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Purification of Biologically Active Globin Messenger RNA by Chromatography on Oligothymidylic Acid-Cellulose

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Abstract

A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.

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... Finally, short RNAs can be removed by denaturing polyacrylamide gel electrophoresis, and long RNAs can be separated by denaturing agarose gel electrophoresis. 108,131 In summary, a variety of methods may be chosen to purify mRNA with different purity requirements and scales, which should be decided by the purpose of the research or application. Apparently, regardless of the method used for purification, strict mRNA quality control standards are the core to ensure the maximum benefits of mRNA therapeutics. ...
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... Cells were harvested at 48 h post-transfection, and TRIzol Reagent (catalog no. T9424, Sigma-Aldrich, Inc., St. Louis, USA) was used to isolate total cellular RNA from the cells (28,29). One mg of RNA was subjected to DNase treatment (catalog no. ...
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A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.
Article
The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.
Article
An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93 ± 2% of EGFP mRNA at a concentration of 10 ng/μL. Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55 ± 27 ng of mRNA from 3.1 ug of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89 ± 24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.
Article
An athermal approach to mRNA enrichment from total RNA using a self‐immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six‐atom spacing between bases which allowed TENA to selectively base‐pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93 ± 2% of EGFP mRNA at a concentration of 10 ng/μL. Self‐immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55 ± 27 ng of mRNA from 3.1 ug of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89 ± 24 ng). Gene expression as measured by RT‐qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT‐qPCR and other downstream molecular biology applications.
Article
Tandemly repeated 72-base-pair (bp) segments located between nucleotides 107 and 250 of the simian virus 40 genome are essential for early region transcription. The functional requirement for the 72-bp repeat was supplied even when that segment was translocated to several locations distant from, and in different orientation, relative to, the promoter. Regardless of the position of the 72-bp enhancer segment, transcription was initiated at the same locations as with the normal promoter. Translocation of the 72-bp repeat segment to other sites in the genome resulted in the appearance of DNase I hypersensitivity at that site in the intranuclear viral minichromosomes. One of the translocations which did not produce enhancement of early- and late-region expression also failed to create a DNase I-hypersensitive site at the translocated 72-bp segment.
Article
cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.
Article
We investigated the expression of cellular sequences c-rasKi and c-fms, which are homologous to the oncogenes of Kirsten rat sarcoma virus and the McDonough strain of feline sarcoma virus, during murine development and in a variety of mouse tissues. The c-rasKi gene was found to be transcribed into two mRNA species of approximately 2.0 and 4.4 kilobases, whereas a single c-fms-related transcript of approximately 3.7 kilobases was identified. The c-rasKi gene appeared to be expressed ubiquitously, since similar levels of transcripts were observed in embryos, fetuses, extraembryonal structures, and a variety of postnatal tissues. In contrast, significant expression of c-fms was found to be confined to the placenta and extraembryonal membranes (i.e., combined yolk sac and amnion). The concentration of c-fms transcripts in the placenta increased approximately 15-fold (relative to day-7 to day-9 conceptuses) during development before reaching a plateau at day 14 to 15 of gestation. The time course of cfms expression in the extraembryonal membranes appeared to parallel the stage-specific pattern observed in the placenta. The level of c-fms transcripts in the extraembryonal tissues reached a level which was approximately 20- to 50-fold greater than that in the fetus. These findings suggest that the c-fms gene product may play a role in differentiation of extraembryonal structures or in transport processes occurring in these tissues. Our results indicate that the c-onc genes analyzed in the present study exert essentially different functions during mouse development.
Article
A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.
Article
Previous analysis of the amdS gene of Aspergillus nidulans has identified multiple regulatory circuits mediated by trans-acting regulatory genes, cis-acting mutations have been identified and shown to specifically affect individual regulatory circuits. Fine-structure genetic mapping of the amdS regions showed that these cis-acting mutations occur in a complex controlling region adjacent to the amdS structural gene. The amdS gene was cloned by differential hybridization, using cDNA probes derived from a high-level-producing strain and from a strain with a large amdS deletion mutation. RNA blotting experiments showed that a single RNA species of 1,600 to 1,700 base pairs is transcribed from the amdS gene. DNA blotting experiments on a large number of amdS mutant strains, including deletions and translocations, allowed the genetic and physical maps of the gene to be correlated. The controlling region of the gene is situated at the 5' end of the gene and the direction of transcription is toward the centromere of chromosome III. The regulatory mutations in the controlling region were found to be due to small-scale alterations in the DNA rather than to large-scale rearrangements resulting in gene fusions.
Article
Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.
Article
This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).
Article
We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt ⁺ colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt ⁺ phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.
Article
In a previous study, we have shown that the three high-G6PD activity mutants are characterized by insertion of the Ins1 sequence consisting of a core sequence flanked by two defective P elements (KP and KP'; the 32nd base of the KP was replaced by guanine in the KP') in front of exonI of the G6PD gene and that the sequence responsible for positive regulation of the G6PD gene expression might be the core sequence but not the flanking KP and KP' elements. The core sequence is composed of either one or two identical units in each mutant. In this report we present evidence (1) that insertion of the Ins1 sequence gives rise to overproduction of G6PD mRNA, (2) that the length and the 5′ end of G6PD mRNA do not differ in wild-type and three mutants, (3) that the insertion site of the Ins1 sequence is the same in the mutants, and (4) that each unit of the core sequence has a pair of DNase I-hypersensitive sites. The possibility exists that the binding of some regulatory proteins to the DNase I-hypersensitive sites might accelerate the transcription rate of the G6PD gene.
Article
The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.
Article
The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs.
Article
Mouse 3T6 cells were transformed with a chimeric DNA plasmid, pSVMgpt, in which the mouse mammary tumor virus (MMTV) promoter was fused to the Escherichia coli gene encoding xanthine-guanine phosphoribosyl transferase (Eco gpt). The transformants exhibited glucocorticoid-inducible expression of Eco gpt. With limiting xanthine concentrations, conditions were established in which cell growth became hormone dependent. Cells selected for their ability to grow in limiting concentrations of both xanthine and glucocorticoids contained amplified levels of Eco gpt DNA, and expression of Eco gpt remained glucocorticoid inducible in these amplified cells. Thus, amplification of the MMTV promoter region in itself did not abolish hormonal responsiveness of a gene. In addition to increased levels of Eco gpt DNA, some of the selected cells also exhibited increased levels (two- to threefold) of glucocorticoid receptors. Lastly, we found that excessive expression of Eco gpt is toxic to 3T6 cells; by maintaining low hormone levels and, therefore, low levels of expression, we were able to select cells with amplified Eco gpt. Thus, the MMTV promoter may be of general utility in expressing genes whose products may be lethal if they are produced in excessive quantities.
Article
The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected under such conditions. A cttl mutant was transformed with an S. cerevisiae gene library in plasmid YEp13. Among the catalase T-positive clones, four contained overlapping DNA fragments according to restriction analysis. Hybridization selection of yeast mRNA binding specifically to one of the cloned DNAs, translation of this mRNA in cell-free protein synthesis systems, and demonstration of catalase T protein formation by specific immunoadsorption showed that the catalase T structural gene had been cloned. By subcloning, the gene was located within a 3.5-kilobase S. cerevisiae DNA fragment. As in wild-type cells, catalase T synthesis in cttl mutant cells transformed with plasmids containing this fragment is sensitive to glucose repression. By DNA-RNA hybridization, catalase T transcripts were shown to be present in oxygen-adapting cells but absent from heme-deficient cells.
Article
Bal31 nuclease was used to resect the herpes simplex virus type 1 thymidine kinase (tk) gene from its 3' end, and a plasmid, pTK206, was isolated that lacked the processing and polyadenylation signals normally found at the 3' end of the gene. The wild-type gene, pTK2, and pTK206 were each transferred to pSV010, a plasmid containing the simian virus 40 (SV40) origin of DNA replication, allowing replication and analysis of the patterns of transcription in Cos-1 cells. Fragments of DNA containing processing and polyadenylation signals from SV40 and polyoma virus were inserted into the 3' end of the resected tk gene, pTK206. We found that tk gene expression requires a processing and polyadenylation signal, that signals from SV40 and polyoma virus could substitute for the herpes simplex virus tk signal, and that considerable differences in the levels of tk mRNA were present in Cos-1 cells transfected by these gene constructs. In addition, tk gene expression was restored to a low level after the insertion of an 88-base-pair fragment from the middle of the SV40 early region. Processing and polyadenylation do not occur in the vicinity of this fragment in SV40, even though it contains the hexanucleotide 5'-AAUAAA-3'.
Article
Somatic cell selective techniques and hybridization analyses with a cloned cDNA probe were used to isolate and identify Chinese hamster cell lines in which the X-linked gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) has been altered. Two of 19 HGPRT-deficient mutants selected were found to have major DNA deletions affecting the HGPRT locus. Cytogenetic studies revealed that the X chromosome of each deletion mutant had undergone a translocation event, whereas those from the remaining 17 mutants were normal. Phenotypic revertants of the thermosensitive HGPRT mutant RJK526 were isolated, and amplification of the mutant allele was shown to be the predominant mechanism of reversion. Comparisons of restriction enzyme fragments of DNA from deletion versus amplification strains identified two regions of the Chinese hamster genome that contained homology to the cDNA probe. One was shown to be much larger than the 1,600-nucleotide mRNA for HGPRT and to be comprised of linked fragments that contained the functional HGPRT gene. The second was neither transcribed nor tightly linked to the functional gene. These initial studies of HGPRT alterations at the level of DNA thus identified molecular mechanisms of phenotypic variation.
Article
Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.
Article
Flagellar radial spokes contribute to the regulation of dynein arm activity and thus the pattern of flagellar bending. We have sequenced the genes for radial spoke protein 4 (RSP4) and RSP6, two of the five proteins that make up the radial spoke head in Chlamydomonas reinhardtii. The two genes, which are tightly linked genetically (B. Huang, G. Piperno, Z. Ramanis, and D.J.L. Luck, J. Cell Biol. 88:80-88, 1981), are separated by only 435 bp. They encode proline-rich polypeptides of 49.8 kDa (RSP4) and 48.8 kDa (RSP6), which are 48% identical to each other but do not resemble any previously sequenced proteins. The transcription start sites of these genes and an additional radial spoke protein gene, that for RSP3, were determined, and patterns of mRNA accumulation during flagellar regeneration were examined for the three radial spoke protein genes. These studies provide the molecular tools for a detailed analysis of radial spoke head function and assembly and for a determination of the mechanism by which the genes required to build a complex organelle are regulated.
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The double-stranded RNA responsible for transmissible hypovirulence in Cryphonectria (Endothia) parasitica was found to affect the accumulation of specific poly(A)+ RNA. Using differential hybridization techniques, two genes were isolated, Vir1 and Vir2, which were specifically expressed as poly(A)+ RNAs in the virulent cells. The highly expressed RNA sequences from these genes were not found in total RNA isolated from either American or European hypovirulent strains, although the genes were present in their genomes. Other virulence- and hypovirulence-specific RNA sequences were also detected. One isolated hypovirulence-specific RNA sequence was expressed in both virulent and hypovirulent cells, but in a two- to fourfold-higher concentration in the hypovirulent cells. The results show that hypovirulence is associated with concurrent changes in a few highly expressed poly(A)+ RNAs, which suggests a specific effect of the double-stranded RNA on fungal gene expression.
Article
To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.
Article
Transformation of the thyroid cell line FRTL-5 results in loss or reduction of differentiation as measured by the expression of thyroglobulin and thyroperoxidase, two proteins whose genes are exclusively expressed in thyroid follicular cells. The biochemical mechanisms leading to this phenomenon were investigated in three cell lines obtained by transformation of FRTL-5 cells with Ki-ras, Ha-ras, and polyomavirus middle-T oncogenes. With the ras oncogenes, transformation leads to undetectable expression of the thyroglobulin and thyroperoxidase genes. However, the mechanisms responsible for the extinction of the differentiated phenotype seem to be different for the two ras oncogenes. In Ki-ras-transformed cells, the mRNA encoding TTF-1, a transcription factor controlling thyroglobulin and thyroperoxidase gene expression, is severely reduced. On the contrary, nearly wild-type levels of TTF-1 mRNA are detected in Ha-ras-transformed cells. Furthermore, overexpression of TTF-1 can activate transcription of the thyroglobulin promoter in Ki-ras-transformed cells, whereas it has no effect on thyroglobulin transcription in the Ha-ras-transformed line. Expression of polyoma middle-T antigen in thyroid cells leads to only a reduction of differentiation and does not severely affect either the activity or the amount of TTF-1. Another thyroid cell-specific transcription factor, TTF-2, is more sensitive to transformation, since it disappears in all three transformed lines, and probably contributes to the reduced expression of the differentiated phenotype.
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4 s + 5 s RNA, 9 s RNA, 18 s RNA, 28 s RNA and polyribosomes, all prepared from rabbit reticulocytes, were injected into living oocytes of the frog Xenopus laevis. Only after injection of 9 s RNA or polyribosomes was haemoglobin-like material formed. Haemoglobin was identified by various criteria: gel filtration, carboxymethyl cellulose chromatography, acrylamide gel electrophoresis and ion exchange chromatography of tryptic peptides. 9 s RNA promoted the formation of globin-like material, whether injected with haemin or without haemin.It is concluded that when injected into a living cell, the 9 s RNA is fairly stable and has the properties of a haemoglobin messenger. The messenger requires no reticulocyte-specific factors for translation, and the translational machinery of the oocyte will accept the messenger RNA from a totally different cell type, from another species. At all stages of assembly and synthesis haemoglobin molecules are reasonably stable in oocytes.
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The rapidly-labeled polyribosomal RNA component from mouse sarcoma 180 cells is retained on nitrocellulose (Millipore) membrane-filters at high ionic strength. This property is due to the presence of a polynucleotide sequence rich in adenylic acid that resists both T(1) and pancreatic RNase digestion. The resistant material shows sedimentation characteristics close to those of transfer RNA. The RNA molecules that contain this material can be separated from the rest of the polysomal RNA by differential phenol extraction with neutral and alkaline Tris buffers. Synthetic poly(A) exhibits the same behavior as the rapidly-labeled polysomal RNA with respect to Millipore binding and phenol fractionation. The characteristics of the rapidly-labeled polysomal RNA component permit its isolation free of ribosomal RNA.
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Polynucleotides containing AMP in excess of 90 mole account for almost 1 of the total RNA synthesized by Ehrlich ascites cells growing either in the mouse or during short incubations in vitro. A method based on the affinity of these AMP-rich polynucleotides for polythymidylate oligomers covalently bound to cellulose has been used to isolate them from the total cellular RNA. The most highly purified fractions are relatively large polynucleotides of the order of 8 to 10 S as judged by their electrophoretic mobility on polyacrylamide gels relative to that of a number of well characterized RNA molecules. They are located primarily in the cell nucleus, for almost 90 of the polyadenylate of whole cells was recovered in nuclei from an aqueous homogenate of these cells. The suppression of polyadenylate synthesis by relatively low doses of actinomycin D suggests that it may be synthesized on a DNA template.
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Several properties of the viral RNA-dependent DNA polymerases and of rabbit globin mRNA make it possible to consider synthesis of the globin gene in vitro. These enzymes copy an RNA template using a short sequence of complementary nucleotides as a primer. Furthermore, globin mRNA has a 3′-terminal sequence of adenylic acid residues that make it particularly suitable as a template, since oligo(dT) can be annealed to a specific site on the mRNA. This small primer could phase the DNA polymerase, possibly ensuring that replication is initiated from that end of the globin message. We have used this approach and find that purified mRNA is an efficient template for the polymerase enzyme. The reaction requires the RNA template and the four deoxyribonucleoside triphosphates, and it is markedly stimulated by the addition of oligo(dT). Consistent with the expectation that the oligo(dT) uniquely phases the polymerase at an adenine-rich region in the globin message, oligo(dG), oligo(dC), and oligo(dA) fail to serve as primers. The product has a density intermediate between that of DNA and RNA, and shifts to a lighter DNA density after treatment with base. Further, it is specifically complementary to globin mRNA and sediments slightly faster in an alkaline sucrose gradient than a DNA standard that has a molecular weight of 129,000. The data suggest that a major portion of the DNA product is a sequence of at least 500 bases, about 50 more than would be necessary to encode rabbit globin. The potential usefulness of this interesting product is discussed.
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A method for the incorporation of chemically synthesized polynucleotides onto cellulose has been developed. By the polymerization of the appropriate mononucleotides, celluloses have been obtained to which thymidine, deoxyadenosine, or deoxycytidine polymers are attached at one of their ends by covalent linkages. Series of oligonucleotides can be bound to columns of these substituted celluloses in base-pairing complexes of different stabilities and subsequently can be fractionally eluted by using a temperature-gradient technique. The application of this method to separation of complex polynucleotides and to the sequence analysis of nucleic acids is discussed.
Article
Immature red blood cells were prepared from phenyl-hydrazine-treated White Leghorn roosters essentially according to the methods of Attardi et al.5 except that, to obtain consistently the characteristic 9S labelling profile reported by these and other investigators, enrichment of the incubation media was required (Fig. 1).Fig.
Article
THE role of formylmethionyl tRNAf (fMet-tRNAf) in the initiation of protein biosynthesis in Escherichia coli is now reasonably well understood1. A similar mechanism involving fMet-tRNAf seems to be common to all 70S type ribosomal systems including those from bacteria, chloroplasts and mitochondria2,3. The corresponding process on 808 type ribosomes is, however, as yet unknown, but there is no evidence to suggest that fMet-tRNAf or any similar blocked aminoacyl-tRNA is involved3. A mammalian cell-free system which synthesizes proteins accurately in response to added messenger RNA would facilitate the study of initiation on 80S ribosomes. An efficient cell-free system derived from mouse ascites tumour cells and primed by encephalomyocarditis (EMC) viral RNA has recently been developed (ref. 4, and M. B. M. and A. Korner, manuscript in preparation). We report here on the nature of the products synthesized.
Article
Small amounts of encephalomyocarditis virus RNA direct a 50-fold increase in amino acid incorporation, in appropriately supplemented ascites tumor cell extracts, under conditions that give rise to authentic viral polypeptides. Incorporation in these crude extracts has a novel characteristic, namely, that it is almost entirely dependent upon the addition of exogenous tRNA. Further, this incorporation is restricted in that tRNA derived from ascites tumor cells or from rat liver permits translation of viral RNA, whereas tRNA from yeast or Escherichia coli does not. These translational barriers are due, at least in part, to an incompatibility between the tRNA of yeast and E. coli and the aminoacyl-tRNA synthetases of the ascites tumor cell. A more extensive basis for this incompatibility is suggested, however, by the failure of the E. coli aminoacyl-tRNA synthetases to restore viral RNA-directed protein synthesis in the presence of tRNA from E. coli, although the coli synthetases fully restore the poly(U)-directed synthesis of polyphenyl-alanine. The possible role that unique or favored codon classes might play in this restriction is considered, together with the implications of the observed requirement for tRNA.
Article
RNA from the encephalomyocarditis virus directs the cell-free synthesis of several discrete, high molecular weight proteins. The largest of these have molecular weights of approximately 110,000, 82,000, 73,000, 61,000 and 44,000 Daltons. In addition, tryptic digestion of the in vitro products gives rise to a number of peptides corresponding to those derived from the viral capsid. The data suggest that approximately one-third of the information encoded by the EMC genome is translated in vitro as a single polypeptide chain, that this translation proceeds in an appropriate phase, and that portions of the genome corresponding to structural proteins of the virus are translated.
Article
Several properties of the viral RNA-dependent DNA polymerases and of rabbit globin mRNA make it possible to consider synthesis of the globin gene in vitro. These enzymes copy an RNA template using a short sequence of complementary nucleotides as a primer. Furthermore, globin mRNA has a 3'-terminal sequence of adenylic acid residues that make it particularly suitable as a template, since oligo(dT) can be annealed to a specific site on the mRNA. This small primer could phase the DNA polymerase, possibly ensuring that replication is initiated from that end of the globin message. We have used this approach and find that purified mRNA is an efficient template for the polymerase enzyme. The reaction requires the RNA template and the four deoxyribonucleoside triphosphates, and it is markedly stimulated by the addition of oligo(dT). Consistent with the expectation that the oligo(dT) uniquely phases the polymerase at an adenine-rich region in the globin message, oligo(dG), oligo(dC), and oligo(dA) fail to serve as primers. The product has a density intermediate between that of DNA and RNA, and shifts to a lighter DNA density after treatment with base. Further, it is specifically complementary to globin mRNA and sediments slightly faster in an alkaline sucrose gradient than a DNA standard that has a molecular weight of 129,000. The data suggest that a major portion of the DNA product is a sequence of at least 500 bases, about 50 more than would be necessary to encode rabbit globin. The potential usefulness of this interesting product is discussed.
Article
An electrophoretic method has been developed for the analysis of ribonucleic acids (RNAs) ranging in size from 104 to 108 daltons. The method depends on the use of acrylamide gels strengthened with agarose for analysis of the larger RNAs. The resolving power of the method permitted individual characterization of RNAs in mixtures containing multiple species of RNA, without prior purification of each species; RNA molecules which differed in molecular weight by only a few per cent could be clearly distinguished, and the molecular weight of each estimated. This unusual application of electrophoretic methods for the determination of molecular weight is based on the observation that, for RNAs, smaller molecules migrate more rapidly than larger ones. The mobility and the logarithm of the molecular weight are inversely related and this relationship is approximately linear. The molecular weights estimated by this technique, although numerically dependent on values assigned to known RNA standards, are highly reproducible in gels of various composition, and are at present the best means of identification of species resolved by gel electrophoresis. By this means, liver 18S RNA is identified as a doublet of RNAs of 0.66 and 0.62 × 106 daltons and the analogous 16 S of Escherichia coli as a doublet of 0.58 and 0.54 × 106 daltons, while liver 5S RNA has a molecular weight of 38,000 daltons.
Article
The presence of a large poly(A) segment has been demonstrated in the heterogeneous rapidly labeled components of polysomal RNA from mammalian cells. Unique properties of poly(A) permit the isolation of this RNA species, which possesses various characteristics of mRNA. The poly-(A) segment causes the transfer of these RNA molecules to he nonaqueous phase during phenol extraction in the presence of Tris-HCl (pH 7.6) and of polysomal proteins or methylated albumin. They are recovered by reextraction with pH 9.0 Tris buffer. The critical factor for this behavior appears to be the ionic concentration during phenol extraction. The poly(A) segment also causes the effective binding of the RNA molecules to nitrocellulose membrane filters (Millipore filters) at high ionic strength. RNA molecules lacking such a poly(A) sequence (such as rRNA) do not share these properties. The RNA adsorbed on Millipore from unlabeled mouse sarcoma 180 polysomal RNA shows the presence of ultraviolet-absorbing material with a 10-30S range of sedimentation values, together with some ribosomal RNA. With rabbit reticulocyte polysomal RNA, the material retained on Millipore consists of a very prominent 10S component, together with some heterogeneous material and a small amount of ribosomal comnonents The phenol fractionation procedure leads to preparations greatly enriched in poly(A)-containing RNA species, but is not as effective in removing rRNA.
Article
Polyadenylic acid [poly(A)] segments containing 150 to 250 nucleotides appear to be covalently linked to heterogeneous nuclear RNA (HnRNA) and messenger RNA (mRNA) in eucaryotic cells. The poly(A) is synthesized in the nucleus, and is probably linked initially to HnRNA that is ultimately transported as mRNA to the cytoplasm. Studies with inhibitors of RNA or poly(A) synthesis indicate that synthesis of poly(A) segments is independent of transcription. The poly(A) marker may prove useful to elucidate mRNA modification and transport in eucaryotic cells.s
Article
We show that globin mRNA is indeed translated by the non-erythropoietic ascites cell system in the absence of any additional reticulocyte components. Our results also confirm the identification11 -13 of the 9S fraction of reticulocyte RNA as the messenger.Messenger RNA was extracted from mouse reticulocytes by sodium dodecyl sulphate (SDS) and was purified on two successive sucrose gradients.
Article
Two eukaryotic mRNAs carry untranslated poly A sequences on the 3′ terminal region, suggesting that they have been conserved during evolution and may play a role in mRNA maturation after transcription.
Article
Polyadenylate sequences have been found covalently linked in heterogeneous DNA-like nuclear RNA of HeLa cells. This poly(A) material seems homogeneous in size and accounts for about 0.5% of such RNA. Similar poly(A) sequences were found in rapidly-labeled polyribosomal RNA, thought to be messenger RNA. A possible model for mRNA synthesis from large heterogeneous nuclear RNA precursor molecules is discussed.
Article
Messenger RNA from HeLa cells contains, as part of the polynucleotide chain, RNase-resistant sequences that are labeled by adenosine but not by uridine. Heterogeneous nuclear RNA also contains adenylate-rich RNase-resistant regions, but in lower proportion than messenger RNA. Hybridization to DNA of (32)P-labeled messenger RNA reveals that some of the adenylate-rich region is included in the rapidly-hybridizing fraction.
Article
An RNA sedimenting at 10 S, with a molecular weight of 2.3 x 10(5), was isolated from rabbit reticylocyte polyribosomes. When this RNA is added to a cell-free extract from Krebs II ascites cells, both alpha and beta chains of rabbit hemoglobin are synthesized; beta chains are made in about 50% excess over alpha chains.
Article
POLYPURINES have been isolated from subcellular fractions of mouse liver by a method which involves a limited nuclease digestion of the RNA, followed by chromatography of the digestion products on polystyrene columns1-3. These polypurines consist of clusters of adenylic acid and guanylic acid in which the A/G ratio is 2-10 the materials isolated from the rough endoplasmic reticulum have sedimentation coefficients of 3-5S, but a molecule containing twenty nucleotides can be obtained from the free polysomes4. The latter polymer is part of a rapidly labelled RNA species that sediments between the 32S subunit and 5S RNA when EDTA-treated polysomes are centrifuged on a sucrose density gradient. With longer times of labelling with 32P the polymer is also found in association with the 32S subunit. It seems likely that the adenine-rich polymer is found in close association with messenger-like RNA. These obssrvations prompted us to investigate the polypurine content of a better characterized mammalian messenger RNA. Such an RNA is the haemoglobin messenger RNA which has been isolated from rabbit reticulocytes5,6, and purified by polyacrylamide gel electrophoresis7. This species of RNA8 and a similar RNA from mouse reticulocytes9 have been demonstrated to initiate specific globin synthesis in vitro. We now describe the isolation of an adenine-rich polymer from purified messenger RNA obtained from rabbit reticulocyte polysomes and its relationship to the size of the ribosomes from which the messenger RNA is derived.
Article
1A cell-free system from mouse Krebs II ascites cells is described which responds by increased amino acid incorporation into protein on addition of encephalomyocarditis virus (EMC-RNA). RNA from other sources does not produce this response.2The stimulation by EMC-RNA occurs over a narrow range of Mg2+ concentrations and is maximal at 5 mM, which is optimal for the endogenous incorporation and lower than that required in the presence of poly(U).3The EMC-RNA-directed product is distinguished from the endogenous products by its size and composition.4After an initial lag of 4 min, during which the nascent chains are too short to be acid-insoluble, EMC-RNA-directed protein synthesis is active for more than 1 h.5EMC-RNA-directed synthesis is particularly sensitive to the inhibitors cycloheximide and dextran sulphate. Using the latter, it has been shown that the initiation of protein synthesis is completed during the first 15 min of incubation.6Only 20–30% of the product of the cell-free system, whether endogenous or EMC-RNA-directed, is released from the ribosomes. Despite the size of the viral RNA, it did not appear to promote the formation of exceptionally large polysomes.7It is concluded that the EMC-RNA is acting as a message for viral proteins in the ascites cell-free system.
Article
When reticulocyte polysomes are dissociated with sodium dodecyl sulfate and analyzed by sucrose density gradient centrifugation, a ribonucleic acid of 9 S (9S ribonucleic acid) is observed. This ribonucleic acid exhibits many of the properties expected for the hemoglobin messenger ribonucleic acid, such as size, per cent of total ribonucleic acid, and involvement in polysomal structure. Because of these unique features it was of interest to study its synthesis in relation to ribosomal ribonucleic acid synthesis during erythroid cell development. This was accomplished by injecting [3H]uridine into anemic mice at various times prior to collecting the circulating reticulocytes. Cells obtained shortly after injection of the ribonucleic acid precursor are those reticulocytes originating from more mature nucleated erythroid cells while those reticulocytes obtained at longer times originate from more immature precursor cells. The data indicate that ribosomal ribonucleic acid is synthesized early in erythroid cell development while 9S ribonucleic acid synthesis is maximal in later cells.
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