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PubMed1983

Identification of the rotaviral gene that codes for hemagglutination and protease-enhanced plaque formation.

Temperature-sensitive mutants of bovine rotavirus, UK Compton strain, and rhesus monkey rotavirus, MMU18006 strain, were used to derive 16 reassortants by coinfection of MA104 cells. The parental viruses differed phenotypically in their neutralization specificity, their ability to hemagglutinate, and their requirement for exogenous trypsin for infectivity. When the reassortants were assayed for neutralization specificity and hemagglutination, four phenotypes were observed, indicating that these two rotaviral functions segregated independently. Protease-enhanced infectivity phenotype segregated with the HA phenotype indicating that these two functions were manifestations of the same gene product. In order to determine the gene responsible for these rotaviral functions, the reassortants were genotyped by hybridizing 32P-labeled parental transcripts and denatured reassortant genomic RNAs and analyzing the resulting hybrids by gel electrophoresis. The fourth RNA segment was clearly shown to code for HA and protease enhanced plaque formation in MA104 cells. The neutralization antigen was linked to the eighth and ninth RNA segments that comigrated during gel electrophoresis and thus could not be differentiated.

Animals↗
PubMed1997

Developmental regulation and cell-specific expression of N-methyl-D-aspartate receptor splice variants in rat hippocampus.

The present study demonstrates cell-specific and developmental regulation of 5' and 3' splicing of the N-methyl-D-aspartate receptor NR1 subunit within specific neuronal populations of the hippocampus. At birth, NR1 transcripts lacking exon 5 (encoding the amino-terminal splice cassette N1) exhibit mature patterns of labelling within the hippocampus, with high levels of expression in the CA1, CA3, and dentate gyrus. In contrast, exon 5-containing (NR1(1XX)) transcripts are expressed at low levels until P8, at which time expression is prominent and essentially uniform in the CA1, CA3, and dentate gyrus. Exon 5 expression increases at a faster rate in CA3 than in CA1 or dentate gyrus. By the third week postnatal (postnatal day P21), exon 5-containing transcripts exhibit a distinct gradient of labelling, with more intense expression in CA3, than in CA1 or dentate gyrus. By P21 pyramidal neurons of the CA1 and granule cells of the dentate gyrus express mainly NR1(0XX) receptor messenger RNAs (lacking exon 5). Because splicing in of the N1 splice cassette confers greater current amplitude and enhanced potentiation by protein kinase C, these observations predict elevated levels of synaptic activity in the CA1 early in postnatal life, a time at which synaptic plasticity is enhanced. The carboxy-terminal splice cassettes C1 and C2 are regulated independently within the hippocampus. Whereas NR1(X11) (C1-, C2-containing) and NR1(X01) (C2 only) receptors exhibit high levels of expression in CA1, CA3, and dentate gyrus, NR1(X00) receptors are expressed more intensely in pyramidal neurons of CA3. NR1(X10) receptor expression is very low in all cells and at all times examined, even in adults. Because splicing in of the C1 cassette is thought to regulate receptor targeting, clustering, and cytoskeletal interactions, N-methyl-D-aspartate receptors in the two hippocampal subfields may play differing roles in synaptogenesis and the formation of new neuronal contacts. Moreover, cell-specific patterns of NR1(X11) receptor messenger RNAs parallel those of NR1(0XX) receptor messenger RNAs; and cell-specific patterns of NR1(1XX) (N1-containing) receptor messenger RNAs parallels those of NR1(X00) (C1-, C2-lacking) receptor messenger RNAs throughout development. These observations suggest that NR1(100) receptors, which exhibits the greatest potentiation by protein kinase C, are likely to be important in CA1 during the second and third weeks postnatal. Cell-specific expression of NR1 splice variants undoubtedly contributes to functional diversity of N-methyl-D-aspartate receptor properties in neuronal populations within the hippocampus. Developmental regulation of NR1 splicing is likely to influence synaptic plasticity and the formation of new synaptic contacts. Moreover, findings from this study suggest that a change in NR1 splicing following a neurological injury could significantly alter glutamate pathogenicity in a particular population of cells.

Animals↗
PubMed1996

Surprising function of the three influenza viral polymerase proteins: selective protection of viral mRNAs against the cap-snatching reaction catalyzed by the same polymerase proteins.

Influenza virus, a negative strand RNA virus, cannibalizes host cell, capped RNA polymerase II transcripts in the nucleus via a process termed "cap-snatching". The viral transcriptase enzyme; which is composed of a complex of the three viral polymerase (P) proteins, contains a cap-dependent endonuclease that cleaves capped cellular RNAs in the nucleus 10-13 nucleotides from their 5' ends. The resulting capped RNA fragments are required as primers for the initiation of viral mRNA synthesis. In the 18 year since the discovery of "cap-snatching" it has not been determined how the viral transcriptase exhibits selectivity and "snatches" caps from cellular, but not viral, mRNAs. Here we elucidate the surprising mechanism of this selectivity: the complex of the same three viral P proteins that catalyzes "cap-snatching" is also responsible for selectivity protecting the 5' ends of viral, but not cellular, mRNAs from "cap-snatching". The viral P protein complex is able to acquire these two very different functions because this complex lacks any detectable activity unless it binds to one or more specific RNA sequences. Here we demonstrate that the viral P protein complex binds to the common sequence in all the viral mRNAs that is immediately 3' to the 5' sequence that is "snatched" from host cell RNAs. This binding activates the cap-binding activity of the P protein complex, thereby enhancing its binding to the capped viral mRNA. We show that these P protein complexes protect the 5' ends of viral mRNAs from endonucleolytic cleavage by the viral transcriptase, whereas the 5' ends of nonviral mRNAs are not protected.

Catalysis↗
PubMed1991

Interference in trans with brome mosaic virus replication by RNA-2 bearing aminoacylation-deficient mutants.

The tRNA-like domain present at the 3' end of each of the three genomic RNAs of brome mosaic virus (BMV) encompasses the (-)-strand promoter essential for replication. The replicative competence of two BMV RNA-2 transcripts bearing mutations delta 5' and 5'AGA in the tRNA-like domain (previously shown by in vitro assays to be deficient in tyrosylation) was evaluated in barley protoplasts. Transfection of protoplasts with low (2 micrograms) amounts of delta 5'RNA-2, together with transcripts of wild-type RNA-1 and -3, not only incapacitated the replication of RNA-2 but also significantly interfered in trans with the synthesis and accumulation of the other viral RNAs. In contrast, RNA-2 mutants bearing either 5'AGA or M4 (a mutation yielding enhanced minus-strand replication activity in vitro) were inhibitory to viral replication only when present at a relatively high level (12 micrograms). Coinoculation of protoplasts with high levels (12 micrograms) of each of the three RNA-2 mutants and transcripts corresponding to wild-type RNA-1, -2 and -3 (2 micrograms each) revealed that the mutants were capable of competing in trans, resulting in greatly reduced accumulation of the viral RNA and suggesting that their expression from constitutive promoters in transgenic plants may provide protection against viral infection.

Base Sequence↗
PubMed2004

Complementarity between RNA dimerization elements favors formation of functional heterozygous murine leukemia viruses.

The cis-elements that direct packaging and dimerization of retroviral RNAs overlap, and it has been suggested that dimerization is required for RNA packaging. This also implies that heterodimerization would be necessary for co-packaging and recombination. Moreover, co-packaging of distinct RNAs may be reduced if incapable of heterodimerizing. In this study, we have designed a novel two-vector rescue system in which co-packaging and interstrand transfer are necessary for transduction. Thus, the rescue titer is a measure of the ability of a given vector combination to co-package and subsequently generate a provirus. In the current MLV-based set-up, we explored Akv- and MLV-like-endogenous virus (MLEV)-derived vectors with modulated dimerization signals. Results show that rescue is influenced by competition at the level of RNA packaging, as well as complementarity between dimerization elements. Altogether, the results support the hypothesis that complementarity between dimerization elements may favor co-packaging of distinct retroviral RNAs.

Animals↗
PubMedDate not supplied

Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.

We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.

Base Sequence↗
PubMed1997

Deletion analysis of Qbeta replicase. Participation of the carboxyl-terminal region of the beta-subunit protein in template recognition.

We have analyzed one of the functional domains of Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta. Deletion mapping analysis of the carboxyl-terminal region of the beta-subunit protein revealed that the terminal 18 amino acid residues (positions 571-588) are dispensable for the replicase reaction. Subsequent deletions up to the Ala-565 residue reduced the RNA polymerizing activity of the replicase in vivo but increased it in vitro. The mutant replicases with enhanced in vitro RNA polymerizing activity were found to have relaxed template specificity for ribosomal RNAs and cellular RNAs as well as Qbeta RNA. Deletions beyond the Ile-564 residue abolished both the RNA polymerizing activity and the binding ability to midivariant (MDV)-poly(+) RNA, a derivative of a natural template for Qbeta replicase, MDV-1 RNA. These results suggest that the carboxyl-terminal part of the beta-subunit participates in RNA recognition of Qbeta replicase.

Amino Acid Sequence↗
PubMed1997

Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin.

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars.

Bacteriorhodopsins↗
PubMed1979

Enhancement of polyribosome formation and RNA synthesis of gibberellic Acid in wounded potato tuber tissue.

As part of a more detailed study on plant tumorigenesis, the action of gibberellic acid (GA(3)) in wounded potato tuber tissues as a model system has been evaluated. GA(3) stimulates total RNA synthesis in wounded tissues, the optimal concentration being 0.1 micromolar. The responsiveness of the tissue toward the hormone develops with time after wounding. Whereas freshly wounded tissue does not respond at all to the hormone, it becomes competent after about 6 hours, the competence being maximal after 1 day of wound healing.GA(3) enhances the formation of polyribosomes in wounded tissues and stimulates the synthesis of both ribosomal RNAs, transfer RNAs, 5S RNA, and a fraction, which in sucrose density gradients sediments between 18S rRNA and 5S RNA. This fraction contains presumptive mRNA.The hormone, then, is somehow recognized by wounded potato tissue in a time-specific way; the signal is transferred to the genome and triggers the synthesis of various RNA species.

Journal Article↗
PubMed2006

Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco.

Plum pox virus (PPV) is able to replicate in inoculated leaves of Nicotiana tabacum, but is defective in systemic movement in this host. However, PPV produces a systemic infection in transgenic tobacco expressing the silencing suppressor P1/HC-Pro from tobacco etch virus (TEV). In this work we show that PPV is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (NahG) that degrades salicylic acid (SA). Replication and accumulation of PPV is higher in the locally infected leaves of plants deficient in SA or expressing TEV P1/HC-Pro silencing suppressor. Accumulation of viral derived small RNAs was reduced in the NahG transgenic plants, suggesting that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco. Besides, expression of SA-mediated defense transcripts, such as those of pathogenesis-related (PR) proteins PR-1 and PR-2 or alternative oxidase-1, as well as that of the putative RNA-dependent RNA polymerase NtRDR1, is induced in response to PPV infection, and the expression patterns of these defense transcripts are altered in the TEV P1/HC-Pro transgenic plants. Long-distance movement of PPV is highly enhanced in NahG x P1/HC-Pro double-transgenic plants and systemic symptoms in these plants reveal that the expression of an RNA-silencing suppressor and the lack of SA produce additive but distinct effects. Our results suggest that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco, and that silencing suppressors, such as P1/HC-Pro, also alter the SA-mediated defense. Both an RNA-silencing and an SA-mediated defense mechanism could act together to limit PPV infection.

Biomarkers↗
PubMed1989

SV-40 large-T immortalization of embryonic bone cells: establishment of osteoblastic clonal cell lines.

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC50, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)2 vitamin D3 treatment. The other cell line (RCT-1), derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 microM retinoic acid (RA). ALP activity increased from 0.003 to 0.25 mumole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC50, 10 nM). Osteopontin mRNA was induced by 1,25 (OH)2D3. This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.

Animals↗
PubMed2002

The effect of swimming on cartilage formation.

Swimming is a non-weight-bearing exercise. Therefore it has the advantage of maintaining skeletal integrity in aged persons with weakened skeletal structures. Unlike other weight-bearing aerobic exercises, however, it does not appear to exert sufficient stimulus on bone-remodeling activities because the local load-bearing on bone tissues is mild. The purpose of this study was to investigate the effect of swimming on bone remodeling, especially with the use of implanted pellets containing bone morphogenetic protein (BMP) and demineralized bone matrix during the initial stages of the differentiation of mesenchymal cells to cartilage cells. Six-week-old female rats were divided into the swimming group and a control, nonswimming group. Test animals were forced to swim in a water bath for 30 min daily for 2 wk. After the swimming protocol, pellets were implanted and harvested. Messenger RNA isolated from pellets was quantified by means of a reverse transcription-polymerase chain reaction. The expression of RNAs for bone sialoprotein and BMP-6 in pellets from the swimming group was apparently enhanced at 7 d after implantation. These results suggested that systemic hormonal and/or metabolic changes that promote cartilage formation might have occurred after swimming because the effect was observed after the swimming protocol had ended and the pellets were implanted at a non-weight-bearing site.

Animals↗
PubMed1994

Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis.

Mouse hepatitis virus (MHV), a coronavirus, utilizes a discontinuous transcription mechanism for subgenomic mRNA synthesis. Previous studies (C.-L. Liao and M. C. C. Lai, J. Virol. 68:4727-4737, 1994) have demonstrated that an upstream cis-acting leader sequence serves as a transcriptional enhancer, but the mechanism of transcriptional regulation is not clear. In this study, we constructed a series of defective interfering (DI) RNAs containing the chloramphenicol acetyltransferase (CAT) gene behind a differentially expressed transcription initiation (intergenic) sequence (for mRNA2-1). These DI RNAs had different copy numbers of the UCUAA pentanucleotide sequence at the 3' end of the leader. Transfection of these DI RNA constructs into cells infected with a helper MHV, which contains either two or three UCUAA copies at the 3' end of the leader, resulted in differential expression of CAT activities. We demonstrated that the copy number of UCUAA repeats in the leaders of both helper viral and DI RNAs affected the level of CAT activity, suggesting that MHV leader RNA could regulate both in trans and in cis the transcription of subgenomic mRNAs. The leader RNA of subgenomic mRNAs was derived from either the trans- or the cis-acting leader. Furthermore, insertion of a UA-rich sequence (UUUAUAAAC) immediately downstream of the leader in DI RNA, to match the sequence of helper viral RNA, enhanced the CAT activity by threefold, suggesting that this nine-nucleotide sequence is a cis-acting element. Interestingly, when the nine-nucleotide sequence was absent in DI RNA, the leaders of subgenomic mRNAs were exclusively derived from the helper virus. In contrast, when the nine-nucleotide sequence was present in DI RNA, the leaders were derived from both helper viral and DI RNAs. These results suggest that the nine-nucleotide sequence either is required for the leader RNA to initiate mRNA synthesis or, alternatively, serves as a transcription terminator for the leader RNA synthesis. However, when a constitutively expressed intergenic sequence (for mRNA7) was used, no difference in transcription efficiency was noted, regardless of the copy number of UCUAA in the DI RNA and helper virus. This study thus indicates that MHV subgenomic RNA transcription requires the interaction among the intergenic (promoter) sequence, a trans-acting leader, and a cis-acting leader sequence. A novel model of transcriptional regulation of coronavirus subgenomic mRNAs is presented.

Cell Line↗
PubMed1998

Oligonucleotide facilitators enable a hammerhead ribozyme to cleave long RNA substrates with multiple-turnover activity.

Trans-acting hammerhead ribozymes are usually efficient in cleaving short RNA model substrates under both single-turnover and multiple-turnover conditions. In contrast, when long RNAs are the substrates, the cleavage efficiency of these ribozymes decreases, including a loss of multiple-turnover activity in many cases. Since target substrates for potential therapeutical purposes are mostly long RNAs, a multiple-turnover cleavage of long RNAs would essentially increase the efficiency of hammerhead ribozymes. Therefore, we explored if oligonucleotide facilitators, capable of enhancing multiple-turnover activity with short substrates, can also affect or cause multiple turnover with long substrates. We examined the effects of 12-base and 24-base oligonucleotide facilitators on the multiple-turnover activity with substrates of different length containing 39-, 452- and 942-base sequences of the human tissue factor (HTF) mRNA. In the absence of facilitator, the ribozyme cleaved only the 39-base substrate with multiple-turnover activity, but not the long 452-base and 942-base substrates. However, facilitator addition enabled the ribozyme to cleave even the 452-base and the 942-base substrates with multiple-turnover activity. All facilitators tested showed a remarkable activating effect with the long substrates. The data demonstrate that a hammerhead ribozyme which, by itself, can only act as a single-turnover catalyst with long substrates, can be switched by facilitators into a multiple-turnover catalyst. Thus, the inactivation of long target RNAs in multiple-turnover reactions may be achieved by addition of oligonucleotide facilitators.

Humans↗
PubMed1985

Sequences involved in determining the locations of the 5' ends of the late RNAs of simian virus 40.

The 5' ends of the simian virus 40 (SV40) late RNAs are heterogeneous in location, spanning a 300-nucleotide region from residues 28 to 325. To examine whether upstream or downstream measuring functions analogous to the TATA box play roles in positioning the 5' ends of these RNAs, we determined by S1 and primer extension mapping the locations of the 5' ends of the late viral RNAs made in monkey cells infected with: (i) three wild-type strains of SV40 that contain tandem duplications of the enhancer region that are 64, 85, and 91, rather than 72, base pairs in length; (ii) four viable mutants that contain alterations in the 21-base-pair tandem repeats; and (iii) four viable mutants that possess small deletions or insertions at or near the major cap site at residue 325. Most of the 5' ends of the RNAs were identical in location to those seen with wild-type strain 776. The only exceptions were the absence of RNAs whose 5' ends mapped to within three bases upstream or downstream of a sequence alteration. In addition, the sequences within residues 251 to 277 that function as transcriptional initiation sites in wild-type strain 776 also did so in their second locations in the wild-type strains in which these sequences are duplicated. Differences were noted in the relative abundances of the numerous 5' ends of the late RNAs, even among the wild-type strains. These findings indicate that many (and likely all) of the approximately two dozen locations of 5' ends of SV40 late RNAs are each determined largely by sequences within their immediate vicinity. However, sequences somewhat removed from these transcriptional initiation sites may modulate the efficiencies with which they are utilized.

Animals↗
PubMed1978

Transcription of host-substituted simian virus 40 DNA in whole cells and extracts.

Viral transcriptional complexes were extracted from the nuclei of monkey kidney cells infected with wild-type simian virus 40 (SV40) or a variant strain containing a high proportion of host-substituted DNA molecules. The RNAs synthesized by these complexes in an in vitro system were analyzed for their content of SV40 and host sequences by a technique of sequential hybridization to plaque-purified and substituted viral DNAs. The relative labeling of the two types of sequences was commensurate with their proportion in the viral DNA (about 20% host). The substituted virus contains both reiterated and unique types of cellular sequences, and both kinds appeared to be transcribed. Transcripts of the substituted sequences formed a much smaller proportion of the virus related RNA recovered from intact infected cells, suggesting that host sequence transcripts are synthesized but rapidly degraded in the whole cell. The alternative, that transcription of these sequences is artificially enhanced in the in vitro system, cannot be rigorously excluded. We compared the self-annealing of viral RNAs from nuclear extracts of cells infected with wild-type and substituted viruses; transcripts labeled both in vivo and in vitro showed a two- to threefold-higher level of self-annealing in the case of the variant than in the case of wild type SV40.

Base Sequence↗
PubMed1997

Characterization of Rev function using subgenomic and genomic constructs in T and COS cells.

The effect of the human immunodeficiency type 1 (HIV-1) Rev protein on the splicing and cytoplasmic accumulation of HIV-1 RNAs was investigated in COS and T cells. Subgenomic and genomic constructs were used which expressed varying levels of complexity in their potential RNA constituents. Using all constructs, in both cell types, an inhibitory effect of Rev on the level of fully spliced HIV-1 RNAs could be demonstrated. An increase in the nuclear level of unspliced pre-mRNA was seen in the presence of Rev with genomic constructs. Thus, the inhibitory effect on splicing was not merely due to enhancement of nuclear export of the pre-mRNA with these constructs. In both cell types, a positive effect of Rev on the cytoplasmic accumulation of HIV-1 RNAs could also be seen. However, in T cells, the Rev-dependent RNAs were still capable of accumulating at a reduced level in the cytoplasmic fraction in the absence of Rev. The identity of the cell type, construct, and RNA species impacted on the phenotypic manifestation of Rev function.

Animals↗
PubMed1986

Heating RNA before cell-free translation gives no selective increases among the translation products of RNA from rat heart and mammary gland, rabbit reticulocyte membranes, or trout liver.

A recent study has reported that the heating of a population of guanidinium-extracted mRNAs prior to translation causes a selective increase in the translation of certain mRNAs. To determine if this phenomenon is a general property of mRNAs, we carried out a comparison of the translation products obtained when phenol-extracted rat heart and mammary gland RNAs, rabbit reticulocyte membrane RNAs, and trout liver RNAs were translated in the reticulocyte translation system, with and without a prior heat treatment. Our results show that no selective increase in the translation of mRNAs was observed for any of these samples. Among the 14 RNA preparations examined, one total mammary RNA preparation did display a twofold increase in the translation of all mRNAs after heat treatment. It is shown that the heat enhancement of translational activity observed for this sample was due to the reversible formation of intermolecular aggregates with a contaminant that can be removed by chromatography on oligo(dT)-cellulose. Since heat treatment did not selectively enhance the activity of any mRNA in these samples, our results show that the current practice of translating phenol-extracted RNAs without a prior heat treatment should be satisfactory for the translation of most mRNA populations.

Animals↗