RESUMO
OBJECTIVE: In humans, the ontogeny of obesity throughout the life course and the genetics underlying it has been historically difficult to study. We compared, in a non-human primate model, the lifelong growth trajectories of obese and non-obese adults to assess the heritability of and map potential genomic regions implicated in growth and obesity. STUDY POPULATION: A total of 905 African green monkeys, or vervets (Chlorocebus aethiops sabaeus) (472 females, 433 males) from a pedigreed captive colony. METHODS: We measured fasted body weight (BW), crown-to-rump length (CRL), body-mass index (BMI) and waist circumference (WC) from 2000 to 2015. We used a longitudinal clustering algorithm to detect obesogenic growth, and logistic growth curves implemented in nonlinear mixed effects models to estimate three growth parameters. We used maximum likelihood variance decomposition methods to estimate the genetic contributions to obesity-related traits and growth parameters, including a test for the effects of a calorie-restricted dietary intervention. We used multipoint linkage analysis to map implicated genomic regions. RESULTS: All measurements were significantly influenced by sex, and with the exception of WC, also influenced by maternal and post-natal diet. Chronic obesity outcomes were significantly associated with a pattern of extended growth duration with slow growth rates for BW. After accounting for environmental influences, all measurements were found to have a significant genetic component to variability. Linkage analysis revealed several regions suggested to be linked to obesity-related traits that are also implicated in human obesity and metabolic disorders. CONCLUSIONS: As in humans, growth patterns in vervets have a significant impact on adult obesity and are largely under genetic control with some evidence for maternal and dietary programming. These results largely mirror findings from human research, but reflect shorter developmental periods, suggesting that the vervet offers a strong genetic model for elucidating the ontogeny of human obesity.
Assuntos
Peso Corporal/fisiologia , Chlorocebus aethiops/crescimento & desenvolvimento , Chlorocebus aethiops/fisiologia , Dieta , Obesidade/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Circunferência da Cintura/fisiologiaRESUMO
We determined the genome sequence of the type strain of Helicobacter canadensis, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places H. canadensis close to Wolinella succinogenes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Helicobacter/genética , Análise de Sequência de DNA , Animais , Infecções por Helicobacter/microbiologia , Humanos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência , Wolinella/genéticaRESUMO
The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.
Assuntos
Genoma Bacteriano , Análise de Sequência de DNA , Treponema pallidum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Grupo Borrelia Burgdorferi/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Reparo do DNA/genética , Replicação do DNA/genética , Enzimas de Restrição do DNA/genética , Metabolismo Energético/genética , Genes Bacterianos , Genes Reguladores , Resposta ao Choque Térmico/genética , Lipoproteínas/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Movimento , Fases de Leitura Aberta , Consumo de Oxigênio/genética , Biossíntese de Proteínas , Recombinação Genética , Origem de Replicação , Transcrição Gênica , Treponema pallidum/metabolismo , Treponema pallidum/patogenicidadeRESUMO
The rat genome project and the resources that it has generated are transforming the translation of rat biology to human medicine. The rat genome was sequenced to a high quality "draft," the structure and location of the genes were predicted, and a global assessment was published (Gibbs RA et al., Nature 428: 493-521, 2004). Since that time, researchers have made use of the genome sequence and annotations and related resources. We take this opportunity to review the currently available rat genome resources and to discuss the progress and future plans for the rat genome.
Assuntos
Genoma , Genômica , Ratos/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Feminino , Biblioteca Genômica , Genômica/organização & administração , Humanos , Camundongos , National Heart, Lung, and Blood Institute (U.S.) , National Human Genome Research Institute (U.S.) , Polimorfismo de Nucleotídeo Único , Estados UnidosRESUMO
The genome of the honeybee fungal pathogen Ascosphaera apis (Maassen) encodes three putative high mobility group (HMG-box) transcription factors. The predicted proteins (MAT1-2, STE11 and HTF), each of which contain a single strongly conserved HMG-box, exhibit high similarity to mating type proteins and STE11-like transcription factors previously identified in other ascomycete fungi, some of them important plant and human pathogens. In this study we characterized the A. apis HMG-box containing genes and analyzed the structure of the mating type locus (MAT1-2) and its flanking regions. The MAT1-2 locus contains a single gene encoding a protein with an HMG-box. We also have determined the transcriptional patterns of all three HMG-box containing genes in both mating type idiomorphs and discuss a potential role of these transcription factors in A. apis development and reproduction. A multiplex PCR method with primers amplifying mat1-2-1 and Ste11 gene fragments is described. This new method allows for identification of a single mating type idiomorph and might become an essential tool for applied and basic research of chalkbrood disease in honeybees.
Assuntos
Abelhas/microbiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Domínios HMG-Box/genética , Onygenales/genética , Sequência de Aminoácidos , Animais , Biologia Computacional , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos Tipo Acasalamento , Dados de Sequência Molecular , Onygenales/metabolismo , Filogenia , Reação em Cadeia da Polimerase/métodos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Treponema pallidum subsp, pallidum, the causative agent of the sexually transmitted disease syphilis, is a fastidious, microaerophilic obligate parasite of humans. This bacterium is one of the few prominent infectious agents that has not been cultured continuously in vitro and consequently relatively little is known about its virulence mechanisms at the molecular level. T. pallidum therefore represented an attractive candidate for genomic sequencing. The complete genome sequence of T. pallidum has now been completed and comprises 1,138,006 base pairs containing 1041 predicted protein coding sequences. An important goal of this project is to identify possible virulence factors. Analysis of the genome indicates a number of potential virulence factors including a family of 12 proteins related to the Msp protein of Treponema denticola, a number of putative hemolysins, as well as several other classes of proteins of interest. The results of this analysis are reviewed in this article and indicate the value of whole genome sequences for rapidly advancing knowledge of infectious agents.
Assuntos
Genoma Bacteriano , Treponema pallidum/genética , Genes Bacterianos , Proteínas Hemolisinas/genética , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Proto-Oncogênicas/genética , Sífilis/microbiologia , Treponema pallidum/patogenicidade , Virulência/genéticaRESUMO
Complete XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 are presented, along with a comparison of the physical map of MG1655 with that of five other K-12 strains. We have mapped 35 XbaI cleavage sites generating 35 fragments ranging in size from 8 kb to 432 kb using methods similar to those used previously for the NotI and SfiI maps of MG1655. The applicability of the MG1655 map to other strains of E. coli K-12 was assessed by comparing the NotI, SfiI and XbaI digestion patterns of EMG2, W1485, W3110, AB1157 and MC4100 with those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the differences between the strains on the basis of simple insertions, deletions and, in one case, an inversion. Insertions and deletions ranging in size from 1 kb to 86 kb are suggested by this model. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains. The various features localized in these strains were used to place 9 of the 17 BlnI fragments on the E. coli physical map. The remaining fragments were placed by hybridization experiments similar to those used for the NotI, SfiI and XbaI maps. In this way, the complete BlnI map was constructed. The cleavage sites for XbaI and BlnI were assigned coordinates based on EcoMap6 developed by Rudd et al. The XbaI and BlnI maps of MG1655 presented here, when combined with the NotI (22 sites) and SfiI (31 sites) maps of MG1655 previously published, bring the total number of mapped rare restriction sites in MG1655 to 105. The strain comparison analysis shows that this map is readily adaptable for use with other K-12 strains.
Assuntos
Escherichia coli/genética , Genoma Bacteriano , Mapeamento por Restrição , Cromossomos Bacterianos , Elementos de DNA Transponíveis/genética , Bases de Dados Factuais , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Variação Genética , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
Insertions of the translocatable ampicillin-resistance element Tn1 were selected in the genome of the temperate Salmonella phage P22 by growing the phage on hosts carrying the resistance plasmid RP4. Insertions of Tn1 into phage P22 are rare (10(-10) per phage) and nonrandomly distributed in the P22 genome. They are found mainly in the vicinity of the P22 ant gene. Insertions within the ant gene are found at many (at least 15) genetically separable sites, are found equally frequently in both orientations and cause irreversible loss of gene function. Some insertions in ant appear to be associated with an adjecent deletion. Prophage deletions were derived from P22::Tn1 phages by two methods. Low multiplicity transductants have nonrandomly distributed endpoints. One end is at or very near the site of the Tn1 insertion, and the other is in the vicinity of gene 12; however, there are many genetically distinguishable endpoints within gene 12. Prophage deletions selected as survivors of induction of a P22Ap mnt-ts lysogen have similarly nonrandom endpoints, with the Tn1-distal end frequently near the ant gene, as well as gene 12. Physical analysis of several prophage deletions suggests that the Tn1 is intact to the resolution of DNA electron microscopy and that the deletions begin at the end of the Tn1 insertion. These results suggest that illegitimate recombination associated with Tn1 shows regional specificity (i.e., preference for some large areas of the P22 genome over other areas), but that within these regions is quite nonspecific.
Assuntos
Ampicilina/farmacologia , Elementos de DNA Transponíveis , Fatores R , Recombinação Genética , Fagos de Salmonella/genética , Escherichia coli/genética , Genes Virais , Ligação Genética , Canamicina/farmacologia , Lisogenia , Neomicina/farmacologia , Salmonella typhimurium/genética , Tetraciclina/farmacologiaRESUMO
Using differential hybridization to screen a rat Sertoli cell cDNA library for hormonally regulated gene products, we isolated a clone, designated 13-10, which contained a 1.0-kilobase insert and hybridized to a 1.7-kilobase message in total testis, Sertoli cells, and peritubular cells. This mRNA was decreased relative to untreated control levels in total testicular RNA from hypophysectomized rats, but was increased by FSH treatment begun on the day of hypophysectomy. FSH caused a transient rise in 13-10 mRNA at 24 h in cultured Sertoli cells. There was no comparable rise in beta-actin RNA or the RNA/DNA ratio at this time, suggesting that the effect on 13-10 was specific. Testosterone had no effect at any time interval studied. The 13-10 mRNA was not increased in peritubular cells treated in vitro with FSH or testosterone. Sequence analysis of 13-10 revealed more than 99% homology with a portion of the sequence of rat liver cytochrome oxidase subunit I (COX I). The clone included 58% of the open reading frame of COX I as well as that for the adjacent Ser-tRNA. COX I is a mitochondrial gene, and Southern analysis confirmed 13-10 sequence in testicular mitochondrial DNA. In addition to FSH, forskolin and (Bu)2cAMP also increased COX I steady state mRNA in Sertoli cells (3.8-, 4.1-, and 9.2-fold, respectively). (Bu)2cAMP increased mRNA for other mitochondrial gene products, COX subunit II and 16S rRNA (6.9- and 5.4-fold, respectively), whereas the smaller effects elicited by forskolin and FSH were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Hormônio Foliculoestimulante/farmacologia , Hipofisectomia , Células de Sertoli/enzimologia , Testículo/enzimologia , Testosterona/farmacologia , Actinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células Cultivadas , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , DNA Mitocondrial/genética , Biblioteca Gênica , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Ribossômico 16S/genética , Ratos , Ratos Endogâmicos , Homologia de Sequência do Ácido Nucleico , Células de Sertoli/efeitos dos fármacos , Testículo/efeitos dos fármacosRESUMO
PURPOSE: The enteric microbiome is known to play a major role in healthy gut homeostasis and several disease states. It may also contribute to both the intestinal recovery and complications that occur in patients with short bowel syndrome. The extent and nature of alterations to the gut microbiota following intestinal resection, however, are not well studied in a controlled setting. The purpose of this investigation is to characterize the effects of massive small bowel resection on the murine enteric microflora. METHODS: Wild-type C57BL6 mice, following a week of acclamation to a liquid rodent diet, underwent either 50% proximal small bowel resection (SBR) or a sham operation. Mice were sacrificed, and enteric contents from the small bowel, cecum, and stool were harvested at 7 and 90 days post-operatively. DNA was isolated, and the V3-V5 regions of the 16s rRNA gene amplified and pyrosequenced on a Roche 454 platform. Sequences were clustered into operation taxonomic units and classified. Communities were then analyzed for diversity and phylogenic composition. RESULTS: In the long-term group, the microbes inhabiting the ileum of mice undergoing SBR and sham operation differed significantly at the genus level (p < 0.001). Small bowel contents collected before and after SBR also differed significantly (p = 0.006). This was driven by an increase in Lactobacillus and decrease in Enterobacteriaceae species in mice undergoing SBR. No difference was seen in the long-term stool or in stool, cecal, or ileal contents in the short-term. No difference in microbial community diversity was found in any group. CONCLUSION: Bowel resection induces long-term changes in the microbial community of the murine ileum, but not at more distal sites of the gastrointestinal tract. The increase in Lactobacillus encountered small bowel of resected mice correlates with limited previous studies. These changes may reflect an adaptive response of the microbiota to maximize energy extraction, but further studies are needed to establish the role played by this altered community.
Assuntos
Bactérias/isolamento & purificação , Mucosa Intestinal/microbiologia , Intestino Delgado/cirurgia , Microbiota/fisiologia , Síndrome do Intestino Curto/microbiologia , Animais , Modelos Animais de Doenças , Seguimentos , Mucosa Intestinal/cirurgia , Intestino Delgado/microbiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.
Assuntos
Bacteriófago lambda/genética , Elementos de DNA Transponíveis , Proteínas Virais/genética , Alelos , Sequência de Bases , DNA Viral/genética , Escherichia coli/genética , Genes Virais , Resistência a Canamicina/genética , Plasmídeos , Mapeamento por RestriçãoRESUMO
The function of many genes cannot be deduced from sequence similarity, and biochemical methods are usually required. Whole genome sequences can be thought of as not only a set of genes but also collections of functional domains. These domains can be studied by affinity methods whereby identification of the ligand can provide information on biochemical function. To take advantage of this method, one must express all functional domains in a form suitable for affinity studies. Phage display technology provides a means for accomplishing this. The pJuFo phage display system, based on the interaction between the leucine zippers Jun and Fos, has been modified and used to create a genomic phage display library from Escherichia coli MG1655. The system has been tested by using the library to map the dominant binding epitopes for an anti-RecA protein polyclonal antibody sera. This methodology provides a general biochemical approach to functional analysis of protein-ligand interactions on a genomewide basis.
Assuntos
Bacteriófagos/genética , Biblioteca Genômica , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Epitopos/imunologia , Epitopos/metabolismo , Escherichia coli/genética , Genes fos/genética , Ligantes , Plasmídeos/genética , Ligação Proteica , Recombinases Rec A/imunologia , Recombinases Rec A/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Regiões Terminadoras Genéticas/genéticaRESUMO
A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59). The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus. Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein. Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum. The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene. This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA. This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA. Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs.
Assuntos
Anticorpos , RNA Polimerases Dirigidas por DNA/genética , Vírus da Hepatite Murina/genética , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/imunologia , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Vetores Genéticos , Camundongos , Dados de Sequência Molecular , Vírus da Hepatite Murina/enzimologia , Plasmídeos , Recombinases Rec A/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Óperon/genética , Proteínas Periplásmicas de Ligação , Fosfoglicerato Mutase/genética , Proteínas Repressoras/genética , Treponema pallidum/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes Bacterianos/genética , Ferro/farmacologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/análise , RNA Mensageiro/análise , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Tandem duplications are caused by unequal crossing over between homologous sequences. Duplications in the lac region of the Escherichia coli chromosome were isolated by two methods. Duplication frequency using a method involving P1 transduction increased from 0.4% with no UV to 2.0% following UV irradiation at 35 J/m2. Duplication frequency in lac using a second generalizable method that does not involve P1 transduction increased from 0.7 to 12% at 35 J/m2 UV. In both cases the duplication frequency began to plateau at UV doses of 12 J/m2 and 24 J/m2. According to segregation analysis of sixteen duplications there may be at least seven classes of duplications isolated by each method. Pulsed-field gel electrophoresis was used to measure the duplications isolated without P1 transduction. The minimum size of the duplications ranged from 30 to 320 kb but could be much larger.
Assuntos
Cromossomos Bacterianos , Escherichia coli/genética , Genes Bacterianos , Óperon Lac , Família Multigênica , Sequências Repetitivas de Ácido Nucleico , Escherichia coli/efeitos da radiação , Plasmídeos , Regiões Promotoras Genéticas , Recombinação Genética , Transdução GenéticaRESUMO
The growth defect of a lambda phage carrying a recA-lacZ fusion was used to select mutations that reduced recA expression. Nine single base changes in the recA promoter were isolated that reduced both induced and basal (repressed) levels of expression. Deletion analysis of the promoter region and mapping of transcripts indicated that there is one main promoter responsible for both basal and induced expression. Some of the mutants displayed a lowered induction ratio, raising the possibility that there is a second, weak promoter that is not regulated by the SOS response. When one of the mutants was examined, it showed normal affinity for LexA repressor binding to the operator site. Binding of RNA polymerase to this mutant promoter, however, was much reduced. Further binding experiments suggested that LexA does not block RNA polymerase binding to the recA promoter, but inhibits a later step in initiation.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Regiões Promotoras Genéticas , Recombinases Rec A/genética , Serina Endopeptidases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Deleção Cromossômica , Mapeamento Cromossômico , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dados de Sequência Molecular , Mutagênese , Recombinases Rec A/metabolismoRESUMO
We describe a modification of an automated kinetic assay for beta-galactosidase (beta-gal) activity. This modification includes an assay to quantitate the amount of protein added to each assay. The determination of specific activity includes the amount of protein in the calculation which produces a specific activity with units of pmol product produced/minute/mg protein. In addition to this modification, we present a series of macros written in Microsoft Excel for either the Macintosh or Windows on the PC. These macros decrease the amount of time required to analyze the data from beta-gal assays.
Assuntos
Autoanálise , beta-Galactosidase/metabolismo , Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Nitrofenilgalactosídeos/metabolismo , Recombinases Rec A/genética , Proteínas Recombinantes de Fusão , Software , Espectrofotometria , beta-Galactosidase/genéticaRESUMO
Whole genome sequences of microbial pathogens present new opportunities for clinical applications. Chief among these are development of antimicrobials, diagnostics, and vaccines. While antimicrobial development is a more difficult, long-term prospect, new diagnostics and vaccines are likely to be the first products of microbial genomics. To take advantage of whole genome sequences, methods for production of gene products in surrogate hosts (heterologous expression) are required that will work for large-scale, high-throughput gene expression. This will allow genomic information from even the most experimentally difficult pathogens to be mined for applications. In addition, screening methods to test gene products for their potential as vaccine candidates are needed for large-scale screening. These areas for technological development should be stimulated by the potential for converting genomic sequence information into applications.
Assuntos
Vetores Genéticos , Genoma Bacteriano , Sífilis/diagnóstico , Sífilis/prevenção & controle , Treponema pallidum/genética , Animais , Vacinas Bacterianas , Genômica , Humanos , Análise de Sequência de DNA , Sorodiagnóstico da Sífilis , Treponema pallidum/imunologia , Treponema pallidum/metabolismoRESUMO
In this study, we define cis-acting elements involved in regulation of the Pasteurella haemolytica leukotoxin promoter. In place of a canonical promoter -35 sequence, the leukotoxin promoter contains four adenine-rich repeats of sequence CA6(C/T)A, phased at approximately 10-base intervals. DNA fragments containing these repeats exhibit retarded mobility in polyacrylamide gels and permitted identification of a static DNA bend in the promoter -70 region. Deletion of the static DNA bend caused a two-fold reduction of leukotoxin transcription in Escherichia coli, suggesting that it is involved in promoter regulation. Three putative upstream activator sites (UAS), similar to those that bind the NifA activator in Klebsiella pneumoniae, are found 130 bp upstream from the transcription start site and are protected from DNase I cleavage by a P. haemolytica-specific factor. The promoter region also binds the DNA bending protein, the integration host factor (IHF), although IHF mutations do not affect its expression in E. coli. The arrangement of these elements suggests that leukotoxin expression is activated by a factor that interacts with the UAS and regulates transcription initiation at a distance via DNA looping. Activation and DNA bending may also influence a second, divergent promoter that lies 340 bp upstream from the leukotoxin start site.
Assuntos
Toxinas Bacterianas/genética , Exotoxinas/genética , Regulação Bacteriana da Expressão Gênica , Mannheimia haemolytica/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Mapeamento por Restrição , Transcrição GênicaRESUMO
The adherence of 44 clinical isolates of Enterococcus faecalis, a common cause of endocarditis, and 13 Enterococcus faecium to substrates of six extracellular matrix (ECM) proteins was examined using 35S-labeled bacteria. One E. faecalis strain, isolated from a patient with endocarditis, adhered to collagen types I and IV and another E. faecalis strain adhered to laminin and to collagen types I and IV. However, most isolates showed little adherence ( < 5% of added cells adhered) when grown at 37 degrees C regardless of their source (endocarditis, urine or fecal sample). When grown at 46 degrees C (but not when grown in CO2 or nutrient limited media), most isolates of E. faecalis increased their adherence to immobilized laminin, collagen types I and IV but not to fibronectin, fibrinogen or bovine serum albumin, whereas none of the E. faecium increased adherence when grown at 46 degrees C or 50 degrees C. The adherence of E. faecalis was eliminated by digestion with trypsin, suggesting that a protein is somehow important, directly or indirectly, for adherence to occur. Pre-incubation of bacteria with soluble collagen types I and IV inhibited the adherence to these ECM proteins. These results demonstrate that in E. faecalis, adherence to ECM proteins is produced during routine in vitro growth conditions by occasional isolates and can be produced during certain stressful growth conditions by others. Whether this adherence relates to the propensity of E. faecalis to cause endocarditis remains to be determined.