The metabolism of a poly(A) minus mRNA fraction in HeLa cells

Milcarek, Christine; Price, Richard; Penman, Sheldon

doi:10.1016/0092-8674(74)90030-0

Cited by 291 publications

(127 citation statements)

References 28 publications

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“…The hybridization of polysomal poly(A)+-mRNA to 3 H-labeled single-copy DNA indicates a complexity which is about 75% of that observed for total polysomal RNA (i.e., 1.2 × 107 nucleotides, or 8900 different mRNAs). Thus, the mRNA population in T. cruzi is similar to that of several higher eukaryotic organisms [6,15,16,[26][27][28] in that it exhibits a distinct population of nonadenylated mRNAs (ca. 3000) which is not represented in the poly(A)+-mRNA class.…”

Section: Discussionmentioning

confidence: 99%

Complexity and content of the DNA and RNA in Trypanosoma cruzi

Lanar

Lévy

Manning

1981

Molecular and Biochemical Parasitology

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The content and sequence complexity of the nuclear DNA and messenger RNA for epimastigotes of Trypanosoma cruzi were determined. From analysis of nuclear DNA reassociation studies and microspectrofluorometric measurements of laser induced fluorescence of cellular DNA, T. cruzi is found to be a diploid organism with a nuclear DNA content of 2.5 x 108 nucleotide pairs (2.8 X 10 -~3 g) and a kinetoplast DNA content of 4.9 X 107 nucleotide pairs (5.4 X 10 -t4 g). Reassoeiation kinetics of nuclear DNA of average length 0.4 kb reveals three kinetic components: a moderately repetitive component with a reiteration frequency of 5.1 X 103 present in 9% of the fragments, a lowly repetitive component with a reiteration frequency of 32 present in 51% of the fragments, and a single-copy component present in 23% of the fragments.By saturation hybridization of total polysomal RNA to 3H-labeled single-copy DNA, it was determined that 68% of the single-copy DNA was represented in the epimastigote polysomal RNA. This corresponds to ca. 12 000 different mRNA species. Of these, ca. 9000 are present as poly(A)÷-RNA, while the remaining 3000 appear not to be polyadenylated. Kinetic analysis of the poly(A)+-RNA population indicates it is composed of at least three classes of RNAs of different abundancy levels: two sequences which occur ca. 3000 times per cell, ca. 750 sequences which occur about 20 times per cell, and ca. 15 500 sequences which occur 1-2 times per cell.

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Section: Discussionmentioning

confidence: 99%

Complexity and content of the DNA and RNA in Trypanosoma cruzi

Lanar

Lévy

Manning

1981

Molecular and Biochemical Parasitology

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“…Post-mitochondrial supernatant from the same number of control cells and HeLa cells infected for 2 hr were layered over sucrose-sodium dodecyl sulfate gradients. After centrifugation the amount of poly(A)-containing RNA was measured by hybridization with radioactive poly(U) (10 This system has been used to measure the activity of purified mRNA. Fig.…”

Section: Methodsmentioning

confidence: 99%

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

Kaufmann¹,

Goldstein²,

Penman³

1976

Proc. Natl. Acad. Sci. U.S.A.

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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 eontrol cells in stimulating incorporation. The native ribosomal subunits and KCI-washed polyribosomes from the infected cells are also active. Only the ribosomal wash fraction prepared from the inhibited polyribosomes has 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. The mechanism by which some infecting viruses effect a selective inhibition of the translation of host cell messenger RNA is still an unsolved problem. One of the most extensively studied examples occurs during poliovirus infection, where host cell protein synthesis is inhibited relatively early in infection, prior to the appearance of appreciable amounts of viral RNA (1). Complete inhibition of host protein synthesis occurs even in the presence of guanidine (1-3), which inhibits viral RNA replication (4,5 (S-200) fractions by centrifugation in a Spinco angle 50 rotor for 50 min at 50,000 rpm. Two-milliliter microtubes, half-filled, were used. These conditions pelleted all polyribosomes mRNA and ribosomal subunits. The polyribosomal pellet was resuspended in standard sucrose solution (0.25 M sucrose, 1 mM dithiothreitol, 0.2 mM EDTA) or in lysing buffer [10 mM K-4-(2-hydroxyethyl)-1-piperazineethanesulfonate (Hepes) (pH 7.1), 0.7 mM Mg acetate, 75 mM KCl, 6 mM 2-mercaptoethanol]. For preparation of salt-washed polyribosomes, 4 M KCl was added dropwise to the polyribosomal suspension to a final concentration of 0.5 M KC1. After shaking for 20 min at 40 the suspension of ribosomes (0.2-0.4 ml) was centrifuged as above. The soluble fraction, used as a ribosomal wash fraction, was dialyzed for 4 hr against 500 volumes of ribosomal wash buffer [5 mM Tris-HCl (pH 7.4), 100 mM KCl, 0.05 mM EDTA, 5 mM 2-mercaptoethanol]. The salt-washed polyribosomal pellet was rinsed and resuspended in the standard sucrose solution. To separate the polyribosomes from the ribosomal subunits, we used a different fractionation procedure. Post-mitochondrial supernatant was centrifuged in 0.7-ml microtubes of a Spinco SW50 rotor for 20 min at 50,000 rpm. These conditions pelleted 85-90% of the polyribosomes and monosomes, whereas 60-0% of the 40S ribosomal subunits were left in the supernatant. Post-mitochondrial supernatant, S-200

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“…Excess poly(A)-containing RNA at concentrations described in the figure legends was mixed with sufficient cDNA to give about 1000 counts/min per sample and overlaid with paraffin oil [19]. Portions of 5-10 pl were removed at intervals ranging from 5 min to 24 h and pipetted into 1.1 ml of S1 nuclease buffer [20] which was subsequently divided into duplicate portions (0.5 ml). To one was added S1 nuclease to hydrolyse unhybridized cDNA and then both were incubated at 46 "C for 2 h. The samples were cooled in ice and adjusted to contain 10 pg/ml bovine serum albumin and 5 "/, trichloroacetic acid.…”

Section: The Hybridization Of Poly(a)-containing Rna With Cdnamentioning

confidence: 99%

The Androgenic Regulation of Abundant mRNA in Rat Ventral Prostate

Parker

Scrace

1978

European Journal of Biochemistry

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The most abundant poly(A)-containing RNA accounting for approximately half the total is regulated by testosterone in rat ventral prostate. A complementary DNA probe to these sequences was isolated by hybridizing the total cellular poly(A)-containing RNA with its complementary DNA to an rotxi, value of 2 x mol I-' s and separating the abundant cDNA from the nonabundant cDNA by hydroxyapatite chromatography. The abundant cDNA, complementary to about three different poly(A)-containing RNA sequences comprising 45 % of the total poly(A)-containing RNA, has been used to investigate the effect of androgens on their metabolism.Following castration there is a progressive decrease in the abundant sequences from 45% of the total in normal animals to less than 0.03% after 14 days; testosterone administration in vivo results in their regeneration. The sequences were not detected in seminal vesicle, liver, heart or spleen but represented about 0.01 % of the poly(A)-containing RNA of kidney.Translation of the poly(A)-containing RNA in vitro in a cell-free system derived from wheat germ resulted in the synthesis of four major proteins which could be separated by polyacrylamide gel electrophoresis. Their synthesis, as measured by [35S]methionine incorporation, accounted for 40 -50 % of the total incorporation and declined after castration but was restored by testosterone treatment. We conclude that a class of abundant poly(A)-containing RNA which codes for four major proteins is regulated by testosterone in rat ventral prostate.The regulation of protein synthesis by steroids is thought to be due to hormonal effects primarily on specific mRNA concentration [l -31. The rat ventral prostate is dependent for its growth and maintenance on androgenic steroids and has been used extensively to study the molecular mechanism of androgen action [4 -71. Much is known about the metabolism of testosterone and its interaction with a cytoplasmic receptor [8 -111 but studies of subsequent events have been hindered by the failure to detect suitable protein markers induced by the hormone.Recently we have approached this problem by examining the effects of androgens on the complexity of prostatic poly(A)-containing RNA [ 121. We demonstrated that prostatic RNA comprised three distinct classes of RNA of different relative abundance and testosterone appeared to have a marked effect on the most abundant class. In order to study the hormonal regulation of this class in more detail we have isolated a cDNA probe specific for the most abundant species of poly(A)-containing RNA. Hybridization of total rH]cDNA with total poly(A)-containing RNA to an rot,,2 value of 2 x mol 1-' s results in the hybridization of the most abundant cDNA whereas the moderate and least abundant species of cDNA will be largely unhybridized. The hybridized and unhybridized cDNA can then be separated by hydroxyapatite chromatography. The abundant cDNA was subsequently used as a probe to investigate the androgenic regulation of the abundant poly(A)-containing RNA sequences.In additio...

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The metabolism of a poly(A) minus mRNA fraction in HeLa cells

Cited by 291 publications

References 28 publications

Complexity and content of the DNA and RNA in Trypanosoma cruzi

Complexity and content of the DNA and RNA in Trypanosoma cruzi

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

The Androgenic Regulation of Abundant mRNA in Rat Ventral Prostate

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