Chromosome location of four genes in Leishmania

Chromosome-sized DNA molecules from Leishmania isolates (L. mexicana amazonensis, L. mexicana mexicana, L. chagasi, L. major, L. donovani, and L. braziliensis) were separated by orthogonal field alternation gel electrophoresis. The chromosome locations of four genes were mapped. The alpha-tubulin and rRNA genes each mapped to a single chromosome size class. The beta-tubulin and the 5'-spliced-leader-sequence genes were found on more than one chromosome size class and showed variation of hybridization profiles across species.     

Fasciola hepatica expresses multiple alpha- and beta-tubulin isotypes

We have identified five alpha-tubulin and six beta-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke alpha-tubulin and between 65 and 97% for beta-tubulin isotypes. Nucleotide sequence identity ranged between 68-77% and 62-80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the alpha-tubulins and two of the beta-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on beta-tubulin, three of the fluke isotypes had tyrosine at position 200 of beta-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.     

Molecular clones of alpha-tubulin genes of Plasmodium yoelii reveal an unusual feature of the carboxy terminus

By using a Trypanosoma brucei alpha-tubulin cDNA probe under reduced stringency hybridization conditions, we have isolated two genomic clones that constitute portions of alpha-tubulin genes of the rodent malarial parasite Plasmodium yoelii. P. yoelii has two alpha-tubulin genes, the 3' portions of which were present in the two clones, Py alpha T1 and Py alpha T2, containing 1.3 kb and 6.6 kb EcoRI fragments respectively. The 1358 bp Py alpha T1 clone was completely sequenced and found to contain 591 nucleotides of uninterrupted coding sequence with a strong bias for AT-rich codons, starting with codon 254 of a consensus alpha-tubulin sequence. Numerous attempts to clone 5' portions of these genes were unsuccessful. A single mRNA of 2.3 kb was recognized by both the clones in the erythrocytic stages of P. yoelii. A probe constituting the untranslated sequences of Py alpha T1 also recognized this RNA but failed to hybridize with Py alpha T2 sequences, indicating that the gene represented by the Py alpha T1 clone was expressed during the erythrocytic stages. The deduced amino acid sequence of the Py alpha T1 gene terminates in Tyr-Glu instead of Glu-Tyr observed in alpha-tubulins of almost all other organisms. The difference observed may have implications for alpha-tubulin metabolism in malarial parasites.     

Detection of two novel large deletions in SLC3A1 by semi-quantitative fluorescent multiplex PCR

Cystinuria is an autosomal recessive aminoaciduria in which two clinical types have been described (type I and non-type I). Cystinuria type I is caused by mutations in SLC3A1, a gene located in 2p16 coding for an amino acid transporter named rBAT. Using multiplex semi-quantitative fluorescent PCR, we amplified the ten exons of SLC3A1 together with exon 5 of DSCR1 (located on chromosome 21) as a double-dose control gene. We detected two large novel deletions in a Belgian family, one comprising exons 2-10 and another one at exon 10. The method described here can be used to detect a range of deletions from single-base differences in size to entire missing exons, making it useful for scanning genes with a small to medium number of exons.     

Alpha-tubulin II is a male-specific protein in Plasmodium falciparum.

The tubulin gene family in Plasmodium falciparum consists of one beta-tubulin and two alpha-tubulin genes (alpha-tubulin I and II). We present here data indicating that alpha-tubulin II is expressed only in male sexual stage parasites. An IgM mAb, 5E7, specifically reacted with stage III (day 4-5) through mature (day 10-11) male gametocytes and with emerging, exflagellating, or freely moving male gametes. No reactivity was detected in female gametocytes, female gametes, sporozoites, or asexual parasites. mAb 5E7 also specifically recognized male gametes of the avian parasite, Plasmodium gallinaceum, and immunoblotted a 50 kDa protein in extracts of male gametes from both species. This 50 kDa antigen was localized by immunoelectron microscopy to axonemes of male gametes in a pattern similar to that obtained with anti-alpha- and anti-beta-tubulin antibodies. Furthermore, mAb 5E7 specifically reacted with recombinant alpha-tubulin II protein obtained using the PCR-amplified alpha-tubulin II gene from a gametocyte-specific cDNA library. The sex-specific expression of alpha-tubulin II and its localization to axoneme of the male parasite suggest a role for this molecule in the morphologic changes that occur during exflagellation and in the motility of the parasite. alpha-Tubulin II and mAb 5E7 may prove useful tools in studies of the biology of sexual stage differentiation and development in P. falciparum in addition to the general understanding of post-translational modifications of tubulin isoforms.     

Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi

A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.     

Cloning, chromosomal mapping and expression pattern of the mouse Brca2 gene

A proportion of human breast cancers result from an inherited predisposition to the disease. Mutations in the BRCA2 gene confer a high risk of breast cancer and are responsible for almost half of these cases. The recent cloning of the human BRCA2 gene has revealed that it encodes a large protein having little significant homology to known proteins. Here we describe the mouse Brca2 gene. The gene maps to mouse chromosome 5, consistent with its location on human chromosome 13q12. We have sequenced cDNA for the entire 3329 amino acid Brca2 protein and this has revealed that, like Brca1, Brca2 is relatively poorly conserved between humans and mice. Brca2 is transcribed in a diverse range of mouse tissues, and the pattern of expression is strikingly similar to that of Brca1. Taken together, our data highlight some intriguing similarities between two genes involved in inherited breast cancer susceptibility.      

Molecular characterisation and structural analysis of an interferon homologue in sea bass (Dicentrarchus labrax L.)

The interferons (IFNs) are a large family of soluble cytokines involved in the immune response against viral pathogens. Three families of IFNs have been identified in mammals (type I, type II and type III) and, recently, homologues of type I and type II genes have been found in various teleost fish species. In this paper we report the cloning of a cDNA encoding an type I IFN molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and gene structure and, finally, its 3D structure obtained by template-based modelling. The sea bass IFN cDNA consists of 1047bp that translates in one reading frame to give the entire molecule containing 185 amino acids. The analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential N-glycosylation sites. The sea bass IFN gene contains four introns as with other type I IFN teleost genes, except medaka that contains three introns. Real time PCR was performed after poly I:C stimulation of DLEC cell line to investigate the expression of sea bass IFN and Mx and an induction was observed for both genes. The predicted 3D structure of sea bass IFN is characterized by an "all-alpha" domain that shows an "up-down bundle" architecture made of six helices (ABB'CDE). The two cysteine residues present in the sequence (i.e. Cys(23) and Cys(126)) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. Our results will give the opportunity to investigate more in detail antiviral immune responses in sea bass and add to studies on the evolution of the IFN system in teleosts and vertebrates more generally.     

KIR specificity and avidity of standard and unusual C1, C2, Bw4, Bw6 and A3/11 amino acid motifs at entire HLA:KIR interface between NK and target cells,the functional and evolutionary classification of HLA class I molecules

Natural killer (NK) cells make vital contributions to the immune system and the reproductive system. Notably, NK cells of donor origin can recognize and kill residual leukaemic cells and cure malignant patients in hematopoietic stem cell (HSC) transplant setting. NK cell function is regulated by KIRs that recognize cognate HLA class I molecules on target cells, depending on their amino acid residues. In review, we addressed the question of binding capacity and avidity of HLA class I molecules to different killer cell immunoglobulin‐like receptors (KIRs) depending on all interacting amino acid residues both on HLA and KIR side. We searched PubMed database and analysed available HLA:KIR crystallographic data for amino acid residues in HLA molecules, those physically involved in binding KIRs (termed here the “entire KIR interface”). Within entire KIR interface, we selected five functional sequence motifs (14–19, 66–76, 77–84, 88–92 and 142–151) and classified them according to the conservation of their amino acid sequences among 8,942 HLA class I molecules. Although some conserved amino acid motifs were shared by different groups of KIR ligands, the HLA motif combinations were exclusive for the ligand groups. In 135 common HLA class I molecules with known HLA:KIR recognition, we found 54 combinations of five motifs in each of the KIR‐binding interfaces (C1, C2, Bw4, A3/11) and conserved non‐KIR‐binding interfaces. Based on the entire KIR interface, this analysis allowed to classify 8,942 HLA class I molecules into KIR specificity groups. This functional and evolutionary classification of entire KIR interfaces provides a tool for unambiguously predicting HLA:KIR interactions for common and those HLA molecules that have not yet been functionally tested. Considering the entire KIR interface in HLA class I molecules, functional interactions of HLA and KIR can be predicted in immune responses, reproduction and allotransplantation. Further functional studies are needed on the HLA:KIR interaction variations caused by the repertoires of peptides presented by HLA molecules and KIR polymorphisms at allelic level.     

Molecular cloning, genomic structure and localization in a blood stage antigen of Plasmodium falciparum characterized by a serine stretch

Two short DNA segments were isolated by screening of a lambda gt11 library from Plasmodium falciparum schizont cDNA with an antiserum against the 140 kDa protein, which confers protective immunity to monkeys. The segments were used to identify a genomic fragment which carries the entire coding sequence for a protein of 113 kDa characterized by a stretch of serine residues (SERP I). We present the complete nucleotide and deduced amino acid sequence as well as the structure of the SERP I gene. The gene consists of four exons interrupted by three short introns located at the amino-terminal half. Exon 1 and the first part of exon 2 code for hydrophobic amino acids of a putative signal sequence. Exon 2 contains two repetitive segments, the first encoding six glycine rich octapeptides and a second region coding for 37 consecutive serine residues. Southern blot analysis demonstrated the conservation of the SERP I gene in four different parasite strains. SERP I could be localized in the parasitophorous vacuole and in the surrounding membranes. We discuss the relationship of this protein to the recently described P126 polypeptide and the possible role of this antigen as a vaccine candidate.     

Nucleotide sequence of the gene for pre-apocytochrome f in the spinach plastid chromosome

Summary We have characterized a cloned fragment of the spinach plastid chromosome encoding the gene for apocytochrome f. Northern blot analysis and hybrid selection translation discloses that the gene is expressed. From the nucleotide sequence, we deduce that the protein contains 285 amino acids and an amino-terminal signal sequence of 35 amino acid residues. The calculated molecular mass of pre-apocytochrome f is 35.3 kd. The clustering of hydrophobic residues indicates that the processed protein (31.3 kd) possesses only a single anchoring transmembrane domain close to the C terminus, and that 75% of the polypeptide chain including the heme-binding site protrudes into the thylakoid lumen. This topology resembles that reported for beef heart mitochondrial cytochrome c₁.