Comparative antiviral efficiency of leukocyte and bacterially produced human alpha-interferon in rhesus monkeys.
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Animals↗
Biomedical subjects
C Weissmann
Publications and source records attributed to C Weissmann.
Molecular analysis of the human interferon-alpha gene family.
Fifteen DNA clones containing sequences related to human interferon-alpha cDNA were isolated from a human chromosomal gene bank (Nagata et al., Nature 287 (1980) 401-408) and characterized by restriction mapping, R-loop and heteroduplex analysis. Nine distinct DNA segments hybridized strongly with interferon-alpha 1 cDNA and formed R-loops with poly(A) RNA from interferon-producing human leukocytes; most if not all of these segments represent functional interferon genes. Five segments hybridized weakly with the probe and did not form R-loops with the poly(A) RNA; one of these was characterized as an interferon-alpha pseudogene. Several DNA segments overlap and define a region of 36 kilobase pairs (kb) that contains three strongly and three weakly hybridizing sequences. From our data and those of Goeddel et al. (Nature 290 (1981) 20-25) we conclude that there exist at least 11 distinct genes of gene-like sequences of the interferon-alpha type in the human genome, of which most likely represents an allelic variant, and at least five pseudogenes distantly related to the interferon-alpha genes.
Chromosome Mapping↗
DNA sequences preceding the rabbit beta-globin gene are required for formation in mouse L cells of beta-globin RNA with the correct 5' terminus.
Mouse thymidine kinase-negative (TK-) L cells were transformed with concatenates of cloned herpes simplex virus 1 TK DNA and different rabbit beta-globin DNAs in which the globin genes were preceded by native flanking sequences of 14, 66, 76, 425, and 1500 nucleotides.l In all cases, selection for TK+ cell lines led to a high yield of lines producing 5-1500 mature rabbit beta-globin-specific RNA strands per cell. The 5' termini of the transcripts mapped to (i) the "cap" nucleotide, (ii) positions 42 to 48 nucleotides downstream from the cap site, or (iii) positions in the vector DNA preceding the gene. In the case of the gene with only 14 base pairs of 5' flanking sequence, a high level of rabbit beta-globin RNA was produced, but none of the transcripts had the correct 5' end; most of them originated in the vector moiety. With 66 base pairs of 5' flanking sequence, 5% of the 5' termini were correct, and with 76 or more base pairs, 30-85% were correct. The region between 14 and 66 base pairs preceding the cap site contains the Hogness box and appears to be essential for correct initiation of transcription. The region between 66 and 76 base pairs before the cap site contains a variant of the canonical sequence G-G-C-T-C-A-A-T-C-T found preceding many other genes at a similar location, and this region may modulate the efficiency of transcription. The sequence of 425 nucleotides preceding the rabbit beta-globin gene is reported.
Animals↗
Target cell specificity of two species of human interferon-alpha produced in Escherichia coli and of hybrid molecules derived from them.
Plasmids containing cDNAs for human interferon (IfN) alpha-1, IFN alpha-2, and several hybrids of the two cDNAs, all joined identically to an Escherichia coli lac promoter fragment gave rise, in E. coli, to fused interferons (fIFNs) that had very different target-cell specificities. fIFN alpha-1 had a lower specific activity on human WISH cells than on bovine MDBK cells, while fIFN alpha-2 showed the opposite behavior. fIFN hybrids with the NH2-proximal half of fIFN alpha-2 behaved qualitatively like fIFN alpha-1, and those with the NH2-proximal half of fIFN alpha-2, behaved like fIFN alpha-2. On mouse L929 cells, fIFN alpha-2 was almost inactive, while fIFN alpha-1 showed relatively high activity. In this case, the fIFN hybrids with the COOH-proximal half of IFN alpha-1 showed activity on mouse cells, while the reciprocal hybrid did not. In many cases, the activity spectrum of the hybrids was very different from that of either parent. We propose that the IFN molecule has either two binding sites or two regions constituting the binding site, one in the COOH- and the other in the NH2-proximal half. The experimental findings can be accounted for if the fits of the two sites to their receptor counterparts on different cell lines are independent of one another.
Animals↗
Partial mapping of ten genes of the human interferon- alpha family.
HulFN-alpha genes form a family of not less than 9-10 distinct members, at least three of which are arranged in tandem. In one case, two variants, believed to be alleles of the same gene, could be identified by sequence analysis. The three gene sequenced so far are devoid in introns.
Alleles↗
The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha.
The 12 interferon (IFN)-related sequences detected in a human gene bank fall into not less than eight distinct classes, indicating that there are at least eight IFN-related genes. Most, if not all, of these direct the synthesis of an IFN in Escherichia coli. The sequence of one chromosomal gene and its flanking regions was identical to that deduced for the cDNA corresponding to IFN-alpha l mRNA. No evidence was found for the existence of an intron, in either the coding or the non-coding segments of the gene.
Base Sequence↗
At least three human type alpha interferons: structure of alpha 2.
The sequence of a human leukocyte-derived complementary DNA (cDNA), Hif-2h, which directs the formation in Escherichia coli of a polypeptide, IFN-alpha 1, with interferon (IFN) activity has been described. A second IFN cDNA, Hif-SN206, which also elicits synthesis of a biologically active IFN, IFN-alpha 2, is described in this article. Whereas IFN-alpha 2 is twice as active on human as on bovine cells, IFN-alpha 1 is 10 to 20 times more active on bovine than on human cells. As deduced from the cDNA's, the messenger RNA's for the two IFN's differ in length and in 20 percent of the nucleotides; the mature IFN polypeptides differ in 17 percent of the amino acids. Both IFN-alpha 1 and IFN-alpha 2 differ from the lymphoblastoid IFN described by others. Therefore, at least three different IFN-alpha genes are expressed in man; studies on genomic DNA reveal the presence of at least eight IFN-related genes.
Amino Acid Sequence↗
Effect of interferon-alpha 1 from E. coli on some cell functions.
Interferon-alpha 1 from Escherichia coli transformed with a hybrid plasmid containing a human leukocyte complementary DNA insert, induces resistance to virus in appropriate target cells. It also shares the following properties with natural leukocyte interferon (IFN). (i) It enhances natural killing activity of human lymphocytes, (ii) it enhances antibody-dependent cell-mediated cytotoxicity, (iii) it suppresses antigen- and mitogen-induced leukocyte migration inhibition, (iv) it inhibits growth of IFN-sensitive Burkitt lymphoma cells. Since these activities are exhibited by a cloned protein species, they are due to IFN itself and not to other human proteins.
Antibody-Dependent Cell Cytotoxicity↗
Gene cloning by press conference.
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Cloning, Molecular↗
Human leukocyte and fibroblast interferons are structurally related.
The coding sequences of the dDNAs of cloned human leukocyte interferon I and human fibroblast interferon show homologies of 45% at the nucleotide and 29% at the amino acid level. We conclude that the two genes were derived from a common ancestor.
Amino Acid Sequence↗
Synthesis in E. coli of a polypeptide with human leukocyte interferon activity.
Double-stranded cDNA prepared from the 12S fraction of poly(A) RNA from interferon (IF)-producing human leukocytes was cloned in Escherichia coli using the pBR322 vector. One of the resulting clones had a 910-base pair insert which could hybridise to IF mRNA and was responsible for the production of a polypeptide with biological IF activity. Up to 10,000 units IF activity per g of cells was obtained from some clones.
Base Sequence↗
Abnormal expression of chromosomal rabbit beta-globin gene in Saccharomyces cerevisiae.
A 5.1 kilobase-pair segment of rabbit chromosomal beta-globin DNA was joined to pJDB219, a plasmid consisting of pMB9, the 2-mu yeast plasmid and the yeast leu-2 gene. Saccharomyces cerevisiae transformed with the globin DNA-containing hybrid produced beta-globin-specific RNA. As compared to mature beta-globin mRNA, these transcripts lacked 20-40 nucleotides from the 5' end, contained all of the small intron and extended to about the middle of the large intron. Thus, no splicing of the primary beta-globin transcript could be detected in the yeast cells.
Animals↗
The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei.
We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.
Animals↗
Comparisons of several biological and physicochemical properties of human leukocyte interferons produced my human leukocytes and by E. coli.
Human interferon (IFN) prepared from virus-induced human leukocyte suspensions (leukocyte-derived interferon) was compared to the IFN extracted from Escherichia coli harboring a human interferon-alpha cDNA hybrid plasmid (Hif-SN35-AH-L6). E coli-derived IFN was 20 to 50 times more active than leukocyte-derived IFN on heterologous bovine, feline, murine and guinea pig cells, relative to the activity on human cells. After partial purification by affinity chromatography on an anti-human lymphoblastoid IFN antibody column, the IFN was analyzed by SDS-polyacrylamide gel electrophoresis. While leukocyte-derived IFN gave a heterogeneous pattern with major peaks of activity of 24000 and 19000 daltons, E. coli-derived IFN gave a heterogeneous peak of activity at about 17-18000 daltons. The leading edge of leukocyte-derived IFN in SDS-polyacrylamide gels was significantly more active on bovine cells than on human cells and coincided in mobility with E. coli-derived IFN, which was also much more active on bone than on human cells. After reduction with mercaptoethanol in SDS, the E. coli-derived IFN lost no activity, whereas the leukocyte-derived IFN lost about 90% of its activity. After reduction, E. coli-derived IFN migrated in SDS-polyacrylamide gels as a single peak at 24000 daltons, as did the residual activity of reduced leukocyte-derived interferon. Out data suggest that the interferon produced by the E. coli harboring the clone Hif-SN35-AH-L6 is analogous in size and cross-species activity to one of the molecular species of leukocyte-derived interferon.
Chromatography, Affinity↗
The nucleotide sequence of a cloned human leukocyte interferon cDNA.
We have determined the nucleotide sequence of the human leukocyte interferon cDNA carried in hybrid plasmid Z-pBR322(Pst)/HcIF-2h, which has been shown to direct the formation of a polypeptide with human leukocyte interferon activity (Nagata et al., 1980). The 910 base pair insert contains a 567 (or 543) base pair coding sequence, which determines a putative preinterferon polypeptide consisting of a signal peptide of 23 (or less likely 15) amino acids, followed by an interferon polypeptide of 166 amino acids (calculated molecular weight, 19 390). The coding sequence is preceded by a (most likely incomplete) 56 bp leader and followed by a 242 bp trailer and seven A residues from the poly(A) tail: A comparison of the sequence of 35 amino terminal amino acids of lymphoblastoid interferon (Zoon et al., 1980; M. Hunkapiller and L. Hood, personal communication) and the corresponding sequence deducted for leukocyte interferon revealed 9 differences. This suggests that these two interferons are encoded by two non-allelic genes.
Amino Acid Sequence↗
Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates.
We have used a modification of the Berk-Sharp technique to determine that the 5' termini of the mouse 15 S beta-globin precursor and the mature mRNA have identical map coordinates. The modification involves the use of 5' (or 3') terminally labeled probes; it allows the detection of the precursor in the presence of excess mature mRNA.
Animals↗
Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1.
The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).
Base Sequence↗
Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence.
The nucleotide sequence of a cloned rabbit chromosomal DNA segment of 1620 nucleotides length which contains a beta-globin gene is presented. The coding regions are separated into three blocks by two intervening sequences of 126 and 573 base pairs, respectively. The rabbit sequence was compared with a homologous mouse sequence. The segments flanking the rabbit gene, as well as the coding regions, the 5' noncoding and part of the 3' noncoding messenger RNA sequences are similar to those of the mouse gene; the homologous introns, despite identical location, are distinctly dissimilar except for the junction regions. Homologous introns may be derived from common ancestral introns by large insertions and deletions rather than be multiple point mutations.
Animals↗