EMBL Data Bank Research Articles

Molecular Cloning and Expression of Cytochrome P450 1C1 in Japanese Eel ( Anguilla Japonica )

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new cDNA of the CYP1C subfamily encoding CYP1C1 was isolated from Japanese eel (Anguilla japonica) liver after intraperitoneal injection with βnaphthoflavone (BNF). The full-length cDNA obtained (3508 bp) contained a 5' noncoding region of 355 bp, an open reading frame of 1581 bp coding for 526 amino acids, a stop codon, and a 3' noncoding region of 1572 bp. The predicted molecular weight of the protein was approximately 59.33 kDa. The deduced amino acid sequence of Japanese eel CYP1C1 had the lower similarity of 70% with that of killifish CYP1C1 while the higher similarity (79 and 81%) was observed with that of rainbow trout CYP1C2 and -1C1 sequences respectively. It exhibited similarities of 71% with that of Indian medaka CYP1C1 and zebrafish CYP1C2. Also the similarity of 74% was registered with the sequence of three-spined stickleback fish CYP1C1, 1C2 and carp CYP1C2. It showed similarity of 77% with that of Nile tilapia CYP1C1, scup CYP1C1 and scup CYP1C2. The phylogenetic tree showed the newly identified Japanese eel CYP1C1 sequence to be clustered with rainbow trout CYP1C1 and -1C2. Japanese eel CYP1C1 was aligned with the CYP1 sequences and has been deposited in the Gen Bank / EMBL data bank with the accession number AY444748. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis of liver, kidney, intestine and gills revealed a distinct induced expression in all organs studied (283.33, 579.35, 20.96 and 3642.32 respectively).

Diabody Mixture Providing Full Protection against Experimental Scorpion Envenoming with Crude Androctonus australis Venom

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.

Book review on Bioinformation Discovery: Data to knowledge in Biology

Researchers in almost all disciplines of Biology use mathematical, statistical models and computer programs to analyze and validate biological data. Systematic data mining is frequently completed in recent years using Bioinformatics soft-wares. The available Bioinformatics techniques and tools help annotate functions for newly generated data in biological investigation. The book (ISBN 978-1-4419-0518-5 e-ISBN 978-1-4419-0519-2) entitled “Bioinformation Discovery: Data to knowledge in Biology” by Dr. Pandjassarame Kangueane is published by Springer USA. This book describes the use of Biological data and Bioinformatics techniques and tools in Biological knowledge discovery. The book contains ten chapters. The first chapter describes concepts, principles and components of Bioinformatics with its various applications in agriculture, healthcare and industrial biotechnology sectors. The author precisely highlights that a sound knowledge in the basic concepts of bioinformatics is the real foundation for Bioinformation discovery from data. In the same chapter he also discussed the importance of Bioinformatics in Drug Discovery. The second chapter describes biological datasets and methods to create data subsets from primary databases like GenBank, EMBL, DDBJ and Protein databank (PDB). The provided dataset examples illustrate the importance of specialized datasets in Biological knowledge discovery. There are number of Bioinformatics tools and techniques freely available online for data mining and knowledge discovery in specific areas of Biology. In this context, Chapter 3 (Tools and Techniques) gives an account of some of the commonly used tools frequently cited in this book. The ongoing trends in biological sciences are to understand the protein-protein interaction within and across cells. The chapter 4 (Protein Subunits Interaction) highlights the use of 3D structure datasets to derive information and geometrical (interface size, planarity, sphericity, and complementarity) and chemical (the types of amino acid chemical groups, hydrophobicity, electrostatic interactions, and H-bonds) properties for protein subunit interactions. The formation and mechanism of folding for homodimers (dimers of identical subunits) is intriguing. The fifth chapter relates structural features to homodimer folding and binding using structural datasets. The provided schematic illustrations help in understanding the homodimer folding and binding mechanism. In Chapter 6 (Fusion Proteins), the author discusses on fusion proteins in one species mimic operons, protein-protein interaction, alternative splicing and multiple functions in another species. This chapter help relate gene fusion in establishing optimal dynamics and kinetics. The study of genetic variation among ethnic groups (American Indian, Australian aboriginal, Black, Caucasoid, Oriental, Hispanic, Mixed race, and Pacific Islander) is fascinating. Chapter 7 (Major Histocompatibility Complex (MHC) and Peptide Binding) discusses major histocompatibility complex (MHC) genes and their sequence polymorphism (sequence level variation). The discussion on issues in human MHC genes and methods for MHC peptide binding prediction helps to get insights into gene polymorphism and its impact in population based medicine. Chapter 8 (HLA Supertypes) gives the precise account of the human leukocyte antigen (HLA) alleles that demonstrates functional overall lap among HLA alleles for population coverage. To take readers further, in Chapter 9 (TEpitope Designer) the author described a web server named ’TEpitope Designer‘ which can be used to facilitate HLA-peptide binding prediction for peptide vaccine design. The last Chapter (Chapter 10: Eukaryotic Genes, Functions, Genomes, Design, and Evolution) has highlighted the main issues associated with the study of eukaryotic genes, genomes, function, design, and their evolution. Database such as U-Genome (database of relevant genome information in unicellular eukaryotes) which plays an important role in the understanding of unicellular genomes, their design and evolution is also discussed along with some other databases like ExInt (database of exon­intron gene structure in eukaryotic genes). This chapter gives a summary of several issues (exons, introns, single exon genes, intron organization and chromosome content) in comparative and functional genomics. In summary, this is a very useful book. It is written meticulously by an expert in the emerging field of Bioinformation discovery. This book provides plenty of interesting tips to use data in knowledge discovery. Each chapter concludes with a set of exercise problems for mastery of the subject. The language used in the book is extremely lucid, and hence book is very useful for undergraduate. This book is highly recommended for post-graduate students in Biology and Life Sciences. It should be noted that the book is available in both hard bound and electronic versions.

Cloning and Sequence Analysis of a Novel Cold-Adapted Lipase Gene from Strain lip35 ( Pseudomonas sp.)

A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 ( Pseudomonas sp.) is described, whereby a lipase gene ( lip) was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.

Cerato-platanin, a phytotoxic protein from Ceratocystis fimbriata: Expression in Pichia pastoris, purification and characterization

Cerato-platanin (CP) is a phytotoxic protein secreted by the Ascomycete Ceratocystis fimbriata f.sp. platani. This Ascomycete causes canker stain which is a severe disease with a high incidence in the European Platanus acerifolia. CP probably plays a role in the disease, eliciting defence-related responses in the host plants. CP is a 120 amino acid protein, containing 40% hydrophobic residues and two S-S bridges. In the EMBL data bank CP is the first member of a new fungal protein family known as the Cerato-Platanin Family. The N-terminal region of CP shows a high similarity with that of cerato-ulmin, a phytotoxic protein produced by the Ophiostoma species and that belongs to the hydrophobin family. Hydrophobins are hydrophobic proteins secreted by many saprophytic or pathogenic fungi and have a remarkable ability to self-assemble into a rodlet structure takes part in physiological and/or pathological processes. The methyltrophic yeast Pichia pastoris was used to obtain a high-level expression of recombinant CP (rCP) and the pPIC9 vector was chosen to bring about extra-cellular secretion of the protein. The preliminary structural and functional characterization presented here reveals no significant differences between the native and the recombinant protein. We also show that CP self-assembles in solution. The availability of rCP will allow its three-dimensional structure to be determined, facilitating an understanding of the role of CP in the pathogenesis of canker stain. It is also an excellent model for investigating the mechanism of action of the other proteins related to CP.

Detection in soil of aerobic hydrogen-oxidizing bacteria related to Alcaligenes eutrophus by PCR and hybridization assays targeting the gene of the membrane-bound (NiFe) hydrogenase

The presence and abundance of the aerobic, chemolithoautotrophic Alcaligenes eutrophus, and related hydrogen-oxidizing bacteria, in soil samples were determined by using a hydrogenase gene probe and two different PCR primer sets that were derived from the alignment of (NiFe) hydrogenase gene sequences available in the EMBL data bank. The specificity of both the gene probe and the primer sets was tested on the genomic DNAs of 19 hydrogenase-containing, or hydrogenase-lacking, bacteria. The hydrogenase gene probe detected hydrogenase-containing Knallgas bacteria, N2-fixing bacteria, and enterobacteria, belonging to the alpha, beta, and gamma subclasses of the Proteobacteria. Hydrogenase-containing sulfate reducers (delta subclass) were not detected. One primer set was degenerated and resulted in the amplification of gene fragments of Knallgas bacteria and N2-fixing bacteria. The other primer set was specifically designed for the gene of the small subunit of the membrane-bound hydrogenase of A. eutrophus. In addition to the target organism, only Variovorax (Alcaligenes) paradoxus gave a gene product of the expected length. A. eutrophus-related bacteria were detected in soil by using two different strategies. The first strategy required cultivation and isolation of soil bacteria on complex media. Colonies were isolated from six different soils and tested by colony hybridization with the hydrogenase gene probe. Fifteen of the positive colonies were chosen for further characterization with the PCR primers. Two of the isolates gave amplification products with the A. eutrophus-specific PCR primers, which were sequenced and found to be 89–90% identical on a nucleotide level to the hydrogenase gene of A. eutrophus H16. The second strategy avoided a cultivation step. Total DNA was isolated from the rhizosphere of rice plants and tested in PCR amplification experiments with the A. eutrophus-specific PCR primers. Two different types of hydrogenase fragments were cloned and partially sequenced. The gene fragment sequences showed 73 and 75% identity to the A. eutrophus hydrogenase sequence. In quantitative PCR amplification assays, the number (about 2.6×107 g−1 fresh roots) of these two species of hydrogenase-containing bacteria was determined in the rice rhizosphere and was found to be similar to the number (about 7.4×106 g−1 fresh roots) of Knallgas bacteria that were determined by enumeration on selective media. Thus, it was apparently possible to detect and enumerate A. eutrophus-related bacteria in soil samples by molecular techniques.

Human papillomavirus infection in actinic keratosis and bowen’s disease: comparative study with expression of cell-cycle regulatory proteins p21 waf1/cip1, p53, pcna, ki-67, and bcl-2 in positive and negative lesions

We examined the presence of human papillomavirus (HPV) DNA in tissues of premalignant skin lesions, i.e., actinic keratosis (n = 13) and Bowen’s disease (n = 62), taken from 69 Japanese immunocompetent and renal transplant recipient patients. Detection and typing of HPV DNA were performed using polymerase chain reaction (PCR) and sequence analysis or restriction fragment length polymorphism (RFLP) analysis, respectively. The positivity rates of HPV DNA in tissues of actinic keratosis and Bowen’s disease were 77% and 65%, respectively. Twenty-seven HPV types were detected in 50 (67%) premalignant skin lesions, in which Z95963 (accession no. in the EMBL Databank), Z95968, AJ010823, and AJ000151 have been described as partial sequences of unknown HPV types. Furthermore, 2 unknown types, HPVX1 and HPVX2, were found in specimens of actinic keratosis. Sequence analysis showed that HPVX1 is related to HPV-37 (86.1% sequence homology) and that HPVX2 is related to HPV-38 (79.7%). These results indicate that various mucosal and epidermodysplasia verruciformis-related HPV types are associated with the pathogenesis of actinic keratosis and Bowen’s disease. In addition, 24 specimens of HPV-positive or HPV-negative premalignant skin lesions were examined immunohistochemically for proliferating cells to determine biological differences between HPV-positive and HPV-negative lesions. Immunohistochemistry for p21 Waf1/Cip1, p53, proliferating cell nuclear antigen (PCNA), Ki-67, and Bcl-2 revealed that there was no significant difference in the cell proliferation activity between HPV-positive and HPV-negative lesions, suggesting that HPV infection alone does not induce cell proliferation in those lesions.

PCR-based genetic evidence for occurrence of Helicobacter pylori and novel Helicobacter species in the canine gastric mucosa

The canine gastric mucosa is known to be a habitat for various Helicobacter species. So far, five Helicobacter species have been described from the canine gastric mucosa, but histological studies have demonstrated a greater variety. In order to gain more information on diversity of canine gastric mucosa colonising helicobacters, biopsy samples of four pet dogs were examined by DNA-based techniques. PCR with a primer pair binding specifically to the 16S rDNA of the species of the genus Helicobacter and generating a fragment of approximately 400 bp indicated the presence of Helicobacter strains in the stomachs of the four dogs. PCR products were cloned into Escherichia coli DH10B™ and PCR-re-amplified 16S rDNA fragments were subjected to amplified ribosomal DNA restriction analysis (ARDRA) employing restriction enzyme HhaI. Restriction profiles indicated the presence of at least two different Helicobacter species in two dogs. Partial sequences of 16S rDNA of six clones were compared with sequences available in the EMBL data bank. Two sequences obtained from different dogs were identical with the corresponding sequences of Helicobacter pylori strains. Three sequences showed highest but moderate similarity values to H. pylori (96.6–98.0%) and one sequence to Helicobacter salomonis (97.3%). In contrast to previous reports our data implicate that the gastric mucosa of dogs may be colonised by strains of H. pylori or a very closely related species but they also confirm indications for the presence of so far uncultivated species of Helicobacter.

A Comprehensive Collection of Chicken cDNAs

Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank. We describe a large-scale expressed-sequence-tag (EST) project aimed at gene discovery in chickens (http://www.chick.umist.ac.uk). In total, 339,314 ESTs have been sequenced from 64 cDNA libraries generated from 21 different embryonic and adult tissues. These were clustered and assembled into 85,486 contiguous sequences (contigs). We find that a minimum of 38% of the contigs have orthologs in other organisms and define an upper limit of 13,000 new chicken genes. The remaining contigs may include novel avian specific or rapidly evolving genes. Comparison of the contigs with known chicken genes and orthologs indicates that 30% include cDNAs that contain the start codon and 20% of the contigs represent full-length cDNA sequences. Using this dataset, we estimate that chickens have approximately 35,000 genes in total, suggesting that this number may be a characteristic feature of vertebrates.

Genomic and pathogenic diversity in Colletotrichum gloeosporioides from wild native Mexican Stylosanthes spp., and taxonomic implications

RAPD amplifications of 119 Mexican Colletotrichum gloeosporioides isolates from wild native Stylosanthes plants, of two Australian (internal reference isolates of type A and type B) and of two African (previously demonstrated as different from the A and B types) allowed the generation of four polymorphic clusters. The RAPD cluster I contained the Australian type A and 103 Mexican isolates from other species than S. guianensis. The cluster II contained the Australian type B isolate and 14 Mexican isolates from S. guianensis. The clusters III and IV contained one African and one Mexican isolate each. A phylogenetic study was performed using the ITS1 sequences from 32 representative Mexican isolates selected in the four RAPD clusters, from the two Australian and the two African used as references, from 13 C. gloeosporioides from various hosts and origins available in the EMBL databank and from three phylogenetically related species of Colletotrichum. Two clusters (S and VHO) were generated. Cluster S contained the 30 isolates clustered in RAPD I and II, and the type A and B isolates from Stylosanthes selected in EMBL. Cluster VHO contained the two isolates belonging to RAPD clusters III and IV as well as the remaining Colletotrichum spp. and C. gloeosporioides isolates from various hosts and origins selected in EMBL. Severe type A lesions were observed on several Stylosanthes species, including S. guianensis, after inoculation with the 26 Mexican isolates selected in RAPD cluster I. The four Mexican isolates selected in RAPD cluster II induced extensive type B lesions only on S. guianensis genotypes. The virulence spectra of the isolates was not linked to variations in their ITS1 region sequence. A wide range of anthracnose lesions was induced by the Mexican isolates in interactions with the various Stylosanthes genotypes. On the basis of results obtained from the molecular and pathogenic experiments in the present study, of morphological and molecular data from previous work, as well as from other authors, a taxonomic infraspecific differentiation was proposed, as C. gloeosporioides f. stylosanthis f. sp. stylosanthis and f. sp. guianensis.

Characterization of a new nepovirus infecting apricot in Southeastern France: apricot latent ringspot virus

A pathogen was transmitted from apricot trees showing symptoms of viral infection to GF305 peach seedlings which reacted by stunting, shortened internodes and chlorotic mottling. The agent was transmitted to cherry, apricot, peach and plum by grafting and to several herbaceous hosts by mechanical inoculation. Isometric nepovirus-like particles of 30–31 nm diameter extracted from infected Chenopodium quinoa sedimented as two peaks in sucrose gradients. These particles contained two single stranded RNAs of approximately 5.9 and 7.9 kb, and a single coat protein subunit of 53.7 kDa. No cross-reactions were observed with a number of nepoviruses infecting fruit trees. Inoculation of purified particles to herbaceous or woody hosts reproduced the same symptoms caused by the original isolate. Sequencing of a 2.2 kbp cDNA clone covering the 3 � end of the small genomic RNA identified an open reading frame encoding a 317 aa N-truncated protein exhibiting significant similarities with the coat protein of nepoviruses. The 1257 nt long 3 � non-coding region showed up to about 65% homology to the equivalent region of members of the subgroup C of nepoviruses. The properties of this pathogen do not match those of any previously described nepovirus. It should therefore be considered as a new member of the subgroup C of nepoviruses, for which the name of Apricot latent ringspot virus (ALRSV) is proposed. The nucleotide sequence reported in this work has been deposited in the EMBL databank under the accession number AJ278875.

Genomic Exploration of the Hemiascomycetous Yeasts: 5. Saccharomyces bayanus var. uvarum

Saccharomyces bayanus var. uvarum investigated here is the species closest to Saccharomyces cerevisiae. Random sequence tags (RSTs) allowed us to identify homologues to 2789 open reading frames (ORFs) in S. cerevisiae, ORFs duplicated in S. uvarum but not in S. cerevisiae, centromeres, tRNAs, homologues of Ty1/2 and Ty4 retrotransposons, and a complete rDNA repeat. Only 13 RSTs seem to be homologous to sequences in other organisms but not in S. cerevisiae. As the synteny between the two species is very high, cases in which synteny is lost suggest special mechanisms of genome evolution. The corresponding RSTs revealed that S. uvarum can exist without any S. cerevisiae DNA introgression. Accession numbers are from AL397139 to AL402278 in the EMBL databank.

Genomic Exploration of the Hemiascomycetous Yeasts: 15. Pichia sorbitophila

This paper reports the genomic analysis of the strain CBS7064 of Pichia sorbitophila, a homothallic diploid yeast. We sequenced 4829 random sequence tags of about 1 kb and compared them to the Saccharomyces cerevisiae gene products. Approximately 1300 nuclear genes, 22 tRNAs, the rDNA locus, and six mitochondrial genes have been identified. The analysis of the rDNA genes has permitted to classify this organism close to the Candida species. Accession numbers from AL414896 to AL419724 at EMBL databank.

Isolation and molecular characterization of KlCOX14, a gene of Kluyveromyces lactis encoding a protein necessary for the assembly of the cytochrome oxidase complex.

The yeast Kluyveromyces lactis was mutagenized with ethyl methane sulphonate and mutants unable to grow on respiratory carbon sources were isolated. Functional complementation of one of these mutants led to the isolation of KlCOX14, a gene encoding a 64 amino acid protein which is the functional homologue of Saccharomyces cerevisiae Cox14p, a protein necessary for the assembly of the cytochrome oxidase holoenzyme (Glerum et al., 1995). The disruption of KlCOX14 resulted in the absence of the absorption bands relative to cytochromes a and a(3) and in the complete loss of respiratory activity. Klcox14 mutants display the typical phenotype of pet mutants and have a reduced growth rate. In addition, unlike the wild-type, Klcox14 mutants are able to grow by fermentation also in the presence of low glucose. The nucleotide sequence of KlCOX14 has been deposited in the EMBL databank with Accession No. AJ238801.

The mutK gene of Vibrio cholerae: a new gene involved in DNA mismatch repair.

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).

Capsid protein-encoding (P1) sequence of foot and mouth disease virus type Asia 1 ind 63/72.

Variations in the amino acid sequence of Foot-and-Mouth Disease Virus (FMDV) structural proteins are the basis for the antigenic diversity of the virus. Majority of antigenic sites for the virus neutralization are present on VP1, the major immunogenic protein. However, a few conformational epitopes are present on the structural proteins VP2 and VP3. The nucleotide sequence encoding all the four structural proteins (P1 region) of FMDV type Asia 1 Ind 63/72 was determined. The nucleotide and the deduced amino acid sequence of P1 of Asia 1 of Indian strain was compared with that of Asia 1 Israel strain. Differences were observed at 284 (14%) nucleotide positions resulting in 69 (10%) amino acid changes. The variation in the derived amino acid sequence is the highest in VP1 (14.4%) followed by VP2 (10%), VP3 (6.4%) and VP4 (3%). Deletion of two amino acids, which was observed in the case of Indian strain as well as in Israel strain indicated that these deletions are specific for type Asia 1. The P1 sequence was also compared with the corresponding region of the other serotypes O1K, A12, Cl and SAT-1. The sequence has been submitted to EMBL data bank, under accession number Y09949.

Method revealing latent periodicity of the nucleotide sequences modified for a case of small samples.

An earlier reported method for revealing latent periodicity of the nucleotide sequences has been considerably modified in a case of small samples, by applying a Monte Carlo method. This improved method has been used to search for the latent periodicity of some nucleotide sequences of the EMBL data bank. The existence of the nucleotide sequences' latent periodicity has been shown for some genes. The results obtained have implied that periodicity of gene structure is projected onto the periodicity of primary amino acid sequences and, further, onto spatial protein conformation. Even though the periodic structure of gene sequences has been eroded, it is still retained in primary and/or spatial structures of corresponding proteins. Furthermore, in a few cases the study of genes' periodicity has suggested their possible evolutionary origin by multifold duplications of some gene's fragments.

Characterization of a new HLA-B39 allele, B*3913, in a Brazilian Caucasian.

A panel of samples, previously typed by serology, was retyped using a line probe assay. One sample from a Brazilian Caucasian individual was serologically typed as B52/B39, but showed an aberrant HLA-B pattern on the diagnostic strip and was typed as B*52012/B*39new. Further analysis by allele-specific amplification and subsequent sequencing of exons 2 and 3 revealed a G(B*3908)-to-T nucleotide substitution at position 467 (codon 156) resulting in an Arg (B*3908)-to-Leu substitution. Furthermore, the sequence revealed a silent mutation at position 174 (codon 58): a G(B*3908)-to-A nucleotide switch. The sequence has been sent to the EMBL databank and the HLA Nomenclature Committee, and the allele was named B*3913.

Primary sequence and molecular model of the variable region of a mouse monoclonal anti-Der p 1 antibody showing a similar epitope specificity as human IgE.

Der p 1, a major mite allergen, elicits IgE antibody responses in 80% of patients suffering from dust mite allergy. Given the potent IgE eliciting properties of Der p 1, there is considerable interest in studying the molecular architecture of the variable (Fv) region of IgE antibodies specific for this allergen. IgE is present in human serum at extremely low concentrations, and as such it is practically impossible to purify sufficient quantities for structural studies. We have therefore sought to sequence and model a representative murine monoclonal (MoAb) anti-Der p 1 antibody, as a surrogate human IgE. The cDNA coding for the Fv region of an anti-Der p 1 MoAb (2C7), that mimics the binding of human IgE to Der p 1, was amplified by PCR, cloned and sequenced. The predicted amino acid sequences were then compared with a directory of human germline V-gene segments. Modelling of the Fv region of MoAb 2C7 was carried out using the extensive database of existing immunoglobulin structures in the Brookhaven PDB. The MoAb 2C7 heavy chain showed greater than 70% homology with three members of the VH3 family, DP-35, DP-53 and DP-54. Similarly, the light chain showed greater than 70% homology with 11 VK sequences, including the VKII sequences DPK18, DPK19 and DPK28. A molecular model of the Fv region of MoAb 2C7 was generated and can be accessed from the EMBL databank. Antibodies similar to MoAb 2C7 could be generated as part of the human repertoire. The availability of 3-dimensional model of MoAb 2C7, as a surrogate human IgE antibody, combined with further data on its epitope specificity, will facilitate studies into IgE antibody responses to Der p 1.

The uracil permease of Schizosaccharomyces pombe: a representative of a family of 10 transmembrane helix transporter proteins of yeasts.

The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w. A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S. cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters. The sequence of the uracil permease gene of S. pombe has been deposited in the EMBL data bank under Accession Number X98696.