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1.
J Cell Biol ; 219(12)2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33053147

RESUMO

Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59 cluster rafts and GM1 cluster rafts were stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK and continually recruited Lyn and H-Ras right beneath them in the inner leaflet with dwell lifetimes <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the nonraftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Because GM1 cluster rafts recruited Lyn and H-Ras as efficiently as CD59 cluster rafts, and because the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft-lipid interactions and thus serve as a key signal transduction platform.

2.
Nat Ecol Evol ; 4(2): 261-269, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907383

RESUMO

Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.


Assuntos
Padronização Corporal , Cílios , Animais , Embrião de Galinha , Madagáscar , Camundongos , Répteis , Peixe-Zebra
3.
J Cell Biol ; 215(5): 705-718, 2016 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-27881714

RESUMO

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be shed from the cell membrane by GPI cleavage. In this study, we report a novel GPI-processing enzyme, termed post-glycosylphosphatidylinositol attachment to proteins 6 (PGAP6), which is a GPI-specific phospholipase A2 mainly localized at the cell surface. CRIPTO, a GPI-AP, which plays critical roles in early embryonic development by acting as a Nodal coreceptor, is a highly sensitive substrate of PGAP6, whereas CRYPTIC, a close homologue of CRIPTO, is not sensitive. CRIPTO processed by PGAP6 was released as a lysophosphatidylinositol-bearing form, which is further cleaved by phospholipase D. CRIPTO shed by PGAP6 was active as a coreceptor in Nodal signaling, whereas cell-associated CRIPTO activity was reduced when PGAP6 was expressed. Homozygous Pgap6 knockout mice showed defects in early embryonic development, particularly in the formation of the anterior-posterior axis, which are common features with Cripto knockout embryos. These results suggest PGAP6 plays a critical role in Nodal signaling modulation through CRIPTO shedding.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Nodal/metabolismo , Fosfolipases A2/metabolismo , Animais , Padronização Corporal , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Knockout , Modelos Biológicos , Mutação/genética , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases/genética , Transdução de Sinais
4.
Dev Dyn ; 245(12): 1176-1188, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27666927

RESUMO

BACKGROUND: Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short-tailed opossum, a metatherian. RESULTS: Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post-coitus. CONCLUSIONS: Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176-1188, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Monodelphis/embriologia , Monodelphis/metabolismo , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Monodelphis/classificação , Proteína Nodal/genética , Proteína Nodal/metabolismo , Filogenia
5.
Dev Biol ; 415(1): 122-142, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27174471

RESUMO

The processes of development leading up to gastrulation have been markedly altered during the evolution of amniotes, and it is uncertain how the mechanisms of axis formation are conserved and diverged between mouse and chick embryos. To assess the conservation and divergence of these mechanisms, this study examined gene expression patterns during the axis formation process in Chinese soft-shell turtle and Madagascar ground gecko preovipositional embryos. The data suggest that NODAL signaling, similarly to avian embryos but in contrast to eutherian embryos, does not have a role in epiblast and hypoblast development in reptilian embryos. The posterior marginal epiblast (PME) is the initial molecular landmark of axis formation in reptilian embryos prior to primitive plate development. Ontogenetically, PME may be the precursor of the primitive plate, and phylogenetically, Koller's sickle and posterior marginal zone in avian development may have been derived from the PME. Most of the genes expressed in the mouse anterior visceral endoderm (AVE genes), especially signaling antagonist genes, are not expressed in the hypoblast of turtle and gecko embryos, though they are expressed in the avian hypoblast. This study proposes that AVE gene expression in the hypoblast and the visceral endoderm could have been independently established in avian and eutherian lineages, similar to the primitive streak that has been independently acquired in these lineages.


Assuntos
Padronização Corporal/fisiologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Lagartos/embriologia , Tartarugas/embriologia , Animais , Blastoderma/fisiologia , Padronização Corporal/genética , Endoderma/metabolismo , Gastrulação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Lagartos/genética , Lagartos/metabolismo , Proteína Nodal/fisiologia , Filogenia , Linha Primitiva/metabolismo , Especificidade da Espécie , Fatores de Transcrição/fisiologia , Tartarugas/genética , Tartarugas/metabolismo
6.
Dev Dyn ; 245(1): 67-86, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26404161

RESUMO

BACKGROUND: Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior-posterior (A-P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A-P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. RESULTS: All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2-positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp-positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A-P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. CONCLUSIONS: Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents.


Assuntos
Ectoderma/fisiologia , Desenvolvimento Embrionário/fisiologia , Endoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Padronização Corporal/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Nodal/genética , Coelhos , Suínos
7.
BMC Genomics ; 16: 977, 2015 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-26581708

RESUMO

BACKGROUND: RNA-seq enables gene expression profiling in selected spatiotemporal windows and yields massive sequence information with relatively low cost and time investment, even for non-model species. However, there remains a large room for optimizing its workflow, in order to take full advantage of continuously developing sequencing capacity. METHOD: Transcriptome sequencing for three embryonic stages of Madagascar ground gecko (Paroedura picta) was performed with the Illumina platform. The output reads were assembled de novo for reconstructing transcript sequences. In order to evaluate the completeness of transcriptome assemblies, we prepared a reference gene set consisting of vertebrate one-to-one orthologs. RESULT: To take advantage of increased read length of >150 nt, we demonstrated shortened RNA fragmentation time, which resulted in a dramatic shift of insert size distribution. To evaluate products of multiple de novo assembly runs incorporating reads with different RNA sources, read lengths, and insert sizes, we introduce a new reference gene set, core vertebrate genes (CVG), consisting of 233 genes that are shared as one-to-one orthologs by all vertebrate genomes examined (29 species)., The completeness assessment performed by the computational pipelines CEGMA and BUSCO referring to CVG, demonstrated higher accuracy and resolution than with the gene set previously established for this purpose. As a result of the assessment with CVG, we have derived the most comprehensive transcript sequence set of the Madagascar ground gecko by means of assembling individual libraries followed by clustering the assembled sequences based on their overall similarities. CONCLUSION: Our results provide several insights into optimizing de novo RNA-seq workflow, including the coordination between library insert size and read length, which manifested in improved connectivity of assemblies. The approach and assembly assessment with CVG demonstrated here would be applicable to transcriptome analysis of other species as well as whole genome analyses.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA , Animais , Benchmarking , Perfilação da Expressão Gênica/normas , Especificidade de Órgãos , Padrões de Referência , Répteis/embriologia , Répteis/genética
8.
Dev Biol ; 347(2): 392-403, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20816794

RESUMO

Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) and examined the TKO (Otx1(-/-)Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Fatores de Transcrição Otx/metabolismo , Animais , Sequência de Bases , Padronização Corporal , Primers do DNA/genética , Diencéfalo/embriologia , Diencéfalo/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Metencéfalo/embriologia , Metencéfalo/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Fatores de Transcrição Otx/deficiência , Fatores de Transcrição Otx/genética , Gravidez
9.
Development ; 137(17): 2939-49, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20667915

RESUMO

We have analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer we identified immediately 3' downstream of the last coding exon is well conserved among tetrapods and unexpectedly directed all the Emx2 expression in forebrain: caudal forebrain primordium at E8.5, dorsal telencephalon at E9.5-E10.5 and the cortical ventricular zone after E12.5. Otx, Tcf, Smad and two unknown transcription factor binding sites were essential to all these activities. The mutant that lacked this enhancer demonstrated that Emx2 expression under the enhancer is solely responsible for diencephalon development. However, in telencephalon, the FB enhancer did not have activities in cortical hem or Cajal-Retzius cells, nor was its activity in the cortex graded. Emx2 expression was greatly reduced, but persisted in the telencephalon of the enhancer mutant, indicating that there exists another enhancer for Emx2 expression unique to mammalian telencephalon.


Assuntos
Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/genética , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Sequência Conservada , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Fatores de Transcrição Otx/metabolismo , Fenótipo , Gravidez , Homologia de Sequência do Ácido Nucleico , Proteínas Smad/metabolismo , Especificidade da Espécie , Fatores de Transcrição TCF/metabolismo , Telencéfalo/embriologia , Telencéfalo/metabolismo , Fatores de Transcrição/deficiência , Xenopus/genética
10.
Dev Biol ; 325(1): 282-95, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18848537

RESUMO

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.


Assuntos
Padronização Corporal , Evolução Molecular , Cabeça/embriologia , Fatores de Transcrição Otx/genética , Homologia de Sequência do Ácido Nucleico , Vertebrados/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Fatores de Transcrição Otx/metabolismo , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Development ; 134(21): 3941-52, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17933795

RESUMO

Mammalian metaphase II (mII) exit and embryogenesis are induced at fertilisation by a signal thought to come from the sperm protein, phospholipase C-zeta (PLCZ1). Meiotic progression can also be triggered without sperm, as in parthenogenesis, although the classic mouse in vivo parthenogenetic model, LT/Sv, fails in meiosis I owing to an unknown molecular etiology. Here, we dissect PLCZ1 specificity and function in vivo and address its ability to interfere with maternal meiotic exit. Wild-type mouse Plcz1 expression was restricted to post-pubertal testes and the brains of both sexes, with region-specifying elements mapping to a 4.1 kb Plcz1 promoter fragment. When broad ectopic PLCZ1 expression was forced in independent transgenic lines, they initially appeared healthy. Their oocytes underwent unperturbed meiotic maturation to mII but subsequently exhibited autonomous intracellular free calcium oscillations, second polar body extrusion, pronucleus formation and parthenogenetic development. Transfer of transgenic cumulus cell nuclei into wild-type oocytes induced activation and development, demonstrating a direct effect of PLCZ1 analogous to fertilisation. Whereas Plcz1 transgenic males remained largely asymptomatic, females developed abdominal swellings caused by benign ovarian teratomas that were under-represented for paternally- and placentally-expressed transcripts. Plcz1 was not overexpressed in the ovaries of LT/Sv or in human germline ovarian tumours. The narrow spectrum of PLCZ1 activity indicates that it is modulated by tissue-restricted accessory factors. This work characterises a novel model in which parthenogenesis and tumourigenesis follow full meiotic maturation and are linked to fertilisation by PLCZ1.


Assuntos
Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Partenogênese , Fosfoinositídeo Fosfolipase C/metabolismo , Espermatozoides/metabolismo , Animais , Sequência de Bases , Transformação Celular Neoplásica , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase , Humanos , Masculino , Meiose , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Neoplasias Ovarianas/genética , Fosfoinositídeo Fosfolipase C/química , Fosfoinositídeo Fosfolipase C/genética , Sensibilidade e Especificidade
12.
EMBO J ; 25(4): 834-45, 2006 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-16456547

RESUMO

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Meiose/fisiologia , Metáfase/fisiologia , Oócitos/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Cdc20 , Células Cultivadas , Ciclina B/metabolismo , Proteínas F-Box/genética , Feminino , Fertilização/fisiologia , Meiose/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Camundongos , Oócitos/citologia , Partenogênese/efeitos dos fármacos , Partenogênese/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos
13.
Biophys J ; 88(5): 3659-80, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15681644

RESUMO

Diffusion of a G-protein coupled receptor, mu-opioid receptor (muOR), in the plasma membrane was tracked by single-fluorescent molecule video imaging and high-speed single-particle tracking. At variance with a previous publication, where gold-tagged muOR was found to be totally confined within a domain, which in turn underwent very slow diffusion itself, we found that muOR undergoes rapid hop diffusion over membrane compartments (210-nm and 730-nm nested double compartments in the case of normal rat kidney cell line), which are likely delimited by the actin-based membrane-skeleton "fence or corrals" and its associated transmembrane protein "pickets", at a rate comparable to that for transferrin receptor (every 45 and 760 ms on average, respectively), suggesting that the fence and picket models may also be applicable to G-protein coupled receptors. Further, we found that strong confinement of gold-labeled muOR could be induced by the prolonged on-ice preincubation of the gold probe with the cells, showing that this procedure should be avoided in future single-particle tracking experiments. Based on the dense, long trajectories of muOR obtained by high-speed single-particle tracking, the membrane compartments apposed and adjoined to each other could be defined that are delimited by rather straight boundaries, consistent with the involvement of actin filaments in membrane compartmentalization.


Assuntos
Biofísica/métodos , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Receptores Acoplados a Proteínas-G/química , Actinas/química , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Células CHO , Linhagem Celular , Coloides/química , Cricetinae , Difusão , Proteínas de Fluorescência Verde/metabolismo , Rim/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Vídeo , Modelos Biológicos , Modelos Moleculares , Modelos Estatísticos , Ratos , Temperatura , Tiazóis/química , Tiazolidinas , Fatores de Tempo
14.
Nucleic Acids Res ; 30(13): 2906-10, 2002 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12087176

RESUMO

Overlapping of genes, especially in an anti-parallel fashion, is quite rare in eukaryotic genomes. We have found a rare instance of exon overlapping involving CHRNE and MINK gene loci on chromosome 17 in humans. CHRNE codes for the epsilon subunit of the nicotinic acetylcholine receptor (AChRepsilon) whereas MINK encodes a serine/threonine kinase belonging to the GCK family. To elucidate the evolutionary trail of this gene overlapping event, we examined the genomes of a number of primates and found that mutations in the polyadenylation signal of the CHRNE gene in early hominoids led to the overlap. Upon extending this analysis to genomes of other orders of placental mammals, we observed that the overlapping occurred at least three times independently during the course of mammalian evolution. Because CHRNE and MINK are differentially expressed, the potentially hazardous mutations responsible for the exon overlap seem to have escaped evolutionary pressures by differential temporo-spatial expression of the two genes.


Assuntos
Evolução Molecular , Homologia de Genes/genética , Proteínas Serina-Treonina Quinases/genética , Receptores Nicotínicos/genética , Animais , Sequência de Bases , Bovinos , Cromossomos Humanos Par 17/genética , DNA/química , DNA/genética , Cães , Genoma , Genoma Humano , Cavalos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites , Dados de Sequência Molecular , Filogenia , Poli A/genética , Primatas , Coelhos , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Suínos , Baleias
15.
Oncogene ; 21(24): 3939-48, 2002 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-12032833

RESUMO

The p21-activated kinase (PAK) family of protein kinases has recently attracted considerable attention as an effector of Rho family of small G proteins and as an upstream regulator of MAPK signalling pathways during cellular events such as re-arrangement of the cytoskeleton and apoptosis. We have cloned a novel human PAK family kinase that has been designated as PAK5. PAK5 contains a CDC42/Rac1 interactive binding (CRIB) motif at the N-terminus and a Ste20-like kinase domain at the C-terminus. PAK5 is structurally most related to PAK4 and PAK6 to make up the PAK-II subfamily. We have shown that PAK5 preferentially binds to CDC42 in the presence of GTP and that CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5 activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly expressed in brain as revealed by Northern blot and in situ hybridization. The expression pattern of PAK5 is distinct from that of PAK4 and PAK6, suggesting a functional division among PAK-II subfamily kinases based on differential tissue distribution.


Assuntos
Encéfalo/enzimologia , Ciclinas/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Northern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 20 , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p21 , DNA Complementar/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Hibridização In Situ , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Distribuição Tecidual , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21 , Proteínas Quinases p38 Ativadas por Mitógeno
16.
J Biol Chem ; 277(8): 5929-39, 2002 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-11741893

RESUMO

We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK does not activate any mitogen-activated protein kinase pathways. Wild type MASK, but not a form lacking the C terminus, exhibits homophilic binding in the yeast two-hybrid system and in coimmunoprecipitation experiments. Additionally, deletion of this C-terminal region of MASK leads to an increased kinase activity toward itself as well as toward an exogenous substrate, myelin basic protein. A potential caspase 3 cleavage site (DESDS) is present in the C-terminal region of MASK, and we show that MASK is cleaved in vitro by caspase 3. Finally, wild type and C-terminally truncated forms of MASK can both induce apoptosis upon overexpression in mammalian cells that is abrogated by CrmA, suggesting involvement of MASK in the apoptotic machinery in mammalian cells.


Assuntos
Apoptose/fisiologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Fragmentação do DNA , Primers do DNA , Éxons , Centro Germinativo/enzimologia , Quinases do Centro Germinativo , Humanos , Íntrons , Mamíferos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia
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