Biomedical subjects

A D Sagar

Publications and source records attributed to A D Sagar.

9 recordsLinked to original sources

PubMed1985

Interferon-beta-related DNA on human chromosome 4.

A DNA subclone (pPE-4000) derived from the lambda B4 interferon-beta-related human genomic DNA clone was used as a probe in blot-hybridization experiments of DNA from a panel of human-rodent somatic cell hybrids containing overlapping subsets of human chromosomes. The DNA hybridization experiments showed that the lambda B4 IFN-beta locus is localized to human chromosome 4. A provisional regional assignment to 4q12-qter was also obtained. Thus available hybridization data implicate human chromosomes 2, 4, and 9 in the human IFN-beta system while the available biological data also implicated human chromosome 5.

Animals↗

PubMed1985

Increased nuclease sensitivity of the human interferon-alpha 1-related genes and the interferon-beta 1 gene during induction by virus.

The chromatin structure of the human interferon (IFN) genes was evaluated during induction of human lymphoblastoid (Namalwa) cells by Sendai virus. Namalwa cells were treated with bromodeoxyuridine (BrdUrd) for 36-48 h and induced with Sendai virus for 7 h; the nuclear fraction was isolated and treated with low levels of either micrococcal nuclease or DNAse I. DNA was extracted from the nuclease-treated chromatin, restricted with Eco RI and analyzed by Southern blotting using IFN-alpha 1 and -beta 1 cDNA probes. An increase in the digestibility of the IFN-alpha 1-related genes and the IFN-beta 1 gene was observed in chromatin prepared from BrdUrd-treated, Sendai virus-induced Namalwa cells as compared with chromatin from uninduced Namalwa cells. Our results indicate that, during IFN induction in Namalwa cells by Sendai virus, the IFN genes assume a more open conformation consistent with increased transcriptional activity across these genes.

Bromodeoxyuridine↗

PubMed1984

Interferon-beta-related DNA is dispersed in the human genome.

Interferon-beta 1 (IFN-beta 1) complementary DNA was used as a hybridization probe to isolate human genomic DNA clones lambda B3 and lambda B4 from a human genomic DNA library. Blot-hybridization procedures and partial nucleotide sequencing revealed that lambda B3 is related to IFN-beta 1 (and more distantly to IFN-alpha 1). Analyses of DNA obtained from a panel of human-rodent somatic cell hybrids that were probed with DNA derived from lambda B3 showed that lambda B3 is on human chromosome 2. Similar experiments indicated that lambda B4 is not on human chromosomes 2, 5, or 9. The finding that DNA related to the IFN-beta 1 gene (and IFN-alpha 1 gene) is dispersed in the human genome raises new questions about the origins of the interferon genes.

Animals↗

PubMed1983

Isolation of novel human genomic DNA clones related to human interferon-beta 1 cDNA.

Southern blot-hybridization analyses of human DNA (from Namalwa lymphoblastoid cells) digested with the restriction endonuclease EcoRI were carried out under optimal conditions with two human fibroblast interferon (IFN-beta 1) cDNA probes, pD19 and pD24, which contain IFN-beta 1 inserts 0.8 and 0.7 kilobase (kb) long, respectively. The analyses revealed the presence of several hybridizable DNA fragments, including two of lengths 6.8 and 5.5 kb, in addition to the classical IFN-beta 1 genomic DNA fragment of length approximately equal to 2.0 kb. We have screened a human DNA library in lambda bacteriophage Charon 4A by using a 32P-labeled IFN-beta 1 insert cDNA (pD24) and thereby isolated six strongly positive human genomic DNA clones. One of these (lambda B37) represents the classical human IFN-beta 1 gene; another (lambda B37) contains a 6.8-kb EcoRI DNA fragment(s) which cross-hybridizes with the IFN-beta 1 cDNA insert probes pD19 and pD24; and the remaining four (which are identical to each other and are exemplified by lambda B4) contain two EcoRI DNA fragments approximately 5.5 and 9 kb long which also cross-hybridize the IFN-beta 1 cDNA probes. A mRNA 0.9 kb long derived from the classical IFN-beta1 gene is expressed in poly(I) . poly(C)-induced human diploid fibroblasts (FS-4 strain). Induced FS-4 cells also contain polyadenylylated RNA 1.8, 3, 5, and approximately equal to 8 kb long derived from the lambda B3 gene, all of which appear to code for biologically active human IFN-beta as tested by using the Xenopus laevis oocyte translation assay. These data strongly indicate that lambda B3 represents a novel functional IFN-beta gene. A 12-kb polyadenylylated RNA, derived from lambda B4, is expressed constitutively at a low level in FS-4 cells, but the amount of this RNA increases 5-7 hr after exposure of the cells to poly(I) . poly(C).

Base Sequence↗

PubMed1982

Multiple human beta interferon genes.

Analysis of human beta interferon (IFN) mRNA preparations obtained from poly(I) . poly (C)-induced human diploid fibroblasts (FS-4) and from several similarly induced human-mouse somatic cell hybrids by electrophoresis through agarose-CH3HgOH tube gels led to the detection of at least five translationally active human IFN-beta mRNA species. The results obtained are consistent with the existence of IFN-beta genes on different human chromosomes. Marked cell-dependent variability in the expression of these IFN mRNA species was observed.

Animals↗

PubMed1981

Further heterogeneity of human alpha interferon mRNA species.

Translationally active (in Xenopus oocytes) human alpha interferon (IFN) messenger RNA's (mRNA's) derived from Sendai virus--induced leukocyte cultures display a bimodal distribution of RNA lengths on electrophoresis through agarose-CH3HgOH gels. The major population (alpha s) consists of mRNA of length 0.7 to 1.4 kilobases, while the minor population (alpha L) consists of RNA of length 1.6 to 3.5 kilobases. Induction of human leukocytes in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB; 100 micromolar) appears to inhibit the accumulation of IFN-alpha s and to enhance that of IFN-alpha L mRNA's (average length about 1.8 kilobases in preparations from DRB-treated cells). Interferons derived from the alpha s mRNA's represent the group of previously recognized alpha interferons while the alpha L interferons are distinguishable from this group by their lower heterospecific activity on bovine cells compared to human cells, their apparent slower mobility in sodium dodecyl sulfate--polyacrylamide gels, and their apparent heteroclitic response toward an antiserum to IFN-alpha.

Dichlororibofuranosylbenzimidazole↗

PubMed1981

Heterogeneity of interferon mRNA species from Sendai virus-induced human lymphoblastoid (Namalva) cells and Newcastle disease virus-induced murine fibroblastoid (L) cells.

Cytoplasmic polyadenylated RNA preparations obtained from Sendai-induced human lymphoblastoid (Namalva) cells and from Newcastle disease virus (NDV)-induced murine (L) cells were denatured in 10-12.5 mM CH3HgOH and then electrophoresed in 2% agarose tube gels containing 10 mM CH3HgOH, the RNA eluted from gel slices and translationally active interferon mRNA species located using the Xenopus oocyte assay. The interferons synthesized were characterized as alpha or beta types based on neutralization tests using specific antisera against human or murine interferon-alpha and interferon-beta. At least two species of mRNA for human interferon-alpha and two for human interferon-beta were detected in RNA from Sendai-induced Namalva cells. These are (approximate mRNA length in parentheses) alpha (1.3 kb), alpha (1.9 kb), beta (1.1 kb) and beta (1.9 kb). Two populations of murine interferon mRNA of lengths approximately 1.4 kb and 3 kb were detected in mRNA preparations from NDV-induced L cells by electrophoresis. However, since the translation products of each of these two populations of mRNA consist of both murine interferon-alpha and murine interferon-beta it is likely that both the 1.4 kb and 3 kb populations contain at least one species each of murine interferon-alpha and murine interferon-beta mRNA.

Animals↗

PubMed1981

Translational activity and functional stability of human fibroblast beta 1 and beta 2 interferon mRNAs lacking 3'-terminal RNA sequences.

Polyadenylylated mRNA was purified from poly(I).poly(C)- and cycloheximide-superinduced human fibroblast (FS-4) cultures. The mRNA was subjected to electrophoresis through an agarose/CH3HgOH gel, and human fibroblast beta 1 and beta 2 interferon mRNAs were isolated. Each mRNA preparation was phosphorolyzed at 0 degrees C for 20 min by using a molar excess of polynucleotide phosphorylase to produce RNAs lacking poly(A) and then incubated at 37 degrees C for varying lengths of time to allow the phosphorylase to further digest the deadenylylated RNA from the 3' end in a processive and synchronous manner. Removal of the poly(A) (less than or equal to 100 residues) and approximately 100 adjacent residues from human fibroblast beta 1 interferon mRNA (native length, 900 residues, including a 3'-noncoding region of 203 residues) did not alter the translational activity or the functional stability of this mRNA in Xenopus oocytes, whereas deletion of the poly(A) and approximately 200 adjacent residues decreased its translational efficiency. On the other hand, removal of the poly(A) (approximately 200 residues) and approximately 200 adjacent residues from human fibroblast beta 2 interferon mRNA (native length, 1300 residues) did not alter the translational activity or the functional stability of this molecule in oocytes. Thus, neither the poly(A) nor large segments of the 3'-noncoding region (which includes the hexanucleotide A-A-U-A-A-A sequence, at least in the case of beta 1 mRNA) are required for the maintenance of the functional stability of human beta 1 and beta 2 interferon mRNAs in Xenopus oocytes.

Animals↗

PubMed1980

Heterogeneity of poly(I) x poly(C)-induced human fibroblast interferon mRNA species.

Three classes of human interferons (IFNs) have been defined on the basis of their immunological properties: the 'Le' or 'alpha' IFN, mainly derived from leukocyte or lymphoblastoid cells; the 'F' or 'beta' IFN, mainly derived from fibroblast cultures; and the 'T', 'immune' or 'gamma' IFN, mainly derived from mitogen- or antigen-stimulated lymphoid cells. Whereas several individual species of Le IFN have been purified to homogeneity, it is generally considered that F IFN represents a single protein. Thus current efforts to clone human fibroblast IFN mRNA sequences are based on the observation that F IFN mRNA sediments in sucrose gradients as a single RNA species of size corresponding to 12-14 S (refs 7-10). We show here, using gel electrohporesis of mRNA, that two populations of translationally active human fibroblast IFN mRNA molecules exist--an abundant '14 S' species and a scarce '11 S' species. Microinjection of either species of mRNA into Xenopus oocytes leads to the synthesis of biologically active F-type human IFN. These data agree with and complement recent RNA hybridization studies of Weissenbach et al.

Animals↗