RNA transcription and translation in the hearts of normal and cardiomyopathic Syrian hamsters

1991 ◽  
Vol 69 (1) ◽  
pp. 88-92 ◽  
Author(s):  
J. D. McCully ◽  
J. D. Mably ◽  
M. J. Sole ◽  
C. C. Liew
Keyword(s):  
Syrian Hamster ◽  
Myocardial Cell ◽  
In Vitro Translation ◽  
Rna Transcription ◽  
Syrian Hamsters ◽  
Rna Translation ◽  
Nuclear Rna ◽  
Reticulocyte Lysate ◽  
Recessive Defect

The cardiomyopathic Syrian hamster has an autosomal recessive defect that results in the development of an early onset cardiac myopathy leading to cardiac dysfunction and, eventually, complete heart failure. To assess the regulatory mechanisms modulating gene expression in the normal and myopathic myocardium, we investigated both RNA transcription and translation. Our results indicated that the incorporation of [3H]UMP into myocardial cell nuclear RNA decreased 10-fold from 7 to 210 days of age in the normal Syrian hamster. The incorporation of [3H]UMP was approximately 50% lower in the cardiomyopathic as compared with the normal Syrian hamster. RNA translation, as assessed by rabbit reticulocyte lysate in vitro translation, indicated that a coordinated 50% decrease in RNA translation occurred in the normal Syrian hamster from 7 to 210 days of age. A further reduction of 20% in translation was found in cardiomyopathic Syrian hamster ventricular RNA translation as compared with matched random bred control groups. Two-dimensional polyacrylamide gel analysis of cell-free translated protein products demonstrated two myocardial peptides that were found to be consistently altered when the normal and cardiomyopathic Syrian hamsters were compared. These results indicate that transcription and translation decrease with age and that these processes are further downregulated, in an additive manner, with the genesis of the disease process.Key words: cardiomyopathy, ribonucleic acid, transcription, translation.

scholarly journals RNA transcription in myocardial-cell nuclei during postnatal development. A study establishing an assay system for transcription in vitro

1988 ◽  
Vol 256 (2) ◽  
pp. 441-445 ◽  
Author(s):  
J D McCully ◽  
C C Liew
Keyword(s):  
Gene Expression ◽  
Rna Synthesis ◽  
Myocardial Cell ◽  
Assay System ◽  
Specific Gene ◽  
Cell Nuclei ◽  
Rna Transcription ◽  
Nuclear Rna ◽  
Transcription In Vitro

A system for RNA transcription in vitro was established in order to determine the relative rate of RNA synthesis in neonatal and adult rat myocardial cells. This assay system optimizes the incorporation of [3H]UMP into RNA by using 3.5 x 10(7) myocardial-cell nuclei, and minimizes RNA degradation for at least 1 h in transcription in vitro, by the addition of human placental RNAase inhibitor. A 100% increase in the incorporation of [3H]UMP into myocardial-cell RNA was found on addition of this inhibitor. Myocardial-cell nuclei derived from 5-, 10-, 15-, 20-, and greater than 100-day-old rat hearts indicated that there is a progressive decrease in RNA synthesis with age. A 3-fold increase in RNA synthesis in 5-day-old myocardial cell nuclei as compared with 20-day-old rat heart was found. RNA synthesis in the adult myocardial cell nuclei decreased more than 10-fold in comparison with the 5-day-old newborn. The incorporation of [3H]UMP into rat liver nuclear RNA was 3-fold greater than in the myocardial-cell nuclear RNA, even when compared with the highly active transcription of 12-day-old heart nuclei. In order to determine the relationship between total RNA synthesis and the extent of specific gene expression in myocardial-cell nuclei during development, two distinct cDNA probes were used for Northern-blot analysis. Our results indicate that myosin-heavy-chain gene expression is remarkably decreased with age, whereas the ‘housekeeping’ gene is continually expressed independently of age.

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes

1987 ◽  
Vol 7 (5) ◽  
pp. 1848-1855
Author(s):  
G M Small ◽  
T Imanaka ◽  
H Shio ◽  
P B Lazarow
Keyword(s):  
Candida Tropicalis ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Moderate Number ◽  
Brij 35 ◽  
Reticulocyte Lysate ◽  
Molecular Information ◽  
Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

scholarly journals U1 small nuclear ribonucleoprotein studied by in vitro assembly.

1983 ◽  
Vol 96 (6) ◽  
pp. 1751-1755 ◽  
Author(s):  
E D Wieben ◽  
S J Madore ◽  
T Pederson
Keyword(s):  
Direct Analysis ◽  
Small Nuclear Rna ◽  
In Vitro System ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
U1 Snrnp ◽  
Nuclear Rna ◽  
Specific Complex ◽  
Reticulocyte Lysate

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

scholarly journals Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system.

1988 ◽  
Vol 107 (2) ◽  
pp. 587-596 ◽  
Author(s):  
M Bouché ◽  
S M Goldfine ◽  
D A Fischman
Keyword(s):  
In Vitro Translation ◽  
Homologous Proteins ◽  
Free System ◽  
Cell Free System ◽  
Reticulocyte Lysate ◽  
Thin Filament Proteins ◽  
A Cell ◽  
The Many ◽  
Kinetic Equilibrium

The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool.

IN VITRO LEUCOCYTE CULTURES OF SYRIAN AND CHINESE HAMSTERS

1972 ◽  
Vol 14 (1) ◽  
pp. 71-76 ◽  
Author(s):  
S. Szajkowski ◽  
M. Ray ◽  
K. L. Moore
Keyword(s):  
Syrian Hamster ◽  
Calf Serum ◽  
Chinese Hamster ◽  
Culture Conditions ◽  
Syrian Hamsters ◽  
Chinese Hamsters ◽  
Mitotic Figures ◽  
Separating Agents ◽  
Made In

An investigation of a variety of different separation procedures and culture conditions was made in order to evaluate the methods for culturing leucocytes in vitro of Syrian and Chinese hamsters. Dextran (6%), human AB serum and fetal calf serum (FCS) were used as separating agents. In the Chinese hamster the best results were obtained with AB serum whereas in the Syrian hamster adequate separation was obtained with all three agents. Supplementation of the culture medium with FCS provided the most favourable conditions for cell survival in culture. Maximal numbers of mitoses were obtained after 4 days of culture at 37 °C with the Syrian hamster (0.5%) and after 5 days with Chinese hamster (0.2%). Leucocytes of Syrian and Chinese hamsters were also cultured following immunization with each of the ABO sera as well as with FCS. Both macro- and micromethods were partially successful. Using the macromethod, no mitoses were observed in the Chinese hamster, but a few in Syrian hamsters immunized with B serum. With the micromethod, mitotic figures were observed in Syrian hamsters immunized with B serum, O serum and FCS, and Chinese hamsters with O serum and FCS. Leucocytes of the Chinese hamster proved difficult to culture successfully by any of the methods used, whereas, with the Syrian hamster, immunization with FCS and culture using micromethod gave the best results.

scholarly journals Ribosome shunting in the cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems

2000 ◽  
Vol 14 (7) ◽  
pp. 817-829
Author(s):  
Lyubov A. Ryabova ◽  
Thomas Hohn
Keyword(s):  
Mosaic Virus ◽  
Landing Site ◽  
Cauliflower Mosaic Virus ◽  
In Vitro Translation ◽  
Wheat Germ Extract ◽  
Reticulocyte Lysate ◽  
Translation Systems ◽  
Small Orfs ◽  
Special Case

The shunt model predicts that small ORFs (sORFs) within the cauliflower mosaic virus (CaMV) 35S RNA leader and downstream ORF VII are translated by different mechanisms, that is, scanning–reinitiation and shunting, respectively. Wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) in vitro translation systems were used to discriminate between these two processes and to study the mechanism of ribosomal shunt. In both systems, expression downstream of the leader occurred via ribosomal shunt under the control of a stable stem and a small ORF preceding it. Shunting ribosomes were also able to initiate quite efficiently at non-AUG start codons just downstream of the shunt landing site in WGE but not in RRL. The short sORF MAGDIS from the mammalian AdoMetDC RNA, which conditionally suppresses reinitiation at a downstream ORF, prevented shunting if placed at the position of sORF A, the 5′-proximal ORF of the CaMV leader. We have demonstrated directly that sORF A is translated and that proper termination of translation at the 5′-proximal ORF is absolutely required for both shunting and linear ribosome migration. These findings strongly indicate that shunting is a special case of reinitiation.

scholarly journals Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

1987 ◽  
Vol 7 (5) ◽  
pp. 1848-1855 ◽  
Author(s):  
G M Small ◽  
T Imanaka ◽  
H Shio ◽  
P B Lazarow
Keyword(s):  
Candida Tropicalis ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Moderate Number ◽  
Brij 35 ◽  
Reticulocyte Lysate ◽  
Molecular Information ◽  
Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

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