Subject(s)
Gene Expression Regulation/immunology , Lymphocyte Activation , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transcription, Genetic/immunology , Animals , Artifacts , Cell Culture Techniques/methods , Clone Cells , Image Processing, Computer-Assisted , Mice , RNA, Messenger/geneticsABSTRACT
Chemokines are key regulators of migration in lymphoid tissues. In the thymus, maturing thymocytes move from the outer capsule to the inner medulla and thereby interact with different types of stromal cells that control their maturation and selection. In the process of searching for molecules specifically expressed at different stages of mouse thymic differentiation, we have characterized the cDNA coding for the thymus-expressed chemokine (TECK) and its receptor CCR9. The TECK receptor gene was isolated and shown to be localized on the mouse chromosome 9F1-F4. Thymic dendritic cells have been initially thought to be a prevalent source of TECK. In contrast, our results indicate that thymic epithelial cells constitute the predominant source of TECK. Consistent with the latter distribution, the TECK receptor is highly expressed by double-positive thymocytes, and TECK can chemoattract both double-positive and single-positive thymocytes. The TECK transcript is also abundantly expressed in the epithelial cells lining the small intestine. In conclusion, the interplay of TECK and its receptor CCR9 is likely to have a significant role in the recruitment of developing thymocytes to discrete compartments of the thymus.
Subject(s)
Chemokines, CC/analysis , Intestinal Mucosa/chemistry , Receptors, Chemokine/analysis , T-Lymphocytes/chemistry , Thymus Gland/chemistry , Animals , Chemokines, CC/genetics , DNA, Complementary/analysis , Epithelial Cells/chemistry , Intestinal Mucosa/immunology , Mice , Mice, Inbred C57BL , Receptors, CCR , Receptors, Chemokine/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3epsilon- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrbeta, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function.
Subject(s)
Gene Expression Regulation , Homeodomain Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Thymus Gland/growth & development , Alternative Splicing , Amino Acid Sequence , Animals , Blotting, Northern , CD3 Complex/metabolism , DNA, Complementary/metabolism , Genes, T-Cell Receptor/genetics , Humans , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Genetic , Molecular Sequence Data , Nucleic Acid Hybridization , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Thymus Gland/metabolism , Tissue DistributionABSTRACT
Modulation of gene expression in mouse thymic epithelium upon culture in the presence of thymocytes (coculture) was studied by comparison of hybridization signatures on a set of nearly 5000 mouse thymus cDNA clones. Forty-nine differentially expressed clones (usually down-regulated in coculture) were characterized by tag sequencing. Many of them corresponded to entities that had not been described previously in the mouse, and were further characterized by genome mapping. This set of genes appears to be involved in growth regulation and differentiation within the thymus.
Subject(s)
Chromosome Mapping , Gene Expression Regulation , RNA, Messenger/genetics , T-Lymphocytes/physiology , Thymus Gland/physiology , Animals , Coculture Techniques , Epithelial Cells , Epithelium/physiology , Genome , Mice , RNA, Messenger/analysis , T-Lymphocytes/cytology , Thymus Gland/cytologyABSTRACT
Physical mapping of the human immunoglobulin lambda locus (IGL) on chromosome 22q11 has shown the existence of at least 52 variable region gene segments. These Vlambda genes are associated with EcoRI fragments detectable in Southern blots of genomic DNA samples. The current physical map of the IGL locus includes a unique Vlambda8 gene (IGL8a, accession no. Z73650) in a 3.7 kb EcoRI fragment. However, our Southern blot-EcoRI-restriction fragment length polymorphism studies on the Brazilian population using a specific probe for the Vlambda8 gene (pVL8 probe) have revealed the presence of two additional fragments bearing Vlambda8 sequences (8.0 kilobase (kb) at 100% frequency and 6.0 kb at 10% frequency). We have used human/rodent somatic cell hybrid DNAs to locate these new Vlambda8 genes outside the major locus on chromosome 22q11.2. Polymerase chain reactions using specific primers for the IGLV8a gene on the somatic hybrid panel showed that chromosome 8 (besides 22q11) also comprises Vlambda8 sequences. This finding represents evidence for the dispersion of the human IGLV8 gene family outside the major locus (orphan genes).
Subject(s)
Chromosomes, Human, Pair 22 , Immunoglobulin Variable Region/genetics , Immunoglobulin lambda-Chains/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Humans , Molecular Sequence Data , Restriction MappingABSTRACT
The hybridization signature approach, using colony filters and labeled complex probes, can provide high throughput measurement of gene activity. We describe here the implementation of this method to follow the expression levels of 47 genes in resting and activated T cells, as well as in epithelial cells. Using 4-fold spotting of colonies, imaging plate detection and various correction and normalization procedures, the technique is sensitive enough to quantify expression levels for sequences present at 0.005% abundance in the probe. Comparison with Northern blotting shows good consistency between the two methods. Upon activation of a T cell clone by an anti-CD3 antibody variations ranging from 2- to 20-fold are measured, some of which had not been reported previously. This 'multiplex messenger assay' method, performed using available commercial apparatus, can be used in many cases where simultaneous assessment of mRNA levels for many genes is of interest.
