scholarly journals The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA

1981 ◽  
Vol 193 (1) ◽  
pp. 209-216 ◽  
Author(s):  
S W Applebaum ◽  
T C James ◽  
D H Wreschner ◽  
J R Tata
Keyword(s):  
Messenger Rna ◽  
Fat Body ◽  
Abundant Species ◽  
Polyadenylated Rna ◽  
High Molecular Weight Species ◽  
Major Species ◽  
Reticulocyte Lysate ◽  
Sepharose 4B ◽  
Monospecific Antibodies

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 × 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 × 10(-3) mol . litre-1 . s. There are 9.1 × 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.

scholarly journals The preparation and characterization of vitellogenin messenger RNA from rainbow trout (Salmo gairdneri)

1984 ◽  
Vol 217 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Y Valotaire ◽  
M Tenniswood ◽  
C Le Guellec ◽  
J R Tata
Keyword(s):  
Rainbow Trout ◽  
Messenger Rna ◽  
Salmo Gairdneri ◽  
Agarose Gel Electrophoresis ◽  
Control Male ◽  
Polyadenylated Rna ◽  
Reticulocyte Lysate ◽  
And Control

Agarose-gel electrophoresis of polyadenylated RNA from livers of oestrogen-treated male rainbow trout revealed a major high-Mr species (7200 nucleotides), which is absent from the polyadenylated RNA isolated from hormonally unstimulated male trout liver. Translation in vitro of the RNA from oestrogen-treated males in a mRNA-dependent rabbit reticulocyte lysate produced a protein (Mr 200 000) that could be immunoprecipitated with antibodies against trout serum vitellogenin, but no immunoprecipitable protein was synthesized with RNA from control animals. DNA complementary to the RNA from oestrogen-stimulated and control male trout liver was synthesized and back-hybridized, with R0t1/2 of 3.8 × 10(-2) and 1 × 10(-1) mol X litre-1 X s for RNA from hormone-treated and control animals respectively. The 9% increase in the abundant mRNA after oestrogen stimulation is due to the induction of vitellogenin mRNA.

A Characterization of Amelogenin Messenger RNA in the Bovine Tooth Germ

1987 ◽  
Vol 1 (2) ◽  
pp. 289-292 ◽  
Author(s):  
M.F. Young ◽  
H.S. Shimokawa ◽  
M.E. Sobel ◽  
J.D. Termine
Keyword(s):  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Matrix Protein ◽  
Tooth Germ ◽  
Northern Analysis ◽  
Incisor Tooth ◽  
Major Species ◽  
Tooth Germs

In order to study the nature of amelogenin mRNA, we isolated ameloblast-rich tissue from the unerupted permanent incisor tooth germs of 18-month-old steers and subjected it to guanidine HC1 solubilization for extraction of mRNA. When poly A+ ameloblast RNA was incubated with radioactive deoxynucleotides and reverse transcriptase, four major transcripts were detected with sizes of 1.9, 1.4, 0.7, and 0.4 kb in length. One of the transcripts (0.7 kb) corresponded precisely in length to that predicted from the size of the major in vitro translated amelogenin proteins (27,000 daltons). To determine whether the transcripts did indeed encode amelogenin mRNA, we constructed a λgt11 cDNA library and isolated several amelogenin cDNA's by screening with amelogenin antibody. Four clones were amplified and insert sizes determined by acrylamide gel electrophoresis. Two of the clones had insert sizes of ~ 0.7 kb (λAm 16, XAm 7), and two had insert sizes of ~ 0.4 kb (λAm 11, λAm 4). When the amelogenin cDNA was radiolabeled and used for northern analysis, two species of amelogenin message (0.75 and 0.45 kb) were evident, both of which showed extensive hybridization to λAm 16 (large) and λAm 11 (small) cDNA. These data indicate that: (1) Amelogenin mRNA is heterogeneous in the bovine tooth germ, having two major species 800 and 400 bases long; and (2) the major species of amelogenin share extensive sequence homology. Based on these data, we suggest that at least part of the heterogeneity of amelogenin matrix protein may arise from the production of heterogeneous amelogenin mRNA's that share some common nucleotide sequences.

Protein synthesis in hyperoxic endothelial cells: evidence for translational defect

1987 ◽  
Vol 63 (2) ◽  
pp. 457-464 ◽  
Author(s):  
L. Jornot ◽  
M. E. Mirault ◽  
A. F. Junod
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Messenger Rna ◽  
Polypeptide Chain ◽  
Primary Cultures ◽  
Chain Elongation ◽  
Aortic Endothelial Cells ◽  
Polyadenylated Rna ◽  
Reticulocyte Lysate ◽  
Porcine Aortic Endothelial Cells

To study the effects of hyperoxia on protein synthesis in primary cultures of porcine aortic endothelial cells, we exposed confluent cells to different O2 concentrations for various durations. Exposure to 95% O2 for 5 days resulted in a 71% inhibition of [3H]phenylalanine incorporation into total proteins. When compared with control cells, we observed no changes in 1) the pool size of free cytoplasmic phenylalanine and of phenylalanine attached to transfer RNA (tRNA), 2) the rate of protein degradation, and 3) the rate of charging of tRNA with phenylalanine. We found that under hyperoxic conditions 1) the incorporation of [3H]-uridine into total and polyadenylated RNA was increased, 2) the efficiency of extracted messenger RNA to direct protein synthesis in a reticulocyte lysate was maintained, 3) the proportion of polymeric to monomeric ribosomes was slightly increased, and 4) the rate of elongation, as measured by the ribosomal transit time, was decreased. Thus the reduction in protein synthesis in hyperoxic cells appears to result primarily from defects at the translational level in polypeptide chain elongation.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

Characterization of striatal neurons expressing high levels of glutamic acid decar☐ylase messenger RNA

1989 ◽  
Vol 492 (1-2) ◽  
pp. 237-244 ◽  
Author(s):  
Marie-Francoise Chesselet ◽  
Elaine Robbins
Keyword(s):  
Glutamic Acid ◽  
Messenger Rna ◽  
Striatal Neurons
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