The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast

The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and cbf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophilamelanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast cbf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.     

The Archaeal Lsm Protein Binds to Small RNAs

Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.     

Synthesis of an unusual α-zein protein is correlated with the phenotypic effects of the floury2 mutation in maize

The soft, starchy endosperm of the maize (Zea mays L)floury2 mutant is associated with a reduction in zein mRNA and protein synthesis, unique protein body morphology, and enhanced levels of a 70 kDa protein, that has been shown to be the maize homolog of a chaperonin found in the endoplasmic reticulum. We found an unusual α-zein protein of 24 kDa to be consistently associated with the zein fraction from floury2 mutants. Three additional α-zein proteins with molecular weights ranging from ca. 25 to 27 kDa are detected in the storage protein fraction of a high percentage of floury2 kernels and a low percentage of normal kernels in a genetically segregating population. The four proteins can be distinguished from one another by immunostaining on Western blots. Synthesis of the 24 kDa protein is regulated by Opaque2, since the 24 kDa protein is lacking in the storage protein fraction of opaque2/floury2 double mutants. The synthesis of an abnormal a-zein protein in floury2 could explain many features of the mutant, such as the abnormal protein body morphology, induction of the 70 kDa chaperonin, and hypostasis to opaque2 (o2). Although we cannot prove that the accumulation of this protein is responsible for the floury2 phenotype, we were able to detect a restriction fragment length polymorphism (RFLP) linked to the floury2 locus with a 22 kDa α-zein probe. We hypothesize that the unique characteristics of the floury2 mutant could be a response to the accumulation of a defective a-zein protein which impairs secretory protein synthesis.     

The DrrC protein of Streptomyces peucetius, a UvrA-like protein, is a DNA-binding protein whose gene is induced by daunorubicin

DrrC, a daunorubicin resistance protein with a strong sequence similarity to the UvrA protein involved in excision repair of DNA, is induced by daunorubicin in Streptomyces peucetius and behaves like an ATP-dependent, DNA binding protein in vitro. The refolded protein obtained from expression of the drrC gene in Escherichia coli was used to conduct gel retardation assays. DrrC bound a DNA segment containing the promoter region of a daunorubicin production gene only in the presence of ATP and daunorubicin. This result suggests that DrrC is a novel type of drug self-resistance protein with DNA binding properties like those of UvrA. Western blotting analysis with a polyclonal antiserum generated against His-tagged DrrC showed that the appearance of DrrC in S. peucetius is coincident with the onset of daunorubicin production and that the drrC gene is induced by daunorubicin. These data also showed that the DnrN and DnrI regulatory proteins are required for drrC expression. The level of DrrA, another daunorubicin resistance protein that resembles ATP-dependent bacterial antiporters, was regulated in the same way as that of DrrC.     

Phylogenetic analysis of protein sequences based on a novel k-mer natural vector method

Based on the k-mer model for protein sequence, a novel k-mer natural vector method is proposed to characterize the features of k-mers in a protein sequence, in which the numbers and distributions of k-mers are considered. It is proved that the relationship between a protein sequence and its k-mer natural vector is one-to-one. Phylogenetic analysis of protein sequences therefore can be easily performed without requiring evolutionary models or human intervention. In addition, there exists no a criterion to choose a suitable k, and k has a great influence on obtaining results as well as computational complexity. In this paper, a compound k-mer natural vector is utilized to quantify each protein sequence. The results gotten from phylogenetic analysis on three protein datasets demonstrate that our new method can precisely describe the evolutionary relationships of proteins, and greatly heighten the computing efficiency.     

Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

Dictyostelium protein kinase C-delta-like protein is localized in the cell nucleus

The molecular mechanism whereby protein kinase C (PKC) molecules transduce signals into the cell nucleus is unknown. In this study, we provide evidence that Dictyostelium discoideum contains PKCδ-like protein that is localized in the nucleus. The Dictyostelium PKCδ-like protein has an apparent molecular mass of 76 kDa. This protein is already highly expressed in vegetative Dictyostelium cells. The expression level remained constant up to 12 h of development, and sharply decreased after 16 h. The PKCδ-like protein is phosphorylated in vivo in response to cAMP and phorbol ester stimulation. Immunofluorescent studies, as well as subcellular fractionation experiments, have indicated that Dictyostelium PKCδ-like protein is permanently located in the nucleus. Our results may indicate that PKCδ-like protein in Dictyostelium functions as a link between cAMP and the tumor-promoting phorbol esters, and events that take place in the nucleus.     

Thioredoxin-interacting Protein (Txnip) Is a Feedback Regulator of S-Nitrosylation

Nitric oxide exerts a plethora of biological effects via protein S-nitrosylation, a redox-based reaction that converts a protein Cys thiol to a S-nitrosothiol. However, although the regulation of protein S-nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus-coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin-interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin-mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S-nitrosylation.     

JcLEA,a Novel LEA-Like Protein from Jatropha curcas,Confers a High Level of Tolerance to Dehydration and Salinity in Arabidopsis thaliana

Jatropha curcas L. is a highly drought and salt tolerant plant species that is typically used as a traditional folk medicine and biofuel crop in many countries. Understanding the molecular mechanisms that underlie the response to various abiotic environmental stimuli, especially to drought and salt stresses, in J. curcas could be important to crop improvement efforts. In this study, we cloned and characterized the gene for a late embryogenesis abundant (LEA) protein from J. curcas that we designated JcLEA. Sequence analyses showed that the JcLEA protein belongs to group 5, a subgroup of the LEA protein family. In young seedlings, expression of JcLEA is significantly induced by abscisic acid (ABA), dehydration, and salt stress. Subcellular localization analysis shows that that JcLEA protein is distributed in both the nucleus and cytoplasm. Moreover, based on growth status and physiological indices, the overexpression of JcLEA in transgenic Arabidopsis plants conferred increased resistance to both drought and salt stresses compared to the WT. Our data suggests that the group 5 JcLEA protein contributes to drought and salt stress tolerance in plants. Thus, JcLEA is a potential candidate gene for plant genetic modification.     

Cloning and sequencing of ribosomal protein L27a and a gene similar to human GS1 in Drosophila

Two closely linked genes were identified and characterized in the 24F region on the left arm of chromosome 2 in Drosophila. One cDNA predicts a protein of 231 amino acids, with a molecular mass of 25.7 kDa. The predicted amino-acid sequence of this protein is 47.2% identical to that of the previously reported human GS1 protein, which is encoded by a gene that is of interest because it is one of the few X-linked genes that escapes X-inactivation. We have accordingly named our gene GS1like (GS1l). The second cDNA begins 383 bp proximal to the first. This cDNA encodes a protein of a predicted 149 amino acids and a molecular mass of 17.0 kDa. This protein represents a homolog of ribosomal protein L27a; thus, we have named the gene RpL27a. This gene might be responsible for the Minute mutation located at 24F. An rpL27a gene was previously localized to 87F/88A; thus, this gene might be present in two locations in Drosophila.     

Identification and mapping of five new genes in bacteriophage T7

Mutations that affect the single-stranded DNA-binding protein of bacteriophage T7 (gene 2.5) and four T7 proteins of unknown function (the gene 4.3, 4.5, 4.7 and 5.5 proteins) are described and mapped by three-factor crosses. An extensive search for mutants defective in the DNA-binding protein (M_r = 25,562) produced several strains in which this protein has an altered electrophoretic mobility but no strains that appear to lack it completely. The gene 2.5 mutation that was mapped produces a slightly short DNA-binding protein that appears functional by tests in vitro. It seems likely that a functional DNA-binding protein is needed for T7 growth but that conditional-lethal amber mutations in it are rare; the nucleotide sequence known to code for the gene 2.5 protein contains only 1 to 3 sites that would be expected to be readily mutable to conditional-lethal amber codons by N-methyl-N?nitro-N-nitrosoguanidine. The gene 4.3, 4.5 and 4.7 proteins (M_r ～ 8000 to 15,000) are eliminated by a deletion mutant that removes most of the DNA between genes 4 and 5. The gene 5.5 protein (M_r ～ 11,700) is made in relatively large amounts and is affected by two different mutations that were mapped between genes 5 and 6. One of these mutations appears to be an amber mutation that eliminates the protein entirely; the other decreases the electrophoretic mobility of the protein (an apparent increase in size). A larger protein (M_r ～ 18,000), found in small amounts and difficult to observe, is also affected by these two mutations; the relationship of this minor protein to the major gene 5.5 protein is not yet known. The genes 2 and 18 proteins have also been identified in patterns of protein synthesis during infection. The proteins specified by at least 34 different T7 genes have now been identified.     

Characterization in vitro and in vivo of the DNA helicase encoded by Deinococcus radiodurans locus DR1572

Deinococcus radiodurans survives extremely high doses of ionizing and ultraviolet radiation and treatment with various DNA-damaging chemicals. As an effort to identify and characterize proteins that function in DNA repair in this organism, we have studied the protein encoded by locus DR1572. This gene is predicted to encode a Superfamily I DNA helicase, except that genome sequencing indicated that it has a one-base frameshift and would not encode a complete helicase. We have cloned the gene from two different D. radiodurans strains and find that the frameshift mutation is not present. The corrected gene encodes a 755 residue protein that is similar to the Bacillus subtilis YvgS protein and to helicase IV of Escherichia coli. The purified protein (helicase IV_Dr) has ATP hydrolysis and DNA helicase activity. A truncated protein that lacks 214 residues from the N-terminus, which precede the conserved helicase domain, has greater ATPase activity than the full-length protein but has no detectable helicase activity. Disruption of locus DR1572 in the D. radiodurans chromosome causes greater sensitivity to hydrogen peroxide and methyl-methanesulfonate compared to wild-type cells, but no change in resistance to gamma and ultraviolet radiation and to mitomycin C. The results indicate that locus DR1572 encodes a complete protein that contributes to DNA metabolism in D. radiodurans.     

Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated

Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure and function of this important enzyme in biomass deconstruction.     

The Crithidia fasciculata RNH1 gene encodes both nuclear and mitochondrial isoforms of RNase H

The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.     

Fusion Protein Strategy to Increase Expression and Solubility of Hypervariable Region of VP2 Protein of Infectious Bursal Disease Virus in Escherichia coli

Infectious bursal disease is one of the most important viral diseases in the young chickens. VP2 protein is the major host protective immunogen of the virus. A hypervariable region is present in VP2 protein (hvVP2) that contains immunodominant epitops. The high hydrophobicity of hvVP2 region causes protein aggregation in Escherichia coli (E. coli). The objective of the present study was to improve the expression and the solubility of the hvVP2 protein in E. coli. The effects of fusion partners on the solubility of hvVP2 protein were studied. The protein was expressed in forms of unfused and N-terminally fused to GST and NusA. The results showed that the unfused hvVP2 protein was expressed in very low level. But, N-terminally fused hvVP2 protein to GST (glutathione-S-transferase) and NusA (N utilization substance A) showed significantly enhanced protein expression. The fusion of GST and hvVP2 was produced in aggregated form while in the presence of NusA, the hvVP2 protein was expressed in a soluble form. The NusA-hvVP2 protein was detected by a neutralizing monoclonal antibody, 1A6, in antigen-capture ELISA. In conclusion, the NusA protein is a suitable fusion partner to improve expression and solubility of the hvVP2 protein in E. coli.     

Babesia bovis expresses a neutralization-sensitive antigen that contains a microneme adhesive repeat (MAR) domain

A gene coding for a protein with sequence similarity to the Toxoplasma gondii micronemal 1 (MIC1) protein that contains a copy of a domain described as a sialic acid-binding micronemal adhesive repeat (MAR) was identified in the Babesia bovis genome. The single copy gene, located in chromosome 3, contains an open reading frame encoding a putative 181 amino acid protein, which is highly conserved among distinct B. bovis strains. Antibodies against both recombinant protein and synthetic peptides mimicking putative antigenic regions in the B. bovis-MIC1 (Bbo-MIC1) protein bind to the parasite in immunofluorescence assays and significantly inhibit erythrocyte invasion in in vitro B. bovis cultures. Bbo-MIC1 is recognized by antibodies in serum from B. bovis infected cattle, demonstrating expression and immunogenicity during infection. Overall, the results suggest that Bbo-MIC1 protein is a viable candidate for development of subunit vaccines.     

The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization,autogenous control,and regions required for transcriptional activation

The positive-acting global sulfur regulatory protein, CYS3, of Neurospora crassa turns on the expression of a family of unlinked structural genes that encode enzymes of sulfur catabolism. CYS3 contains a leucine zipper and an adjacent basic region (b-zip), which together constitute a bipartite sequence-specific DNA-binding domain. Specific anti-CYS3 antibodies detected a protein of the expected size in nuclear extracts of wild-type Neurospora under conditions in which the sulfur circuit is activated. The CYS3 protein was not observed in cys-3 mutants. Nuclear extracts of wild type, but not cys-3 mutants, also showed specific DNA-binding activity identical to that obtained with a CYS3 protein expressed in Escherichia coli. A truncated CYS3 protein that contains primarily the b-zip domain binds to DNA with high specificity and affinity in vitro, yet fails to activate gene expression in vivo, and instead inhibits the function of the wild-type CYS3 protein. Amino-terminal, carboxy-terminal, and internal deletions as well as alanine scanning mutagenesis were employed to identify regions of the CYS3 protein that are required for its trans-activation function. Regions of CYS3 carboxy terminal to the b-zip motif are not completely essential for function although loss of an alanine-rich region results in decreased activity. All deletions amino terminal to the b-zip motif led to a complete loss of CYS3 function. Alanine scanning mutagenesis demonstrated that an unusual proline-rich domain of CYS3 appears to be very important for function and is presumed to constitute an activation domain. It is concluded that CYS3 displays nuclear localization and positive autogenous control in Neurospora and functions as a trans-acting DNA-binding protein.     

Binding ofNicotiana nuclear proteins to the subterminal regions of theAc transposable element

Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.