Percent Amino Acid Identity Research Articles

Comparative sequence analysis of SARS nCoV and SARS CoV genomes for variation in structural proteins

SARS-nCoV was identified as corona virus had spread worldwide very quickly and affected more than million people worldwide. To halt this acceleration and for efficient control the knowledge on genomic information is of utmost importance. We attempted to determine the nature of variation i.e., insertion, deletion, substitution, among structural sequences required to code for membrane, spike, nucleocapsid, envelope protein and glycosylation variation between SARS CoV and SARS nCoV spike glycoproteins, respectively. Comparative sequence analysis was performed by using retrieved sequences from the NCBI database. The analyzed sequences revealed, that the sequences coding for envelope protein show minor substituting amino acids. SARS CoV showed 94.74 percent amino acid identities with SARS nCoV amino acid sequences coding for envelope protein. In comparison to SARS nCoV, distinct amino acid residues vary in SARS CoV sequences coding for membrane, nucleocapsid, and spike proteins, respectively. S protein coding sequences of SARS CoV exhibited one deletion, six insertion and six hundred three substitutions in SARS nCoV sequence. Insertion of valine was found in receptor binding domain of SARS nCoV at position 487, and NSPR amino acid residues at position 683–686. Deletions and substitutions were also found in nucleotide sequences of strain B.1.617.2 of SARS nCoV. Additionally, binding interaction pattern of ACE2 receptor protein with original wild-type SARS-CoV-2 strain with the recently evolved Omicron variant was also evaluated. The docking results substantiated that the specific variation in binding residues is likely to impact virulence pattern of both variants.

Characterization of plant glutamine synthetase S-nitrosation

The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non-conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.

Suppression of argonautes compromises viral infection in Penaeus monodon

Argonaute (Ago) proteins, the catalytic component of an RNA-induced silencing complex (RISC) in RNA interference pathway, function in diverse processes, especially in antiviral defense and transposon regulation. So far, cDNAs encoding four members of Argonaute were found in Penaeus monodon (PmAgo1-4). Two PmAgo proteins, PmAgo1 and PmAgo3 shared high percentage of amino acid identity to Ago1 and Ago2, respectively in other Penaeid shrimps. Therefore, the possible roles of PmAgo1 and PmAgo3 upon viral infection in shrimp were characterized in this study. The level of PmAgo1 mRNA expression in shrimp hemolymph was stimulated upon YHV challenge, but not with dsRNA administration. Interestingly, silencing of either PmAgo1 or PmAgo3 using sequence-specific dsRNAs impaired the efficiency of PmRab7-dsRNA to knockdown shrimp endogenous PmRab7 expression. Inhibition of yellow head virus (YHV) replication and delayed mortality rate were also observed in both PmAgo1-and PmAgo3-knockdown shrimp. In addition, silencing of PmAgo3 transcript, but not PmAgo1, revealed partial inhibition of white spot syndrome virus (WSSV) infection and delayed mortality rate. Therefore, our study provides insights into PmAgo1and PmAgo3 functions that are involved in a dsRNA-mediated gene silencing pathway and play roles in YHV and WSSV replication in the shrimp.

HIV drug resistance testing among patients failing second line antiretroviral therapy. Comparison of in-house and commercial sequencing

IntroductionHIV genotyping is often unavailable in low and middle-income countries due to infrastructure requirements and cost. We compared genotype resistance testing in patients with virologic failure, by amplification of HIV pol gene, followed by “in-house” sequencing and commercial sequencing. MethodsRemnant plasma samples from adults and children failing second-line ART were amplified and sequenced using in-house and commercial di-deoxysequencing, and analyzed in Harare, Zimbabwe and at Stanford, U.S.A, respectively. HIV drug resistance mutations were determined using the Stanford HIV drug resistance database. ResultsTwenty-six of 28 samples were amplified and 25 were successfully genotyped. Comparison of average percent nucleotide and amino acid identities between 23 pairs sequenced in both laboratories were 99.51 (±0.56) and 99.11 (±0.95), respectively. All pairs clustered together in phylogenetic analysis. Sequencing analysis identified 6/23 pairs with mutation discordances resulting in differences in phenotype, but these did not impact future regimens. ConclusionsThe results demonstrate our ability to produce good quality drug resistance data in-house. Despite discordant mutations in some sequence pairs, the phenotypic predictions were not clinically significant.

Percent amino-acid identity thresholds are not necessarily conservative for predicting allergenic cross-reactivity

International guidance on assessing amino-acid sequences for allergenic cross reactivity, such as that provided by Codex, relies on percent amino-acid identity across local alignments. Previous work has shown this approach to exhibit poor selectivity for true allergen cross-reactivity. Here we provide a hypothetical example illustrating that this approach is also not necessarily conservative in detecting high similarity across amino acid sequences, and thus, is not necessarily conservative in detecting potential allergenic cross reactivity among proteins. It is recommended that an established local-alignment bioinformatic tool like FASTA be used in conjunction with statistical measures of similarity to detect the risk of a protein cross-reacting with an allergen.

Functional Characterization of the Plastidial 3-Phosphoglycerate Dehydrogenase Family in Arabidopsis

This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of eda9 was complemented with an EDA9 complementary DNA under the control of a 35S promoter (Pro-35S:EDA9). However, this construct, which is poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in eda9.1eda9.1 Pro-35S:EDA9 was arrested at the polarized stage. Pollen from these lines lacked tryphine in the interstices of the exine layer, displayed shrunken and collapsed forms, and were unable to germinate when cultured in vitro. A metabolomic analysis of PGDH mutant and overexpressing plants revealed that all three PGDH family genes can regulate Ser homeostasis, with PGDH being quantitatively the most important in the process of Ser biosynthesis at the whole-plant level. By contrast, the essential role of EDA9 could be related to its expression in very specific cell types. We demonstrate the crucial role of EDA9 in embryo and pollen development, suggesting that the phosphorylated pathway of Ser biosynthesis is an important link connecting primary metabolism with development.

Isolation and molecular characterization of a second serotype of the encephalomyocarditis virus

Encephalomyocarditis virus (EMCV) and Theilovirus are the two species of the Cardiovirus genus. Whereas theiloviruses comprise several sero-/genotypes, all known EMCV isolates are serologically very similar and are thought to belong to one serotype, named EMCV-1. Here, a novel EMCV type is described. Strain RD 1338 (D28/05) was isolated from a wood mouse (Apodemus sylvaticus) in Germany and can be distinguished from EMCV-1 by serological and molecular means. Failure of EMCV-1 specific hyperimmune sera to neutralize RD 1338 suggests a distinct serotype. The viral genome was de novo sequenced using next-generation Illumina/Solexa technologies. Considerable differences of the BC-loop/loop I/loop II sequences of VP1, the VP2 puff and the VP3 knob provide a structural basis for deviant serological properties. Sequence alignments reveal amino acid identities of 75 percent for the P1 region and 84 percent for the P2 and P3 regions when comparing RD 1338 to EMCV-1 strains and some 60 percent and less than 50 percent amino acid identities, respectively, for comparisons with theilovirus strains. Phylogenetic analyses of the P1, 2C and 3CD gene regions support the establishment of an EMCV-2 serotype. In contrast to the theilovirus sero-/genotypes that show a narrow host range, EMCV-1 infects a wide variety of hosts. The host range of EMCV-2 remains to be determined.

Biochemical and Molecular Characterization of Secreted α-Xylosidase from Aspergillus niger

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.

An integrative approach to ortholog prediction for disease-focused and other functional studies

BackgroundMapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward.ResultsWe report a simple but effective tool, the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist).ConclusionsDIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

Isolation and Partial Characterization of an R2R3MYB Transcription Factor from the Bamboo Species Fargesia fungosa

Formation of the woody stems of bamboo requires the coordinated regulation of cellulose, xylan, and lignin biosynthesis. Lignin contributes 25–30% of harvested bamboo constituents, and it strongly influences the physical properties of bamboo. In this study, we report on cloning and partial characterization of an R2R3MYB transcription factor gene (FfMYB1) from the bamboo species Fargesia fungosa. FfMYB1 consists of a coding region of 813 bp, corresponding to a predicted peptide of 270 amino acids, and an upstream promoter sequence of 1.46 kb. The deduced peptide sequence of FfMYB1 has the highest percent amino acid identity to NtMYBGR1 of tobacco, as well as to both AtMYB20 and AtMYB43 of Arabidopsis. Both NtMYBGR1 and AtMYB20/43 are putative activators of the phenylpropanoid pathway for lignin production. Histochemical analysis of F. fungosa stems showed rapid production of lignin during stem maturation, and FfMYB1 transcript was detected in leaves and stems of 1-year old shoots. A phylogenetic study of R2R3MYB sequences available for the bamboo subfamily identified other potential lignin-related R2R3MYBs, in particular bphylf044c24 of the bamboo genera Phyllostachys.

Molecular characterization of MHC class II region in guinea fowl

1. The MHC class II gene was amplified, cloned and sequenced in guinea fowl. 2. The NumeMHC II sequence of 754 nucleotides included complete exon 1 (91 nt), exon 2 (270 nt), exon 3 (282 nt) and exon 4 (110 nt). 3. The size of β 1 and β 2, domains were 89 and 93 amino acids, respectively in guinea fowl. 4. High amino acid variability (38·2%) was observed within guinea fowl in β 1 domain, while in β 2 domain, amino acid variability (6·3%) was low. 5. Among poultry species, the percent amino acid identity between guinea fowl and chicken, quail, pheasant and duck was 38·8, 42·2, 44·4 and 58·8 in β 1 domain; and 13·8, 17·0, 13·8 and 27·6 in β 2 domain, respectively. 6. Sequence alignment with mammalian and avian MHC showed that many of the conserved features of MHC class II glycoprotein was conserved in guinea fowl. 7. Within-species genetic distances (Poisson correction) based on cumulative amino acid variability in β 1 domain and β 2 domains was 0·141 in guinea fowl. 8. Guinea fowl showed low and similar genetic distances with all the poultry species (0·255–0·268) except duck (0·456). 9. Guinea fowl made separate branch within the major cluster having chicken, quail and pheasant, showing equal distance from these poultry species, whereas duck MHC II clustered separately.

Phylogenetic diversity of stress signalling pathways in fungi.

BackgroundMicrobes must sense environmental stresses, transduce these signals and mount protective responses to survive in hostile environments. In this study we have tested the hypothesis that fungal stress signalling pathways have evolved rapidly in a niche-specific fashion that is independent of phylogeny. To test this hypothesis we have compared the conservation of stress signalling molecules in diverse fungal species with their stress resistance. These fungi, which include ascomycetes, basidiomycetes and microsporidia, occupy highly divergent niches from saline environments to plant or mammalian hosts.ResultsThe fungi displayed significant variation in their resistance to osmotic (NaCl and sorbitol), oxidative (H2O2 and menadione) and cell wall stresses (Calcofluor White and Congo Red). There was no strict correlation between fungal phylogeny and stress resistance. Rather, the human pathogens tended to be more resistant to all three types of stress, an exception being the sensitivity of Candida albicans to the cell wall stress, Calcofluor White. In contrast, the plant pathogens were relatively sensitive to oxidative stress. The degree of conservation of osmotic, oxidative and cell wall stress signalling pathways amongst the eighteen fungal species was examined. Putative orthologues of functionally defined signalling components in Saccharomyces cerevisiae were identified by performing reciprocal BLASTP searches, and the percent amino acid identities of these orthologues recorded. This revealed that in general, central components of the osmotic, oxidative and cell wall stress signalling pathways are relatively well conserved, whereas the sensors lying upstream and transcriptional regulators lying downstream of these modules have diverged significantly. There was no obvious correlation between the degree of conservation of stress signalling pathways and the resistance of a particular fungus to the corresponding stress.ConclusionOur data are consistent with the hypothesis that fungal stress signalling components have undergone rapid recent evolution to tune the stress responses in a niche-specific fashion.

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.

BackgroundVisceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced.ResultsWe have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (ΔLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of ΔLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving ΔLicpa::CPA) was sufficient to complement the reduced infectivity of both ΔLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone ΔLicpaC1::CPA compared with the CPA-deficient mutant ΔLicpaC1.ConclusionThe data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the ΔLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.

The G Protein–Coupled Receptor Subset of the Chicken Genome

G protein–coupled receptors (GPCRs) are one of the largest families of proteins, and here we scan the recently sequenced chicken genome for GPCRs. We use a homology-based approach, utilizing comparisons with all human GPCRs, to detect and verify chicken GPCRs from translated genomic alignments and Genscan predictions. We present 557 manually curated sequences for GPCRs from the chicken genome, of which 455 were previously not annotated. More than 60% of the chicken Genscan gene predictions with a human ortholog needed curation, which drastically changed the average percentage identity between the human–chicken orthologous pairs (from 56.3% to 72.9%). Of the non-olfactory chicken GPCRs, 79% had a one-to-one orthologous relationship to a human GPCR. The Frizzled, Secretin, and subgroups of the Rhodopsin families have high proportions of orthologous pairs, although the percentage of amino acid identity varies. Other groups show large differences, such as the Adhesion family and GPCRs that bind exogenous ligands. The chicken has only three bitter Taste 2 receptors, and it also lacks an ortholog to human TAS1R2 (one of three GPCRs in the human genome in the Taste 1 receptor family [TAS1R]), implying that the chicken's ability and mode of detecting both bitter and sweet taste may differ from the human's. The chicken genome contains at least 229 olfactory receptors, and the majority of these (218) originate from a chicken-specific expansion. To our knowledge, this dataset of chicken GPCRs is the largest curated dataset from a single gene family from a non-mammalian vertebrate. Both the updated human GPCR dataset, as well the chicken GPCR dataset, are available for download.

Cloning and expression analysis of goldfish ( Carassius auratus L.) prominin

The integral membrane protein known as prominin was first identified on the apical surface of mouse neural epithelial cells as well as on the surface of human haematopoietic progenitor cells. This report describes a prominin-like sequence and expression analysis of the prominin in the goldfish. The predicted amino acid sequence for goldfish prominin shares all of the hallmark structural characteristics of the prominin family, however the relatedness assessed using the percent amino acid identity indicated that goldfish prominin cannot be placed into the current mammalian dichotomy of type 1 or 2. The real time PCR analyses indicated that prominin was broadly expressed in different tissues with particularly high levels observed in the kidney and gill of the goldfish. Goldfish prominin was also found to be differentially expressed in subpopulations of in vitro-derived goldfish macrophages, with the highest expression observed in progenitor cells.

Sequences of avian reovirus M1, M2 and M3 genes and predicted structure/function of the encoded μ proteins

We report the first sequence analysis of the entire complement of M-class genome segments of an avian reovirus (ARV). We analyzed the M1, M2 and M3 genome segment sequences, and sequences of the corresponding μA, μB and μNS proteins, of two virus strains, ARV138 and ARV176. The ARV M1 genes were 2283 nucleotides in length and predicted to encode μA proteins of 732 residues. Alignment of the homologous mammalian reovirus (MRV) μ2 and ARV μA proteins revealed a relatively low overall amino acid identity (∼30%), although several highly conserved regions were identified that may contribute to conserved structural and/or functional properties of this minor core protein (i.e. the MRV μ2 protein is an NTPase and a putative RNA-dependent RNA polymerase cofactor). The ARV M2 genes were 2158 nucleotides in length, encoding predicted μB major outer capsid proteins of 676 amino acids, more than 30 amino acids shorter than the homologous MRV μ1 proteins. In spite of the difference in size, the ARV/MRV μB/μ1 proteins were more conserved than any of the homologous proteins encoded by other M- or S-class genome segments, exhibiting percent amino acid identities of ∼45%. The conserved regions included the residues involved in the maturation- and entry- specific proteolytic cleavages that occur in the MRV μ1 protein. Notably missing was a region recently implicated in MRV μ1 stabilization and in forming “hub and spokes” complexes in the MRV outer capsid. The ARV M3 genes were 1996 nucleotides in length and predicted to encode a μNS non-structural protein of 635 amino acids, significantly shorter than the homologous MRV μNS protein, which is attributed to several substantial deletions in the aligned ARV μNS proteins. Alignments of the ARV and MRV μNS proteins revealed a low overall amino acid identity (∼25%), although several regions were relatively conserved.

Characterization of SpAPETALA3 and SpPISTILLATA, B class floral identity genes in Spinacia oleracea, and their relationship to sexual dimorphism

Floral organ identity B class genes are generally recognized as being required for development of petals and stamens in angiosperm flowers. Spinach flowers are distinguished in their complete absence of petals in both sexes, and the absence of a developed stamen whorl in female flowers. As such, we hypothesized that differential expression of B class floral identity genes is integral to the sexual dimorphism in spinach flowers. We isolated two spinach orthologs of Arabidopsis B class genes by 3' and 5' RACE. Homology assignments were tested by comparisons of percent amino acid identities, searches for diagnostic consensus amino acid residues, conserved motifs, and phylogenetic groupings. In situ hybridization studies demonstrate that both spinach B class genes are expressed throughout the male floral meristem in early stages, and continue to be expressed in sepal primordia in reduced amounts at later stages of development. They are also highly expressed in the third whorl primordia when they arise and continue to be expressed in these tissues through the development of mature anthers. In contrast, neither gene can be detected in any stage in female flowers by in situ analyses, although northern blot experiments indicate low levels of SpAP3 within the inflorescence. The early, strong expressions of both B class floral identity genes in male floral primordia and their absence in female flowers demonstrate that B class gene expression precedes the origination of third whorl primordia (stamen) in males and is associated with the establishment of sexual floral dimorphism as it initiates in the first (sepal) whorl. These observations suggest that regulation of B class floral identity genes has a role in the development of sexual dimorphism and dioecy in spinach rather than being a secondary result of organ abortion.

Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.

Alteration of amino acid residues at the L-chain N-terminus and in complementarity-determining region 3 increases affinity of a recombinant F(ab) for the human N blood group antigen.

To examine the fine specificity of glycopeptide-specific antibodies, this study focused on the human MN blood group system. F(ab) phage display methods were previously used to construct an F(ab) family in which the H-chain Fd fragment was held constant whereas the L chains were "shuffled." This yielded two related F(ab), N92 and NNA7, with low and high affinity for N, respectively. Although their L-chain sequences are very similar, sharing 92 percent amino acid identity, there are intriguing differences at the N-terminus and in complementarity-determining region 3 (CDR3) at positions 89, 91, 92, and 96. Site-directed mutagenesis, ELISA, and hemagglutination were used to examine the contributions of these variations to antibody affinity. Studies with the N92-S91G and NNA7-G91S mutants demonstrated that the Gly at position 91 was critically important for ensuring high affinity. Indeed, the affinity of N92-S91G was almost as high as N92TM, in which all four CDR3 residues were changed to match NNA7. N-terminal L-chain differences were surprisingly important in determining affinity. For example, when the N-terminus of N92 was changed to match that of NNA7, affinity increased approximately 30-fold. Specific residues at the L-chain N-terminus and in CDR3 significantly affected F(ab) affinity for N. Future structural studies of these F(ab), alone and complexed with this glycopeptide antigen, will provide further insights into these phenomena.

Molecular phylogenetic relationship between two symbiotic photo-oxygenic prokaryotes, Prochloron sp. and Synechocystis trididemni

Synechocystis trididemni is a photo-oxygenic prokaryotic symbiont in some didemnid ascidians. Although the cells are pink and contain phycoerythrin, they morphologically resemble those of another symbiont of other didemnids, Prochloron didemni, which has no phycobilin pigments but has both chlorophyll a and chlorophyll b. The cytological similarities suggest that they might have evolved from a common ancestor. We examined this hypothesis by comparing nucleotide sequences in gene fragments of the 16S ribosomal RNA (16S rRNA) gene and in the large subunit of Rubisco gene (rbcL). ‘Blast’ search of GenBank showed that the most similar sequences to the 16S rRNA gene and rbcL of S. trididemni were those of Prochloron sp. The percent nucleotide and amino acid identities of the 16S rRNA genes, rbcL and the deduced Rubisco amino acid sequences between Prochloron sp. and S. trididemni were 94%, 90% and 99%, respectively. In trees based on the 16S rRNA gene and rbcL sequences, S. trididemni formed a clade with Prochloron. These data thus support the hypothesis that Prochloron sp. and S. trididemni arose from a common cyanobacterial ancestor and that the ancestor of Prochloron sp. somehow acquired the ability to synthesize chlorophyll b, as well as chlorophyll a, and subsequently lost the ability to synthesize phycobiliproteins.