François Meyer

Reversible silencing of enhancers by sequences derived from the human IFN-α promoter

Abstract The virus-responsive element of the IFN-α 1 promoter, VRE(IFNα), comprises two imperfect 19 bp repeats, repA and repB. VRE(IFNα), tetrameric repA, and tetrameric GAAAGT (a subsequence of repB) or tetrameric AAGTGA conferred inducibility on a reporter gene when placed upstream of a complete or truncated promoter. Induced transcription was weak with a minimal promoter (TATA box only), but was strongly stimulated by the SV40 enhancer placed immediately upstream of the inducible element. Surprisingly, under noninduced conditions, tetrameric repA, GAAAGT, and AAGTGA (but not VRE(IFNα)) completely silenced enhancement of constitutive transcription by the SV40 72 bp repeat when interposed between the latter and the TATA box; silencing was fully abrogated by induction.

Interactions of Qβ replicase with Qβ RNA

Abstract The interactions of Qβ replicase with Qβ RNA were investigated by treating replicase-Qβ RNA complexes under various conditions with ribonuclease T 1 , and by characterizing enzyme-bound RNA fragments recovered by a filter binding technique. Evidence for replicase binding at two internal regions of Qβ RNA was obtained. One region (at about 1250 to 1350 nucleotides from the 5′ end) overlaps with the initiation site for coat protein synthesis; this interaction is thought to be inessential for template activity but rather to be involved in the regulation of protein synthesis. Binding to this site (called the S-site) requires moderate concentrations of salt but no magnesium ions. The other region (at about 2550 to 2870 nucleotides from the 5′ end) is probably essential for template activity; binding to this site (called the M-site) is dependent on the presence of magnesium ions. The nucleotide sequences of the RNA fragments from the two sites were determined and found to have no common features. Under the conditions tested, replicase binding at the 3′ end of Qβ RNA could not be demonstrated, except when initiation of RNA synthesis was allowed to occur in the presence of GTP and host factor. If instead of intact Qβ RNA, a complete RNAase T 1 digest of Qβ RNA was allowed to bind to replicase, oligonucleotides from the S-site and the M-site, and oligonucleotides from a region close to the 3′ end, were found to have the highest affinity to the enzyme. The RNA fragments recovered in highest yield, M-2 and S-3 from the M and S-site, respectively, were isolated on a preparative scale and their enzyme binding properties were studied. In competition assays with random RNA fragments of the same size, selective binding was observed both for the M and the S-site fragment. Partial competition for replicase binding was found if M-2 and S-3 were presented simultaneously to the enzyme. Either fragment, if preincubated with replicase, caused a specific inhibition of initiation of Qβ RNA-directed RNA synthesis, without inhibiting the poly(rC)-directed reaction. The results are discussed in terms of a model of replicase-Qβ RNA recognition. Template specificity is attributed to binding of internal RNA regions to replicase, resulting in a specific spatial orientation of the RNA by which the inherently weak, but essential, interaction at the 3′ end is allowed to occur and to lead to the initiation of RNA synthesis.

Site-directed mutagenesis in DNA: Generation of point mutations in cloned β globin complementary DNA at the positions corresponding to amino acids 121 to 123

Abstract We have utilized the principle of site-directed mutagenesis, previously applied to the RNA of bacteriophage Qβ, to generate nucleotide transitions in a predetermined region of DNA. Plasmid PβG, which contains an almost complete DNA copy of rabbit β globin messenger RNA, was nicked at the Eco RI site which is located within the globin gene, in a region corresponding to amino acids 121 and 122. Substrate-limited nick translation using DNA polymerase I and N 4 -hydroxydCTP, dCTP and dATP led to the replacement of TMP residues by the nucleotide analog in the immediate vicinity of the nicks. The substituted DNA was amplified in vivo , treated with Eco RI and retransfected. 1.9% of the amplified DNA was found to be Eco RI-resistant. Nucleotide sequence analysis of the critical region of six Eco RI-resistant isolates revealed that two plasmids had one, three had two and one had three A · T → G · C transitions, all located within the substituted region. No point mutations ( −3 %) were found in control preparations; however, a small number of deletion mutants, lacking the Eco RI site, were isolated.

The Use of Site-Directed Mutagenesis in Reversed Genetics

In classical genetics, the relationship between genotype and phenotype is explored by selecting or screening for organisms with deviant properties and subsequently mapping the cognate lesion in their genome. This approach has been immensely fruitful in correlating structure-function relationships at the molecular level, particularly in the case of microorganisms and viruses. It encounters limitations when, for example, the role of noncoding, functionally undefined segments of a genome are to be studied since we do not know what properties to screen or select for, or when a lesion leads to an unconditionally lethal mutation. Several years age we developed a new methodology to deal with such difficulties in the case of phage Qβ (1–4). In this approach, which we have called “reversed genetics” (5,6), a mutation is first generated in a predetermined area of the genome by site-directed mutagenesis (1) and the effect of the lesion is then studied either in vivo or in vitro. A similar approach has been applied to the study of SV40 by Nathans, Berg and their colleagues (7–10). In the first part of this article, we shall describe the application of reversed genetics to phage Qβ; in the second part, we shall describe the extension of site-directed mutagenesis to cloned eukaryotic DNA.

Vom nativen zum rekombinanten Humanerythropoietin

Human erythropoietin (EPO), which occurs only in minute quantities in human plasma, has been isolated from urine of patients and purified to homogeneity. A partial amino acid sequence from tryptic digests of the purified protein was selected for oligodeoxynucleotide probe synthesis. These probes were used to isolate the EPO gene from a human gene bank. The isolated gene was engineered into a eukaryotic expression vector and introduced into chinese hamster ovary cells commonly used for recombinant protein production. A specific amplification of the EPO gene copy number in these cells led to the secretion of milligram quantities of EPO per liter of cell supernatant. This protein has similar characteristics to native urinary EPO, regarding its electrophoretic mobility, glycosylation pattern and biologic activities in vitro and in vivo. A scaled-up industrial process provides recombinant EPO for its clinical use.

CRYPTIC SPLICE SITES IN THE RABBIT ß-GLOBIN GENE ARE REVEALED FOLLOWING INACTIVATION OF AN AUTHENTIC 5′ SPLICE SITE BY MUTAGENESIS IN VITRO

ABSTRACT. The processing of transcripts of genes altered by restructuring or site-directed mutagenesis was studied in a transient (acute) transformation system.