RESUMO
In this study, infants' visual processing of depth-inducing stimuli was tested using a new method suitable for experimental settings. Stereograms of the Lang-Stereopad® were presented in a timed preferential-looking paradigm to determine infants' preference for a stereogram as compared to a stimulus not inducing an impression of depth. A total of 80 infants were tested at 7 months of age; of these, a sub-sample of 41 infants were tested longitudinally at 4 and 7 months to characterize the developmental trajectory of their preference. Infants were simultaneously presented with a card showing a random-dot stereogram (800" disparity) and a similar looking dummy card without stereogram. In the total sample, 7-month-olds showed a clear preference for the stereogram regardless of sex. In the longitudinal sample, 7-month-olds but not 4-month-olds looked significantly longer to the stereogram as compared to the dummy card. On individual level, 56% of the 4-month-olds and 85% of the 7-month-olds predominantly looked at the stereogram. The findings yield evidence for a clear developmental progression and show that the test cards of the Lang-Stereopad® prototype provide a viable instrument to determine the preference for depth-inducing stimuli in young infants when used in a controlled experimental setting.
Assuntos
Desenvolvimento Infantil , Percepção de Profundidade , Feminino , Humanos , Lactente , Estudos Longitudinais , Masculino , Visão BinocularRESUMO
The ventral occipitotemporal (vOT) cortex serves as a core region for visual processing, and specific areas of this region show preferential activation for various visual categories such as faces and print. The emergence of such functional specialization in the human cortex represents a pivotal developmental process, which provides a basis for targeted and efficient information processing. For example, functional specialization to print in the left vOT is an important prerequisite for fluent reading. However, it remains unclear, which processes initiate the preferential cortical activations to characters arising in the vOT during child development. Using a multimodal neuroimaging approach with preschool children at familial risk for developmental dyslexia, we demonstrate how varying levels of expertise modulate the neural response to single characters, which represent the building blocks of print units. The level of expertise to characters was manipulated firstly through brief training of false-font speech-sound associations and secondly by comparing characters for which children differed in their level of familiarity and expertise accumulated through abundant exposure in their everyday environment. Neural correlates of character processing were tracked with simultaneous high-density electroencephalography and functional magnetic resonance imaging in a target detection task. We found training performance and expertise-dependent modulation of the visual event-related potential around 220â¯ms (N1) and the corresponding vOT activation. Additionally, trained false-font characters revealed stronger functional connectivity between the left fusiform gyrus (FFG) seed and left superior parietal/lateral occipital cortex regions with higher training performance. In sum, our results demonstrate that learning artificial-character speech-sound associations enhances activation to trained characters in the vOT and that the magnitude of this activation and the functional connectivity of the left FFG to the parieto-occipital cortex depends on learning performance. This pattern of results suggests emerging development of the reading network after brief training that parallels network specialization during reading acquisition.
Assuntos
Aprendizagem por Associação , Desenvolvimento Infantil/fisiologia , Potenciais Evocados Visuais/fisiologia , Neuroimagem Funcional/métodos , Rede Nervosa/fisiologia , Lobo Occipital/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Prática Psicológica , Leitura , Reconhecimento Psicológico/fisiologia , Fala/fisiologia , Lobo Temporal/fisiologia , Criança , Pré-Escolar , Dislexia/fisiopatologia , Eletroencefalografia/métodos , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Rede Nervosa/diagnóstico por imagem , Rede Nervosa/crescimento & desenvolvimento , Lobo Occipital/diagnóstico por imagem , Lobo Occipital/crescimento & desenvolvimento , Fonética , Risco , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/crescimento & desenvolvimentoRESUMO
Learning letter-speech sound correspondences is a major step in reading acquisition and is severely impaired in children with dyslexia. Up to now, it remains largely unknown how quickly neural networks adopt specific functions during audiovisual integration of linguistic information when prereading children learn letter-speech sound correspondences. Here, we simulated the process of learning letter-speech sound correspondences in 20 prereading children (6.13-7.17 years) at varying risk for dyslexia by training artificial letter-speech sound correspondences within a single experimental session. Subsequently, we acquired simultaneously event-related potentials (ERP) and functional magnetic resonance imaging (fMRI) scans during implicit audiovisual presentation of trained and untrained pairs. Audiovisual integration of trained pairs correlated with individual learning rates in right superior temporal, left inferior temporal, and bilateral parietal areas and with phonological awareness in left temporal areas. In correspondence, a differential left-lateralized parietooccipitotemporal ERP at 400 ms for trained pairs correlated with learning achievement and familial risk. Finally, a late (650 ms) posterior negativity indicating audiovisual congruency of trained pairs was associated with increased fMRI activation in the left occipital cortex. Taken together, a short (<30 min) letter-speech sound training initializes audiovisual integration in neural systems that are responsible for processing linguistic information in proficient readers. To conclude, the ability to learn grapheme-phoneme correspondences, the familial history of reading disability, and phonological awareness of prereading children account for the degree of audiovisual integration in a distributed brain network. Such findings on emerging linguistic audiovisual integration could allow for distinguishing between children with typical and atypical reading development. Hum Brain Mapp 38:1038-1055, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Percepção Auditiva/fisiologia , Mapeamento Encefálico , Dislexia/fisiopatologia , Potenciais Evocados/fisiologia , Percepção Visual/fisiologia , Estimulação Acústica , Criança , Dislexia/diagnóstico por imagem , Eletroencefalografia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Oxigênio/sangue , Estimulação Luminosa , Aprendizagem VerbalRESUMO
Anatomical plasticity such as fibre growth and the formation of new connections in the cortex and spinal cord is one known mechanism mediating functional recovery after damage to the central nervous system. Little is known about anatomical plasticity in the brainstem, which contains key locomotor regions. We compared changes of the spinal projection pattern of the major descending systems following a cervical unilateral spinal cord hemisection in adult rats. As in humans (Brown-Séquard syndrome), this type of injury resulted in a permanent loss of fine motor control of the ipsilesional fore- and hindlimb, but for basic locomotor functions substantial recovery was observed. Antero- and retrograde tracings revealed spontaneous changes in spinal projections originating from the reticular formation, in particular from the contralesional gigantocellular reticular nucleus: more reticulospinal fibres from the intact hemicord crossed the spinal midline at cervical and lumbar levels. The intact-side rubrospinal tract showed a statistically not significant tendency towards an increased number of midline crossings after injury. In contrast, the corticospinal and the vestibulospinal tract, as well as serotonergic projections, showed little or no side-switching in this lesion paradigm. Spinal adaptations were accompanied by modifications at higher levels of control including side-switching of the input to the gigantocellular reticular nuclei from the mesencephalic locomotor region. Electrolytic microlesioning of one or both gigantocellular reticular nuclei in behaviourally recovered rats led to the reappearance of the impairments observed acutely after the initial injury showing that anatomical plasticity in defined brainstem motor networks contributes significantly to functional recovery after injury of the central nervous system.
Assuntos
Tronco Encefálico/fisiopatologia , Locomoção/fisiologia , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Tronco Encefálico/patologia , Modelos Animais de Doenças , Feminino , Lateralidade Funcional/fisiologia , Ratos , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/patologiaRESUMO
Rodents are frequently used to model damage and diseases of the central nervous system (CNS) that lead to functional deficits. Impaired locomotor function is currently evaluated by using scoring systems or biomechanical measures. These methods often suffer from limitations such as subjectivity, nonlinearity and low sensitivity, or focus on a few very restricted aspects of movement. Thus, full quantitative profiles of motor deficits after CNS damage are lacking. Here we report the detailed characterization of locomotor impairments after applying common forms of CNS damage in rodents. We obtained many objective and quantitative readouts from rats with either spinal cord injuries or strokes and from transgenic mice (Epha4−/−) during skilled walking, overground walking, wading and swimming, resulting in model-specific locomotor profiles. Our testing and analysis method enables comprehensive assessment of locomotor function in rodents and has broad application in various fields of life science research.
Assuntos
Sistema Nervoso Central/fisiopatologia , Locomoção , Desempenho Psicomotor , Animais , Sistema Nervoso Central/lesões , Feminino , Marcha , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Transtornos Psicomotores/diagnóstico , Transtornos Psicomotores/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Natação , CaminhadaRESUMO
The Visual Word Form Area (VWFA) is a key region of the brain's reading network and its activation has been shown to be strongly associated with reading skills. Here, for the first time, we investigated whether voluntary regulation of VWFA activation is feasible using real-time fMRI neurofeedback. 40 adults with typical reading skills were instructed to either upregulate (UP group, N = 20) or downregulate (DOWN group, N = 20) their own VWFA activation during six neurofeedback training runs. The VWFA target region was individually defined based on a functional localizer task. Before and after training, also regulation runs without feedback ("no-feedback runs") were performed. When comparing the two groups, we found stronger activation across the reading network for the UP than the DOWN group. Further, activation in the VWFA was significantly stronger in the UP group than the DOWN group. Crucially, we observed a significant interaction of group and time (pre, post) for the no-feedback runs: The two groups did not differ significantly in their VWFA activation before neurofeedback training, but the UP group showed significantly stronger activation than the DOWN group after neurofeedback training. Our results indicate that upregulation of VWFA activation is feasible and that, once learned, successful upregulation can even be performed in the absence of feedback. These results are a crucial first step toward the development of a potential therapeutic support to improve reading skills in individuals with reading impairments.
Assuntos
Dislexia , Neurorretroalimentação , Autocontrole , Adulto , Humanos , Imageamento por Ressonância Magnética/métodos , Neurorretroalimentação/métodos , Aprendizagem/fisiologia , Mapeamento EncefálicoRESUMO
We assessed the Swiss-German version of GraphoLearn, a computer game designed to support reading by training grapheme-phoneme correspondences. A group of 34 children at risk for dyslexia trained three times a week during 14 weeks, on top of their standard school instruction. The sample was divided into two groups of 18 and 16 children, who started training at either the middle or the end of first grade. We found beneficial training effects in pseudoword reading in both training groups and for rapid automatized naming skills in the group that trained earlier. Our results suggest that both the efficiency in phonological decoding and rapid access to verbal representations are susceptible to facilitation by GraphoLearn. These findings confirm the utility of the training software as a tool to support school instruction and reading-related abilities in beginning readers. We discuss ideas to improve the content and outcomes of future versions of the training software.
RESUMO
The level of reading skills in children and adults is reflected in the strength of preferential neural activation to print. Such preferential activation appears in the N1 event-related potential (ERP) over the occipitotemporal scalp after around 150-250â¯ms and the corresponding blood oxygen level dependent (BOLD) signal in the ventral occipitotemporal (vOT) cortex. Here, orthography-sensitive (print vs. false font) processing was examined using simultaneous EEG-fMRI in 38 first grade children with poor and typical reading skills, and at varying familial risk for developmental dyslexia. Coarse orthographic sensitivity was observed as an increased activation to print in the N1 ERP and in the BOLD signal of individually varying vOT regions in 57% of beginning readers. Finer differentiation in processing orthographic strings (words vs. nonwords) further occurred in specific vOT clusters. Neither method alone showed robust differences in orthography-sensitive processing between typical and poor reading children. Importantly, using single-trial N1 ERP-informed fMRI analysis, we found differential modulation of the orthography-sensitive BOLD response in the left vOT for typical readers only. This result, thus, confirms subtle functional alterations in a brain structure known to be critical for fluent reading at the very beginning of reading instruction.
Assuntos
Mapeamento Encefálico/psicologia , Encéfalo/fisiopatologia , Eletroencefalografia/métodos , Imageamento por Ressonância Magnética/métodos , Leitura , Criança , Feminino , Humanos , Masculino , Lobo Occipital , Lobo Temporal/fisiologiaRESUMO
During reading acquisition, neural reorganization of the human brain facilitates the integration of letters and speech sounds, which enables successful reading. Neuroimaging and behavioural studies have established that impaired audiovisual integration of letters and speech sounds is a core deficit in individuals with developmental dyslexia. This longitudinal study aimed to identify neural and behavioural markers of audiovisual integration that are related to future reading fluency. We simulated the first step of reading acquisition by performing artificial-letter training with prereading children at risk for dyslexia. Multiple logistic regressions revealed that our training provides new precursors of reading fluency at the beginning of reading acquisition. In addition, an event-related potential around 400 ms and functional magnetic resonance imaging activation patterns in the left planum temporale to audiovisual correspondences improved cross-validated prediction of future poor readers. Finally, an exploratory analysis combining simultaneously acquired electroencephalography and hemodynamic data suggested that modulation of temporoparietal brain regions depended on future reading skills. The multimodal approach demonstrates neural adaptations to audiovisual integration in the developing brain that are related to reading outcome. Despite potential limitations arising from the restricted sample size, our results may have promising implications both for identifying poor-reading children and for monitoring early interventions.
Assuntos
Encéfalo/fisiologia , Dislexia/fisiopatologia , Aprendizagem/fisiologia , Leitura , Estimulação Acústica , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico , Criança , Dislexia/diagnóstico por imagem , Eletroencefalografia , Potenciais Evocados/fisiologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Fonética , Estimulação Luminosa , Percepção da Fala/fisiologia , Percepção Visual/fisiologiaRESUMO
Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.
Assuntos
Alcanos/metabolismo , Membrana Celular/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Bactérias Gram-Positivas/enzimologia , Oxigenases de Função Mista/metabolismo , Proteobactérias/enzimologia , Composição de Bases , Biodegradação Ambiental , Citocromo P-450 CYP4A , Sistema Enzimático do Citocromo P-450/genética , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Proteobactérias/genética , Proteobactérias/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNARESUMO
Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.
Assuntos
Citocromo P-450 CYP4A/química , Halomonadaceae/enzimologia , Pseudomonas putida/enzimologia , Alcanos/metabolismo , Sequência de Aminoácidos , Citocromo P-450 CYP4A/metabolismo , Hidroxilação , Modelos Moleculares , Dados de Sequência Molecular , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg(-1) (k(cat) = 1.6 s(-1)) and K(m) values for styrene of 0.45 +/- 0.05 mM (wild type) and 0.38 +/- 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg(-1) (k(cat) [NADH] = 60 s(-1)). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.
Assuntos
FMN Redutase/isolamento & purificação , FMN Redutase/metabolismo , Oxigenases/isolamento & purificação , Oxigenases/metabolismo , Pseudomonas/enzimologia , Estireno/metabolismo , Clonagem Molecular , Dimerização , Transporte de Elétrons , Compostos de Epóxi/metabolismo , Escherichia coli/genética , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Peso Molecular , NAD/metabolismo , Subunidades Proteicas/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Riboflavina/metabolismo , Especificidade por SubstratoRESUMO
The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS:
Assuntos
Alcanos/metabolismo , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/genética , Elementos de DNA Transponíveis , Oxigenases de Função Mista/genética , Família Multigênica , Pseudomonas putida/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Quimiotaxia , Citocromo P-450 CYP4A , Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pseudomonas putida/metabolismo , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The marine gamma-Proteobacterium Alcanivorax borkumensis is highly specialized in the assimilation of aliphatic hydrocarbons, and makes up a large part of the biomass in oil-polluted marine environments. In addition to the previously identified alkane hydroxylase AlkB1, a second alkane hydroxylase (AlkB2) showing 65% identity to the Pseudomonas aeruginosa AlkB2 alkane hydroxylase was identified. Unlike alkB1, alkB2 is not flanked by genes involved in alkane metabolism. Heterologous expression of the A. borkumensis AP1 alkB1 and alkB2 genes showed that they encode functional alkane hydroxylases with substrate ranges similar to those of their P. putida and P. aeruginosa homologues. The transcription initiation sites and levels of the alkB1, alkB2 and alkS mRNA transcripts were determined. Expression of both alkB1 and alkB2 was induced by alkanes, but transcripts corresponding to alkB1 were much more abundant than those of alkB2. An inverted repeat similar to the binding site for the P. putida GPo1 transcriptional activator AlkS was present upstream of the promoters for alkB1 and alkB2, although that of alkB2 was less well conserved, and only the transcriptional fusion of promoter PalkB1 to the reporter gene lacZ efficiently responded to n-octane. Contrary to what has been found for the P. putida GPo1 alkane degradation pathway, expression of the A. borkumensis AP1 alkS gene was not induced by alkanes, and an AlkS binding site was not present upstream of the promoter for alkS. This indicates that, in spite of the clear similarities, the A. borkumensis alk-genes are regulated by a strategy different from that of the P. putida GPo1 alk genes.
Assuntos
Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Halomonadaceae/enzimologia , Halomonadaceae/genética , Alcanos/metabolismo , Fusão Gênica Artificial , Biodegradação Ambiental , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Genes Reporter , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência , Especificidade por Substrato , Sítio de Iniciação de Transcrição , Poluição Química da Água , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC, HbpA) was changed by directed evolution using error-prone PCR. In situ screening of mutant libraries resulted in the identification of proteins with increased activity towards 2-tert-butylphenol and guaiacol (2-methoxyphenol). One enzyme variant contained amino acid substitutions V368A/L417F, which were inserted by two rounds of mutagenesis. The double replacement improved the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the two substitutions increased V(max) from 0.22 to 0.43 units mg(-1) protein and decreased the K'(m) from 588 to 143 microm, improving k'(cat)/K'(m) by a factor of 8.2. With 2-tert-butylphenol as the substrate, k'(cat) was increased more than 5-fold. Another selected enzyme variant contained amino acid substitution I244V and had a 30% higher specific activity with 2-sec-butylphenol, guaiacol, and the "natural" substrate 2-hydroxybiphenyl. The K'(m) for guaiacol decreased with this mutant, but the K'(m) for 2-hydroxybiphenyl increased. The primary structure of HbpA shares 20.1% sequence identity with phenol 2-monooxygenase from Trichosporon cutaneum. Structure homology modeling with this three-domain enzyme suggests that Ile(244) of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol. In contrast, Val(368) and Leu(417) are not close to the active site and would not have been obvious candidates for modification by rational design.
Assuntos
Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Mutagênese , Pseudomonas/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Variação Genética , Cinética , Leucina/química , Oxigenases de Função Mista/metabolismo , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenóis/farmacologia , Reação em Cadeia da Polimerase , Ligação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Valina/químicaRESUMO
We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene. Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.