Superinduction of the human gene encoding low density lipoprotein receptor

Expression of human LDL receptor mRNA and protein is induced in human glioblastoma-astrocytoma cells upon transfer into lipoprotein-deficient medium, a mode of induction likely to be physiological. The presence of cycloheximide (CHX) leads to up to 7.5-fold superinduction of LDL receptor mRNA within 4 hr and, upon removal of this inhibitor, to superinduction of LDL receptor protein as well. The extent of superinduction of LDL receptor mRNA reaches over 40-fold beyond the level expressed in medium containing regular serum. Despite its extensive superinduction, LDL receptor mRNA decays rapidly in the presence of CHX. Stabilization of LDL receptor mRNA is thus unlikely to account for the observed superinduction. These results show that normally the LDL receptor gene is expressed to only a small fraction of its full potential.     

Superinduction of the human interferon-beta promoter.

Superinduction, that is induction with simultaneous blocking of mRNA translation, enhances the induction of interferon-beta in response to virus or double-stranded RNA in human fibroblasts. Expression of the cloned IFN-beta gene upon transfer into heterologous cells reflects the endogeneous interferon expression with respect to induction or superinduction indicating the involvement of cellular mediators in this mode of gene regulation. Expression from gene hybrids in mouse Mo57/2 cells reveals that 5' flanking DNA sequences from the human IFN-beta gene are responsible for induction and superinduction. Superinduction of the human IFN-beta promoter is demonstrated in several rodent and primate cell lines. In addition, expression from promoter mutants in mouse cells indicates that DNA sequences responsible for induction and superinduction are identical.     

Expression in Escherichia coli of chemically synthesized gene for the human immune interferon.

A 454 base pair fragment of double stranded DNA consisting of a gene for a human immune interferon (hIFN-gamma), initiation and termination signals plus appropriate restriction endonuclease sites, was totally synthesized. The synthesis involved preparation of 62 oligodeoxyribonucleotides by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. This synthetic gene was expressed in E. coli under the control of the lac UV5 promoter. The product has antiviral activity which was acid labile and completely neutralized by antiserum to hIFN-gamma but not by antiserum to hIFN-alpha or hIFN-beta. Molecular weight of hIFN-gamma produced by E. coli was estimated to be about 32,000 and 17,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively.     

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E. coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively. The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.     

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.     

Superinduction of interferon and and a study of its messenger RNA]

Combined use of interferon inductor poly-IC and antibiotics (cycloheximide and actinomycin D) provided a significant increase (up to 1000 times) in interferon production by chick, mouse, monkey and human cells. Messenger RNA with matrix activity for interferon (mRNA-IF) was isolated from superinduced cells. On translation of mRNA-IF in homogenous and heterogenous cells the specificity of interferons produced was determined by the type of the cells from which mRNA-IF was isolated. Sedimentation analysis of various mRNA-IF revealed 2 peaks of activity: major (5--15S) and minor (25--30S).     

Characterization of receptors for immune interferon in U937 cells with 32P-labeled human recombinant immune interferon

Recombinant human immune interferon (HuIFN-gamma) was labeled with [gamma-32P]ATP and cyclic-AMP-dependent protein kinase from bovine heart to a specific radioactivity of 11,000 Ci/mmol. At least two molecules of phosphate were incorporated per molecule of interferon. The binding of [32P]HuIFN-gamma to human U937 histiocytic lymphoma cells was time dependent, and displaceable by HuIFN-gamma but not by HuIFN-alpha A or HuIFN-beta. The specific binding was saturable with less than 10% nonspecific binding. The dissociation constant of [32P]HuIFN-gamma for U937 interferon receptors was calculated to be 1.5 X 10(-10) M with a total of 1,800 binding sites/cell. Dissociation of bound [32P]IFN-gamma at 24 degrees C exhibited two distinct rates. A fast dissociation with a specific rate constant of 0.141 min-1, and a slow dissociation with a specific rate constant of 0.0027 min-1. The Kd for [32P]HuIFN-gamma was calculated from kinetic constants to be 5.4 X 10(-10) M.     

Characteristics of immune interferon produced by human lymphocyte cultures compared to other human interferons.

A factor with antiviral activity has been produced in vitro by combined macrophage-lymphocyte cultures from patients with recent herpes labialis in response to HSV antigen stimulation. It has been designated "immune interferon" and characterized in comparison to several other human interferons. It was shown to be relatively unstable at pH 2 and at 56 degrees C. Rabbit anti-human leukocyte interferon serum was shown to be less active against immune interferon than against diploid cell interferon or against vesicle fluid interferon. The possibility of immune interferon being a totally different anti-viral protein or a protein with certain shared antigen determinants or structures with classical viral interferon is discussed. A simplified method for the assay of anti-interferon sera with microtiter plates is also described.     

Identification of a gene encoding for the human formyl peptide receptor.

The neutrophil FMLP receptor is involved in activation and subsequent response to certain chemotactic stimuli. The normal receptor has been reported to consist of several components, ranging in size from 43-94 kDa, and to contain both high and low affinity states. However, limited information is available on the gene/s which encode for the receptor. In this study, we have generated oligonucleotide probes derived from a published cDNA sequence encoding for one of the components of the FMLP receptor, and used these probes to amplify genomic DNA from HL-60 cells as well as normal human neutrophils, using the polymerase chain reaction. Such procedure resulted in the amplification of a single, approximately 1 kb fragment of genomic DNA identical in sequence to the cDNA described in the literature for one of the isoforms of the receptor. This finding supports the notion that the human FMLP receptor is encoded by at least one, intronless gene.     

The absence of introns within a human fibroblast interferon gene.

Experiments in which immobilised restriction fragments of genomic DNA were hybridised with a cloned human fibroblast interferon cDNA indicate that the homologous chromosomal genes exist in only one basic arrangement. This is in marked contrast to recent studies by Nagata et al. (1) showing that there are at least eight gene arrangements for human leukocyte interferon. Having isolated a chromosomal human fibroblast interferon gene from a gene bank, we conclude from nucleotide sequencing studies that there is a complete absence of introns within the RNA-coding region. In view of a similar observation recently made for a human leukocyte interferon gene (1), it would appear as if interferon genes in general are unlike the vast majority of eukaryote genes in this respect.     

Cloning and structure of the human immune interferon-gamma chromosomal gene.

Two clones containing the human immune interferon-gamma (IFN-gamma) chromosomal gene were isolated from a human DNA library present in lambda Charon4A phage. DNA from these clones specified biologically active interferon upon injection into the nuclei of Xenopus laevis oocytes. Analysis of the clones revealed that they were derived from the same chromosomal segment. Restriction fragments that hybridized with 32P-labeled cDNA probes were subcloned into plasmids and the complete sequence of the IFN-gamma gene was determined. Unlike IFNs-alpha and -beta, IFN-gamma does contain introns. Their presence was also revealed by electron microscopy. It is intriguing that the smallest of the three introns is located just in the middle of the Glu-Glu sequence which is conserved among all three forms of interferon at approximately the same position. The promoter region was found to contain a prototype TATA box, many palindromic structures and several repeating sequences and two symmetrical structures. Particularly interesting was the existence of two sequences homologous to those present in the chicken albumin and the human IFN-beta gene promoter region. A sequence GTGTTG common to several other genes was found in the region approximately 10 nucleotides downstream from the polyadenylation site.