Secondary structure model for mouse beta Maj globin mRNA derived from enzymatic digestion data, comparative sequence and computer analysis.

A model for the secondary structure of mouse beta Maj globin messenger RNA is presented based on enzymatic digestion data, comparative sequence and computer analysis. Using 5'-32P-end-labeled beta globin mRNA as a substrate, single-stranded regions were determined with S1 and T1 nucleases and double-stranded regions with V1 ribonuclease from cobra venom. The structure data obtained for ca. 75% of the molecule was introduced into a computer algorithm which predicts secondary structures of minimum free energy consistent with the enzymatic data. Two prominent base paired regions independently derived by phylogenetic analysis were also present in the computer generated structure lending support for the model. An interesting feature of the model is the presence of long-range base pairing interactions which permit the beta globin mRNA to fold back on itself, thereby bringing the 5'- and 3'-noncoding regions within close proximity. This feature is consistent with data from other laboratories suggesting an interaction of the 5'- and 3'-domains in the mammalian globin mRNAs.     

Rabbit oviductal glycoprotein 1 gene: genomic organization polymorphism analysis and mRNA expression

The OVGP1 is an oviductal glycoprotein that has positive effects on fertilization and early embryo development. We have amplified and sequenced the rabbit OVGP1 gene, which spans 13 kb and it is formed by 11 exons and 10 introns. To find polymorphisms, a region encompassing the promoter to intron 1 has been sequenced in 22 rabbits of the H, V, R, and A Spanish lines. We have identified five SNPs and one triallelic microsatellite in the promoter region, and three SNPs and one dinucleotide INDEL in intron 1. The 10 polymorphic sites cosegregate forming two haplotypes. The allelic frequencies of the microsatellite have been analyzed in 98 rabbits belonging to the four lines and the three alleles were found in all the lines. The relative quantification of the OVGP1 mRNA in liver, kidneys, lungs, skeletal muscle, ovary, uterus, and oviduct reveals that the OVGP1 expression in the oviduct is 5,500 higher than in the uterus or ovary, whereas a low level of basal expression is detected in non-reproductive tissues. We have also compared the mRNA expression between samples of oviducts taken from non-mated rabbit and samples of oviducts at different stages of the early pregnancy, but no significant differences were found.     

Sequence analysis of T1 ribonuclease fragments of 18S ribosomal RNA by 5''-terminal labeling, partial digestion, and homochromatography fingerprinting.

The method employed to determine the sequence of a T1 RNase fragment, A-A-A-A-A-U-A-A-C-A-A-U-A-C-A-Gp, from Novikoff rat hepatoma 18S ribosomal RNA is described. This method is applicable to any oligoribonucleotide produced by specific endonucleases that leave the newly cleaved 5'-end free for labeling with polynucleotide kinase and gamma-(32p)-ATP. The (32p)-labeled oligoribonucleotide is subjected to partial endonucleolytic digestion and fractionated by two-dimensional homochromatography fingerprinting. The nucleotide sequence is determined by following mobility shifts of the labeled and partially digested oligoribonucleotides in homochromatography fingerprinting.     

Organization of sequences of avian globin mRNA.

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 6.50 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions. 1. 45-50 of these untranslated nucleotides are present as poly (A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lenghts of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules; The combined data is sued to formulate a regional map of chicken globin mRNA,     

mRNA quantitation by a simple and sensitive RNAse protection assay.

An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

7-Methylguanosine-dependent inhibition of globin mRNA translation by methylglyoxal.

Studies on the inhibition of translation by methylglyoxal of capped and chemically decapped globin mRNAs in the rabbit reciculocyte system strongly suggest that it is cap-dependent. Concentrations of methylglyoxal (0.2 mM), which effected substantial inhibition (80%) of capped mRNA, were only slightly inhibitory (10%) to the decapped species. In addition, the inhibition was K+-dependent with maximal inhibition occurring at the K+ optimum for translation of the capped species, suggesting cap recognition is required for the effect. Results with endogenous mRNA further substantiate that initiation and not elongation is the site of action. These results are consistent with an inhibition due to a newly discovered, rapid reaction of methylglyoxal with the 7-methylguanosine of the cap structure.     

The number of globin gene sequences in "cytoplasmic" DNA fragments.

DNA was isolated from the cytoplasm of primary cultures of mouse foetal liver cells. The proportion of globin genes was determined by two methods of cDNA-DNA hybridisation in globin complementary DNA excess. The proportion was similar in ‘cytoplasmic’ DNA and nuclear DNA. This argues against an informational role for this class of ‘cytoplasmic’ DNA, which has all of the characteristics of nuclear DNA arising from nucleosomes derived from chromatin.     

Binding of globin mRNA, beta-globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein

The binding of rabbit globin mRNA, in-vitro-generated beta-globin mRNA segments, and RNA homopolymers by proteins of rabbit reticulocyte polysomal messenger ribonucleoproteins (mRNP) after SDS gel electrophoresis and electroblotting was examined. The polysomal mRNP proteins have a higher affinity for mRNA than for rRNA and tRNA while having a higher affinity for polypurine than polypyrimidine homopolymers. Binding experiments with synthetic poly(A) and with segments of beta-globin mRNA transcribed from a cDNA in vitro revealed a set of polysomal mRNP proteins which preferentially bind the poly(A)-free beta-globin mRNA. A protein of Mr 90,000 binds specifically the 3'-nontranslated trailer of the poly(A)-free beta-globin mRNA and not the poly(A)-containing globin mRNA. Another set of proteins preferentially binds poly(A). The latter group of proteins contains a prominent species of Mr 72,000, which is most likely the rabbit poly(A)-binding protein. Three polysomal mRNP proteins which bound rabbit globin mRNA did not bind preferentially any of the other RNA probes used.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.