The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poirier, G. M. C.
Right arrow Articles by Erlander, M. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poirier, G. M. C.
Right arrow Articles by Erlander, M. G.
The Journal of Immunology, 1999, 163: 4960-4969.
Copyright © 1999 by The American Association of Immunologists

Immune-Associated Nucleotide-1 (IAN-1) Is a Thymic Selection Marker and Defines a Novel Gene Family Conserved in Plants1

Ghislaine M. C. Poirier2,*, Graham Anderson*, Arne Huvar{dagger}, Pamela C. Wagaman{dagger}, John Shuttleworth*, Eric Jenkinson*, Michael R. Jackson{dagger}, Per A. Peterson{dagger} and Mark G. Erlander{dagger}

* Anatomy Department, Medical School, Birmingham University, Edgbaston, United Kingdom; and {dagger} Pharmaceutical Research Institute R.W. Johnson, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell development in the thymus is a multistep differentiation process that is phenotypically defined by the cell surface expression of CD4 and CD8 leukocytes markers. During this process, CD4-CD8- double negative (DN)3 thymocytes differentiate into CD4+CD8+ double positive (DP) thymocytes, which in turn mature into fully functional helper CD4+CD8- or cytotoxic CD4-CD8+ single positive (SP) thymocytes. Additional heterogeneity divides the DN phase into four phenotypically and functionally distinct stages that are distinguished by the cell surface expression of the leukocyte markers CD25 (IL-2R {alpha}-chain) and CD44 (phagocyte glycoprotein-1, pgp-1). These stages succeed each other in the following order: 1) CD44+CD25- (DN1 stage), 2) CD44+CD25+ (DN2 stage), 3) CD44-CD25+ (DN3 stage), and 4) CD44-CD25- (DN4 stage) (1). The thymocyte production line is controlled by two major checkpoints, one occurring at the DN2 stage, called ß selection (reviewed in Ref. 2), the other occurring at the DP stage, represented by positive and negative selections (reviewed in Ref. 3). These checkpoints ensure the selective production of thymocytes that are able to mount immune responses against pathogens but unable to react against self-Ags. At the DN3 checkpoint, thymocytes that have successfully rearranged TCR ß genes and express a properly folded TCR ß-chain are rescued from cell death, enter the cell cycle, and pursue their maturation program (4). In contrast, thymocytes that fail to express the TCR ß-chain at their cell surface undergo programmed cell death unless they are able to enter the {gamma}{delta} TCR lineage. Thus, thymocytes isolated from recombinase activating gene-2 (RAG-2) mice are blocked at the DN3 stage (5). At the DP checkpoint, the fate of thymocytes is determined by the specificity of their TCR-{alpha}ß complex. Thus, thymocytes whose TCR recognizes peptide/MHC complexes with a high avidity/affinity undergo activation-induced apoptosis (negative selection), whereas those that are unable to recognize peptide/MHC complexes undergo apoptosis (death by neglect). In contrast, thymocytes bearing a TCR able to recognize peptide/MHC complexes with a moderate affinity/avidity further differentiate (positive selection) (reviewed in Ref. 6). Positive selection marks the initiation of various cellular changes including acquisition of a functional competence, lineage commitment (7, 8, 9), cell activation (10), cell survival (11), cell migration (12), and also increase of cell size. Hence positive selection ensures the development of mature T lymphocytes able to mount immune responses against a wide variety of Ags presented by MHC molecules. The characterization of the molecular basis of positive selection may therefore have implications in both thymic T cell development and peripheral immune responses. Although the importance of positive selection has long been recognized and many of the cellular changes that it mediates have been extensively characterized, its molecular basis is still poorly understood. To gain further insight into the molecular mechanisms of positive selection we have used the differential display technique (13) and compared gene expression patterns between freshly purified DP and SP thymocytes. To limit the extent of cell purification, we took advantage of the MHC class I-restricted 2C TCR transgenic mouse model (14). This model represents a source of thymocytes enriched in SP or DP subsets depending on whether the mice express (SP subset) or not (DP subset) H-2b MHC class I molecules (15). The application of the differential display technique to DP and SP cells allowed us to identify known and unknown genes showing raised or lower regulation between these two cell populations. These genes include the transcription factor ets2 (16), the serine protease granzyme A (17), cyclin D, and the recently cloned gene CSA-19 (18), as well as a novel gene, which we called mIAN-1 (murine immune-associated nucleotide-1 binding). This report focuses on the characterization of mIAN-1 and the novel family proteins to which it belongs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

ß2m-/- (15) and 2C TCR transgenic mice (14) were provided by J. Sprent (Scripps Research Institute, San Diego, CA). TCR{alpha}-/- mice (19) were obtained from A. Hayday (Yale University, New Haven, CT). RAG-1 knockout (RAG-/-) mice (20) and MHC class I/class II double knockout mice (MHC DK) were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6J mice were obtained from the breeding stock of R.W. Johnson PRI (La Jolla, CA), and BALB/c mice were from the breeding stock of the Biomedical Sciences Unit of Birmingham University. Mice were kept in a specific pathogen-free environment. Day 15 and day 17 embryos were obtained from timed mating of mice showing a vaginal plug at day 0.

Cell purification and flow cytometry

For the differential display analysis, CD4-CD8+ thymocytes from 2C TCR H-2b mice and CD4+CD8+ thymocytes from 2C TCR H-2b ß2-m-/- mice were purified using a FACS (Becton Dickinson, Mountain View, CA). In all other experiments cells were purified using magnetic beads (Dynal, Merseyside, U.K.). Flow cytometry analysis was performed with the following mAbs: PE anti-CD4 (clone RM4-5, PharMingen, San Diego, CA), FITC anti-CD8 (clone 53-6.7, PharMingen), PE anti-B220 (clone RA3-6B2, PharMingen), PE anti-F4/80 (clone A3-1 Serotec, Oxford, U.K.), PE anti-CD44 (clone IM7, PharMingen), and biotin anti-CD25 (clone 7D4, PharMingen).

Cell culture

Foetal thymic organ culture was performed as described elsewhere (21) in the presence or absence of anti-CD3 mAb (clone 145.2C11, PharMingen) at a final concentration of 5 µg/ml.

RNA extraction

Total RNA was extracted using the RNAzol B solution (Tell-Test, Friendswood, TX) or the RNeasy Total RNA kit (Qiagen, Santa Clarita, CA). Poly(A) RNA was purified from total RNA using the FastTrack Kit (Invitrogen, Carlsbad, CA). Total RNA was treated with DNase I (Life Technologies, Grand Island, NY) before differential display and RNA amplification.

Differential display

A detailed protocol for differential display has been published elsewhere (22). Briefly, differential display PCRs were run on denaturing acrylamide gel, and differentially expressed bands were excised from the gel. DNA from these bands was reamplified using the same sets of primers that were used for the differential display PCR and the resulting PCR products were inserted into T-overhang vector using the TA Cloning Kit (Invitrogen). Differentially expressed candidate genes were screened by reverse Northern blot using amplified RNA-derived radiolabeled cDNA probes as described elsewhere (23).

Full-length cDNA cloning

Full-length cDNA clones of mIAN-1 were obtained by screening a C57BL/6J mouse spleen {lambda} phage cDNA library (Stratagene, San Diego, CA). Plaques were transferred onto nitrocellulose filters and DNA was denatured in 1.5 M NaCl, 0.5 M NaOH and then neutralized in 1.5 M NaCl and 0.5 M Tris-HCl (pH 8.0). Filters were rinsed in 2x SSC, and DNA was UV cross-linked once (UV-Stratalinker, Stratagene). Filters were incubated at 42°C in 1x Southern prehybridization buffer (5 Prime -> 3 Prime, Boulder, CO) containing 50% formamide and 100 µg/ml of sheared salmon sperm DNA (5 Prime -> 3 Prime). Plasmid DNA derived from the W12G2 differential display band was digested with EcoRI and the resulting insert was radiolabeled in low melting point agarose in the presence of [{alpha}-32P]dCTP using the oligolabeling kit (Pharmacia, Piscataway, NJ). Hybridization was performed at 42°C in 1x Southern hybridization buffer containing 50% formamide and 100 µg/ml of sheared salmon sperm in the presence of 8 x 105 cpm/ml of radiolabeled DNA. Filters were washed once in 2x SSC and 0.1% SDS at room temperature for 20 min, once in 1x SSC and 0.1% SDS at 65°C for 1 h, and once in 0.2x SSC and 0.1% SDS at 65°C for 15 min. Secondary and tertiary screening of positive plaques were then performed, and four plaques each originating from a distinct primary plaque were isolated. Phages were excised from the {lambda}ZAP II vector using the ExAssist helper phage (Stratagene) according to the manufacturer’s instructions and SOLR cells (Stratagene) were transformed with the rescued recombinant pBluescript double-stranded phagemid. Sequencing of plasmid DNA derived from each of the four plaques was performed on either a model 373 or 377 DNA sequencer (Applied Biosystems, Foster city, CA) according to the manufacturer’s protocol and assembled using the Sequencher program (Gene Codes, Ann Arbor, MI).

Northern blot

Poly(A) RNA derived from thymocytes of 2C TCR H-2b mice and TCR{alpha}-/- mice was electrophoresed on a formaldehyde-agarose gel, transferred onto maximum strength NYTRAN 0.45 µm (Schleicher & Schuell, Keene, NH), and RNA was UV cross-linked. Mouse Multiple Tissue Northern Blot was obtained from Clontech (Palo Alto, CA). Membranes were prehybridized in 1x Northern prehybridization buffer (50% formamide and 100 µg/ml of sheared salmon sperm; 5 Prime -> 3 Prime) for 6 h at 42°C. Hybridization was performed in 1x Northern hybridization buffer (50% formamide and 100 µg/ml of sheared salmon sperm DNA) at 42°C in the presence of 3 x 106 cpm/ml of [{alpha}-32P]-labeled cDNA probes ([{alpha}-32P]dCTP, Amersham, Arlington Heights, IL) synthesized using the oligolabeling kit (Pharmacia). Membranes were subsequently washed once in 2x SSC and 0.2% SDS for 5 min at room temperature, once in 2x SSC and 0.2% SDS for 15 min at 55°C, and finally once in 0.2x SSC and 0.2% SDS for 30 min at 55°C. Finally, membranes were placed onto a phosphor screen for 1 h to 1 day and results were analyzed using a PhosphorImager 445SI (Molecular Dynamics, Sunnyvale, CA).

In situ hybridization

In situ hybridization was performed in the presence of 35S-radiolabeled RNA probes as detailed elsewhere (24) with some modifications. An RNA probe was synthesized from a 400-bp PstI restriction fragment of mIAN-1 full-length cDNA cloned into the pBluescript II SK(+) vector (Stratagene). The transcription reaction was performed in the presence of 50 U of T3 (for the sense probe) or T7 (for the antisense probe) RNA polymerase (Life Technologies), 10 U RNasin (Life Technologies), 200 µCi [35S]UTP per probe (DuPont-NEN, Boston, MA), 3.3 mM each GTP, CTP, ATP, and 100 mM DTT for 1 h at 37°C. The reaction was stopped by adding 5 volumes of 1% SDS, 10 mM Tris (pH 7.4), 1 mM EDTA, and 10 mM DTT and heating at 65°C for 5 min. Unincorporated nucleotides were removed by passing through a G-50 Sephadex column (Boehringer Mannheim, Indianapolis, IN).

Tissues obtained from perfused animals were embedded in OCT and stored at -70°C. Frozen sections (10 µm thick) were digested in the presence of 2 µg/ml proteinase K, then rinsed in 0.1% triethanolamine (pH 8.0), acetylated in the presence of acetyl anhydride, rinsed in 2x SSC, and progressively dehydrated with increasing concentrations of ethanol. Hybridization was performed at 58°C overnight in the presence of 8 x 105 cpm RNA probe per slide. Slides were then rinsed in 4x SSC, digested in the presence of 5 mg RNase A (Sigma, St. Louis, MO), and gradually desalted in the presence of decreasing concentrations of SSC. Slides were finally dehydrated in the presence of increasing concentrations of ethanol. Radioactivity was detected using NTB-2 liquid autoradiography emulsion (Eastman Kodak, Rochester, NY) and slides were counterstained with hematoxylin and eosin (Shandon, Pittsburgh, PA).

Rabbit anti-serum production

A NH2-terminal synthetic peptide of mIAN-1 (MEVQCGGAGFIPESSRSSHELGC-COOH; Biosynthesis, Lewisville, TX) was conjugated to a carrier protein using maleimide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL), and rabbits were injected three times with the conjugated peptide in Freund’s adjuvant.

Western blot

Cells were lysed in 1x SDS reducing buffer and proteins from 5 x 104 cells were resolved on discontinuous SDS-polyacrylamide gel. Proteins were then transferred onto Hybond ECL nitrocellulose membrane (Amersham). Membranes were incubated for 1.5 h at room temperature in the presence of rabbit antiserum against mIAN-1 (1/2000), or mouse ascites fluid containing mAb anti-ß-actin (1/1000; Sigma). After washing they were incubated for 40 min at room temperature in the presence of HRP-conjugated donkey anti-rabbit Ig (Amersham) or sheep anti-mouse Ig (Amersham). Blotted proteins were detected using the ECL Plus Detection System (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and sequence analysis of a full-length cDNA clone encoding mIAN-1

Differential display was applied to CD4+CD8+ (DP) and CD4-CD8+ (SP) thymocytes isolated from H-2b 2C TCR transgenic mice that were back-crossed (DP) or not (SP) onto ß2m knockout mice, and candidate cDNA clones were tested by differential screening. Among the truly differentially expressed clones, W12G2 was selected for further analysis because it had a novel sequence and showed strong up-regulation from DP to SP thymocytes. As shown on Fig. 1GoA, a cDNA probe derived from W12G2 hybridizes to a 1.8-kb mRNA transcript isolated from thymocytes derived from H-2b 2C TCR transgenic mice (80% SP; data not shown). However, the W12G2 cDNA probe does not recognize any mRNA transcript from TCR{alpha}-/- thymocytes (98% DP; data not shown), thus confirming that expression of the W12G2 gene in this TCR transgenic model requires positive selection to occur. The W12G2 cDNA probe was then used to screen a mouse spleen cDNA phage library and four cDNA clones of 1.8 kb were isolated. Nucleotide sequencing revealed that these clones share the same sequence referred as to mIAN-1 in the rest of the manuscript. The sequence contains an open reading frame encoding a protein of 328 aa with a predicted molecular mass of 38 kDa (Fig. 1GoB). Scanning of mIAN-1 protein against the Prosite database of patterns resulted in the identification of the ATP/GTP-binding site motif A (P-loop; pattern PDQC00017) located in the NH2-terminal region of the protein. Protein primary structure analysis with various programs including Coils (25), Paircoil (26), and Multicoil (27) predicts the presence of a short coiled-coil between aa 254 and 282 of mIAN-1. This suggests that the COOH-terminal region of mIAN-1 may participate in the formation of a protein complex. Finally, mIAN-1 is predicted to localize in the cytoplasm with a certainty of 0.65 by the Psort II program (28).



View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 1. Isolation and description of mIAN-1 full-length cDNA. A, Northern blot analysis of mIAN-1 expression during positive selection of thymocytes. The blot was prepared with 2 µg of mRNA derived from 2C TCR H-2b or TCR{alpha}-/- thymocytes and hybridized with a radiolabeled cDNA probe derived from the W12G2 clone. After removal of the probe, the blot was hybridized with a mouse cytoplasmic ß actin cDNA probe as a control. The blot was exposed to a phosphorscreen for 1 day (mIAN-1) or 1 h (ß actin) before analysis on a PhosphorImager. B, Nucleotide and deduced amino acid sequence of mIAN-1. The nucleotide sequence of mIAN-1 is derived from a cDNA clone isolated by plaque hybridization with the W12G2 cDNA probe. Both strands of the cDNA were sequenced. The stop codon and the putative polyadenylation site are typed in bold characters. The open reading frame is indicated below the nucleotide sequence in single-letter amino acid code. The boxed amino acid segment indicates the GTP-binding site motif A (P-loop). Underlined is the polypeptide region predicted to form a coiled coil.

 
MIAN-1 belongs to a novel family of at least three members and is homologous to the pathogen-induced plant protein aig1

To identify cDNA clones sharing sequence homology with mIAN-1 GenBank and EST databases were screened with the tblastn program (29) using the predicted 328-aa sequence of mIAN-1. This analysis showed that mIAN-1 shares protein sequence homology with the plant protein aig1 (30), previously described as being induced upon bacterial infection. High scoring segment pairs were also obtained with the immune specific mouse cDNA iap38 (31). A distinct mouse cDNA clone (EST AA197670), designated mIAN-3, was also identified and subsequently sequenced. The nucleotide sequence of mIAN-3 (Fig. 2Go) has an open reading frame that is preceded by a stop codon and encodes a protein of 293 aa with a predicted molecular mass of 33.8 kDa. Protein sequence analysis reveals the presence of a P-loop in the NH2-terminal region and predicts that mIAN-3 is localized in the cytoplasm (65% probability). In contrast to mIAN-1, mIAN-3 has a low probability of forming a coiled coil as determined by the Paircoil and Multicoil programs.



View larger version (69K):
[in this window]
[in a new window]
  		FIGURE 2. Nucleotide and deduced amino acid sequence of mIAN-3. The nucleotide sequence of mIAN-1 is derived from EST AA197670. Both strands of the cDNA were sequenced. The stop codon and the putative polyadenylation site are typed in bold characters. The open reading frame is indicated below the nucleotide sequence in single-letter amino acid code. The boxed amino acid segment indicates the GTP-binding site motif A (P-loop).

 
Analysis of the multiple sequence alignment of aig1 and mouse IAN proteins (Fig. 3Go) reveals that the NH2-terminal region of the proteins is highly conserved, whereas the COOH-terminal region appears to be specific to each protein. Overall, mIAN-1 shares 35% amino acid identity with mIAN-2, 38% with mIAN-3, and 29% with aig1.



View larger version (96K):
[in this window]
[in a new window]
  		FIGURE 3. Alignment of aig1, mIAN-1, mIAN-3, and iap38 protein sequences. aig1 (GenBank accession number U40856) and iap38 were identified as mIAN-1 protein homologues by tblastn analysis. The analysis also revealed a protein reading frame shift in the sequence of iap38 (GenBank accession number Y08026). The shift was corrected by inserting a nucleotide between base 607 and 608. Protein sequence alignment was performed using the protein weight matrix BLOSUM 30. The alignment was analyzed using the Multiple Sequence Alignment Editor GeneDoc. Amino acids conserved in all sequences are shaded in black and amino acids conserved in at 80–100% of the sequences are shaded in gray. Shading areas allow for similarity groups.

 
IANs are putative GTP-binding proteins

As shown in Fig. 3Go, the P-loop motif, GxxxxGKS ("x" equals any amino acid), is conserved in all IAN family members, suggesting that they may represent GTP- and/or ATP- binding proteins. To identify proteins sharing additional sequence elements with IANs, protein databases were searched using the PSI-blast program (32). Protein sequences having an E value lower than 0.024 were selected and then analyzed using the multiple alignment program ClustalX and the Multiple Sequence Alignment Editor GeneDoc (33). These proteins include three known GTP-binding proteins, era (34), the chloroplast outer envelope protein (OEP) 34 (35, 36) and OEP 86 (35). They also include two proteins predicted to bind GTP (gi 1001345 and gi 2145946) for their expression of the GTP-binding motifs GxxxGK[S,T], DxxG, and [N,T]KxD (37), and a hypothetical protein from Arabidopsis thaliana (gi 2244936) expressing the motifs GxxxGK[S,T] and DxxG. This result supports the idea that IAN proteins may bind GTP. Interestingly, multiple sequence alignment analysis (Fig. 4Go) shows that the GTP-binding motifs mentioned above are aligned with identical or closely related sequence elements found in IAN proteins and that the dis- tance between these motifs is conserved. This further supports the suggestion that the IAN family defines a novel type of GTP-binding proteins.



View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 4. Identification of proteins sharing motifs with IAN family members. The protein sequence of mIAN-1 was used to perform PSI-blast searches against nonredundant CDS sequences of GenBank database. Sequences with an E value smaller than 0.024 were selected and aligned with IAN proteins using the ClustalX program (protein weight matrix: BLOSUM 30). Areas shaded in black show residues conserved in 100% of the sequences and areas shaded in gray show residues conserved in 80% of the sequences. Similarity groups are allowed in both areas. "x" refers to any amino acid, and "{theta}" refers to apolar or hydrophobic amino acids. Positions where several amino acids are allowed are indicated by square brackets. gi 510190, Pisum s; (P.s.) chloroplast OEP34; gi 1151244, Arabidopsis thaliana (A.t.) protein similar to P.s. chloroplast OEP34; gi 2317911, A.t. protein similar to P.s. chloroplast OEP 34; gi 710465, P.s. chloroplast OEP86; gi 3193301, A.t. putative protein similar to P.s. chloroplast OEP86; gi 1001345, Synechocystis sp. hypothetical protein; gi 2984183, Aquifex a. GTP binding protein era; gi 2145946, Mycobacterium l. probable GTP-binding protein; gi 3415109, Homo sapiens (H.s.) era GTPase A protein; gi 3415111, H.s. era GTPase B protein; gi 2244936, A.t. protein similar to chloroplast OEP8.

 
mIAN-1 is predominantly expressed in lymphoid cells

The tissue distribution of mIAN-1 mRNA was analyzed by Northern blots assay and in situ hybridization. Northern blot analysis reveals high levels of expression in the spleen and low levels in the lung, kidney and heart (Fig. 5GoA). In contrast, other tissues, including testis, skeletal muscle, liver, and the brain, show barely detectable or undetectable levels of expression. In situ hybridization of mIAN-1 on thymic sections derived from a wild-type adult mouse shows a strong signal in the medulla, where SP thymocytes reside, but not in the cortex, which is the site of DP thymocytes (Fig. 5GoB). This is consistent with the induction of mIAN-1 expression as a result of positive selection in nontransgenic animals. High levels of hybridization are also detected in the white pulp of the spleen (Fig. 5GoB). In addition, in situ hybridization on tissue sections of a perfused kidney reveals high levels of expression in the glomeruli, indicating that mIAN-1 may be expressed in glomeruli resident cells (Fig. 5GoB). Taken together, these results show that mIAN-1 mRNA is predominantly expressed in the immune system.



View larger version (96K):
[in this window]
[in a new window]
  		FIGURE 5. Tissue distribution of mIAN-1 mRNA. A, Northern blot analysis of mIAN-1 tissue distribution. Multiple tissue Northern blot was hybridized with the W12G2 and ß actin cDNA probes as described in Fig. 1Go. B, Expression of mIAN-1 in adult mouse tissues by in situ hybridization. Tissue sections from thymus (A and B), spleen (C), and kidney (cortical area) (D) from adult mice were hybridized with a mIAN-1 specific anti-sense probe (A, C, and D) or sense probe (B). Hybridization was performed as indicated in Materials and Methods. Thymus and spleen, x10 magnification; kidney, x20 magnification.

 
To confirm protein expression, a rabbit antiserum was raised against a peptide encompassing aa 1–22 of mIAN-1. Immunoblotting of a lysate of CD4+ T lymphocytes shows that this anti-serum recognizes a 42-kDa protein (Fig. 6Go). The specificity of the recognition was confirmed by two results: 1) recognition was inhibited by the peptide used for immunization but not by an irrelevant peptide (data not shown); and 2) the antiserum did not detect any protein in a cell line that does not express mIAN-1 mRNA (data not shown). This antiserum was used to study the expression of mIAN in other immune cell populations and revealed that mIAN-1 is expressed in T helper and cytotoxic lymphocytes and, to a lower extent, in B lymphocytes, but not in peritoneal macrophages (Fig. 6Go).



View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 6. Western blot analysis of mIAN-1 expression in the hematopoietic system. mIAN-1 protein expression in purified CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and peritoneal macrophages was analyzed by immunoblotting using a rabbit antiserum against a NH2-terminal peptide of mIAN-1. T and B lymphocytes were purified from lymph nodes obtained from an adult BALB/c mouse, and peritoneal macrophages were obtained from a RAG-/- mouse. Cell purity was analyzed by cytofluorometry using anti-CD4, anti-CD8, anti-B220, or F4/80 mAbs.

 
mIAN-I expression in the thymus correlates with CD3-mediated signaling

To determine whether expression of mIAN-1 protein correlates with TCR-regulated events, we analyzed its expression patterns during the early stages of T cell development in BALB/c mice. Because the percent of DN thymocytes in the thymus of adult mice is low (~2–5% of the total thymocyte population), the analysis was performed using day 15 and day 17 embryos whose thymuses contain a substantially higher proportion of DN cells. At day 15 of embryogenesis, T cell development has not yet reached the DP stage and 62% of the thymocytes are in the DN1 or DN2 stage (Fig. 7GoA). To obtain a cell population enriched in DN2 cells, thymocytes isolated from day 15 embryos were purified with anti-CD25-coated magnetic beads (95% purity; assessed by counting rosettes). To obtain a cell population enriched in DN3 cells, thymocytes derived from day 17 embryos were depleted of CD8+ and CD44+ cells using magnetic beads (Fig. 7GoA) and then rosetted to CD25-coated magnetic beads yielding 95% rosetted cells. DN4 cells were not included in this analysis because of the difficulty of isolating these cells in large numbers. Expression of mIAN-1 in DN1/DN2, DN2, and DN3 enriched cells was then compared with the expression in DP and CD4 SP cells by Western blot analysis. As shown on Fig. 7GoB, cell populations enriched in DN1/DN2 (day 15 embryo total thymocytes), DN2 (day 15 embryo CD25+ purified thymocytes), and DN3 (day 17 embryo CD8-CD44-CD25+ purified thymocytes) do not express detectable levels of mIAN-1 protein, whereas mIAN-1 protein can be detected in purified CD4 SP thymocytes. As expected from the results of the Northern blot and in situ hybridization analysis, mIAN-1 protein is not expressed in DP cells. To assess expression in DN4 cells, we used the RAG-/- fetal thymic organ culture (FTOC) model. RAG-/- thymocytes are blocked at the DN3 stage, but can be induced to differentiate into the DN4 stage, up to the DP stage, by treating FTOC with anti-CD3 mAb (38). As shown in Fig. 8Go, after 4 days of anti-CD3 treatment, the percent of CD25+ cells decreased from 86 to 28%, and the percent of CD8 SP cells increased from 2 to 21%, whereas the percent of DP cells remains essentially unchanged at 1–2%. These results indicate that thymocytes have differentiated toward the DN4 and CD8+ intermediate cells but have not yet reached the DP stage. As expected, mIAN-1 protein is not expressed in thymocytes derived from untreated FTOC. However, it can be readily detected in the total thymocyte population derived FTOC treated with anti-CD3 mAb for 4 days, suggesting that mIAN-1 is expressed in DN4 cells and/or CD8+ intermediate cells. After 7 days of anti-CD3 treatment, mIAN-1 expression can be detected in CD4- cells but not in DP cells. This indicates that the DP cells generated in this model are similar to those isolated from a wild-type mouse and suggests that mIAN-1 is transiently expressed during the transition between DN3 and DP RAG-/- thymocytes.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. Western blot analysis of mIAN-1 expression in thymocyte subsets. A, Purification and phenotypic analysis of thymocytes subsets. Thymocytes were isolated from BALB/c embryos (E day 15, E day 17) or new-born animals (NB) and stained with PE anti-CD4, FITC anti-CD8, PE anti-CD44, and biotin anti-CD25-conjugated mAbs. FACS analysis was performed with light scatter gates set to exclude nonviable cells and cell doublets. E day 15 total represent the total cell population obtained by teasing the thymus of day 15 embryos. DN thymocytes from day 17 embryo (E day 17 DN) were obtained after cell depletion using anti-CD8- and anti-CD44-coated magnetic beads. NB day 5 CD4 SP cells were obtained by rosetting cells to anti-CD4-coated magnetic beads followed by bead removal with Detachbeads. B, Expression of mIAN-1 protein in thymocyte subpopulations. E day 15 CD25+ DN cells and E day 17 CD44-CD25+ DN cells were obtained by rosetting E day 15 total cells and E day 17 DN cells, respectively, to anti-CD25-coated magnetic beads, and the rosetted cells were used without bead removal. CD25-coated beads were used as a control. NB DP cells were obtained from MHC DK mice without cell purification (~85% purity). Western blot analysis was performed as on Fig. 6Go.

 


View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of anti-CD3 treatment on mIAN-1 expression in RAG-/- FTOC culture. FTOC culture of fetal RAG-/- thymocytes were maintained in culture medium for 4 days to enrich for DN CD44-CD25+ thymocytes, and then for 4–7 days in the presence or absence of anti-CD3 mAb. Thymocytes were recovered by teasing the thymuses and then used for Western blot and FACS analysis, either immediately (Total), or after enrichment in DP thymocytes or CD4- thymocytes with anti-CD4 coated magnetic beads. FACS analysis was performed with PE-conjugated anti-CD4, FITC-conjugated anti-CD8, and biotin-conjugated anti-CD25. Proteins were detected as in Fig. 5Go.

 
This finding suggested that mIAN-1 expression in thymocytes could be related to CD3-mediated selection events. To further check this hypothesis, we analyzed mIAN-1 expression during another CD3-mediated thymic selection event that can occur at the CD4+CD8+CD3low stage, negative selection, using an in vitro model of negative selection. Thymuses from day 15 BALB/c embryo were maintained in FTOC containing culture medium only for 7 days to allow thymocytes to reach the double positive stage, and then treated with anti-CD3 Abs for 12 h. Thymocytes undergoing apoptosis were purified using anti-annexin Abs and magnetic beads, and analyzed for mIAN mRNA expression by RT-PCR. Using this model we found that annexin positive thymocytes up-regulate mRNA although to a somewhat lower extent than SP thymocytes (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the process of isolating genes that are differentially regulated upon positive selection of thymocytes, we have identified a novel gene, mIAN-1, that is switched on during this process and predominantly expressed in the immune system. mIAN-1 protein shares sequence homology with the pathogen-induced plant protein aig1 (24) and defines a novel family of at least three members in mice and one in plants. However, search for IAN family members in the full genome databases of bacteria, yeast, and Caenorhabditis elegans did not yield any match suggesting that IAN proteins are not expressed in prokaryotes, unicellular fungi, and at least some lower eukaryotes.

Search for IAN-related proteins using the PSI-blast analysis yielded several proteins known or expected to have a GTP/GDP specificity, suggesting that IANs are GTP/GDP-binding proteins. A tentative consensus sequence derived from the alignment of these proteins reveals the presence of three motifs, {theta}{theta}{theta}{theta}GxxxxGKS ({theta} = apolar or hydrophobic amino acid; x = any amino acid), {theta}{theta}DTx[G,D], and {theta}{theta}{theta}{theta}[T,N]xx[D,E] that are identical or closely related to the motifs known to participate in the formation of the GTP-binding site of small GTPases, {theta}{theta}{theta}{theta}GxxxxGK[S,T] (motif G-1), DxxG (motif G-3), and {theta}{theta}{theta}{theta}[T,N]KxD (motif G-4) (37). Although the overall similarity of these proteins is low, the alignment is likely to be reliable because it involves only a small number of gaps. In addition, the amino acid segments located between the motifs have a conserved length. The G-1 motif which is known to form bonds with the {alpha}- and ß-phosphates of GTP/GDP in Ras proteins (37) is entirely conserved in IAN proteins. IANs also have a conserved threonine located between motifs G-1 and G-3. Interestingly, a conserved threonine located at a similar position in p21ras and other nucleotide-binding proteins is known to establish a bond with Mg2+ and is required for GTP hydrolysis (39). Finally, the amino acid that is believed to give the high specificity of Ras proteins for GTP/GDP (D in motif G-4) (40) is conserved in IAN proteins (D or E).

Altogether, these observations support the idea that IANs may be GTP-binding proteins with an unconventional GTP-binding site. This hypothesis is further supported by the fact that one of the proteins found in the gapped blast analysis, era, has already been shown to bind specifically to GTP/GDP (34). In addition, the presence of a perfect G-4 motif does not seem to be an absolute requirement to confer GTP/GDP-binding specificity. For example, IFN-induced guanylate binding proteins, which do express motifs G-1, G-2, and G-3 but lack the G-4 motif, have been shown to bind GTP/GDP/GMP exclusively (41). Interestingly, the sequence of these proteins contains a similar motif to that found in IAN proteins, {theta}{theta}TxxD, which could be responsible for the guanine specificity. Experiments aiming at determining the nucleotide specificity of IAN proteins are currently in progress.

Within the immune system, high levels of mIAN-1 protein are found in T cells and low levels in B cells, whereas peritoneal macrophages do not show any protein expression. Interestingly, another member of the IAN family, iap38, has been reported to be predominantly expressed in B cells and macrophages, but not in resting T cells (31), suggesting that mIAN-1 could represent the T cell protein homologue of iap38.

The up-regulation of mIAN-1 during thymic selection events could be the result of signaling through the TCR and/or the receptors of accessory molecules or soluble factors present in the thymic microenvironment. A role for CD3-mediated signaling in mIAN-1 expression in thymocytes is supported by the fact that EL4 6.1 thymic lymphoma cells, which do not express mIAN-1 constitutively, can be induced to express mIAN-1 mRNA by treatment with immobilized anti-CD3 mAb (data not shown). However, this does not indicate whether mIAN-1 expression is a direct result or a secondary effect of CD3 stimulation. In addition, our results do not exclude the possibility that other cell surface molecules may also participate in the control of mIAN-1 expression in normal thymocytes.

Our results show that the mIAN-1 protein is not expressed in DN1, DN2, and DN3 thymocytes isolated from wild-type mice. In contrast, DN4 and/or CD8+ intermediate cells generated by anti-CD3 treatment of RAG-/- thymocytes in FTOC do express mIAN-1. Several pieces of evidence indicate that RAG-/- FTOC model mimics normal T cell development between the DN3 and DP stages, and that the DN3 to DP transition is mediated by CD3 signaling. First, FACS staining analysis shows that in vivo and in vitro anti-CD3 induced differentiation of RAG-/- thymocytes cannot be distinguished from the DN3 to DP transition in thymuses derived from wild-type animals (38, 42, 43). In addition, both wild-type DN3 cell differentiation and CD3-mediated restoration of RAG-/- DN3 cells differentiation are lck-dependent events (44). Finally, we have shown that DP thymocytes isolated from both wild-type mice and anti-CD3-treated RAG-/- FTOC do not express mIAN-1 protein. Altogether, these results suggest that mIAN-1 protein is transiently expressed as a result of ß selection as well as upon positive selection in wild-type mice.

Although mIAN-1 expression in thymocytes is limited to CD3-activated cells, it is readily detectable in CD4 and CD8 mature T lymphocytes isolated from a nonimmune mouse, indicating that it is constitutively expressed in naive T lymphocytes. It is not yet clear whether expression occurs irrespectively of TCR stimulation or whether it is the result of low affinity TCR/MHC interactions such as the one thought to be required for the survival of naive T lymphocytes (45). Our preliminary results indicate that mIAN-1 protein levels in CD4+ T lymphocytes do not change after incubating the cells for 24 h in culture medium (data not shown), suggesting that, in contrast to thymocytes, lymphocytes may not require TCR/MHC interactions to express mIAN-1 protein. However, this could also be interpreted in term of a long protein half-life. A longer-term analysis using purified lymphocytes able to survive in culture medium over a sustained period of time, such as lymphocytes derived from bcl-2 transgenic mice (46) will be required to answer this issue.

The function of the known IAN family members, aig1 and iap38, is currently unknown. In Arabidopsis, aig1 is progressively expressed after the first 9 h following a virulent infection. However, when the plant is infected with an avirulent pathogen expressing the AvrRpt2 gene product, aig1 expression is seen between 6 and 15 h following infection, after which time gene expression drops. The down-regulation of aig1 expression has been shown to coincide with the onset of a group of defense mechanisms, collectively known as hypersensitive response, which culminates in localized programmed cell death (47). Our results show that mIAN-1 expression in thymocytes is switched on during ß selection, positive selection and to some extent negative selection. Because positive and negative selection are functionally opposed (cell survival and maturation vs induced cell death), mIAN-1 expression patterns do not offer a straightforward idea of its function. However, interestingly a recent study has showed that the plant specific receptor of AvrRpt2 gene product, RPS2 (48), shares sequence homology with CED-4 and its human homologue APAF-1 (49). Taken together with the fact that APAF-1 participates in the control of the caspase cascade (50), van der Biezen and Jones’ study (49) suggests that RPS2-mediated signals, including the expression of aig1, may also participate in the control of apoptosis. It is not yet clear how such a function would account for the expression patterns of mIAN-1 in thymocytes. Additional experiments aimed at analyzing the function of mIAN-1 are currently in process.


   Acknowledgments
 
We thank Joseph Trotter and Tom Knapp for FACS sorting; Hongquing Guo for help with the differential display; James Chambers, Anton Bittner, and Kathy Witmeyer for nucleotide sequencing; Lin Luo for help with in situ hybridization; and Terry Jones and John Shuttleworth for advice for the Western blot analysis.


   Footnotes
 
1 The work was supported by a R.W. Johnson PRI Fellowship (to G.M.C.P.) and a Wellcome Trust project grant (to G.A.). Back

2 Address correspondence and reprint requests to Dr. Ghislaine Poirier, Anatomy Department, Medical School, Birmingham University, Edgbaston, B152TT, U.K. E-mail address: Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; RAG-2, recombinase activating gene-2; IAN-1, immune-associated nucleotide-1; mIAN-1, murine IAN-1; FTOC, fetal thymic organ culture; ß2m, ß2-microglobulin. Back

Received for publication March 22, 1999. Accepted for publication August 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Godfrey, D. I., J. Kennedy, T. Suda, A. Zlotnik. 1993. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150:4244.[Abstract]
  2. Fehling, H. J., H. von Boehmer. 1997. Early {alpha}ß T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[Medline]
  3. Kisielov, P., H. von Boehmer. 1995. Development and selection of T cells: facts and puzzles. Adv. Immunol. 58:87.[Medline]
  4. Mallick, C. A., E. C. Dudley, J. L. Viney, M. J. Owen, A. C. Hayday. 1993. Rearrangement and diversity of T cell receptor ß chain genes in thymocytes: a critical role for the ß chain in development. Cell 73:513.[Medline]
  5. Penit, C., B. Lucas, F. Vasseur. 1995. Cell expansion and growth arrest phases during the transition from precursor (CD4-CD8-) to immature (CD4+CD8+) thymocytes in normal and genetically modified mice. J. Immunol. 154:5103.[Abstract]
  6. Ohashi, P.. 1996. T cell selection and autoimmunity: flexibility and tuning. Curr. Opin. Immunol. 8:808.[Medline]
  7. Kaye, J., M. L. Hsu, M. E. Sauron, S. C. Jameson, N. R. J. Gascoigne, S. M. Hedrick. 1989. Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor. Nature 341:746.[Medline]
  8. Kisielov, P., H. S. Teh, H. Blüthmann, H. Von Boehmer. 1988. Positive selection of antigen-specific T cells in thymus by restricting MHC molecules. Nature 335:730.[Medline]
  9. Fowlkes, B. J., E. Schweighoffer. 1995. Positive selection of T cells. Curr. Opin. Immunol. 7:188.[Medline]
  10. Bendelac, A., P. Matzinger, R. A. Seder, W. E. Paul, R. H. Schwartz. 1992. Activation events during thymic selection. J. Exp. Med. 175:731.[Abstract/Free Full Text]
  11. Huesmann, M., B. Scott, P. Kisielow, H. von Boehmer. 1991. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Cell 66:533.[Medline]
  12. Salomon, D. R., C. F. Mojcik, A. C. Chang, S. Wadsworth, D. H. Adams, J. E. Coligan, E. M. Shevach. 1994. Constitutive activation of integrin {alpha}4ß1 defines a unique stage of human thymocytes development. J. Exp. Med. 179:1573.[Abstract/Free Full Text]
  13. Liang, P., A. B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967.[Abstract/Free Full Text]
  14. Sha, W. C., C. A. Nelson, R. D. Newberry, D. M. Kranz, J. H. Russell, D. Y. Loh. 1988. Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature 335:271.[Medline]
  15. Maarten, Z., M. Bix, N. E. Simister, J. M. Loring, D. H. Raulet, R. Jaenish. 1990. ß2-microglobulin deficient mice lack CD4-8+ cytolytic cells. Nature 344:742.[Medline]
  16. Bhat, N. K., K. L. Komschlies, S. Fujiwara, R. J. Fisher, B. J. Mathieson, T. A. Gregorio, H. A. Young, J. W. Kasik, K. Ozato, T. Papas. 1989. Expression of ets genes in mouse thymocytes subsets and T cells. J. Immunol. 142:672.[Abstract]
  17. Ebnet, K., C. N. Levelt, T. T. Tran, K. Eichmann, M. M. Simon. 1995. Transcription of granzyme A and B genes is differentially regulated during lymphoid ontogeny. J. Exp. Med. 181:755.[Abstract/Free Full Text]
  18. Fisicaro, N., M. Katerelos, J. Williams, D. Power, A. D’Apice, M. Pearse. 1995. Identification of genes down-regulated in the thymus by cyclosposrin-A: preliminary characterization of clone CSA-19. Mol. Immunol. 32:556.
  19. Philpott, K. L., J. L. Viney, G. Kay, S. Rastan, E. M. Gardiner, S. Chae, A. C. Hayday, M. J. Owen. 1992. Lymphoid development in mice congenitally lacking T cell receptor {alpha}ß-expressing cells. Science 256:1448.[Abstract/Free Full Text]
  20. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. Rag-1 deficient mice have no mature B and T lymphocytes. Cell 68:869.[Medline]
  21. Anderson, K. L., G. Anderson, R. H. Michell, E. J. Jenkinson, J. J. T. Owen. 1996. Intracellular signaling pathways involved in the induction of apoptosis in immature thymic T lymphocytes. J. Immunol. 156:4083.[Abstract]
  22. Galindo, J. E., G. M.-C. Poirier, H. Guo, A. Huvar, P. C. Wagaman, J. Zhu, J. Tench, J. S. Wan, M. G. Erlander. 1998. Differential display of RNA. ed. DNA Markers: Protocols, Applications and Overviews 225.-236. John Wiley & Sons, New York.
  23. Poirier, G. M., J. Pyati, J. S. Wan, M. G. Erlander. 1997. Screening differentially expressed cDNA clones obtained by differential display using amplified RNA. Nucleic Acids Res. 25:913.[Abstract/Free Full Text]
  24. Simmons, D. M., J. L. Arriza, L. W. Swanson. 1989. A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes. J. Histotechnol. 12:169.
  25. Lupas, A., M. van Dyke, J. Stock. 1991. Predicting coiled coils from protein sequences. Science 252:1162.[Free Full Text]
  26. Berger, B., D. B. Wilson, E. Wolf, T. Tonchev, M. Milla, P. S. Kim. 1995. Predicting coiled coils by use of pairwise residue correlations. Proc. Natl. Acad. Sci. USA 92:8259.[Abstract/Free Full Text]
  27. Wolf, W., P. S. Kim, B. Berger. 1997. Multicoil: a program for predicting two- and three-stranded coiled coils. Protein Sci. 6:1179.[Medline]
  28. Nakai, K., M. Kanehisa. 1992. A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14:897.[Medline]
  29. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403.[Medline]
  30. Reuber, T. L., F. M. Ausubel. 1996. Isolation of Arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes. Plant Cell 8:241.[Abstract]
  31. Krücken, J., H.-P. Schmitt-Wrede, U. Markmann-Mulish, F. Wunderlich. 1997. Novel gene expressed in spleen cells mediating acquired testosterone-resistant immunity to Plasmodium chabaudi malaria. Biochem. Biophys. Res. Commun. 230:167.[Medline]
  32. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389.[Abstract/Free Full Text]
  33. Nicholas, K. B., H. B. Nicholas, D. W. I. Deerfield. 1997. GeneDoc: analysis and visualization of genetic variation. EMBNET. News 4:1.
  34. Ahnn, J., P. E. March, H. E. Takiff, M. Inouye. 1986. A GTP-binding protein of Escherichia coli has homology to yeast Ras proteins. Proc. Natl. Acad. Sci. USA 83:8849.[Abstract/Free Full Text]
  35. Kessler, F. B., G. Blobel, H. A. Patel, D. J. Schnell. 1994. Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science 266:1035.[Abstract/Free Full Text]
  36. Seedorf, M., K. Waegemann, J. Soll. 1995. A constituent of the chloroplast import complex represents a new type of GTP-binding protein. Plant J. 7:401.[Medline]
  37. Bourne, H. R., D. A. Sanders, F. McCormick. 1991. The GTPase superfamily: conserved structure and molecular mechanism. Nature 349:117.[Medline]
  38. Levelt, C. N., P. Mombaerts, A. Iglesias, S. Tonegawa, K. Eichmann. 1993. Restoration of early thymocyte differentiation in T-cell receptor ß-chain-deficient mutant mice by transmembrane signaling through CD3{epsilon}. Proc. Natl. Acad. Sci. USA 90:11401.[Abstract/Free Full Text]
  39. Traut, T. W.. 1994. The functions and consensus motifs of nine types of peptide segments that form different type of nucleotides-binding sites. Eur. J. Biochem. 222:9.[Medline]
  40. Sander, C., A. Valencia. 1995. Three-dimensional structure of ras and ras-related proteins. ed. Guidebook to the Small GTPases 20. Oxford University Press, Oxford.
  41. Cheng, Y. S., C. E. Patterson, P. Staeheli. 1991. Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP. Mol. Cell. Biol. 11:4717.[Abstract/Free Full Text]
  42. Shinkai, Y., F. W. Alt. 1994. CD3{epsilon}-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR ß chain expression. Int. Immunol. 6:995.[Abstract/Free Full Text]
  43. Jacobs, H., D. Vandeputte, L. Tolkamp, E. de Vries, J. Borst, A. Berns. 1994. CD3 components at the surface of pro-T cells can mediate pre-T cell development in vivo. Eur. J. Immunol. 24:934.[Medline]
  44. Wu, G., J. S. Danska, C. Guidos. 1996. Lck dependence of signaling pathways activated by {gamma}-irradiation and CD3{epsilon} engagement in RAG-1-/--immature thymocytes. Int. Immunol. 8:1159.[Abstract/Free Full Text]
  45. Kirberg, J., A. Berns, H. von Boehmer. 1997. Peripheral T cell survival requires continual ligation of the T cell receptor to major histocompatibility complex-encoded molecules. J. Exp. Med. 186:1269.[Abstract/Free Full Text]
  46. Cory, S.. 1995. Regulation of lymphocyte survival by the BCL-2 gene family. Annu. Rev. Immunol. 13:513.[Medline]
  47. Staskawicz, B. J., F. M. Ausubel, B. J. Baker, J. G. Ellis, J. G. Jones. 1995. Molecular genetics of plant disease resistance. Science 268:661.[Abstract/Free Full Text]
  48. Mindrinos, M., F. Katagiri, G.-L. Yu, F. M. Ausubel. 1994. The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell 78:1089.[Medline]
  49. van der Biezen, E. A., J. D. G. Jones. 1998. The NB-ARC domain: a novel signaling motif shared by plant resistance gene products and regulators of cell death in animals. Curr. Biol. 8:R226.[Medline]
  50. Hu, Y., M. A. Benedict, D. Wu, N. Inohara, G. Nunez. 1998. Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation. Proc. Natl. Acad. Sci. USA 95:4386.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Cell. ProteomicsHome page
K. A. Duthie, L. C. Osborne, L. J. Foster, and N. Abraham
Proteomics Analysis of Interleukin (IL)-7-induced Signaling Effectors Shows Selective Changes in IL-7R{alpha}449F Knock-in T Cell Progenitors
Mol. Cell. Proteomics, October 1, 2007; 6(10): 1700 - 1710.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Carter, C. Dion, S. Schnell, W. J. Coadwell, M. Graham, L. Hepburn, G. Morgan, A. Hutchings, J. C. Pascall, H. Jacobs, et al.
A Natural Hypomorphic Variant of the Apoptosis Regulator Gimap4/IAN1
J. Immunol., August 1, 2007; 179(3): 1784 - 1795.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
U. Dalberg, H. Markholst, and L. Hornum
Both Gimap5 and the diabetogenic BBDP allele of Gimap5 induce apoptosis in T cells
Int. Immunol., April 1, 2007; 19(4): 447 - 453.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Schnell, C. Demolliere, P. van den Berk, and H. Jacobs
Gimap4 accelerates T-cell death
Blood, July 15, 2006; 108(2): 591 - 599.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Chtanova, R. Newton, S. M. Liu, L. Weininger, T. R. Young, D. G. Silva, F. Bertoni, A. Rinaldi, S. Chappaz, F. Sallusto, et al.
Identification of T Cell-Restricted Genes, and Signatures for Different T Cell Responses, Using a Comprehensive Collection of Microarray Datasets
J. Immunol., December 15, 2005; 175(12): 7837 - 7847.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
C. Dion, C. Carter, L. Hepburn, W. J. Coadwell, G. Morgan, M. Graham, N. Pugh, G. Anderson, G. W. Butcher, and J. R. Miller
Expression of the Ian family of putative GTPases during T cell development and description of an Ian with three sets of GTP/GDP-binding motifs
Int. Immunol., September 1, 2005; 17(9): 1257 - 1268.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
V. E. Mick, T. K. Starr, T. M. McCaughtry, L. K. McNeil, and K. A. Hogquist
The Regulated Expression of a Diverse Set of Genes during Thymocyte Positive Selection In Vivo
J. Immunol., November 1, 2004; 173(9): 5434 - 5444.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
M. Michalkiewicz, T. Michalkiewicz, R. A. Ettinger, E. A. Rutledge, J. M. Fuller, D. H. Moralejo, B. Van Yserloo, A. J. MacMurray, A. E. Kwitek, H. J. Jacob, et al.
Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat
Physiol Genomics, October 4, 2004; 19(2): 228 - 232.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
T. Sandal, L. Aumo, L. Hedin, B. T. Gjertsen, and S. O. Doskeland
Irod/Ian5: An Inhibitor of {gamma}-Radiation- and Okadaic Acid-induced Apoptosis
Mol. Biol. Cell, August 1, 2003; 14(8): 3292 - 3304.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
A. Kanapin, S. Batalov, M. J. Davis, J. Gough, S. Grimmond, H. Kawaji, M. Magrane, H. Matsuda, C. Schonbach, R. D. Teasdale, et al.
Mouse Proteome Analysis
Genome Res., June 1, 2003; 13(6): 1335 - 1344.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
A. J. MacMurray, D. H. Moralejo, A. E. Kwitek, E. A. Rutledge, B. Van Yserloo, P. Gohlke, S. J. Speros, B. Snyder, J. Schaefer, S. Bieg, et al.
Lymphopenia in the BB Rat Model of Type 1 Diabetes is Due to a Mutation in a Novel Immune-Associated Nucleotide (Ian)-Related Gene
Genome Res., July 1, 2002; 12(7): 1029 - 1039.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
L. Hornum, J. Romer, and H. Markholst
The Diabetes-Prone BB Rat Carries a Frameshift Mutation in Ian4, a Positional Candidate of Iddm1
Diabetes, June 1, 2002; 51(6): 1972 - 1979.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Cambot, S. Aresta, B. Kahn-Perles, J. de Gunzburg, and P.-H. Romeo
Human Immune Associated Nucleotide 1: a member of a new guanosine triphosphatase family expressed in resting T and B cells
Blood, May 1, 2002; 99(9): 3293 - 3301.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poirier, G. M. C.
Right arrow Articles by Erlander, M. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poirier, G. M. C.
Right arrow Articles by Erlander, M. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS