Haim Aviv

Biosynthesis and stability of globin mRNA in cultured erythroleukemic friend cells

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with 3H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA convalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2-4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16-17 hr. The rest of the poly(A)-containing RNA was composed to two kinetic populations: 85-90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but it subsequently declined gradually. Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.

Messenger RNA population analysis during erythroid differentiation: A kinetical approach

The poly(A) + RNA of cultured spleen cells (derived from anaemic mice), pulse-labelled with [ 3 H]uridine, decays with the characteristics of a population composed of two major kinetical groups, one with a half-life of three hours, the other with one of 35 hours. The poly(A) + RNA also contains globin RNA, with a half-life of 17 hours. After a 30-hour labelling period, a steady-state level of globin RNA is reached within the poly(A) + population corresponding to about 10%. On the other hand, the poly(A) + RNA population of circulating mature reticulocytes of anaemic mice labelled in vivo for 24 hours contains more than 98% globin RNA. The half-life of globin RNA in these cells, however, is not changed from the value observed in the cultured spleen cells (17 h). Possible mechanisms to account for the population-shift between these two cell species are discussed.

Effects of interferon on hemoglobin synthesis and leukemia virus production in Friend cells

Establishment of the antiviral state by interferon does not impair differentiation of Friend cells. Interferon actually produces an increase in dimethylsulfoxide-induced hemoglobin synthesis. However, both the constitutive production and the induction of leukemia virus in these cells are inhibited by interferon.

Theoretical analysis of a model for globin messenger RNA accumulation during erythropoiesis.

The data presented in the preceding paper (Bastos et al. , 1977) are analysed mathematically and a model is built which describes the accumulation of the three labelled poly(A)-containing RNA populations of the erythroid cells. The same model can also simulate the change in the relative proportions of globin mRNA in these cells after chase of the labelled nucleotide. The implications of this model for the later stages of erythropoiesis are discussed: it is shown that, if no change in the kinetical parameters occurs at the point of RNA synthesis cessation, globin RNA within the poly(A)-containing population of these cells cannot reach the level observed in reticulocytes. Among the various possible changes examined, destabilization of the most stable RNA species in this population (M2) is the only mechanism which is able to drive the erythroblast to reticulocyte transition in a time-span compatible with the actual erythropoietic process.

Preferential synthesis of viral late RNA by nuclei isolated from SV40 lytically infected cells

Nuclei from SV40-infected monkey cells were isolated late in lytic infection and their cell-free transcriptional activity was characterized. 3H-RNA synthesized in vitro was hybridized to excess quantities of separated SV40 DNA strands which were each covalently bound to Sepharose. It was found that 3-5% of the newly synthesized RNA is virus-specific and that the plus-strand DNA, coding for late RNA sequences, is transcribed at a rate about 15 times higher than that of the minus-strand DNA, which codes for early RNA sequences. This indicates that transcriptional control has a major role in determining the relative abundancy of early and late RNA classes in lytically infected cells.

Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell

A method is provided for recovering a purified animal growth hormone or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, the corresponding naturally-occurring animal growth hormone from a bacterial cell in which the animal growth hormone or polypeptide analog has been produced by means of expression of a plasmid encoding the hormone or polypeptide analog which comprises: (a) disrupting the cell wall of the bacterial cell in a buffered neutral pH solution so as to produce a lysate containing precipitated hormone or polypeptide analog; (b) recovering the resulting precipitated hormone or polypeptide analog; (c) suspending the precipitated hormone or polypeptide analog so recovered in distilled water; (d) treating the resulting precipitate-containing suspension with a sodium hydroxide solution having an alkaline pH of about 11.8 so as to solubilize the precipitate and thus the hormone or polypeptide analog contained therein; (e) separating the solubilized hormone or polypeptide analog from other soluble components by gel filtration chromatography; and (f) subjecting the hormone or polypeptide analog thus separated to ion exchange chromatography to purify the hormone or analog and thereby recover purified hormone or polypeptide analog.

EVALUATION OF SEVERAL ENRICHMENT PROCEDURES FOR THE ISOLATION OF RECOMBINANT PLASMID DNA

A number of methods for the selective enrichment of recombinant plasmids were examined; these include alkaline phosphatase treatment of the restricted pBR322 vector, as well as a combination of this and S1 nuclease treatment of the ligated mixture of pBR322 and pCR1 plasmids orS. griseus DNA followed by D-cycloserine treatment to enrich for cells carrying recombinant molecules. The relative efficiencies of these methods were compared.

Evaluation of immunological methods for detection of bovine growth hormone (BGH) produced in E. Coli

The use of several immunological methods for studies on synthesis of bovine growth hormone (BGH) by E. coli is described here. The ELISA procedure was shown to be the least sensitive and unfit for assaying BGH in E. coli extracts. The solid-phase radioimmunoassay (RIA) proved to be highly sensitive, but since E. coli extract itself (not containing BGH) interfered with the immunological reaction, its use for measuring BGH was practically limited. The best adequate procedure proved to be radioimmunoassay in solution, which was not adversely affected by the E. coli extract and was sufficiently sensitive to detect nanogram quantities of BGH. The size of the BGH produced by normal bacterial cells was investigated by protein fractionation, transfer to nitrocellulose paper and detection by anti-BGH serum. This method was also served for semi-quantitative determination of BGH in the bacterial extract.

Isolation of viable deletion mutants of Streptomyces actinophage (Pal 6) and their molecular characterization.

SummaryDeletion mutants of bacteriophage Pal 6 were isolated by successive treatments of either heat (60° C) or pyrophosphate (10 mM). These mutants were characterized by restriction enzyme cleavage analysis. The pyrophosphate resistant clones lost the whole Eco R1 fragment in which the Sal I site is located, as well as an unrelated Hind III fragment. These results show that the region containing the Sal I site in the phage genome is not essential for phage viability. This single Sal I site is therefore suitable as a potential insertion site for DNA cloning. On the other hand, the heat resistant clones that were isolated and characterized do not appear to have detectable deletions as indicated by their Eco R1 DNA digestion pattern.

Screening for Highly Active Plasmid Promoters via Fusion to β-Galactosidase Gene

Abstract A plasmid containing promoter-deleted inactive β-galactosidase gene [1] was used to select promoters of the pEP121 plasmid [2]. Colonies of cells harboring reactivated β-galactosidase gene were identified by their red color on McConkey plates. The quantitative amounts of β-galactosidase produced in each clone were estimated by assaying enzyme activity and by measuring the specific β-galactosidase protein following fractionation of total cells′ proteins on polyacrylamide gel. A wide range of enzyme activities was observed. The most active promoter isolated was shown to promote β-galactosidase production more efficiently, compared with the original β-galactosidase promoter, amounting to 20% of all cell proteins. Such highly active promoters may be utilized in the future, to promote expression of cloned genes in bacteria.