Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity.

Class I release factors in ciliates with variant genetic codes

In eukaryotes with the universal genetic code a single class I release factor (eRF1) most probably recognizes all stop codons (UAA, UAG and UGA) and is essential for termination of nascent peptide synthesis. It is well established that stop codons have been reassigned to amino acid codons at least three times among ciliates. The codon specificities of ciliate eRF1s must have been modified to accommodate the variant codes. In this study we have amplified, cloned and sequenced eRF1 genes of two hypotrichous ciliates, Oxytricha trifallax (UAA and UAG for Gln) and Euplotes aediculatus (UGA for Cys). We also sequenced/identified three protist and two archaeal class I RF genes to enlarge the database of eRF1/aRF1s with the universal code. Extensive comparisons between universal code eRF1s and those of Oxytricha, Euplotes and Tetrahymena, which represent three lineages that acquired variant codes independently, provide important clues to identify stop codon-binding regions in eRF1. Domain 1 in the five ciliate eRF1s, particulary the TASNIKS heptapeptide and its adjacent region, differs significantly from domain 1 in universal code eRF1s. This observation suggests that domain 1 contains the codon recognition site, but that the mechanism of eRF1 codon recognition may be more complex than proposed by Nakamura et al. or Knight and Landweber.

Splicing of constitutive upstream introns is essential for the recognition of intra-exonic suboptimal splice sites in the thrombopoietin gene

The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon–intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.

Conserved stem–loop structures in the HIV-1 RNA region containing the A3 3′ splice site and its cis-regulatory element: possible involvement in RNA splicing

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3′ splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem–loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem–loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3′ A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

GeneSplicer: a new computational method for splice site prediction

GeneSplicer is a new, flexible system for detecting splice sites in the genomic DNA of various eukaryotes. The system has been tested successfully using DNA from two reference organisms: the model plant Arabidopsis thaliana and human. It was compared to six programs representing the leading splice site detectors for each of these species: NetPlantGene, NetGene2, HSPL, NNSplice, GENIO and SpliceView. In each case GeneSplicer performed comparably to the best alternative, in terms of both accuracy and computational efficiency.

AsMamDB: an alternative splice database of mammals

The objective of database AsMamDB is to facilitate the systematic study of alternatively spliced genes of mammals. Version 1.0 of AsMamDB contains 1563 alternatively spliced genes of human, mouse and rat, each associated with a cluster of nucleotide sequences. The main information provided by AsMamDB includes gene alternative splicing patterns, gene structures, locations in chromosomes, products of genes and tissues where they express. Alternative splicing patterns are represented by multiple alignments of various gene transcripts and by graphs of their topological structures. Gene structures are illustrated by exon, intron and various regulatory elements distributions. There are 4204 DNAs, 3977 mRNAs, 8989 CDSs and 126 931 ESTs in the current database. More than 130 000 GenBank entries are covered and 4443 MEDLINE records are linked. DNA, mRNA, exon, intron and relevant regulatory element sequences are provided in FASTA format. More information can be obtained by using the web-based multiple alignment tool Asalign and various category lists. AsMamDB can be accessed at http://166.111.30.6 5/ASMAM DB.html.

SpliceDB: database of canonical and non-canonical mammalian splice sites

A database (SpliceDB) of known mammalian splice site sequences has been developed. We extracted 43 337 splice pairs from mammalian divisions of the gene-centered Infogene database, including sites from incomplete or alternatively spliced genes. Known EST sequences supported 22 815 of them. After discarding sequences with putative errors and ambiguous location of splice junctions the verified dataset includes 22 489 entries. Of these, 98.71% contain canonical GT–AG junctions (22 199 entries) and 0.56% have non-canonical GC–AG splice site pairs. The remainder (0.73%) occurs in a lot of small groups (with a maximum size of 0.05%). We especially studied non-canonical splice sites, which comprise 3.73% of GenBank annotated splice pairs. EST alignments allowed us to verify only the exonic part of splice sites. To check the conservative dinucleotides we compared sequences of human non-canonical splice sites with sequences from the high throughput genome sequencing project (HTG). Out of 171 human non-canonical and EST-supported splice pairs, 156 (91.23%) had a clear match in the human HTG. They can be classified after sequence analysis as: 79 GC–AG pairs (of which one was an error that corrected to GC–AG), 61 errors corrected to GT–AG canonical pairs, six AT–AC pairs (of which two were errors corrected to AT–AC), one case was produced from a non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two other cases left of supported non-canonical splice pairs. The information about verified splice site sequences for canonical and non-canonical sites is presented in SpliceDB with the supporting evidence. We also built weight matrices for the major splice groups, which can be incorporated into gene prediction programs. SpliceDB is available at the computational genomic Web server of the Sanger Centre: http://genomic.sanger.ac.uk/spldb/SpliceDB.html and at http://www.softberry.com/spldb/SpliceDB.html.

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

Competition for Microtubule-binding with Dual Expression of Tau Missense and Splice Isoforms

How tau mutations lead to neurodegeneration is unknown but may be related to altered microtubule binding properties of mutant tau protein. The tendency for the mutations to cluster around the microtubule-binding domain of tau or to alter the ratios of those splice isoforms that affect binding supports the view that the tau/microtubule interaction is critical and finely regulated. In cells transfected with both mutant and wild-type tau isoforms fused to either yellow fluorescent protein or cyan fluorescent protein we can observe tau fusion proteins that differ by a single amino acid or by the inclusion or exclusion of exon 10. With coexpression of mutant and wild-type tau, the mutant isoform appears diffuse throughout the cytoplasm; however, when mutant tau is expressed alone, it appears mostly bound to the microtubules. Dual imaging of the three- and four-repeat tau isoforms indicated that the expression of four-repeat tau displaced three-repeat tau from the microtubules. These results suggest that altered kinetic competition among the isoforms for microtubule binding could be a disease precipitant.

Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression

Although neurogenesis in the embryo proceeds in a region- or lineage-specific fashion coincident with neuropeptide expression, a regulatory role for G protein-coupled receptors (GPCR) remains undefined. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates sympathetic neuroblast proliferation, whereas the peptide inhibits embryonic cortical precursor mitosis. Here, by using ectopic expression strategies, we show that the opposing mitogenic effects of PACAP are determined by expression of PACAP receptor splice isoforms and differential coupling to the phospholipase C (PLC) pathway, as opposed to differences in cellular context. In embryonic day 14 (E14) cortical precursors transfected with the hop receptor variant, but not cells transfected with the short variant, PACAP activates the PLC pathway, increasing intracellular calcium and eliciting translocation of protein kinase C. Ectopic expression of the hop variant in cortical neuroblasts transforms the antimitotic effect of PACAP into a promitogenic signal. Furthermore, PACAP promitogenic effects required PLC pathway function indicated by antagonist U-73122 studies in hop-transfected cortical cells and native sympathetic neuroblasts. These observations highlight the critical role of lineage-specific expression of GPCR variants in determining mitogenic signaling in neural precursors.

Naturally variant autosomal and sex-linked loci determine the severity of iron overload in β2-microglobulin-deficient mice

Hereditary hemochromatosis (HH) is a common chronic human genetic disorder whose hallmark is systemic iron overload. Homozygosity for a mutation in the MHC class I heavy chain paralogue gene HFE has been found to be a primary cause of HH. However, many individuals homozygous for the defective allele of HFE do not develop iron overload, raising the possibility that genetic variation in modifier loci contributes to the HH phenotype. Mice deficient in the product of the β2-microglobulin (β2M) class I light chain fail to express HFE and other MHC class I family proteins, and they have been found to manifest many characteristics of the HH phenotype. To determine whether natural genetic variation plays a role in controlling iron overload, we performed classical genetic analysis of the iron-loading phenotype in β2M-deficient mice in the context of different genetic backgrounds. Strain background was found to be a major determinant in iron loading. Sex played a role that was less than that of strain background but still significant. Resistance and susceptibility to iron overload segregated as complex genetic traits in F1 and back-cross progeny. These results suggest the existence of naturally variant autosomal and Y chromosome-linked modifier loci that, in the context of mice genetically predisposed by virtue of a β2M deficiency, can profoundly influence the severity of iron loading. These results thus provide a genetic explanation for some of the variability of the HH phenotype.