Isolation and identification of separated messenger RNAs for rabbit alpha and beta globin.

Rabbit globin messenger RNA was separated into two species by polyacrylamide gel electrophoresis in formamide. The two species were isolated from the gel and assayed for messenger activity in the ascites cell-free system. The product of the cell-free system was analysed by column chromatography and by finger-printing. The RNA species with the lower mobility (mol. wt 227,000) codes mainly for β globin, whilst the RNA with the higher mobility (mol. wt 202,000) codes mainly for α globin. Fingerprint analysis of DNA copies of the separated mRNA species may be distinguished, and suggest that the polynucleotide sequences are of homogeneity comparable to the messenger activity. We conclude therefore that a physical separation of the two messenger species has been obtained.     

Polypeptide synthesis directed by DNA as a messenger in cell-free polypeptide synthesis by extreme thermophiles,Thermus thermophilus HB27 and Sulfolobus tokodaii strain 7

Polypeptide synthesis at high temperature directed by single strand DNA as a messenger was investigated using cell-free extracts of an extremely thermophilic bacterium, Thermus thermophilus strain HB27, and a hyperthermophilic, acidophilic archaeon, Sulfolobus tokodaii strain 7. Aminoglycoside antibiotics enhanced the reaction; neomycin stimulated it most effectively when the extract of the thermophilic bacterium was used, and paromomycin was the best among the antibiotics tested for the extract of the hyperthermophilic archaeon. A common correlation was found between the stimulation of DNA-directed polypeptide synthesis and the misreading rate in RNA-directed polypeptide synthesis. Spermine stimulated the reaction directed by DNA like in the case of poly(Phe) synthesis directed by poly (rU). The cell-free systems can be used for direct production of proteins from genes in high throughput studies on the structural genomics of thermophilus.     

Reiteration frequency of the gene coding for the small subunit of ribulose--1,5--bisphosphate carboxylase.

The messenger RNA coding for the precursor of the small subunit of ribulose--1,5--bisphosphate (Ru-P2) carboxylase has been partially purified from pea leaves. The mRNA has a size of 11.5S, is approximately 800 nucleotides long and, on cell-free translation, directs the synthesis of a single major polypeptide of 20,000 daltons. Antiserum for the small subunit of Ru-P2 carboxylase immunoprecipitated 52% of the 35S--methionine-labeled cell-free translation products. The RNA hybridizes with pea DNA with monophasic kinetics corresponding to the presence of one, or very few, gene copies per haploid genome.     

Induction of DNA replication-mediated double strand breaks by psoralen DNA interstrand cross-links

The effect of DNA interstrand cross-links (cross-links) on DNA replication was examined with a cell-free SV40 origin-dependent DNA replication system. A defined template DNA with a single psoralen cross-link and the SV40 origin of replication was replicated by HeLa cell-free extract in the presence of SV40 large T antigen. The psoralen cross-link inhibited DNA replication by terminating chain elongation at 1-50 nucleotides before the cross-linked sites. The termination of DNA replication by the cross-links mediated the generation of double strand breaks near the cross-linked sites. These results are the first biochemical evidence of the generation of double strand breaks by DNA replication.     

Immunochemical Isolation of γ-Globulin mRNA and Estimation of Immunoglobulin Gene Reiteration

The polyribosomes synthesizing γ-globulin have been isolated by the achievement of specific precipitation using bentonite-treated anti-IgG antibody. The RNA extracted from the immunochemically precipitated polysomes was tested for its ability to direct the synthesis of proteins in a cell-free system. The specific γ-globulin-synthesizing activity (cpm of γ-globulin synthesized/μg RNA) of this RNA was 10-fold greater than that from total polysomes. γ-globulin mRNA (messenger RNA) isolated by immunoprecipitation was more than 89% pure with respect to contamination by other species of mRNA. The products synthesized by the cell-free system were also analyzed by sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis. This RNA has been hybridized with mouse myeloma DNA. The estimation of immunoglobulin gene reiteration was carried out using hybridization kinetics with consideration given to the DNA/RNA ratio since the estimation from the “half Cot value” is not accurate. The results suggest that in the mouse there are about 20 copies per subgroup of genes coding for the variable region of the H and L chains.     

Transport of functional messenger RNA from liver nuclei in a reconstituted cell-free system

The reliability of a reconstituted cell-free system for messenger RNA processing and transport, consisting of isolated nuclei in fortified cytosol, has been evaluated in terms of the functionality and regulated release of the transported product. The poly(A) messenger RNA transport in vitro formed appropriate initiation complexes with ribosomes in an optimized translation system and had template activity comparable to that transported in vivo. The intra-nuclear origin of this messenger RNA is supported by pulse-labeling studies, its transport from detergent-treated nuclei and the absence of the release under non-transport conditions. Serum albumin was identified by immunoprecipitation and electrophoresis as one of the products synthesized when the transported RNA was translated in vitro. The transport of messenger RNA in the cell-free system was dependent on specific cytosol (soluble cytoplasmic) proteins. These proteins, which constitutes less than 0.1% of the total cytosol proteins, are precipitated wtih streptomycin with high specificity.     

Absence of translational control of histone synthesis during the HeLa cell life cycle

The cell-free synthesis of histone-like polypeptides has been achieved using a selected class of small polyribosomes as the only particulate fraction. This synthesis is prevented if the deoxyribonucleic acid (DNA) inhibitor, cytosine arabinoside, is added to the cells prior to disruption, and it is not detected when the cytoplasm used is derived from postmitotic (G₁) cells. When the 100,000 g supernate from pure metaphase populations was compared with that from S phase cells, the cell-free synthesis of histone-like polypeptides in the presence of S phase polyribosomes remained unchanged. These data suggest that, except for the histone messenger RNA-ribosome complex, the cytoplasmic factors requisite for histone synthesis are present throughout the cycle, and that the shut-off of this synthesis is not under translational control.     

Possible involvement of messenger RNA-associated proteins in protein synthesis

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization. The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system. Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP; deproteinized mRNA however was not translated. Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated. Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP.     

Parallel assessment of circulatory cell-free DNA by PCR and nucleosomes by ELISA in breast tumors

OBJECTIVES: In order to assess the potential biomolecules for breast cancer, we analyzed in parallel the levels of cell-free glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cell-free nucleosomes in serum samples from patients with benign and malignant breast tumors. The levels of cell-free DNA obtained by quantitative PCR were compared with those obtained by enzyme-linked immunosorbent assay (ELISA). METHODS: Twenty-three patients with benign breast tumors, 27 patients with breast cancer, and 32 age-matched healthy women were recruited. The amounts of serum nucleosomes were analyzed by ELISA and the levels of cell-free GAPDH were measured by real-time quantitative PCR. The correlation between nucleosome and cell-free GAPDH levels was examined using the Spearman rank test. RESULTS: The levels of cell-free GAPDH were significantly higher in the serum samples of patients with benign and malignant breast tumors than in those of the control group (median 37,966 GE/mL, range 3,802-130,104 versus 11,770 GE/mL, range 2,198-73,522, p=0.035 and median 40,698 GE/mL, range 3,644-192,482 versus 11,770 GE/mL range 2,198-73,522, p=0.001). The concentration of cell-free GAPDH correlated significantly with the quantities of nucleosomes in serum samples (r=0.451, p=0.000). There was, however, no significant difference between healthy individuals and women with benign breast tumors or breast cancer in terms of nucleosomes determined by ELISA. CONCLUSION: Our data suggest that the cell-free serum GAPDH DNA assayed by quantitative PCR is a better biomarker than nucleosomes assayed by ELISA in patients with breast tumors.     

Isolation and in vitro translation of human placental lactogen messenger RNA from human term placenta.

A messenger activity for HPL was identified in normal human term placentas. The mRNA was translated in rabbit reticulocyte cell-free system. The HPL synthesized was quantified by a specific immunoprecipitation and further identified by electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The HPL synthesized in the reticulocyte lysate exhibited a molecular weight between 20,000 and 22,000 daltons similar to the active hormone. The messenger RNA activity for HPL corresponded to a sedimentation coefficient of 11-12 S. Furthermore the messenger activity for HPL was preferentially associated with membrane bound polyribosomes than with free polyribosomes.     

Agarose gel electrophoretic analysis of damage to supercoiled DNA by adriamycin in the presence of beta-NADH dehydrogenase

In a cell-free system, the anticancer anthracycline antibiotic adriamycin was able to convert purified covalently closed circular, superhelical, form I bacteriophage PM2 DNA to relaxed circular form II DNA in the presence of either sodium borohydride (NaBH4), NADPH cytochrome P-450 reductase or beta-NADH dehydrogenase isolated from myocardial cells. There was no detectable increase in the amount of form III linear duplex DNA formed during the reaction even at high drug concentrations. Less drug was required for the conversion of form I to form II DNA in the presence of the enzymic reducing agents than in the presence of NaBH4. Form II DNA, prepared by irradiation using a Cs-137 source, was not degraded to form III linear duplex DNA. However, form I0 DNA, covalently closed circular DNA without superhelical turns, freshly prepared using topoisomerase I, was converted to form II DNA similar to the conversion of superhelical form I to form II DNA. Again, no increase in the amount of form III linear duplex DNA could be detected.     

Preparation of Efficient Excision Repair Competent Cell-Free Extracts from C. reinhardtii Cells

Chlamydomonas reinhardtii is a prospective model system for understanding molecular mechanisms associated with DNA repair in plants and algae. To explore this possibility, we have developed an in vitro repair system from C. reinhardtii cell-free extracts that can efficiently repair UVC damage (Thymine-dimers) in the DNA. We observed that excision repair (ER) synthesis based nucleotide incorporation, specifically in UVC damaged supercoiled (SC) DNA, was followed by ligation of nicks. Photoreactivation efficiently competed out the ER in the presence of light. In addition, repair efficiency in cell-free extracts from ER deficient strains was several fold lower than that of wild-type cell extract. Interestingly, the inhibitor profile of repair DNA polymerase involved in C. reinhardtii in vitro ER system was akin to animal rather than plant DNA polymerase. The methodology to prepare repair competent cell-free extracts described in the current study can aid further molecular characterization of ER pathway in C. reinhardtii.     

Transcription of polyoma virus DNA in vitro. II. Transcription of superhelical and linear polyoma DNA by RNA polymerase II.

The protein product of the bacteriophage T4 gene 32 is a single-stranded DNA binding protein which functions during phage DNA repair, replication and recombination. Recently the gene 32 protein was shown to participate in the regulation of its own expression. Although the purified protein is known to interact with DNA, the autoregulation was shown to occur at the translational level. The previous analysis in vivo, although coherent, was indirect. We report here direct cell-free experiments in which purified gene 32 protein specifically represses translation of gene 32 messenger RNA.     

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.     

Recovery of biologically functional messenger RNA from agarose gels by passive elution

A general method for isolating biologically active messenger RNA (mRNA) from agarose gels is reported. Purified cellular RNA is resolved by preparative agarose gel electrophoresis and recovered in high yields (80%) by passive diffusion. Polyadenylated mRNA isolated from the eluted RNA is functionally intact based on the ability of the RNA to serve as a template in cell-free translation systems and complementary DNA synthesis reactions. The entire procedure is simple and rapid. A substantial purification of the mRNAs coding for skeletal muscle myosin heavy chain, light chain subunits and carbonic anhydrase III has been achieved employing this method.     

SV40 DNA replication inhibition by the monofunctional DNA alkylator Et743

Ecteinascidin 743 (Et743) is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC-rich sequences resulting in an unusual bending toward the major groove. The ability of Et743 to block DNA replication was studied using the well-established simian virus (SV40) model for mammalian DNA replication in cells and cell-free extracts. Intracellular SV40 DNA isolated from Et743-treated BSC-1 cells was analyzed by native, two-dimensional agarose gel electrophoresis. A low frequency of Et743 adducts detected at 30-100 nM drug concentrations inhibited SV40 origin activity and induced formation of unusual DNA replication intermediates. Under cell-free conditions, only a high Et743 adduct frequency reduced SV40 DNA synthesis. Comparative studies involving related DNA alkylators, tomamycin and saframycin A, revealed inhibition of SV40 DNA replication in cells at concentrations approximately 10 times higher than Et743. Under cell-free conditions tomamycin- or saframycin-A-adducted DNA templates inhibited DNA synthesis similarly to Et743. Et743 appears to be unusual among other alkylators, because its adducts strongly inhibit intracellular SV40 DNA replication but are relatively weak as cis inhibitors as measured under cell-free conditions.     

Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase.

Yeast poly(adenylic acid)-containing messenger ribonucleic acid isolated from two strains of Saccharomyces cerevisiae was fractionated by preparative polyacrylamide gel electrophoresis in the presence of formamide. Three messenger ribonucleic acids, present at high intracellular concentration, were electrophoretically eluted from the polyacrylamide gels and translated in a wheat germ cell-free extract. The in vitro synthesized polypeptides were identified by tryptic peptide analysis. Messenger ribonucleic acids coding for enolase and glyceraldehyde-3-phosphate dehydrogenase were isolated from commercially grown baker's yeast (strain F1), and messenger ribonucleic acid coding for phosphoglycerate kinase was isolated from Saccharomyces cerevisiae (ATCC 24657). Significant differences in the spectrum of abundant messenger ribonucleic acids isolated from commercially grown baker's yeast (strain F1) and strain 24657 were observed. When both strains were grown under identical conditions, however, the spectrum of messenger ribonucleic acid isolated from the cells is indistinguishable.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.