ABSTRACT
INTRODUCTION: In 20% of children with febrile syndrome, it appears as fever of unknown origin (FUO) syndrome. Management strategies in this group have high sensitivity but low specificity. OBJECTIVES: To cha racterize serious bacterial infections (SBI) in children younger than three months old hospitalized because of FUO syndrome and to evaluate the utility of clinical and laboratory parameters in the identification of patients that are at high risk of SBI. PATIENTS AND METHOD: Prospective study in patients aged < 3 months hospitalized due to FUO syndrome between January 2014 and November 2015 in two pediatric hospitals in the Metropolitan Region. INCLUSION CRITERIA: age 4 days - 3 months, fever > 38°C longer than 72 hours after onset without demonstrable cause. EXCLUSION CRITERIA: anti microbial use up to 7 days before admission, preterm infants < 34 weeks, birth weight < 2 kg, and im munocompromised. Demographic, clinical, and laboratory tests data were recorded as well as blood count and CRP, discharge diagnosis, and ruled out, probable or confirmed SBI. RESULTS: 32% of the patients were discharged with diagnosis of SBI, 28% with diagnosis of viral or probably viral infec tion, 34% with diagnosis of not specified FUO syndrome, and 6% due to other causes. There were no significant differences in the CRP value, altered WBCs count, toxic aspect, or hours of fever at the admission when comparing groups with and without SBI (p < 0.05). The combination of clinical and laboratory parameters showed 27% of sensitivity, 90% of specificity, 60% of PPV, and 71% of NPV. CONCLUSION: It was not possible to establish clinical and laboratory parameters that allow the identifi cation of children younger than 3 months old at high risk of SBI, however, they maintain their value as low risk indicators. It is necessary further investigation of other clinical and laboratory elements that allow discriminating SBI from viral infections.
Subject(s)
Bacterial Infections/complications , Bacterial Infections/diagnosis , Clinical Decision Rules , Fever of Unknown Origin/etiology , Hospitalization , Severity of Illness Index , Bacterial Infections/blood , Bacterial Infections/epidemiology , Biomarkers/blood , Female , Humans , Infant , Infant, Newborn , Logistic Models , Male , Prevalence , Prospective Studies , Risk Assessment , Sensitivity and Specificity , SyndromeABSTRACT
Resumen: Introducción: Un 20% de los niños con síndrome febril se presenta como síndrome febril sin foco (SFSF). Las es trategias de manejo en este grupo presentan alta sensibilidad, pero baja especificidad. Objetivos: Ca racterizar las infecciones bacterianas serias (IBS) en menores de 3 meses hospitalizados por SFSF, y evaluar utilidad de parámetros clínicos y de laboratorio en la identificación de pacientes con alto riesgo de IBS. Pacientes y Método: Estudio prospectivo en pacientes < 3 meses hospitalizados entre enero 2014 y noviembre 2015 por SFSF en dos hospitales pediátricos de la Región Metropolitana. Criterios de inclusión: edad 4 días - 3 meses, fiebre > 38°C de < 72 h de evolución sin causa demostra ble. Criterios de exclusión: uso de antimicrobianos hasta 7 días previo a su ingreso, prematuros < 34 semanas, peso de nacimiento < 2 kg e inmunocomprometidos. Se registraron datos demográficos, clínicos, y exámenes de laboratorio, hemograma y PCR, diagnóstico de egreso, IBS descartada, IBS probable o confirmada. Resultados: 32% de los pacientes egresó con diagnóstico de IBS, 28% con diagnóstico de infección viral o probablemente viral, 34% con diagnóstico de SFSF no especificado y 6% SFSF por otras causas. No se encontraron diferencias significativas en PCR, leucocitosis, aspecto tóxico ni horas de fiebre al ingreso al comparar los grupos con y sin IBS (p > 0,05). La combinación de parámetros clínicos y de laboratorio mostro sensibilidad de 27%, especificidad de 90%, VPP 60% y VPN 71%. Conclusión: No fue posible establecer que parámetros clínicos y de laboratorio permitan identificar menores de 3 meses con alto riesgo de IBS, manteniendo su utilidad como indicadores de bajo riesgo. Es necesario contar con otros elementos clínicos y de laboratorio que permitan discrimi nar IBS de infecciones virales.
Abstract: Introduction: In 20% of children with febrile syndrome, it appears as fever of unknown origin (FUO) syndrome. Management strategies in this group have high sensitivity but low specificity. Objectives: To cha racterize serious bacterial infections (SBI) in children younger than three months old hospitalized because of FUO syndrome and to evaluate the utility of clinical and laboratory parameters in the identification of patients that are at high risk of SBI. Patients and Method: Prospective study in patients aged < 3 months hospitalized due to FUO syndrome between January 2014 and November 2015 in two pediatric hospitals in the Metropolitan Region. Inclusion criteria: age 4 days - 3 months, fever > 38°C longer than 72 hours after onset without demonstrable cause. Exclusion criteria: anti microbial use up to 7 days before admission, preterm infants < 34 weeks, birth weight < 2 kg, and im munocompromised. Demographic, clinical, and laboratory tests data were recorded as well as blood count and CRP, discharge diagnosis, and ruled out, probable or confirmed SBI. Results: 32% of the patients were discharged with diagnosis of SBI, 28% with diagnosis of viral or probably viral infec tion, 34% with diagnosis of not specified FUO syndrome, and 6% due to other causes. There were no significant differences in the CRP value, altered WBCs count, toxic aspect, or hours of fever at the admission when comparing groups with and without SBI (p < 0.05). The combination of clinical and laboratory parameters showed 27% of sensitivity, 90% of specificity, 60% of PPV, and 71% of NPV. Conclusion: It was not possible to establish clinical and laboratory parameters that allow the identifi cation of children younger than 3 months old at high risk of SBI, however, they maintain their value as low risk indicators. It is necessary further investigation of other clinical and laboratory elements that allow discriminating SBI from viral infections.
Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Bacterial Infections/complications , Bacterial Infections/diagnosis , Severity of Illness Index , Fever of Unknown Origin/etiology , Clinical Decision Rules , Hospitalization , Syndrome , Bacterial Infections/blood , Bacterial Infections/epidemiology , Biomarkers/blood , Logistic Models , Prevalence , Prospective Studies , Sensitivity and Specificity , Risk AssessmentABSTRACT
RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggyBac-mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA (dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodies or hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection.
Subject(s)
Animals, Genetically Modified/virology , Bombyx/virology , Genes, Viral , Nucleopolyhedroviruses/genetics , Animals , Animals, Genetically Modified/immunology , Base Sequence , Blotting, Western , Bombyx/genetics , Bombyx/immunology , Cell Line , Gene Targeting , Molecular Sequence Data , Nucleopolyhedroviruses/physiology , Polymerase Chain Reaction , Pupa/genetics , Pupa/virology , RNA Interference , Transformation, Genetic , Transgenes , Viral Fusion Proteins/analysis , Viral Plaque AssayABSTRACT
Serial analysis of gene expression (SAGE) was used to examine the profile of expressed genes during embryonic development in the domesticated silkworm, Bombyx mori, after irradiation with Cobalt-60. A comparison of the SAGE sequence tags derived from irradiated embryos with those from normal embryos revealed 673 differentially expressed genes (P < 0.01 and at least three folds change). Of these, 292 genes were highly expressed in normal embryos and 381 genes were highly expressed in irradiated embryos. These results provide valuable information for understanding the mechanisms of radiation-induced changes in gene expression. In addition, it was noted that the generation of longer cDNA fragments from SAGE tags is an efficient way to identify genes, thereby facilitating the analysis of large numbers of unknown genes.
Subject(s)
Bombyx/genetics , Bombyx/radiation effects , Gene Expression Profiling , Gene Expression Regulation/radiation effects , Genes, Insect/genetics , Animals , Gene Library , Molecular Sequence DataABSTRACT
Background: There are a few studies examining the etiology of community acquired pneumonia (CAP) in Chile. Aim: To evaluate the etiology of CAP in hospitalized adults. Method: We prospectively studied 130 patients (mean age +/- SD: 68 +/- 18 y.o.; overall hospital mortality: 6.2 percent), over a 16 month period. Microbiological evaluation included blood and sputum cultures for bacteria; serology for C. pneumoniae, C. psittaci and M. pneumoniae; urine antigen for L. pneumophila; and nasopharyngeal swab for respiratory viruses. Results: Etiology was identified from 64 (49 percent) patients (two or more pathogens in 6). The most frequent microorganisms were S. pneumoniae (34 percent), Parainfluenza types 1 to 3 (22 percent), Influenza A or B (14 percent ), C. pneumoniae (6 percent), M. pneumoniae (6 percent), H. influenzae (5 percent) and S. marcescens (5 percent). Twenty-five of 27 (93 percent) respiratory viruses were identified in autumn or winter. Pneumococcal pneumonia patients (19) compared to those infected with respiratory virus (23) were younger (59 +/- 18 versus 72 +/- 17 y.o.; p = 0.021) and had less comorbidities (47 versus 87 percent; p = 0.0001). No patients with bacteremia (13 of 121: 11 percent) died. Conclusions: S. pneumoniae remains the most important pathogen to cover by initial antibiotic therapy; the second most frequent etiological agents were respiratory viruses followed by "atypical pathogens". Recommendations for the management of patients infected with these two last categories of agents should be included in future national guidelines.
Fundamento: Hay escasos estudios que examinen la etiología de la neumonía adquirida en la comunidad (NAC) en población adulta chilena. Objetivo: Identificar la etiología de la NAC en adultos inmunocompetentes hospitalizados. Método: Estudiamos, prospectiva y consecutivamente durante 16 meses, a 130 pacientes (edad promedio +/ - DS: 68 +/ - 18 años; letalidad en el hospital: 6,2 por ciento). La evaluación microbiológica incluyó cultivo de expectoración y hemocultivos para bacterias; Financiamiento: Fondo de Investigación de la Sociedad Chilena de Enfermedades Respiratorias (2002) y fondo de la Dirección de Investigación de la Pontificia Universidad Católica de Chile (DIPUC 2003/10E). Serología para C. pneumoniae, C. psittaci, M. pneumoniae; antígeno urinario para L. pneumophila e hisopado nasofaríngeo para virus respiratorios. Resultados: Se identificó la etiología en 64 (49 por ciento) pacientes (dos o más patógenos en 6). Los principales microorganismos fueron: S. pneumoniae (34 por ciento), virus Parainfluenza 1 a 3 (22 por ciento), virus Influenza A o B (14 por ciento), C. pneumoniae (6 por ciento), M. pneumoniae (6 por ciento), H. influenzae (5 por ciento) y S. marcescens (5 por ciento). El 93 por ciento (25/27) de los virus respiratorios se identificaron en otoño-invierno. Los pacientes con neumonía neumocócica (19) comparados con aquéllos infectados por virus respiratorios (23) eran más jóvenes (59 + /- 18 versus 72 +/ - 17 años; p = 0,021) y tenían menos comorbilidades (47 por ciento versus 87 por ciento; p = 0,0001). Ninguno de los 13 (11 por ciento) pacientes con bacteremia falleció en el hospital. Conclusiones: S. pneumoniae sigue siendo el principal patógeno a cubrir por el tratamiento antibiótico empírico; los virus respiratorios y los agentes atípicos fueron los que siguieron en frecuencia. Las futuras guías clínicas nacionales deberían incluir recomendaciones para el manejo de los pacientes infectados por estos dos últimos grupos de...
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Aged, 80 and over , Community-Acquired Infections/microbiology , Pneumonia/epidemiology , Pneumonia/microbiology , Anti-Bacterial Agents/therapeutic use , Gram-Negative Bacteria/isolation & purification , Gram-Positive Bacteria/isolation & purification , Chile/epidemiology , Hospitalization , Pneumonia/mortality , Pneumonia/drug therapy , Prospective Studies , Seasons , Viruses/isolation & purificationABSTRACT
There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.
Subject(s)
Body Patterning/physiology , Cilia/ultrastructure , DNA-Binding Proteins/physiology , Transcription Factors/physiology , Animals , Base Sequence , Body Patterning/genetics , DNA/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Dyneins/genetics , Female , Fetal Death/genetics , Gene Expression Regulation, Developmental , In Situ Hybridization, Fluorescence , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Scanning , Regulatory Factor X Transcription Factors , Regulatory Factor X1 , Transcription Factors/deficiency , Transcription Factors/geneticsABSTRACT
Here, we identified the main transactivator of fhx, the gene encoding the silk protein fibrohexamerin in posterior silk gland cells (PSG), as the homeotic SGF1/fork head factor. The same factor also stimulates sericin-1, another silk protein encoding gene, in the middle silk gland cells. SGF1/fork head is present in the silk gland nuclei during the whole course of larval life, but its binding to the fhx promoter occurs at intermolt and not during molt, when fhx is respectively turned on and off. The alternative binding of the factor is associated with specific changes in the fhx chromatin topology in PSG cells. Taken together, our results show that stabilization of SGF1/fork head to its target sequence is critical to promote fhx transcription at each intermolt. We also found that fhx is characterized by a PSG-specific DNase I hypersensitive site in the first intron, present during molt and intermolt, i.e. independent of the transcriptional status of the gene. All these data suggest that differential chromatin accessibility and fork head activation are crucial in controlling the spatial and temporal regulation of the fhx gene in the posterior silk gland cells.
Subject(s)
Fibroins/genetics , Glycoproteins/genetics , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Animals , Bombyx/genetics , Bombyx/growth & development , Bombyx/metabolism , Chromatin/metabolism , Forkhead Transcription Factors , Gene Expression , Larva/growth & development , Nuclear Proteins/genetics , Nucleosomes/metabolism , Peptides, Cyclic/genetics , Protein Binding , Sericins , Trans-Activators/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
The spermatozoon features an extremely condensed and inactive nucleus. The unique sperm chromatin organization is acquired during the late stages of spermatid differentiation by the replacement of somatic histones with sperm-specific chromosomal proteins. At fertilization, the inactive sperm nucleus must be rapidly transformed into a DNA replication competent male pronucleus before the formation of the zygote. The sequential events of this crucial process are well conserved among animals and are controlled by molecules present in the egg. We have previously identified a Drosophila maternal effect mutation called sésame, which specifically arrests male pronucleus formation at a late stage of chromatin decondensation. In this study, we show that sésame affects maternal histone incorporation in the male pronucleus, a situation that is expected to prevent nucleosomal organization of the paternal chromatin. As an apparent consequence, the male pronucleus is arrested before the first S-phase and does not condense mitotic chromosomes. However, centromeric heterochromatin is present on paternal centromeres, which occasionally interact with microtubules. The abnormal chromatin organization of the male pronucleus does not prevent the formation of a male pronuclear envelope, which breaks down and reassembles in synchrony with maternally derived nuclei present in the same cytoplasm.
Subject(s)
Chromatin/genetics , Chromatin/metabolism , Chromatin/physiology , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Drosophila/genetics , Fertilization , Spermatozoa/physiology , Animals , Basement Membrane/metabolism , Cell Cycle , Cell Cycle Proteins , Cell Nucleus/metabolism , Centromere/metabolism , Centromere Protein A , Chromosomal Proteins, Non-Histone , Cytoplasm/metabolism , DNA Polymerase I/metabolism , DNA-Binding Proteins , Epitopes , Female , Heterochromatin/metabolism , Histone Chaperones , Histones/biosynthesis , Male , Microscopy, Confocal , Microscopy, Fluorescence , Mitosis , Mothers , Mutation , Proliferating Cell Nuclear Antigen/biosynthesis , S Phase , Testis/metabolism , Transcription FactorsABSTRACT
The Lepidopteran densovirus-derived vector, pJlacZDeltaNS3, is a defective virus genome with an insertion of lacZ DNA in the viral structural protein coding sequence, and a deletion of the sequence coding the non-structural polypeptide NS3. pJlacZDeltaNS3 was injected into Drosophila eggs and the maintenance of the viral genome was monitored by expression of beta-galactosidase and by Southern blot hybridizations. Intense beta-galactosidase activity was observed in many somatic tissues of third-instar larvae and adult flies, in more than 60% of the injected animals. DNA analyses showed that staining in adult tissues correlated with the amplification of the vector. Together, these results suggest the occurrence of early events of integration of the vector into the Drosophila host genome.
Subject(s)
Densovirus/genetics , Genetic Vectors/genetics , Animals , Blotting, Southern/methods , DNA/analysis , Drosophila , Gene Amplification , Gene Expression , Larva , beta-Galactosidase/geneticsABSTRACT
We report the expression pattern of a Drosophila transcription factor, Drosophila regulatory factor X (dRFX), which belongs to the RFX winged-helix transcription factor family. dRFX is distributed in type I sensory neuron lineage of the peripheral nervous system throughout Drosophila development and thus represents the first described type I lineage characteristic marker in Drosophila. In addition, dRFX is also detected in the brain throughout development and in spermatids in adult flies.
Subject(s)
Brain/embryology , Brain/metabolism , DNA-Binding Proteins/biosynthesis , Drosophila/embryology , Neurons/metabolism , Spermatids/metabolism , Transcription Factors/biosynthesis , Animals , Cell Lineage , Gene Expression , Immunohistochemistry , In Situ Hybridization , Male , Nervous System/embryology , Regulatory Factor X Transcription Factors , Time FactorsABSTRACT
maternal haploid (mh) is a strict maternal effect mutation that causes the production of haploid gynogenetic embryos (eggs are fertilized but only maternal chromosomes participate in development). We conducted a cytological analysis of fertilization and early development in mh eggs to elucidate the mechanism of paternal chromosome elimination. In mh eggs, as in wild-type eggs, male and female pronuclei migrate and appose, the first mitotic spindle forms, and both parental sets of chromosomes congress on the metaphase plate. In contrast to control eggs, mh paternal sister chromatids fail to separate in anaphase of the first division. As a consequence the paternal chromatin stretches and forms a bridge in telophase. During the first three embryonic divisions, damaged paternal chromosomes are progressively eliminated from the spindles that organize around maternal chromosomes. A majority of mh embryos do not survive the deleterious presence of aneuploid nuclei and rapidly arrest their development. The rest of mh embryos develop as haploid gynogenetic embryos and die before hatching. The mh phenotype is highly reminiscent of the early developmental defects observed in eggs fertilized by ms(3)K81 mutant males and in eggs produced in incompatible crosses of Drosophila harboring the endosymbiont bacteria Wolbachia.
Subject(s)
Cell Nucleus/metabolism , Chromosomes/metabolism , Mitosis , Zygote/physiology , Aneuploidy , Animals , Cell Movement , Cell Nucleus/ultrastructure , Chromosomes/ultrastructure , Drosophila , Female , Fertilization/physiology , Haploidy , Heterochromatin/physiology , Male , Microscopy, Confocal , Mutation , Phenotype , Sex Factors , Time Factors , Zygote/ultrastructureABSTRACT
After entering the oocyte and before the formation of the diploid zygote, the sperm nucleus is transformed into a male pronucleus, a process that involves a series of conserved steps in sexually reproducing animals. Notably, a major modification of the male gamete lies in the decondensation of the highly compact sperm chromatin. We present here the phenotype of sésame (ssm), a maternal effect mutation which affects the formation of the male pronucleus in Drosophila melanogaster. Homozygous ssm(185b) females produce haploid embryos which develop with only the maternally derived chromosomes. These haploid embryos die at the end of embryogenesis. Cytological analyses of the fertilization in eggs laid by ssm(185b) mutant females showed that both pronuclear migration and pronuclear apposition occurred normally. However, a dramatic alteration of the male pronucleus by which its chromatin failed to fully decondense was systematically observed. Consequently, the affected male pronucleus does not enter the first mitotic spindle, which is organized around only the maternally derived chromosomes. Immunodetection of lamina antigens indicates that a male pronuclear envelope is able to form around the partially decondensed paternal chromatin. This suggests that the maternally provided sésame(+) function is required for a late stage of sperm chromatin remodeling.
Subject(s)
Cell Nucleus/physiology , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Embryo, Nonmammalian/physiology , Genomic Imprinting , Mutation , X Chromosome , Zygote/physiology , Animals , Blastoderm/physiology , Chromosome Mapping , Diploidy , Embryo, Nonmammalian/cytology , Female , Gastrula/physiology , Male , Y ChromosomeABSTRACT
The RFX family of transcription factors is characterized by a unique DNA binding domain. Five genes have been isolated in mammals, one gene in Caenorhabditis elegans and in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Whereas the roles of the RFX genes are beginning to be understood in yeasts, no clear function has been reported in multicellular organisms, except for RFX5, the most divergent member of the family. To study the physiological role of RFX transcription factors using an alternative multicellular model, we report the isolation and characterization of the Drosophila RFX gene (dRFX). The fruit fly protein shares highly conserved domains with the mammalian factors RFX1 to 3 and is more closely related to this subgroup. It binds DNA with the same target specificity as mammalian factors RFX1 to 3. dRFX is located on chromosome III and we characterized the entire locus. dRFX expression was analyzed during embryogenesis. dRFX mRNAs are detected only in the peripheral nervous system and in the brain of the embryo.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA-Binding Proteins/metabolism , Drosophila/embryology , Embryo, Nonmammalian/metabolism , Exons , Gene Expression Regulation, Developmental , Genes, Insect/genetics , In Situ Hybridization , Introns , Molecular Sequence Data , Regulatory Factor X Transcription Factors , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription Factors/metabolism , VertebratesABSTRACT
We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.
Subject(s)
Bombyx/genetics , DNA Transposable Elements/genetics , Genetic Vectors/genetics , Germ-Line Mutation/genetics , Transformation, Genetic/genetics , Actins/genetics , Aging/metabolism , Animals , Animals, Genetically Modified , Base Sequence , Bombyx/embryology , Bombyx/growth & development , Bombyx/metabolism , Crosses, Genetic , Female , Green Fluorescent Proteins , Larva/metabolism , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Male , Moths/enzymology , Moths/genetics , Mutagenesis, Site-Directed/genetics , Promoter Regions, Genetic/genetics , Pupa/metabolism , Recombination, Genetic/genetics , Sequence Analysis, DNA , Terminal Repeat Sequences/genetics , Transgenes/genetics , Transposases/genetics , Transposases/metabolismABSTRACT
Tooth organogenesis is dependent on reciprocal and sequential epithelial-mesenchymal interactions and is marked by the appearance of phenotypic matrix macromolecules in both dentin and enamel. The organic matrix of enamel is composed of amelogenins, ameloblastin/amelin, enamelins and tuftelin. Dentin is mainly composed of type I collagen, but its specificity arises from the nature of the non-collagenous proteins (NCPs) involved in mineralization, phosphophoryn (DPP), dentin sialoprotein (DSP), osteocalcin, bone sialoprotein and dentin matrix protein-1 (Dmp1). In this paper, we studied the pattern of expression of four mineralizing protein genes (type I collagen, amelogenin, DSPP and osteocalcin) during the development of rat teeth by in situ hybridization on serial sections. For this purpose, we used an easy and rapid procedure to prepare highly-specific labeled single-stranded DNA probes using asymmetric polymerase chain reaction (PCR). Our results show that type I collagen is primarily expressed in polarizing odontoblasts, followed by the osteocalcin gene expression in the same polarized cells. Concomitantly, polarized ameloblasts start to accumulate amelogenin mRNAs and transiently express the DSPP gene. This latter expression switches over to odontoblasts whereas mineralization occurs. At the same time, osteocalcin gene expression decreases in secretory odontoblasts. Osteocalcin may thus act as an inhibitor of mineralization whereas DSP/DPP would be involved in more advanced steps of mineralization. Amelogenin and type I collagen gene expression increases during dentin mineralization. Their expression is spatially and temporally controlled, in relation with the biological role of their cognate proteins in epithelial-mesenchymal interactions and mineralization.
Subject(s)
Extracellular Matrix Proteins/genetics , Gene Expression Regulation, Developmental , Tooth/metabolism , Amelogenin , Animals , Animals, Newborn , Collagen/genetics , Dental Enamel Proteins/genetics , In Situ Hybridization , Osteocalcin/genetics , Phosphoproteins/genetics , Protein Precursors , RNA, Messenger , Rats , Rats, Sprague-Dawley , Sialoglycoproteins/genetics , Tooth/embryologyABSTRACT
The gene encoding the silk protein P25 in Bombyx mori is expressed in the posterior silk gland (PSG) cells and repressed in the middle silk gland (MSG) cells. To identify the factors involved in this transcription-dependent spatial restriction, we examined the P25 chromatin in PSG and MSG nuclei by DNase I-aided ligation-mediated PCR and analyzed the expression of various P25-lacZ constructs in biolistically treated silk glands. P25 promoter activation depends on two cis-acting elements. One coincides with the target sequence of SGFB, a silk gland-specific factor present in all silk gland nuclei, but bound to its target DNA sequence in only PSG cells. The interaction of the other element with a factor that we named PSGF is also exclusive to PSG cells. Placed ahead of a non-P25-related basal promoter, the SGFB and PSGF elements are sufficient to drive posterior-cell transcription. Collectively, our data support the hypothesis that the spatial restriction of P25 expression is driven by the stabilization of SGFB onto its target sequence by the action of PSGF.
Subject(s)
Bombyx/genetics , Chromatin/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Insect Proteins/genetics , Animals , Base Sequence , DNA/metabolism , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins/analysis , Sequence DeletionABSTRACT
To identify the functional regulatory elements of the promoter of the cytoplasmic actin A3 gene in Bombyx mori, transient expression of A3-LacZ mutants was assayed in cultured Lepidoptera cells. This led to the recognition of two proximal and contiguous domains exerting strong negative and positive effects, respectively on promoter activity. The negative region contains a ten-base-pair sequence that binds Bombyx silk gland cell nuclear proteins in vitro. The positive regulatory element was identified as a serum response element (SRE) by its sequence, and its in vitro binding properties. Moreover, structural analysis of posterior and median silk gland cell chromatin by dimethyl sulfate-aided LMPCR revealed that SRE is bound to its cognate factor in situ, in most, if not all, the approximately 100,000 A3 copies of the polyploid DNA stock. The regulation of the A3 promoter in the silk gland would thus result from the combined action of these two antagonist factors.
Subject(s)
Actins/genetics , Bombyx/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , Nuclear Proteins/metabolism , Transcription, Genetic , Actins/metabolism , Animals , Base Sequence , Binding Sites , Binding, Competitive , Cells, Cultured , Chromatin/genetics , Cytoplasm/genetics , DNA Footprinting , Exocrine Glands/cytology , Exocrine Glands/metabolism , Gene Expression Regulation , Genes, Insect , Molecular Sequence Data , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Serum Response Factor , Spodoptera/cytology , TransfectionABSTRACT
By screening cDNA and genomic libraries, we have cloned A4, the fourth and last actin gene of Bombyx mori, which encodes a typical cytoskeleton actin and is expressed in all larval tissues. A4 is closely related to A3, another cytoplasmic actin gene of the silkworm, in its encoded amino-acid sequence, and the location as well as the sequence of a single intron. Both A3 and A4 have possibly arisen from the recent duplication of an intron-containing ancestral gene. The two genes display different organization of their 5' untranslated and flanking sequences. In contrast to A3, which harbours a single promoter, A4 exhibits two leader exons transcribed by the use of alternative promoters. A3 and A4 actins differ only by two amino acids at positions known to vary among cytoplasmic actins of other species, and are likely to be functionally equivalent. We speculate that transcriptional constraints are actually the target of a selective pressure that maintains two distinct cytoplasmic actin genes in insects, as well as in other animals.
Subject(s)
Actins/genetics , Bombyx/genetics , Gene Expression Regulation, Developmental/physiology , Promoter Regions, Genetic/genetics , Amino Acid Sequence , Amino Acids/genetics , Animals , Base Sequence , Cloning, Molecular , Cytoplasm , Exons/genetics , Genes, Insect/genetics , Genetic Variation , Molecular Sequence Data , Multigene Family/genetics , Phylogeny , RNA, Messenger/analysisABSTRACT
The gene encoding the silk protein P25 is specifically transcribed in the posterior silkgland of Bombyx during larval intermoults and is repressed during moults. By performing in vitro DNA-protein interactions, at least five putative regulatory elements were localized in the 5' flanking region of the gene. The most proximal element, close to the TATA box, interacts with SGFB, a silkgland-specific factor which could be involved in the tissue-specific expression of the gene. A more upstream sequence is recognized by an ubiquitous factor, BMFA, which exhibits cyclical modifications in relation to the moulting cycle and could thus be involved in the temporal control of the gene during the development. A construct containing a reporter gene fused to 1450 bp of P25 5' flanking sequences was integrated into the Drosophila genome and shown to be specifically expressed in the larval salivary gland, the organ homologous to the silkgland. Recurrent deletions of this construct showed that the proximal 254 bp contain all the sequences required for this specific expression. Similar foreign constructs introduced in the silkgland in vivo by a particle delivery system were specifically transcribed in the posterior silkgland but remained silent in the middle silkgland as the endogenous genes. This methodology will be used to assay the function of the defined cis-acting elements in the spatial regulation of expression of P25.
Subject(s)
Bombyx/genetics , Insect Proteins , Transcription, Genetic/genetics , Animals , Animals, Genetically Modified , Base Sequence , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila , Gene Expression Regulation/genetics , Larva/genetics , Microinjections , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of non-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75NGFR and p140prototrk) on responsive cells. The continuously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF, proNGF and p75NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75NGFR was correlated with differentiation event only in dental mesenchyme: polarizing odontoblasts expressed p75NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.
