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2.
Mol Microbiol ; 85(4): 669-83, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22686427

RESUMO

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived 'MgPar' donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10(-4) events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption-deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C-MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.


Assuntos
Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Variação Antigênica , Reparo do DNA , Mycoplasma genitalium/genética , Mycoplasma genitalium/metabolismo , Recombinases Rec A/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Deleção de Genes , Teste de Complementação Genética , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Mitomicina/toxicidade , Dados de Sequência Molecular , Taxa de Mutação , Mycoplasma genitalium/fisiologia , Reação em Cadeia da Polimerase , Recombinases Rec A/genética , Recombinação Genética , Análise de Sequência de DNA , Raios Ultravioleta
3.
ACS Synth Biol ; 1(7): 267-73, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-23651249

RESUMO

The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ~200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Composição de Bases , Cromossomos Artificiais de Levedura/química , Cromossomos Artificiais de Levedura/genética , Clonagem Molecular , DNA Recombinante/química , DNA Recombinante/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Synechococcus/genética , Synechococcus/metabolismo , Biologia Sintética
4.
Methods Mol Biol ; 634: 147-55, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20676982

RESUMO

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (e.g. cDNA, genomic DNA, and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3' sequences of the primers match the template perfectly. When two outer mutagenic oligonucleotides (oligos) are incorporated together with the desired mutagenic oligos into the newly synthesized mutant strand, they serve as anchors for PCR primers which have 3' sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.


Assuntos
Mutagênese Sítio-Dirigida , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Primers do DNA , Reação em Cadeia da Polimerase , Moldes Genéticos
5.
Science ; 329(5987): 52-6, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20488990

RESUMO

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.


Assuntos
Bioengenharia , Engenharia Genética , Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Proteínas de Bactérias/análise , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/síntese química , DNA Bacteriano/genética , Escherichia coli/genética , Deleção de Genes , Genes Bacterianos , Dados de Sequência Molecular , Mycoplasma mycoides/crescimento & desenvolvimento , Mycoplasma mycoides/fisiologia , Mycoplasma mycoides/ultraestrutura , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Saccharomyces cerevisiae/genética , Transformação Bacteriana
6.
Nat Methods ; 6(5): 343-5, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19363495

RESUMO

We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.


Assuntos
DNA Recombinante/biossíntese , DNA Recombinante/química , Engenharia Genética/métodos , Clonagem Molecular/métodos , DNA Ligases/metabolismo , DNA Circular/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Genes/genética , Técnicas Genéticas , Vetores Genéticos/biossíntese , Genoma/genética , Mycoplasma genitalium/genética , Fosfodiesterase I/metabolismo , Plasmídeos/biossíntese
7.
J Histochem Cytochem ; 57(6): 599-604, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19255251

RESUMO

Inhibitor of DNA-binding-1 (ID1) negatively regulates cell differentiation and senescence, and enhances cellular proliferation and angiogenesis. Elevated levels of ID1 have been found in a variety of cancers, including prostate cancer, but whether ID1 has a tumourigenic role remains to be established. We established heterozygous and homozygous ID1-transgenic mouse lines driven by the prostate-specific probasin promoter (-426 to +28 bp). Although elevated levels of ID1 were confirmed by RT-PCR, immunohistochemistry, and Western blot analysis, there were no morphological changes identified in the prostate of transgenic mice at 26 and 52 weeks. Thus, overexpression of ID1 alone is not sufficient to drive neoplastic change in mouse prostate.


Assuntos
Proteína de Ligação a Androgênios/genética , Biomarcadores Tumorais/biossíntese , Transformação Celular Neoplásica/metabolismo , Proteína 1 Inibidora de Diferenciação/biossíntese , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Transformação Celular Neoplásica/patologia , Proteína 1 Inibidora de Diferenciação/genética , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Próstata/patologia , Neoplasias da Próstata/patologia
8.
Science ; 319(5867): 1215-20, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18218864

RESUMO

We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.


Assuntos
Clonagem Molecular , DNA Bacteriano/síntese química , Genoma Bacteriano , Genômica/métodos , Mycoplasma genitalium/genética , Sequência de Bases , Cromossomos Artificiais Bacterianos , Cromossomos Artificiais de Levedura , DNA Recombinante , Escherichia coli/genética , Vetores Genéticos , Oligodesoxirribonucleotídeos/síntese química , Plasmídeos , Recombinação Genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Transformação Genética
9.
J Histochem Cytochem ; 54(2): 223-9, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16234506

RESUMO

Ornithine decarboxylase (ODC) is the key enzyme in the polyamine synthesis pathway and is overexpressed in a variety of cancers. We have performed a detailed immunostaining analysis of the expression of ODC in normal, benign prostatic hyperplasia (BPH), and cancerous prostate tissues. We conclude that ODC is overexpressed in both BPH and neoplastic tissues and that ODC overexpression appears to be an early event in prostate carcinogenesis. The extent of overexpression decreases as cancer progresses. Interestingly, ODC overexpression was also detected in patients who underwent androgen ablation therapy, suggesting ODC overexpression may contribute to the androgen-independent survival of prostate cancer cells. ODC is perinuclear localized in BPH samples but is diffusely cytoplasmic in cancer samples. Having shown ODC overexpression in human prostate cancer, we developed prostate-specific ODC transgenic mice to further investigate whether ODC overexpression alone is a causal factor in prostate carcinogenesis. RT-PCR and immunostaining confirmed that ODC was overexpressed in a subset of prostate epithelial cells. Although minor nucleoli enlargements in some tissues were detected, gross morphological changes were not observed in transgenic prostates. Therefore, overexpression of ODC alone in this subset of prostate epithelial cells is not sufficient to induce prostate carcinogenesis.


Assuntos
Ornitina Descarboxilase/biossíntese , Próstata/enzimologia , Proteína de Ligação a Androgênios/genética , Animais , Epitélio/enzimologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Ornitina Descarboxilase/genética , Regiões Promotoras Genéticas , Hiperplasia Prostática/enzimologia , Neoplasia Prostática Intraepitelial/enzimologia , Neoplasias da Próstata/enzimologia , RNA Mensageiro/biossíntese , Ratos
10.
Biotechnol Lett ; 27(12): 823-8, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16086242

RESUMO

Transgenic research often suffers from the lack of strong tissue specific promoters and the lack of suitable antibodies for transgene detection. We have now constructed a novel prostate specific 2-in-1 Tetracycline-off (Tet-off), bicistronic expression vector, designated PbTetOIE, for transgenic research. The vector allows potent induction as well as inducible suppression of transgene expression in the prostate epithelial cells, and also allows the detection of transgene expression at the cellular level through the detection of the internal enhanced green fluorescent protein (EGFP) downstream of the transgene.


Assuntos
Proteína de Ligação a Androgênios/genética , Vetores Genéticos/genética , Regiões Promotoras Genéticas/genética , Próstata/metabolismo , Transgenes/genética , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Clonagem Molecular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos , Microscopia de Fluorescência , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Ribossomos/metabolismo , Tetraciclina/farmacologia , Transfecção
11.
Gene ; 348: 55-63, 2005 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-15777716

RESUMO

Prostate cancer deaths are due to functional escape of prostate cancer cells from their original androgen-dependent growth. To better understand the origin and evolution of hormone-refractory prostate cancer, it is important to identify and characterize genes expressed in the androgen-deprived prostate. We have verified that the rudimentary prostate of congenital androgen deficient mice (hpg) is indeed androgen independent. Using suppression subtractive hybridization between mRNA derived from prostates of hypogonadal (hpg) with or without 14 days of testosterone replacement we have cloned a novel gene from the hpg prostate, termed ADMP (for androgen down regulated gene expressed in mouse prostate), that is down regulated by androgens. ADMP expression is strong in hpg mouse prostate, weak in mature castrated mouse prostate and absent in normal intact or androgen-replaced hpg mouse prostates. While ADMP expression is androgen independent in the hpg prostate, it appears to be androgen-dependent in the kidney and brain of normal intact mouse suggesting tissue specific regulation of ADMP by androgens. Human ADMP mRNA expression is suppressed by androgens in the androgen-sensitive LNCaP cell line. The predicted mouse and human protein of 76 amino acids shares sequence similarity to a putative G-protein coupled receptor indicating its possible role in signal transduction. Human ADMP expression was seen predominantly in the prostate epithelium with weaker expression in the fibroblasts and endothelial cells. Cloning and characterization of ADMP has made it feasible to determine its prospective role in the absence of androgens in prostate cancer.


Assuntos
Androgênios/farmacologia , Proteínas de Membrana/genética , Próstata/metabolismo , Adrenalectomia , Sequência de Aminoácidos , Androgênios/deficiência , Animais , Sequência de Bases , Linhagem Celular Tumoral , Cromossomos Humanos Par 3/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Epitélio/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização In Situ , Masculino , Metribolona/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Dados de Sequência Molecular , Orquiectomia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Serina C-Palmitoiltransferase
12.
Cancer Res ; 64(8): 2766-73, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15087392

RESUMO

A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.


Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Neoplasias da Glândula Tireoide/metabolismo , Apoptose/fisiologia , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Conformação Proteica , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Neoplasias da Glândula Tireoide/química , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Transfecção
13.
Nucleic Acids Res ; 32(7): e59, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15087491

RESUMO

In the post-genomic era, the ability to synthesize any arbitrary DNA sequence is increasingly in demand. A bottleneck in current gene synthesis technologies is the associated cost, due primarily to the high cost of oligonucleotides synthesis and post-synthesis sequencing. In the present paper, an improved method for low-cost gene synthesis that combines dual asymmetrical PCR and overlap extension PCR is presented, which enables any DNA sequence to be synthesized error free. Additionally, the method is easily amenable to automation.


Assuntos
Genes Sintéticos/genética , Técnicas Genéticas , Códon/genética , Eletroforese em Gel de Ágar , Escherichia coli/genética , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , Proinsulina/genética , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes
14.
Nucleic Acids Res ; 31(3): e11, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12560514

RESUMO

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3' sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3' sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.


Assuntos
Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Sequência de Bases , Biotecnologia , DNA/genética , Primers do DNA/química , Moldes Genéticos
15.
J Androl ; 23(5): 652-60, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12185099

RESUMO

To better understand the role of androgens in prostate development and disease it is important to characterize androgen-regulated genes in the prostate. Using suppression subtractive hybridization between congenitally androgen-deficient (hpg) and androgen-replaced hpg mouse prostates, we have cloned a novel androgen up-regulated gene from mouse prostate (AUMP). The messenger RNA sequence of AUMP consists of 805 nucleotides with an open reading frame of 408 base pairs. In non-hpg mice with normal androgen levels, AUMP is selectively expressed in the prostate, as shown by reverse transcriptase-polymerase chain reaction and Northern blot analysis of 9 organs. Depletion of androgens via castration of mature mice resulted in loss of AUMP expression, whereas testosterone replacement restored it. Tissue in situ hybridization localized AUMP expression to the luminal epithelial cells of the androgen-sufficient prostate. Database searches indicate that AUMP codes for a novel protein that shares approximately 65% similarity and 35% identity to palmitoyl protein thioesterase of human, rat, mouse, and bovine. A motif for protein-transport protein, which promotes translocation as well as integration of secretory proteins into membrane, is also present. Further efforts will be made to obtain the human homologue of AUMP that will enable evaluation of its role in normal and diseased human prostate.


Assuntos
Androgênios/genética , Clonagem Molecular , Próstata/metabolismo , Regulação para Cima , Sequência de Aminoácidos/genética , Androgênios/fisiologia , Animais , Sequência de Bases/genética , Epitélio/metabolismo , Previsões , Expressão Gênica , Masculino , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Distribuição Tecidual
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