High Degree Of Identity Research Articles

De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome

Many of the complement regulatory genes within the RCA cluster (1q32) have arisen through genomic duplication and the resulting high degree of sequence identity is likely to predispose to gene conversion events. The highest degree of identity is between the genes for factor H (CFH) and five factor H-related proteins--CFHL1, CFHL2, CFHL3, CFHL4, and CFHL5. CFH mutations are associated with atypical hemolytic uremic syndrome (aHUS). In the Newcastle cohort of 157 aHUS patients we have identified CFH mutations in 25 families or individuals. Eleven of these 25 independent mutations are either c.3226C>G,Q1076E; c.3572C>T,S1191L; c.3590T>C,V1197A or combined c.3572C>T,S1191L/c.3590T>C,V1197A. Sequence analysis shows that all four of these changes could have arisen as a result of gene conversion between CFH and CFHL1. Analysis of parental samples in two patients with S1191L/V1197A has shown that the changes are de novo thus providing conclusive evidence that gene conversion is the mutational mechanism in these two cases. To confirm that S1191L and V1197A are disease predisposing we examined their functional significance in three ways - analysis of the C3b/C3d binding characteristics of recombinant mutant S1191L/V1197A protein, heparin affinity chromatography and haemolytic assays of serum samples from aHUS patients carrying these changes. The results showed that these changes resulted in impaired C3b binding and a defective capacity to control complement activation on cellular surfaces. We, therefore, provide conclusive evidence that gene conversion is responsible for functionally significant CFH mutations in aHUS.

Immunization with antigen-pulsed dendritic cells significantly improves the immune response to weak self-antigens

Dendritic cells (DCs) are the only professional antigen-presenting cells endowed with the ability to stimulate naïve T cells and initiate a primary immune response. For this reason, DC-based immunization has been shown to be highly effective in eliciting CTL responses to viruses and tumor-associated antigens. Here we report on the use of DC immunization to enhance the B cell-mediated humoral immune response to highly conserved proteins and the application of this approach to the generation of monoclonal antibodies (mAbs) against these proteins. To illustrate the technique we describe the production of mAbs to class II transactivator (CIITA), the major histocompatibility complex (MHC) CIITA, a difficult immunogen owing to its high degree of identity among species. We show that mice immunized with a combination of an intravenous injection of DCs pulsed with recombinant fragments of CIITA followed by intraperitoneal injection of the antigen in incomplete Freund's adjuvant induced a detectable antibody response against CIITA, while sera from mice immunized using the traditional method (i.e. intraperitoneal immunization with 50 μg of protein in complete Freund's adjuvant) gave an almost undetectable response. Furthermore, a total of four fusion experiments demonstrate that immunization with Ag-pulsed DCs is necessary for the efficient generation of hybridomas and a good yield of mAbs specific for the recombinant and the native endogenous CIITA protein. Conversely, four independent fusions carried out with splenocytes from mice immunized using the traditional method failed to produce anti-CIITA hybridomas. We propose that immunization with antigen-loaded DCs should be the method of preference when attempting to raise mAbs against weak self-immunogens.

Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

BackgroundThe isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes.In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity.ResultsOf 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP.ConclusionThe results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.

Homology Models of the Yersinia Pseudotuberculosis and Yersinia Pestis General Porins and Comparative Analysis of Their Functional and Antigenic Regions

The amino acid sequences of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) have recently deduced but their three-dimensional structures were not known. These sequences were analyzed using the servers 3D-PSSM and PredPort. The YPS and YPT porins were shown to have a high degree of identity (above 50%) in primary and secondary structures. The three-dimensional models of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) were obtained using the homology modeling approach, SWISS-MODEL Protein Modeling Server and 3-D structure of PhoE porin from E. coli as template. The superposition of the Ca-atoms of the monomers of the Yersinia porins and PhoE porin gave a root mean square deviations of 0.47 A and 0.43 A for YPS and YPT respectively. Yersinia porins were found to be very similar in their three-dimentional structure to other non-specific enterobacterial porins, having the same features of overall fold and disposition of loop L3. The intrinsic structures of the monomer pores of YPS and YPT were investigated and their conductances were predicted with the program HOLE. The good correspondence between the theoretical and experimental magnitudes of YPS conductance was found. The Yersinia porins were determined to be unusual in containing the substitution, Glu replaced by Val, in a highly conserved pentapeptide (Pro-Glu-Phe-Gly-Gly-Asp), located in the loop L3 tip that disturbs the functionally important cluster of the acidic amino acids in the constriction site. Comparative analysis of structural organization of YPS and E. coli OmpF porin in the regions involved in subunit association and pore lumen was performed. The YPS porin functional properties were predicted. The differences between these porins in polar interactions playing a signifiant role in stabilization of the porin trimers were found and discussed in term of the variations in trimer stability. The Yersinia porins were shown to have the highest degree of the structural similarity. The differences between the porins were observed in their external loops. Their loops L6 and loops L8 showed 71.4 and 52.9% of sequence identity, respectively. The arrangement of charged residues clustered in the channel external vestibule of these porins was found to be also different sugesting the possible differences in their functional properties. The surface exposed regions of Yersinia porins involved in their potential sequential antigenic determinants were compared. The structural basis of their cross reactivity and antigenic differences is discussed.

Cloning, sequence analysis, and characterization of a novel β-glucosidase-like activity from Pichia etchellsii

Genomic DNA fragment encoding a novel β-glucosidase-like activity of the yeast Pichia etchellsii was cloned and expressed in Escherichia coli. An open-reading frame of 1515 bp, termed mugA, coding for a protein of predicted molecular mass of approximately 54 kDa was confirmed for this activity. The sequence of the deduced protein did not show homology with the generic β-glucosidases but a high degree of identity was seen with several Ser-Asp (SD)-rich cell-surface-associated proteins. The secondary structure prediction program 3D-PSSM indicated the protein to be composed of largely helical and coiled structures, which was confirmed by circular dichroism spectroscopy. The encoded protein, MUGA, was purified by about 53-fold and characterized as a monomer of 52.1 kDa by SDS–PAGE and MALDI-TOF. The protein displayed high hydrolytic activity on methylumbelliferyl β- d-glucoside but relatively very little hydrolysis of p-nitrophenyl β- d-glucoside and gentiobiose, characteristic substrates for β-glucosidases. The binding experiments performed between P. etchellsii cells and the purified E. coli expressed MUGA indicated binding with the cell surface, which was monitored by fluorescence microscopy. In competition experiments with the SD dipeptide, less protein was shown to bind to the cell surface, in a concentration-dependent manner.

Selection of a Macaque Fab with Framework Regions Like Those in Humans, High Affinity, and Ability To Neutralize the Protective Antigen (PA) of Bacillus anthracis by Binding to the Segment of PA between Residues 686 and 694

Human anthrax infection cannot always be treated successfully by antibiotics, as highlighted by recent bioterrorist attacks; thus, adjunct therapies are clearly needed for the future. There is a particular need to further develop adjunct therapies that can neutralize secreted toxins, such as antibodies directed towards the 83-kDa protective antigen (PA(83)). In the absence of human donors, we immunized a macaque (Macaca fascicularis) with PA(83) to obtain such antibodies suitable as an adjunct therapy for human anthrax infection. By using bone marrow as a template, we PCR amplified specific Fab-encoding genes and cloned them as an immune library (10(7) clones). We isolated a high-affinity (equilibrium dissociation constant [K(D)], 3.4 nM), highly neutralizing (50% inhibitory concentration, 5.6 +/- 0.13 nM) Fab (designated 35PA(83)) from this library by panning. Its epitope was localized by Pepscan analysis between residues 686 and 694 of PA(83) and is part of the region which directly interacts with the cell receptor. 35PA(83) may thus neutralize the anthrax toxin by competing directly for its receptor. The genes encoding 35PA(83) were similar to those of a human immunoglobulin germ line and were assigned to subgroups of human V, (D), or J genes by IMGT/V-QUEST analysis. The 35PA(83) framework regions were 92% identical to a representative allele of each subgroup. When compared to framework regions coded by related human germ line genes, only 2 of 74 (VH) or 75 (VK) analyzed amino acids of 35PA(83) have different chemical characteristics. A very high degree of identity with human framework regions makes 35PA(83) well suited for expression as a whole primatized immunoglobulin G and demonstrates the practicality of using macaque Fabs when immunized human plasma cell donors are not available.

Characterization of Gonadotrophin‐Releasing Hormone Precursor cDNA in the Old World Mole‐Rat Cryptomys Hottentotus Pretoriae: High Degree of Identity with the New World Guinea Pig Sequence

Regulation of pituitary gonadotrophins by the decapeptide gonadotrophin-releasing hormone 1 (GnRH1) is crucial for the development and maintenance of reproductive functions. A common amino acid sequence for this decapeptide, designated as 'mammalian' GnRH, has been identified in all mammals thus far investigated with the exception of the guinea pig, in which there are two amino acid substitutions. Among hystricognath rodents, the members of the family Bathyergidae regulate reproduction in response to diverse cues. Thus, highveld mole-rats (Cryptomys hottentotus pretoriae) are social bathyergids in which breeding is restricted to a particular season in the dominant female, but continuously suppressed in subordinate colony members. Elucidation of reproductive control in these animals will be facilitated by characterization of their GnRH1 gene. A partial sequence of GnRH1 precursor cDNA was isolated and characterized. Comparative analysis revealed the highest degree of identity (86%) to guinea pig GnRH1 precursor mRNA. Nevertheless, the deduced amino acid sequence of the mole-rat decapeptide is identical to the 'mammalian' sequence rather than that of guinea pigs. Successful detection of GnRH1-synthesizing neurones using either a guinea pig GnRH1 riboprobe or an antibody against the 'mammalian' decapeptide is consistent with the guinea pig-like sequence for the precursor and the classic 'mammalian' form for the decapeptide. The high degree of identity in the GnRH1 precursor sequence between this Old World mole-rat and the New World guinea pig is consistent with the theory that caviomorphs and phiomorphs originated from a common ancestral line in the Palaeocene to mid Eocene, some 63-45 million years ago.

Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases

Cloning of cDNA encoding an alpha-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative alpha-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of alpha-glucosidase, EC. 3.2.1.20.

Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Ggamma-globin gene.

The human gamma-globin genes form part of a 5-kb tandem duplication within the beta-globin gene cluster on chromosome 11. Despite a high degree of identity between the two genes, we show that while the upstream Ggamma-globin gene terminates transcription efficiently, termination in the Agamma gene is inefficient. This is primarily due to the different strengths of the polyA signals of the two genes; Ggamma-globin has a functionally stronger polyA signal than the Agamma gene. The probable cause of this difference in polyA efficiency characteristics lies with a number of base changes which reduce the G/U content of the GU/U-rich region of the Agamma polyA signal relative to that of Ggamma. The 3' flanking regions of the two gamma-globin genes have similar abilities to promote transcription termination. We found no evidence to suggest a cotranscriptional cleavage event, such as that seen in the human beta-globin gene, occurs in either gamma-globin 3' flank. Instead we find evidence that the 3' flank of the Ggamma-globin gene contains multiple weak pause elements which, combined with the strong polyA signal the gene possesses, are likely to cause gradual termination across the 3' flank.

Production and characterization of recombinantly derived peptides and antibodies for accurate determinations of somatolactin, growth hormone and insulin-like growth factor-I in European sea bass ( Dicentrarchus labrax)

A specific radioimmunoassay (RIA) for European sea bass ( Dicentrarchus labrax) growth hormone (GH) was developed and validated. For this purpose, a stable source of GH was produced by means of recombinant DNA technology in a bacteria system. The identity of the purified protein (ion exchange chromatography) was demonstrated by Western blot and a specific GH antiserum was raised in rabbit. In Western blot and RIA system, this antiserum recognized specifically native and recombinant GH, and it did not cross-react with fish prolactin (PRL) and somatolactin (SL). In a similar way, a specific polyclonal antiserum against the now available recombinant European sea bass SL was raised and used in the RIA system to a sensitivity of 0.3 ng/ml (90% of binding of tracer). Further, European sea bass insulin-like growth factor-I (IGF-I) was cloned and sequenced, and its high degree of identity with IGF-I peptides of barramundi, tuna, and sparid fish allowed the use of a commercial IGF-I RIA based on barramundi IGF-I antiserum. These assay tools assisted for the first time accurate determinations of SL and GH–IGF–I axis activity in a fish species of the Moronidae family. Data values were compared to those found with gilthead sea bream ( Sparus aurata), which is currently used as a Mediterranean fish model for growth endocrinology studies. As a characteristic feature, the average concentration year round of circulating GH in growing mature males of European sea bass was higher than in gilthead sea bream. By contrast, the average concentration of circulating SL was lower. Concerning to circulating concentration of IGF-I, the measured plasma values for a given growth rate were also lower in European sea bass. These findings are discussed on the basis of a different energy status that might allowed a reduced but more continuous growth in European sea bass.

Cloning and characterisation of an almond 9-lipoxygenase expressed early during seed development

Lipoxygenases (LOXs) are a class of non-heme iron containing dioxygenases which catalyse the hydroperoxidation of polyunsaturated fatty acids such as linoleic and linolenic acids. Plant LOXs are commonly believed to play an important role in fundamental plant processes such as defence, development and senescence. However, the physiological role of these enzymes in seed development is still largely unknown. In a previous work we showed that LOXs are highly expressed in almond seed at early developmental stages and that 9-LOX accounts for most of the LOX activity detected in almonds. To elucidate the role of 9-LOXs in seed development and in influencing seed quality, we carried out the molecular cloning and characterisation of a cDNA encoding the almond ( Prunus dulcis) seed 9-LOX. The predicted protein comprises 862 amino acids with a calculated molecular mass of 97.7 kDa and an isoelectric point of 5.53. The almond LOX shows 79% identity towards a LOX previously reported from hazelnut seed. A high degree of identity was also found towards cotton (75%) and tobacco (73%) LOXs involved in plant defence responses. The cDNA was expressed in E. coli and produced an active enzyme showing a strict product specificity, 9-hydroperoxides being produced almost exclusively with either linoleic or linolenic acid as substrates. The biochemical characterisation of the main LOX isoforms detected at different seed physiological stages indicated that 9-LOX metabolism is specific for seed development and could have important implications for seed quality.

Biochemical Characterization and Quantitative Gene Expression Analysis of the Multi-Stress Inducible Metallothionein from Tetrahymena thermophila

A cadmium-binding protein with biochemical features of a metallothionein (MT) has been isolated and purified to homogeneity from the ciliate Tetrahymena thermophila. N-terminal sequencing revealed the posttranslational cleavage of the first two amino acids and, in general, a high degree of identity with known MTs from other ciliates. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis of the apothionein revealed a molecular mass (16,763 Da) higher to those of mammals and of other protozoa. Finally, quantitative real-time PCR has been used to investigate the susceptibility of this ciliate MT to gene activation in response to heavy metals and to other stressors. Our data indicate that while zinc is not effective at all and cadmium is the best inducer, other stress factors, such as mercury, copper, heat and hydrogen peroxide, also activated gene transcription. As in vertebrate cells, interleukin-6 (IL-6) that stimulates ciliate growth, was able to enhance MT gene synthesis. This complex of data seems to indicate a general role of this protein in stress response.

Molecular cloning of a new laccase from the edible straw mushroom Volvariella volvacea: possible involvement in fruit body development

Cloning of a laccase-encoding cDNA from the edible straw mushroom, Volvariella volvacea, was performed using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The cDNA of the putative laccase gene ( lac4) consisted of 1689 bp, including an open reading frame encoding a 23-amino acid signal peptide at the N-terminal end and a 540-amino acid mature protein with a predicted molecular mass of 58 173 Da and a p I value of 6.1. The 10 histidine residues and one cysteine residue required to co-ordinate the four copper atoms at the active site of the protein were all conserved. The amino acid sequence of V. volvacea lac4 has a high degree of identity with other basidiomycete laccases. Transcription of the laccase gene was analysed by RT-PCR and, unlike many other laccase genes, shown to be regulated independently of either copper or aromatic compounds under the test conditions. However, the laccase gene is strongly expressed during that part of the mushroom developmental cycle involving fruit body morphogenesis.

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (Phaseolus vulgaris L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome.

An atypical topoisomerase II sequence from the slime mold Physarum polycephalum.

We have determined the complete nucleotide sequence of the cDNA encoding DNA topoisomerase II from Physarum polycephalum. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic enzymes, a 250-bp fragment was polymerase chain reaction (PCR) amplified. This fragment was used as a probe to screen a Physarum cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. Rapid amplification of cDNA ends (RACE)-PCR was employed to isolate the remaining portion of the gene. The complete sequence of 4613 bp contains an open reading frame of 4494 bp that codes for 1498 amino acid residues with a theoretical molecular weight of 167 kDa. The predicted amino acid sequence shares similarity with those of other eukaryotes and shows the highest degree of identity with the enzyme of Dictyostelium discoideum. However, the enzyme of P. polycephalum contains an atypical amino-terminal domain very rich in serine and proline, whose function is unknown. Remarkably, both a mitochondrial targeting sequence and a nuclear localization signal were predicted respectively in the amino and carboxy-terminus of the protein, as in the case of human topoisomerase III alpha. At the Physarum genomic level, the topoisomerase II gene encompasses a region of about 16 kbp suggesting a large proportion of intronic sequences, an unusual situation for a gene of a lower eukaryote, often free of introns. Finally, expression of topoisomerase II mRNA does not appear significantly dependent on the plasmodium cycle stage, possibly due to the lack of G1 phase or (and) to a mitochondrial localization of the enzyme.

Abundant gene conversion between arms of palindromes in human and ape Y chromosomes

Eight palindromes comprise one-quarter of the euchromatic DNA of the male-specific region of the human Y chromosome, the MSY. They contain many testis-specific genes and typically exhibit 99.97% intra-palindromic (arm-to-arm) sequence identity. This high degree of identity could be interpreted as evidence that the palindromes arose through duplication events that occurred about 100,000 years ago. Using comparative sequencing in great apes, we demonstrate here that at least six of these MSY palindromes predate the divergence of the human and chimpanzee lineages, which occurred about 5 million years ago. The arms of these palindromes must have subsequently engaged in gene conversion, driving the paired arms to evolve in concert. Indeed, analysis of MSY palindrome sequence variation in existing human populations provides evidence of recurrent arm-to-arm gene conversion in our species. We conclude that during recent evolution, an average of approximately 600 nucleotides per newborn male have undergone Y-Y gene conversion, which has had an important role in the evolution of multi-copy testis gene families in the MSY.

Cloning and detection of metallothionein mRNA by RT-PCR in mangrove oysters ( Crassostrea rhizophorae)

A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters ( Crassostrea rhizophorae), using β-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction.

Naturally occurring amino acid substitutions in the HIV-2 ROD envelope glycoprotein regulate its ability to augment viral particle release

The envelope glycoprotein of HIV-2 ROD10 has the intriguing ability to enhance the rate of viral particle release from infected cells. However, not all HIV-2 envelope glycoproteins are active in this regard. Indeed, we have previously noted that, despite a high degree of identity with that of ROD10, the envelope protein of the ROD14 isolate was unable to enhance virus production. In this study, site-directed mutagenesis was employed to reveal that a single naturally occurring alanine-to-threonine substitution at position 598, located in the extracellular part of the TM subunit, fully accounted for the lack of activity of the ROD14 Env in HeLa and 12D7 cells. A second mutation at position 422, substituting a lysine residue in ROD10 for an arginine in ROD14, was additionally required for efficient virus release from infected H9 cells, suggesting cell-type-specific requirements for this activity. Interestingly, the ROD14 Env protein exhibited a trans-dominant negative effect on particle release by ROD10 Env, suggesting that the viral release activity of the HIV-2 ROD envelope protein may be regulated by its ability to assemble into functional oligomeric structures.

Primary structure and compartmentalization of Drosophilamelanogaster host cell factor

Human host cell factor-1 (HCF-1) is a large, 2035-residue nuclear protein that interacts with cellular and viral transcription factors. It contains an N-terminal kelch domain, C-terminal fibronectin type III (FnIII) domain, and a central region including tandem repeats which act as cleavage sites. A second human HCF-1 related gene encodes a protein with a high degree of homology in both the N-terminal kelch domain and C-terminal FnIII domain, but lacks the central portion and as a result is considerably smaller at 792 residues. A unique HCF orthologue has been found in Caenorhabditis elegans which is structurally more related to HCF-2 than HCF-1. Here we report the cloning and expression of the single Drosophila melanogaster host cell factor orthologue (dHCF). The dHCF is 1500 residues in size, intermediate between HCF-1 and HCF-2 and contains an N-terminal kelch domain, and C-terminal FnIII domain both of which show a very high degree of identity, and a central region of some 700 residues with more limited homology. Despite containing a central region no repeat-related motifs were apparent. The dHCF is expressed as a single unprocessed polypeptide consistent with the lack of the internal HCF-1 processing sites, and exhibits a predominantly nuclear localization. We show that this nuclear localization is dependent on a bipartite nuclear localization signal at the C-terminus of the protein, which contains a long spacer of 20 amino acids between two basic clusters. Finally, we also show that dHCF is unable to rescue the tsBN67 cell cycle arrest phenotype. These results indicate that dHCF is an orthologue of HCF-1, although both proteins might not be functionally exchangeable.

An aspartate aminotransferase of Wolbachia endobacteria from Onchocerca volvulus is recognized by IgG1 antibodies from residents of endemic areas

Wolbachia are intracellular alpha-proteobacteria, closely related to Rickettsia, that infect various arthropods and filarial parasites. In the present study, the cDNA encoding the aspartate aminotransferase (AspAT) of Wolbachia from the human pathogenic filarial parasite Onchocerca volvulus (Ov-WolAspAT) was identified. At the amino acid level, the identity of the Ov-WolAspAT was 56% to Rickettsia prowazekii AspAT and 54% to the AspAT of the nitrogen-fixing bacterium Sinorhizobium meliloti, but the highest degree of identity was found to the putative AspAT of Wolbachia from Brugia malayi and Drosophila melanogaster (85%). All of these bacterial AspATs are members of the AspAT subclass Ib. A 35 kDa fragment of the Ov-WolAspAT was expressed in Escherichia coli, and immunolocalization using polyclonal antibodies against this antigen revealed that Ov-WolAspAT is present in a considerable proportion of the Wolbachia from O. volvulus, as well as in the endobacteria of several other filarial parasites. Western blot analysis using recombinant Ov-WolAspAT as antigen showed that IgG1 antibodies were present in 70 (51%) individuals living in areas endemic for O. volvulus, B. malayi or Wuchereria bancrofti and no IgG4 or IgE antibodies were found. Among 40 sera of persons from Uganda and Liberia who were putatively not infected with human filarial parasites, 11 (28%) individuals presented IgG1 antibodies, while none of the 33 sera from healthy Europeans and none of the 14 sera from patients with proven Rickettsia or Brucella infections reacted with the antigen. These results also show that an intracellular protein of Wolbachia endobacteria (WolAspAT) acts as antigen in human filariasis.