Gérard Marbaix

The Search for the Messenger RNA of Hemoglobin

Publisher Summary This chapter describes the search for the messenger RNA (mRNA) of hemoglobin. Synthetic polynucleotides can certainly fulfill the main function of natural mRNA. A viral RNA is a homogeneous molecular species that contains the information for several proteins, three in the simplest viruses and in some defective viruses perhaps only one. Reticulocyte RNA was centrifuged on a sucrose gradient and separated into four fractions that were assayed for stimulatory activity. The fraction containing 23 S ribosomal RNA was slightly inhibitory, those containing either 16 S or 4S RNA gave a slight stimulation, but fraction 111 that contains a part of the light side of the 16s peak and the region of the gradient between 16s and 4S, did stimulate hemoglobin synthesis in the cell-free reticulocyte system. The fact that the polypeptides made in vitro correspond to hemoglobin, shows that the system is correctly phased. A rough estimate of the amount of mRNA in reticulocyte polyribosomes as compared with ribosomal RNA (rRNA) indicated that it should be possible to detect its ultraviolet light absorption, by merely scaling up the operations, by a factor of ten.

Release of messenger RNA from rabbit reticulocyte polyribosomes at low concentration of divalent cations

Abstract 1. 1. An RNA that we identify as the 9-S messenger RNA previously studied can be released from rabbit reticulocyte polyribosomes by EDTA or CM-cellulose treatment. 2. 2. The criteria for this identification are: high specific radioactivity as compared with ribosomal RNA; high sensitivity to ribonuclease when polyribosomes are treated with minute quantities of this enzyme; a sedimentation velocity identical to that of 9-S messenger RNA after sodium dodecyl sulfate treatment. 3. 3. However, this RNA liberated from reticulocyte polyribosomes by EDTA or CM-cellulose treatment is probably associated with proteins. Indeed, it sediments more rapidly than purified 9-S mRNA if sodium dodecyl sulfate treatment is omitted. 4. 4. 5-S RNA is also released from polyribosomes by the procedure used to liberate messenger RNA.

Genomic integration of bovine leukemia provirus and lack of viral RNA expression in the target cells of cattle with different responses to BLV infection.

Enzootic bovine leukosis (EBL) is a contagious lymphoproliferative disease whose etiological agent is a retrovirus, the bovine leukemia virus (BLV). EBL is a complex disease. Soon after infection a strong humoral antibody response develops and persists for the animal’s entire life. Such BLV-infected cattle can remain asymptomatic virus carriers for many years. They can also at a given time develop persistent lymphocytosis (PL) characterized by a permanent large number of peripheral lymphocytes. A variable but always significant percentage of PL animals develop lymphoid tumors, the terminal tumor phase of EBL. The remnant tumor cases develop suddenly in BLV carriers without any previous hematologic disorder. In general, the fate of BLV-infected animals is variable and depends upon several factors, including age, genetic make-up, environmental factors, and immunologic surveillance (see Burny et al. 1980 for a review).

Effect of aurintricarboxylic acid and of NaF on the binding of globin messenger RNA to reticulocyte 40S ribosomal subunits

Abstract Purified globin messenger RNAs bind selectively to 40S ribosomal subunits from reticulocytes in an energy-free system. Attachment is totally inhibited by aurintricarboxylic acid whereas NaF does not prevent binding.

Separation of the messenger RNA of reticulocyte polyribosomes

Abstract Attempts at isolating reticulocyte messenger RNA have been briefly reported before : Arnstein & Cox (1963) found that a RNA fraction which strongly stimulates amino acid incorporation sediments with ribosomal RNA. Cohn (1964) detected two RNA fractions which are more rapidly labeled than the average RNA of the cells. Hemoglobin synthesis in reticulocytes occurs in polyribosomes, structures made of several 80 S ribosomes attached to a threadlike molecule of RNA which is most probably the informational or messenger RNA (Warner et al. 1963, Gierer, 1963 , Hardesty et al. 1963 ). The present communication describes the labeling and complete separation of this RNA from the other RNA fractions of the cells.

Alcohol dehydrogenase in Arabidopsis: analysis of the induction phenomenon in plantlets and tissue cultures

SummaryAlcohol dehydrogenase (ADH) activity is expressed in Arabidopsis seeds and tissue cultures. During the germination process, ADH activity declines rapidly and is no longer detectable in 9- to 10-day-old seedlings. The synthesis of ADH could be demonstrated in seedlings submitted to anaerobiosis by 35S-methionine incorporation studies.Callus, induced from seeds or leaves on a 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, and cell suspension cultures are characterized by a high level of ADH activity. The incorporation of 35S-methionine and two-dimensional electrophoresis indicated that ADH induction was due to de novo synthesis of the polypeptides. In vitro translation of total poly (A)+-RNA from seedlings and callus showed that only callus mRNA was able to direct the synthesis of ADH polypeptides. This demonstrates the de novo synthesis of ADH mRNA during callus induction.Northern blot hybridization, using in vitro labelled ADH1-F DNA from maize as a probe, revealed sequence homology at the mRNA level between Arabidopsis and maize.

Isolement du rna messager des réticulocytes de lapin

Abstract Isolation of messenger ribonucleic acid of rabbit reticulocytes The thread of RNA which connects the ribosomes in reticulocyte polyribosomes has been isolated. 250 μg of this RNA fraction can be obtained from one rabbit. The sedimentation constant (about 9 S) indicates that the molecular weight is approx. 150 000, i.e., the size predicted for the messengers of each hemoglobin chain if they are monocistronic. The ratio ( G + C ) ( A + U ) equals 1.1, as compared with 1.84 for ribosomal RNA. On methylalbumin columns, this RNA is eluted in the same region of the gradient as 26-S ribosomal RNA, in spite of the large difference in size and composition of these two RNAs.

Degradation of deadenylated rabbit α globin mRNA in Xenopus oocytes is associated with its translation

THE 3′-OH poly(A) segment of globin mRNA ensures the stability of the message when injected into Xenopus oocytes1–4. We recently developed5 a specific method for the removal of the poly(A) tail from globin mRNA, based on synchronous processive phosphorolysis of mRNA using molar excess of E. coli polynucleotide phosphorylase at 0 °C. When injected into Xenopus oocytes, deadenylated globin mRNA is translated for a relatively short period and then rapidly degraded1,2. Native poly(A)-containing mRNA, however, is considerably more stable in the same conditions and is translated for extended periods of time1,2,7. The poly (A) segment itself is responsible for the stability of native globin mRNA in Xenopus oocytes since poly(A) re-addition to previously deadenylated mRNA restores the functional stability of the message3. We do not yet know, however, how poly(A) exerts its protective function; furthermore, the mechanism of degradation of poly(A)-free mRNA is not known. We report here the use of the enhancing effects of haemin on the translation of α-globin mRNA in frog oocytes6 to establish that, in these cells, the degradation of injected poly(A)-free α-globin mRNA is linked to its translation.

Polyribosome formation from haemoglobin ribonucleoprotein in vitro.

When reticulocyte polyribosomes are treated with EDTA, an RPN particle containing 9 S RNA is released [l-4] . This species of RNA is the mRNA for globin [5]. One of the first steps in the initiation of protein synthesis involves the binding of mRNA to the smalI subparticle [6] . This binding has been demonstrated both on native [7] and derived subparticles [8]. In order to demonstrate the subsequent steps in initiation of protein synthesis it is necessary to show formation of an initiation complex from both subparticles with mRNA and subsequent build up of disomes and larger polyribosomes as a result of repeated initiation on the added mRNA. We have found that this is possible, using a preparation of ribosomes and enzymes obtained from a rabbit reticulocyte lysate by pH 5.4 precipitation, which is capable of efficient initiation [9] . Furthermore, aurlne tricarboxylic acid (ATA) and cycloheximide are found to inhibit different steps in this process.

Effect of tRNA pool balance on rate and uniformity of elongation during translation of fibroin mRNA in a reticulocyte cell-free system.

Unsuccessful attempts to synthesize complete fibroin chains in vitro were previously made in heterologous cell-free system [3]. In the present work, we succeeded to obtain complete translation of purified fibroin mRNA in a rabbit reticulocyte lysate. Whilst this work was being completed [1], similar results were published by Lizardi et al. [4]. The synthesis of full-sized molecules of fibroin (M.W. 360,000) was achieved by adding tRNA from the posterior silk gland to the cell-free system. With tRNA from other sources, both the translation rate and the amount of complete fibroin chains dropped. This effect of tRNA is situated at the elongation levels. Analysis of cell-free synthesized products by polyacrylamide gel electrophoresis shows that smaller discrete polypeptides are accumulated after 120 minutes of incubation. These polypeptides correspond to growing fibroin chains. This pattern of translation products suggests that elongation might decelerate at specific sites of the fibroin mRNA. These results show that a tRNA pool adjusted to mRNA codon frequency is required to obtain the maximal average elongation rate. A stochastic model based on random acceptance of tRNA at the ribosomal A site for the codon-anticodon recognition process can explain this phenomenon. It can also explain the occurrence of the unfinished discrete fibroin polypeptides during in vitro translation.