scholarly journals The nature of the 5'-linked 5' nucleotide sequence at the 5' end of rabbit globin messenger ribonucleic acid

1976 ◽  
Vol 155 (3) ◽  
pp. 637-644 ◽  
Author(s):  
J A Hunt ◽  
G N Oakes
Keyword(s):  
Messenger Rna ◽  
Periodate Oxidation ◽  
Hydroxyl Group ◽  
Globin Mrna ◽  
Pancreatic Ribonuclease ◽  
Phosphodiester Bond ◽  
Messenger Ribonucleic Acid ◽  
End Groups ◽  
A Charge ◽  
End Group

Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one of the 2′,3′ hydroxyl end-groups is linked through the normal 3′-5′ phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2′,3 hydroxyl group is revealed. This structure could be a 5′-linked 5′-phospho di- or tri-ester. On digestion with ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3′ end plus fragments that are not found after stepwise degradation. These fragments have a charge of -6 and -8 from pancreatic ribonuclease or -7 from ribonuclease T1 digestion. These charges are changed to -3.4 and -4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5′ sequences m7G(5′)ppp′AmpYpGp ... and m7G(5′)pppAmpApGpYp.

scholarly journals The Enzymatic Phosphorylation of Nucleic Acids and Its Application to End-Group Analysis

1966 ◽  
Vol 49 (6) ◽  
pp. 81-97 ◽  
Author(s):  
Charles C. Richardson ◽  
Bernard Weiss
Keyword(s):  
Molecular Weight ◽  
Group Analysis ◽  
Single Strand ◽  
Selective Labeling ◽  
Phosphodiester Bond ◽  
Polynucleotide Kinase ◽  
End Groups ◽  
Hydroxyl Termini ◽  
End Group

Polynucleotide kinase catalyzes the transfer of a phosphate group from ATP to the 5'-hydroxyl termini of polynucleotides. Selective labeling of the 5'-hydroxyl termini of DNA with polynucleotide kinase has been used to study the number and the identity of the 5'-terminal residues of bacteriophage DNA's, and to examine the nature of the phosphodiester bond cleavages produced by endonucleases and by sonic irradiation. The intact strands of T7 DNA bear 5'-phosphoryl end-groups; only deoxyadenylate and deoxythymidylate are present as 5'-terminal residues. The intact strands of native λ-DNA bear 5'-hydroxyl end-groups. M13 DNA, a circular molecule, cannot be phosphorylated. End-group labeling of DNA provides a method for determination of molecular weight; calibration against other DNA preparations is not required. The molecular weight of a single strand of T7 DNA, determined by end-group labeling, is 13.1 x 106; the molecular weight of a single strand of λ-DNA is 16.0 x 106. These values are in agreement with molecular weight estimates by sedimentation analysis and electron microscopy. Sonic irradiation of DNA has been shown to favor the production of polynucleotides terminated by 5'-phosphomonoester groups. All four deoxyribonucleotides are present as 5'-terminal residues of sonicated DNA.

scholarly journals Terminal sequence studies of high-molecular-weight ribonucleic acid. The 3′-termini of rabbit globin messenger ribonucleic acid

1973 ◽  
Vol 131 (2) ◽  
pp. 315-325 ◽  
Author(s):  
J. A. Hunt
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Ribonucleic Acid ◽  
Periodate Oxidation ◽  
Apparent Molecular Weight ◽  
Paper Electrophoresis ◽  
Terminal Sequence ◽  
Globin Mrna ◽  
Messenger Ribonucleic Acid ◽  
Oxidation Analysis

Haemoglobin mRNA isolated from EDTA-treated polyribosomes has an apparent molecular weight of 120000–180000 estimated by condensation with 3H-labelled isoniazid after periodate oxidation. Analysis of the ribonuclease digests of isoniazid-labelled RNA by paper electrophoresis and column chromatography enables the amount of contaminating 18S, 7S, 5S and 4S RNA to be estimated, and a corrected molecular weight of globin mRNA as the acid is 161000 or 500 nucleotides in length. This molecule contains two groups of 3′-terminal sequences in equal yield; G-Y-A6 and G-Y-A7 in the ratio 3:2, and G-N9–16-Y-A2 and G-N9–16-Y-N3 in the ratio 3:2. The significance of these sequences is discussed in relation to the poly(A) content of globin mRNA, the specificity of the sequences, and possible function in processing and biosynthesis of mRNA.

scholarly journals Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic Acid Levels in Vitro*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1224-1231 ◽  
Author(s):  
Ursula B. Kaiser ◽  
Andrzej Jakubowiak ◽  
Anna Steinberger ◽  
William W. Chin
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Pulse Frequency ◽  
Gnrh Receptor ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Subunit Gene ◽  
Differential Effects ◽  
Messenger Ribonucleic Acid

Abstract The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin α, LHβ, and FSHβ subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nm GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. α, LHβ, and FSHβ messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies. α and LHβ mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSHβ mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of α and LHβ but was different from that of FSHβ. This suggests that α and LHβ mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSHβ mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate α, LHβ, and GnRHR gene expression are similar, whereas the stimulation of FSHβ mRNA levels may be different.

scholarly journals The identification of globin messenger ribonucleic acid in newt erythropoietic cells

1979 ◽  
Vol 183 (1) ◽  
pp. 105-114
Author(s):  
J A Grasso ◽  
G P Casale
Keyword(s):  
Ionic Strength ◽  
Mrna Translation ◽  
High Ionic Strength ◽  
Globin Mrna ◽  
Triturus Cristatus ◽  
Messenger Ribonucleic Acid ◽  
Reticulocyte Lysate ◽  
Globin Synthesis ◽  
26S Rrna ◽  
Mrna Precursor

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.

scholarly journals Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells

Blood ◽  
1979 ◽  
Vol 54 (4) ◽  
pp. 933-939
Author(s):  
R Gambari ◽  
RA Rifkind ◽  
PA Marks
Keyword(s):  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Globin Mrna ◽  
Induced Differentiation ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Mrna Content ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.

scholarly journals Microbubbles Decorated with Dendronized Magnetic Nanoparticles for Biomedical Imaging. Effective Stabilization via Fluorous Interactions

2019 ◽  
Author(s):  
Da Shi ◽  
Justine Wallyn ◽  
Dinh-Vu Nguyen ◽  
Francis Perton ◽  
Delphine Felder-Flesch ◽  
...  
Keyword(s):  
Molar Ratio ◽  
Aqueous Dispersions ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Investigation ◽  
End Groups ◽  
Mixed Films ◽  
Effective Stabilization ◽  
Effective Result ◽  
End Group

Dendrons fitted with three oligoethylene glycol (OEG) chains, one of which carrying a fluorinated or hydrogenated end group, and bearing a bisphosphonate polar head (C n X2 n +1OEG8Den, X = F or H; n= 2 or 4) were synthesized and grafted on the surface of iron oxide nanoparticles (IONPs) for microbubble-mediated imaging and therapeutic purposes. The size and stability of the dendronized IONPs (IONP@C n X2 n +1OEG8Den) in aqueous dispersions were monitored by dynamic light scattering. Investigation of the spontaneous adsorption of IONP@C n X2 n +1OEG8Den at the interface between air - or air saturated with perfluorohexane - and an aqueous phase establishes that exposure to the fluorocarbon gas markedly increases the rate of adsorption of the dendronized IONPs to the gas/water interface and decreases the equilibrium interfacial tension. This suggests that fluorous interactions are at play between the supernatant fluorocarbon gas and the fluorinated end groups of the dendrons. Furthermore, small, stable perfluorohexane-stabilized microbubbles (MBs) with a dipalmitoylphosphatidylcholine (DPPC) shell that incorporates IONP@C n X2 n +1OEG8Den (DPPC/Fe molar ratio 28:1) were prepared and characterized using both optical microscopy and an acoustical method of size determination. The dendrons fitted with fluorinated end groups lead to smaller and more stable MBs than those fitted with hydrogenated groups. The most effective result is already obtained with C2F5, for which MBs, ~1.0mm in radius, reach a half-life of ~6.0 h. An atomic force microscopy investigation of spin-coated mixed films of DPPC/IONP@C2X5OEG8Den combinations (molar ratio 28:1) shows that the IONPs grafted with the fluorinated dendrons are located within the phospholipid film, while those grafted with the hydrocarbon dendrons are completely absent from the phospholipid film.

scholarly journals Chronic Exposure to High Glucose Concentrations Increases Proglucagon Messenger Ribonucleic Acid Levels and Glucagon Release from InR1G9 Cells1

Endocrinology ◽  
1999 ◽  
Vol 140 (10) ◽  
pp. 4644-4650 ◽  
Author(s):  
Eric Dumonteil ◽  
Beate Ritz-Laser ◽  
Chistophe Magnan ◽  
Iléana Grigorescu ◽  
Alain Ktorza ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Glucose ◽  
Messenger Rna ◽  
Cell Function ◽  
High Plasma ◽  
Glucagon Release ◽  
Cell Content ◽  
Messenger Ribonucleic Acid ◽  
Elevated Glucose ◽  
Rna Levels

Abstract α cell function is impaired in diabetes. In diabetics, plasma levels of glucagon are high despite persistently elevated glucose levels and may even rise paradoxically in response to a glucose load; high plasma glucagon levels are accompanied by increased proglucagon gene expression. We have investigated the effects of high glucose concentrations on InR1G9 cells, a glucagon-producing cell line. We show here that chronically elevated glucose concentrations increase glucagon release by 2.5- to 4-fold, glucagon cell content by 2.5- to 3-fold, and proglucagon messenger RNA levels by 4- to 8-fold, whereas changes for 24 h have no effect on proglucagon messenger RNA levels. Persistently elevated glucose affects proglucagon gene expression at the level of transcription and insulin is capable of preventing this effect. We conclude that chronically elevated glucose may be an important factor in the α cell dysfunction that occurs in diabetes and thus that glucose may not only affect the β cell but also the α cell.

scholarly journals Transforming Growth Factor-βs Inhibit Somatostatin Messenger Ribonucleic Acid Levels and Somatostatin Secretion in Hypothalamic Cells in Culture*

Endocrinology ◽  
1997 ◽  
Vol 138 (10) ◽  
pp. 4401-4409 ◽  
Author(s):  
M. Quintela ◽  
R. M. SeñarÍs ◽  
C. Diéguez
Keyword(s):  
Growth Factor ◽  
Messenger Rna ◽  
Transforming Growth Factor ◽  
Inhibitory Effect ◽  
Mrna Levels ◽  
Messenger Ribonucleic Acid ◽  
Cells In Culture ◽  
Somatostatin Secretion ◽  
Hypothalamic Cells ◽  
The Stability

Abstract Treatment of hypothalamic cells in monolayer culture with transforming growth factor-β1 (TGFβ1) significantly reduced both basal and cAMP-induced somatostatin messenger RNA (mRNA) levels and somatostatin secretion. This inhibitory effect was dose- and time-dependent and not mediated by glial cells, as it was also observed in glial-free hypothalamic cell cultures treated with cytosine arabinonucleoside. TGFβ2 and -β3 mimicked the actions of TGFβ1, which indicated that the three isoforms of the TGFβ family expressed in the central nervous system displayed similar effects on the somatostatinergic neurons. The blockade of synthesis of proteins with either cycloheximide or puromycin for 24 h prevented the inhibitory effect of TGFβ1 on somatostatin mRNA. This implied that the reduction of this mRNA by TGFβ1 required de novo protein synthesis. We next studied whether TGFβ1 acted at the transcriptional or posttranscriptional level by altering the stability of somatostatin mRNA. Examination of the rate of disappearance of somatostatin mRNA by Northern blot, after inhibition of mRNA transcription with either actinomycin D (AcD) or 5,6-dichloro-1β-ribofuranosyl benzimidazole revealed that TGFβ1 did reduce the stability of somatostatin mRNA. This effect was observed when we pretreated the cultures with TGFβ1 4 h before the addition of AcD, but not when we administered TGFβ1 simultaneously with AcD or 5,6-dichloro-1β-ribofuranosyl benzimidazole. Altogether these results demonstrated that the treatment of hypothalamic cells in culture with TGFβ1, TGFβ2, or TGFβ3 resulted in a decrease in somatostatin mRNA levels and somatostatin secretion. TGFβ1 reduced the steady state levels of somatostatin mRNA by inducing the synthesis of a protein (s), that appears to accelerate the degradation of the mRNA of somatostatin. Whether TGFβ1 has additional effects on the transcription of the somatostatin gene will require further study.

scholarly journals Structure of the endonuclease IV homologue fromThermotoga maritimain the presence of active-site divalent metal ions

2010 ◽  
Vol 66 (9) ◽  
pp. 1003-1012 ◽  
Author(s):  
Stephen J. Tomanicek ◽  
Ronny C. Hughes ◽  
Joseph D. Ng ◽  
Leighton Coates
Keyword(s):  
Metal Ions ◽  
Active Site ◽  
Excision Repair ◽  
Divalent Metal ◽  
Hydroxyl Group ◽  
Divalent Metal Ions ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Dna Base ◽  
Ap Site

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically,Thermotoga maritimaendonuclease IV is a member of the second conserved AP endonuclease family that includesEscherichia coliendonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of theT. maritimaendonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of theT. maritimaendonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.

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