measured over the range of pH 4--7. The depurination following reaction at the N7-position of guanine was found to have a half-life of 3 h. Reaction of the isomeric 7 alpha,8 beta-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (syn-BPDE) with DNA gave the expected N2- and no N7-guanine product. When either benzo[a]pyrene or anti-BPDE was added to mouse embryo or Chinese hamster V79 cells respectively, a major N2-guanine product and a very minor adenine product were isolated from the DNA, but no N7-guanine product was detected.
Assuntos
Benzopirenos/metabolismo , Guanina/metabolismo , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cricetinae , DNA/metabolismo , Técnicas In Vitro , CamundongosRESUMO
Two metabolites of 3-methylcholanthrene (3MC), previously shown to be precursors of the DNA-bound form of 3MC observed in embryo cells in culture, were prepared from 3MC by microsomal metabolism and isolated by high pressure liquid chromatography (HPLC). From HPLC analysis of the metabolites of 3MC, from mass spectrometric analysis and from comparison with the fluorescence spectra of all 5 possible dihydrodiols of the alkylated benzanthracenes, it was deduced that one of the precursor metabolites was a 9,10-dihydrodiol of 3MC while the other was a 1 or 2-hydroxy derivative thereof.
Assuntos
DNA/metabolismo , Metilcolantreno/metabolismo , Animais , Células Cultivadas , Fenômenos Químicos , Química , Hidroxilação , Técnicas In Vitro , Microssomos/metabolismo , RatosRESUMO
Tritiated 3-methylcholanthrene (3MC) was administered to mouse embryo cells in culture from which DNA was later isolated and hydrolysed to deoxyribonucleosides. The hydrolysate was analysed by LH20 chromatography, HPLC and fluorescence spectroscopy. The 3MC-nucleoside products were compared with the nucleoside derivatives obtained from 3MC-11,12-oxide-treated DNA and found to differ in both fluorescence spectra and chromatographic behaviour. Microsome-catalyzed DNA binding of primary metabolites of 3MC identified two metabolites for which DNA reaction was extensive. These metabolites were characterized by their chromatographic and spectrophotometric properties. The products of their microsome-induced DNA binding were isolated, and comparison with the products derived from cell-mediate 3MC-DNA reaction suggested that these metabolites might be precursors of this latter reaction. The chemical nature of the metabolites and of their DNA reaction products is discussed in relation to the "bay region" and carbonium ion concepts of the ultimate carcinogen of polycyclic hydrocarbons.
Assuntos
DNA/metabolismo , Metilcolantreno/metabolismo , Animais , Células Cultivadas , Fenômenos Químicos , Química , Cromatografia em Gel , Desoxirribonucleosídeos , Hidrólise , Camundongos , Microssomos Hepáticos/metabolismo , Espectrometria de FluorescênciaRESUMO
Diethylstillboestrol, a synthetic and carcinogenic hormone, binds to DNA as a result of incubation with a liver microsomal preparation in vitro and on incubation with primary mouse foetal cells in culture. Enzymic digestion of DNA samples thus prepared gives several covalent deoxyribonucleoside-diethylstilboestrol products from the microsomal system. One of these is produced in small but significant yield in the tissue-culture system.
Assuntos
DNA/metabolismo , Dietilestilbestrol/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Desoxirribonucleosídeos/metabolismo , Masculino , CamundongosRESUMO
A study of the liver microsome-mediated binding to added DNA of the phenol metabolites of benzo(a)pyrene (BP-OH) and of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (BP-7,8-diol) suggested that as in the case of BP itself the reaction was catalysed by the enzyme aryl hydrocarbon hydroxylase. The addition of glutathione to the microsomal incubation inhibited the binding of BP and BP-OH more than that of BP-7,8-diol. Analysis by LH20 chromatography of the deoxyribonucleoside products from BP-DNA showed greater inhibition by glutathione of formation of the major product believed to result from further metabolism of BP-OH, than of the product arising by metabolism of BP-7,8-diol. The chromatographic behaviour and fluorescence spectrum of this major product were consistent with its derivation from 9-hydroxybenzo(a)pyrene (BP-9-OH) and furthermore suggested that BP-9-OH-4,5-oxide was the derivative whose reaction with DNA yielded this microsome-mediated BP-DNA product.
Assuntos
Benzopirenos/metabolismo , DNA/metabolismo , Microssomos Hepáticos/metabolismo , Alquilação , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cromatografia em Gel , DNA/análise , Fluorescência , Fluorometria , Glutationa/metabolismo , Glutationa/farmacologia , Purinas/análise , RatosRESUMO
The addition of borate buffer to the aqueous methanol used to elute hydrocarbon-deoxyribonucleoside derivatives from an LH 20 Sephadex column resulted in the separation of the products of reaction with DNA of the stereoisomers, (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy- and (+/-)-7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrenes, i.e., the syn- and anti-benzo(a)pyrene-diolepoxide, respecitvely. By this technique it was shown that the microsome-mediated binding to DNA of benzo(a)pyrene-7,8-dihydrodiol involved exclusively the anti-benzo(a)pyrene-diolepoxide. The benzo(a)pyrene binding to DNA that resulted on exposure of BHK21/C13 cells to this carcinogen was also shown to result predominantly by reaction of the anti-benzo(a)pyrene-diolepoxide. However, in this case other derivatives, including the syn-benzo(a)pyrene diolepoxide, might also be involved.
Assuntos
Benzopirenos/metabolismo , DNA/metabolismo , Animais , Microssomos Hepáticos/metabolismo , Ligação Proteica , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Metabolism of 7-methylbenz[a]anthracene (7MeBA) by 3-methylcholanthrene-induced rat liver microsomes in the presence of added native or denatured DNA resulted in covalent binding of the hydrocarbon to the nucleic acid. Enzymatic degradation and column chromatographic fractionation showed that the hydrocarbon-deoxyribonucleoside products were separable from the products similarly obtained from DNA having 7MeBA bound following treatment of mouse embryo cells in culture with this hydrocarbon. Comparison of the microsome catalysed hydrocarbon-deoxyribonucleoside products with those obtained by reaction with DNA of 7MeBA-5,6-oxide suggested that this K-region epoxide made a significant contribution to the liver microsome-induced DNA binding.
Assuntos
Benzo(a)Antracenos/metabolismo , DNA/metabolismo , Desoxirribonucleosídeos/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cromatografia em Gel , Peso Molecular , RatosRESUMO
Administration of 3-methylcholanthrene (3MC) to rats greatly enhanced the aryl hydrocarbon hydroxylase (AHH) activity of liver nuclei. However, the binding in vitro [3H]benzo[alpha]pyrene (BP) to DNA within the nuclei which occurred at the same time as hydroxylation of BP was much less enhanced. Thin layer chromatography of the metabolites of BP produced by these nuclei revealed the same metabolites in similar relative amounts as were produced by rat liver microsomes prepared from rats which had received 3MC. The binding to DNA was further analysed by hydrolysis of the DNA and fractionation on a Sephadex column. This analysis revealed that the binding to DAN in nuclei was very similar in nature to that which occurred when calf-thymus DNA was added to microsomes metabolising BP.
Assuntos
Benzopirenos/metabolismo , DNA/metabolismo , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação , Fígado/efeitos dos fármacos , Masculino , Metilcolantreno/farmacologia , Microscopia Eletrônica , Microssomos Hepáticos/efeitos dos fármacos , NADP/farmacologia , RatosRESUMO
Individual metabolites of benzo (a) pyrene were isolated from rat liver microsomal incubation of the parent hydrocarbon, and subsequently bound to DNA in separate incubations with microsomes. Of the metabolites examined, by far the greatest binding resulted with 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (BP-7,8-diol); the binding od the benzo (a) pyrene phenols (BP-OH) was about 50% that of the 7,8-diol. Analysis of the hydrocarbon-deoxyribonucleoside derivatives obtained by enzymic degradation of these DNA samples revealed that the binding of BP-7,8-diol was accounted for mainly by a single product identical to one of the product identical to one of the products obtained from DNA with bound benzo(a)pyrene. Furthermore, the microsome-induced binding of BP-OH to DNA yielded mainly a single product identical in chromatographic behaviour to the major product derived from benzo(a)pyrene when bound to DNA during incubation with microsomes.
Assuntos
Benzopirenos/metabolismo , DNA/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Benzopirenos/análise , Sítios de Ligação , Desoxirribonucleosídeos/metabolismo , História do Século XVIII , RatosRESUMO
Chemical conversion of generally tritiated benzo(a)pyrene to 6 and 1,6-substituted derivatives resulted in 30% and 48% loss of tritium respectively. Metabolism of [3H], [14C]-benzo(a)pyrene by rat liver microsomes yielded 3-hydroxybenzo(a)pyrene with 30% loss of tritium, a mixture of quinones with 50% loss of tritium and three dihydrodiol metabolites which had retained all the tritium of the parent hydrocarbon. DNA isolated from mouse embryo cells which had been exposed to [3H], [14C]-benzo(a)pyrene, and DNA with this hydrocarbon bound following in vitro rat liver microsomal incubation were degraded enzymically and the hydrocarbon-deoxyribonucleoside products isolated. The tritium contents of the products obtained from both DNA samples were very close to those of the original double labelled benzo(a)pyrene. These results are inconsistent with a phenol or quinone intermediate being responsible for the reaction with DNA, but fully consistent with a diol epoxide intermediate as proposed by Sims et al. (1974).