Comparison of complexity and diversity of polyadenylated polysomal and informosomal messenger ribonucleic acid from Chinese hamster cells

Biochemistry ◽  
1979 ◽  
Vol 18 (19) ◽  
pp. 4254-4261 ◽  
Author(s):  
Ronald A. Walters ◽  
Paul M. Yandell ◽  
M. Duane Enger
Keyword(s):  
Ribonucleic Acid ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
Messenger Ribonucleic Acid

scholarly journals Large heterogeneous nuclear ribonucleic acid has three times as many 5' caps as polyadenylic acid segments, and most caps do not enter polyribosomes.

1981 ◽  
Vol 1 (2) ◽  
pp. 179-187 ◽  
Author(s):  
M Salditt-Georgieff ◽  
M M Harpold ◽  
M C Wilson ◽  
J E Darnell
Keyword(s):  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Turnover Rate ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
Rna Molecules ◽  
Rapid Turnover ◽  
Polyadenylic Acid ◽  
Nuclear Rna ◽  
Unstable Molecules

The rate of synthesis in Chinese hamster cells of 5' cap structures, m7 GpppNmp, in large (greater than 700 bases) heterogeneous nuclear ribonucleic acid (RNA) molecules is two to three times faster than the synthesis of 3'-terminal polyadenylic acid segments. As judged by presence of caps, newly synthesized polysomal messenger RNA, exclusive of messenger RNA the size of histone messenger RNA, is more than 90% in the polyadenylated category. It appears, therefore, that between half and two-thirds of the long capped heterogeneous nuclear RNA molecules do not contribute a capped polysomal derivative to the cytoplasm. There are capped, nonpolysomal, non-polyadenylated molecules with a rapid turnover rate that fractionate with the cytoplasm. These metabolically unstable molecules either could represent leakage into the cytoplasm during fractionation or could truly spend a brief time in the cytoplasm before decay.

Chinese hamster polyadenylated messenger ribonucleic acid: relationship to non-polyadenylated sequences and relative conservation during messenger ribonucleic acid processing

1981 ◽  
Vol 1 (2) ◽  
pp. 188-198
Author(s):  
M M Harpold ◽  
M C Wilson ◽  
J E Darnell
Keyword(s):  
Cho Cells ◽  
Ribonucleic Acid ◽  
Half Life ◽  
Large Fraction ◽  
Chinese Hamster ◽  
Great Majority ◽  
Molecular Probes ◽  
Processing Efficiency ◽  
Messenger Ribonucleic Acid ◽  
Nuclear Processing

We have further analyzed the metabolism of specific messenger ribonucleic acid (mRNA) sequences within the cytoplasmic and nuclear RNA of Chinese hamster ovary (CHO) cells by using a set of previously constructed complementary deoxyribonucleic acid (DNA) clones (Harpold et al., Cell 17:1025-1035, 1979) as specific molecular probes in a variety of RNA:DNA hybridization experiments. The majority of the labeled mRNA complementary to each of the nine clones was found in the polyribosomes, with some variation between individual sequences. The great majority of each specific mRNA labeled for 3 h or less was in the polyadenylated [poly(A)+] fraction. However, the amount of each sequence increased in the non-poly(A)+ [poly(A)-] fraction after very long label times, suggesting the derivation of the poly(A)- RNA from the poly(A)+ RNA. Eight of the nine mRNA's have cytoplasmic half-lives ranging from 8 to 14 h, whereas one of the mRNA's, the scarcest in the group, has a somewhat shorter half-life of approximately 3 h. The proportion of each of the specific long-lived mRNA's within the total labeled mRNA increased as a function of labeling time, indicating that a large fraction, probably greater than 50%, of the initially labeled poly(A)+ mRNA in CHO cells has a half-life of less than 3 h. A quantitative analysis of the kinetics of labeling of specific nuclear and cytoplasmic sequences indicated that a significant fraction of the mRNA sequences transcribed from genes containing these nine CHO sequences were successfully processed into mRNA. However, two of the CHO mRNA sequences were only partially conserved during nuclear processing to yield mRNA. These studies demonstrated that events at two post-transcriptional levels, differential nuclear processing efficiency of different primary transcripts and cytoplasmic stability of different mRNA's, can be involved in the determination of the cytoplasmic concentrations of different mRNA's.

scholarly journals Chinese hamster polyadenylated messenger ribonucleic acid: relationship to non-polyadenylated sequences and relative conservation during messenger ribonucleic acid processing.

1981 ◽  
Vol 1 (2) ◽  
pp. 188-198 ◽  
Author(s):  
M M Harpold ◽  
M C Wilson ◽  
J E Darnell
Keyword(s):  
Cho Cells ◽  
Ribonucleic Acid ◽  
Half Life ◽  
Large Fraction ◽  
Chinese Hamster ◽  
Great Majority ◽  
Molecular Probes ◽  
Processing Efficiency ◽  
Messenger Ribonucleic Acid ◽  
Nuclear Processing

We have further analyzed the metabolism of specific messenger ribonucleic acid (mRNA) sequences within the cytoplasmic and nuclear RNA of Chinese hamster ovary (CHO) cells by using a set of previously constructed complementary deoxyribonucleic acid (DNA) clones (Harpold et al., Cell 17:1025-1035, 1979) as specific molecular probes in a variety of RNA:DNA hybridization experiments. The majority of the labeled mRNA complementary to each of the nine clones was found in the polyribosomes, with some variation between individual sequences. The great majority of each specific mRNA labeled for 3 h or less was in the polyadenylated [poly(A)+] fraction. However, the amount of each sequence increased in the non-poly(A)+ [poly(A)-] fraction after very long label times, suggesting the derivation of the poly(A)- RNA from the poly(A)+ RNA. Eight of the nine mRNA's have cytoplasmic half-lives ranging from 8 to 14 h, whereas one of the mRNA's, the scarcest in the group, has a somewhat shorter half-life of approximately 3 h. The proportion of each of the specific long-lived mRNA's within the total labeled mRNA increased as a function of labeling time, indicating that a large fraction, probably greater than 50%, of the initially labeled poly(A)+ mRNA in CHO cells has a half-life of less than 3 h. A quantitative analysis of the kinetics of labeling of specific nuclear and cytoplasmic sequences indicated that a significant fraction of the mRNA sequences transcribed from genes containing these nine CHO sequences were successfully processed into mRNA. However, two of the CHO mRNA sequences were only partially conserved during nuclear processing to yield mRNA. These studies demonstrated that events at two post-transcriptional levels, differential nuclear processing efficiency of different primary transcripts and cytoplasmic stability of different mRNA's, can be involved in the determination of the cytoplasmic concentrations of different mRNA's.

Large heterogeneous nuclear ribonucleic acid has three times as many 5' caps as polyadenylic acid segments, and most caps do not enter polyribosomes

1981 ◽  
Vol 1 (2) ◽  
pp. 179-187
Author(s):  
M Salditt-Georgieff ◽  
M M Harpold ◽  
M C Wilson ◽  
J E Darnell
Keyword(s):  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Turnover Rate ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
Rna Molecules ◽  
Rapid Turnover ◽  
Polyadenylic Acid ◽  
Nuclear Rna ◽  
Unstable Molecules

The rate of synthesis in Chinese hamster cells of 5' cap structures, m7 GpppNmp, in large (greater than 700 bases) heterogeneous nuclear ribonucleic acid (RNA) molecules is two to three times faster than the synthesis of 3'-terminal polyadenylic acid segments. As judged by presence of caps, newly synthesized polysomal messenger RNA, exclusive of messenger RNA the size of histone messenger RNA, is more than 90% in the polyadenylated category. It appears, therefore, that between half and two-thirds of the long capped heterogeneous nuclear RNA molecules do not contribute a capped polysomal derivative to the cytoplasm. There are capped, nonpolysomal, non-polyadenylated molecules with a rapid turnover rate that fractionate with the cytoplasm. These metabolically unstable molecules either could represent leakage into the cytoplasm during fractionation or could truly spend a brief time in the cytoplasm before decay.

scholarly journals Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

1989 ◽  
Vol 9 (4) ◽  
pp. 1635-1641 ◽  
Author(s):  
S G Grant ◽  
R G Worton
Keyword(s):  
Cell Lines ◽  
X Chromosome ◽  
Gene Activation ◽  
Chinese Hamster ◽  
Fibroblast Cell ◽  
Dna Demethylation ◽  
Hprt Gene ◽  
Primary Fibroblast ◽  
Chinese Hamster Cells ◽  
Inactive X Chromosome

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.

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