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At least 685 records · Page 38Linked to original sources
PubMed2000

Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: a crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue.

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5'-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions -3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position -3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.

Anticodon↗
PubMed2002

Pokeweed antiviral protein binds to the cap structure of eukaryotic mRNA and depurinates the mRNA downstream of the cap.

Several cap-binding proteins from both the nucleus and cytosol have been identified that mediate processes such as pre-mRNA splicing, translation initiation, and mRNA turnover. Here we describe a novel cap-binding protein, pokeweed antiviral protein (PAP), a 29-kDa type I ribosome-inactivating protein (RIP) isolated from Phytolacca americana. In addition to depurinating the sarcin/ricin loop of the large rRNA, an activity common to all RIPs, we have reported recently that PAP depurinates capped, but not uncapped RNAs in vitro. Here we characterize this activity further and, using affinity chromatography, show that PAP binds to the m7Gppp cap structure. PAP UV-crosslinks to m7GpppG-capped luciferase mRNA more efficiently than GpppG-capped luciferase mRNA, indicating specificity for the methylated guanosine. We present evidence that PAP does not remove the cap structure or depurinate the m7Gppp as shown by primer extension of capped and uncapped luciferase transcripts incubated with PAP. Modeling studies of cap interaction with PAP predict that the cap structure would bind to the active site of PAP in a similar manner to guanine. We map the depurination sites on the capped luciferase RNA and illustrate that depurination occurs at specific adenine and guanine residues throughout the RNA sequence. Incubation of isolated ribosomes with PAP and increasing molar concentrations of m7GpppG relative to PAP resulted in a decrease in the level of rRNA depurination. Therefore, at elevated concentrations, the methylated cap structure competes with the adenine or guanine for binding to PAP, even though the affinity of PAP for capped message is almost fourfold lower than for rRNA. These results demonstrate that the activity of PAP is not limited to rRNA depurination, but that PAP binds to the cap structure and depurinates mRNAs downstream of the cap in vitro. These findings may have implications for understanding PAP activity in vivo.

Antiviral Agents↗
PubMed1998

Phosphorylation stabilizes alternatively spliced CD44 mRNA transcripts in breast cancer cells: inhibition by antisense complementary to casein kinase II mRNA.

CD44, the predominant vertebrate cell surface receptor for hyaluronan, exists in a variety of isoforms resulting from alternative splicing of a single gene. Particular spliced variants of CD44 correlate with increased cell motility, and with poor clinical prognosis in several kinds of carcinomas. Combinations of 9 variant exons that confer this enhanced motility on tumor cells are inserted into a single site in the middle of the extracellular domain of CD44. Evidence suggests that phosphorylation of 2 serine residues in the intracellular domain of CD44 are involved in controlling these events. However, evidence is lacking as to the nature of such kinases. Acidic amino acids in close proximity to these 2 serine residues suggests casein kinase II (CKII) is involved. We now show an antisense phosphorothioate oligonucleotide designed to hybridize to the AUG translation initiation codon of subunit CKII alpha' mRNA blocks in vivo phosphorylation of CD44 in MDA231 breast tumor cells, and at the protein level decreases ectopic expression of total CD44 as well as the metastatic v-7 CD44 isoform. Furthermore subplateau RT-PCR analysis demonstrated antisense transfected MDA231 tumor cells had significant down-regulated or eliminated mRNA transcripts of metastatic CD44 isoforms. CKII as a CD44-associated serine kinase therefore may serve as an important molecule in a signaling cascade that produces a variety of cellular responses in MDA231 breast cancer cells. Since the 3'-untranslated region of CD44 mRNA contain 4 dispersed AUUUA sequences which serve as signals targeting mRNA for rapid turnover, a mechanism is proposed by which CD44 phosphorylation mediates labile message stabilization, hence providing insights into the processes involved in cancer cell growth, invasion and metastasis.

Alternative Splicing↗
PubMed1995

Reexpression of developmentally regulated MAP2c mRNA after ischemia: colocalization with hsp72 mRNA in vulnerable neurons.

Levels of mRNAs encoding the microtubule-associated proteins MAP2b and MAP2c as well as the 70-kDa stress protein [72-kDa heat shock protein (hsp72)] were evaluated in postischemic rat brain by in situ hybridization with oligonucleotide probes corresponding to the known rat sequences. Rats were subjected to 10-min cardiac arrest, produced by compression of major thoracic vessels, followed by resuscitation. The normally expressed MAP2b mRNA showed transient twofold elevations in all hippocampal neuron populations at 6-h recirculation, followed by a return to control levels by 24 h. MAP2b hybridization was progressively lost thereafter from the vulnerable CA1 and outer cortical layers, preceding both the fall in immunoreactive MAP2b and the eventual cell loss in these regions. The depletion of MAP2b mRNA coincided with an increase in the alternatively spliced MAP2c in vulnerable regions during 12-48 h of recirculation, precisely overlapping the late component of hsp72 expression that persisted in these cell populations. Previous studies have suggested that the initial induction of hsp72 provides an index of potential postischemic injury in neuron populations that may or may not be injured, while lasting hsp72 mRNA expression is associated with cell damage. In contrast, the present results demonstrate that MAP2c expression under these conditions occurs uniquely in neuron populations subject to injury. Available evidence suggests that MAP2c expression represents a plastic response in subpopulations of neurons that will survive in these regions, although it remains to be explicitly determined whether it may also be transiently expressed in dying cells. In any case, these observations demonstrate that reexpression of developmentally regulated MAP2c mRNA is a relatively late postischemic response in vulnerable cell populations, indicating that pathways regulating MAP2 splicing may be closely associated with mechanisms of neuron injury and/or recovery.

Animals↗
PubMed2002

The effect of obesity on the ratio of type 3 17beta-hydroxysteroid dehydrogenase mRNA to cytochrome P450 aromatase mRNA in subcutaneous abdominal and intra-abdominal adipose tissue of women.

OBJECTIVES: To investigate (1) whether type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), the enzyme which catalyzes the conversion of androstenedione to testosterone in the testis, is co-expressed with P450aromatase in the preadipocytes of women, and (2) whether the relative expression of type 3 17beta-HSD and aromatase varies in subcutaneous abdominal vs intra-abdominal adipose tissue of women. SUBJECTS: Subcutaneous abdominal and intra-abdominal adipose tissue was obtained from women undergoing elective abdominal surgery (age 22-78 y, body mass index (BMI) 22.4-52.9 kg/m(2)). MEASUREMENTS: Expression of type 3 17beta-HSD in adipose cell fractions was determined using RT-PCR. Preadipocyte steroidogenesis was investigated in primary cultures using androstenedione as substrate. Messenger RNA levels for type 3 17beta-HSD and aromatase were measured in adipose tissue from the subcutaneous abdominal and intra-abdominal depots using a quantitative multiplex competitive RT-PCR assay. RESULTS: Type 3 17beta-HSD is co-expressed with aromatase in the abdominal preadipocytes of women. Cultured preadipocytes from both subcutaneous abdominal (n=5) and intra-abdominal (n=5) sites converted androstenedione to testosterone, and there was minimal conversion of androstenedione to estrone. Consistent with this, the levels of type 3 17beta-HSD mRNA were significantly higher than aromatase mRNA at both sites (P<0.05; n=8 subcutaneous abdominal, n=12 intra-abdominal adipose tissue). The ratio of levels of 17beta-HSD mRNA to aromatase mRNA in intra-abdominal adipose tissue was positively correlated with BMI (n=11, r=0.61, P<0.05) and waist circumference (n=10, r=0.65, P<0.05). The converse was found in subcutaneous abdominal adipose tissue. CONCLUSION: The intra-abdominal adipose tissue of women may be substantially androgenic, increasingly so with increasing obesity, particularly central obesity. While androgen production by this adipose tissue deposit may not contribute to circulating testosterone levels due to hepatic clearance, it may have hitherto unrecognised local effects in the intra-abdominal adipose tissue and also on the liver via the hepatic portal system. These studies suggest a mechanism linking central obesity with insulin resistance and dyslipidaemia.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

Muscarinic1 and 2 receptor mRNA in the human caudate-putamen: no change in m1 mRNA in schizophrenia.

Studies using tissue obtained at autopsy suggest that changes in cholinergic neurons could be important in the pathology of schizophrenia.1-4 We have previously reported a decrease in [3H]pirenzepine binding5 and [3H]AF-DX 384 binding6 to caudate-putamen (CP) from subjects who had schizophrenia. Under the conditions chosen, [3H]pirenzepine would predominately bind to muscarinic1 (M1) and muscarinic4 (M4) receptors,7whereas [3H]AF-DX 384 would mainly bind to muscarinic2 (M2) and M4 receptors.8 Given the relative concentrations of M1, M2 and M4 receptors in the human CP and the magnitude of the decreases in radioligand binding in schizophrenia, our results most likely reflected a change in the density of M1 and M2 receptors in the CP from the schizophrenic subjects. In situ hybridisation has now been used to determine levels of m1 and m2 mRNA in CP from 14 schizophrenic and 16 control subjects previously used for radioligand binding. m2 mRNA in the CP from the schizophrenic and control subjects was below the sensitivity of in situhybridisation. There was no difference in the levels of m1 mRNA in CP from schizophrenic and control subjects (mean +/- SEM: 103 +/- 16 vs106 +/- 17 fmol [35S]oligonucleotide probe g-1estimated tissue equivalents, P = 0.91). In conclusion, data from our radioligand binding studies show decreases in [3H]pirenzepine binding that are likely to reflect a decrease in the density of M1 receptors in CP from schizophrenic subjects. Our data in this study show the absence of a concomitant change in mRNA coding for that receptor.

Adult↗
PubMed1988

Cloning of cDNA to rat mammary-gland fatty acid synthase mRNA. Evidence for the expression of two mRNA species during lactation.

A cDNA library was constructed in the expression vector lambda gt11, by synthesizing cDNA from size-selected poly(A) RNA from lactating rat mammary gland, using random hexanucleotide primers. Using this library we identified two recombinants which, on addition of a lac z inducer, produced proteins recognized by affinity-purified anti-fatty-acid synthase antibody, and which, therefore, contained fatty acid synthase coding sequences. The inserts were subcloned, were shown to be between 500 and 600 base pairs in size, and to cross-hybridize. The cloned DNA was then used in Northern hybridizations with mRNA isolated at various stages throughout lactation. Two mRNA species were identified of approx. 9.7 and 10.4 kilobases, which increased and decreased in parallel during lactation, reaching a peak at 12-13 days. Both mRNA species disappeared rapidly if the pups were removed prematurely. This study provides evidence that, during hormonal induction in lactation, regulation of the level of fatty acid synthase protein can be accounted for by variation in the level of mRNA.

Animals↗
PubMed1994

Regulation of mammalian ribonucleotide reductase R1 mRNA stability is mediated by a ribonucleotide reductase R1 mRNA 3'-untranslated region cis-trans interaction through a protein kinase C-controlled pathway.

Ribonucleotide reductase catalyses the reaction that eventually provides the four deoxyribonucleotides required for the synthesis and repair of DNA. U.v.-cross-linking and band-shift experiments have identified in COS 7 monkey cells an approx. 57 kDa ribonucleotide reductase R1 mRNA-binding protein called R1BP, which binds specifically to a 49-nt region of the R1 mRNA 3'-untranslated region (3'UTR). The R1BP-RNA binding activity was down-regulated by the tumour promoters phorbol 12-myristate 13-acetate (PMA; 'TPA') and okadaic acid, and up-regulated by the protein kinase C inhibitor staurosporine, in a dose-dependent fashion. Furthermore, staurosporine treatment decreased the stability of R1 and CAT (chloramphenicol acetyltransferase)/R1 hybrid mRNAs, whereas PMA and okadaic acid increased the stability of these messages, in a dose-dependent manner. In contrast, treatment of cells with forskolin, a protein kinase A inhibitor, did not alter either R1BP-RNA binding or R1 mRNA-stability characteristics. Transfectants containing R1 or CAT/R1 cDNA constructs with a deletion of the 49-nt 3'UTR sequence failed to respond in message-stability studies to the effects of PMA, staurosporine or okadaic acid. These observations indicate that a protein kinase C signal pathway regulates ribonucleotide reductase R1 gene expression post-transcriptionally, through a mechanism involving a specific cis-trans interaction at a 49-nt region within the R1 mRNA 3'UTR.

3T3 Cells↗
PubMed1994

Sodium status affects GC-B natriuretic peptide receptor mRNA levels, but not GC-A or C receptor mRNA levels, in the sheep kidney.

1. In humans and experimental animals the natriuresis and diuresis resulting from infusion of atrial natriuretic peptide varies with the sodium status of the subject. Tissue binding studies have suggested that this may be related to changes in the renal receptors for the hormone. 2. In order to establish whether these changes are under transcriptional control, we examined the levels of mRNA for the three natriuretic peptide receptors [GC-A, GC-B and clearance (C) receptors] in renal cortex and medulla from six sodium-loaded, six sodium-depleted and four control sheep. cDNA probes specific to each receptor were generated using the polymerase chain reaction. 3. GC-B receptor mRNA levels were increased approximately two-fold in the renal cortex of sodium-depleted animals, whereas there was no influence on GC-B receptor mRNA levels in the renal medulla. There was no significant difference in mRNA levels for the GC-A and C receptors. 4. At present the role of the GC-B receptor and its natural ligand C-type natriuretic peptide in the control of renal function is unknown. The present experiments imply some intrarenal function for the GC-B receptor and its natural ligand, although the site of any such function, e.g. renal vasculature or tubules, remains unclear. In addition, we have shown that if GC-A and C receptor levels in the sheep are modulated by sodium, the regulation occurs beyond the level of gene transcription.

Animals↗
PubMed1997

Increased levels of IL-13 mRNA, but not IL-4 mRNA, are found in vivo in peripheral blood mononuclear cells (PBMC) of patients with atopic dermatitis (AD).

Previous studies using in vitro systems with various stimuli have shown that PBMC from patients with AD show increased levels of IL-4 but decreased levels of interferon-gamma (IFN-gamma) compared with PBMC from normal controls. However, in vitro conditions do not always mimic the in vivo condition. We therefore believe that it is important to quantify the expression of these cytokines in freshly isolated PBMC. This study examines the expression of IFN-gamma, IL-4 and IL- 13 mRNA in freshly isolated PBMC from adult patients with AD, from patients with psoriasis vulgaris and from healthy adults, using the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Levels of IFN-gamma mRNA were significantly lower in PBMC of patients with AD than in controls. IL-4 mRNA levels did not differ significantly between groups. Conversely, levels of mRNA for IL-13 were significantly greater in PBMC of patients with AD than in controls. An increase in IL-13 expression may regulate the in vivo synthesis of IgE in patients with AD.

Adolescent↗
PubMed1999

Ontogeny of diazepam binding inhibitor/acyl-CoA binding protein mRNA and peripheral benzodiazepine receptor mRNA expression in the rat.

The Diazepam Binding Inhibitor/Acyl-CoA Binding Protein (DBI/ACBP) has been implicated in different functions, as acyl-CoA transporter and as an endogenous ligand at the GABA(A) receptor and the peripheral benzodiazepine receptor (PBR). The latter is thought to be involved in control of steroidogenesis. We studied the ontogeny of DBI/ACBP and PBR mRNA expression in embryos and offspring of time-pregnant Long Evans rats by in-situ hybridization with 33P-endlabelled oligonucleotides. Both mRNAs were present in embryo and placenta at gestational day (G)11, the earliest stage studied. DBI/ACBP mRNA was strongly expressed from embryonic through mid-foetal stages in central nervous system (maximum in neuroepithelium), cranial and sympathetic ganglia, anterior pituitary, adrenal cortex, thyroid, thymus, liver and (late foetal) brown adipose tissue, moderately in testis, heart, lung and kidney. In brain, a late foetal decrease of DBI/ACBP mRNA was followed by an increase at postnatal day 6. Peripheral benzodiazepine receptor mRNA expression started very low and increased to moderate levels in adrenal cortex and medulla, testis, thyroid, brown adipose tissue, liver, heart, lung, salivary gland at mid- to late-foetal stages. Data suggest a significant role of DBI/ACBP at early developmental stages. Both proteins may be involved in the control of foetal steroidogenesis. However, differences in developmental patterns indicate that additional functions may be equally important during ontogeny, such as the involvement in lipid metabolism in the case of DBI/ACBP.

Acyl Coenzyme A↗
PubMed2001

An element within the 5' untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation.

The untranslated regions of mRNAs encoding heat-shock proteins have been reported to contain elements important to the post-transcriptional regulation of these key components of the stress response. In this report we describe an element from the 5'UTR of human Hsp70 mRNA that increases the efficiency of mRNA translation. Cloning of this region upstream of the coding sequence of two different reporter genes (firefly luciferase and chloramphenicol acetyltransferase) increases expression of the reporter under normal cell culture conditions by up to an order of magnitude. This effect was observed in three different promoter contexts (HSP, SV40 and CMV) and in six cell lines. The increase in protein production is not accompanied by any alteration in mRNA levels, suggesting that the element facilitates translation. 5' or 3' truncated sequences are ineffective in enhancing reporter expression, suggesting that the activity arises from the secondary structure of the element, rather than from some smaller defined motif. Computer analysis of this region revealed that it is able to form stable secondary structures (DeltaG approximately -292.6 kJ x mol(-1)). The Hsp70 element does not seem to act as an internal ribosome entry site. Incorporation of the sequence into plasmids used for DNA vaccination produces increased antibody responses, confirming that the sequence is functional in primary cells. These data suggest that the 5'UTR of human Hsp70 mRNA plays an important role in determining Hsp70 expression levels, and that it contains an element of general utility in enhancing recombinant protein expression systems.

5' Untranslated Regions↗
PubMed2002

Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection.

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) (32)P-labeled riboprobe generated by in vitro transcription of the U(S)8.5 ORF bound three proteins identified as the products of U(S)11, U(L)47, and U(L)49 (VP22) genes. (ii) Viral RNA was bound to U(L)47 or U(S)11 proteins immune precipitated from cells transduced with baculoviruses expressing U(L)47 or U(S)11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a U(S)8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the U(S)8-12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in approximately 1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-U(S)8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the U(L)47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.

Animals↗
PubMed1977

Presence of a putative 15S precursor to beta-globin mRNA but not to alpha-globin mRNA in Friend cells.

Dimethyl sulfoxide-induced Friend cells were labeled for periods of 5-60 min. The denatured RNA was fractionated by sucrose gradient centrifugation and the distribution of alpha- and beta-globin-specific [(3)H]RNA was determined by hybridization to hybrid plasmids containing mouse alpha- and beta-globin DNA, respectively. After 5 min of labeling, a 15S peak of beta-globin-specific (but not alpha-globin-specific) [(3)H]RNA was detected, next to an equal amount of 10S beta-globin [(3)H]RNA. With increasing periods of labeling, the amount of 15S beta-globin [(3)H]RNA remained constant but the amount 10S beta-globin [(3)H]RNA increased steadily. alpha-Globin-specific [(3)H]RNA sedimented at 11 S after 5 min of labeling and at 9.5 S after longer labeling periods. Analysis of 15S globin-specific [(3)H]RNA purified by the poly(dC)-cDNA method [Curtis, P. J. & Weissmann, C. (1976) J. Mol. Biol. 106, 1061-1075] showed oligonucleotides characteristic of beta-globin mRNA but not of alpha-globin mRNA, as well as about 20 new oligonucleotides. Our results suggest that 10S beta-globin mRNA arises via a 15S precursor that has a half-life of 5 min or less; 9.5S alpha-globin mRNA may be derived from an 11S precursor.

Animals↗
PubMed1979

Hormone-induced increase in levels of functional mRNA and alpha-amylase mRNA in barley aleurones.

Incubation of barley aleurone cells with gibberellic acid produces a progressive increase in the RNA content of the cells. The activity of poly(A)-containing RNA, measured in an in vitro wheat germ protein-synthesizing system, reaches a maximum approximately 12 hr after hormone addition and declines thereafter. The structurally intact functional mRNA content in these cells, measured as poly(A)-RNA with 5' "caps," also shows a maximum at 12 hr and correlates with the translational capacity of poly(A)-RNA. Activation of mRNA by guanylylation or methylation after addition of gibberellic acid is ruled out. Available evidence indicates that gibberellic acid stimulates protein synthesis by increasing the synthesis of mRNA. Studies with cycloheximide suggest that the induction of synthesis of alpha-amylase mRNA by gibberellic acid requires protein synthesis after hormone addition.

Journal Article↗
PubMed1979

Parvovirus RNA transcripts containing sequences not present in mature mRNA: a method for isolation of putative mRNA precursor sequences.

We report here a method of RNA preparation that may enrich for precursor RNA sequences and the results of an investigation of adeno-associated virus (AAV) RNA transcription that used this method. Whole cells were lysed with detergent and high salt and separated into supernatant and pellet (crude chromatin) fractions. These fractions were then separately deproteinized by proteolytic digestion and phenol extractions. DNA was removed from the preparation by two cycles of pancreatic DNase digestion and phenol extraction. Hybridization analyses of the RNA obtained from AAV/adenovirus-infected KB (human) cells revealed some AAV-specific RNA sequences that were not present in the mature 20S mRNA. These additional sequences were contained in AAV RNA molecules present in the pellet fraction, whereas the 20S AAV mRNA accumulated in the supernatant. A species of AAV-specific RNA (about 22S), which was associated only with the pellet fraction and was labeled only after a short pulse, appeared to have a kinetic relationship with the more stable cytoplasmic 20S mRNA. These putative AAV mRNA "precursors" and precursor sequences were not observed previously when conventional methods were used to obtain RNA from either whole cells or isolated nuclei.

Base Sequence↗
PubMed1984

Anti-mRNA: specific inhibition of translation of single mRNA molecules.

A plasmid was constructed to generate RNA complementary to the beta-galactosidase mRNA under control of the phage lambda PL promoter. When this anti-mRNA was produced, synthesis of beta-galactosidase was dramatically inhibited (98%). Syntheses of galactoside permease and transacetylase, whose coding sequences are downstream of the beta-galactosidase coding region, are inhibited to a lesser degree, 80% and 55%, respectively. The generation of anti-mRNA that can be targeted to inhibit a single species of mRNA molecule within cells provides a potent mechanism by which specific transcripts can be translationally inactivated. This can be used to determine the function of proteins as well as to select cloned genes in a single rapid and convenient step.

Acetyltransferases↗
PubMed1990

Regulation of mRNA abundance in activated T lymphocytes: identification of mRNA species affected by the inhibition of protein synthesis.

Inhibition of protein synthesis has often been observed to increase the concentration of mRNAs that encode proteins associated with the regulation of cell division. As two-dimensional gel electrophoresis permits the simultaneous monitoring of individual elements in large populations of gene products, we have used this technique to assess the effect of cycloheximide treatment on the mRNA complement of activated mouse T cells in an objective fashion. Two-dimensional gels of proteins generated by cell-free translation of mRNA from T-cell blasts display about 400 spots; only 5 of these are reproducibly enhanced by cycloheximide treatment and about 4 are diminished. The cDNA cloning vector lambda jac allows analysis of large arrays of molecular clones by cell-free expression, and we have used it in a sibling selection scheme to isolate a clone of one of the prominently induced mRNA species, which we refer to as chx1. chx1 mRNA concentration is increased by cycloheximide treatment of activated B cells, as well as T cells, and it is rapidly and transiently induced, in a cycloheximide-enhanced manner, upon serum stimulation of resting 3T3 fibroblastoid cells. The chx1 protein is hydrophilic, is slightly basic, and has patches of homology with the Jun-D gene product. The chx1 gene is remarkable in its lack of detectable introns and of strong bias against CpG dinucleotides.

Amino Acid Sequence↗