Distinct T-lymphocyte subsets recognize antigens in conjunction with different classes of major histocompatibility complex (MHC) glycoproteins using the T-cell receptor (TCR), a disulphide-linked heterodimer associated with the CD3 complex on the cell surface. In general, class I and class II MHC products provide a context for the recognition of foreign antigens by CD8+ and CD4+ T cells, respectively. This recognition seems to be largely dependent on alpha beta TCR heterodimers, whereas the function of the second gamma delta TCR, present on a minor subpopulation of cells, is still unknown. In the mouse, the existence of six cell-surface MHC class I products (K, D, L, Qa-1, Qa-2 and Tla) has been firmly established by serological, biochemical and genetic evidence. So far, only the most polymorphic of them, K, D and L ('classical' class I) have been reported as restriction elements for T-cell recognition of foreign antigens. The function of the relatively invariant Qa and Tla molecules remains unknown. We have made a T-helper cell hybridoma clone (DGT3) that recognizes synthetic copolymer poly(Glu50Tyr50) in the context of Qa-1 cell surface product, and has a CD4-CD8- phenotype. Our studies indicate that DGT3 cells express the gamma delta TCR on the cell surface, implicating its role in Qa-1-restricted antigen recognition. This is the first evidence that T cells can recognize foreign antigen in association with self Qa product, confirming that Qa molecules not only topologically, but also functionally, belong to the MHC.
The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.
The Saccharomyces cerevisiae RNA polymerase II subunit gene RPB7 was isolated and sequenced. RPB7 is a single copy gene whose sequence predicts a 19,000 Dalton protein of 171 amino acids. RPB7 is known to dissociate from RNA polymerase II as an RPB4/RPB7 subcomplex in vitro. RPB7 also appears to interact with RNA polymerase II in a manner dependent upon RPB4, since RNA polymerase II purified from cells lacking RPB4 also lacks RPB7. Previous results have demonstrated that deletion of the RPB4 results in slow growth and cold- and temperature-sensitivity. In contrast, deletion of the RPB7 gene revealed that it is essential for cell growth and viability. Loss of both the RPB4 and the RPB7 genes causes lethality. These results suggest that RPB7 contributes to the function of RNA polymerase II in the absence of RPB4 either in a manner independent of its association with the enzyme or by directly binding to the enzyme in a manner independent of its association with RPB4.
To assess functional relatedness of individual components of the eukaryotic transcription apparatus, three human subunits (hsRPB5, hsRPB8, and hsRPB10) were tested for their ability to support yeast cell growth in the absence of their essential yeast homologs. Two of the three subunits, hsRPB8 and hsRPB10, supported normal yeast cell growth at moderate temperatures. A fourth human subunit, hsRPB9, is a homolog of the nonessential yeast subunit RPB9. Yeast cells lacking RPB9 are unable to grow at high and low temperatures and are defective in mRNA start site selection. We tested the ability of hsRPB9 to correct the growth and start site selection defect seen in the absence of RPB9. Expression of hsRPB9 on a high-copy-number plasmid, but not a low-copy-number plasmid, restored growth at high temperatures. Recombinant human hsRPB9 was also able to completely correct the start site selection defect seen at the CYC1 promoter in vitro as effectively as the yeast RPB9 subunit. Immunoprecipitation of the cell extracts from yeast cells containing either of the human subunits that function in place of their yeast counterparts in vivo suggested that they assemble with the complete set of yeast RNA polymerase II subunits. Overall, a total of six of the seven human subunits tested previously or in this study are able to substitute for their yeast counterparts in vivo, underscoring the remarkable similarities between the transcriptional machineries of lower and higher eukaryotes.The eukaryotic mRNA transcription apparatus comprises RNA polymerase II, general transcription factors and their associated factors, and gene-specific factors (reviewed in references 6, 13, and 23). RNA polymerase II (pol II) is a multisubunit enzyme with ϳ12 to 15 subunits, depending on the organism, that plays a major role in mRNA synthesis since it possesses the catalytic machinery for the formation of the 3Ј-5Ј phosphodiester bonds between ribonucleoside triphosphates and presumably responds to signals from the multiple factors involved in regulating its function during initiation and elongation of mRNA synthesis.Yeast Saccharomyces cerevisiae pol II has been a useful paradigm for enzyme function since its subunit structure and amino acid sequences are strikingly similar to pol II subunits from a variety of eukaryotes and even archaebacteria. S. cerevisiae pol II has 12 subunits, designated RPB1-RPB12, ranging in size from ϳ190 to ϳ8 kDa (reviewed in references 26 and 27). Five of these subunits (RPB5, RPB6, RPB8, RPB10, and RPB12) are also present in RNA polymerases I and III and are usually referred to as the common subunits.Since the functions of most pol II subunits are unclear, one approach to understanding their role in transcription has been to isolate the genes encoding human RNA polymerase subunits and test for functional similarities by determining if the human subunit can function in place of its yeast homolog. Only a few of the human subunit genes have been tested for heterocomplementation in S. cerevisiae. hsRPB6 has a highly conserve...
In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.
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