Differential gene expression in CD3e- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation

 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), CD3e- 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., Tcrβ, 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. Received: 3 June 1999 / Revised: 3 August 1999     

Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations

The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15–16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.     

Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations

The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15-16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.     

Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.     

Structure of extrachromosomal circular DNAs excised from T-cell antigen receptor alpha and delta-chain loci

Small polydisperse circular (spc) DNA was isolated from mouse thymocytes, fragmented by HindIII digestion and cloned into the vector. Sixty DNA clones were randomly selected from the 10,400 phage library. The average size of insert was one-fifth of the original circular molecule. Twenty spc-DNA clones were homologous to DNA probes derived from T-cell antigen receptor (TCR) alpha-chain loci. We have characterized nine clones by DNA sequencing; they contain new germline sequences of the TCR alpha-chain variable (V alpha) and joining (J alpha) gene segments and the products out of the recombination of a V alpha with a J alpha gene segment. An additional four spc-DNA clones carried a new rearranging gene of the TCR delta-chain that is located between V alpha and J alpha genes. At least nine of 60 DNA clones carried the recombination junction of a heptamer-heptamer head-to-head structure expected from an excised product of V-J joining. This shows that most extrachromosomal circular DNAs in the thymus are formed by a sequence-dependent recombination mechanism. We suggest that a functional T-cell receptor V alpha gene can be constructed by somatic random rearrangements through successive looping-out, excision and deletion.     

Thymic lesions in cats infected with a pathogenic molecular clone or an ORF-A/2-deficient molecular clone of feline immunodeficiency virus

Previous studies using feline immunodeficiency virus (FIV) molecular clones lacking the putative transactivator gene (ORF-A/2) failed to address the issue of thymus pathogenesis or investigate the levels of viral replication in separate lymphoid compartments (Y. Inoshima, et al., J. Virol. 70:8518-8526, 1996; E. E. Sparger, et al., Virology 205:546-553, 1994). Using a highly pathogenic molecular clone of FIV, JSY3, and an ORF-A/2-deficient mutant, JSY3DeltaORF-A/2, we compared viral replication and the extent of thymic dysfunction as measured by the formation of lymphoid follicles and alteration of the thymocyte subsets. Viral replication was reduced in JSY3DeltaORF-A/2-infected cats as measured by lymphocyte coculture, immunohistochemistry, and quantitative PCR. Cell-associated viral load measured by lymphocyte coculture varied in a tissue-dependent manner with replication highest in lymphocytes isolated from the thymus, lower in those from the peripheral blood, and lowest in those from lymph node. Thymic proviral load and the number of viral p24 Gag-positive cells within the thymus detected by immunohistochemistry were also reduced. In addition, the onset of a reduced peripheral blood CD4/CD8 ratio was delayed in JSY3DeltaORF-A/2-infected cats. The formation and extent of thymic lymphoid follicular hyperplasia were similar in JSY3 and JSY3DeltaORF-A/2-infected cats as measured by anticytokeratin immunohistochemistry and flow cytometry for percent pan T-negative, immunoglobulin G-positive cells within the thymus. In contrast, comparison of thymocyte subpopulations demonstrated a reduced expansion of single-positive CD4(-) CD8(+) thymocytes in JSY3DeltaORF-A/2-infected cats. Level of viral replication, therefore, may not correlate with the formation of thymic lymphoid follicles but may correlate with the expansion of the single-positive CD4(-) CD8(+) thymocyte subpopulation.     

Molecular cloning and characterization of the complementary DNA coding for the B-chain of murine Clq

cDNA clones coding for the B-chain of murine Clq were isolated from a mouse macrophage library. The characterized clones include the total coding region plus a leader sequence. High homology was found with human Clq B-chain in the coding region (81%). Northern blot analysis of total RNA from different tissues of Balb/c mice showed one band of approximately 1.2 kb. The highest signal was found in RNA preparations of thioglycolate-activated peritoneal macrophages. The probe also hybridized with mRNA from spleen, thymus and heart. Extremely weak signals were found in liver, kidney, lung and intestine tissues.     

Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid fibroblasts

Hybrids were generated between mouse hepatoma cells which exhibit a transformed phenotype, and rat normal diploid fibroblasts. Most isolated hybrid clones contain a single set of chromosomes from each parent. Such clones grow to low saturation densities and are unable to grow or to form colonies in soft agar. The transformed phenotype of the parental hepatoma cells is thus suppressed in these hybrids. Suppression is very stable; however, subclones which have regained a transformed phenotype could be selected; these subclones show a significant reduction of their chromosome number. Amongst the hybrid clones isolated after fusion, a few are characterized by an excess of mouse chromosomes and a reduced number of rat chromosomes. Such clones exhibit a transformed phenotype. Our results show that, provided the hybrids contain an almost complete single set of chromosomes of each parent, spontaneous transformation behaves as a recessive trait in hybrids formed with normal diploid cells.     

Long-term proliferation of mouse thymic epithelial cells in culture

Summary Epithelial cells from the normal mouse thymus were successfully cultivated on tissue culture plastic when plated with lethally irradiated support cells of the LA7 rat mammary tumor line. As the irradiated LA7 cells slowly decreased in number the thymus cells proliferated concomitantly to form a confluent monolayer. The cells now in culture have been subcultured 8 times, have doubled in number at least 30 times, and are still proliferating vigorously. The culture technique also supported clonal growth from a single cell, and nine clones have been isolated. The colony-forming efficiency of thymic cells plated at low concentrations was about 8%. These cultures were never overgrown by fibroblasts. The thymus cells were characterized as epithelial by the presence of cytoplasmic keratin and numerous desmosomes and tonofilaments. They were shown to be mouse cells by immunocytochemistry with species specific antibodies, by isoenzyme analysis, and by karyology. The cells stained when reacted with antibodies to tubulin, vimentin, and actin, but not with antibodies to Thy-1.2, Lyt-1, Lyt-2, Ia, or H-2 proteins. More than 85% of the cells had a normal mouse diploid chromosome number of 40. This culture technique opens the way for future studies of T-cell education with homogeneous thymic epithelial cell populations both in vitro and after reimplantation into genetically defined strains of mice. This work was supported by the Veterans Administration, Washington, D.C.     

Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method.

Production of germ-line competent chimeric mice from embryonic stem (ES) cells is an inevitable step in establishing gene-manipulated mouse lineages. A common method used for creating chimeric mice is the injection of ES cells into the blastocoelic cavity (blastocyst injection). The aggregation method is an alternative way to introduce ES cells to the host embryo which is less difficult than blastocyst injection. Here we re-examined the condition of embryo-ES cell coculture on the aggregation method and found improvement of germ-line competent chimeric production by a simple modification of the coculture medium. Moreover, R1 ES cell and its 10 gene-manipulated subclones were tested by this method. Although all ES cell clones showed good morphology and a normal karyotype, the efficiency of chimeric development and germ-line transmission varied among clones and were classified into three grades according to germ-line competency. In the first group (class A), both the incidence of chimera with high ES cell contribution and the rate of germ-line transmission were fairly high. Germ-line competent chimeras were obtained but with rather low efficiency in the second group (class B), while another group (class C) showed an absence of high ES cell-contributed chimeras and no germ-line transmission. These results suggest the usefulness of this modified aggregation method to predict the potency of ES cell clones for germ-line competency.     

Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm.     

Nearly identical members of the heterogeneous IAP gene family are expressed in thymus of different mouse strains.

Poly(A)RNAs prepared from the thymuses of C57BL/6J and DBA/2J mice were used to construct cDNA libraries in the bacterial expression vector lambda gt11. The libraries were scanned first for protein production with polyvalent antiserum prepared against the 73kDa gag protein of mouse intracisternal A-particles (IAP). Reactive plaques were crossed-screened by hybridization with an IAP-specific DNA probe. Two IAP-specific protein-producing plaques were obtained from the C57BL/6 library and 4 from the DBA/2 library. One C57BL/6 cDNA clone (B12) and two DBA cDNA clones (D8 and D20) were sequenced in their entirety. Clones B12 and D8 were remarkably similar, particularly when compared to the 6 other IAP elements that have been sequenced thus far. We discuss the evidence which leads us to suggest that these clones may be derived from allelic IAP elements expressed in mouse thymus.     

Brain-specific expression of MAP2 detected using a cloned cDNA probe

We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.     

Multilineage hemopoiesis induced by cloned stromal cells

Long-term hemopoiesis in culture depends upon the presence of an adherent layer composed of a variety of stromal cells. A subtype of endothelial-adipocytes from the bone marrow stroma (clone 14F1.1) was previously shown to induce long-term myelopoiesis and renewal of pluripotent stem cells. One of a series of stromal cell lines and clones from mouse thymus stroma (STAC-1.2) has now been found to support long-term hemopoiesis. These marrow- and thymus-derived stromal cell clones also have lymphopoietic activities: precursor T cells, or pre-B cells accumulated in co-cultures of thymus cells and the stromal clones, as indicated by cell surface markers, T cell receptor and immunoglobulin gene rearrangements. The predominance of a cell type in these cultures depended upon the serum used to supplement the medium. Recombinant interleukin 2 (IL-2) and the 14F1.1 clone synergistically promoted the proliferation of thymocytes, while a thymus hormone, THF-gamma 2, shifted the population to a relatively mature phenotype. It is proposed that one major function of stromal cells, whether from the bone marrow or thymus, is to restrain the maturation flow and preferentially support the accumulation of cells at early differentiation stages.     

Class I major histocompatibility complex cDNA clones from sheep thymus: alternative splicing could make a long cytoplasmic tail

To investigate the class I major histocompatibility complex (MHC) genes expressed in the young sheep thymus, a cDNA library was screened with a human HLA-B7 cDNA probe under conditions of relaxed stringency. Thirteen clones were isolated and found by partial sequences to fall into five classes, requiring the expression of at least three loci. One sequence was found six times, almost half of the total, and may thus represent the major message expressed in the young sheep thymus. One of the clones was found to have failed to excise the intron between cytoplasmic exons 7 and 8, leading to the predicted synthesis of a cytoplasmic domain 23 amino acids longer than the other sheep sequences, and 15 amino acids longer than any cytoplasmic domain previously described. The sequences of all the clones were found to be most similar to bovine, and least similar to mouse class I MHC sequences.The nucleotide sequence data reported in this paper have been sunmitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M 34672-6.