Organization, structure and expression of murine interferon alpha genes.

Using a human interferon-alpha probe we have isolated recombinant phages containing murine interferon-alpha (Mu IFN-alpha) genes from a genomic library. One of these phages contained two complete Mu IFN-alpha genes and part of a third gene. The insert of a second phage held two IFN genes. This indicates that the Mu IFN-alpha genes are clustered in the genome as is the case for the analogous human genes. The nucleotide sequences of these 5 genes were determined. They show that the genes are all different, albeit highly homologous. The deduced amino acid sequences show that four of the five genes contain a putative glycosylation site. Three genes were transiently expressed in COS cells and they gave rise to protein products showing antiviral properties. The expression of the five Mu IFN-alpha genes and the Mu IFN-beta gene was studied in virus-induced mouse L cells. The individual mRNAs were visualized in a nuclease S1 experiment, using a specific probe for each gene. In RNA preparations from induced cells mRNAs for each of the five alpha genes and the beta gene were present. However, substantial differences in the amounts of the individual mRNAs were observed.     

Repression of the murine interferon alpha 11 gene: identification of negatively acting sequences.

The uninducible murine interferon alpha 11 gene (Mu IFN-alpha 11) shows strong homology with the highly inducible Mu IFN-alpha 4 gene in the promoter region. Negative regulatory sequences located between positions -470 and -145 were characterized in the Mu IFN-alpha 11 promoter. The removal of these sequences leads to virus-inducibility of Mu IFN-alpha 11 while their insertion in Mu IFN-alpha 4 corresponding region significantly reduced the inducibility of Mu IFN-alpha 4 promoter. On the other hand, the virus-responsive element (VRE) of the Mu IFN-alpha 11 differs by a single nucleotide substitution at position -78 from the VRE alpha 4. Constructions carrying either VRE alpha 11 or VRE alpha 4 upstream a heterologous promoter displayed different virus inducibilities. The -78 A/G substitution affects the inducibility by decreasing the affinity of VRE-binding trans-regulators. Our results suggest that the combined effect of the negative regulatory sequences and of the mutation in the VRE alpha 11, completely silences the Mu IFN-alpha 11 gene.     

Characterization of the murine alpha interferon gene family

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-alpha) subtypes. IFN-alpha, as well as IFN-beta, IFN-kappa, IFN-epsilon, and limitin, are thought to bind the same receptor, raising the question of whether different IFN subtypes possess specific functions. As some confusion existed in the identity and characteristics of mouse IFN-alpha subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-alpha family. A total of 14 IFN-alpha genes were detected in the mouse genome, in addition to three IFN-alpha pseudogenes. Four IFN-alpha genes (IFN-alpha1, IFN-alpha7/10, IFN-alpha8/6, and IFN-alpha11) exhibited surprising allelic divergence between 129/Sv and C57BL/6 mice. All IFN-alpha subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-alpha4, IFN-alpha11, IFN-alpha12, IFN-beta, and limitin) showed higher biological activity levels than others, whereas IFN-alpha7/10 exhibited lower activity. Most murine IFN-alpha turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-alpha subtypes did not diverge primarily to acquire specific biological activities but probably evolved to acquire specific expression patterns. In L929 cells, IFN genes activated in response to poly(I*C) transfection or to viral infection were, however, similar.     

Sequence and expression of a novel murine interferon alpha gene--homology with enhancer elements in the regulatory region of the gene

A murine interferon gene (MuIFN alpha) has been isolated from a cosmid library. The sequence of a 1.2-kb HindIII-PstI fragment revealed a new MuIFN alpha gene which has not yet been described and which was termed MuIFN alpha 7. The coding sequence produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter. A comparison of the MuIFN alpha 7 gene with the known interferon genes in their coding and flanking sequences shows homologies between enhancer elements found in the 5' upstream region of the coding gene. The core element common to all known viral enhancers, GTGG(AAA/TTT)G is repeated four times in the MuIFN alpha 7 5'-flanking region, as in all known MuIFN alpha genes.     

Extensive linkage disequilibrium,a common 16.7-kilobase deletion,and evidence of balancing selection in the human protocadherin alpha cluster

Regions of extensive linkage disequilibrium (LD) appear to be a common feature of the human genome. However, the mechanisms that maintain these regions are unknown. In an effort to understand whether gene density contributes to LD, we determined the degree of promoter sequence variation in a large tandem-arrayed gene family, the human protocadherin alpha cluster, on chromosome 5. These genes are expressed at synaptic junctions in the developing brain and the adult brain and may be involved in the determination of synaptic complexity. We sequenced the promoters of all 13 alpha protocadherin genes in 96 European Americans and identified polymorphisms in the promoters alpha 1, alpha 3, alpha 4, alpha 5, alpha 7, alpha 9, alpha 11, and alpha 13. In these promoters, 11 common SNPs are in extensive LD, forming two 48-kb haplotypes of equal frequency, in this population, that extend from the alpha1 through alpha 7 genes. We sequenced these promoters in East Asians and African Americans, and we estimated haplotype frequencies and calculated LD statistics for all three populations. Our results indicate that, although extensive LD is an ancient feature of the alpha cluster, it has eroded over time. SNPs 3' of alpha 7 are involved in ancestral recombination events in all populations, and overall alpha-cluster LD is reduced in African Americans. We obtained significant positive values for Tajima's D test for all alpha promoter SNPs in Europeans (D=3.03) and East Asians (D=2.64), indicating an excess of intermediate-frequency variants, which is a signature of balancing selection. We also discovered a 16.7-kb deletion that truncates the alpha 8 gene and completely removes the alpha 9 and alpha 10 genes. This deletion appears in unaffected individuals from multiple populations, suggesting that a reduction in protocadherin gene number is not obviously deleterious.     

Genomic structure of murine macrophage inflammatory protein-1 alpha and conservation of potential regulatory sequences with a human homolog, LD78

The gene for a murine macrophage inflammatory cytokine, MIP-1 alpha, belongs to a newly recognized superfamily encoding small, inducible peptides shown to be up-regulated in association with cellular activation or transformation (tentatively designated the scy, or small cytokine, gene family). Secreted scy family peptides as a group, and MIP-1 alpha in particular, have inflammatory and mitogenic activities, and the family has been divided into CXC and CC subfamilies according to the spacing of conserved cysteine residues in the primary amino acid sequences. We have isolated and characterized a genomic clone encoding the CC subfamily member MIP-1 alpha. The organization of the murine MIP-1 alpha gene into three exons interrupted by two introns is identical to that found for other members of the CC subfamily (e.g., huLD78, muJE, huJE/MCP-1, muTCA3, and hul-309), which has been taken as evidence of evolution from a common ancestral gene. With the exception of the ratPF4 gene, which shares the two-intron/three-exon pattern typical of the CC subfamily, sequenced genes encoding CXC subfamily peptides (e.g., hulL-8 and hulP-10) include an additional intervening sequence that creates a fourth exon. Genomic nucleotide sequences 5' of the MIP-1 alpha cap site are highly homologous to corresponding regions of the human gene encoding a CC peptide variously designated as LD78/GOS19/pAT464, including consensus regulatory motifs in common, reinforcing the contention that MIP-1 alpha and LD78 may be interspecies homologs.     

Segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine IFN-beta gene to the murine IFN-alpha genes.

Southern blot analysis with murine (Mu) interferon (IFN)-alpha cDNA of restricted genomic DNA of three inbred strains of mice belonging to the species Mus musculus domesticus (BALB/c, C57BL/6, and DBA/2) revealed only a limited degree of polymorphism. For example, with HindIII there were only two polymorphic bands out of 14 hybridizing fragments. With Mu IFN-beta cDNA there was no polymorphism at all between BALB/c and C57BL/6 in DNA restricted with seven different enzymes. In contrast, HindIII-restricted DNA of an inbred strain of wild mice (M. spretus Lataste) hybridized with the IFN-alpha probe displayed a high degree of polymorphism compared with the three strains of laboratory mice and was also polymorphic when probed with IFN-beta cDNA. Although M. musculus domesticus and M. spretus Lataste represent different species, certain interspecies crosses are possible in the laboratory. This enabled us to follow segregation of restriction fragment length polymorphism in HindIII-restricted DNA obtained from 18 backcross progeny of a (DBA/2 X M. spretus)F1 X DBA/2 interspecies cross. There was complete coincidence between the segregation of parental (DBA/2) and (DBA/2 X M. spretus)F1-type IFN-beta and IFN-alpha restriction fragment length polymorphism, indicating tight linkage of the IFN-beta and IFN-alpha genes. In addition, in 15 of 18 progeny the segregation coincided with that of the brown locus on chromosome 4, in accord with previous results obtained with the IFN-alpha probe in strains derived from crosses between BALB/c and C57BL/6 mice. Thus, the Mu IFN-beta gene is tightly linked to the Mu IFN-alpha gene cluster on chromosome 4 near the brown locus.     

Tumor necrosis factor alpha enhances influenza A virus-induced expression of antiviral cytokines by activating RIG-I gene expression

Epithelial cells of the lung are the primary targets for respiratory viruses. Virus-carried single-stranded RNA (ssRNA) can activate Toll-like receptors (TLRs) 7 and 8, whereas dsRNA is bound by TLR3 and a cytoplasmic RNA helicase, retinoic acid-inducible protein I (RIG-I). This recognition leads to the activation of host cell cytokine gene expression. Here we have studied the regulation of influenza A and Sendai virus-induced alpha interferon (IFN-alpha), IFN-beta, interleukin-28 (IL-28), and IL-29 gene expression in human lung A549 epithelial cells. Sendai virus infection readily activated the expression of the IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas influenza A virus-induced activation of these genes was mainly dependent on pretreatment of A549 cells with IFN-alpha or tumor necrosis factor alpha (TNF-alpha). IFN-alpha and TNF-alpha induced the expression of the RIG-I, TLR3, MyD88, TRIF, and IRF7 genes, whereas no detectable TLR7 and TLR8 was seen in A549 cells. TNF-alpha also strongly enhanced IKK epsilon mRNA and protein expression. Ectopic expression of a constitutively active form of RIG-I (deltaRIG-I) or IKK epsilon, but not that of TLR3, enhanced the expression of the IFN-beta, IL-28, and IL-29 genes. Furthermore, a dominant-negative form of RIG-I inhibited influenza A virus-induced IFN-beta promoter activity in TNF-alpha-pretreated cells. In conclusion, IFN-alpha and TNF-alpha enhanced the expression of the components of TLR and RIG-I signaling pathways, but RIG-I was identified as the central regulator of influenza A virus-induced expression of antiviral cytokines in human lung epithelial cells.     

Isolation of novel GRO genes and a phylogenetic analysis of the CXC chemokine subfamily in mammals

Approximately 15 different alpha, or CXC, chemokines have thus far been isolated from 11 species of mammals. Among the best studied chemokines are the 12 human proteins that are encoded by 11 paralogous genes. In order to better understand the evolution and function of this group of genes, we isolated and characterized six novel GRO and GRO-related cDNA sequences from the cow (Bos taurus), the sheep (Ovis aries), the rabbit (Oryctolagus cuniculus), and the guinea pig (Cavia porcellus). The amino acid sequence of the diverged guinea pig GRO or KC gene is only 50%-60% similar to presumed orthologs from other species, while the sheep and cow GRO proteins are 90%-99% similar to each other. The presence of multiple GRO genes in the cow, the rabbit, and the sheep is consistent with what has been observed for humans. Phylogenetic analyses of amino acid sequences from 44 proteins indicate that genes orthologous to many of the 11 known from humans exist in other species. One such gene, interleukin 8, or IL8, has been isolated from nine species, including the rodent guinea pig; however, this gene is absent in the rat and the mouse, indicating a unique gene loss event in the rat/mouse (muroid rodent) lineage. The KC (or MIP2) gene of rodents appears to be orthologous to the GRO gene found in other taxonomic orders. Combined evidence from different sources suggests that IP10 and MIG share sister taxon relationships on the evolutionary tree, while the remaining paralogous genes represent independent lineages, with limited evidence for kinship between them. This observation indicates that these genes originated nearly contemporaneously via a series of gene duplication events. Relative-rate tests for synonymous and nonsynonymous nucleotide substitutions in the KC and IL8 genes did not detect rate heterogeneity; however, there are several notable features regarding the IL8 genes. For example, the IL8 proteins from two Old World monkeys are as similar to one another as they are to the IL8 protein from humans, and all observed nucleotide differences between the IL8 genes of the two monkeys cause amino acid changes; in other words, there are no synonymous differences between them.