Interferon-alpha Gene Research Articles

Use of a new technique to map the porcine alpha interferon gene to chromosome 1.

Use of a new technique to map the porcine alpha interferon gene to chromosome 1.

Replication-incompetent herpesvirus vector delivery of an interferon alpha gene inhibits human immunodeficiency virus replication in human monocytes.

Human monocytes and macrophages are nondividing cells that serve as a major reservoir for human immunodeficiency virus (HIV) at all stages of infection. To investigate viral-mediated gene delivery as a means of inhibiting HIV replication in human monocytes, a replication-incompetent herpes simplex virus vector was developed that expressed human interferon alpha. Monocytes infected with this herpes simplex virus vector and then challenged with HIV showed dramatically reduced cytopathic effects and HIV replication compared to control treated monocytes. Similar effects on HIV replication were observed if monocytes were first infected with HIV and then treated with the recombinant vectors. These results demonstrate that replication-incompetent herpes simplex virus gene delivery of interferon alpha directly to human monocytes can greatly decrease HIV replication and suggest that such a vector might deliver therapeutically important genes directly to sites of HIV infection.

Human interferon-alpha A, -alpha 2, and -alpha 2(Arg) genes in genomic DNA

The frequency in human genomic DNA of three human interferon (IFN) alpha genes which differ from each other by a single base substitution was examined in 11 normal individuals. The polymerase chain reaction (PCR) was used to amplify the coding sequence for the Hu-IFN-alpha 2, Hu-IFN-alpha A, and Hu-IFN-alpha 2(Arg) sequences from genomic DNA. The PCR products were then cloned and individual clones were sequenced. PCR products were also analyzed by restriction endonuclease analysis for the IFN-alpha 2 and IFN-alpha A genes by use of a HinfI site which is eliminated by the substitution of an A for a G in IFN-alpha A. The IFN-alpha A gene which was cloned from the myeloblastoid cell line KG-1 was not observed in any of the 201 clones sequenced from normal individuals or in the Namalwa cell line. It was detected in KG-1 cell genomic DNA where it represented 49% of the clones sequenced. Similarly the IFN-alpha 2(Arg) gene was not detected in normal individuals or in the KG-1 cell line but only in the lymphoblastoid cell line from which it was cloned. In Namalwa cells the IFN-alpha 2(Arg) sequence represented 35% of the clones sequenced while the IFN-alpha 2 sequence comprised 59%. Therefore, both the IFN-alpha A and IFN-alpha 2(Arg) sequences represent alleles of the IFN-alpha 2 gene.

Gene expression regulation for interferon-alpha in hepatocellular carcinoma

We investigated whether regulation of interferon-alpha gene expression could be involved in liver tumor biology and the role, if any, of hepatitis B virus in the regulation of tumor cytokine gene expression. Gene expression was investigated at the transcriptional level using 'in situ' hybridization of cytokine message with an interferon-alpha cDNA probe and at the translational level with immunohistochemistry using an immunoperoxidase technique. Compared to histologically normal liver, a greater percentage of tumor and non-tumor-involved liver tissue sections (67-80% vs. 17%) contained cells positive for interferon-alpha messenger RNA, many of which were also seen to contain an increased number of transcripts (> 100 grains/cell). Hepatitis B infection did not appear to play a role in gene activation, at the hepatocellular level, in liver disease. Except for sinusoidal cells, cells containing cytokine transcripts also produced mature immunoreactive protein. Absence of interferon-alpha protein within mononuclear and sinusoidal cells in seronegative hepatocellular carcinoma tissue with/without underlying liver disease suggested deficient cytokine gene expression, at the post transcriptional level, within these cells in this group. Bile duct epithelia in tumor tissue were found to contain immunoreactive protein for interferon-alpha. In summary our results suggest that interferon-alpha gene activation in hepatocellular carcinoma occurs as a result of the liver cell damage and does not play a dominant role in tumor biology.

Assignment of a locus for familial melanoma, MLM, to chromosome 9p13-p22.

Linkage analysis of ten Utah kindreds and one Texas kindred with multiple cases of cutaneous malignant melanoma (CMM) provided evidence that a locus for familial melanoma susceptibility is in the chromosomal region 9p13-p22. The genetic markers analyzed reside in a candidate region on chromosome 9p21, previously implicated by the presence of homozygous deletions in melanoma tumors and by the presence of a germline deletion in an individual with eight independent melanomas. Multipoint linkage analysis was performed between the familial melanoma susceptibility locus (MLM) and two short tandem repeat markers, D9S126 and the interferon-alpha (IFNA) gene, which reside in the region of somatic loss in melanoma tumors. An analysis incorporating a partially penetrant dominant melanoma susceptibility locus places MLM near IFNA and D9S126 with a maximum location score of 12.71. Therefore, the region frequently deleted in melanoma tumors on 9p21 presumably contains a locus that plays a critical role in predisposition to familial melanoma.

Homozygous deletions within human chromosome band 9p21 in melanoma.

Genetic studies have implicated the early involvement of a gene on chromosome arm 9p in the development of cutaneous melanoma. We have performed loss-of-heterozygosity studies to confirm these original findings and identify the most frequently rearranged or deleted region of 9p. Eight markers were analyzed, including (from 9pter to proximal 9q) D9S33, the beta-interferon (IFNB1) locus, the alpha-interferon (IFNA) gene cluster, D9S126, D9S3, D9S19, the glycoprotein 4 beta-galactosyltransferase (GGTB2) gene, and the argininosuccinate synthetase pseudogene 3 (ASSP3). Two or more of these loci were found to be hemizygously reduced in 12 of 14 (86%) informative metastatic melanoma tumor and cell line DNAs, and homozygous deletions of the marker D9S126 were observed in 2 of 20 (10%) melanoma cell lines. These findings have resulted in the identification of a small critical region of 2-3 megabases on 9p21 in which a putative melanoma tumor-suppressor gene appears likely to reside. Several 9p candidate genes, including IFNB1, the IFNA gene cluster, GGTB2, and the tyrosinase-related protein (TYRP) locus, have all been eliminated as potential targets because they are located outside of the homozygously deleted regions.

Genetic and physical map of the interferon region on chromosome 9p

A region of chromosome 9, surrounding the interferon-beta (IFNB1) locus and the interferon-alpha (IFNA) gene cluster on 9p13-p22, has been shown to be frequently deleted or rearranged in a number of human cancers, including leukemia, glioma, non-small-cell lung carcinoma, and melanoma. To assist in better defining the precise region(s) of 9p implicated in each of these malignancies, a combined genetic and physical map of this region was generated using the available 9p markers IFNB1, IFNA, D9S3, and D9S19, along with a newly described locus, D9S126. The relative order and distances between these loci were determined by multipoint linkage analysis of CEPH (Centre d'Etude du Polymorphisme Humain) pedigree DNAs, pulsed-field gel electrophoresis, and fluorescence in situ hybridization. All three mapping approaches gave concordant results and, in the case of multipoint linkage analysis, the following gene order was supported for these and other closely linked chromosome 9 markers present in the CEPH database: pter-D9S33-IFNB1/IFNA-D9S126-D9S3-D9S19 -D9S9/D9S15-ASSP3-qter. This map serves to extend preexisting chromosome 9 maps (which focus primarily on 9q) and also reassigns D9S3 and D9S19 to more proximal locations on 9p.

Spontaneous Transformation of Rat Ovarian Surface Epithelial Cells: Association With Cytogenetic Changes and Implications of Repeated Ovulation in the Etiology of Ovarian Cancer

Ovarian surface epithelial cells undergo several rounds of division to repair the wound created by follicular rupture at the time of ovulation. This cyclical requirement for cell division, when not interrupted by the long anovulatory rest periods that occur during pregnancy and lactation, may contribute to the development of ovarian cancer. To test this hypothesis, we isolated rat ovarian surface epithelial cells from 10 adult female Fisher rats, initiated two mixed-population and seven clonal cell lines, and repeatedly subcultured these cells in vitro for more than 20 passages. We then tested them for the acquisition of the following four features associated with transformation: 1) the loss of contact inhibition, 2) the capacity for substrate-independent growth, 3) the ability to form tumors when injected subcutaneously and/or intraperitoneally into athymic mice, and 4) cytogenetic abnormalities. Loss of contact inhibition was observed in all nine late-passage cell lines. Six of the nine late-passage, but none of the early-passage, cell lines tested exhibited a capacity for substrate-independent growth that was augmented in a dose-dependent manner by epidermal growth factor. Two late-passage cell lines (clone 2 and mixed-population 2) generated tumors in athymic BALB/c mice within 3 weeks following subcutaneous injection of 5 x 10(6) cells, whereas similar numbers of early-passage cells from the same cell lines failed to generate palpable tumors. Late-passage clone 7 cells were tumorigenic when 5 x 10(7) cells were injected intraperitoneally. Two of the cell lines analyzed exhibited alterations involving losses of part or all of one member of the chromosome 5 pair. Clone 2 possessed an interstitial deletion, del(5)(q21.3q24), consistent with the loss of an uncloned putative tumor suppressor gene at 5q22q23 previously reported to reside near the loci for the interferon alpha, interferon beta, and c-jun genes. Early-passage clone 7 cells exhibited chromosome 5 monosomy, while late-passage cells contained one normal chromosome 5 and a derivative (5q12q). Southern analysis of the three cell lines revealed no consistent loss of loci for the interferon and c-jun genes, although early-passage clone 7 cells had one half the gene copy number for the interferon beta and c-jun genes and both early- and late-passage clone 7 cells lacked DNA sequences hybridizing with the probe for interferon alpha. This pattern of passage-dependent spontaneous transformation of rat ovarian surface epithelial cells in vitro supports the hypothesis that repetitious ovulation contributes to the etiology of human ovarian cancer.

Specific interferon genes are expressed in individual cells in the peritoneum and bone marrow of normal mice.

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of IFN mRNA per cell showed that from 20-50% of the cells from the peritoneum and bone marrow of both normal pathogen-free and axenic mice exhibited grain counts significantly greater than background levels following hybridization with riboprobes specific for the mouse interferon-alpha (IFN-alpha), IFN-beta, or IFN-gamma genes. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Grain counts were reduced to background levels when cells were pretreated with ribonuclease prior to in situ hybridization. The extent of labeling with either IFN-alpha or IFN-beta-specific probes increased following i.v. inoculation of mice with the IFN-inducer Newcastle disease virus (NDV) whereas the degree of labeling observed with a probe specific for beta-actin remained unchanged. No significant differences were observed in the number of bone marrow or peritoneal cells that expressed IFN-alpha or IFN-beta mRNA from either high (C57B1/6) or low (BALB/c) IFN-producing strains of mice. The majority of IFN-alpha and IFN-beta-containing cells from both the bone marrow and peritoneum of normal pathogen-free and axenic mice resembled monocytes morphologically, whereas the majority of IFN-gamma mRNA-containing cells resembled small lymphocytes. In addition, in the bone marrow a number of large cells which resembled megacaryocytes were found to express high levels of IFN-alpha mRNA. Nuclear run-on assays showed that IFN-alpha and IFN-beta genes were actively transcribed in both bone marrow and peritoneal cells from normal and axenic mice. Low levels of de novo IFN-gamma RNA synthesis were detected in the nuclei of peritoneal cells only. The expression of IFN genes in individual cells in the tissues of normal animals may constitute a basis for the regulation of both homeostasis and host defense against virus infection and neoplastic cells.

Identification of a novel virus-responsive sequence in the promoter of murine interferon-alpha genes

We have previously shown that the infection of mouse L-cells with Newcastle disease virus activates transcription of the alpha 4 but not the alpha 6 interferon gene and that the induction is mediated by a 35-base pair inducible element (IE) found in the alpha 4 promoter (-109 to -75). In the present study, we show that the inactivity of the alpha 6 promoter can be mapped to 2 out of 6 nucleotides in which the alpha 6 differs from alpha 4 IE. The symmetrical sequence, GTAAAGAAAGT (-103 to -93), present in the alpha 4 IE is essential for its inducibility and binding of nuclear protein(s) to the alpha 4 IE.

Differential Modulation of Specific Gene Expression Following High- and Low-LET Radiations

Experiments were designed to examine the effects of radiation quality on specific gene expression within the first 3 h following radiation exposure in Syrian hamster embryo (SHE) cells. Preliminary work demonstrated the induction of c-fos and alpha-interferon genes following exposure to low-linear-energy-transfer (low-LET) radiations (X rays or gamma rays). More detailed experiments revealed induction of c-fos mRNA within the first 3 h following exposure to either X rays (75 cGy) or gamma rays (90 cGy). We could not detect induction of c-fos following exposure of SHE cells to fission-spectrum neutrons (high-LET) from the JANUS reactor administered at either high (12 cGy/min) or low (0.5 cGy/min) dose rates. Expression of alpha-interferon mRNA was similarly induced by low-LET radiations but only modestly by JANUS neutrons. The induction by gamma rays was dose-dependent, while induction by neutrons was specific for low doses and low dose rates. These experiments demonstrate the differential gene inductive response of cells following exposure to high- and low-LET radiations. These experiments suggest that these different qualities of ionizing radiation may have different mechanisms for inducing many of the cellular consequences of radiation exposure, such as cell survival and cell transformation.

Deletions of Interferon Genes in Acute Lymphoblastic Leukemia

Structural rearrangements involving the short arm of chromosome 9, including bands 9p21 and 22, are found in the leukemia cells of 7 to 13 percent of patients with acute lymphoblastic leukemia. The interferon-alpha gene cluster and the interferon-beta 1 gene have been localized to this chromosomal region. We have previously demonstrated deletions of these genes in several cell lines established in vitro from patients with lymphoblastic leukemia. We report here homozygous or hemizygous deletions of the interferon-alpha and interferon-beta 1 genes in samples of leukemia cells from patients with lymphoblastic leukemia. Of 62 patients examined, 18 (29 percent) had such deletions. Four patients (7 percent) had homozygous deletions of the interferon-alpha gene cluster; of these, one also had a homozygous deletion and three had hemizygous deletions of the interferon-beta 1 gene. Fourteen patients (23 percent) had hemizygous deletions of both the interferon-alpha gene cluster and the interferon-beta 1 gene. In 8 of the 18 patients with deletions, the deletions of interferon genes were submicroscopic; in the 11 other patients, chromosomal rearrangements of 9p, including translocations or deletions, were visible on light microscopy. These chromosomal and molecular deletions are likely to be related to the loss of a tumor-suppressor gene (or genes) located on 9p, which may be an interferon gene or an unrelated but closely linked gene.

Identification of a Functional Allele of a Human Interferon-α Gene Previously Characterized as a Pseudogene

Three recombinant phage lambda L47 clones containing 4 alpha interferon (IFN) genes have been isolated from a newly constructed human genomic library. Each gene is an allele of a previously described IFN gene, three being only minor variants. The fourth gene SMTIII.1A is a functional allele of the psi LeIF-L gene which previously has been described only as a pseudogene. Therefore, it appears likely that other variant alleles may remain to be described and that the IFN system may be able to tolerate some degeneracy as a consequence of the large number of members of the family.

Isolation and Characterization of a Functional Murine Interferon Alpha Gene which Is Not Expressed in Fibroblasts upon Virus Induction

A mouse genomic segment containing three new members of the murine interferon alpha (MuIFN-alpha) gene family was isolated from a fibroblastic cosmid library. A 4 kb EcoRI fragment contained a new MuIFN-alpha gene named MuIFN-alpha 8. The nucleotide sequence of the coding and flanking regions of this gene showed a high level of homology to those of known members of the MuIFN-alpha family. Transient expression of the MuIFN-alpha 8 gene in COS cells and oocyte translation of in vitro transcripts both led to a biologically active protein. The antiviral activity was neutralized by monoclonal and polyclonal MuIFN-alpha antibodies. Although the 5' flanking sequence shows features characteristic of an IFN regulatory region, the MuIFN-alpha 8 gene is not expressed in murine fibroblasts treated with Newcastle disease virus or poly(I).poly(C).

Induction of human interferon gene expression is associated with a nuclear factor that interacts with the NF-kappa B site of the human immunodeficiency virus enhancer

The relationship between transcription of alpha and beta interferon (IFN-alpha and IFN-beta) genes and the interaction of IFN promoter-binding transcription factors has been examined in monoblastoid U937 cells following priming with recombinant IFN-alpha 2 (rIFN-alpha 2) and Sendai virus induction. Pretreatment of U937 cells with rIFN-alpha 2 prior to Sendai virus infection increased the mRNA levels of IFN-alpha 1, IFN-alpha 2, and IFN-beta as well as the final yield of biologically active IFN. Analysis of nuclear protein-IFN promoter DNA interactions by electrophoretic mobility-shift assays demonstrated increased factor binding to IFN-alpha 1 and IFN-beta regulatory domains, although no new induction-specific complexes were identified. On the basis of competition electrophoretic mobility-shift assay results, factors interacting with the IFN-alpha 1 and IFN-beta promoters appear to be distinct DNA-binding proteins. U937 factor binding was localized to the P2 domain (-64 to -55) of the IFN-beta regulatory element, a sequence motif with 80% homology to the recognition site of transcription factor NF-kappa B. Protein-DNA interactions within the IFN-beta P2 domain were, in fact, specifically competed by either excess homologous P2 fragment or the human immunodeficiency virus enhancer element which contains two duplicated NF-kappa B recognition sites. Hybrid promoter-chloramphenicol acetyltransferase fusion plasmids, containing either the IFN-beta regulatory element or the human immunodeficiency virus enhancer element linked to the simian virus 40 promoter, were analyzed for virus and phorbol ester inducibility in epithelial and lymphoid cells, respectively. In the 293 cell line, both plasmids were constitutively expressed but not virus inducible, while in Jurkat cells, chloramphenicol acetyltransferase activity from these plasmids was induced by tumor-promoting agent treatment. These experiments suggest that induction of IFN gene expression may be controlled in part by transcription regulatory proteins binding to an NF-kappa B-like site within the IFN-beta promoter.

Structure-Function Analysis of Mouse Interferon Alpha Species: MuIFN- 10, a Subspecies with Low Antiviral Activity

Journal of General Virology (1988), 69, 67–75 It has been drawn to our attention that the DNA sequence of the MuIFN-α10 gene published in this paper is almost identical to one previously published for the MuIFN-α7 gene [Dion, M., Vodjdani, G. & Doly, J. (1986). Sequence and expression of a novel murine interferon alpha gene — homology with enhancer elements in the regulatory region of the gene. Biochemical and Biophysical Research Communications 138, 826–834]. There is one sequence difference in the 5′ non-coding region and one in the 3′ non-coding region, but the protein-coding region is identical in both analyses. It is proposed that MuIFN-α10 be renamed MuIFN-α7(10).

Differential and cell type specific expression of murine alpha-interferon genes is regulated on the transcriptional level.

In mouse cells induced with virus infection or dsRNA, the relative levels of alpha-4 interferon mRNA were higher than the levels of alpha-1 and alpha-6 mRNAs; the ratio between relative levels of alpha-4 and alpha-1 or alpha-6 mRNA was, however, dependent on the cell type. Recombinant plasmids, in which the expression of chloramphenicol acetyltransferase (CAT) gene was directed by the promoter regions of alpha-1, alpha-4 or alpha-6 interferon genes were constructed and their inducible expression was studied either in transient assay or in permanently transfected mouse cells. The highest levels of CAT activity and CAT mRNA were observed with alpha-4 CAT plasmid, while the expression of alpha-1 CAT was consistently higher than that coded by alpha-6 CAT plasmid; the ratio between CAT activities coded by alpha-4 CAT and alpha-1 CAT was dependent on cell type. However, in heterologous Vero cells, the transfected alpha-1 and alpha-4 genes were expressed constitutively, and the levels of mRNAs were comparable. These results show that the difference in the relative levels of individual alpha-1 and alpha-4 mRNAs reflects the transcriptional inducibility of the respective promoter regions.

Somatic cell mapping and restriction fragment analysis of bovine alpha and beta interferon gene families.

DNA from bovine x hamster hybrid cells preferentially segregating bovine chromosomes has been analyzed by blot hybridization with alpha and beta interferon probes. Retention or loss of bovine interferon genes was compared to segregation of bovine isozyme loci representing previously described syntenic groups. Families of bovine alpha (IFNA) and beta (IFNB) interferon genes were segregated in concordance with each other and with aconitase-1 (ACO1) on bovine syntenic group U18. This syntenic relationship is conserved on human chromosome 9p and on the portion of mouse chromosome 4 proximal to the centromere. In addition, cattle restriction fragment length polymorphisms were identified with both IFNA and IFNB probes. Of particular interest is a polymorphism apparently due to duplication of IFNB genes.

Structure-function analysis of mouse interferon alpha species: MuIFN-alpha 10, a subspecies with low antiviral activity.

A mouse interferon alpha gene (MuIFN-alpha 10) was isolated from a BALB/c cosmid genomic library. The gene was located on a 1.8 kb HindIII fragment and a 5.1 kb EcoRI fragment. The coding region and parts of the 5' and 3' non-coding regions were sequenced. The results showed that the MuIFN-alpha 10 gene encoded a protein of 167 amino acids. Like most other MuIFN-alpha species it contained a putative N-glycosylation site at amino acid positions 78 to 80. It also possessed cysteine residues at positions 1, 29, 86, 99 and 129. In the signal peptide, in addition to cysteine 21, which is present in all MuIFN-alpha species sequenced so far, a cysteine was found at position 22. At the amino acid level MuIFN-alpha 10 showed strong homology to MuIFN-alpha 1 (only 15 out of 167 amino acids were different). The MuIFN-alpha 10 gene was transiently expressed in monkey COS cells under the direction of the simian virus 40 early promoter. The protein product secreted by COS cells was equally active on mouse (L929) and hamster (CHO) cells. However, as compared to MuIFN-alpha 1 and MuIFN-alpha 4 the specific activity on mouse cells of the protein was 10- to 100-fold lower. To find out which region of its structure was responsible for this low activity, hybrids of the genes encoding MuIFN-alpha 10 and MuIFN-alpha 1 were constructed using the two common XmmI sites which correspond to positions between amino acids 67 and 68 and 123 and 124, respectively. The data showed that hybrid constructs which were MuIFN-alpha 1-like from amino acid 68 or MuIFN-alpha 10-like from position 124 to the C terminus possessed high antiviral activity. Other hybrid constructs were hardly active at all. This implied that the amino acid 68 to 123 region was mainly responsible for the low antiviral activity of MuIFN-alpha 10. In this part of the molecule MuIFN-alpha 1 and MuIFN-alpha 10 differed in only five amino acids. A serine at position 110 and a valine at 85 were unique to MuIFN-alpha 10 as compared to all known MuIFN-alpha and human IFN-alpha subspecies.

Linkage analysis of the murine interferon alpha locus (Ifa) on chromosome 4.

We performed linkage analysis of the murine interferon alpha gene cluster, Ifa, with three different marker genes on chromosome 4: the major urinary protein locus (Mup-1), the brown locus (b) and the misty locus (m). The gene order (from centromere) with intervening percent recombination derived from a first three-point cross is Mup-1--13.6 (+/- 3.6)--Ifa--7.9 (+/- 2.8)--m and from a second three-point cross Mup-1--1.7 (+/- 1.7)--b--3.5 (+/- 2.4)--Ifa. The combined results indicate that the gene order, from the centromere, is Mup-1--b--Ifa--m.