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PubMed1995

The dimerization/packaging sequence is dispensable for both the formation of high-molecular-weight RNA complexes within retroviral particles and the synthesis of proviruses of normal structure.

Retroviral particles contain a dimer of two genomic RNA molecules, linked by noncovalent intermolecular bonds. Studies by electron microscopy of viral RNA extracted from virions as well as in vitro studies have implicated a sequence, designated the dimer linkage sequence (DLS), in the dimerization process. The DLS has been localized within a short region encompassing the psi packaging sequence, between nucleotides 212 and 563 for the Moloney murine leukemia retrovirus (MoMLV) RNA. In this report, we show that viral RNAs lacking both the DLS and psi packaging sequences--and even an RNA lacking the first 6,537 nucleotides of MoMLV--can assemble within retroviral particles as high-molecular-weight, slow-migrating, heat-sensitive complexes closely related to those observed for wild-type viral RNAs. Furthermore, we show that proviruses of normal structure are generated upon infection of test cells with retroviral particles which contain the DLS/psi-deleted viral RNAs. These observations demonstrate that the DLS and psi packaging sequences are not essential in cis to form a functional RNA complex for reverse transcription and integration.

3T3 Cells↗

PubMed2003

[Competitive RT-PCR assay for quantification of GPI-PLD gene expression].

OBJECTIVE: To establish a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to quantify the expression of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) gene. METHODS: A competitive GPI-PLD cDNA fragment was constructed by site-directed mutagenesis by PCR method. The competitive cDNA fragment had the same primer binding sites as the target cDNA, but was 20 bp shorter in length. The competitive cDNA was transcripted into the competitive RNA in vitro as the internal standard. Then the competitive RNA was reversely transcripted and amplified together with total RNA extracted from K562 cells. The two amplified cDNA fragments could be distinguished by 10% polyacrylamide gel electrophoresis. The concentration of cellular GPI-PLD mRNA was derived from the ratio between the intensities of the bands corresponding to the amplified products. RESULTS: A 198 bp GPI-PLD competitive RNA was constructed and prepared. The competitive RNA could compete well with the target RNA in the RT-PCR reaction. The expression of GPI-PLD gene in K562 cells was (440 +/- 20) copies/ng. CONCLUSION: A competitive RT-PCR assay for the quantification of GPI-PLD gene expression may be established successfully. This method is highly sensitive, specific, and accurate.

Gene Expression Regulation, Neoplastic↗

PubMed1993

Cytochrome P-450 17 alpha-hydroxylase protein and mRNA in the testis of the testicular feminized (Tfm) mouse.

The testicular feminized (Tfm) mouse lacks functional androgen receptors and develops with a female external phenotype and internal testes. The testes of these animals contain normal, or close to normal, numbers of Leydig cells but secrete very low amounts of androgen due to a lack of 17 alpha-hydroxylase activity. To determine whether this loss of activity is due to a lack of enzyme synthesis or a change in catalytic activity we have examined 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha)) protein and mRNA levels in the testes of Tfm mice. Levels of P-450(17 alpha) protein were measured by immunoblotting, while mRNA was measured following reverse transcription (RT) and amplification by the polymerase chain reaction (PCR). Conditions for RT-PCR were determined which allowed semiquantification of P-450(17 alpha) mRNA relative to beta-actin mRNA. In extracts of Tfm testes P-450(17 alpha) protein was undetectable using antiserum against porcine P-450(17 alpha). In contrast, a protein of around 54 kDa was clearly detectable in extracts of control cryptorchid testes. Using RT-PCR, P-450(17 alpha) mRNA was detectable in both control and Tfm testes but, expressed in terms of beta-actin mRNA, levels of P-450(17 alpha) mRNA in control testes were 40-fold higher than those in Tfm testes. If the total amount of RNA extracted from each testis is taken into account then P-450(17 alpha) mRNA levels per testis were up to 400-fold higher in control testes. These results show that the reduced level of 17 alpha-hydroxylase activity in Tfm testes is related to reduced protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Type-1 plasminogen activator inhibitor in human breast carcinomas.

We have investigated the occurrence of type-1 inhibitor of plasminogen activators (PAI-1) in human breast tumors. PAI-1 levels, measured by enzyme-linked immunosorbent assay, were significantly higher in malignant breast carcinomas (n = 178) than in benign breast tumors (n = 25). The levels of PAI-1 were found to be correlated with those of urokinase-type plasminogen activator (u-PA). The presence of PAI-1 in tumor extracts was also demonstrated by immunoblotting analysis. Immunohistochemical investigations by the use of monoclonal and polyclonal antibodies showed that PAI-1 was mostly localized in the tumor islands, associated with the tumor cells; in addition, it was present in vessel walls and in normal duct epithelia, but absent from the stroma. Analysis of RNA extracted from tumors by polymerase chain reaction revealed the presence of PAI-1 mRNA. We conclude that PAI-1 is present in human breast carcinoma cells, and that it is--at least partially-- produced locally, either by the cancer cells or by other cells in the tumors. We have previously demonstrated that a high level of u-PA in human breast carcinomas is associated with poor prognosis. These results, combined with our present findings, present 2 possibilities: either the cancer cells need PAI-1 in order to utilize the u-PA-mediated pathway of plasminogen activation for invasion and metastasis; or PAI-1 represents a defense mechanism against tumor invasion.

Animals↗

PubMed2000

Hormonal regulation and cellular distribution of connexin 32.2 and connexin 32.7 RNAs in the ovary of Atlantic croaker.

The in vitro effects of human chorionic gonadotropin (hCG) on ovarian connexin (Cx) 32.2 and 32.7 RNA levels and ovarian follicle maturation were assessed, and the cellular distribution of Cx transcripts in the ovary was determined. hCG caused a concentration-dependent induction of Cx32.2 RNA, which peaked coincidentally with the appearance of morphological indices of oocyte maturational competence (OMC). Cx32.2 RNA levels declined thereafter in all treatment groups, although this decline was not accompanied by the onset of germinal vesicle breakdown (GVBD) at the lowest hCG concentration used. The levels of Cx32.7 RNA initially declined and subsequently increased to preincubation values after hCG treatment, but these changes were not dependent on hCG concentration. In a separate experiment, the decline in Cx32.7 RNA occurred in the presence or absence of hCG and was prevented by low (physiological) concentrations of estradiol-17beta (E2) or by protein kinase C (PKC) inhibitor, but was enhanced in the presence of high E2 concentrations or of PKC activator. These changes in Cx32. 7 RNA abundance were not associated with any indices of oocyte maturation. In situ hybridization of tissue sections showed the presence of Cx32.2 and Cx32.7 RNA in somatic cells of the ovarian follicle but not in oocytes. Cx32.2 RNA seemed to be present in granulosa and thecal cells, but the assay resolution was insufficient to reliably determine the distribution of Cx32.7 transcript by somatic cell type. In view of earlier findings that Cx32.2-based (but not Cx32.7-based) connexons can form functional homotypic channels, these results indicate that Cx32.2 gene expression in granulosa cells is sufficient for the formation of homologous gap junctions (GJ). Northern blot of RNA extracts from ovulated eggs, which are free of follicle cells, showed the presence of relatively low levels of both Cx RNAs. Thus, it is possible that Cx32.2 is present in oocytes and that it participates in heterologous (homotypic) GJ formation between the oocyte and the granulosa cells. In conclusion, Cx32.2 RNA levels in somatic cells of the ovarian follicle correlated positively with morphological indices of OMC acquisition, but subsequently declined during GVBD. These changes in Cx32.2 RNA may function in the regulation of GJ contacts during follicular maturation.

Animals↗

PubMed2007

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in Aspergillus flavus NRRL 3357 and Aspergillus parasiticus SRRC 143.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B(1) and B(2) biosynthesis, while A. parasiticus cultures had significantly increased B(1) and G(1) biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.

Aflatoxins↗

PubMed1991

Nucleotide sequence and RNA hybridization analyses reveal an ambisense coding strategy for maize stripe virus RNA3.

The 2357-nt sequence of maize stripe virus (MStV) RNA3 was determined. Two nonoverlapping open reading frames (ORFs) of opposite polarities are contained in RNA3. A 591-nt ORF is located near the 5' end of MStV RNA3, while a second ORF of 948 nt is located near the 3' end in the viral complementary RNA (vcRNA). In vitro translation of transcripts derived from cDNA clones representing regions of each ORF was used to identify the respective proteins. A ca. 22,000 Mr protein was translated from the 591-nt ORF transcript. This protein comigrated in SDS-PAGE with a protein produced by in vitro translation of MStV RNA and is referred to as the NS3 protein. The protein from the 948-nt ORF transcript was specifically immunoprecipitated by antiserum to the MStV nucleocapsid protein (N protein). RNA hybridization analyses identified two subgenomic RNAs in total RNA extracts from MStV-infected Zea mays plants. These RNAs are ca. 650 and 1350 nt in size, are of opposite polarities, and correspond to regions of RNA3 containing the two ORFs. These data suggest that MStV RNA3 has an ambisense coding strategy similar to that found for the S RNA of the vertebrate-infecting phleboviruses, uukuviruses, and arenaviruses, as for tomato spotted wilt virus.

Amino Acid Sequence↗

PubMed1999

Breast tumour cell-induced down-regulation of type I collagen mRNA in fibroblasts.

This study investigated the modulation of type I collagen gene expression in normal fibroblasts by breast tumour cells. Northern analysis of total RNA extracted from stages I, II and III breast tumour tissue revealed that collagen mRNA levels were elevated in stage I tumours compared to the adjacent normal breast tissues, whereas they were decreased in stages II and III breast tumours. This aberrant collagen gene expression was confirmed by non-radioactive RNA:RNA in situ hybridization analysis of 30 breast carcinomas which localized the production of type I collagen mRNA to the stromal fibroblasts within the vicinity of the tumour cells. In order to determine whether the tumour cells were directly responsible for this altered collagen production by the adjacent fibroblasts, breast tumour cell lines were co-cultured with normal fibroblasts for in vitro assessment of collagen and steady-state collagen RNA levels. Co-culture of tumour cells and normal fibroblasts in the same dish resulted in down-regulation of collagen mRNA and protein. Treatment of the fibroblasts with tumour-cell conditioned medium also resulted in decreased collagen protein levels but the mRNA levels, however, remained unaltered. These results suggested that the tumour cells either secrete a labile 'factor', or express a cell surface protein requiring direct contact with the fibroblasts, resulting in down-regulation of collagen gene expression. Modulation of the ECM is a common characteristic of invading tumour cells and usually involves increased production of collagenases by the tumour cells or stromal fibroblasts. This study showed that tumour cells were also able to modulate collagen mRNA production by stromal fibroblasts, which may facilitate tumour cell invasion and metastasis.

Blotting, Northern↗

PubMed1999

Comparative evaluation of hepatitis C virus infection in serum and liver tissue of patients with chronic liver disease by reverse transcription-polymerase chain reaction.

OBJECTIVE: To undertake a comparative evaluation of diagnostic modalities used in the detection of hepatitis C virus (HCV) infection by serum antibody in ELISA and HCV-RNA in the serum as well as in the liver tissue of the same patients with chronic liver disease by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Thirty-five subjects comprising 30 cases of cirrhosis of the liver and five cases of chronic hepatitis were included in the study group. The criteria for including the subjects in the study were the availability and quality of serum and liver samples. Serologic ELISA tests for HCV antibody as well as HBsAg and anti-HBc IgG for hepatitis B virus (HBV) were performed on the patient's sera, while RT-PCR was carried out with RNA extracted from both serum and liver biopsies of the patients. Primer sequences selected from the conserved NS5 region of the HCV genome were used in RT-PCR. RESULTS: Of 30 cases of cirrhosis studied, five (16.6%) were anti-HCV antibody positive, while seven (23.3%) were positive for HCV RNA in serum and 10 (33.3%) were positive in liver tissue. One of the anti-HCV antibody-positive patients was negative for HCV RNA in both liver and serum by PCR. All seven patients who showed positivity for HCV RNA in serum were also positive for HCV RNA in the liver. Another three cirrhosis cases (10%) found to be HCV positive in liver tissue were negative for both antibody and HCV RNA in serum. Of the five cases of chronic hepatitis, two (40%) were found to be positive by all three tests. HCV was found to be more prevalent in HBV-infected cases (8/18; 44.5%). However, two of the three cirrhotic patients who showed HCV positivity only in liver PCR tested negative for HBV surface antigen. CONCLUSIONS: RT-PCR detection of HCV RNA in liver tissue is of significant clinical importance and is more reliable than RT-PCR in serum or antibody tests by ELISA. RT-PCR can effectively complement antibody tests for reliable diagnosis, since assessment of HCV positivity by a single test seems to be inadequate. The frequent association of HCV infection with HBV suggests that hepatitis B and C viruses may share similar modes of transmission in India.

Journal Article↗

PubMed1984

Induction of cytochrome P-450 isozymes in rat hepatoma-derived cell cultures.

We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P-450. One Reuber hepatoma-derived line (Fu5-C8), which under normal culture conditions produces no detectable cytochrome P-450(MC) or cytochrome P-450(PB)--the major cytochrome P-450 isozymes induced by 3-methylcholanthrene and phenobarbital, respectively--was tested for the ability to accumulate either cytochrome P-450 isozyme in response to treatment with various xenobiotics. By immune-precipitation from [35S]-methionine-labeled cell extracts, using monospecific anticytochrome P-450(MC) antibody or monoclonal anticytochrome P-450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P-450(MC) in response to 3-methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P-450(PB). RNA extracted from Fu5-C8 cells directed the in vitro synthesis of immune-precipitable cytochrome P-450(MC) only after treatment of the cells with 3-methylcholanthrene. Kinetic analysis of the response of these cells to 3-methylcholanthrene induction revealed detectable levels of immune-precipitable cytochrome P-450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P-450(MC) and also cytochrome P-450(PB) constitutively, as determined by specific immune-precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3-methylcholanthrene resulted in an induction of cytochrome P-450(PB) or cytochrome P-450(MC), respectively. Double-labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P-450(MC), but only a subset of cells synthesize cytochrome P-450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P-450(PB).

Animals↗

PubMed1989

Tissue-specific regulation of the concentration of calbindin D28K mRNA in the developing chicken.

A BamHI-HindIII restriction fragment containing the 5'-terminal portion of the gene encoding chicken Calbindin D28K was sequenced and used as a probe in Northern-blot hybridizations to RNA extracted from the brain and intestine of chickens at various stages of development. In both tissues Calbindin D28K mRNA consists of a family of three species, which differ by size. In the intestine Calbindin D28K mRNAs appear at hatching and reach a peak at day 7. In the brain the same RNA species are easily detected at least 7 days before hatching, show a moderate increase at hatching and remain essentially constant during the first 10 days of adult life. The concentration of Calbindin D28K mRNAs in the intestine is strictly dependent on Vitamin D, while it is not in the cerebellum.

Animals↗

PubMed1995

Quantitative analysis of hepatitis C virus RNA in liver biopsies by competitive reverse transcription and polymerase chain reaction.

BACKGROUND/AIMS: The determination of HCV-RNA concentration in liver samples is likely to provide interesting insights for the study of disease progression and for evaluation of the efficacy of anti-viral therapy. METHODS: A procedure was developed for the precise quantification of HCV-RNA in liver biopsies, based on the competitive reverse transcription-polymerase chain reaction technology. This competitive assay consists of the co-amplification of the target RNA with known amounts of a competitor RNA molecule containing the same sequence as the target plus an insertion in the middle, allowing resolution of the two amplification products by gel electrophoresis. RESULTS: The amounts of HCV-genomic-RNA and beta-actin mRNA (the latter being used as an internal standard to overcome the problem of reproducibility of quantitative RNA extraction) were evaluated in liver biopsies of 15 patients affected by hepatitis C virus-positive chronic liver disease at the time of diagnosis. All the patients underwent alpha-interferon therapy for 6 months and were subsequently followed for at least 1 further year after the end of treatment. Viral RNA concentration (which ranged from 2 to 2.7 x 10(5) HCV-RNA molecules per 10(6) beta-actin molecules) directly correlated with the efficacy of treatment, indicating that low levels of viral replication in the liver are associated with a poor response to therapy. CONCLUSIONS: This study suggests that the determination of viral load in the liver is an important prognostic tool for the prediction of the efficacy of alpha-interferon therapy.

Adult↗

PubMed2002

Activation of RNA-dependent protein kinase and nuclear factor-kB by regulatory RNA from lipopolysaccharide-stimulated macrophages: implications for cytokine production.

Our previous results showed that L-RNA, extracted from lipopolysaccharide-stimulated macrophages, induces interleukin-1, interleukin-8 and tumor necrosis factor-alpha (TNF-alpha) in resident macrophages. It was demonstrated the promoter of these cytokine genes contain nuclear factor-kB (NF-kappa B) binding sites. We hypothesized that this effect of L-RNA is mediated by RNA-dependent protein kinase (PKR) through NF-kappa B activation. We found that L-RNA activates PKR and induces NF-kappa B activation through degradation of its inhibitor I-kappa B alpha. These data support the idea that L-RNA acts as a regulatory RNA. A model for the mechanism of action of L-RNA is proposed.

Animals↗

PubMed1994

Coat protein of cucumber necrosis virus is not required for efficient generation or accumulation of defective interfering RNAs.

It is generally assumed that defective interfering (DI) forms of viruses are encapsidated in structural proteins encoded by the helper virus. Virion RNA extracts from cucumber necrosis virus (CNV) infections showing high levels of cellular DI RNAs contain barely detectable levels of DI RNAs, suggesting that DI RNAs are encapsidated very inefficiently. In addition, accumulation of CNV DI RNAs occurs at equal efficiency in coinoculated plants using either synthetic wild-type CNV genomic RNA as helper or a mutant of CNV which lacks the coat protein-coding sequence. Together this shows that the CNV coat protein is not required for efficient accumulation of CNV DI RNA in plants. Factors that could account for the high level of CNV DI RNAs in plants are discussed.

Capsid↗

PubMed2007

Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification.

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

Animals↗

PubMed2006

Retinal growth hormone in perinatal and adult rats.

Growth hormone (GH) mRNA and protein have recently been localized in the neural retina of embryonic chicks, in which exogenous GH promotes cell survival. GH is also expressed in the rat CNS, in which it has neuroprotective roles, although its presence in the rat neural retina is unknown and is the focus of the present study. GH immunoreactivity, to a 22-kDa protein, was present in extracts of fetal (embryonic day [ED]17) eyes and in extracts from the neural retinas of newborn pups, comparable to GH immunoreactivity in pituitary extracts. The GH immunoreactivity in the neural retina was widespread but was most intense in large rounded cells in the retinal ganglion cell (RGC) layer and in the optic fiber layer derived from the axons of the RGCs. A 693-bp cDNA was also generated by the RT-PCR of RNA extracted from the eyes of ED17 rats and from the neural retinas and eyes of newborn rats, when amplified in the presence of oligonucleotide primers for the rat GH cDNA. Expression of the GH gene in the neural retina was also shown by specific in situ hybridization of an antisense GH riboprobe to cells in the neural retina, particularly those in the RGC layers of fetal and adult rat eyes. These results demonstrate GH expression in the neural retinas of fetal, newborn, and adult rats, in which retinal GH might have neuroprotective roles.

Animals↗

PubMedDate not supplied

Messenger ribonucleoprotein particles from plant cell cultures.

Messenger ribonucleoprotein particles were isolated from the polysomes of logarithmically growing plant cell cultures, pulse-labeled with [3H] adenosine for 30 min. More than 80% of the labeled RNP was present in particles sedimenting between 80 S and 30 S on sucrose density gradients, but was not associated with ribosomal subunits. The size distribution differs from those reported for polysomal mRNP particles to date. After fixation with glutaraldehyde the labeled RNP particles had a buoyant density of 1.38 g/cm3 in CsCl gradients. Radioactively labeled RNA extracted from the RNP particles showed a heterodisperse size distribution and contained poly (A) stretches as determined by affinity chromatography and ribonuclease digestion experiments.

Cells, Cultured↗

PubMed1975

Different binding of poly(A)-containing and poly(A)-free fractions of nuclear ribonucleic acid to ribosomes from rat liver.

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0 degrees C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A)-45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A)-45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.

Animals↗