Constitutive expression of the pre-TCR enables development of mature T cells.

Expression and signalling through the pre-TCR and the TCRalphabeta resemble two critical checkpoints during T cell development. We investigated to which extent a pre-TCR can functionally replace mature TCRalpha chains during T cell development. For this purpose, transgenic mice were generated expressing the pre-TCRalpha (pTalpha) under the transcriptional control of TCRbeta regulatory elements.

Gimap4 accelerates T-cell death.

Gimap4, a member of the newly identified GTPase of the immunity-associated protein family (Gimap), is strongly induced by the pre-T-cell receptor in precursor T lymphocytes, transiently shut off in double-positive thymocytes, and reappears after TCR-mediated positive selection. Here, we show that Gimap4 remains expressed constitutively in the cytosol of mature T cells. A C-terminal IQ domain binds calmodulin in the absence of calcium, and conserved PKC phosphorylation motifs are targets of concanavalin A (ConA)- or PMA/ionomycin-induced PKC activation.

DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation.

How rearranged immunoglobulin (Ig) genes are further diversified by somatic hypermutation is unknown. Using VDJ passenger Ig heavy chain (IgH) knockin mouse strains, we now demonstrate a high frequency of DNA double-strand breaks (DSBs) in the targeted VDJ passenger gene of germinal center (GC) B cells. These DSBs parallel the distribution of mutations in the targeted hypermutation domain and are found preferentially at RGYW motifs, the intrinsic hot spots of somatic hypermutation.

PIM1 reconstitutes thymus cellularity in interleukin 7- and common gamma chain-mutant mice and permits thymocyte maturation in Rag- but not CD3gamma-deficient mice.

The majority of lymphomas induced in Rag-deficient mice by Moloney murine leukemia virus (MoMuLV) infection express the CD4 and/or CD8 markers, indicating that proviral insertions cause activation of genes affecting the development from CD4(-)8(-) pro-T cells into CD4(+)8(+) pre-T cells. Similar to MoMuLV wild-type tumors, 50% of CD4(+)8(+) Rag-deficient tumors carry a provirus near the Pim1 protooncogene. To study the function of PIM proteins in T cell development in a more controlled setting, a Pim1 transgene was crossed into mice deficient in either cytokine or T cell receptor (TCR) signal transduction pathways.

New COP1-binding motifs involved in ER retrieval.

Coatomer-mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins. For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER). Making use of the two-hybrid system, we characterized a new sequence (deltaL) which interacts specifically with the delta-COP subunit of the COP1 complex.

The Sec20/Tip20p complex is involved in ER retrieval of dilysine-tagged proteins.

Sec20p and Tip20p were previously identified as two interacting proteins involved in early steps of the secretory pathway in Saccharomyces cerevisiae. Here we describe a novel temperature-sensitive allele of TIP20 and analyze its phenotype. While sec20 and tip20 mutants exhibited a defect in forward ER-to-Golgi transport at the non-permissive temperature, both were also defective for retrieval of various dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum (ER) at lower temperature.

Sec12p requires Rer1p for sorting to coatomer (COPI)-coated vesicles and retrieval to the ER.

In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER. The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins. In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi.

Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval.

Two new thermosensitive yeast mutants defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2-1 and ret3-1 were defective for ER retrieval, only ret2-1 exhibited a defect in forward ER-to-Golgi transport at the non-permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro.

Steric masking of a dilysine endoplasmic reticulum retention motif during assembly of the human high affinity receptor for immunoglobulin E.

Signals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the alpha chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the gamma chain, allowing transport out of the ER of the fully assembled receptor.

Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum.

Dilysine motifs in cytoplasmic domains of transmembrane proteins are signals for their continuous retrieval from the Golgi back to the endoplasmic reticulum (ER). We describe a system to assess retrieval to the ER in yeast cells making use of a dilysine-tagged Ste2 protein. Whereas retrieval was unaffected in most sec mutants tested (sec7, sec12, sec13, sec16, sec17, sec18, sec19, sec22, and sec23), a defect in retrieval was observed in previously characterized coatomer mutants (sec21-1, sec27-1), as well as in newly isolated retrieval mutants (sec21-2, ret1-1).

Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA.

A quantitative PCR assay for the detection of HIV-1 nucleic acids is described. The assay is based on a competitive internal standard nucleic acid which can be discriminated from target sequences by the presence of a new restriction enzyme site. The method was used to quantitate plasmid molecules containing HIV-1 sequences, HIV-1 DNA and HIV-1 RNA purified from HIV-1-infected tissue culture cells as well as HIV-1 DNA present in the peripheral blood mononuclear cells of an AIDS patient.