Streptomyces aureofaciens sporulation-specific sigma factor σ directs expression of a gene encoding protein similar to hydrolases involved in degradation of the lignin-related biphenyl compounds

A previously established method for the identification of promoters recognized by a heterologous RNA polymerase holoenzyme containing a particular sigma factor was used to identify promoters dependent upon a sporulation specific sigma factor, σ^RpoZ, of Streptomyces aureofaciens. Three new positive DNA fragments were identified, and these putative rpoZ-dependent promoters, P_ren24, P_ren57, and P_ren71, contained sequences similar to the consensus sequence of flagellar and chemotaxis promoters. However, only P_ren71 was active in S. aureofaciens. The promoter was induced at the time of aerial mycelium formation, and was inactive in an S. aureofaciens strain with an rpoZ-disrupted gene. The results suggest that the P_ren71 promoter is recognized by an RNA polymerase holoenzyme containing σ^RpoZ in S. aureofaciens. Sequence analysis of the region directed by P_ren71 revealed a gene, ren71, encoding a protein of 358 amino acids with an M_r 37 770. The deduced protein product showed end-to-end sequence similarity to the meta-cleavage compound hydrolase of Sphingomonas paucimobilis.     

Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein.

Septicemia due to Staphylococcus aureus often begins as a focal infection (e.g., colonized wounds or catheters) from which the organism gains access to the bloodstream. On the basis of recent data from this laboratory, it is likely that S. aureus colonizes catheters and endothelium by using a fibrinogen-binding protein to mediate adhesion to fibrinogen-coated surfaces. To characterize the fibrinogen-reactive protein, we screened a lambda Zap library of S. aureus DB, a clinical isolate, for clones that were reactive with fibrinogen. Of 100,000 plaques screened, 3 were found to react with fibrinogen on immunoblots. Plasmid DNA prepared from clones 14, 30, and 36, upon digestion with EcoR1, which released the insert, revealed fragments of 4.6, 3.6, and 3.2 kb, respectively. To identify the cloned protein expressed in E. coli, cells were fractionated into periplasmic, membrane, and cytoplasmic fractions. Expression studies of clone 14, which comprised approximately two-thirds of the mature molecule, including the C terminus, revealed a 34-kDa fibrinogen-reactive protein in both the periplasmic and membrane fractions. This protein, designated FbpA, could be partially purified on a fibrinogen column. By using both clones 14 and 36 as templates, the complete DNA sequence of the fibrinogen-binding protein was obtained, yielding a molecule with a predicted size of 69,991 Da. Although sequence analysis revealed a high degree of homology with coagulase, there is a unique sequence of 11 amino acids that is not found in three known coagulases as well as two recently cloned fibrinogen-binding proteins. This unique sequence shares homology with a cell wall anchor motif found in other gram-positive surface proteins.     

Functional expression in Escherichia coli of cloned reovirus S1 gene encoding the viral cell attachment protein sigma 1

A cDNA clone encompassing the complete reovirus (serotype 3) S1 gene was constructed using two partial clones containing overlapping sequences. The gene (with the first 15 bases at the 5' end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter. The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-sigma 1 antibodies. Like authentic soluble sigma 1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors. Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E. coli cells, were also found to exhibit hemagglutinating (HA) activity. Such hemagglutination was inhibited by a monoclonal anti-sigma 1 antibody previously shown to inhibit reovirus HA activity. It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact.     

Cloning, sequencing, and expression of a gene from Campylobacter jejuni encoding a protein (Omp18) with similarity to peptidoglycan-associated lipoproteins.

A Campylobacter jejuni genomic plasmid library was screened with antiserum generated against whole C. jejuni, revealing two immunoreactive clones. Sequence analysis of the recombinant plasmids revealed a common open reading frame of 498 nucleotides encoding a protein of 165 amino acids with a calculated molecular mass of 18,018 Da. The recombinant product partitioned to the outer membrane fractions of Escherichia coli transformants and has been designated Omp18. The deduced amino acid sequence of the cloned C. jejuni gene exhibits considerable similarity to peptidoglycan-associated lipoproteins from other gram-negative bacteria.     

Disruption of the hup gene encoding a histone-like protein HS1 and detection of HS12 of Streptomyces lividans.

When the latter half of the hup gene encoding a histone-like protein HS1 of Streptomyces lividans TK24 was replaced by the kanamycin resistance gene, the hup mutant EY1 grew slowly in liquid medium and this delay was overcome by introduction of the complete hup. EY1 sporulated normally on solid medium, with no serious defects as observed in hupAB mutants of Escherichia coli. Therefore, HS1 probably has a role in growth in the presence of liquid medium and this organism may possess another histone-like protein with functions overlapping those of HS1. We cloned the hup2 gene encoding another histone-like protein HS12, which has two motifs of prokaryotic histone-like protein and eukaryotic histone H1. The amount of HS12 increased in EY1, determined by western blotting analysis using an anti-His-tagged HS12 polyclonal antibody. We are entertaining the notion that the increased amount of HS12 partially suppressed the defects caused by the hup mutation.     

Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein.

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.     

大鼠IgE依赖组胺释放因子基因的克隆表达及其功能研究

目的:获得原核表达的可溶性大鼠IgE依赖组胺释放因子(rHRF),并检测其诱发大鼠致敏肥大细胞释放组胺的功能.方法:克隆Wistar大鼠rHRF基因完整编码区序列并在BL21细胞中进行原核表达,纯化的可溶性重组rHRF刺激卵清蛋白变应原致敏的大鼠肺肥大细胞,利用荧光分光光度法测定组胺释放量.结果:测序证实克隆基因与GenBank中大鼠IgE依赖组胺释放因子(又称翻译控制肿瘤蛋白)mRNA序列的一致性为97%,推测的氨基酸序列一致性为95%.SDS-PAGE显示重组rHRF相对分子质量(Mr)约为24 000;重组rHRF诱发大鼠致敏肥大细胞释放组胺不依赖变应原,且呈剂量依赖关系. 结论:rHRF具有不依赖变应原诱发大鼠致敏肥大细胞释放组胺的功能,可能在Ⅰ型超敏反应过程中发挥重要作用,为进一步研究rHRF的免疫学功能打下基础.     

Rapid changes in the expression of a gene encoding a calcium-binding protein in Schistosoma mansoni

Genes expressed in a stage-specific manner may help us understand the molecular events controlling the complex life cycle of schistosomes. cDNA and genomic clones encoding a calcium-binding protein (CaBP) were obtained from cercariae and their sequence determined. The encoded protein (69 amino acids long) shows clear resemblance to the domain structure and organization of CaBP molecules. It contains two typical calcium-binding loops, the distance between which is identical to the length conserved in other CaBP molecules. In addition, the schistosome CaBP shows Ca2+-dependent electrophoretic mobility (increased with Ca2+-ions and decreased with EGTA). Northern blots revealed expression of the CaBP gene in cercariae but not in sporocyst or worm (developmental stages preceding and following cercaria). The preferential expression of this CaBP in the cercaria raises questions as to what cercaria-specific function(s) it performs. The structure of the gene is similar to that in other eukaryotes, and one intron interrupts the coding sequence. The region of the cap site was determined, and there was no evidence of the spliced leader sequence found in the mRNAs of other parasites. The CaBP reveals a rapid change in gene expression, since the mRNA is missing in the parasite residing in infected snails, but is readily detected in cercariae 1 h after shedding. We identified other genes which are turned on (like the CaBP) or shut off within the short period of transition from cercariae in the snail to free-swimming cercariae.     

Identification and expression of a Fasciola hepatica gene encoding a gut antigen protein bearing repetitive sequences.

A Fasciola hepatica cDNA clone of about 2 kb was isolated from an expression library by immunological screening using blood serum from an experimentally infected calf. The cDNA clone hybridised to a RNA of about 3 kb in a Northern blot experiment. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 1636 bp which encoded 24 tandemly arranged 20-amino acid-long repeats, followed by 65 non-repeated residues preceding the stop codon. This antigen was expressed in Escherichia coli as beta-galactosidase fusion proteins which were used for the production of specific antibodies. Immunofluorescence studies using specific antifusion sera revealed that the antigen was specifically expressed in the parasite intestine epithelial cells. Due to its early appearance it might be possible to design diagnostic assays based on this repeated antigen for identification of recently infected animals.     

Identification of the gene encoding the FhbB protein of Treponema denticola, a highly unique factor H-like protein 1 binding protein

The gene encoding the Treponema denticola factor H-like protein 1 (FHL-1) binding protein, FhbB, was recovered and characterized. Sequence conservation, expression, and properties of FhbB were analyzed. The identification of FhbB represents an important step in understanding the contribution of FHL-1 binding in T. denticola pathogenesis and in development of periodontal disease.     

Characterization of the gene encoding a 26-kilodalton protein (OMP26) from nontypeable Haemophilus influenzae and immune responses to the recombinant protein

A 26-kDa protein (OMP26) isolated and purified from nontypeable Haemophilus influenzae (NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the H. influenzae Rd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.     

Analysis of a scrambled gene: the gene encoding alpha-telomere-binding protein in Oxytricha nova.

Following cell mating in ciliates, a copy of the micronuclear genome is processed into a new macronucleus through massive cutting, reordering, splicing, elimination, and amplification of the DNA. DNA processing includes the deletion of short interrupting elements called internal eliminated sequences (IESs), followed by the splicing of the remaining segments, known as macronuclear destined sequences (MDSs). The MDSs in some micronuclear genes, such as actin I, are scrambled and must be reordered during IES removal and MDS splicing to yield a functional gene. Here, we describe the cloning, sequencing, and characterization of a different scrambling pattern for the gene that encodes the alpha subunit of the telomere-binding protein of Oxytricha nova. The micronuclear gene is made up of 14 MDSs in the scrambled order 1-3-5-7-9-11-2-4-6-8-10-12-13-14. Only the scrambled version is present in the micronucleus, and only the unscrambled version is present in the macronucleus. We propose that unscrambling occurs by homologous recombination guided by pairs of direct repeats at MDS-IES junctions. The patterned array of scrambling may be a clue to the origin of scrambling in this gene.     

Influence of the alternative sigma(28) factor on virulence and flagellum expression of Legionella pneumophila

The fliA gene of Legionella pneumophila encoding the alternative sigma(28) factor was inactivated by introducing a kanamycin resistance cassette. Electron microscopy and Western blot analysis revealed that the fliA mutant strain is aflagellate and expresses no flagellin. Reporter gene assays indicated that the flaA promoter is not active in the fliA mutant strain. The fliA mutant strain multiplied less effectively in coculture with amoebae than the wild-type strain and was not able to replicate in coculture with Dictyostelium discoideum.     

deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix protein similar to the hairy gene product.

Neural precursor cells in Drosophila acquire their identity early during their formation. In an attempt to determine whether all neural precursors share a set of genetic machinery, perhaps to control properties of differentiation common to all neurons, we used the enhancer-trap method to identify several genes (pan-neural genes) that are expressed in all neurons and/or their precursors. One of the pan-neural genes is deadpan, which encodes a helix-loop-helix protein closely related to the product of the segmentation gene hairy. The function of deadpan is essential for viability and is likely to be involved in the functional rather than the morphological differentiation of neurons.