ABSTRACT
OBJECTIVES: The aim of our study was to compare the mode of delivery after a caesarean performed before 28 weeks of gestation compared to a control group of patients who had a caesarean at term. Our secondary objective was to compare the risk of uterine rupture in these both groups. PATIENTS AND METHODS: This retrospective case-control was realised in a level III maternal center between the 1st of January 2001 and the 31th of December 2010. All patients who underwent caesarean section between 22 and 28 weeks of gestation were included, regardless of the indication of caesarean section, their gender and type of pregnancy (single or multiple). The only exclusion criterion was the existence of a scarred uterus. RESULTS: Seventy-four patients were included in the case group and 144 in the control group. On the 74 patients who had a Caesarean before 28 weeks of gestation, 33 had a subsequent pregnancy and 31 have been studied. Twenty-four patients (77.4%) had a caesarean. Of the 7 patients (22.6%) having a vaginal attempt, the success rate was 100%. In the control group, 44 patients (30.6%) had a caesarean and 100 patients (69.4%) having a vaginal attempt, the success rate was 80%. No uterine rupture was found in both groups. Two cases of incomplete uterine rupture were found in each group. In our small series, the risk of uterine rupture was not increased compared to the group of patients having a caesarean section at term. CONCLUSION: In a subsequent pregnancy, the caesarean rate was significantly higher after a caesarean section before 28 weeks after a caesarean section performed at term. However, there is no contraindication against a vaginal attempt after a caesarean section performed before 28 weeks, except in the case of a corporeal cesarean section proved. It is therefore important to refer to the operative report of cesarean section to know whether there was or not the lower segment and the hysterotomy realized.
Subject(s)
Cesarean Section, Repeat/statistics & numerical data , Cesarean Section/adverse effects , Cesarean Section/statistics & numerical data , Premature Birth , Vaginal Birth after Cesarean/statistics & numerical data , Adult , Case-Control Studies , Female , Gestational Age , Humans , Pregnancy , Retrospective StudiesABSTRACT
OBJECTIVE: To describe the epidemiological, clinical and prognostic factors of placental abruption and fetal death in utero and to investigate possible risk factors for their occurrence. PATIENTS AND METHODS: Observational retrospective study including the women having presented a placental abruption between January 2001 and January 2012, in a IIB maternity. Women's sociodemographic characteristics, clinical symptoms and the method used to detect placental abruption were collected. Patient data of those whose pregnancy resulted in fetal death were compared to those with more favorable outcomes. RESULTS: There were 171 cases of placental abruption among 21,913 patients having delivered, which represents a 0.78% incidence. Diagnosis was rarely based on clinical data (30%). The rate of fetal death in utero represented 25% of the pregnancy's outcomes. A history of fetal death in utero increased the risk of placental abruption (P<0.001). This complication was more frequent for patients who did not have pregnancy monitoring (P=0.054) and before 37 weeks of amenorrhoea (P=0.005). CONCLUSION: Placental abruption is an important cause of perinatal mortality and maternal morbidity. Among the observed risk factors, only regular pregnancy monitoring can be an easy way to prevent these complications.
Subject(s)
Abruptio Placentae/diagnosis , Abruptio Placentae/therapy , Pregnancy Outcome , Abruptio Placentae/epidemiology , Adolescent , Adult , Cohort Studies , Female , French Guiana/epidemiology , Humans , Infant, Newborn , Pregnancy , Pregnancy Outcome/epidemiology , Premature Birth/epidemiology , Prognosis , Retrospective Studies , Risk Factors , Young AdultABSTRACT
OBJECTIVES: Women who had severe preeclampsia are at high risk of gestational vascular complications (preeclampsia, gestational hypertension, fetal death, small for gestational age, placenta abruptio) in subsequent pregnancies. The aim of this study was to describe outcomes of subsequent pregnancy after severe preeclampsia with delivery before 34 weeks of gestation during the first pregnancy. PATIENTS AND METHODS: One hundred and thirty-four primiparous women delivered before 34 weeks of gestation resulting in severe preeclampsia between January 2002 and December 2009. The data of the index pregnancy were identified from the medical record of our maternity, those of the subsequent pregnancy from paper or computerized medical records of the hospitals where deliveries took place. Our study ended on December 31 2011 for a decrease of at least 2 years after the index pregnancy. RESULTS: Of the 75 subsequent pregnancies, 59 have been studied. Twenty patients (34%) had gestational vascular complications, in type of gestational hypertension alone (10%), preeclampsia (65%), isolated small for gestational age (20%) or fetal death (5%). Thirty-nine patients (66%) showed no recurrence of gestational vascular complications but only 33 patients (56%) had a pregnancy of course totally physiological. The only risk factor for recurrent gestational vascular complications in subsequent pregnancy was a long time interval between two pregnancies, with an increased risk in case of delay beyond 26 months. CONCLUSION: Women with a history of severe preeclampsia with delivery before 34 weeks of gestation during first pregnancy are at increased risk for gestational vascular complications during the next pregnancy. A close obstetrical monitoring is recommended during a subsequent pregnancy.
Subject(s)
Gestational Age , Pre-Eclampsia/physiopathology , Pregnancy Outcome , Premature Birth/physiopathology , Abruptio Placentae/epidemiology , Birth Intervals , Female , Fetal Death/epidemiology , Humans , Hypertension, Pregnancy-Induced/epidemiology , Infant, Newborn , Infant, Small for Gestational Age , Parity , Pregnancy , Premature Birth/epidemiology , Premature Birth/etiology , Recurrence , Risk FactorsABSTRACT
OBJECTIVES: To examine the efficiency on early-onset neonatal infections of the ANAES guidelines for early-onset infections prophylaxis, based on a systematic prenatal vaginal swab aiming Group B Streptococcus and/or infection risk factors during delivery. PATIENTS AND METHODS: This is a retrospective cohort study of early-onset infections during a period of 28 months (6125 deliveries) compared to an earlier period (6141 deliveries). RESULTS: The number of newborns admitted for suspected infection and the rate of sepsis have been unchanged. But the total number of infections has decreased (1.50 vs 2.02 %, p=0,024), without increase of the number of infections due to other germs such as Escherichia coli. Furthermore, a strategy based only on risk factors would not have allowed the early screening and treatment of 23 Streptococcus B infected newborns. CONCLUSION: We have proved the efficiency of the protocol in terms of prevention of early-onset infections. However, it has led to a dramatic increase in the consumption of antibiotics, which is worrying concerning maternal and neonatal bacterial ecology.
Subject(s)
Infectious Disease Transmission, Vertical/prevention & control , Practice Guidelines as Topic , Pregnancy Complications, Infectious , Streptococcal Infections/prevention & control , Streptococcal Infections/transmission , Streptococcus agalactiae , Adult , Anti-Bacterial Agents/administration & dosage , Cohort Studies , Female , France , Health Policy , Humans , Infant, Newborn , Pregnancy , Pregnancy Complications, Infectious/drug therapy , Retrospective Studies , Streptococcal Infections/epidemiology , Streptococcus agalactiae/isolation & purificationABSTRACT
OBJECTIVES: To examine adherence to the Anaes guidelines for early-onset infections prophylaxis, based on a systematic prenatal vaginal swab aiming group B Streptococcus and/or infection risk factors during delivery. PATIENTS AND METHODS: Retrospective study of 6125 consecutive deliveries through a 28-month period. Data were collected from the patients files recorded in a computer database. RESULTS: Overall, a vaginal swab was performed on 88.3% of the patients. Among these, 79.1% were performed according to the guidelines. Among the patients, 90.2% with a positive swab received intrapartum antibiotics. In accordance to the national guidelines, we noticed a 40% increase in the consumption of antibiotics, without any severe complication during the study. Certain points must be improved though: time of the sampling, antibiotic therapy in case of quick delivery, management of patients with allergy to penicillin. CONCLUSION: Concordance with the national guidelines is feasible in a department with a high obstetrical activity.
Subject(s)
Infectious Disease Transmission, Vertical/prevention & control , Practice Guidelines as Topic , Pregnancy Complications, Infectious , Streptococcal Infections/prevention & control , Streptococcal Infections/transmission , Streptococcus agalactiae , Anti-Bacterial Agents/administration & dosage , Female , France , Health Policy , Humans , Infant, Newborn , Pregnancy , Pregnancy Complications, Infectious/diagnosis , Pregnancy Complications, Infectious/drug therapy , Prenatal Care , Retrospective Studies , Streptococcal Infections/diagnosis , Streptococcus agalactiae/isolation & purification , Vagina/microbiologyABSTRACT
The class VI POU domain family member known as Emb in the mouse (rat Brn5 or human mPOU/TCFbeta1) is present in vivo as a protein migrating at about 80 kDa on western blots, considerably larger than that predicted (about 42 kDa) from previously cloned coding sequences. By RT-PCR and 5' RACE strategies a full-length Emb sequence, Emb FL, is now identified. Shorter sequences encoding the -COOH terminal, and an -NH(2) terminal isoform, EmbN, were also isolated. Comparisons of Emb coding sequences between species, including the full-length zebra fish, POU(c), are presented, together with a compilation of the multiple transcripts produced by alternative splicing and the presence of different transcriptional start and stop sites, from the Emb gene.
Subject(s)
DNA-Binding Proteins/genetics , Transcription Factors/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cell Line , Cell Line, Tumor , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA-Binding Proteins/metabolism , Genes/genetics , Humans , Molecular Sequence Data , POU Domain Factors , Protein Isoforms/genetics , Rats , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription Factors/metabolism , Transcription, Genetic/geneticsABSTRACT
Expression of the mouse cardiac actin gene depends on a distal enhancer (-7 kbp) which has been shown, in transgenic mice, to direct expression to embryonic skeletal muscle. The presence of this distal sequence is also associated with reproducible expression of cardiac actin transgenes. In differentiated skeletal muscle cells, activity of the enhancer is driven by an E box, binding MyoD family members, and by a 3' AT-rich sequence which is in the location of a DNase I-hypersensitive site. This sequence does not bind MEF2 proteins, or other known muscle transcription factors, directly. Oct1 and Emb, a class VI POU domain protein, bind to consensus sites on the DNA, and it is the binding of Emb which is important for activity. Emb binds as a major complex with MEF2D and the histone transacetylase p300. The form of Emb present in this complex and as a major form in muscle cell extracts is longer (80 kDa) than that previously described. These results demonstrate the importance of this novel complex in the transcriptional regulation of the cardiac actin gene and suggest a potential role in chromatin remodeling associated with muscle gene activation.
Subject(s)
Acetyltransferases/metabolism , Actins/genetics , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation , Histones/metabolism , Myocardium/metabolism , Transcription Factors/metabolism , Actins/metabolism , Animals , Base Sequence , Cell Line , DNA Footprinting , Histone Acetyltransferases , MEF2 Transcription Factors , Macromolecular Substances , Mice , Mice, Inbred C3H , Molecular Sequence Data , Myogenic Regulatory Factors , Sequence Alignment , Transcriptional Activation , p300-CBP Transcription FactorsABSTRACT
The SopA protein plays an essential, though so far undefined, role in partition of the mini-F plasmid but, when overproduced, it causes loss of mini-F from growing cells. Our investigation of this phenomenon has revealed that excess SopA protein reduces the linking number of mini-F. It appears to do so by disturbing the partition complex, in which SopB normally introduces local positive supercoiling upon binding to the sopC centromere, as it occurs only in plasmids carrying sopC and in the presence of SopB protein. SopA-induced reduction in linking number is not associated with altered sop promoter activity or levels of SopB protein and occurs in the absence of changes in overall supercoil density. SopA protein mutated in the ATPase nucleotide-binding site (K120Q) or lacking the presumed SopB interaction domain does not induce the reduction in linking number, suggesting that excess SopA disrupts the partition complex by interacting with SopB to remove positive supercoils in an ATP-dependent manner. Destabilization of mini-F also depends on sopC and SopB, but the K120Q mutant retains some capacity for destabilizing mini-F. SopA-induced destabilization thus appears to be complex and may involve more than one SopA activity. The results are interpreted in terms of a regulatory role for SopA in the oligomerization of SopB dimers bound to the centromere.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/physiology , F Factor/genetics , F Factor/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Culture Media , Escherichia coli/genetics , PhenotypeABSTRACT
PURPOSE: To analyze retrospectively survival and prognostic factors of patients with non-Hodgkin's lymphoma (NHL) autografted from 1979 to 1995 in a single institution. PATIENTS AND METHODS: A total of 120 patients, 64 with aggressive and 56 with low-grade NHL, were autografted. The carmustine (BCNU), etoposide, cytarabine, and melphalan (BEAM) regimen was used in 104. The autograft was marrow in 101 patients. Marrow was purged in vitro by mafosfamide for 63 patients (adjusted dose [AD] in 32; unique dose [UD] in 31); 27 patients received a CD34+-selected graft. Following intensification, 45 patients received additional radiotherapy on previous sites of involvement. RESULTS: Outcome at 5 years for patients transplanted with low-grade NHL in first complete remission (CR1), in first partial remission (PR1), and in second complete remission (CR2) or beyond showed an event-free survival (EFS) of 75% +/- 12%, 46% +/- 18%, and 57% +/- 24%, a relapse incidence (RI) of 21% +/- 12%, 49% +/- 19%, and 43% +/- 25%, and a transplant-related mortality (TRM) of 5% +/- 5%, 10% +/- 7%, and 0%, respectively. For patients with aggressive NHL transplanted in CR1, in PR1, in CR2 or beyond, and in resistant relapse or in primary refractory disease, the EFS was of 73% +/- 9%, 58% +/- 19%, 29% +/- 16%, and 10% +/- 9%, the RI 22% +/- 9%, 14% +/- 9%, 77% +/- 18%, and 66% +/- 20%, and the TRM 6% +/- 6%, 32% +/- 21%, 11% +/- 10%, and 71% +/- 22%, respectively. In patients autografted upfront in first remission, additional radiotherapy was associated with a higher EFS, in univariate (P = .03) and multivariate analysis (P = .02, relative risk [RR] = .021). The role of graft purging with mafosfamide on the outcome reflected by the dose of colony-forming unit-granulocyte-macrophage (CFU-GM) per kilogram infused postpurging was assessed by univariate analysis: patients in first remission who received lower doses of CFU-GM had a lower RI and a higher EFS. CONCLUSION: This retrospective analysis suggests that marrow purging and posttransplant radiotherapy improve the outcome of patients with NHL autografted in first remission.
Subject(s)
Bone Marrow Purging , Hematopoietic Stem Cell Transplantation , Lymphoma, Non-Hodgkin/therapy , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carmustine/administration & dosage , Combined Modality Therapy , Cyclophosphamide/analogs & derivatives , Cytarabine/administration & dosage , Disease-Free Survival , Etoposide/administration & dosage , Female , Humans , Lymphoma, Non-Hodgkin/pathology , Lymphoma, Non-Hodgkin/radiotherapy , Male , Melphalan/administration & dosage , Middle Aged , Prognosis , Recurrence , Remission Induction , Retrospective StudiesABSTRACT
The mbrC17 mutation in Escherichia coli had been shown to cause conditional growth defects and an increase in the quantity of DNA per cell. The present work was aimed at identifying the mutation. Sequencing showed that the MbrC17 phenotype does not involve glr (murI), as previously suggested. P1 transduction data indicated that the mbrC17 mutation is closely linked to rpoB, and allele exchange showed it to lie within the secE-nusG operon. A single change relative to wild type was found in the secE-nusG region from the mbrC17 strain, a G-->A mutation 23 bp upstream of the secE coding sequence. This mutation causes a two-fold increase in the concentration of secE-nusG mRNA.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial , Mutation , Ploidies , Amino Acid Isomerases/genetics , Cell Division , Chromosome Mapping , Phenotype , Time FactorsABSTRACT
The question of whether or not toluene diisocyanate (TDI)-induced airway hyperresponsiveness in the guinea-pig is accompanied by neutrophil influx into bronchoalveolar lavage fluid (BALF) was addressed. Two modes of exposure were studied; (1) acute exposures where animals were exposed to 3 ppm TDI for 1 h and experiments were carried out 30 min, 4 h, 24 h, 48 h and 1 week after the TDI exposures; (2) subacute exposures where animals were exposed to 0.080 and 0.046 ppm TDI for 48 h 1 week, respectively, and experiments were carried out 24 h after the TDI exposures. The changes in airway responsiveness to increasing doses of intravenous acetylcholine (ACh) in anaesthetized and tracheotomized spontaneously breathing guinea-pigs were examined. In order to elucidate the possible relationships of airway responsiveness to cellular infiltration, bronchoalveolar lavage was performed in additional group of guinea-pigs exposed to the same conditions. After acute exposure to 3 ppm TDI, increased bronchial responsiveness was evident within 30 min, lasted 48 h, but had vanished 1 week after the exposure. An influx of neutrophils occurred into the BALF within 1 h after exposure. The influx of neutrophil into BALF lasted 48 h and vanished 1 week after the end of exposure. After 48 h of exposure to TDI at 0.080 ppm, or 0.046 ppm for 1 week, increased bronchial responsiveness was evident 24 h after the end of the both modes of exposure, but no influx of neutrophils was observed into the BALF. It was concluded that even though the neutrophil influx and hyperresponsiveness evolve in the same way after acute exposure to a high concentration of TDI (3 ppm), this is not the case after subchronic exposure to low concentrations of TDI, where a bronchial hyperresponsiveness is observed without detectable neutrophil influx.
Subject(s)
Bronchi/drug effects , Bronchial Hyperreactivity/chemically induced , Inflammation/chemically induced , Neutrophils/drug effects , Toluene 2,4-Diisocyanate/toxicity , Acetylcholine/administration & dosage , Administration, Inhalation , Animals , Bronchoalveolar Lavage Fluid/cytology , Female , Guinea Pigs , Neutrophils/physiology , Time Factors , Toluene 2,4-Diisocyanate/administration & dosageABSTRACT
OBJECTIVE: To determine the incidence of carbohydrate intolerance during pregnancy and its impact on neonatal outcomes, which have not been formally established in Canada. DESIGN: Chart review. PARTICIPANTS: All 1432 women who delivered a single newborn in the months of May and September 1992 in Hôpital Sainte-Justine, Montreal, Hôpital Maisonneuve-Rosemont, Montreal, and Centre hospitalier de Rouyn-Noranda, Rouyn, Que. Women with multiple births or pre-existing diabetes mellitus were excluded. OUTCOME MEASURES: Carbohydrate intolerance, including gestational glucose intolerance (one abnormal result of an oral glucose tolerance test) and gestational diabetes mellitus (two or more abnormal results of the test); neonatal outcomes, including birth weight, hypoglycemia and hyperbilirubinemia. RESULTS: Of the 1074 women tested, 72 (6.7%) had gestational glucose intolerance and 98 (9.1%) had gestational diabetes mellitus. Of those women with carbohydrate intolerance, 120 were treated and 50 were not. Women who were not treated had higher rates of poor neonatal outcomes than women without carbohydrate intolerance (p < 0.05) or than women with carbohydrate intolerance who had received treatment (p < 0.05). Women who were not treated had higher rates of newborns with a birth weight of more than 4000 g (18.0%), large-for-gestational-age newborns (20.0%) and newborns with hypoglycemia (24.0%) or hyperbilirubinemia (40.0%) than women without carbohydrate intolerance (for whom the rates were 6.7%, 10.0%, 10.0% and 22.8%, respectively) or than women with carbohydrate intolerance who received treatment (for whom the rates were 6.7%, 6.7%, 10.0% and 21.7%, respectively). CONCLUSIONS: In this population, carbohydrate intolerance during pregnancy is a significant problem. Not only is the incidence rate higher than that usually estimated, but the impact on neonatal morbidity is also significant.
Subject(s)
Diabetes, Gestational/epidemiology , Glucose Intolerance/epidemiology , Pregnancy Outcome , Adult , Age Factors , Body Mass Index , Diabetes Mellitus/genetics , Diabetes, Gestational/drug therapy , Female , Humans , Hyperbilirubinemia/epidemiology , Infant, Newborn , Insulin/therapeutic use , Obesity , Pregnancy , Risk FactorsABSTRACT
Gene therapy defines a new therapeutic avenue whose site of action is at the level of the gene itself; viral vectors (adenovirus, retrovirus, herpes virus) or non-viral (liposomes, plasmids) enable the transfer of a fraction of DNA (transgenic) to the target itself. In this review, we present recently acquired data on the mechanisms of oncogenesis and anti-tumor immunity which have enabled the application of several therapeutic strategies in oncology; the transfer of gene(s), coding for cytokines or for coactivation factors in order to develop active immunotherapy; the transfer of suicides genes; the transfer of multidrug resistance gene (MDR1); the transfer of tumor suppressor genes or of cDNA coding for antisense oligonucleotides in order to correct genomic anomalies which are responsible for the malign phenotype. The development of gene therapy demands the resolution of a number of technical difficulties such as vectorisation, targeting, and the expression of the stability of the trans-gene. Phase 1 trials in man have established the innocuity of certain vectors and have confirmed the expression of trans-genes (marker genes). Compared to monogenic hereditary diseases, the "molecular heterogenetic" of bronchial tumours, the consequence of the instability of the genome and the diversity of amplified oncogenes are a major difficulty. In addition, each one of these approaches prevents limiting factors: for example the exclusive targeting of malign cells is an indispensable pre-requisite for the transfer of suicide genes and in the same way the expression the tumour in antigens is the pre-requisite for the development of active immunotherapy. We report the overall results of applied trials for pulmonary carcinomas on murine models and present their applications which are underway in men.
Subject(s)
Genetic Therapy , Lung Neoplasms/therapy , Animals , Bronchial Neoplasms/genetics , Carcinoma/genetics , Clinical Trials, Phase I as Topic , Cytokines/genetics , DNA/genetics , DNA, Antisense/genetics , DNA, Complementary/genetics , Disease Models, Animal , Gene Amplification , Gene Expression , Genes, MDR/genetics , Genes, Tumor Suppressor/genetics , Genetic Vectors/therapeutic use , Humans , Immunotherapy, Active , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Mice , Oncogenes/genetics , Phenotype , TransfectionABSTRACT
Nested PCR (NPCR), a two-step procedure in which the products of a first PCR using 'outer' primers are reamplified using 'inner primers', has been successfully used to test for the chronic myeloid leukemia (CML)-specific bcr-abl transcripts. A major drawback of the conventional nesting strategy is linked to the opening of the reaction tube between the two successive PCR reactions, giving a risk of contaminating the second mix with amplicons. In this paper, the application of a new protocol for NPCR without reopening the reaction tube between the two steps of the procedure is described for the research of residual leukemic cells in the peripheral blood of 14 CML patients treated by bone marrow transplantation (BMT) or interferon (IFN). This assay which is both highly specific and sensitive, offers several advantages over the use of conventional NPCR: it is more sensitive, faster and decreases the risk of false-positive results. In addition, chemiluminescent detection of amplified DNA after transfer onto a nylon membrane, although comparable with radioactive hybridization in terms of sensitivity and speed, is more advantageous in safety and convenience. In conclusion, this assay could be adapted to a number of clinical diagnostic uses.
Subject(s)
Fusion Proteins, bcr-abl/biosynthesis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Polymerase Chain Reaction/methods , Transcription, Genetic , Base Sequence , Bone Marrow Transplantation , DNA Primers , Exons , Gene Expression , Humans , Interferons/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/therapy , Molecular Sequence Data , Reference ValuesABSTRACT
The chicken beta tropomyosin (beta TM) gene has two alternative transcription start sites (sk and nmCAP sites) which are used in muscle or non muscle tissues respectively. In order to understand the mechanisms involved in the tissue-specific and developmentally-regulated expression of the beta TM gene, we have analyzed the 5' regions associated with each CAP site. Truncated regions 5' to the nmCAP site were inserted upstream to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and these constructs were transfected into avian myogenic and non myogenic cells. The maximum transcription is driven by the CAT construct (-168/ + 216 nt) in all cell types. Previous deletion analysis of the region 5' to the beta TMskCAP site has indicated that 805 nt confer myotube-specific transcription. In this work, we characterized an enhancer element (-201/-68 nt) which contains an E box (-177), a variant CArG box (-104) and a stretch of 7Cs (-147). Mutation of any of these motifs results in a decrease of the myotube-specific transcriptional activity. Electrophoretic mobility shift assays indicate that these cis-acting sequences specifically bind nuclear proteins. This enhancer functions in an orientation-dependent manner.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Muscles/metabolism , Promoter Regions, Genetic/genetics , Transcription, Genetic/genetics , Tropomyosin/genetics , Animals , Base Sequence , Cell Line , Chickens , DNA/genetics , DNA/metabolism , DNA, Complementary/analysis , Fibroblasts , Molecular Sequence Data , Muscles/cytology , Mutagenesis, Site-Directed , Nuclear Proteins/metabolism , Protein Binding , Quail/embryology , RNA Caps/genetics , RNA, Messenger/analysis , RNA, Messenger/biosynthesisABSTRACT
The chick beta tropomyosin (TM) gene has two alternative transcription initiation start sites which are used in muscle or non muscle tissue. A recombinant plasmid containing 805 nucleotides (nt) of the sequence upstream to the muscle CAP site driving the bacterial chloramphenicol acetyltransferase gene is sufficient for muscle specific expression. Of the two E boxes present in this construct, only the E box proximal to the CAP site is functional since deletion or mutation of this E box causes a decrease of CAT activity (about 40%). Separate mutation of Sp1 motifs also reduces the transcription driven by the 805nt fragment. Double mutation of E box and Sp1 motifs show that helix-loop-helix muscle regulatory factors and ubiquitous Sp1 transcription factor are required in the initiation of the transcription of the chick beta TM gene in muscle tissue. Our results also suggest that other factors may participate to this process.