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Biomedical subjects

S Penman

Publications and source records attributed to S Penman.

At least 109 records · Page 6Linked to original sources
PubMed1977

A cytoskeletal structure with associated polyribosomes obtained from HeLa cells.

A method is described by which HeLa cells can be fractionated to reveal a skeletal-like structure in the cytoplasm. This cytoskeleton has many of the cell's ultrastructural features, such as 100A filaments, microfilaments, centrioles, and microspikes, although most of the cellular protein, membranes, and microtubules have been extracted. Associated with the cytoskeleton are most of the polysomal, but not the monomeric, ribosomes. These polysomes are distributed throughout the cytoskeleton except in the region of the 100A filaments, which resembles the distribution in intact cells. Degradation of mRNA with low levels of ribonuclease releases most ribosomes from the cytoskeleton. Prior disaggregation of polyribosomes in vivo releases ribosomes but not mRNA. Cytochalasin B administered in vivo releases the mRNA from the cytoskeleton. These results suggest an attachment of polyribosomes to the cytoskeleton via mRNA.

Cell Fractionation↗
PubMed1977

The extensive homology between mRNA sequences of normal and SV40-transformed human fibroblasts.

The poly(A)-containing messenger RNA of normal diploid fibroblast and SV40-transformed progeny cells are compared by cross-hybridizing cDNA. We find a high degree of homology between the mRNA from normal and transformed cells. Despite imperfections in the procedure, the technique permits the conclusion that, at most, 3% of the mRNA in the transformed cell has sequences not present in the normal parental cell. Furthermore, much of the difference appears to occur in low and intermediate complexity classes of mRNA molecules. Extension homology in the mRNA sequences of disparate cell lines may be a general phenomenon, and even HeLa cell mRNA is nearly identical to that of diploid human fibroblasts.

Base Sequence↗
PubMed1977

HeLa cell poly(A)- mRNA codes for a subset of poly(A)+ mRNA-directed proteins with an actin as a major product.

Poly(A)+ and poly(A)- mRNA from HeLa cells were separated and translated in heterologous messenger-dependent protein synthesizing systems. Two-dimensional electrophoretic analysis revealed three classes of polypeptides. At the level of detectability in the electropherograms, a small number (about 10) of proteins were detected only among the poly(A)- mRNA products, a larger number (about 40) were produced by both poly(A)- and poly(A)+ mRNA, and a large number of polypeptides were found exclusively in the poly(A)+ mRNA products. The major product of both poly(A)+ and poly(A)- mRNA was shown to be the beta form of actin.Previous cross hybridization measurements suggested little homology between poly(A)+ and poly(A)- mRNA populations. In view of the apparent identity of many poly(A)- products with those of poly(A)+, the homology between poly(A)+ and poly(A)- mRNA sequences was examined in greater detail. cDNA complementary to only the most abundant poly(A)+ message sequences was prepared. About 10% of this cDNA hybridized to abundant sequences in the poly(A)- fraction. This corresponded to only 2% of the total mass of poly(A)+ mRNA and accounted for the failure to detect cross hybridization in previous experiments. Thus, a small number of poly(A)+ sequences appear to be present in relatively high concentration in poly(A)- mRNA as evidenced by both the translation products and the cross hybridization results.

Actins↗
PubMed1976

Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclear RNA of HeLa cells.

Highly purified steady state heterogeneous nuclear RNA from HeLa cells has been prepared by a new procedure. Detergent-washed nuclei are disrupted in 0.4 M ammonium sulfate, which also disociated contamination polysomes. The hnRNA remains bound to chromatin, which can be pelleted by gentle centrifugation. Ribonuclease inhibitors permit the preparation of very high molecular weight nuclear RNA. The hnRNA was cleaved with alkali. The poly(A)-containing fragments were separated from those containing oligo(A), and a cDNA copy was prepared. Hybridization of this nuclear cDNA to cytoplasmic mRNA showed that the scarce (complex) message sequences make up a larger proportion of nuclear RNA than of cytoplasmic RNA. In addition, at least 30% of the poly(A)-adjacent sequences in nuclear RNA have no apparent counterparts in the cytoplasm. cDNA prepared from hnRNA sedimenting faster than 45S under denaturing conditions gives similar results, showing the presence of both message and non-message sequences in very large transcripts. cDNA complementary to mRNA was separated into the abundant and scarce sequences, and hybridized separately to the poly(A)-adjacent sequences in nuclear RNA. The hybridization of the abundant sequence cDNA was used to set an upper limit on possible cytoplasmic contamination. Hybridization of the scarce cytoplasmic sequences are represented in nuclear RNA approximately once per cell.

Cell Nucleus↗
PubMed1976

Small RNA species of the HeLa cell: metabolism and subcellular localization.

The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the "nuclear skeleton." SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.

Cell Fractionation↗
PubMed1976

Poliovirus-induced inhibition of polypeptide initiation in vitro on native polyribosomes.

The inhibition of HeLa cell protein synthesis by poliovirus was studied by examining initiation in vitro on endogenous host polyribosomes. At an early stage, before major viral RNA replication and protein synthesis begins, the initiation of translation on cellular mRNA is strongly inhibited. Fractionation of extracts from infected cells shows that the lesion is associated mainly with the crude polyribosome fraction. The cellular mRNA appears unchanged and is as active as mRNA from control cells in stimulating incorporation. The native ribosomal subunits and KCl-washed polyribosomes from the infected cells are also active. Only the ribosomal wash fraction prepared from the inhibited polyribosomes had reduced activity. However, the reduction in the ribosomal wash activity measured in a reconstructed system is not as large as the inhibition seen with "native" polyribosomes. The results indicate that a viral induced inhibition is probably associated with the ribosomal wash fraction, but the reconstructed system is not equivalent to the "native" inhibited system.

Cell-Free System↗
PubMed1976

Changes in RNA in relation to growth of the fibroblast. IV. Alterations in theproduction and processing of mRNA and rRNA in resting and growing cells.

In previous reports, it was shown that both the concentration and rate of production of rRNA and mRNA were greater in growing than in resting 3T6 fibroblasts. Studies on isolated nuclei indicated that ribosomal RNA production is apparently controlled at the level of transcription. In contrast, hnRNA, the putative precursor of mRNA, appeared to be synthesized at the same rate in resting and growing cells. This finding was unexpected and has been tested in several ways. In this report, we show by an independent method that the relative rate of production of mRNA compared to hnRNA is several-fold higher in growing than in resting cells. However, the kinetics of processing of mRNA appear unchanged. This result suggests either that mRNA arises from a small subfraction of hnRNA or that the efficiency of processing of the hnRNA precursor is an important control mechanism which determines mRNA production ingrowing and resting states. Comparison of the initial rates of labeling of hnRNA and cytoplasmic message gives the efficiency with which the cytoplasmic mRNA is produced from nucleoplasmic RNA. The very low efficiency (3-4% in growing and 1-2% in resting cells) suggests that not every hnRNA molecule gives rise to a cytoplasmic message. In contrast to the similar kinetics of mRNA production in resting and growing states, processing of ribosomal RNA is much slower in the resting state and the emergence time for 28S RNA from nucleolus is greatly lengthened.

Cell Division↗
PubMed1975

Increasing content of poly A(+) mRNA of serum-stimulated cells in the absence of ribosome synthesis.

When 3T6 cells undergo a serum-induced transition from resting to growing state, the number of ribosomes and the amounts of mRNA increase as the cells prepare for DNA synthesis. We have examined the effect of preventing ribosome synthesis during this transition. When resting cells are stimulated to grow in the presence of 5-fluorouridine, mRNA accumulates normally during the first eight hours, though new ribosome formation is completely blocked by the drug. At later times, mRNA continues to accumulate, but at a reduced rate. The ratio of poly A(+) mRNA to rRNA increases from the value characteristic of resting 3T6 (1.8%) to that of growing 3T6 (2.7%) by five hours, and continues to increase to abnormally high values after this time. Although labelling of tRNA is not affected after brief exposure of cells to fluorouridine, the drug prevents the later accumulation of tRNA that ordinarily occurs following serum stimulation of resting cells. This failure of accumulation is not the result of increased lability of fluorinated tRNA, but is probably due to failure of the transcription rate of pre-tRNA to increase. It is possible that this effect might be due to a regulatory system coupling tRNA content to ribosome content. In cultures stimulated with serum in the presence of fluorouridine the rate of protein synthesis increases with poly A(+) mRNA content during the first eight hours; it then fails to increase further, possibly because ribosomes become rate-limiting.

Blood↗
PubMed1975

The messenger RNA sequences in growing and resting mouse fibroblasts.

The sequences present in messenger RNA in resting and growing 3T6 cells have been examined. First, the abundance and complexity classes of mRNA in growing 3T6 were compared to those in other established cell lines. The overall complexities measured for mRNA from HeLa cells and the three mouse fibroblast lines, 3T6, SV-PY-3T3, and L, are qualitatively similar and correspond to approximately 10,000 sequences. The relative amount of the two major abundance classes and their complexities appear identical in the three mouse fibroblast lines despite their different histories. HeLa cell mRNA is significantly different both in the amount and the complexity of the two major classes. The complexity of the two mRNA classes appears the same in resting and growing 3T6, although there is a small difference in relative amounts. Cross hybridizing cDNA and mRNA from resting and growing cells shows that the majority of mRNA sequences are the same in the two states. However, cross hybridization after the common sequences are removed shows that about 3% of the mRNA in resting cells is not present in the growing state, while the opposite cross shows 3% of the mRNA in growing cells is not present in resting cells. These differences may result from alterations in gene expression which are related to the growth state of the cell.

Base Sequence↗
PubMed1975

hnRNA size and processing as related to different DNA content in two dipterans: Drosophila and Aedes.

The size of hnRNA transcripts and the fraction of hnRNA that is converted to mRNA in cell lines of Drosophila melanogaster and Aedes albopictus are compared. Both insects belong to the order Diptera, but Aedes has a 5-6 fold larger genome than does Drosophila. The Aedes line produces significantly (2-2.5 fold) larger hnRNA than does the Drosophila line, even though the two cell lines grow under similar conditions and produce mRNA of the same size and sequence complexity. These data suggest that within a given taxonomic order, the size of hnRNA increases with increasing genome size. The fraction of hnRNA converted to mRNA [cytoplasmic poly(A)+ RNA] has been measured for the two cell types by comparing initial rates of labeling of hnRNA with initial rates of appearance of labeled mRNA in the cytoplasm. While 20of the Drosophila hnRNA is converted to mRNA, only 3.3% of the Aedes hn %RNA is converted to mrRNA. The poly(A) content of the hnRNA from the two species is also different; Drosophila hnRNA has approximately three times as much poly(A) as does Aedes hnRNA. The data show-at least for these two species-that the average amount of hnRNA transcribed relative to the amount of mRNA formed increases as genome size increases. The data are consistent with the interpretation that more DNA is transcribed into hnRNA in Aedes, the organism with the larger genome, than in Drosophila.

Adenine Nucleotides↗
PubMed1975

Two very different components of messenger RNA in an insect cell line.

The messenger RNA lifetimes have been measured in a cell line derived from an invertebrate source, the mosquito Aedes albopictus. The experiments were made possible by a new technique for obtaining undegraded cytoplasmic RNA from cells with high endogenous nuclease levels. There are two components to the decay kinetics of Aedes mRNA. The major fraction of the steady state message population has a half-life of 20 hr which is, as in mammalian cells, comparable to the cell generation time. The short-lived component turns over very rapidly with a half-life estimated to be about 1.2 hr. The difference in lifetime between the short and long-lived components is about 15 fold in these cells, compared to 3-4 fold in mammalian cells. This may reflect the need for a more responsive mRNA regulating system in poikilothermic organisms. The great disparity between the principle messenger lifetimes permits a more definite assignment of a two component behavior to message decay. The data in the case of mammalian cells could not rule out a family of intermediate lifetimes. The long-lived mRNA has a much smaller average sedimentation value than the short-lived material. The effect is similar to, but much larger than, that seen in mammalian cells. Although the lifetime difference is much greater in the insect cells than in human (HeLa) cells, the fast and slow components comprise about the same proportion of the steady state mRNA population: 30 percent and 70 percent, respectively.

Aedes↗
PubMed1975

Changes in RNA in relation to growth of the fibroblast. III. Posttranscriptional regulation of mRNA formation in resting and growing cells.

The proportion of hnRNA processed into cytoplasmic mRNA in resting and growing 3T3 and 3T6 cells has been investigated by measuring the efficiency of transfer of nuclear poly(A) to the cytoplasm. When nuclear poly(A) is either labeled continuously with precursor or pulse labeled and then chased with cordycepin, growing cells transfer approximately twice as much of the poly(A) from the nucleus to the cytoplasm as do resting cells. When cells undergo a serum-induced transition from the resting to growing state, the efficiency of poly(A) transfer is increased to that characteristic of growing cells by 3 hr after the addition of the serum. On the other hand, the proportion of hnRNA which is polyadenylated and the total nuclear poly(A) content are the same in resting and growing cells. It appears that the principal means by which the cell regulates its poly(A)+ mRNA content in the growing and resting states is by regulating the efficiency which nuclear poly(A)+ RNA is converted to cytoplasmic mRNA.

Adenine↗
PubMed1975

Analysis of drosophila mRNA by in situ hybridization: sequences transcribed in normal and heat shocked cultured cells.

Messenger RNA transcribed in cultured Drosophila cells adapted for growth under conditions permitting labeling to high specific acitivty has been analyzed by the technique of in situ hybridization. Poly(A)-containing cytoplasmic RNA binds specifically and reproducibly to about 50 bands in the salivary gland polytene chromosomes. In addition heavy labeling of the beta-heterochromatin associated with each of the chromosome arms is observed. The species which are detected probably belong to the more abundant classes of RNA. When the cultured Drosophila cells are subjected to heat shock immediately before labeling with 3H-uridine, there is a drastic alteration in the pattern of gene transcription detected by in situ hybridization. Most of the mRNA synthesis which could be detected in the normal cell is shut off. Newly synthesized RNA hybridizes strongly to seven new sites which do not bind mRNA from control cells. The new loci correspond almost exactly to the regions of Drosophila polytene chromosomes which puff when intact larvae are subjected to an identical heat treatment.

Animals↗