RESUMO
Intact acetylcholine receptors have been purified on a novel affinity resin from three electric fish endemic to Australian waters. Their binding properties and morphology are compared with those of their northern hemisphere homolog, Torpedo marmorata. All four exhibit apparent dissociation constants, Kd, in the nanomolar range for the snake neurotoxin alpha-bungarotoxin and have a distinctive rosette-like appearance when viewed in negative stain under the electron microscope. Furthermore, these rosettes are paired, indicating that acetylcholine receptors from southern ocean electric fish exist as dimers, in the same fashion as their northern hemisphere counterparts. The cDNAs of the receptor's four subunits were sequenced from Hypnos monopterigium and the northern hemisphere counterpart, Torpedo marmorata, while cDNAs from only two subunits, alpha and delta, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the delta subunit of each of the newly sequenced fish species is a cysteine residue. Its conservation suggests that the mechanism for the observed dimerization of acetylcholine receptors is disulfide bond formation between the delta subunit of adjacent receptors, analogous to acetylcholine receptor dimers observed in other electric fish. It appears that this mechanism for receptor clustering is unique to acetylcholine receptors packed and organized in the specialized organs of electric fish. Alignment of the deduced protein sequences with the equivalent sequences from Torpedo californica and humans reveals a high degree of homology.
Assuntos
Evolução Molecular , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Torpedo/genética , Animais , Austrália , Sequência de Bases , Cromatografia em Camada Fina , Primers do DNA , DNA Complementar/genética , Dimerização , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Dados de Sequência Molecular , Oceanos e Mares , Filogenia , Ligação Proteica , Receptores Colinérgicos/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
We have characterized the molecular organization and expression of four proline-rich protein genes from Arabidopsis (AtPRPs). These genes predict two classes of cell wall proteins based on DNA sequence identity, repetitive motifs, and domain organization. AtPRP1 and AtPRP3 encode proteins containing an N-terminal PRP-like domain followed by a C-terminal domain that is biased toward P, T, Y, and K. AtPRP2 and AtPRP4 represent a second, novel group of PRP genes that encode two-domain proteins containing a non-repetitive N-terminal domain followed by a PRP-like region rich in P, V, K, and C. Northern hybridization analysis indicated that AtPRP1 and AtPRP3 are exclusively expressed in roots, while transcripts encoding AtPRP2 and AtPRP4 were most abundant in aerial organs of the plant. Histochemical analyses of promoter/beta-glucuronidase fusions localized AtPRP3 expression to regions of the root containing root hairs. AtPRP2 and AtPRP4 expression was detected in expanding leaves, stems, flowers, and siliques. In addition, AtPRP4 expression was detected in stipules and during the early stages of lateral root formation. These studies support a model for involvement of PRPs in specifying cell-type-specific wall structures, and provide the basis for a genetic approach to dissect the function of PRPs during growth and development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Peptídeos/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Sequência de Bases , Parede Celular/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Fases de Leitura Aberta , Peptídeos/química , Proteínas de Plantas/química , Prolina , Domínios Proteicos Ricos em Prolina , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
SbPRP1 is a member of the soybean (Glycine max L. Merr) proline-rich cell wall protein family and is expressed at high levels in root tissue. To characterize the sequences required for this expression, we have fused 1.1 kb of upstream flanking DNA sequence from an SbPRP1 genomic clone to a gene encoding beta-glucuronidase (GUS). This construct was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. Histochemical staining of GUS activity in transgenic tobacco indicated that SbPRP1 is expressed in the apical and elongating region of both primary and lateral roots, most strongly in the epidermis. A similar localization pattern was found in transformed hairy roots when this construct was introduced into cowpea (Vigna aconitifolia) using Agrobacterium rhizogenes-mediated transformation. Nested 5'-deletion analysis of the SbPRP1 promoter indicated that a minimal promoter for SbPRP1 expression in roots is located within the first 262 bases of upstream flanking DNA and that the region between -1080 and -262 is required for maximal expression of this gene. Gel retardation assays showed that nuclear factors can be detected in soybean roots which specifically bind to sequences located between -1080 and -623, a region which is needed for maximal expression of the SbPRP1 promoter. Northern hybridization analysis was also used to show that little SbPRP1 mRNA was present in roots during the first 24 h after imbibition. These studies indicate that SbPRP1 expression is localized to the actively growing region of the root and that this expression is temporally regulated during very early stages of seedling growth.
Assuntos
Fabaceae/genética , Glycine max/genética , Proteínas de Membrana/genética , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plantas Medicinais , Plantas Tóxicas , Sequência de Bases , Northern Blotting , Parede Celular/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Glucuronidase/genética , Glucuronidase/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA/genética , RNA/isolamento & purificação , Mapeamento por Restrição , Deleção de SequênciaRESUMO
A carrot (Daucus carota, L.) genomic clone (DcPRP1) was isolated on the basis of its homology to previously described cDNAs encoding a wound-inducible, proline-rich cell wall protein. DNA sequence analysis showed that DcPRP1 contains a single open-reading frame encoding a 235-amino acid protein that is colinear with that predicted from the cDNA sequence with the exception of four amino acids at the N terminus and a 60-nucleotide insertion present within the genomic clone. Genomic Southern hybridization analysis showed that the cloned sequence hybridized with a single restriction enzyme fragment using several restriction enzymes. Primer extension and northern hybridization analysis indicated that the expression of DcPRP1 is developmentally regulated and linked to the formation of storage roots, where this gene is expressed at high levels after wounding. The level of DcPRP1 mRNA was greatest in tissue immediately adjacent to the wound site. Treatment of unwounded carrot storage roots with 10 microM 2,4-dichlorophenoxy-acetic acid, indoleacetic acid, or naphthalene-1-acetic acid also resulted in the accumulation of DcPRP1 transcripts to a level equal to that seen in wounded tissue.
Assuntos
Regulação da Expressão Gênica , Ácidos Indolacéticos/farmacologia , Peptídeos/genética , Proteínas de Plantas/genética , Verduras/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Primers do DNA , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Biblioteca Genômica , Dados de Sequência Molecular , Biossíntese Peptídica , Proteínas de Plantas/biossíntese , Domínios Proteicos Ricos em Prolina , RNA Mensageiro/análise , Análise de Sequência de DNA , Distribuição Tecidual , Verduras/efeitos dos fármacosRESUMO
Processing of the 58 kDa to the 31 kDa form of inhibin (Inh) involves cleavage of the amino-terminal peptide (alpha N) from the alpha 43-subunit. We show that active immunisation of female sheep against a recombinant bovine alpha N impairs their fertility. In Exp 1, 5 treated (Group 1; 300 micrograms alpha N) and 6 control ewes (Group 2; adjuvant only) were immunized (Day 1) and given boosters on Days 22 and 56. In Group 1, mean +/- SEM binding of 125I-31 kDa Inh was less than 0.5% on Days 33 and 44, whereas binding of 125I-58 kDa Inh was 4.9 +/- 0.7 and 6.2 +/- 0.6%, respectively. In Group 2 binding of both tracers was less than 0.5%. The corpora lutea (CL)/ewe in Group 1 on Days 44 and 82 were 1.8 +/- 0.2 and 2.8 +/- 0.9, respectively, and were not different from those in Group 2 (1.7 +/- 0.3 and 1.5 +/- 0.2, respectively). One ewe in Group 1 versus 5/6 ewes in Group 2 were diagnosed pregnant. In Exp 2, 18 treated and 16 controls were immunized as in Exp 1. The binding of 125I-58 kDa Inh in treated ewes (2.4 +/- 0.3%) was greater than in controls (less than 0.5%) on Day 56. The CL/ewe in treated ewes (1.8 +/- 0.2) was similar to that in controls (2.0 +/- 0.1) on Day 76. All 16 control ewes but only 7/17 treated ewes were subsequently diagnosed pregnant. The plasma progesterone concentrations were similar in treated ewes which did (7.6 +/- 1.2 nmol/L) and did not (7.0 +/- 0.7) become pregnant. Neither basal nor GnRH-stimulated concentrations of LH, nor basal concentrations of Inh differed between treated and controls in Exp 2. Similarly, there were no differences in FSH, except that basal concentrations were higher in the luteal phase of treated ewes. We conclude that immunisation of ewes against alpha N results in a significant reduction in fertility.
Assuntos
Fertilidade , Imunização , Inibinas/fisiologia , Prenhez/fisiologia , Animais , Corpo Lúteo/fisiologia , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Liberador de Gonadotropina/farmacologia , Inibinas/imunologia , Hormônio Luteinizante/sangue , Substâncias Macromoleculares , Gravidez , Proteínas Recombinantes/imunologia , OvinosRESUMO
ß-Conglycinin, and abundant storage protein in soybean (Glycine max (L.) Merr.) seeds, is a trimeric protein consisting of various isomers containing the three subunits, α', α and ß. Accumulation of the ß-subunit is unique because it appears to be regulated by a variety of developmental and environmental signals. In this paper we describe the isolation and characterization of a genomic clone encoding the ß-subunit of ß-conglycinin. The genomic clone was characterized by restriction-enzyme mapping and partial DNA sequence analysis, by immunoprecipitation of a hybrid-selected invitro translation product, and by RNA blot hybridization reactions. An mRNA of approx. 1700 nucleotides hybridized to an internal 2-kilobase (kb) region of this 4.4-kb cloned DNA restriction fragment and was translated to yield a polypeptide with an approximate molecular weight of 48 kilodalton. This polypeptide is immunoprecipitable by antibody against ß-conglycinin and is of appropriate size to represent the precursor polypeptide of the ß-subunit. When this sequence was used as a probe in RNA blot hybridization experiments, the ß-gene transcript was first detected by stage K and accumulated through stage O during soybean seed development, coincident with appearance of the ß-subunit. Partial DNA sequence analysis of the 5' end of the gene confirmed that the isolated gene encoded a ß-subunit, based upon the previously reported amino terminal sequence for this protein. Genomic DNA blot hybridization analyses indicate that multiple DNA restriction fragments are highly homologous to this cloned ß-gene sequence.