The human T cell receptor gamma genes are transcribed from TATA-less promoters containing a conserved heptamer sequence.

We have used the anchored polymerase chain reaction (A-PCR) to clone and compare the 5' upstream regions of the human T cell receptor gamma (TRG) genes. Whereas little homology was found among subgroups I, II, III and IV, sequence alignment of TRG subgroup I members revealed a high degree of homology in the 5' sequences. A conserved heptamer sequence (CTGCAGG), which was found upstream from the translation initiation site of all TRG genes in our analysis. Determination of the transcription initiation site located the conserved heptamer 65 base pairs upstream from the cap sites of V5. No TATA box or other cis-acting promoter sequences could be identified in any of the human TRG upstream sequences.

RNA switches regulate initiation of translation in bacteria.

A large variety of RNA-based mechanisms have been uncovered in all living organisms to regulate gene expression in response to internal and external changes, and to rapidly adapt cell growth in response to these signals. In bacteria, structural elements in the 5' leader regions of mRNAs have direct effects on translation initiation of the downstream coding sequences. The docking and unfolding of these mRNAs on the 30S subunit are critical steps in the initiation process directly modulating and timing translation. Structural elements can also undergo conformational changes in response to environmental cues (i.e., temperature sensors) or upon binding of a variety of trans-acting factors, such as metabolites, non-coding RNAs or regulatory proteins. These RNA switches can temporally regulate translation, leading either to repression or to activation of protein synthesis.

Viral alteration of cellular translational machinery increases defective ribosomal products.

Here we show that cells expressing genes inserted into Semliki Forest virus (SFV) vectors generate a large fraction of defective ribosomal products (DRiPs) due to frequent initiation on downstream Met residues. In monopolizing the host cell translational machinery, SFV reduces levels of translation eukaryotic initiation factor 4E (eIF4E), diminishes phosphorylation of ribosome subunit S6, and phosphorylates translation initiation factor eIF2alpha. We show that the last event is required for SFV mistranslation of inserted genes. Downstream initiation is suppressed by fusing inserted genes with the open reading frame encoding the SFV capsid, demonstrating that one function of the capsid element is to enable ribosomes to initiate translation in the proper location. These results show that in modifying translation, viral vectors can unpredictably increase the generation of truncated polypeptides and thereby the DRiP fraction of inserted gene products, which can potentially affect their yield, therapeutic efficacy, and immunogenicity.

Structure of the human CD94 C-type lectin gene.

The genomic structure of the human CD94 gene was obtained by analyzing genomic clones obtained from two different libraries. The CD94 gene contains six exons separated by five introns. The carbohydrate-recognition domain (CRD) is encoded by three exons, and the conservation of intron positions within the CRD indicated that CD94 is closely related to group V of C-type lectins. Primer extension and S1 nuclease protection assays showed that initiation of transcription in the CD94 gene is heterogeneous, but restricted to a 60 base pair region around the major initiation site enclosed within a putative initiator element (TTA+1TTCA). The study of the promoter region of CD94 may help to understand the selective expression of this C-type lectin glycoprotein on NK cells and subsets of cytotoxic T cells.

Regulatory elements in the promoter of a murine TCRD V gene segment.

TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human gammadelta lymphoma, and necessary elements fall between -86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at -62, an Ets site at -39, a TATA box at -26, the major transcriptional start site sequence (-8 to -5 and -2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at -52, and GATA-3 binds to a GATA motif at -5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.

A developmental stage-specific promoter directs germline transcription of D beta J beta gene segments in precursor T lymphocytes.

The tissue- and stage-specific assembly of Ag receptor genes is regulated by transcriptional control elements positioned within Ig and TCR loci. To further understand the role of cis-acting elements in these regulatory mechanisms, we have characterized a transcriptional promoter that drives germline expression of TCR beta gene segments in vivo. The activity of this promoter, termed PD beta, is restricted to a highly conserved 400-bp region located directly upstream from D beta 1-coding sequences. Maximal PD beta activity requires a TATA element situated within the D beta 1 recombination signal sequences and consensus binding sites for the ubiquitous SP1 and the T cell-specific GATA-3 transcription factors. When linked to active enhancer elements, PD beta directs transcription in most cell types; however, the TCR beta enhancer (E beta) stimulates PD beta function specifically in precursor T lymphocytes. These findings suggest that PD beta/E beta interactions may contribute to differential regulation of regions within the TCR beta locus during thymocyte development.