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1.
Genesis ; 62(3): e23611, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38888221

RESUMO

Olfactory sensory neurons (OSNs) are one of a few neuron types that are generated continuously throughout life in mammals. The persistence of olfactory sensory neurogenesis beyond early development has long been thought to function simply to replace neurons that are lost or damaged through exposure to environmental insults. The possibility that olfactory sensory neurogenesis may also serve an adaptive function has received relatively little consideration, largely due to the assumption that the generation of new OSNs is stochastic with respect to OSN subtype, as defined by the single odorant receptor gene that each neural precursor stochastically chooses for expression out of hundreds of possibilities. Accordingly, the relative birthrates of different OSN subtypes are predicted to be constant and impervious to olfactory experience. This assumption has been called into question, however, by evidence that the birthrates of specific OSN subtypes can be selectively altered by manipulating olfactory experience through olfactory deprivation, enrichment, and conditioning paradigms. Moreover, studies of recovery of the OSN population following injury provide further evidence that olfactory sensory neurogenesis may not be strictly stochastic with respect to subtype. Here we review this evidence and consider mechanistic and functional implications of the prospect that specific olfactory experiences can regulate olfactory sensory neurogenesis rates in a subtype-selective manner.


Assuntos
Neurogênese , Neurônios Receptores Olfatórios , Receptores Odorantes , Animais , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Neurônios Receptores Olfatórios/fisiologia , Neurogênese/genética , Olfato/fisiologia , Olfato/genética , Humanos
2.
bioRxiv ; 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38405728

RESUMO

In mammals, olfactory sensory neurons (OSNs) are born throughout life, presumably solely to replace neurons lost via turnover or injury. This assumption follows from the hypothesis that olfactory neurogenesis is strictly stochastic with respect to neuron subtype, as defined by the single odorant receptor allele that each neural precursor stochastically chooses out of hundreds of possibilities. This hypothesis is challenged by recent findings that the birthrates of a fraction of subtypes are selectively diminished by olfactory deprivation. These findings raise questions about how, and why, olfactory stimuli are required to promote the neurogenesis of some OSN subtypes, including whether the stimuli are generic (e.g., broadly activating odors or mechanical stimuli) or specific (e.g., discrete odorants). Based on RNA-seq and scRNA-seq analyses, we hypothesized that the neurogenic stimuli are specific odorants that selectively activate the same OSN subtypes whose birthrates are accelerated. In support of this, we have found, using subtype-specific OSN birthdating, that exposure to male and musk odors can accelerate the birthrates of responsive OSNs. Collectively, our findings reveal that certain odor experiences can selectively "amplify" specific OSN subtypes, and that persistent OSN neurogenesis may serve, in part, an adaptive function.

3.
STAR Protoc ; 4(3): 102432, 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37436902

RESUMO

Mammals typically have hundreds of distinct olfactory sensory neuron subtypes, each defined by expression of a specific odorant receptor gene, which undergo neurogenesis throughout life at rates that can depend on olfactory experience. Here, we present a protocol to quantify the birthrates of specific neuron subtypes via the simultaneous detection of corresponding receptor mRNAs and 5-ethynyl-2'-deoxyuridine. For preparation prior to beginning the protocol, we detail procedures for generating odorant receptor-specific riboprobes and experimental mouse olfactory epithelial tissue sections. For complete details on the use and execution of this protocol, please refer to van der Linden et al. (2020).1.


Assuntos
Neurônios Receptores Olfatórios , Receptores Odorantes , Animais , Camundongos , Mucosa Olfatória/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Neurogênese/genética , Odorantes , Mamíferos/metabolismo
4.
Cell Rep ; 33(1): 108210, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33027656

RESUMO

In mammals, olfactory sensory neurons (OSNs) are born throughout life, ostensibly solely to replace damaged OSNs. During differentiation, each OSN precursor "chooses," out of hundreds of possibilities, a single odorant receptor (OR) gene, which defines the identity of the mature OSN. The relative neurogenesis rates of the hundreds of distinct OSN "subtypes" are thought to be constant, as they are determined by a stochastic process in which each OR is chosen with a fixed probability. Here, using histological, single-cell, and targeted affinity purification approaches, we show that closing one nostril in mice selectively reduces the number of newly generated OSNs of specific subtypes. Moreover, these reductions depend on an animal's age and/or environment. Stimulation-dependent changes in the number of new OSNs are not attributable to altered rates of cell survival but rather production. Our findings indicate that the relative birth rates of distinct OSN subtypes depend on olfactory experience.


Assuntos
Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Animais , Diferenciação Celular , Camundongos
5.
Sci Data ; 5: 180260, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30512012

RESUMO

Olfactory experience can alter the molecular and cellular composition of chemosensory neurons within the olfactory sensory epithelia of mice. We sought to investigate the scope of cellular and molecular changes within a mouse's olfactory system as a function of its exposure to complex and salient sets of odors: those emitted from members of the opposite sex. We housed mice either separated from members of the opposite sex (sex-separated) or together with members of the opposite sex (sex-combined) until six months of age, resulting in the generation of four cohorts of mice. From each mouse, the main olfactory epithelium (MOE), vomeronasal organ (VNO), and olfactory bulb (OB) were removed and RNA-extracted. A total of 36 RNA samples, representing three biological replicates per sex/condition/tissue combination, were analyzed for integrity and used to prepare RNA-seq libraries, which were subsequently analyzed via qPCR for the presence of tissue- or sex-specific markers. Libraries were paired-end sequenced to a depth of ~20 million fragments per replicate and the data were analyzed using the Tuxedo suite.


Assuntos
Perfilação da Expressão Gênica , Bulbo Olfatório , Mucosa Olfatória , Receptores Odorantes/genética , Caracteres Sexuais , Órgão Vomeronasal , Animais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Odorantes , Bulbo Olfatório/fisiologia , Mucosa Olfatória/fisiologia , Órgão Vomeronasal/fisiologia
6.
Nat Commun ; 9(1): 5081, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514924

RESUMO

Within the mammalian olfactory sensory epithelium, experience-dependent changes in the rate of neuronal turnover can alter the relative abundance of neurons expressing specific chemoreceptors. Here we investigate how the mouse olfactory sensory receptor repertoire changes as a function of exposure to odors emitted from members of the opposite sex, which are highly complex and sexually dimorphic. Upon housing mice either sex-separated or sex-combined until six months of age, we find that sex-separated mice exhibit significantly more numerous differentially expressed genes within their olfactory epithelia. A subset of these chemoreceptors exhibit altered expression frequencies following both sex-separation and olfactory deprivation. We show that several of these receptors detect either male- or female-specific odors. We conclude that the distinct odor experiences of sex-separated male and female mice induce sex-specific differences in the abundance of neurons that detect sexually dimorphic odors.


Assuntos
Neurônios Receptores Olfatórios/metabolismo , Caracteres Sexuais , Comportamento Sexual Animal/fisiologia , Olfato/fisiologia , Animais , Comportamento Animal , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Odorantes , Mucosa Olfatória/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo
7.
Dev Neurobiol ; 78(12): 1171-1190, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30246932

RESUMO

As the catalytic component of γ-secretase, presenilin (PS) has long been studied in the context of Alzheimer's disease through cleaving the amyloid precursor protein. PS/γ-secretase, however, also cleaves a multitude of single-pass transmembrane proteins that are important during development, including Notch, the netrin receptor DCC, cadherins, drebrin-A, and the EphB2 receptor. Because transgenic PS-KO mice do not survive to birth, studies of this molecule during later embryonic or early postnatal stages of development have been carried out using cell cultures or conditional knock-out mice, respectively. As a result, the function of PS in synapse formation had not been well-addressed. Here, we study the role of PS in the developing Xenopus tadpole retinotectal circuit, an in-vivo model that allows for protein expression to be manipulated specifically during the peak of synapse formation between retinal ganglion cells and tectal neurons. We found that inhibiting PS in the postsynaptic tectal neurons impaired tadpole visual avoidance behavior. Whole cell recordings indicated weaker retinotectal synaptic transmission which was characterized by significant reductions in both NMDA receptor (NMDAR)- and AMPA receptor (AMPAR)-mediated currents. We also found that expression of the C-tail fragment of the EphB2 receptor, which is normally cleaved by PS/γ-secretase and which has been shown to upregulate NMDARs at the synapse, rescued the reduced NMDAR-mediated responses. Our data determine that normal PS function is important for proper formation and strengthening of retinotectal synapses through cleaving the EphB2 receptor.


Assuntos
Comportamento Animal/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Presenilinas/metabolismo , Receptor EphB2/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Células Ganglionares da Retina/fisiologia , Colículos Superiores/fisiologia , Sinapses/fisiologia , Percepção Visual/fisiologia , Animais , Larva , Colículos Superiores/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Xenopus
8.
Trends Genet ; 31(9): 516-27, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26299477

RESUMO

The broad diversity of cell types within vertebrates arises from a unique genetic blueprint by combining intrinsic cellular information with developmental and other extrinsic signals. Lying at the interface between cellular signals and the DNA is the chromatin, a dynamic nucleoprotein complex that helps to mediate gene regulation. The most basic subunit of chromatin, the nucleosome, consists of DNA wrapped around histones, a set of proteins that play crucial roles as scaffolding molecules and regulators of gene expression. Growing evidence indicates that canonical histones are commonly replaced by protein variants before and during cellular transitions. We highlight exciting new results suggesting that histone variants are essential players in the control of cellular plasticity during development and in the adult nervous system.


Assuntos
Plasticidade Celular/genética , Variação Genética/fisiologia , Histonas/genética , Adulto , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Epigênese Genética , Humanos , Proteínas Mutantes/fisiologia
9.
Elife ; 4: e07860, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-26140685

RESUMO

The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased--rather than monoallelic--expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.


Assuntos
Alelos , Cerebelo/fisiologia , Perfilação da Expressão Gênica , Impressão Genômica , Animais , Bioestatística , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Análise de Sequência de DNA
10.
Elife ; 1: e00070, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23240083

RESUMO

We have identified a replication-independent histone variant, Hist2h2be (referred to herein as H2be), which is expressed exclusively by olfactory chemosensory neurons. Levels of H2BE are heterogeneous among olfactory neurons, but stereotyped according to the identity of the co-expressed olfactory receptor (OR). Gain- and loss-of-function experiments demonstrate that changes in H2be expression affect olfactory function and OR representation in the adult olfactory epithelium. We show that H2BE expression is reduced by sensory activity and that it promotes neuronal cell death, such that inactive olfactory neurons display higher levels of the variant and shorter life spans. Post-translational modifications (PTMs) of H2BE differ from those of the canonical H2B, consistent with a role for H2BE in altering transcription. We propose a physiological function for H2be in modulating olfactory neuron population dynamics to adapt the OR repertoire to the environment.DOI:http://dx.doi.org/10.7554/eLife.00070.001.


Assuntos
Histonas/genética , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Processamento de Proteína Pós-Traducional , Olfato/genética , Sequência de Aminoácidos , Animais , Morte Celular/genética , Expressão Gênica , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Odorantes/análise , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/citologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
Bioorg Med Chem ; 14(9): 3081-9, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16412655

RESUMO

Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.


Assuntos
DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Integrases/genética , Integrases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Sequência de Bases , Simulação por Computador , Humanos , Camundongos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Ratos , Especificidade por Substrato
12.
Proc Natl Acad Sci U S A ; 101(20): 7566-71, 2004 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-15138302

RESUMO

With few exceptions the genetic codes of all known organisms encode the same 20 amino acids, yet all that is required to add a new building block are a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique codon that specifies the amino acid. For example, the amber nonsense codon, TAG, together with orthogonal Methanococcus jannaschii or Escherichia coli tRNA/synthetase pairs have been used to genetically encode a variety of unnatural amino acids in E. coli and yeast, respectively. However, the availability of noncoding triplet codons ultimately limits the number of amino acids encoded by any organism. Here, we report the design and generation of an orthogonal synthetase/tRNA pair derived from archaeal tRNA(Lys) sequences that efficiently and selectively incorporates an unnatural amino acid into proteins in response to the quadruplet codon, AGGA. Frameshift suppression with L-homoglutamine (hGln) does not significantly affect protein yields or cell growth rates and is mutually orthogonal with amber suppression, permitting the simultaneous incorporation of two unnatural amino acids, hGln and O-methyl-L-tyrosine, at distinct positions within myoglobin. This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.


Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Códon , Código Genético , Pyrococcus horikoshii/genética , Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Evolução Molecular , Lisina-tRNA Ligase/metabolismo , Pyrococcus horikoshii/enzimologia , RNA de Transferência/metabolismo
13.
Chem Biol ; 10(11): 1085-94, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14652076

RESUMO

The basis for the altered DNA specificities of two Cre recombinase variants, obtained by mutation and selection, was revealed by their cocrystal structures. The proteins share similar substitutions but differ in their preferences for the natural LoxP substrate and an engineered substrate that is inactive with wild-type Cre, LoxM7. One variant preferentially recombines LoxM7 and contacts the substituted bases through a hydrated network of novel interlocking protein-DNA contacts. The other variant recognizes both LoxP and LoxM7 utilizing the same DNA backbone contact but different base contacts, facilitated by an unexpected DNA shift. Assisted by water, novel interaction networks can arise from few protein substitutions, suggesting how new DNA binding specificities might evolve. The contributions of macromolecular plasticity and water networks in specific DNA recognition observed here present a challenge for predictive schemes.


Assuntos
Integrases/metabolismo , Proteínas Virais/metabolismo , Água/química , Integrases/química , Modelos Moleculares , Proteínas Virais/química
14.
Nucleic Acids Res ; 31(23): 6700-9, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-14627803

RESUMO

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is 'orthogonal', meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA(Glu) sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.


Assuntos
Substituição de Aminoácidos , Archaea/enzimologia , Archaea/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutamato-tRNA Ligase/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , Acilação , Sequência de Bases , Códon sem Sentido/genética , Sequência Consenso , Proteínas de Escherichia coli/biossíntese , Glutamato-tRNA Ligase/genética , Dados de Sequência Molecular , Mutagênese , Conformação de Ácido Nucleico , Pyrococcus horikoshii/enzimologia , RNA de Transferência de Ácido Glutâmico/genética
16.
J Am Chem Soc ; 125(4): 935-9, 2003 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-12537491

RESUMO

We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.


Assuntos
Bactérias/genética , Genoma Bacteriano , Fenilalanina/análogos & derivados , Fenilalanina/genética , Aminoacil-tRNA Sintetases/biossíntese , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Mathanococcus/enzimologia , Mathanococcus/genética , Mioglobina/biossíntese , Mioglobina/genética , Fenilalanina/biossíntese , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência Aminoácido-Específico/genética
17.
Nat Biotechnol ; 20(10): 1044-8, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12244330

RESUMO

A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cm(r)) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E. coli.


Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Evolução Molecular Direcionada/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Engenharia de Proteínas/métodos , Aminoácidos/metabolismo , Linhagem Celular , Escherichia coli/classificação , Regulação Bacteriana da Expressão Gênica , Controle de Qualidade , Especificidade da Espécie , Especificidade por Substrato , Tirosina/metabolismo
18.
J Am Chem Soc ; 124(31): 9026-7, 2002 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-12148987

RESUMO

We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-l-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.


Assuntos
Azidas/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Reagentes de Ligações Cruzadas , Mathanococcus/enzimologia , Mioglobina/biossíntese , Espectrometria de Massas por Ionização por Electrospray , Tirosina-tRNA Ligase/metabolismo
19.
Proc Natl Acad Sci U S A ; 99(7): 4185-90, 2002 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-11904359

RESUMO

Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites. To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid. Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry. Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel loxP site, or negatively for variants that cannot recognize the wild-type loxP site. The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity. One of the identified recombinases selectively recombines a novel recombination site and operates at a rate identical to that of wild-type Cre. Analysis of the sequences of the resulting Cre variants provides insight into the evolution of these altered specificities. This and other systems should contribute to our understanding of protein-DNA recognition and may eventually be used to evolve custom-tailored recombinases that can be used for gene study and inactivation.


Assuntos
Integrases/genética , Recombinação Genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Biblioteca Gênica , Integrases/química , Proteínas Virais/química
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