HLA-DRA promoter polymorphism and diversity generation within the immune system

Multiple major histocompatibility complex (MHC) alleles exist at most class I and II loci. Polymorphism of MHC polypeptides may reflect either different levels of selective pressure operating on each molecule or different mutation rates at different loci. To gain further insight into this issue, we sequenced the non-coding promoter region of the HLA-DRA gene from several Epstein-Barr virus-transformed B cell lines and compared the extent of polymorphism found in this region with the known polymorphism of the HLA-DQB promoter. Our results indicate that the HLA-DRA promoter displays a low level of polymorphism while the promoter of HLA-DQB exhibits a nucleotide substitution rate fivefold greater than that of DRA. Moreover, through phylogenetic analysis, the HLA-DRA promoter was found to have diverged much less than the associated alleles of HLA-DRB1 and -DQA1. Taken together, these results suggest that the HLA-DRA promoter is highly conserved and may be under a stronger functional constraint than the promoter regions of other MHC class II genes. Received: 30 May 1996 / Revised: 24 January 1997     

Structures of wild-type and mutant signal sequences of Escherichia coli ribose binding protein.

The structure of a chemically synthesized 25-residue-long functional signal peptide of Escherichia coli ribose binding protein was compared with that of a nonfunctional mutant-signal peptide using circular dichroism and two-dimensional 1H NMR in solvents mimicking the amphiphilic environments. The functional peptide forms an 18-residue-long alpha-helix starting from the NH2-terminal region and reaching to the hydrophobic stretch in a solvent consisting of 10% dimethylsulfoxide, 40% water, and 50% trifluoroethanol (v/v). The nonfunctional mutant peptide, which contains a Pro at position 9 instead of a Leu in the wild-type peptide, does not have any secondary structure in that solvent but forms a 12-residue-long alpha-helix within the hydrophobic stretch in water/trifluoroethanol (50:50, v/v) solvent. It seems that the Pro-9 residue in the nonfunctional peptide disturbs the helix propagation from the hydrophobic stretch to the NH2-terminal region. Because both of these peptides have stable helices within the hydrophobic stretch, it may be concluded that the additional 2 turns of the alpha-helix in the NH2-terminal region of the wild-type signal peptide is important for its function.     

Trifluoperazine binding to mutant calmodulins

Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.     

Usage of primary cells to delineate IFN-gamma-responsive DNA elements in the HLA-DRA promoter and to identify a novel IFN-gamma-enhanced nuclear factor.

The IFN-gamma regulation of the HLA-DRA gene was examined in a primary cell type, the astrocyte. Site-specific mutagenesis of the DRA promoter reveals that three known sequences, S, X, and Y, are required for an optimal IFN-gamma response. Specifically for the X sequence, the X1, but not the X2, site is involved in IFN-gamma regulation of HLA-DRA in the astrocyte. Most interesting, a novel IFN-gamma-enhanced protein (IFNEX) with specificity for the X element has consistently been observed in nuclear extracts made from primary astrocytes. The correlation of the functional importance of X1 in IFN-gamma-regulated DRA expression and the enhancement of IFNEX by IFN-gamma strongly suggests that IFNEX may play a crucial role in IFN-gamma-regulated class II MHC gene expression.     

A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter.

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.