RESUMO
The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay.
Assuntos
Indutores da Angiogênese/metabolismo , Meios de Cultivo Condicionados/farmacologia , Expressão Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Regeneração Óssea/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/farmacologia , Meios de Cultivo Condicionados/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Expressão Gênica/genética , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Interleucina-8/farmacologia , Células-Tronco Mesenquimais/citologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The nucleotide sequence of five genes from the major Bacillus subtilis chemotaxis locus has been determined. Four of these genes encode proteins that are homologous to the Salmonella typhimurium FlgB, FlgC, FlgG and FliF proteins. One gene encodes a protein that is homologous to the Escherichia coli FliE protein. The data from S. typhimurium and E. coli suggest that all of these proteins form part of the hook-basal body (HBB) complex of the bacterial flagella. The FlgB, FlgC and FlgG proteins are components of the proximal and distal rods. The FliF protein forms the M-ring that anchors the rod assembly to the membrane. The role of the FliE protein within the HBB complex has not yet been determined. The similarity between the B. subtilis and S. typhimurium proteins suggests that the structure of the M-ring and the rod may be similar in the two species. However, we observed some differences in size and amino acid composition between some of the corresponding homologues that suggest the basal body proteins may be organized slightly differently within B. subtilis.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Fases de Leitura Aberta , Mapeamento por Restrição , Salmonella typhimurium/genética , Homologia de Sequência do Ácido NucleicoRESUMO
The effects of TGF-beta on expression of the platelet-derived growth factor-induced KC protein were explored in mouse mesenchymal C3H10T1/2 and pre-osteoblastic MC3T3-E1 cells to identify a potential role for TGF-beta in expression of angiogenic cytokines during osteogenic differentiation. KC is a member of the CXC chemokine family with homology to human IL-8, a potent neutrophilic chemotactic cytokine. TGF-beta treatment results in increased KC mRNA and protein secretion in C3H10T1/2 induced towards the osteoblastic lineage with all-trans-retinoic acid. This is due to up-regulated transcription rather than enhanced mRNA stability. No induction of KC expression was seen in untreated C3H10T1/2 or MC3T3-E1 upon TGF-beta stimulation. Use of the translational inhibitor cycloheximide results in mRNA "superinduction" suggesting other factors are involved that normally function to down-regulate KC expression. TGF-beta-stimulated conditioned media were a potent chemostimulant for human microvascular endothelial cells (HMEC-1). This activity could be inhibited by pre-incubation with anti-KC neutralizing antibodies.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Células Cultivadas , Quimiocina CXCL1 , Quimiocinas , Quimiocinas CXC , Quimiotaxia/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
The Bacillus subtilis gene encoding FliY has been cloned and sequenced. The gene encodes a 379-amino-acid protein with a predicted molecular mass of 41,054 daltons. FliY is partly homologous to the Escherichia coli and Salmonella typhimurium switch proteins FliM and FliN. The N-terminus of FliY has 33% identity with the first 122 amino acids of FliM, whereas the C-terminus of FliY has 52% identity with the last 30 amino acids of FliN. The middle 60% of FliY is not significantly homologous to either of the proteins. A fliY::cat null mutant has no flagella. Motility can be restored to the mutant by expression of fliY from a plasmid, although chemotaxis is still defective since the strain exhibits smooth swimming behaviour. fliY::cat is in the cheD complementation group. One of the cheD point mutants does not switch although the population grown from a single cell has both smooth swimming and tumbling bacteria, implying that the switch is locked. Expression of fliY in wild-type B. subtilis makes the cells more smooth-swimming but does not appear to affect chemotaxis. Expression of fliY in wild-type S. typhimurium severely inhibits chemotaxis and also makes the cells smooth swimming. Expression in a non-motile S. typhimurium fliN mutant restores motility but not chemotaxis, although expression in a non-motile E. coli fliM mutant does not restore motility. The homology, multiple phenotypes, and interspecies complementation suggest that FliY forms part of the B. subtilis switch complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Flagelos/metabolismo , Genes Bacterianos , Proteínas de Membrana , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Quimiotaxia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Salmonella typhimurium/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
In Escherichia coli, chemotactic sensory transduction is believed to involve phosphoryl transfer for excitation, and changes in receptor methylation for adaptation. In Bacillus subtilis, changes in degree of receptor methylation do not bring about adaptation. Novel methylation reactions are believed to be involved in excitation in B. subtilis. The main chemotaxis proteins of E. coli--CheA, CheB, CheR, CheW and CheY--are present in B. subtilis but play somewhat different roles in the two organisms. Several unique chemotaxis proteins are also present in B. subtilis. Some of the properties of B. subtilis chemotaxis are also seen in Halobacterium halobium, suggesting that there may be a similar underlying mechanism that predates the evolutionary separation of the bacteria from the archaea and eucarya.
Assuntos
Bacillus subtilis/fisiologia , Quimiotaxia/fisiologia , Escherichia coli/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Bactérias/metabolismo , Fatores Quimiotáticos/metabolismo , Genes Bacterianos/genética , MetilaçãoRESUMO
The Bacillus subtilis gene encoding CheB, which is homologous to Escherichia coli CheY, the regulator of flagellar rotation, has been cloned and sequenced. It has been verified, using a phage T7 expression system, by showing that a small protein, the same size as E. coli CheY, is actually made from this DNA. Despite the fact that the two proteins are 36% identical, with many highly conserved residues, they appear to play different roles. Unlike CheY null mutants, which swim smoothly, CheB null mutants tumble incessantly. However, a CheB point mutant swims smoothly, even in the presence of a plasmid bearing cheB, which restores the null mutants to wild type. Expression of CheB in wild type B. subtilis makes the cells exhibit more tumbling. Since both absence of CheB and presence of high levels of CheB cause tumbling, CheB appears to be required, in certain circumstances, for both smooth swimming and tumbling. Expression in wild type E. coli makes the cells smooth swimmers and strongly inhibits chemotaxis.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Quimiotaxia , Genes Bacterianos , Proteínas de Membrana/genética , Sequência de Aminoácidos , Bacillus subtilis/fisiologia , Sequência de Bases , Southern Blotting , DNA Bacteriano/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Proteínas Quimiotáticas Aceptoras de Metil , Dados de Sequência Molecular , Mutação , Plasmídeos , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J. M. Slavicek, N. Hayes-Plazolles, and M. E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants exhibited the traits of few polyhedra (FP) mutants; however, no large DNA insertions or deletions that correlated with the appearance of the FP phenotype were found. In this study, we have characterized several of the PF mutants at the phenotypic and genetic levels. Genetic techniques were used to group the mutations in the LdMNPV PF mutants to the same or closely linked genes. Wild-type viruses were recovered after coinfection of L. dispar 652Y cells with certain combinations of PF mutants. These viruses were analyzed by restriction endonuclease analysis and found to be chimeras of the original PF mutants used in the coinfections. Marker rescue experiments localized the mutations in one group of PF isolates to the region containing the LdMNPV 25K FP gene. The mutations in these PF mutants were identified. Four of five of the LdMNPV FP mutants contain small insertions or deletions within the 25K FP gene. The fifth LdMNPV FP mutant analyzed contained a large deletion that truncated the C terminus of the 25K FP gene product. All of the deletions occurred within the same potential hairpin loop structure, which had the lowest free energy value (most stable hairpin) of the five potential hairpin loop structures present in the 25K FP gene. One of the insertion mutants contained an extra base within a repetitive sequence. These types of mutations are likely caused by errors that occur during DNA replication. The relationship between the types of mutations found within the LdMNPV 25K FP gene and DNA replication-based mutagenesis is discussed.
Assuntos
Replicação do DNA/genética , DNA Viral/genética , Genes Virais , Insetos/virologia , Nucleopoliedrovírus/genética , Animais , Sequência de Bases , Dados de Sequência Molecular , MutaçãoRESUMO
The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding vPK has been cloned and sequenced. LdMNPV vPK shows a 24% amino acid identity to the catalytic domains of the eucaryotic protein kinases nPKC from rabbits, HSPKCE from humans, APLPKCB from Aplysia californica, and dPKC98F from Drosophila melanogaster, and homology to several other protein kinases from yeasts, mice, and bovines. The homology suggests that vPK is a serine/threonine protein kinase as defined by Hanks et al. (S.K. Hanks, A.M. Quinn, and T. Hunter, Science 241:42-52, 1988). Temporal expression studies indicate that vPK is expressed throughout the infection cycle beginning at 4 h postinfection, first as a delayed-early gene and subsequently as a late gene. Sequence analysis and primer extension reactions confirm the presence of distinct early and late transcription initiation regions. Expression of vPK with a rabbit reticulocyte system generated a 31-kDa protein, which is in close agreement with the predicted size of 32 kDa from the amino acid sequence. Phosphorylation activity of in vitro-expressed vPK was demonstrated by using calf thymus histones.
Assuntos
Genes Virais/genética , Mariposas/microbiologia , Nucleopoliedrovírus/genética , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Genoma Viral , Histonas/metabolismo , Dados de Sequência Molecular , Nucleopoliedrovírus/enzimologia , Fosforilação , Proteína Quinase C/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) gene has been identified that encodes a homolog to the granulovirus (GV) enhancin proteins that are capable of enhancing the infection of other baculoviruses. Enhancin genes have been identified and sequenced for three species of GVs but have not been found in any other nuclear polyhedrosis virus to date. The LdMNPV enhancin gene is located between 67.6 and 70.1 kbp on the viral genome. Northern and primer extension analyses of viral RNAs indicate that the enhancin gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The LdMNPV enhancin exhibits 29% amino acid identity to the enhancin proteins of the Trichoplusia ni, Pseudaletia unipuncta, and Helicoverpa armigera GVs. All four proteins contain a conserved zinc-binding domain characteristic of metalloproteases. A recombinant virus (enhancin::cat) was constructed in which the LdMNPV enhancin gene was inactivated by insertion mutagenesis in order to ascertain the effect of the enhancin protein on viral potency. The bioassay results indicate that disruption of the enhancin gene in the LdMNPV results in a reduction in viral potency.
Assuntos
Genes Virais , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Viral/genética , Regulação Viral da Expressão Gênica , Dados de Sequência Molecular , Mutagênese Insercional , Filogenia , Biossíntese de Proteínas , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The Lymantria dispar nucleopolyhedrovirus (LdMNPV) gene encoding the 25K FP protein has been cloned and sequenced. The 25K FP gene codes for a 217 amino acid protein with a predicted molecular mass of 24870 Da. Expression of the 25K FP protein in a rabbit reticulocyte system generated a 27 kDa protein, in close agreement with the molecular mass predicted from the nucleotide sequence. The gene is located between 40.3 and 40.8 map units on the viral genome. It is transcribed in a counterclockwise direction with respect to the circular map at late times during the infection cycle from a consensus baculovirus late promoter. The LdMNPV and Autographa californica nucleopolyhedrovirus (AcMNPV) 25K FP proteins exhibit 52% amino acid identity with several regions showing greater than 75% identity. Homologues to the AcMNPV orf59 and orf60 were also identified upstream (with respect to the genome) of the 25K FP gene in LdMNPV and exhibit 52% and 45% amino acid identity, respectively.
Assuntos
Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Cosmídeos , Genes Virais , Marcadores Genéticos , Dados de Sequência Molecular , Mariposas , Mutação , Biossíntese de Proteínas , Coelhos , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding G22 was cloned and sequenced. The G22 gene codes for a 191 amino acid protein with a predicted M(r) of 22,000. Expression of G22 in a rabbit reticulocyte system generated a protein with an M(r) of 24,000, in close agreement with the molecular mass predicted from the nucleotide sequence. G22 is not significantly homologous to any known protein, nor is a G22 homologue present in the Autographa californica MNPV (AcMNPV). Temporal expression studies indicated that the G22 gene was transcribed at readily detectable levels in the presence of cycloheximide. Transcripts were detected immediately after the virus adsorption period and throughout the infection cycle. The early transcriptional start sites of G22 map to a sequence that resembles a subset of RNA polymerase II promoters/start sites that are found upstream of Drosophila melanogaster developmental and retrotransposon genes which lack TATA box motifs. Several consensus late baculovirus promoter/mRNA start site sequences (ATAAG) were identified upstream of the G22 gene start codon.
Assuntos
Genes Precoces/genética , Genes Virais/genética , Mariposas/virologia , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Livre de Células , Células Cultivadas , Clonagem Molecular , Cicloeximida/farmacologia , Regulação Viral da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Viral/biossíntese , RNA Viral/genética , Coelhos , Mapeamento por Restrição , Análise de Sequência de DNA , Proteínas Virais/químicaRESUMO
Three genes from the Bacillus subtilis major che-fla operon have been cloned and sequenced. Two of the genes encode proteins that are homologous to the Escherichia coli and Salmonella typhimurium flagellar biosynthetic proteins FliP and FliQ. The third gene, designated fliZ, encodes a 219-amino-acid protein with a predicted molecular mass of 24,872 Da. FliZ is not significantly homologous to any known proteins. Null mutants in fliP and fliZ do not have flagella; however, motility can be restored to the fliZ null mutant by expression of fliZ from a plasmid. FliZ has a conventional N-terminal signal sequence that does not direct secretion of the protein but appears to target the protein to the membrane. Two possible models of insertion of FliZ into the membrane are described.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Flagelos/metabolismo , Proteínas de Membrana , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Southern Blotting , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação/genética , Óperon/genética , Plasmídeos/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Enhancins are a group of proteins first identified in granuloviruses (GV) that have the ability to enhance nuclear polyhedrosis virus potency. We had previously identified an enhancin gene (E1) in the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) (D. S. Bischoff and J. M. Slavicek, J. Virol. 71:8133-8140, 1997). Inactivation of the E1 gene product within the viral genome lowered viral potency by an average of 2.9-fold. A second enhancin gene (E2) was identified when the entire genome of LdMNPV was sequenced (Kuzio et al., Virology 253:17-34, 1999). The E2 protein exhibits approximately 30% amino acid identity to the LdMNPV E1 protein as well as the enhancins from Trichoplusia ni GV, Pseudaletia unipuncta GV, Helicoverpa armigera GV, and Xestia c-nigrum GV. Northern analysis of viral RNA indicated that the E2 gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The effect of the enhancin proteins on viral potency was investigated through bioassay using two recombinant viruses, one with a deletion in the E2 gene (E2del) and a second with deletion mutations in both enhancin genes (E1delE2del). The enhancin gene viral constructs were verified by Southern analysis and shown not to produce enhancin gene transcripts by Northern analysis. The E2del virus exhibited an average decrease in viral potency of 1.8-fold compared to wild-type virus. In the same bioassays, the recombinant virus E1cat, which does not produce an E1 gene transcript, exhibited an average decrease in viral potency of 2.3-fold compared to control virus. The E1delE2del virus exhibited an average decrease in viral potency of 12-fold compared to wild-type virus. Collectively, these results suggest that both LdMNPV enhancin genes contribute to viral potency, that each enhancin protein can partially compensate for the lack of the other protein, and that both enhancin genes are necessary for wild-type viral potency.
Assuntos
Genes Virais/fisiologia , Mariposas/virologia , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Bioensaio , Dados de Sequência Molecular , Nucleopoliedrovírus/fisiologia , RNA Viral/análise , Recombinação Genética , Homologia de Sequência de AminoácidosRESUMO
The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Sequência de Aminoácidos , Bacillus subtilis/fisiologia , Sequência de Bases , Movimento Celular , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Teste de Complementação Genética , Genótipo , Dados de Sequência Molecular , Plasmídeos , Conformação Proteica , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transformação BacterianaRESUMO
Amino acid sequence comparison suggests that numerous proteins are common to the signal transduction pathways controlling chemotaxis in Bacillus subtilis and Escherichia coli. However, previous work has indicated several differences between the two systems. We have undertaken a comparative study of the roles of the CheY protein in chemotaxis by B. subtilis and E. coli. Although CheY from the two species share only 36% amino acid sequence identity, purified B. subtilis CheY was phosphorylated in vitro by E. coli CheA, and dephosphorylation of CheY-P was enhanced by E. coli CheZ. Alteration of the putative site of phosphorylation in B. subtilis CheY, Asp54, eliminated chemotaxis in vivo, further confirming that phosphorylation is important for B. subtilis chemotaxis. Loss of CheY function resulted in tumbling behavior in B. subtilis. Introduction of positively charged residues in place of Asp10 of B. subtilis CheY abolished function, whereas the corresponding changes in E. coli CheY apparently result in constitutive activation. The B. subtilis CheY Asp10 mutant proteins also failed to cause tumbling in E. coli, consistent with a different interaction between CheY and the flagellar switch in the two species. Finally, B. subtilis adapted more rapidly to positive stimuli than negative stimuli, whereas the opposite is true of E. coli. We conclude that B. subtilis regulates its response to positive chemotactic stimuli by enhancing phosphorylation of chemotaxis proteins, whereas E. coli reduces phosphorylation in the same circumstance.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias , Quimiotaxia/genética , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Bacillus subtilis/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Genes Bacterianos/genética , Genes de Troca/genética , Histidina Quinase , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Quimiotáticas Aceptoras de Metil , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in the polyhedrin or 25K FP gene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in the polyhedrin gene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include the polyhedrin or 25K FP genes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.