1-like Sequence Research Articles

Proteomic investigation of the blue mussel larval shell organic matrix

Shell matrix proteins (SMPs) are occluded within molluscan shells and are fundamental to the biological control over mineralization. While many studies have been performed on adult SMPs, those of larval stages remain largely undescribed. Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell proteomes. Following development of a method for cleaning larval shells of tissue contaminants, 49 SMPs were identified using shotgun proteomics. Twenty-one proteins were independently identified in all samples indicating that they form a subset of the core larval shell proteome. These included: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP domain-containing sequence and a ferric-chelate reductase 1-like sequence. Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. While key predictable molluscan shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized, indicating that this shell proteome is unique to mussel larvae. Specifically, comparison with adult mytilids reveals that nine domains are exclusive to the larval shell proteome and only four domains are conserved among species and developmental stages. Thus, strong species-specific and ontogenetic variation exists in shell proteome composition.

The duck EB66® cell substrate reveals a novel retrotransposon

During the establishment of the duck EB66® cell line as a new cell substrate for vaccine production in industry, a very low level of reverse transcriptase (RT) activity was detected in the culture supernatant by product-enhanced RT assay but a whole battery of tests failed to evidence infectious particles. Results from extensive biochemical and physical testing demonstrated that RT activity was associated to an intracellular, non-enveloped and dense structure different from an infectious retroviruses.In silico analysis of Anas platyrhynchos genome revealed that the most likely candidates for encoding a ribonucleoprotein (RNP)-associated RT were nine copies of chicken repeat 1 (CR1)-like elements, belonging to the non-long terminal repeat retrotransposons. The presence of the full length Anas platyrhynchos chicken repeat 1-like sequence (APCR1) was confirmed in EB66® cells and the related ribonucleic acid was present in the RT-containing fraction of EB66® cells.

Finding RB/Rpi-blb1/Rpi-sto1-like sequences in conventionally bred potato varieties

The main objectives in potato breeding are increasing yield abilities and improving resistance to numerous pathogens and pests. Among them, the late blight caused by the Phytophthora infestans oomycete is one of the most destructive potato diseases both in Russia and worldwide. Wild relatives of cultivated potato are traditionally used in breeding as the source of valuable R genes conferring resistance to pathogens. Of particular interest are Mexican wild species because Mexico is the centre of origin and diversity of P. infestans and at the same time, it is the centre of potato species diversity. Mexican wild potato species S. bulbocastanum and S. stoloniferum are an important source of the R genes conferring broad-spec trum resistance against various isolates of P. infestans (Rpi-blb1, Rpi-blb2, Rpi-sto1). Recently these genes have been transferred into cultivated potato gene pool using the cisgene approach. At the same time there is a high probability of finding geno types with the Rpi-sto1 gene (functional homologues of Rpi-blb1) among conventionally bred varieties because for about 40 years S. stoloniferum has been used in breeding as a source of the Rysto and Ry-fsto genes of the extreme resistance to the most important viral pathogen PVY. In this study 188 potato varieties bred in Russia and in near-abroad countries were screened for the presence of six gene-specific markers of the RB/Rpi-blb1 = Rpi-sto1 and Rpi-blb2 genes conferring broad-spectrum resistance against P. infestans, and for the markers linked to the Rysto and Ry-fsto genes conferring extreme resistance to PVY. In addition, a marker for detecting male sterile mitochondrial DNA type gamma derived from S. stoloniferum was used. The genotypes selected through the molecular markers were divided into four groups: (A) 13 PVY resistant varieties carrying diagnostic markers of the Rysto, Ry-fsto genes and having sterile mt-type gamma; (B) four varieties possessing mt-type gamma and not having the markers of the R genes introgressed from S. stoloniferum; (C) eight genotypes carrying five gene-specific markers for the RB/Rpi-blb1/= Rpi-sto1; (D) the rest 166 (86.9 %) varieties not possessing any of the diagnostic markers associated with the S. stoloniferum genetic material. The sequences of the Rpi-sto1- and BLB1 F/R-amplicons were identical in all the genotypes of group ‘C’ and showed respective 99 % and 100 % similarity to the corresponding fragments of the Rpi-sto1 and Rpi-blb1 genes from the GenBank database. Among the genotypes of group ‘C’ various mt-types were detected, and some of them were male fertile.

Differential expression and sequence polymorphism of the olive pollen allergen Ole e 1 in two Iranian cultivars.

Molecular evidence on the heterogeneity present in the Ole e 1 allergen of the olive pollen is emerging. Such polymorphism is dependent on the cultivar origin of pollen, which also determines wide differences in the expression of this protein. Determination of biochemical and molecular characteristics of Ole e 1 pollen allergen in two Iranian olive cultivars, namely 'Rowghani' and 'Zard' is necessary to assess their allergenicity potential. SDS-PAGE and immunoblotting analysis of pollen extracts showed that both cultivars present high and low expression of Ole e 1, respectively. These protein levels correlated with similarly different levels of transcripts, as determined by RT-PCR. Two-dimensional protein profiles also showed conspicuous differences in the distribution and the level of expression of those spots reacting to an anti-Ole e 1 antibody. Bioinformatic analysis of four Ole e 1 sequences corresponding to 'Rowghani' and two sequences for 'Zard', showed numerous heterogeneities when compared with those Ole e 1 and Ole e 1-like sequences present in databases. Nucleotide substitutions resulted in many cases in changes over the predicted amino acid sequences. A cladistic analysis of the sequences showed Iranian entries in a central position between West-European sequences, and Ole e 1-like sequences from other Oleaceae species. Moreover, amino acid changes affected key epitopes of the protein involved in the recognition of the protein by the human immune system. Putative implications of polymorphism in both the biological role and the allergic reactivity of Ole e 1 are discussed.

Changes in the community structure and activity of betaproteobacterial ammonia‐oxidizing sediment bacteria along a freshwater–marine gradient

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.

Differences between betaproteobacterial ammonia-oxidizing communities in marine sediments and those in overlying water.

To assess links between betaproteobacterial ammonia-oxidizing bacteria (AOB) in marine sediment and in overlying water, communities in Loch Duich, Scotland, were characterized by analysis of clone libraries and denaturant gradient gel electrophoresis of 16S rRNA gene fragments. Nitrosospira cluster 1-like sequences were isolated from both environments, but different sequence types dominated water and sediment samples. Detailed phylogenetic analysis of marine Nitrosospira cluster 1-like sequences in Loch Duich and surrounding regions suggests the existence of at least two different phylogenetic subgroups, potentially indicative of new lineages within the betaproteobacterial AOB, representing different marine ecotypes.

Genomic Organization of the Chicken T-Cell Receptor .BETA. Chain D-J-C Region

We examined overlapping genomic clones containing the chicken T cell receptor (TCR) Dbeta-Jbeta-Cbeta complex, which contains a single diversity segment, four joining segments and four exons that encode the constant region. This sequence comprised 18.3 kb. All four Jbeta sequences possessed typical recombination signal sequences (RSS) with intervening 12-bp spacers at their 5'-ends and splice sites at their 3'-ends. No Jbeta-pseudogenes were identified. TGTG sequences in the RSS heptamer sequences were well conserved, as is the case in mammals. A chicken repeat 1-like sequence was found in the intron region between Jbeta-1336 and Cbeta, and several small repeat sequences were identified in intron regions throughout this cloned genome. As germline sequences revealed complete Jbeta sequences, the CDR3 (complementarity-determining region) sequences of TCRbeta from non-immunized splenocytes were analyzed. Non-coding (N) and palindromic (P) nucleotides were frequently observed at the Dbeta-Jbeta recombination sites. There were differences in length of deletion at the 5'-end of each Jbeta. Deletion of the 5'-end of Jbeta-1280 was particularly short when compared with that of Jbeta-1336, but there were no changes in the length of the CDR3 using any of the four Jbeta sequences.

ISMmy 1, a novel insertion sequence of Mycoplasma mycoides subsp. mycoides small colony type

A new insertion sequence, ISMmy 1, has been identified in the bovine pathogen Mycoplasma mycoides subsp. mycoides biotype small colony ( MmymySC). The occurrence of ISMmy 1 in 15 MmymySC strains and 12 other mycoplasmas was examined by Southern blotting. All MmymySC strains showed identical hybridisation patterns except for the type strain PG1 T, the vaccine strain T1Sr49, and the strain Afadé, which all had unique patterns. ISMmy 1-like sequences were also found in the bovine pathogen Mycoplasma bovis strain Donetta T while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy 1. This observation suggests horizontal transfer between MmymySC and M. bovis.

Immunological detection of alkaline-diaminobenzidine-negativeperoxisomes of the nematode Caenorhabditis elegans purification and unique pH optima of peroxisomal catalase.

We purified catalase-2 of the nematode Caenorhabditis elegans and identified peroxisomes in this organism. The peroxisomes of C. elegans were not detectable by cytochemical staining using 3, 3'-diaminobenzidine, a commonly used method depending on the peroxidase activity of peroxisomal catalase at pH 9 in which genuine peroxidases are inactive. The cDNA sequences of C. elegans predict two catalases very similar to each other throughout the molecule, except for the short C-terminal sequence; catalase-2 (500 residues long) carries a peroxisomal targeting signal 1-like sequence (Ser-His-Ile), whereas catalase-1 does not. The catalase purified to near homogeneity from the homogenate of C. elegans cells consisted of a subunit of 57 kDa and was specifically recognized by anti-(catalase-2) serum but not by anti-(catalase-1) serum. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected catalase-2 inside vesicles judged to be peroxisomes using morphological criteria. The purified enzyme (220 kDa) was tetrameric, similar to many catalases from various sources, but exhibited unique pH optima for catalase (pH 6) and peroxidase (pH 4) activities; the latter value is unusually low and explains why the peroxidase activity was undetectable using the standard alkaline diaminobenzidine-staining method. These results indicate that catalase-2 is peroxisomal and verify that it can be used as a marker enzyme for C. elegans peroxisomes.

Diversity of GB virus C/hepatitis G virus isolates in Singapore: predominance of group 2a and the Asian group 3 variant.

The occurrence of GBV-C/HGV infection was studied in 160 individuals from two high-risk groups in Singapore. RT-PCR of the 5'-UTR detected GBV-C/HGV RNA in 3/73 (4.1%) of patients undergoing maintenance hemodialysis for chronic renal failure, and in 17/87 (19.5%) of patients coinfected with HCV who tested positive for HCV RNA. Phylogenetic analysis of 5'-UTR sequences from these 20 samples showed that the Asian or group 3 variant was found in 45% of the samples sequenced, thus confirming the high frequency of this variant in the region. Group 2a variants accounted for 50% of the samples with a complete absence of group 2b. Our analysis also provided strong bootstrap support for the subdivision of group 2 into subgroups 2a and 2b. This study shows that isolates belonging to all three main groups of GBV-C/ HGV can be detected in Singapore, with the large majority belonging to groups 2a (50%) and 3 (45%). Only a single group 1-like sequence was detected within the 20 isolates. Of interest also is that all group 3 isolates were identified in Chinese patients while group 2a was found in both Chinese and Malay.

Characterization of the Gene for HRG4 (UNC119), a Novel Photoreceptor Synaptic Protein Homologous to Unc-119

HRG4 (HGMW-approved symbol UNC119) is a novel human photoreceptor-enriched gene coding for a 240-amino-acid protein. Initially, HRG4 was shown to be 57% homologous to a newly discoveredCaenorhabditis elegansgene, mutated in a coordination mutant and involved in chemosensation. Recently, HRG4 has been localized to the photoreceptor synapses in the outer plexiform layer of the retina. The HRG4 gene was cloned and characterized to facilitate its analysis as a potential pathogenic gene. The gene consisted of five coding exons, spread over approximately 8 kb of genomic DNA. The transcriptional start site was 14 bp upstream of the cDNA, 68 bp upstream of the putative translational initiation codon. Five GC boxes were identified in a 100-bp upstream region, along with a photoreceptor conserved element 1-like sequence at −603. Another photoreceptor gene-associated sequence, Ret-1, was present in intron 1, 71 bp downstream of the exon 1/intron 1 border. A CpG island encompassing exon 1 and sequences just before and after it was present. The gene was fine mapped to 17q11.2, facilitating its future consideration as a candidate for retinal diseases mapped to the same region.

An onion isolate of tobacco rattle virus: reactivity with an antiserum to Hypochoeris mosaic virus, a putative furovirus, and molecular analysis of its RNA 2.

A tubular virus from onion was found to react with an antiserum to Hypochoeris mosaic virus (HyMV), a putative furovirus. Sequence analysis of its genomic RNAs and further serological tests, however, indicated it to be a tobravirus rather than a furovirus. The reactivity of the HyMV antiserum with several isolates of tobacco rattle virus (TRV) suggests that HyMV itself may be a tobravirus. The deduced amino acid sequences of the putative proteins encoded on RNA 2 of the onion virus isolate (ON) suggest close evolutionary relationships to the TRV isolate TCM from tulip. However, RNA 2 of the ON isolate contains a shorter RNA 1-like sequence on its 3'-end and an additional small ORF upstream of its RNA 1-like part. The sequence of its 315 5'-terminal nucleotides is more similar to that of RNA 2 of the PLB isolate from potato than to that of TCM RNA 2.

Nonidentity between plasmid and chromosomal copies of ISS 1-like sequences in Lactococcus lactis subsp. lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes †

The nucleotide sequence of an insertion sequence (IS) observed during mating experiments using the lactose-protease plasmid, pUCL22, of Lactococcus (Lc.) lactis subsp. lactis CNRZ270, was found to be similar to that of ISS 1 from Lc. lactis subsp. lactis ML3. The IS was named ISS 1RS. The chromosome of this strain contains several copies of ISS 1-like IS as assessed by hybridization. One of these copies was cloned and named ISS 1CH. Its sequence differs from that of the plasmid-borne copy, and appears to be more closely related to ISS 1N from Lc. lactis subsp. cremoris SK11. This suggests independent introduction of both ISS 1 elements. Moreover, the observation of plasmid genes integrated in the CNRZ270 chromosome near ISS 1CH suggests that their presence is the result of integration by a Campbell mechanism using both IS homologies. ISS 1-like sequences were also found on plasmids of numerous Lc. lactis strains, as well as one out of seven Lactobacillus (Lb.) casei and one out of three Lb. plantarum strains examined.

Characterization of the promoter/regulatory region of the bovine CYP11A (P-450scc) gene. Basal and cAMP-dependent expression.

The promoter/regulatory region of the bovine CYP11A (P-450scc) gene was cloned from a bovine genomic library. One major start site of transcription was identified by primer extension analysis with a minor start site four nucleotides further upstream. A putative TATA box is located at position -31, and at position -68 resides a putative binding site for the transcription factor Sp1. Transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells was used to locate regions within the P-450scc 5'-flanking sequences that are important for basal and cAMP-dependent transcription of the reporter genes. While cAMP-dependent accumulation of mRNA derived from expression of the endogenous bovine P-450scc gene can be inhibited by protein synthesis inhibitors, transcription of reporter gene constructs containing the promoter/regulatory region of the P-450scc gene is not affected by cycloheximide following transient transfection of Y1 cells or primary bovine adrenocortical cells. Basal expression of these constructs as well as cAMP responsiveness is reduced upon deletion of sequences between -186 and -101, further deletion to -50 leading to loss of virtually all the remaining cAMP responsiveness. The sequence between -183 and -83 alone will direct both basal and cAMP-enhanced transcription when fused to a heterologous promoter and is equally active in either the correct or reverse orientation. No homology to the consensus cAMP-responsive element (CRE) or AP-2 binding site is found in this region whereas an activator protein 1-like sequence is found at position -116. It is concluded that the cAMP responsiveness of P-450scc gene expression is mediated by sequences different from canonical consensus regulatory elements. Whether or not there are sequences conferring cAMP responsiveness which are common both to P-450scc and the other steroidogenic P-450 genes remains to be established.

Small nuclear RNA-associated proteins are immunologically related as revealed by mapping of autoimmune reactive B-cell epitopes.

Autoantibodies from a patient with systemic lupus erythematosus, which recognize U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), were used to map B-cell autoepitopes on the U1 snRNP-specific A protein. This protein contains two regions that are highly similar to regions in the U2 snRNP-specific B" protein. A site termed epitope 2 maps in one such region and was found to react with antibodies cross-reactive between A and B". A second site, epitope 1, is situated in a proline-rich region that shows no homology with B". This epitope can bind three different autoantibodies with distinct specificities. Epitope 1-affinity-purified antibodies from different patients react with either (i) the A protein exclusively; (ii) proteins A, B'/B, a synthetic peptide for part of the N polypeptide, and an unidentified protein with a molecular mass of 50 kDa; or (iii) proteins A, B'/B, C, and the N-derived peptide. Comparison of the primary structures of proteins B'/B, N, and C reveals multiple epitope 1-like sequences in all of them. The possibility that these repeating regions act as immunogens in patients with autoimmune disease is discussed.