多聚谷氨酰胺延伸蛋白募集细胞内正常蛋白质或RNA的分子机制

多聚谷氨酰胺(PolyQ)疾病,是一类由编码蛋白质的基因中CAG三核苷酸重复序列的异常延伸所引发的神经退行性疾病.CAG三核苷酸重复序列导致所编码蛋白质的PolyQ序列的异常延伸,使蛋白质发生错误折叠和积聚,并在细胞内形成包涵体.包涵体的形成是神经退行性疾病的一个重要特征.PolyQ蛋白在积聚过程中,可以将细胞内与其特异相互作用的蛋白质或RNA募集到包涵体中.被募集的其他蛋白质或RNA不仅自身的可溶性组分减少,而且由于被"挟持"到包涵体中其在细胞内的有效组分也相应地减少,从而影响其正常的生物功能.根据特异相互作用的模式,我们将募集作用分为以下几种类型:蛋白质(含Poly Q蛋白)的共积聚;特定结构域或模体介导的募集作用(包括泛素等修饰介导的募集作用);RNA介导的募集作用;以及对分子伴侣蛋白的募集作用.PolyQ延伸蛋白的积聚和对其他组分的募集可能是引发细胞毒性和神经退行性病变的重要原因.     

Overlapping Genes Produce Proteins with Unusual Sequence Properties and Offer Insight into De Novo Protein Creation

It is widely assumed that new proteins are created by duplication, fusion, or fission of existing coding sequences. Another mechanism of protein birth is provided by overlapping genes. They are created de novo by mutations within a coding sequence that lead to the expression of a novel protein in another reading frame, a process called “overprinting.” To investigate this mechanism, we have analyzed the sequences of the protein products of manually curated overlapping genes from 43 genera of unspliced RNA viruses infecting eukaryotes. Overlapping proteins have a sequence composition globally biased toward disorder-promoting amino acids and are predicted to contain significantly more structural disorder than nonoverlapping proteins. By analyzing the phylogenetic distribution of overlapping proteins, we were able to confirm that 17 of these had been created de novo and to study them individually. Most proteins created de novo are orphans (i.e., restricted to one species or genus). Almost all are accessory proteins that play a role in viral pathogenicity or spread, rather than proteins central to viral replication or structure. Most proteins created de novo are predicted to be fully disordered and have a highly unusual sequence composition. This suggests that some viral overlapping reading frames encoding hypothetical proteins with highly biased composition, often discarded as noncoding, might in fact encode proteins. Some proteins created de novo are predicted to be ordered, however, and whenever a three-dimensional structure of such a protein has been solved, it corresponds to a fold previously unobserved, suggesting that the study of these proteins could enhance our knowledge of protein space.Since their discovery (76), overlapping genes, i.e., DNA sequences simultaneously encoding two or more proteins in different reading frames, have exerted a fascination on evolutionary biologists. Among several mechanisms, they can be created by a process called “overprinting” (43), in which a DNA sequence originally encoding only one protein undergoes a genetic modification leading to the expression of a second reading frame in addition to the first one (Fig. ​(Fig.1).1). The resulting overlap encodes an ancestral, “overprinted” protein region and a protein region created de novo (i.e., not by duplication) called an “overprinting” or “novel” region (Fig. ​(Fig.1).1). At present, it is widely thought that the creation of proteins de novo is very rare, contrary to their emergence by gene duplication, which is thought to be the major factor (for reviews, see references 55 and 94). However, this belief might actually reflect the fact that proteins created de novo are in general very difficult to identify (55). Indeed, a long-standing question is whether a protein that has no detectable homolog in other organisms (called an “orphan” protein or “ORFan” [27] or “taxonomically restricted” [110]) represents a protein created de novo in a particular organism or merely a protein that is a member of a larger family whose other members have diverged beyond recognition or have become extinct (115). Proteins created de novo by overprinting provide a valuable opportunity to address these questions, and this constitutes one of the two strands of our study.Open in a separate windowFIG. 1.Creation of a novel protein region (C-terminal extension) by overprinting. Top, a DNA sequence encodes two proteins in different reading frames. Notice the potential, unused stop codon downstream of protein X. Middle, a mutation abolishes the stop codon of protein X, causing its elongation (“overprinting”) to the preexisting stop codon. This results in a gene overlap. Bottom, the overlap encodes an overprinted (ancestral) protein region (dark gray) and an overprinting (novel) protein region (light gray).Practically all studies of overlapping genes have been focused on evolutionary constraints and informational characteristics at the DNA level (see, e.g., references 46, 71, 75, 84, 85, and 114). However, very little has been done to assess potential effects of the overlap on the corresponding protein products. Two studies reported that overlapping proteins are enriched in amino acids with a high codon degeneracy (arginine, leucine, and serine) (68) and that they often simultaneously encode a cluster of basic amino acids in one frame and a stretch of acidic amino acids in the other frame (66).The other strand of the present study is based on earlier observations of the overlapping gene set of measles virus (41), which suggested that protein regions encoded by overlapping genes might have a propensity toward structural disorder.Structural disorder is an essential state of numerous proteins, in which it is associated mostly with signaling and regulation roles (21, 96, 111). The key feature of intrinsically disordered proteins (also called “unstructured” or “natively unfolded”) is that under physiological conditions, instead of a particular three-dimensional (3D) structure, they adopt ensembles of rapidly interconverting structural forms. Different degrees of disorder exist, from random coils to molten globules (100), and some disordered regions can become ordered under certain conditions (21, 96, 117). A variety of computer programs have been developed to predict these regions (19, 23, 101). Each predictor typically differs in what kind of “disorder” it identifies (23, 78), matching only some of the types of disorder mentioned above. Therefore, in order to choose a proper predictor, it was necessary to define precisely what kind of structural disorder we expected to find in proteins encoded by overlapping genes.At least two nonexclusive hypotheses can explain why overlapping genes might encode disordered proteins: (i) the newly created (overprinting) protein of each overlap might tend to be disordered, and (ii) structural disorder in proteins encoded by overlapping genes might alleviate evolutionary constraints imposed on their sequence by the overlap. These hypotheses are clarified below.Intuitively, the conditions required for a protein to fold into a stable 3D configuration, including sequence composition, periodicity, and complexity, are such that structurally ordered proteins represent a vanishingly small fraction of all possible amino acid sequences. Indeed, proteins artificially created from random nucleotide sequences generally have a low secondary structure content (107, 112). Hence our first hypothesis: novel, overprinting proteins are not expected to have a fixed 3D structure at birth, given the low probability of generating structure from a completely new sequence.Disordered proteins are generally subject to less structural constraint than ordered ones (13). Hence our second hypothesis: the presence of disorder in one or both products of an overlapping gene pair could greatly alleviate evolutionary constraints imposed by the overlap, allowing both protein products to scan a wider sequence space without losing their function.Both hypotheses suppose only the lack of a rigid structure, as opposed to a total lack of structure (e.g., some proteins created de novo from a random nucleotide sequence, though lacking secondary structure, have a certain degree of order [112]). For that reason, in this work, we use the widest possible definition of disorder, i.e., the lack of a rigid 3D structure, and we use a program whose predictions of disorder correspond to this definition, PONDR VSL2 (69) (see Results).In this work, we collected a large number of experimentally proven cases of proteins encoded by overlapping genes in unspliced eukaryotic RNA viruses and analyzed their sequence properties.     

De Novo Synthesis of Steroids and Oxysterols in Adipocytes

Local production and action of cholesterol metabolites such as steroids or oxysterols within endocrine tissues are currently recognized as an important principle in the cell type- and tissue-specific regulation of hormone effects. In adipocytes, one of the most abundant endocrine cells in the human body, the de novo production of steroids or oxysterols from cholesterol has not been examined. Here, we demonstrate that essential components of cholesterol transport and metabolism machinery in the initial steps of steroid and/or oxysterol biosynthesis pathways are present and active in adipocytes. The ability of adipocyte CYP11A1 in producing pregnenolone is demonstrated for the first time, rendering adipocyte a steroidogenic cell. The oxysterol 27-hydroxycholesterol (27HC), synthesized by the mitochondrial enzyme CYP27A1, was identified as one of the major de novo adipocyte products from cholesterol and its precursor mevalonate. Inhibition of CYP27A1 activity or knockdown and deletion of the Cyp27a1 gene induced adipocyte differentiation, suggesting a paracrine or autocrine biological significance for the adipocyte-derived 27HC. These findings suggest that the presence of the 27HC biosynthesis pathway in adipocytes may represent a defense mechanism to prevent the formation of new fat cells upon overfeeding with dietary cholesterol.     

Transferable Coarse-Grained Potential for De Novo Protein Folding and Design

Protein folding and design are major biophysical problems, the solution of which would lead to important applications especially in medicine. Here we provide evidence of how a novel parametrization of the Caterpillar model may be used for both quantitative protein design and folding. With computer simulations it is shown that, for a large set of real protein structures, the model produces designed sequences with similar physical properties to the corresponding natural occurring sequences. The designed sequences require further experimental testing. For an independent set of proteins, previously used as benchmark, the correct folded structure of both the designed and the natural sequences is also demonstrated. The equilibrium folding properties are characterized by free energy calculations. The resulting free energy profiles not only are consistent among natural and designed proteins, but also show a remarkable precision when the folded structures are compared to the experimentally determined ones. Ultimately, the updated Caterpillar model is unique in the combination of its fundamental three features: its simplicity, its ability to produce natural foldable designed sequences, and its structure prediction precision. It is also remarkable that low frustration sequences can be obtained with such a simple and universal design procedure, and that the folding of natural proteins shows funnelled free energy landscapes without the need of any potentials based on the native structure.     

Genome-Wide Prediction and Validation of Peptides That Bind Human Prosurvival Bcl-2 Proteins

Programmed cell death is regulated by interactions between pro-apoptotic and prosurvival members of the Bcl-2 family. Pro-apoptotic family members contain a weakly conserved BH3 motif that can adopt an alpha-helical structure and bind to a groove on prosurvival partners Bcl-x_L, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. Peptides corresponding to roughly 13 reported BH3 motifs have been verified to bind in this manner. Due to their short lengths and low sequence conservation, BH3 motifs are not detected using standard sequence-based bioinformatics approaches. Thus, it is possible that many additional proteins harbor BH3-like sequences that can mediate interactions with the Bcl-2 family. In this work, we used structure-based and data-based Bcl-2 interaction models to find new BH3-like peptides in the human proteome. We used peptide SPOT arrays to test candidate peptides for interaction with one or more of the prosurvival proteins Bcl-x_L, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. For the 36 most promising array candidates, we quantified binding to all five human receptors using direct and competition binding assays in solution. All 36 peptides showed evidence of interaction with at least one prosurvival protein, and 22 peptides bound at least one prosurvival protein with a dissociation constant between 1 and 500 nM; many peptides had specificity profiles not previously observed. We also screened the full-length parent proteins of a subset of array-tested peptides for binding to Bcl-x_L and Mcl-1. Finally, we used the peptide binding data, in conjunction with previously reported interactions, to assess the affinity and specificity prediction performance of different models.     

De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls

Actinomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycine max [L.] Merr. cv. Harosoy 63) hypocotyls and in hypocotyls inoculated with the pathogenic fungus Phytophthora megasperma var. sojae A. A. Hildb., but had little effect on protein synthesis within 6 hours. Blasticidin S, conversely, inhibited protein synthesis in the hypocotyls without exhibiting significant effects on messenger RNA synthesis. The normal cultivar-specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely diminished by actinomycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceollin was concomitantly reduced. The effects of actinomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceollin both required de novo messenger RNA and protein synthesis early after infection. Furthermore, actinomycin D and blasticidin S also were effective in suppressing resistance expression and glyceollin production in soybean hypocotyls when inoculated with various Phytophthora species that were normally nonpathogenic to the plants. This indicated that the mechanism of general resistance to these normally nonpathogenic fungi also involves de novo messenger RNA and protein synthesis and production of glyceollin.     

Design and Synthesis of De Novo Peptide for Manganese Binding

The de novo peptide with 63-residues (MHB) has been synthesized biochemically and used for the binding of manganese (II) ions. In designed peptide, the leucine of the peptide dA1 (prototype) was replaced by His27 and Asp41 for binding the manganese (II) ions. The different chromatography studies and mass determination showed that new peptide folds into a monomeric, highly helical with a active site structure similar to the native Mn–SOD in an aqueous solution. Electron paramagnetic resonance (EPR) study suggested that the peptide binds single manganese (II) ion per molecule loosely with K _D value of about 36 μM. The circular dichroism (CD) studies demonstrated that the helical contents of the peptide did not change significantly even after binding the metal ions. The SOD activity study of the Mn–peptide complex showed that the IC₅₀ values is 8.08 μM.     

Emergence,Retention and Selection: A Trilogy of Origination for Functional De Novo Proteins from Ancestral LncRNAs in Primates

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.     

Characterization of Herpes Simplex Virus Type 1 RNA Present in the Absence of De Novo Protein Synthesis

We used herpes simplex virus type 1 (HSV-1) DNA and restriction fragments of HSV-1 DNA covalently coupled to cellulose as a reagent to isolate for further characterization the major and minor HSV-1 immediate-early mRNA species in HeLa cells infected and maintained in the absence of de novo protein synthesis. Five major and several minor immediate-early mRNA species were characterized. One major species was a 4.2-kilobase mRNA mapping in the TR(S)/IR(S) region with its 3' end distal to the U(S) region; this mRNA encoded a 170,000-dalton polypeptide in vitro. A 2.8-kilobase mRNA, encoding a 120,000-dalton polypeptide, was mapped in the TR(L)/IR(L) region with its 3' end directed toward the U(L) region. Three 1.8-kilobase mRNA species were mapped. One, mapping in the IR(S) region with its 3' end in the U(S), encoded a 68,000-dalton polypeptide. One mapped in the TR(S) region and had its 3' end in the U(S) region; the third one encoded a 64,000-dalton polypeptide and mapped in the U(L) region near the IR(L) region. One minor species 5.2 kilobases in size was clearly detectable mapping in the U(L) region. Furthermore, there were indications that one or more immediate-early mRNA species approximately 3 kilobases in size hybridized to regions near the TR(L) and in or near the TR(S)/IR(S) regions. Nuclear immediate-early RNA mapped only in those regions where polyribosomal immediate-early mRNA mapped, although minor differences were seen. Finally, we demonstrated that at least three major immediate-early mRNA's-4.2 kilobases, 2.8 kilobases, and the 1.8-kilobase one mapping in the IR(S)/U(S) region-continued to appear on polyribosomes as functional mRNA late after infection.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway

The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An Escherichia coli strain carrying a defect in NAD de novo biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of E. coli or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue E. coli host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As the QAPRTase gene is ubiquitous in species ranging from bacteria to mammals, the potential environmental biosafety problems caused by horizontal gene transfer can be eliminated.Antibiotic resistance genes are the most commonly used markers for selecting and maintaining recombinant plasmids in hosts, such as Escherichia coli. However, the use of these genes has several drawbacks. For example, horizontal transfer of the antibiotic resistance gene can potentially contribute to the rapid emergence of multidrug-resistant organisms (e.g., superbacteria) (11, 29). Another significant concern is that the antibiotic resistance genes in DNA vaccines may become integrated into human chromosomes (23). The possibility arises, although the probability is low, that once the antibiotic resistance gene is translated into a functional protein, the vaccinee might be resistant to the corresponding antibiotic. This would add to the difficulty of curing diseases caused by infectious pathogens. Accordingly, the use of antibiotic resistance genes is undesirable in many areas of biotechnology, especially in gene therapy products and genetically engineered microorganisms (17, 23, 28). Furthermore, the addition of antibiotics is costly in large-scale cultivation, and there are risks of contamination of the final product with antibiotics (2, 3). Finally, the constitutively expressed antibiotic resistance genes impose a metabolic burden on the host cells, resulting in reduced growth rate and cell density (4, 27). An alternative strategy is to utilize antibiotic-free host-plasmid balanced lethal systems to select and maintain the recombinant plasmids.To date, several such systems have been developed to replace traditional antibiotic selection systems. They include auxotrophy complementation (AC), postsegregational killing (PSK), and operator-repressor titration (ORT) (8). The AC system is based on a strain auxotrophic for an essential metabolite, obtained by mutating or knocking out the corresponding chromosomal gene, which can be complemented with the plasmid-borne selection gene. The choice of the essential gene used for complementation of host auxotrophy is critical, and it is mainly involved in DNA precursor, amino acid, or cell wall biosynthetic pathways. Various essential genes, such as asd, thyA, and glnA, have been utilized to construct AC systems (5, 9, 21, 22, 24, 26, 28). However, all of these systems require extra nutrients or expensive reagents. The PSK system relies on the balance between toxin and antitoxin, expressed from genome and plasmid, respectively. If a cell loses the plasmid, the corresponding antitoxin is degraded and the toxin then kills the cell. Unfortunately, this system has proven ineffective for plasmid maintenance during prolonged culture (6, 14). The ORT system utilizes plasmids with the lac operator to derepress a modified essential chromosomal gene. Loss of these types of plasmids no longer titrates the repressor and leads to the death of the bacterium. This system requires short, nonexpressed lac operator functions as the vector-borne selection marker and enables the selection and maintenance of plasmids free from expressed selectable marker genes (7, 8, 15, 30). Additionally, several other nonantibiotic selection systems (e.g., the fabI-triclosan system) have recently been developed (12, 17, 18).Among the antibiotic-free selection systems that have been developed, the AC system has drawn much attention and has now been applied in numerous bacterial species, such as Lactococcus lactis, Salmonella spp., Vibrio cholerae, Mycobacterium bovis, and E. coli (5, 16, 21, 22, 24). However, all of the AC systems utilize plasmid-borne bacterial-origin genes to complement the auxotrophy. These systems may suffer from a potential risk that the bacterial-origin genes may be integrated into human chromosome when they are used in transgenic products, such as DNA vaccines. Therefore, a better strategy would be to use the genes of the vaccinees themselves to construct an AC system. Not only would this type of approach select and maintain plasmids in bacteria, but it could also be widely applied in the production of safer DNA vaccines.In the present study, we successfully developed a novel antibiotic-free plasmid selection system based on complementation of host auxotrophy in the NAD synthesis pathway. The NAD synthesis pathway, including de novo and salvage pathways, differs among species. However, by comparison of NAD metabolism in different species, quinolinic acid phosphoribosyltransferase (QAPRTase) appears to be a common enzyme for de novo NAD biosynthesis in both prokaryotes and eukaryotes (13). Therefore, the QAPRTase gene was viewed as a favorable candidate that could potentially be utilized to construct a new AC system.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

Naegleria gruberi De Novo Basal Body Assembly Occurs via Stepwise Incorporation of Conserved Proteins

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, γ-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally γ-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.The beautiful and enigmatic pinwheel structures of centrioles and basal bodies have captured the imaginations of cell biologists for over a century. These small (∼1-μm) organelles are composed largely of a cylinder of nine microtubule triplets (11). The surrounding amorphous material harbors the microtubule-organizing activities of the centrosome, placing centrioles at the hub of the microtubule cytoskeleton. Metazoan centrosomes define mitotic spindle poles, and their centrioles are called basal bodies when used to form cilia (29). Moreover, in 1900 Meeves showed in a series of classical experiments that centrioles and basal bodies are interconvertible structures (34). Centrioles must replicate exactly once per cell cycle, as duplication errors can lead to problems with chromosome segregation and cell morphology (17).Virtually all animal cells have a pair of centrosomal centrioles that duplicate via “templated” assembly, with the new centriole developing perpendicular and attached to a preexisting centriole (4). Centrioles can also be formed “de novo” in cytosol devoid of preexisting centrioles and basal bodies (20). In addition to many in vivo examples (20), terminally differentiated fibroblasts held in S phase can assemble centrioles de novo after removal of preexisting centrioles by laser microsurgery (15).The amoeboflagellate Naegleria gruberi grows as an amoeba that completely lacks a cytoplasmic microtubule cytoskeleton. However, when exposed to stressors such as temperature, osmotic, or pH changes, Naegleria rapidly differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton from scratch, including two basal bodies and flagella (8). This differentiation occurs synchronously, with approximately 90% of cells growing visible flagella in a 15-min window (T₅₀ = 65 min after initiation of differentiation). As part of this differentiation, Naegleria has been shown to assemble the pinwheel structure of the basal bodies de novo, about 10 min before flagella are seen (11).Two centrosomal proteins that have been studied during Naegleria differentiation are centrin and γ-tubulin. Centrin is a calcium-binding phosphoprotein that is an integral component of the wall and lumen of basal bodies and of the pericentriolar lattice in many organisms (4, 19). During differentiation, Naegleria induces synthesis of centrin protein, which then localizes specifically to basal body structures throughout differentiation (18). γ-Tubulin is a general microtubule nucleation factor that localizes to microtubule-organizing centers (MTOCs) of many types. Surprisingly, Naegleria''s γ-tubulin homolog has been reported to localize to basal body precursor complexes and then move to the other end of the cell before disappearing completely (32).A third protein that has come under recent scrutiny for its role in centriole duplication is SAS-6, a functionally conserved coiled-coil protein required for the formation of diverse basal body precursor structures (7, 21,–23, 31). In Caenorhabditis elegans and Drosophila melanogaster, SAS-6 is recruited at S phase to form the “central tube,” a cylindrical basal body precursor that lacks microtubules (22, 23). SAS-6 is also required for the formation of the flat ring or cartwheel with nine radiating spokes, which is the first structure to be formed in the Chlamydomonas basal body (21).To determine if Naegleria is likely to have typical basal body components, we identified conserved basal body genes in the Naegleria genome. We also made antibodies to and localized Naegleria''s homologs of SAS-6 and γ-tubulin. Finally, we have determined the order of expression and incorporation of these proteins, as well as centrin, during Naegleria de novo basal body assembly.     

Outer Membrane Proteins of Escherichia coli VII. Evidence That Bacteriophage-Directed Protein 2 Functions as a Pore

Protein 1, a major protein of the outer membrane of Escherichia coli, has been shown to be the pore allowing the passage of small hydrophilic solutes across the outer membrane. In E. coli K-12 protein 1 consists of two subspecies, 1a and 1b, whereas in E. coli B it consists of a single species which has an electrophoretic mobility similar to that of 1a. K-12 strains mutant at the ompB locus lack both proteins 1a and 1b and exhibit multiple transport defects, resistance to toxic metal ions, and tolerance to a number of colicins. Mutation at the tolF locus results in the loss of 1a, in less severe transport defects, and more limited colicin tolerance. Mutation at the par locus causes the loss of protein 1b, but no transport defects or colicin tolerance. Lysogeny of E. coli by phage PA-2 results in the production of a new major protein, protein 2. Lysogeny of K-12 ompB mutants resulted in dramatic reversal of the transport defects and restoration of the sensitivity to colicins E2 and E3 but not to other colicins. This was shown to be due to the production of protein 2, since lysogeny by phage mutants lacking the ability to elicit protein 2 production did not show this effect. Thus, protein 2 can function as an effective pore. ompB mutations in E. coli B also resulted in loss of protein 1 and similar multiple transport defects, but these were only partially reversed by phage lysogeny and the resulting production of protein 2. When the ompB region from E. coli B was moved by transduction into an E. coli K-12 background, only small amounts of proteins 1a and 1b were found in the outer membrane. These results indicate that genes governing the synthesis of outer membrane proteins may not function interchangeably between K-12 and B strains, indicating differences in regulation or biosynthesis of these proteins between these strains.