Neuritogenic Lewis rat T cells use Tcrb chains that include a new Tcrb-V8 family member

The P2 protein obtained from Schwann cells induces a population of T cells which, upon adoptive transfer, causes the disease experimental allergic neuritis (EAN), an animal model for Guillain-Barre syndrome. In this report, a truncated peptide, FR22, derived from a previously reported neuritogenic T-cell determinant, was used to generate from Lewis rats T cells that were shown to cause EAN. Since our previous studies showed that Tcrb-V8 was used by a majority of T-cell hybridomas specific for the neuritogenic peptide P26, which contains the FR22 sequence, we sequenced the Tcrb-V8 ⁺ mRNA from FR22-specific T-cell lines, and compared the sequences obtained with those obtained from similarly generated myelin basic protein (MBP) 68–88-specific Lewis rat T-cell lines. We found that in the EAN lines, several members of the Tcrb-V8 family were used, including a new family member, Tcrb-V8E. This was more diverse than the MBP-68–88-specific response in which only a single Tcrb-V8 family member was used. Also, in the EAN lines, the beta chain sequences did not show the same conserved junctional regions seen in the MBP lines. Thus, T-cell receptor beta chain usage in the response to this dominant neuritogenic peptide appears to be less restricted than the response to the dominant encephalitogenic determinant of MBP both in V region usage and in CDR3 usage.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databse and have been assigned the accession numbers UO6100 (Tcrb-V8A), UO6101 (Tcrb-V8B), UO6102 (Tcrb-V8C), UO6103 (Tcrb-V8D), and UO6104 (Tcrb-V8E)     

Positive and negative selection of Tcrb-V6+ T cells

Tcrb-V6⁺ T cells are deleted by an endogenous superantigen probably encoded by a mouse mammary tumor provirus (Mtv), Mtv-7, in association with major histocompatibility complex (MHC) class II molecules. In contrast, Tcrb-V6⁺CD4⁺ T cells are positively selected by MHC class II E molecules in Mtv-7 ^– mice. We have examined the levels of Tcrb-V6⁺CD4⁺ and Tcrb-V6⁺CD8⁺ T cells from six combinations of backcross mice. In this paper we show that: 1) Tcrb-V6⁺CD8⁺ T cells can be positively selected by MHC class I molecules; 2) MHC class II A molecules can also influence the levels of Tcrb-V6⁺CD4⁺ T cells; 3) Mtv-7 ^– NZW mice have a new Mtv, Mtv-44, which co-segregates with a gene encoding the partial deletion ligand for Tcrb-V6⁺ T cells; 4) the remaining Tcrb-V6⁺ T cells from mice with partial deletion of these T cells appear not to be anergized in the periphery. Address correspondence and offprint requests to: K. Tomonari.     

Phylogenetic signals from point mutations and polymorphic Alu

Allelic frequency data derived from five polymorphic Alu insertion loci and five point mutation polymorphic loci were compared to determine their ability to infer phylogenetic relationships among human populations. While point mutation polymorphisms inferred a monophyletic Caucasian clade that is corroborated by other studies, these data failed to support the generally accepted monophyly of Orientals with native Americans. In addition, there is less statistical bootstrap support for the maximum-likelihood tree derived from the point mutation polymorphisms as compared to those generated from either the Alu insertion data or the combined Alu insertion+point mutation data. The Alu data and the combined Alu insertion+point mutation data inferred a monophyletic relationship among the Oriental and native American populations. The Alu insertion data and the combined Alu insertion+point mutation data also displayed two separate, well defined, tight clusters of the Caucasian and the Oriental+native American populations which was not inferred from the point mutation data. These findings indicate greater phylogenetic information contained in Alu insertion frequencies than in allelic frequencies derived from point-mutations. This revised version was published online in July 2006 with corrections to the Cover Date.     

The association between HLA-A alleles and an Alu dimorphism near HLA-G

The AluYb8 sequences are a subfamily of short interspersed Alu retroelements that have been amplified within the human genome during recent evolutionary time and are useful polymorphic markers for studies on the origin of human populations. We have identified a new member of the Yb8 subfamily, AluyHG, located between the HLA-H and -G genes and 88-kb telomeric of the highly polymorphic HLA-A gene within the alpha block of the major histocompatibility complex (MHC). The AluyHG element was characterised with a view to examining the association between AluyHG and HLA-A polymorphism and reconstructing the history of the MHC alpha block. A specific primer pair was designed for a simple PCR assay to detect the absence or presence (dimorphism) of the AluyHG element within the DNA samples prepared from a panel of 46 homozygous cell-lines containing complete or recombinant ancestral haplotypes (AH) of diverse ethnic origin and 92 Caucasoid and Asian subjects on which HLA-A typing was available. The AluyHG insertion was most strongly associated with HLA-A2 and, to a lesser degree with HLA-A1, -A3, -A11, and A-19. The gene frequency of the AluyHG insertion for 146 Caucasians and 94 Chinese-Han was 0.30 and 0.32 and there was no significant difference between the observed and expected frequencies. The results of the association studies and the phylogenetic analysis of HLA-A alleles suggest that the AluyHG sequence was integrated within the progenitor of HLA-A2, but has been transferred by recombination to other human ancestral populations. In this regard, the dimorphic AluyHG element is an important diagnostic marker for HLA association studies and could help in elucidating the evolution and functions of the MHC alpha block and polymorphism within and between ancestral haplotypes. Received: 7 December 2000 / Accepted: 28 February 2001     

Genetic analysis of the Mls system. Formal Mls typing of the commonly used inbred strains

In order to elucidate the biological role of minor lymphocyte stimulating (Mls) gene products, we have been investigating the fundamental immunogenetic characteristics of the Mls system. In this report, describe the distribution of stimulatory Mls products, Mls^a and Mls^c, in a panel of laboratory inbred strains based on the response pattern of H-2-compatible naive T-cell populations as well as monospecific Mls^a- or Mls^c-reactive T-cell clones. In addition, the expression of four different T-cell receptor (Tcr) b-V segment Tcrb-V3, –V6, –V8.1, and –V9, which were recently reported to be associated with T-cell recognition of Mls gene products in these strains, was examined. The results indicate that the majority of commonly used laboratory strains including those originally typed as Mls ^aare also expressing Mls^c determinants and that very few independent inbred strains are non-Mls ^c. Moreover, the pattern of Tcrb-V expression in spleen as well as in thymus suggests that the association between Mls expression and clonal deletion of self Mls-reactive T cells appears to be the general rule in inbred strains. Based on these results, implications for the nondetectable Mls-like gene products in other species besides the mouse are discussed.     

Study of Alu sequences at the hypoxanthine phosphoribosyltransferase (hprt) encoding region of man

The hypoxanthine phosphoribosyltransferase (hprt) encoding region of man is considered rich in Alu sequences; with 49 sequences present within 57 kilobases. Subfamily classification of the Alu sequences and identification of flanking direct repeats has been carried out to detect past rearrangements associated with their insertion into the region. Members of the Alu-J and three Alu-S subfamilies are present, along with the existence of free left arm sequences. Using available data, a comparison is made of the Alu subfamilies present at different gene regions. The heterogeneity in the number of each subfamily present at different genes shows that no one particular subfamily attained saturation in the genome. Several adjacent insertions of Alu sequences are seen at the hprt region. Furthermore two novel sequences are described, there is an incident where one Alu sequence has inserted into the middle poly(A) tract of an existing sequence at the hprt region; while another resulted from an Alu/Alu cross-over event elsewhere in the genome, before insertion into the hprt region. Once inserted, the Alu sequences are rarely subject to loss or rearrangement.     

A genetic and functional analysis of the unusually large variable region in the M·Alu I DNA-(cytosine C5 )-methyltransferase

The M·AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5′-AGCT-3′. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more “target-recognizing domains” (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M·AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M·AluI is in fact monospecific it was found that AGCT methylation represents only 80–90% of the methylating activity of this enzyme, while control experiments with the enzyme M·HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M·AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M·AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M·AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.     

Influence of the CCR2-V64I Polymorphism on Human Immunodeficiency Virus Type 1 Coreceptor Activity and on Chemokine Receptor Function of CCR2b, CCR3, CCR5, and CXCR4

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at similar levels, and neither molecule affected the expression or coreceptor activity of CCR3, CCR5, or CXCR4 in cotransfected cell lines. Peripheral blood mononuclear cells (PBMCs) from CCR2-V64I heterozygotes had normal levels of CCR2b and CCR5 but slightly reduced levels of CXCR4. CCR2b and CCR2b-V64I functioned equally well as HIV-1 coreceptors, and CCR2-V64I PBMCs were permissive for HIV-1 infection regardless of viral tropism. The MCP-1-induced calcium mobilization mediated by CCR2b signaling was unaffected by the polymorphism, but MCP-1 signaling mediated by either CCR2b- or CCR2-V64I-encoded receptors resulted in heterologous desensitization (i.e., limiting the signal response of other receptors) of both CCR5 and CXCR4. The heterologous desensitization of CCR5 and CXCR4 signaling by both CCR2 allele receptor types provides a mechanistic link that might help explain the in vivo effects of CCR2 gene variants on progression to AIDS as well as the reported antiviral activity of natural CCR2 ligands.     

Recombinations between Alu repeat sequences that result in partial deletions within the C1 inhibitor gene

Genomic DNA sequence analysis was used to define the extent of deletions within the C1 inhibitor gene in two families with type I hereditary angioneurotic edema. Southern blot analysis initially indicated the presence of the partial deletions. One deletion was approximately 2 kb and included exon VII, whereas the other was approximately 8.5 kb and included exons IV–VI. Genomic libraries from an affected member of each family were constructed and clones containing the deletions were analyzed. Sequence analysis of the deletion joints of the mutants and corresponding regions of the normal gene in the two families demonstrated that both deletion joints resulted from recombination of two Alu repetitive DNA elements. Alu repeat sequences from introns VI and VII combined to make a novel Alu in family A, and Alu sequences in introns III and VI were spliced to make a new Alu in family B. The splice sites in the Alu sequences of both mutants were located in the left arm of the Alu element, and both recombination joints overlapped one of the RNA polymerase III promoter sequences. Because the involved Alu sequences, in both instances, were oriented in the same direction, unequal crossingover is the most likely mechanism to account for these mutations.     

Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

Why Are Young and Old Repetitive Elements Distributed Differently in the Human Genome?

Alu elements are not distributed homogeneously throughout the human genome: old elements are preferentially found in the GC-rich parts of the genome, while young Alus are more often found in the GC-poor parts of the genome. The process giving rise to this differential distribution remains poorly understood. Here we investigate whether this pattern could be due to a preferential degradation of Alu elements integrated in GC-poor regions by small indel mutations. We aligned 5.1 Mb of human and chimpanzee sequences and examined whether the rate of insertion and deletion inside Alu elements differed according to the base composition surrounding them. We found that Alu elements are not preferentially degraded in GC-poor regions by indel events. We also looked at whether very young L1 elements show the same change in distribution compared to older ones. This analysis indicated that L1 elements also show a shift in their distribution, although we could not assess it as precisely as for Alu elements. We propose that the differential distribution of Alu elements is likely to be due to a change in their pattern of insertion or their probability of fixation through evolutionary time.Reviewing Editor: Dr. Stephen Freeland     

Structure and sequence of the human α- -iduronidase gene

In humans, a deficiency of the lysosomal hydrolase α- -iduronidase (IDUA; EC 3.2.1.76) results in the lysosomal storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, thereby causing the lysosomal storage disorder mucopolysaccharidosis type I. The gene for IDUA is split into 14 exons spanning approximately 19 kb. We report the sequence of two noncontiguous segments of the IDUA gene, one 1.8-kb segment containing exons 1 and 2 and surrounding sequences and a second segment of 4.5 kb containing the last 12 exons. The potential promoter for IDUA has only GC box type consensus sequences consistent with a housekeeping promoter and is bounded by an Alu repeat sequence. The first two exons of IDUA are separated by an intron of 566 bp, then there is a large intron of approximately 13 kb, and the last 12 exons are clustered within 4.5 kb. No consensus polyadenylation signal was found in the 3′ untranslated region, although two variant polyadenylation signals are proposed.     

Inter-Alu-like species-specific sequences in the Saccharum complex

Alu sequences constitute the most abundant family of short interspersed nuclear elements, SINEs, in the primate genome. The Alu-PCR method, which consists of amplification between Alu sequences, is usually applied in human genetics to provide polymorphic markers. Here we report the presence of Alu-like sequences in sugarcane and related species by applying the Alu-PCR-like method. Amplifications using a PCR primer defined in conserved regions of Alu human sequences lead to specific complex multiband profiles in all the Saccharum and related genera clones surveyed. The isolation and characterisation of the amplified genus-specific inter-Alu-like fragments allowed us to isolate repeated sequences that are specific for different genera of the Saccharum complex: MsCIR2 from Miscanthus, EaCIR6 and EaCIR7 from Erianthus, and SrCIR2 from Saccharum. Two PCR diagnostic tests were developed from the inter-Alu-like sequences MsCIR2 and EaCIR6, and proved efficient in identifying intergeneric hybrids Saccharum×Miscanthus or Saccharum×Erianthus, respectively. The present study illustrates how the Alu-PCR-like method could help investigating the origin of amphiploid species and monitoring introgression in plants. Received: 7 March 1999 / Accepted: 25 March 1999     

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M67511 for V6.1A and M67512 for V6.1B.     

Comparative Analysis of <Emphasis Type="Italic">Alu</Emphasis> Insertion Sequences in the APP 5′ FlankingRegion in Humans and Other Primates

Overexpression of the amyloid precursor protein gene (APP) may play a role in the neuropathology of Alzheimers disease. Therefore, elucidating the mechanisms involved in APP gene regulation is of primary importance, and various cis-acting regulatory elements located in 5 distal regions are known to play a main role. Some of them lie within Alu elements, one of which (Alu1) is only found in humans and apes while the other (Alu2) has a much older history and is also found in rhesus. These Alu insertions harbor sequence motifs that may act as cis-regulatory elements, which may cause differences in APP regulation among primate species and whose functionality may be ascertained through their conservation in a comparative analysis. We have performed a comparative analysis of the region comprising the two Alu elements of the APP promoter in several primates, including humans. We have found a significant decrease in nucleotide diversity in the Alu2 element (inserted in all the species analyzed) compared to the Alu1 (inserted only in apes). This finding can be interpreted as a constriction in the Alu2 sequence variation as a consequence of a functional role of this element in the APP expression. The present results suggest a wider extension of the regulatory elements than the known short consensus regulatory sequences. Moreover, the different conservation of two highly similar and neighboring sequences suggests that, besides the importance of the sequence motifs, their position in relation to the gene suggests that they have played a role in being recruited as regulatory elements.     

De novo insertion of an<Emphasis Type="Italic"> Alu</Emphasis> sequence in the coding region of the<Emphasis Type="Italic"> CLCN5</Emphasis> gene results in Dent's disease

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and eventual renal failure. Various types of mutations in the renal chloride channel gene, CLCN5, have been identified in patients with this disease. We studied a Spanish patient with Dent's disease and found, by polymerase chain reaction amplification of the CLCN5 exons, an abnormally large exon 11. Sequencing analysis revealed that this was attributable to the insertion in codon 650 of an Alu element of the "young" Ya5 subfamily. The Alu element was inserted with the same orientation as the CLCN5 gene and arose de novo on the maternal chromosome. Polymorphism analysis indicated that the insertion occurred in the germline of the maternal grandfather. The presence of a long poly(A) tract and evidence for a 16-bp target-site duplication implied that the Alu element was integrated by retrotransposition. This mutation predicts a truncated ClC-5 protein that lacks part of the carboxy-terminus and is likely to result in loss of function of the chloride channel. Insertions of Alu sequences, which are rarely found in coding regions, have occasionally been reported to cause other genetic diseases. However, this is the first report of a retrotransposon insertion in the CLCN5 gene associated with Dent's disease.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

The interleukin-6 gene locus seems to be a preferred target site for retrotransposon integration

Recently, we have observed the insertion of a retrotransposon into the interleukin-6 (II-6) locus of a mouse somatic cell line. Here we report the characterization of Il-6 genomic regions from both mouse and rat. Restriction site analysis, DNA sequence analysis, and computer-assisted search revealed eight retrotransposon-like elements distributed over a 25 kilobase (kB) mouse Il-6 region. In the rat, five different retrotransposons have been identified within a 17 kb Il-6 region. The retrotransposons belong to the LINE-, Alu I or Alu II families, or to a rat specific class of retrotransposons. Some of the retrotransposons class of retrotransposons. Some of the retrotransposons exhibit characteristic features such as target site duplication and a poly A-tract. Remarkably, several retrotransposons map to different chromosomal locations in the mouse and rat. A genealogical tree of mouse, rat, and human Il-6 loci demonstrates a series of retrotranspositions that recently occurred in evolution. These results suggest that the Il-6 locus serves as a preferred target site for retrotransposon integration during evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M36993-4 (L.1.R3), M36995 (L1.R2), and M36996 (L1.M1/L1.M2).This work contains part of the doctoral thesis of Z. Qin and I. Schuller.     

Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene in five patients with Lesch-Nyhan syndrome

Mutations identified in the hypoxanthine phosphoribosyltransferase (HPRT) gene of patients with Lesch-Nyhan (LN) syndrome are dominated by simple base substitutions. Few hotspot positions have been identified, and only three large genomic rearrangements have been characterized at the molecular level. We have identified one novel mutation, two tentative hot spot mutations, and two deletions by direct sequencing of HPRT cDNA or genomic DNA from fibroblasts or T-lymphocytes from LN patients in five unrelated families. One is a missense mutation caused by a 610C→T transition of the first base of HPRT exon 9. This mutation has not been described previously in an LN patient. A nonsense mutation caused by a 508C→T transition at a CpG site in HPRT exon 7 in the second patient and his younger brother is the fifth mutation of this kind among LN patients. Another tentative hotspot mutation in the third patient, a frame shift caused by a G nucleotide insertion in a monotonous repeat of six Gs in HPRT exon 3, has been reported previously in three other LN patients. The fourth patient had a tandem deletion: a 57-bp deletion in an internally repeated Alu-sequence of intron 1 was separated by 14 bp from a 627-bp deletion that included HPRT exon 2 and was flanked by a 4-bp repeat. This complex mutation is probably caused by a combination of homologous recombination and replication slippage. Another large genomic deletion of 2969 bp in the fifth patient extended from one Alu-sequence in the promoter region to another Alu-sequence of intron 1, deleting the whole of HPRT exon 1. The breakpoints were located within two 39-bp homologous sequences, one of which overlapped with a well-conserved 26-bp Alu-core sequence previously suggested as promoting recombination. These results contribute to the establishment of a molecular spectrum of LN mutations, support previous data indicating possible mutational hotspots, and provide evidence for the involvement of Alu-mediated recombination in HPRT deletion mutagenesis. Received: 21 April 1998 / Accepted: 16 July 1998