GBP1 Research Articles

Compartmentalization of genes coding for immunodominant antigens to fragile chromosome ends leads to dispersed subtelomeric gene families and rapid gene evolution in Plasmodium falciparum

Recent studies on the chromosome structure of Plasmodium falciparum have led to two observations: chromosome breakage occurs frequently in subtelomeric regions and the genes coding for a number of immunodominant parasite proteins are located in these fragile chromosomal segments. Toward understanding the biological significance of these observations, we have been studying the variability of a number of these telomeric genes in parasite lines isolated in different regions of the world. In this report, we present evidence that the telomeric location of the resa and the gbp genes of P. falciparum has allowed their dispersion to other chromosomes and eventual alteration. In the first example it is shown that the resa gene has been dispersed to subtelomeric positions on chromosomes 1, 2, 11 and 14 in clinical isolates from West African patients, giving rise to new parasite genotypes and gene linkage groups. Cloning and molecular analysis of the newly detected resa-related sequences reveal that two of the members of the family have diverged from the ancestral copy on chromosome 1, while the third member on chromosome 14 is very homologous to the ancestral copy indicating that it arose from a recent translocation event. In the second example, we show that the gbp genes form a dispersed gene family that maps to at least three different chromosome extremities. The data suggest that the compartmentalization of P. falciparum antigen genes to the chromosome ends lead to gene families scattered on several chromosome extremities. We propose that the generation of segmental aneuploidy is a specific mechanism of genome adaptation of P. falciparum to its host environment. We present a model to explain the duplicative translocation of chromosome termini.

Interferon regulatory factor 1 is required for mouse Gbp gene activation by gamma interferon.

Full-scale transcriptional activation of the mouse Gbp genes by gamma interferon (IFN-gamma) requires protein synthesis in embryonic fibroblasts. Although the Gbp-1 and Gbp-2 promoters contain binding sites for transcription factors Stat1 and IFN regulatory factor 1 (IRF-1), deletion analysis revealed that the Stat1 binding site is dispensable for IFN-gamma inducibility of Gbp promoter constructs in transfected fibroblasts. However, activation of the mouse Gbp promoter by IFN-gamma requires transcription factor IRF-1. Transient overexpression of IRF-1 cDNA in mouse fibroblasts resulted in high-level expression of Gbp promoter constructs. Unlike wild-type cells, IRF-1% embryonic stem cells lacking functional transcription factor IRF-1 contained very low levels of Gbp transcripts that were not increased in response to differentiation or treatment with IFN-gamma. Treatment of IRF-1% mice with IFN-gamma resulted in barely detectable levels of Gbp RNA in spleens, lungs, and livers, whereas such treatment induced high levels of Gbp RNA in the organs of wild-type mice. These observations suggest two alternative pathways for transcriptional induction of genes in response to IFN-gamma: immediate response that results from activation of preformed Stat1 and delayed response that results from induced de novo synthesis of transcription factor IRF-1.

The interferon-inducible GBP1 gene: structure and mapping to human chromosome 1

We have isolated DNA clones containing the interferon (IFN)-inducible guanylate-binding protein-1-encoding gene ( GBP1) from a human genomic library. Two overlapping phage clones contained the entire GBP1 gene. The 2880 bp corresponding to the GBP1 cDNA were subdivided into eleven exons which were interrupted by a total of 9500 bp of intron DNA. All exon/intron junctions contained consensus splice donor and acceptor sequences. Using hybrid rodent cell lines containing human chromosomes, GBP1 was mapped to human chromosome 1. In addition to GBP1, we detected and partially characterized a novel gene, GBP3, with a structure related to GBP1 and a high degree of sequence homology to both GBP1 and GBP2. Our data suggest that the GBP family of genes contains members other than the previously characterized GBP1 and GBP2.

Tyrosine phosphorylated p91 binds to a single element in the ISGF2/IRF-1 promoter to mediate induction by IFN alpha and IFN gamma, and is likely to autoregulate the p91 gene.

ISGF2 was initially identified, purified and cloned as an interferon-alpha (IFN alpha) induced transcription factor that binds to the IFN-stimulated response element (ISRE) of IFN alpha/beta-stimulated genes (ISGs). It was reported to be transcriptionally regulated by several cytokines including IFN alpha and IFN gamma. IFN alpha and IFN gamma inducibility is mediated by a single element: a high affinity, nearly palindromic version of the IFN gamma activation site (GAS). The ISGF2 GAS is bound specifically by p91, which was previously identified as a subunit of the ISG activator ISGF3, and shown to mediate IFN gamma induction of the GBP gene via a GAS. Tyrosine phosphorylation and DNA binding activity of p91 parallel transcription of ISGF2 in response to IFN alpha and/or IFN gamma, consistent with induction mediated by only a GAS. Transcription of the genes that encode p91 and p113, another subunit of ISGF3, is activated only by IFN alpha. This result suggests induction mediated by an ISRE, and implies autoregulation, requiring the products of both genes. Specificity of the ISRE is the basis for the previous conclusion. In contrast, it appears likely that the ISGF2 GAS, and p91 or related factors, also mediate induction of ISGF2 by IL-6 and prolactin. Convergence of signalling pathways from at least four cytokines on this single site would thus be a key aspect of a general role for ISGF2 in cellular growth control.

Interferon gamma-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3.

A 39-nt DNA sequence, the interferon gamma (IFN-gamma) response region (GRR), is necessary for the IFN-gamma-induced transcription of the high-affinity Fc receptor for IgG (Fc gamma RI) and sufficient for the IFN-gamma-induced transcription of transfected plasmids. By using extracts from IFN-gamma-treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the 3' domain of the GRR. The sequence of this core resembles the IFN-gamma-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-gamma induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-gamma induction and for formation of one of the DNA-protein complexes. By antibody reactivity, we show that a 91-kDa protein, first identified as a component of ISGF3, the IFN-alpha-induced transcription complex, is present in at least two of the DNA-protein complexes. IFN-alpha can induce the formation of the faster-migrating 91-kDa protein-GAS complex but not the slower-migrating complex. Furthermore, IFN-alpha does not result in appreciable transcriptional activation of Fc gamma RI or constructs containing the GRR. Thus, these data demonstrate that the IFN-gamma-activated 91-kDa protein is required for IFN-gamma induction of Fc gamma RI and suggest that an additional complex may be required for optimal expression and specificity.

Dissection of the interferon gamma-MHC class II signal transduction pathway reveals that type I and type II interferon systems share common signalling component(s).

We have used a herpes virus thymidine kinase (HSV-TK) based metabolic selection system to isolate mutants defective in the interferon gamma mediated induction of the MHC class II promoter. All the mutations act in trans and result in no detectable induction of MHC and invariant chain (Ii) gene expression. Scatchard analysis indicates that the mutants have a normal number of surface IFN gamma receptors with the same affinity constant. The mutants fall into two broad categories. One class of mutants is still able to induce MHC class I, IRF-1, 9-27, 1-8 and GBP genes by IFN gamma. A second class of mutants is defective for the IFN gamma induction of all the genes tested; surprisingly, the IFN alpha/beta induction of MHC class I, 9-27, ISG54 and ISG15 genes is also defective in these mutants, although different members of this class can be discriminated by the response of the GBP and IRF-1 genes to type I interferons. These data demonstrate that the signalling pathways of both type I and type II interferon systems share common signal transduction component(s). These mutants will be useful for the study of IFN gamma regulation of class II genes and Ii chain, and to elucidate molecular components of type I and type II interferon signal transduction.

Transcriptional induction of IFN-gamma-responsive genes is modulated by DNA surrounding the interferon stimulation response element.

The 9/27 and GBP mRNAs are both inducible by Interferon-gamma (IFN-gamma). The promoters of both genes contain an Interferon Stimulation Response Element (ISRE), but while the GBP gene is strongly induced transcriptionally by IFN-gamma the response of the 9/27 promoter is very weak. We investigated the molecular basis for this difference. The different IFN-gamma-responsiveness was found to have more than one reason. First, 9/27 promoter DNA was unable to bind the Gamma Interferon Activation Factor (GAF) with a single high affinity site. It efficiently competed for the association of the GAF with the GBP promoter but this competition was due to the presence of two low affinity sites, the ISRE and an ISRE-like sequence, suggesting that the GAS and ISRE, though both having clear preferences for specific proteins, may nevertheless share a certain degree of structural homology. Second, the 9/27 and GBP ISREs differed markedly in their affinities for regulatory proteins (ISGFs 1,2,3) and the GBP ISRE was more potent in mediating IFN-gamma-induced promoter activity in transient transfection. Third and most importantly, however, the strong difference between the IFN-gamma response of the two promoters was mainly due to the sequences surrounding the ISRE: the positive-acting GAS on one side and sequences with silencing properties 5' and 3' of the 9/27 ISRE on the other side. The data thus show mechanisms to both up- and down-regulate the activity of the ISRE.

Interferon induction of gene transcription analyzed by in vivo footprinting.

The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.

Interferon Induction of Gene Transcription Analyzed by In Vivo Footprinting

The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.

Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt

The nucleotide sequence of the gbp gene, which encodes the glucan-binding protein (GBP) of Streptococcus mutans, was determined. The reading frame for gbp was 1,689 bases. A ribosome-binding site and putative promoter preceded the start codon, and potential stem-loop structures were identified downstream from the termination codon. The deduced amino acid sequence of the GBP revealed the presence of a signal peptide of 35 amino acids. The molecular weight of the processed protein was calculated to be 59,039. Two series of repeats spanned three-quarters of the carboxy-terminal end of the protein. The repeats were 32 to 34 and 17 to 20 amino acids in length and shared partial identity within each series. The repeats were found to be homologous to sequences hypothesized to be involved in glucan binding in the GTF-I of S. downei and to sequences within the protein products encoded by gtfB and gtfC of S. mutans. The repeated sequences may represent peptide segments that are important to glucan binding and may be distributed among GBPs from other bacterial inhabitants of plaque or the oral cavity.

Classics of Soviet Satire, Compiled and edited by Peter Henry, Volume I, London & Wellingborough: Collet's (Publishers) Ltd. 1972. xxiv, 235 pp. GBP2.25.

Classics of Soviet Satire, Compiled and edited by Peter Henry, Volume I, London & Wellingborough: Collet's (Publishers) Ltd. 1972. xxiv, 235 pp. GBP2.25.