Bacteriophage T4 self-assembly: localization of gp3 and its role in determining tail length

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.     

Programmed cell death in Xenopus laevis spinal cord, tail and other tissues, prior to, and during, metamorphosis

Programmed cell death is necessary for the shaping and remodelling of nervous and non-nervous tissues during development. Amphibia, whose body undergoes profound modifications during metamorphosis, are particularly useful models for studying the relationship between cell death in muscles and other non-nervous tissues on the one hand, and in the nervous system connected with these tissues on the other hand. We checked the occurrence of apoptotic cells (identified by TUNEL labelling) in different organs and regions from hatching (stages 35-36) to climax (stages 63-64) in the African Clawed Frog Xenopus laevis. Some organs (e.g., skin and digestive tract) contained apoptotic cells during the entire period studied. In transitory organs (cement gland and gills), a single wave of cell death occurred during the regression of these tissues. In order to compare the timing of cell death in the spinal cord with that of tail regression, we counted the number of TUNEL-positive cells in spinal cord sections taken from animals between stages 54 and 64. Three-dimensional reconstructions using confocal microscopy of vibratome slices immunostained for the detection of c-Jun-like protein accumulated in the cytoplasm of apoptotic cells showed numerous cells at various degrees of degeneration. Many of these cells still presented the morphological characteristics of neurones. The peak of apoptosis was found at stage 58, preceding tail regression. This suggests that neural cell death is not a consequence but rather an element upstream in the chain of events leading to tail degeneration.     

Changes of the Golgi apparatus and lysosomes during decidualization in mice.

The Golgi apparatus of the endometrial stromal cells of pregnant mice increases in size simultaneously with the differentiation of stromal cells into decidual cells. The activity of acid phosphatase in this organelle increases during this stage. On the other hand, the involuting decidual cells show morphological and cytochemical signs of Golgi regression (dilated cisternae, lack of enzymatic activity) together with the finding of numerous, pleomorphic lysosomes that have intense cytochemical label. These results confirm morphological data suggesting that decidual cell death occurs by autophagic degeneration.     

Identification and characterization of cytochrome bc(1) subcomplexes in mitochondria from yeast with single and double deletions of genes encoding cytochrome bc(1) subunits

We have examined the status of the cytochrome bc(1) complex in mitochondrial membranes from yeast mutants in which genes for one or more of the cytochrome bc(1) complex subunits were deleted. When membranes from wild-type yeast were resolved by native gel electrophoresis and analyzed by immunodecoration, the cytochrome bc(1) complex was detected as a mixed population of enzymes, consisting of cytochrome bc(1) dimers, and ternary complexes of cytochrome bc(1) dimers associated with one and two copies of the cytochrome c oxidase complex. When membranes from the deletion mutants were resolved and analyzed, the cytochrome bc(1) dimer was not associated with the cytochrome c oxidase complex in many of the mutant membranes, and membranes from some of the mutants contained a common set of cytochrome bc(1) subcomplexes. When these subcomplexes were fractionated by SDS/PAGE and analyzed with subunit-specific antibodies, it was possible to recognize a subcomplex consisting of cytochrome b, subunit 7 and subunit 8 that is apparently associated with cytochrome c oxidase early in the assembly process, prior to acquisition of the remaining cytochrome bc(1) subunits. It was also possible to identify a subcomplex consisting of subunit 9 and the Rieske protein, and two subcomplexes containing cytochrome c(1) associated with core protein 1 and core protein 2, respectively. The analysis of all the cytochrome bc(1) subcomplexes with monospecific antibodies directed against Bcs1p revealed that this chaperone protein is involved in a late stage of cytochrome bc(1) complex assembly.     

Leishmania major: identification of developmentally regulated proteins in procyclic and metacyclic promastigotes

The differentiation from procyclic to metacyclic promastigotes (metacyclogenesis) has been correlated with an increased infectivity in a number of Leishmania species. We compared the proteomes of procyclic and metacyclic promastigotes of L. major. Lysates from either life cycle stage were resolved by 2D-PAGE, followed by Coomassie brilliant blue staining. Spots were analyzed by MALDI-TOF MS. 25 protein spots were found to be differentially expressed during metacyclogenesis. We found that proteins involved in protein synthesis were less abundant in metacyclic promastigotes, while proteins involved in motility, including paraflagellar rod protein 1D, α-tubulin and β-tubulin were more abundant. Also, two mitochondrial enzymes (succinyl-CoA synthetase β subunit and cytochrome c oxidase subunit IV) were differentially expressed in both life cycle stages. Down-regulation of proteins related to synthetic pathway in metacyclic promastigotes is consistent with the arrested growth in this life cycle stage, while up-regulation of proteins related to motility in metacyclic promastigotes is in agreement with the high motility observed in this stage.     

Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.     

EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. I. STIMULATORY EFFECT OF PROLACTIN ON THE COLLAGEN SYNTHESIS OF THE TADPOLE TAIL FIN

Bovine prolactin stimulated the growth of connective tissues both in the tail fins and in other regions of the tadpole tail. Correlated with the morphological effect of the hormone on the tadpole tails, protein synthesis in tail fins was promoted about 2 times by prolactin. Experiments performed to determine the kind of protein, the synthesis of which was stimulated by prolactin, revealed that the hormone specifically enhanced collagen synthesis about 40 folds as compared to untreated animals.     

The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution of the activated 45,000-dalton (alpha) subunit

Activation of the stimulatory guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by guanine nucleotides or by Al3+, Mg2+, and F-stabilizes the protein to thermal denaturation or to inactivation by LiBr, guanidine HCl, or urea. Such activation allows the resolution of the active 45,000-Da alpha subunit from the 35,000-Da beta subunit by a high performance gel filtration procedure. Separation of the active alpha subunit has allowed definitive evaluation of the subunit dissociation model for the activation of G/F. The resolved alpha subunit is sufficient to reconstitute the adenylate cyclase activity of the cyc-S49 cell mutant. The alpha subunit alone is also sufficient to activate a preparation of the catalyst of adenylate cyclase that had been resolved from all other identified components of the enzyme system. The resolved alpha subunit displays hydrodynamic properties characteristic of activated G/F. The alpha subunit contains a high affinity guanine nucleotide-binding site. Activation of G/F by guanine nucleotides or by Al3+ + Mg2+ + F- allows resolution of the activated alpha subunit. Reversal of the activated state of the resolved alpha subunit occurs only slowly. Addition of beta subunit enhances the rate of deactivation. Deactivation of the activated alpha subunit by the beta subunit changes the S20,w for G/F activity from 2.0 to 4.0 (in Lubrol), consistent with a formation of the alpha X beta heterodimer. These data, taken in aggregate, constitute proof for the proposed mechanism of activation of G/F by non-hydrolyzable analogs of GTP and by Al3+, Mg2+, and F-. They are analogous to data obtained for transducin, the GTP-binding regulatory protein from vertebrate rod outer segment discs, and for the putative inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (the substrate for islet-activating protein). The model provides several powerful tests for study of mechanisms of hormonal regulation of adenylate cyclase in membranes.     

A G protein gamma subunit peptide stabilizes a novel muscarinic receptor state

A prenylated peptide specific to the C terminal tail of a G protein gamma subunit type, gamma5, inhibits activation of a G protein by the M2 muscarinic receptor. The gamma5 peptide was tested for direct effects on the M2 receptor's properties. The wild type gamma5 peptide reduced the affinity of M2 for the agonist, carbachol, more than 5-fold in an antagonist displacement assay. The peptide was inactive when its amino acid sequence was scrambled or when it was unprenylated. Although the wild type peptide reduced the affinity of M2 for the antagonist QNB, it had no effect on the antagonists NMS or atropine. These results suggest that in the presence of the peptide the M2 receptor adopts a novel conformational state that affects the ligand binding surface. The results also suggest that the G protein gamma5 subunit tail interacts with a receptor.     

Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins

The association of agonists with muscarinic receptors in membranes from bovine brain was affected only slightly by guanine nucleotides. However, solubilization of these membranes with deoxycholate and subsequent removal of detergent resulted in a preparation of receptors with increased affinity for agonists and a large increase in response to guanine nucleotides. Chromatography of deoxycholate extracts of membranes on DEAE-Sephacel resulted in the separation of receptors from 95% of the guanine nucleotide-binding activity. Guanine nucleotides had no effect on the binding of agonists to these resolved receptors. The effect of guanine nucleotides was restored after the addition of either of two purified guanine nucleotide-binding proteins from bovine brain. One of these proteins, presumably brain GI, is composed of subunits with the same molecular weights (alpha, 41,000; beta, 35,000; gamma, 11,000) and functions as the inhibitory guanine nucleotide-binding protein isolated from liver. The other protein, termed Go, is a novel guanine nucleotide-binding protein that possesses a similar subunit composition (alpha, 39,000; beta, 35,000; gamma, 11,000) but whose function is not yet known. Addition of either protein to the resolved receptor preparation increased agonist affinity by at least 10-20-fold, and low concentrations of guanine nucleotides specifically reversed this effect. Reconstitution of receptors with the resolved subunits of Go demonstrates that the beta subunit alone had no effect on agonist binding, but that this subunit does appear to enhance the effects observed with the alpha subunit alone.     

Truncation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum affects the holoenzyme assembly and activity.

Truncations of the subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum were generated by site-directed mutagenesis to examine the role of the C-terminal tail section. Removal of the last and the penultimate alpha-helices in the tail section changes the quaternary structure of the protein. Electrophoretic and electron microscope analysis revealed that the truncated subunits assemble into an octamer, whereas the wild-type enzyme has a dimeric structure. The octomerization of the mutant protein is due to a hydrophobic patch exposed to the solvent by truncation of the subunit. The mutant protein thus consists of four dimers, bound end-to-end by hydrophobic interactions. Insertion of a polar amino acid in the hydrophobic patch by a L424 to N424 substitution restores the familiar dimeric structure. Truncation of the subunit is associated with a considerable decrease in catalytic activity. The mutants undergo carbamylation but bind the reaction intermediate analog, 2-carboxy arabinitol-1,5-bisphosphate, poorly. This indicates that loss of activity in the mutant is due to weakened substrate binding. These findings suggest that the mutations in the tail section of the subunit are transmitted to the active site, although the C-terminal region is far from the active site. On the basis of the crystal structure of Rubisco, we propose a model for how the truncations of the enzyme subunit induce conformational changes in one of the two phosphate binding sites.     

Electron microscopy and histochemistry of tail regression in the brachyury mouse

The dominant Brachyury allele (T), nonlethal in the heterozygous condition, leads to a shortening of the mouse tail by resorption of distal structures and tissues in the caudal axis during development. Regressing Brachyury tail tissues of embryos prepared for conventional electron microscopy contained cells with numerous lysosome-like organelles and pseudopodal cytoplasmic extensions, but nonregressing tail tissues had few comparable structures. Such morphological evidence, when correlated with observations of high levels of acid phosphatase activity detected in resorbing Brachyury tail tissues with light microscope histochemistry, suggests that tail regression is mediated by considerable autophagy and/or phagocytosis.     

The largest subunit of RNA polymerase II in Dictyostelium: conservation of the unique tail domain and gene expression.

cDNAs encoding the largest subunit of RNA polymerase II were isolated from a Dictyostelium cDNA library. A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced. Similar to other eukaryotic RNA polymerases II, the largest subunit of Dictyostelium RNA polymerase II contains a unique repetitive tail domain at its carboxyl-terminal region. It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein. RNA blots show that cDNA probes hybridized with a single mRNA species of approximately 6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons. Interestingly, expression of the largest subunit of RNA polymerase II appears to be under developmental regulation. The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h. Cells began to accumulate a higher level of the RNA polymerase II mRNA after 9 h of development. When cells were treated with low concentrations of cAMP pulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Phylogenetic relationships of Pestalotiopsis and allied genera inferred from ribosomal DNA sequences and morphological characters

The taxonomy of the coelomycetous fungus Pestalotiopsis and other closely related genera based on morphological characters has been equivocal. To gain insight in the phylogenetic relationships of Pestalotiopsis and its allies, part of the large subunit (28S) ribosomal DNA region was examined and compared with existing morphological information. Phylogenetic analyses were conducted using parsimony, distance, and likelihood criteria. Results of the analyses showed that Bartalinia, Pestalotiopsis, Seimatosporium, and Seiridium represent distinct monophyletic groups with high bootstrap support. However, Truncatella species are paraphyletic with Bartalinia, sharing a common ancestor. Pestalotia species sequenced clustered together with Pestalotiopsis. These genera should be recognized as distinct natural groups except for Monochaetia and Discosia, which need to be further resolved. Tree topologies are generally in concordance with previous morphological hypotheses, most notably the placement of all Pestalotia species, except the type P. pezizoides, in Pestalotiopsis. Well-supported clades corresponding to groupings based on conidial morphology were resolved and the relative importance of morphological characters for generic delimitation is discussed. Molecular data also provide further evidence to support the association of these coelomycetes with the Amphisphaeriaceae.     

Differential regulation of G protein subunit expression in mouse oocytes, eggs, and early embryos

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.     

青海沙蜥的两性异型和雌性繁殖

作者研究了青海沙蜥(Phrynocephalus vlangalii)形态特征的两性异形和雌体繁殖特征。蜥蜴于2005年5月初捕自西宁以西约150km的倒淌河,被检形态特征包括体色、体长、腹长、尾长、头长和头宽,新排卵雌体维持在实验室梯度热环境中直至产仔。成体两性异形显著,而性未成熟个体缺乏两性异形。最大的成年雄体和雌体分别为70.2mmSVL(snout-vent length)和82.8mmSVL。雄性成体具有相对较大的头长、头宽和尾长,雌性成体SVL大于雄体且具有相对较大的腹长。对4个形态特征进行主成分分析(特征值≥0.5)区分出2个主成分,共解释83.9%的两性相关形态特征的变异。去除SVL差异的影响后,尾长、头长和头宽在第一主成分有较高的正负载系数(解释57.8%的变异),腹长在第二主成分有较高的负负载系数(解释26.1%的变异)。实验室梯度热环境下的雌体于6月下旬至7月中旬产单窝、2-6个后代。窝仔数和窝仔重与母体SVL呈正相关,幼仔重与母体SVL无关。未在青海沙蜥中检测到后代数量与大小之间的权衡。     

Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase triggers acute morphological changes in thyroid epithelial cells

In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP-dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP-dependent protein kinases, is responsible for cAMP-dependent morphological transformation.