RESUMEN
OBJECTIVE: Duchenne muscular dystrophy (DMD) is a musculo-degenerative disease characterized by lack of dystrophin production with no definite cure available currently. Discarded umbilical cord is a potential source of mesenchymal stem cells which are non-immunogenic and can be used for transplantation in allogenic set ups. Given the regenerative and anti-inflammatory properties of mesenchymal stem cells (MSCs), here we investigated its role in the cellular therapy of DMD patients. DESIGN: This is a single-blinded study conducted in various hospitals of India situated in Mumbai, Delhi, and Lucknow. Inclusion criteria for enrolling the patients in the study were boys aged between 5 to 18 years, absence of dystrophin in the immunohistochemistry of muscle biopsy and mutation in dystrophin gene in cytogenetic analysis. The exclusion criteria were presence of dystrophin in the muscle biopsy, patients on corticosteroids etc. UC-MSCs (2 millions/kg body weight) were administered through IV and IM injection. Muscle power in muscles of proximal upper limb, distal upper limb, proximal lower limb, distal lower limb, hip flexors, hip extensors, hip abductors, and paraspinal muscles were measured in 11 DMD patients after UC-MSCs transplantation and were followed for up to 3 years (average follow up 1.5 years). 5 DMD patients did not receive any UC-MSCs transplantation and served as the control group. RESULTS: The treatment group (N = 11 at baseline) had a pretransplantation strength of 3.45 ± 1.0357 and 4.090 ± 0.8312 in muscles of proximal upper limb and distal upper limb respectively. After 1 year (N = 9) these strengths remained stable with an average of 3.78 (1.03) and 4.22 (0.83). In contrast, the control group (N = 5) has a pre-transplantation strength of 3.6 (0.54) and 4 (1) in the proximal and distal upper limb respectively. After 1 year, (N = 5) 3/5 subjects had a slight but not statistically significant decrease in the proximal upper limb, mean 3.0 (1.0) and 5/5 had a lunit decrease in strength, mean 3.0 (1.0). The treatment group had a pre-transplantation strength of 2.0909 ± 0.8312 and 3.1181 ± 0.8738 in muscles of distal and proximal lower limbs respectively. At 1 year (N = 9), 4/9 subjects had a 1 unit increase in strength in the distal lower limb (mean 3.78 (0.97)) and 8/9 subjects had a lunit increase in strength in the proximal lower limb, mean 3.11 (1.05). The control group has a mean of 3.41 (0.54) and 3.0 (1.0) at baseline in the distal and proximal lower limb respectively. By 1 year, 3/5 subjects had a 1 unit decrease (mean 2.8 (0.45)) and 5/5 had a lunit decrease, mean 2.0 (1.0) in distal and proximal lower limb strength. Stability in muscle function was also achieved in muscles of hip flexors, hip extensors, hip abductors, and paraspinal muscles at one year as compared to untreated group. CONCLUSION: UC-MSCs administration not only resulted in the stabilization of muscle power but also did not show GVHD or any deleterious effects on the patients and thus may be considered as safe option for treatment of DMD as compared to control untreated group although further larger double-blinded studies are needed.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Trasplante de Células Madre Mesenquimatosas , Distrofia Muscular de Duchenne/terapia , Adolescente , Niño , Preescolar , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Distrofina/genética , Estudios de Factibilidad , Humanos , India , Masculino , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Fuerza Muscular , Distrofia Muscular de Duchenne/genética , Resultado del Tratamiento , Extremidad SuperiorRESUMEN
Driver posture is an important factor to be considered in the ergonomics design process of automobiles. Most decisions during automobile design and manufacture are informed by studying the intricate biomechanical components of human musculoskeletal systems to ensure maximum comfort, safety and well-being during driving. A case study is presented that confirms inappropriate foot position as a causative factor for the development of abnormal lateral/plantar heel callosities when driving a 4 x 4-style vehicle. The driver's foot position was influenced by the seat geometry of the vehicle. Cessation of driving the 4 x 4-style vehicle and driving of an alternative automobile while on holiday for a period of 4 weeks resolved the condition. On return to the 4 x 4-style vehicle, however, the abnormal callus patterns redeveloped while using the same footwear and no change in any other parameters. It is therefore suggested that seat and consequent foot position is an important ergonomic factor that should be addressed in the future design of automobile seating.
Asunto(s)
Conducción de Automóvil , Callosidades/etiología , Talón , Adulto , Automóviles/normas , Diseño de Equipo , Ergometría , Humanos , Masculino , Presión/efectos adversosRESUMEN
Overproduction of mucus and of mucin glycoproteins and goblet cell hyperplasia occurs in chronic obstructive airway diseases, including asthma and cystic fibrosis. Mucus overproduction results from alterations in several cellular processes, including altered regulation of airway mucin genes on exposure to environmental and infectious agents and to inflammatory mediators. Seven of the nine identified MUC genes (which encode the protein backbone of mucins) are normally expressed in human respiratory tract tissues. Several inflammatory mediators have now been shown to regulate expression of MUC2, MUC5AC, and MUC5B genes. Importantly, mucin gene expression can be regulated both transcriptionally and posttranscriptionally. Current information on airway mucin gene expression is summarized in this review along with an overview of airway epithelial model systems. In vitro model systems include airway epithelial carcinoma cell lines and primary normal human bronchial epithelial (NHBE) cells. In vivo systems include human respiratory tract tissues and rodent airways. Our laboratory has begun to investigate the role of cytokines on mucin gene expression in vitro and in vivo and on goblet cell metaplasia in vivo. Because cytokines can alter cell proliferation, we characterized the effect of interleukin (IL)-4 and IL-13 on the proliferation of NHBE cells and three human lung carcinoma cell lines--A549, NCI-H292, and Calu-3--that are frequently used for analyses of airway mucin gene expression. Both IL-4 and IL-13 had cell-specific effects. They increased proliferation moderately (1.2-3.0-fold) in NHBE and Calu-3 cells, but markedly inhibited proliferation of A549 cells in a dose-dependent manner. IL-4 increased proliferation of NCI-H292 cells moderately, although IL-13 had no significant effect. We also examined the role of IL-13 and IL-4 on MUC5AC messenger RNA (mRNA) expression in A549, Calu-3, and H292 cell lines and did not observe any significant effect. However, we recently showed an increase in Muc-5ac mRNA and protein expression in a murine model of ovalbumin-induced allergic asthma and in murine airways when IL-13 was delivered intranasally (Alimam, N.Z., et al. Am J. Respir. Cell Mol. Biol. 22:253--260). Thus, we speculate that IL-13 plays a role in the differentiation of murine airway epithelial cells into goblet cells, which then express Muc-5ac mRNA. A detailed analysis of the role of cytokines in airway cell differentiation and mucin gene expression both in vitro and in vivo is required to elucidate the roles of mucins in airway health and diseases. Identification of Muc-5ac as a major gene and gene product in goblet cell metaplasia should facilitate delineation of the molecular mechanisms underlying the induction and reversal of airway goblet cell metaplasia and goblet cell hyperplasia.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-13/farmacología , Interleucina-4/farmacología , Mucinas/genética , Animales , Northern Blotting , Tumor Carcinoide/metabolismo , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares/metabolismo , Células Tumorales CultivadasRESUMEN
During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras , Espermatocitos/enzimología , Espermatogénesis/genética , Transactivadores/metabolismo , beta-N-Acetilglucosaminilglicopéptido beta-1,4-Galactosiltransferasa/biosíntesis , Animales , Secuencia de Bases , Sitios de Unión , Modulador del Elemento de Respuesta al AMP Cíclico , Huella de ADN , Proteínas de Unión al ADN/fisiología , Escherichia coli/genética , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Genéticos , Regiones Promotoras Genéticas , Isoformas de Proteínas/fisiología , Espermátides/enzimología , Factores de Transcripción/clasificación , Factores de Transcripción/metabolismo , Transcripción Genética , beta-N-Acetilglucosaminilglicopéptido beta-1,4-Galactosiltransferasa/genéticaRESUMEN
beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glándulas Mamarias Animales/enzimología , N-Acetil-Lactosamina Sintasa/biosíntesis , N-Acetil-Lactosamina Sintasa/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Huella de ADN , Desoxirribonucleasa I , Femenino , Células L , Lactancia , Ratones , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Especificidad de Órganos , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Mapeo Restrictivo , Factor de Transcripción Sp1/metabolismo , Transactivadores/aislamiento & purificación , Transactivadores/metabolismo , Factores de Transcripción/aislamiento & purificaciónRESUMEN
The developmental and hormonal regulation of UDP-GlcNA:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme which initiates the biosynthesis of asparagine-linked glycoproteins, was investigated in mouse mammary glands. An anti-peptide antibody raised against the carboxyl-terminal 11 amino acids of mouse GPT, immunoadsorbed GPT activity and recognized a protein of expected size (approximately 48 kDa) on Western blots. Mouse mammary glands at different stages of development were examined for GPT activity, immunoreactive protein, and GPT mRNA. All three parameters showed a similar trend, i.e. they were low in tissues from virgin and pregnant animals, increased steadily during lactation, reaching a peak around mid to late lactation, and declined thereafter in glands from post-lactating animals. At mid-lactation, the increase in GPT activity, immunoreactive protein, and GPT mRNA relative to the virgin stage was 9.5-, 3.3- and 5.4-fold respectively, on a per cell basis. These data suggest possible transcriptional and post-transcriptional modulation of GPT gene expression during development of mouse mammary gland. The results on hormonal regulation of GPT in mouse mammary explants and primary mouse mammary epithelial cells showed that all three parameters cited above were stimulated maximally by the combined presence of insulin, hydrocortisone, and prolactin, indicating that the hormonal regulation of GPT expression is also mediated at the level of RNA.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hormonas/farmacología , Glándulas Mamarias Animales/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Animales , Femenino , Hidrocortisona/farmacología , Insulina/farmacología , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Fragmentos de Péptidos/inmunología , Prolactina/farmacología , ARN Mensajero/análisis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/inmunologíaRESUMEN
The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, was isolated and characterized. Southern blot analyses demonstrated a single copy gene for GPT. The gene spans about 7.5 kilobase pairs of DNA and is divided into 9 exons by 8 introns. All the introns are found in the coding region, and most of them occur in segments separating the putative membrane-spanning domains. The exon/intron organization of the gene also correlates with the presence of several highly conserved regions of potential functional importance among yeast, leishmania, hamster, and mouse enzymes. Primer extension and reverse transcription-polymerase chain reaction analyses suggested the presence of several potential transcription start sites, with the closest one being approximately 200 base pairs upstream from the translation initiation codon. The 5'-flanking region lacks a typical TATA box, but is high in GC content and contains two putative Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 3'-end reverse transcription-polymerase chain reaction analysis indicated that the first of the two polyadenylation sites was used predominantly, in agreement with a approximately 2.0-kilobase pair GPT message seen on Northern blots of RNA from a wide variety of mouse tissues. This is the first report of cloning of a gene for an enzyme of the dolichol cycle in higher eukaryotes. A novel finding of this study is the observation of a G-->A change between the genomic sequence and nucleotide 280 in the cDNA. This could have important implications as an RNA editing mechanism for regulating the expression of the gene and therefore, protein N-glycosylation. A previous study (11) had shown that the activity of GPT was developmentally regulated in mouse mammary gland, with possible involvement by the hormone prolactin. The availability of the GPT gene with its promoter should facilitate future studies on delineating the mechanism for the hormonal regulation of GPT.
Asunto(s)
Ratones/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN/genética , ADN/metabolismo , Cartilla de ADN , Exones , Femenino , Biblioteca de Genes , Intrones , Hígado/enzimología , Glándulas Mamarias Animales/enzimología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Mapeo RestrictivoRESUMEN
A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.
Asunto(s)
Mapeo Cromosómico , Clonación Molecular , ADN/genética , Fosfotransferasas/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos) , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , ADN/química , ADN/aislamiento & purificación , Femenino , Lactancia , Glándulas Mamarias Animales/química , Ratones , Datos de Secuencia Molecular , Fosfotransferasas/química , Fosfotransferasas/inmunología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
Urokinase-type plasminogen activator (uPA) is expressed at higher levels in many transformed cells as compared with their non-transformed counterparts. The transformed phenotype is associated with changes in the cytoskeleton. Therefore, we have investigated whether alterations in the cytoskeleton can trigger changes in the expression of the uPA gene. To this end we analyzed the expression of the uPA gene following exposure of porcine kidney cells, LLC-PK1, to agents that modify the organization of specific components of the cytoskeleton. These cells exhibited increased uPA mRNA and protein after disruption of microtubules by colchicine or nocodazole treatment or after disruption of microfilaments by cytochalasin B treatment. Colchicine, nocodazole, and cytochalasin B did not cause alterations in the level of cAMP-dependent protein kinase in LLC-PK1 cells. In contrast, down-regulation of protein kinase C by phorbol myristate acetate, reduced, but did not fully prevent the induction of uPA mRNA when LLC-PK1 cells were subsequently exposed to colchicine, nocodazole, or cytochalasin B. Apparently, a signal transduction pathway in part involving protein kinase C but not cAMP-protein kinase mediates the regulatory changes at the transcriptional level of the uPA gene. Inhibition of protein synthesis by cycloheximide prior to the exposure of LLC-PK1 cells to colchicine, nocodazole, or cytochalasin B, largely prevented the induction of uPA mRNA.
Asunto(s)
Colchicina/farmacología , Citocalasina B/farmacología , Citoesqueleto/efectos de los fármacos , Precursores Enzimáticos/genética , Nocodazol/farmacología , Activadores Plasminogénicos/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Citoesqueleto de Actina/efectos de los fármacos , Animales , Línea Celular , Núcleo Celular , Citoesqueleto/ultraestructura , Inducción Enzimática , Precursores Enzimáticos/biosíntesis , Técnica del Anticuerpo Fluorescente , Expresión Génica , Microtúbulos/efectos de los fármacos , Activadores Plasminogénicos/biosíntesis , Proteínas Quinasas/metabolismo , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/genética , Mapeo Restrictivo , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
To characterize proteins that bind to the cyclic AMP inducible promoter of the urokinase-type plasminogen activator gene, we performed a DNAase I footprinting analysis. Within 500 nucleotides upstream of the transcription start site we found eight protected regions due to at least four different binding proteins. Among these is a single binding site for the transcription factor CTF/NF1, which is flanked on each side by two conserved binding sites for the transcription factor Sp1. A region at -380, which shares a similarity with sequences observed in the corresponding regions of other cyclic AMP regulated genes, was protected. This binding site contains a sequence of ten nucleotides which is repeated further upstream at -480 and also protected against DNAase I digestion. Comparisons of extracts from four different cell lines revealed that all DNA binding factors are present in nuclei of uPA expressing and nonexpressing cells. Mechanism underlying hormonal regulation of the gene is discussed.
Asunto(s)
Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Proteínas de Unión al ADN/fisiología , Desoxirribonucleasa I , Datos de Secuencia Molecular , Peso Molecular , PorcinosRESUMEN
The genes encoding the two plasminogen activators, tissue plasminogen activator and urokinase, were mapped to mouse chromosomes using probes derived from the respective mouse cDNAs. DNA from mouse-Chinese hamster and mouse-rat somatic cell hybrids was digested with BamHI and EcoRI, respectively, and analyzed by Southern blot hybridization for the segregation of the two genes. Tissue plasminogen activator and urokinase cosegregated with mouse chromosomes 8 and 14, respectively. The plasminogen activator genes thus fall into two syntenic groups that are conserved in human and mouse.
Asunto(s)
Mapeo Cromosómico , Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , ADN/genética , Enzimas de Restricción del ADN , Ligamiento Genético , Humanos , Células Híbridas , Ratones , Hibridación de Ácido Nucleico , RatasRESUMEN
The nucleotide sequence of the human tissue plasminogen activator (t-PA) gene has been established. A total of 36,594 base pairs (bp) was sequenced; this included 32,720 bp from the site of initiation of transcription to the polyadenylation site, in addition to 3,530 and 344 bp of 5' and 3' flanking DNA, respectively. Thirteen intervening sequences divide the gene into 14 coding regions; the size range for exons is 43-914 bp, while that for introns is 111-14,257 bp. The gene and 5' flanking region contain 28 copies of Alu repetitive DNA and a single KpnI repeat. The transcription initiation site was identified by S1 nuclease, exonuclease VII, and primer extension analysis as an A residue; "TATA" and "CAAT" boxes are located in the expected positions upstream of this proposed site. Results of the analysis of the gene sequence and its comparison with data banks are described. The protein and gene structures of tissue and urokinase plasminogen activator are compared; based on these features the evolutionary relationship of the two human plasminogen activators appears to be close.
Asunto(s)
Genes , Activador de Tejido Plasminógeno/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/metabolismo , Enzimas de Restricción del ADN , Humanos , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
A panel of human-mouse somatic cell hybrids and specific complementary DNA probes were used to map the human tissue plasminogen activator and urokinase genes to human chromosomes 8 and 10, respectively. This result is in contrast to a previous assignment of a plasminogen activator gene to chromosome 6. As neoplastic cells produce high levels of plasminogen activator, it is of interest that aberrations of chromosome 8 have been linked to various leukemias and lymphomas and that two human oncogenes, c-mos and c-myc, have also been mapped to chromosome 8.
Asunto(s)
Mapeo Cromosómico , Activadores Plasminogénicos/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Cromosomas Humanos 6-12 y X , ADN/genética , Genes , Humanos , Células Híbridas , Leucemia/genética , Linfoma/genética , Ratones , Hibridación de Ácido Nucleico , OncogenesRESUMEN
Cloned Drosophila melanogaster tRNA genes have been transcribed in a homologous cell-free extract isolated from a Schneider II cell line. The major product of the reaction is a tRNA precursor which is processed to a tRNA sized species. The kinetics of transcription has been followed for 5 different valine tRNA gene clones. The results demonstrate formation of stable transcription complex with at least two kinetic steps. While the rate of formation of the transcription complex is similar to different clones, the ultimate rate of transcription varies dramatically. Comparison of the DNA sequence of the tRNA genes suggests that rate determining nucleotides lie outside the canonical tRNA split-internal promoters.
Asunto(s)
Clonación Molecular , Drosophila melanogaster/genética , Drosophila/genética , Genes , ARN de Transferencia/genética , Transcripción Genética , Animales , Línea Celular , Sistema Libre de Células , Cinética , PlásmidosRESUMEN
Segments of cloned Drosophila DNA from four recombinant plasmids that hybridize with tRNA4Val have been sequenced. The segments from pDt92R and pDt120R that hybridize to 90C on the third polytene chromosome appear to be either repeats or alleles. They contain one structural gene each of identical sequence but differ at eight sites in 506 base pairs. The structural genes differ at four sites from the sequence expected from that of tRNA4Val. A third plasmid, pDt14, which hybridizes to 89BC on the third chromosome, also contains a structural gene with the same sequence as those in pDt92R and pDt120R. In addition, pDt14 has a gene for tRNA2Phe 214 base pairs upstream and with the same polarity as the tRNA4Val gene. The tRNA2Phe gene contains a 23-base pair segment identical with the corresponding segment in the tRNA4Val genes except for one base pair. The fourth plasmid investigated, pDt55, hybridizes to 70BC. It contains two tRNA4Val genes 525 base pairs apart with opposite polarity. These genes have identical sequences, which corresponds to that expected from the sequence of tRNA4Val. There is no evidence that the first three tRNA4Val genes are expressed at any stage during the development of Drosophila.