A human T cell-specific molecule is a member of a new gene family

We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.     

Genomic structure of the human ezrin gene

The ERM proteins, ezrin, radixin, and moesin, act as linkers between the plasma membrane and actin cytoskeleton. They are involved in a variety of cellular functions, such as cell adhesion, migration, and the organization of cell surface structures, and are highly homologous, both in protein sequence and in functional activity, with merlin/schwannomin, a neurofibromatosis-2-associated tumor-suppressor protein. We report here the genomic structure and intron junction sequences of the human ezrin gene. Ezrin consists of 13 exons and spans approximately 24 kb genomic DNA. The coding parts of the exons range in size from 12 bp to 275 bp and the introns from 182 bp to 7 kb. The genomic structures of ezrin and moesin are highly conserved, suggesting their recent divergence. Radiation hybrid mapping has refined the location of ezrin to the interval between D6S442 and D6S281. Received: 1 June 1998 / Accepted: 25 August 1998     

Costimulation of multiple NK cell activation receptors by NKG2D

The activation of NK cells is mediated through specific interactions between activation receptors and their respective ligands. Little is known, however, about whether costimulation, which has been well characterized for T cell activation, occurs in NK cells. To study the function of NKG2D, a potential NK costimulatory receptor, we have generated two novel hamster mAbs that recognize mouse NKG2D. FACS analyses demonstrate that mouse NKG2D is expressed on all C57BL/6 IL-2-activated NK (lymphokine-activated killer (LAK)) cells, all splenic and liver NK cells, and approximately 50% of splenic NKT cells. Consistent with limited polymorphism of NKG2D, its sequence is highly conserved, and the anti-NKG2D mAbs react with NK cells from a large number of different mouse strains. In chromium release assays, we show that stimulation of NK cells with anti-NKG2D mAb can redirect lysis. Also, enhanced lysis of transfected tumor targets expressing NKG2D ligand could be inhibited by addition of anti-NKG2D mAb. Interestingly, stimulation of LAK cells via NKG2D alone does not lead to cytokine release. However, stimulation of LAK via both an NK activation receptor (e.g., CD16, NK1.1, or Ly-49D) and NKG2D leads to augmentation of cytokine release compared with stimulation through the activation receptor alone. These results demonstrate that NKG2D has the ability to costimulate multiple NK activation receptors.     

Genomic structure of the human PLZF gene.

The human PLZF (promyelocytic leukaemia zinc finger) gene encodes a Krüppel-like zinc finger protein, which was identified via the reciprocal translocation t(11;17)(q23;q21) fusing it to the retinoic acid receptor alpha (RARalpha) gene in promyelocytic leukaemia. To determine its complete genomic organisation, we constructed a cosmid-map fully containing the hPLZF gene. The gene has seven exons, including a novel 5' untranslated exon, varying in size from 87 to 1358bp and spans at least 120kb. Flanking intronic sequences were identified and all splice acceptor and donor sites conformed to the gt/ag rule. Five polymorphic markers could be fine located in its vicinity. These data will facilitate mutation analysis of hPLZF in t(11;17) leukaemia cases, as well as assist mapping and loss-of-heterozygosity analysis. Here we have tested hPLZF as a possible candidate for the PGL1 locus involved in hereditary head and neck paragangliomas. However, mutation analysis revealed no aberration in 12 paraganglioma patients from different families.     

Genomic organization of the human CD5 gene

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.     

Genomic structure and domain organisation of the human Bak gene

The Bcl-2 homologue, Bak, is a potent inducer of apoptosis. FISH data presented here located the gene to 6p21.3. Mapping was consistent with its location centromeric of the HSET locus and approximately 400 kb from the MHC. The construction of a contig of genomic clones across the locus facilitated the sequencing of a PAC containing the gene. Comparison of the gene structure to functional and physical domains revealed a good agreement between the physical structure and the intron–exon organisation. The position of a single intron was conserved in comparison to other members of the Bcl-2 family, namely Bax, CED-9, Bcl-X and Bcl-2, but all other introns were displaced, consistent with a divergent phylogeny.     

Genomic structure of the human mitochondrial aldehyde dehydrogenase gene

We have isolated and characterized four overlapping clones from two cosmid human genomic libraries, which span about 90 kilobase pairs (kbp) and contain the entire human mitochondrial aldehyde dehydrogenase (ALDH2) gene. Restriction maps of the genomic clones were elucidated utilizing cDNA probes and specific oligonucleotide probes. The organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The ALDH2 gene is about 44 kbp in length and contains at least 13 exons which encode 517 amino acid residues. Except for the signal NH2-terminal peptide, which is absent in the mature enzyme, the amino acid sequence deduced from the exons coincided with the reported primary structure of human liver ALDH2 (J. Hempel, R. Kaiser, and H. J?rnvall, 1985, Eur. J. Biochem. 153: 13-28). Several introns contain Alu repetitive sequences. A TATA-like sequence (TTATAAAA) and a CAAT-like sequence (GTCATCAT) are located 473 and 515 bp, respectively, upstream from the translation initiation codon. Primer extension and S1 nuclease mapping were performed to characterize the 5'-region of the gene.     

Genomic structure of the human unconventional myosin VI gene

Mutations in myosin VI (Myo6) cause deafness and vestibular dysfunction in Snell's waltzer mice. Mutations in two other unconventional myosins cause deafness in both humans and mice, making myosin VI an attractive candidate for human deafness. In this report, we refined the map position of human myosin VI (MYO6) by radiation hybrid mapping and characterized the genomic structure of myosin VI. Human myosin VI is composed of 32 coding exons, spanning a genomic region of approximately 70 kb. Exon 30, containing a putative CKII site, was found to be alternatively spliced and appears only in fetal and adult human brain. D6S280 and D6S284 flank the myosin VI gene and were used to screen hearing impaired sib pairs for concordance with the polymorphic markers. No disease-associated mutations were identified in twenty-five families screened for myosin VI mutations by SSCP analysis. Three coding single nucleotide polymorphisms (cSNPs) were identified in myosin VI that did not alter the amino acid sequence. Myosin VI mutations may be rare in the human deaf population or alternatively, may be found in a population not yet examined. The determination of the MYO6 genomic structure will enable screening of individuals with non-syndromic deafness, Usher's syndrome, or retinopathies associated with human chromosome 6q for mutations in this unconventional myosin.     

Genomic structure,gene expression,and promoter analysis of human multidrug resistance-associated protein 7

The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5'-flanking region at -1,780-1,287 bp, and at -611 to -208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226% by genotoxic 2-acetylaminofluorene and 347% by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.     

The Pmed1 gene promoter of human Fc gamma RIIIA can function as a NK/T cell-specific restriction element, which involves binding of Sp1 transcription factor

The low-affinity receptor for IgG (human FcgammaRIIIA) is selectively expressed by a subset of T lymphocytes, NK cells, and macrophages. To understand the mechanisms underlying this pattern of cell type-specific expression, we initially identified alternative promoters, Pmed1/2 and Pprox, in the 5' end of the FcgammaRIIIA gene. In this study, we focused on the Pmed1 promoter and demonstrated this 93-bp region to be highly specific in governing restriction to NK/T cell lines. This property of Pmed1 is context independent and can extend to a disparate promoter. Deletion analysis defined a contribution of two separate elements located to the 5' 21-bp (-942/-922) and 3' 72-bp (-921/-850) regions of Pmed1 in conferring NK/T cell specificity. The 5' part of Pmed1 contains binding sites for Sp1 and NK element-recognizing factors and substitution mapping studies revealed a critical requirement of the Sp1-I site. The importance of Sp1 protein to regulate maximal Pmed1 promoter activity was further established by EMSAs and cotransfection experiments in Sp1-null Drosophila SL2 cells. Our data suggest that Sp1 can contribute, in part, to NK/T cell restriction and further indicate that the FcgammaRIIIA Pmed1 sequence might be useful to direct the NK/T cell-specific expression of heterologous genes.