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1.
Braz. j. biol ; Braz. j. biol;79(3): 488-494, July-Sept. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1001450

RESUMO

Abstract In this study we evaluate the biotic potential and reproductive parameters of Spodoptera cosmioides (Walker, 1858) under controlled conditions (25 ± 1 °C, 70 ± 10% RH and 14 hour photophase). Females, on average, (14.433 days) did not live significantly longer than their male counterparts (13.100 days). The mean durations of the pre-oviposition, oviposition and post-oviposition periods were 2.767, 10.600 and 1.067 days, respectively. The mean fecundity of females was 5,073.533 eggs and the mean fertility was 5,021.027 larvae. On average, females copulated 1.167 times. Fecundity was positively correlated with the number of copulations (r = 0.583, P <0.001). The number of copulations, however, was negatively correlated whit the duration of the pre-oviposition (r = -0.560, P = 0.007) and oviposition (r = -0.479, P = 0.048) periods, and overall longevity (r = -0.512, P = 0.031). The biotic potential was estimated at 6.547 x 1021 individuals/female/year. The net reproductive rate (Ro) was 2,193.722 times per generation and the mean generation time (T) was 46.407 days. The intrinsic rate of increase (rm) was 0.166, with a finite rate of increase (λ) of 1.180, per week.


Resumo Neste estudo avaliamos o potencial biótico e parâmetros reprodutivos de Spodoptera cosmioides (Walker, 1858) em condições controladas (25 ± 1 °C, 70 ± 10% UR e 14 horas de fotofase). Em média, as fêmeas (14,433 dias) não viveram significativamente mais do que os machos (13,100 dias). A duração média dos períodos de pre-oviposição, oviposição e pós-oviposição foram 2,767; 10,600 e 1,067 dias, respectivamente. A fecundidade média das fêmeas foi de 5.073,533 ovos e a fertilidade média foi de 5.021,027 larvas. Em média, as fêmeas copularam 1,167 vezes. A fecundidade correlacionou-se positivamente com o número de cópulas (r = 0,583; P <0,001). Entretanto, o número de cópulas correlacionou-se negativamente com a duração dos períodos de pré-oviposição (r = -0,560; P = 0,007), oviposição (r = -0,479; P = 0;048) e a longevidade (r = -0,512; P = 0,031). O potencial biótico foi estimado em 6,547 x 1021 indivíduos/fêmea/ano. A taxa líquida de reprodução (Ro) foi de 2.193,722 vezes por geração e o tempo médio de cada geração (T) foi de 46,407 dias. A taxa intrínseca de crescimento (rm) foi 0,166, com uma taxa finita de aumento (λ) de 1,180, por semana.


Assuntos
Animais , Masculino , Feminino , Oviposição , Comportamento Sexual Animal , Spodoptera/fisiologia , Características de História de Vida , Reprodução , Tábuas de Vida , Spodoptera/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/fisiologia , Longevidade
2.
Neotrop Entomol ; 48(3): 433-448, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30515720

RESUMO

Spodoptera marima (Schaus, 1904) and Spodoptera ornithogalli (Guenée, 1852) are noctuid moths recognized for their economic importance in the New World. Historically, they have been considered cryptic species, being supposedly differentiated from each other by the absence of sexual dimorphism in S. marima, subtle differences in coloration on the hindwings and scale tufts of the ovipositor and the shape of male valva. The species are allopatric: North and Central America for S. ornithogalli and South America for S. marima. Previous phylogenetic studies using multilocus DNA sequences including a few specimens of S. marima suggested these species merge into a single clade. Here, we performed a comparative analysis to elucidate further their taxonomic status, using specimens of S. marima from various localities in Brazil. These species were recovered in the phylogenetic analysis as consistently mixed with each other in a major well-supported clade within Spodoptera. The existence of a spatial pattern for genetic structure was not clearly supported when two biogeographic regions (Neotropics and Neartic) were taken in separation; however, an individual-level isolation by distance was significant. The morphological characters traditionally used to differentiate the species were also not consistent. Therefore, we now consider S. marima a new junior synonym of S. ornithogalli. Results on host plant use demonstrated that it is a polyphagous species using more than 200 plants, which may mediate phenotypic variation along its geographical range.


Assuntos
Variação Genética , Spodoptera/genética , Distribuição Animal , Animais , Teorema de Bayes , Brasil , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Genes Mitocondriais , Masculino , América do Norte , Filogenia , América do Sul , Spodoptera/classificação , Asas de Animais/anatomia & histologia
3.
Neotrop Entomol ; 48(1): 98-110, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29873032

RESUMO

Feltia submontana (Köhler, 1961) is redescribed based on specimens from Northwestern Argentina and Central and Southeastern Brazil. Taxonomic comments, photographs of the adults, characters of taxonomic importance, and illustrations of structures of the labial palpus, legs, and male and female genitalia are provided. The species is compared with similar-looking and supposedly closely related species, such as F. hispidula (Guenée, 1852) and F. lilacina (Zerny, 1916). The species, originally described for Argentina, is reported for Brazil for the first time. Most Brazilian specimens come from the "Cerrado" but also from Southeastern Atlantic Forests. The life cycle of F. submontana specimens collected in Planaltina, Distrito Federal, Brazil, is described; the species probably has only a single generation per year and imagines are on the wing in the late autumn and early winter months; the last instar prepupa and pupa pass through aestival diapause. The abundance of F. submontana relative to other species of Agrotis Ochsenheimer, 1816, and Feltia Walker, 1856, in the above-cited locality is accessed through 4 years of standardized collecting with light trap; the species is the second most abundant species of these genera in the area, with about one fifth of the captures, second only to A. ipsilon (Hufnagel, 1766), with about two thirds of the captures, and about two times more abundant than F. subterranea (Fabricius, 1794); the latter two are regarded as important pest species in South America.


Assuntos
Mariposas/classificação , Distribuição Animal , Animais , Argentina , Brasil , Feminino , Genitália Feminina , Genitália Masculina , Estágios do Ciclo de Vida , Masculino , Mariposas/anatomia & histologia
4.
Braz J Biol ; 79(3): 488-494, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30304293

RESUMO

In this study we evaluate the biotic potential and reproductive parameters of Spodoptera cosmioides (Walker, 1858) under controlled conditions (25 ± 1 °C, 70 ± 10% RH and 14 hour photophase). Females, on average, (14.433 days) did not live significantly longer than their male counterparts (13.100 days). The mean durations of the pre-oviposition, oviposition and post-oviposition periods were 2.767, 10.600 and 1.067 days, respectively. The mean fecundity of females was 5,073.533 eggs and the mean fertility was 5,021.027 larvae. On average, females copulated 1.167 times. Fecundity was positively correlated with the number of copulations (r = 0.583, P <0.001). The number of copulations, however, was negatively correlated whit the duration of the pre-oviposition (r = -0.560, P = 0.007) and oviposition (r = -0.479, P = 0.048) periods, and overall longevity (r = -0.512, P = 0.031). The biotic potential was estimated at 6.547 x 1021 individuals/female/year. The net reproductive rate (Ro) was 2,193.722 times per generation and the mean generation time (T) was 46.407 days. The intrinsic rate of increase (rm) was 0.166, with a finite rate of increase (λ) of 1.180, per week.


Assuntos
Características de História de Vida , Oviposição , Comportamento Sexual Animal , Spodoptera/fisiologia , Animais , Feminino , Larva/crescimento & desenvolvimento , Larva/fisiologia , Tábuas de Vida , Longevidade , Masculino , Reprodução , Spodoptera/crescimento & desenvolvimento
5.
Neotrop Entomol ; 47(3): 344-351, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28484967

RESUMO

This study aimed to evaluate the biotic potential and life table of individuals of Helicoverpa armigera (Hübner) from different host plants (citrus, corn, and cotton) and Brazilian states (São Paulo, Distrito Federal, and Bahia) in artificial diet, under laboratory conditions (25 ± 1°C, 70 ± 10% RH, 14 h photophase). The longevity, pre-, post- and oviposition periods, fecundity, and fertility of 15 mating pairs per origin were evaluated. We also compared the reproductive parameters of each group of insects (São Paulo (SP), Distrito Federal (DF), and Bahia (BA)), including the net reproductive rate (Ro), mean generation time (T), intrinsic rate of increase (r m), and finite rate of increase (λ). Microsatellite analysis from individuals collected in different locations and host plants did not show differences among the parental insects. It was verified that parental progeny collected in cotton fields from Bahia had a higher biotic potential, a higher reproductive rate (Ro), and a better fecundity compared to the insects from remaining regions. The life table charts indicate that the highest values for the reproductive parameters of the Bahia progeny are associated with higher specific fertility, particularly in early adulthood. The greatest biotic potential of the Bahia progeny may be due to increased selection pressure from the insecticide used (organophosphate and pyrethroid) on cotton crops compared to that of other crops, as well due to the massive adoption of Bt cotton-producing areas of that state from 2013 outbreaks.


Assuntos
Lepidópteros/fisiologia , Animais , Brasil , Citrus , Dieta , Feminino , Fertilidade , Gossypium , Tábuas de Vida , Longevidade , Masculino , Repetições de Microssatélites , Oviposição , Reprodução , Zea mays
6.
Braz. j. biol ; Braz. j. biol;2017.
Artigo em Inglês | LILACS-Express | LILACS, VETINDEX | ID: biblio-1467214

RESUMO

Abstract In this study we evaluate the biotic potential and reproductive parameters of Spodoptera cosmioides (Walker, 1858) under controlled conditions (25 ± 1 °C, 70 ± 10% RH and 14 hour photophase). Females, on average, (14.433 days) did not live significantly longer than their male counterparts (13.100 days). The mean durations of the pre-oviposition, oviposition and post-oviposition periods were 2.767, 10.600 and 1.067 days, respectively. The mean fecundity of females was 5,073.533 eggs and the mean fertility was 5,021.027 larvae. On average, females copulated 1.167 times. Fecundity was positively correlated with the number of copulations (r = 0.583, P 0.001). The number of copulations, however, was negatively correlated whit the duration of the pre-oviposition (r = -0.560, P = 0.007) and oviposition (r = -0.479, P = 0.048) periods, and overall longevity (r = -0.512, P = 0.031). The biotic potential was estimated at 6.547 x 1021 individuals/female/year. The net reproductive rate (Ro) was 2,193.722 times per generation and the mean generation time (T) was 46.407 days. The intrinsic rate of increase (rm) was 0.166, with a finite rate of increase () of 1.180, per week.


Resumo Neste estudo avaliamos o potencial biótico e parâmetros reprodutivos de Spodoptera cosmioides (Walker, 1858) em condições controladas (25 ± 1 °C, 70 ± 10% UR e 14 horas de fotofase). Em média, as fêmeas (14,433 dias) não viveram significativamente mais do que os machos (13,100 dias). A duração média dos períodos de pre-oviposição, oviposição e pós-oviposição foram 2,767; 10,600 e 1,067 dias, respectivamente. A fecundidade média das fêmeas foi de 5.073,533 ovos e a fertilidade média foi de 5.021,027 larvas. Em média, as fêmeas copularam 1,167 vezes. A fecundidade correlacionou-se positivamente com o número de cópulas (r = 0,583; P 0,001). Entretanto, o número de cópulas correlacionou-se negativamente com a duração dos períodos de pré-oviposição (r = -0,560; P = 0,007), oviposição (r = -0,479; P = 0;048) e a longevidade (r = -0,512; P = 0,031). O potencial biótico foi estimado em 6,547 x 1021 indivíduos/fêmea/ano. A taxa líquida de reprodução (Ro) foi de 2.193,722 vezes por geração e o tempo médio de cada geração (T) foi de 46,407 dias. A taxa intrínseca de crescimento (rm) foi 0,166, com uma taxa finita de aumento () de 1,180, por semana.

7.
Braz. j. biol ; Braz. j. biol;76(2): 526-530, Apr.-June 2016. tab, graf
Artigo em Inglês | LILACS | ID: lil-781395

RESUMO

Abstract This study aimed to evaluate the effect of keeping three couples in the same cage, and the size of adults emerged from small, medium-sized and large pupae (278.67 mg; 333.20 mg and 381.58 mg, respectively), on the reproductive potential of S. eridania (Stoll, 1782) adults, under controlled conditions (25 ± 1 °C, 70% RH and 14 hour photophase). We evaluated the survival, number of copulations, fecundity and fertility of the adult females. The survival of females from these different pupal sizes did not differ statistically, but the survival of males from large pupae was statistically shorter than from small pupae. Fecundity differed significantly and correlated positively with size. The number of effective copulations (espematophores) and fertility did not vary significantly with pupal size. Our results emphasize the importance of indicating the number of copulations and the size of the insects when reproductive parameters are compared.


Resumo Este estudo objetivou avaliar o efeito de confinar três casais em cada gaiola e o tamanho de adultos emergidos de pupas pequenas, medias e grandes (278,67 mg, 333,20 mg e 381,58 mg, respectivamente), sobre o potencial reprodutivo de S. eridania (Stoll, 1782), em condições controladas (25 ± 1 °C, 70% UR e 14 horas de fotofase). Avaliou-se a sobrevivência, o número de cópulas, fecundidade e fertilidade dos adultos. A sobrevivência não diferiu significativamente entre fêmeas provenientes de pupas de diferentes tamanhos, mas os machos oriundos de pupas grandes tiveram sobrevivência significativamente menor que os demais tamanhos. A fecundidade diferiu significativamente e correlacionou-se positivamente com o tamanho. O número de cópulas (espematóforos) e a fertilidade não variaram em função do peso pupal. Os resultados enfatizam a importância de indicar o número de cópulas e o tamanho dos insetos estudados para que comparações entre os parâmetros reprodutivos possam ser efetuadas.


Assuntos
Animais , Masculino , Feminino , Spodoptera/crescimento & desenvolvimento , Spodoptera/fisiologia , Pupa/fisiologia , Meio Ambiente , Fenômenos Reprodutivos Fisiológicos , Fertilidade , Larva/fisiologia
8.
Braz J Biol ; 76(2): 526-30, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26959952

RESUMO

This study aimed to evaluate the effect of keeping three couples in the same cage, and the size of adults emerged from small, medium-sized and large pupae (278.67 mg; 333.20 mg and 381.58 mg, respectively), on the reproductive potential of S. eridania (Stoll, 1782) adults, under controlled conditions (25 ± 1 °C, 70% RH and 14 hour photophase). We evaluated the survival, number of copulations, fecundity and fertility of the adult females. The survival of females from these different pupal sizes did not differ statistically, but the survival of males from large pupae was statistically shorter than from small pupae. Fecundity differed significantly and correlated positively with size. The number of effective copulations (espematophores) and fertility did not vary significantly with pupal size. Our results emphasize the importance of indicating the number of copulations and the size of the insects when reproductive parameters are compared.


Assuntos
Spodoptera , Animais , Meio Ambiente , Feminino , Fertilidade , Larva/fisiologia , Masculino , Pupa/fisiologia , Fenômenos Reprodutivos Fisiológicos , Spodoptera/crescimento & desenvolvimento , Spodoptera/fisiologia
9.
Neotrop Entomol ; 45(1): 22-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26429580

RESUMO

We provide detailed temporal and morphological parameters of the immature stages of Spodoptera dolichos (Fabricius) larvae fed on artificial diet under controlled conditions (25 ± 1 °C, 70 ± 10% RH, and 14 h photophase). The viability of the egg, larval, pupal, and prepupal stages was 97.5%, 97.0%, 93.1%, and 98.9%, respectively. The average duration of the egg, larval, prepupal, and pupal stages was 5.0, 23.4, 3.2, and 21.5 days, respectively. Females took longer at the larval stage than males, with 10.5% of them having seven instars. The growth rate of female larvae that developed through six and seven instars was 1.72 and 1.54, respectively. Female pupae were significantly larger, exhibiting slower development than males.


Assuntos
Spodoptera/crescimento & desenvolvimento , Animais , Feminino , Larva/crescimento & desenvolvimento , Masculino , Pupa/crescimento & desenvolvimento
10.
Braz J Biol ; 74(1): 205-11, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25055103

RESUMO

We studied the biology of Tiracola grandirena (Herrich-Schäffer, 1868) (Lepidoptera: Noctuidae: Hadeninae) at 25 ± 1 °C, 70 ± 10% RH and 14 hours of photo phase. Three experiments, using 150 larvae each, were conducted for the larval stage. In the first, used to assess the duration and survival of all stages, insects were reared individually and fed an artificial diet (Grenee). In the second, individuals were also reared separately, but were fed leaves of 10 plants from different families. In the third, the larvae were not individualised, the food plants were rotated such as to provide three plant species every 48 hours. In the first experiment, the viability of the eggs, larvae, pupae and prepupae was 91.9, 94.7, 32.49 and 43.5%, respectively. The average duration of the egg, larvae, prepupae, pupae and adult were 6.0, 25.3, 25.7, 21.4 and 12.7 days, respectively. The prolonged prepupal period indicates that T. grandirena can develop larval (prepupal) diapause. The results of the experiments with different host plants showed that T. grandirena is polyphagous at species, population and individual level.


Assuntos
Lepidópteros/fisiologia , Estágios do Ciclo de Vida/fisiologia , Animais , Feminino , Larva/crescimento & desenvolvimento , Lepidópteros/classificação , Lepidópteros/crescimento & desenvolvimento , Masculino , Análise de Sobrevida
11.
Eur J Cell Biol ; 80(8): 527-38, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11561904

RESUMO

Phytoalexins originating from plant tissues may cause within cells of fungi or oomycetes a change in the localization of actin, tubulin and chaperones. To test the hypothesis in a filamentously growing oomycete, we compared the distribution of cellular markers in the presence and absence of hydroxystilbene phytoalexins. Using cDNA from the phytopathogenic organism Phytophthora megasperma, the causal agent of root rot on soybean and many other plants, and including probes for Hsp70 and Hsp40, we cloned a DnaJ-protein (Jcp) with the capacity of interacting with both a particular Hsp70 isoform via its J-domain and with other proteins via its tetratricopeptide repeat (TPR) domain. Antisera raised against the bacterially expressed protein Jcp allowed the analysis of its intracellular localization during hyphal growth. Following the subfractionation of cell homogenates, we detected virtually all immunoreactive Jcp in the plasma membrane-enriched fraction and as constituent of a membrane-associated protein complex. In agreement with the biochemical findings, immunocytochemical stains of hyphae showed Jcp as part of cortical patches positioned along the plasma membrane similar to the distribution of actin patches. Confocal microscopy, however, revealed that the Jcp-containing patches did not generally co-localize with the patches visualized by the actin stain. The 59-kDa Jcp, characterized by a large 8-fold TPR domain at the N-terminal region and a J-domain close to the C-terminus, is a good candidate for bridging the gap between Hsp70 and Hsp90 by protein-protein interactions. By administration of plant-derived phytoalexins it was shown that the presence of resveratrol or piceatannol significantly reduces the amount of the Jcp-containing patches, but does not lead to a relocalization of intracellular Jcp.


Assuntos
Proteínas de Choque Térmico/química , Phytophthora/química , Sequência de Aminoácidos , Membrana Celular , Clonagem Molecular , Escherichia coli/genética , Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico/genética , Hifas/efeitos dos fármacos , Proteínas de Membrana/química , Dados de Sequência Molecular , Micélio/química , Extratos Vegetais/farmacologia , Sequências Repetitivas de Aminoácidos , Sesquiterpenos , Frações Subcelulares/química , Terpenos , Fitoalexinas
12.
J Biol Chem ; 275(50): 39193-9, 2000 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-10973973

RESUMO

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.


Assuntos
Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/fisiologia , Coração/embriologia , Miocárdio/citologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Bário/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Éxons , Vetores Genéticos , Genótipo , Homozigoto , Concentração Inibidora 50 , Íons , Cinética , Camundongos , Camundongos Knockout , Modelos Genéticos , Nisoldipino/farmacologia , Fenótipo , RNA/metabolismo , Fatores de Tempo
13.
FEBS Lett ; 470(2): 189-97, 2000 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-10734232

RESUMO

The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel.


Assuntos
Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/metabolismo , Família Multigênica/genética , Sequência de Aminoácidos , Animais , Bário/metabolismo , Western Blotting , Encéfalo/metabolismo , Cálcio/metabolismo , Canais de Cálcio Tipo T/genética , Linhagem Celular , Clonagem Molecular , Condutividade Elétrica , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ , Cinética , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transfecção
14.
Yeast ; 14(5): 419-30, 1998 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-9559550

RESUMO

DnaJ proteins have been localized in different intracellular compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)(+)-RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39.5 kDa protein. The putative protein contains neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p.


Assuntos
Candida/genética , Candida/metabolismo , Proteínas Fúngicas/biossíntese , Resposta ao Choque Térmico , Ribossomos/química , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Candida/química , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Citosol/química , DNA Complementar/isolamento & purificação , Proteínas Fúngicas/análise , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico , Dados de Sequência Molecular , Biossíntese de Proteínas , Análise de Sequência de DNA , Frações Subcelulares
15.
Eur J Biochem ; 224(2): 533-40, 1994 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-7925369

RESUMO

The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site-directed mutagenesis, chemical modification of wild-type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active-site residue Y25 has a normal pKa while the C-terminal Y326 is unusually acidic. 1H-NMR signals of a peculiar ligand-sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14-27) which shields the active site during catalysis is highly flexible in the free enzyme.


Assuntos
Asparaginase/química , Asparaginase/metabolismo , Escherichia coli/enzimologia , Estrutura Secundária de Proteína , Tirosina , Asparaginase/biossíntese , Sequência de Bases , Bromosuccinimida , Primers do DNA , Guanidina , Guanidinas , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinâmica
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