Amount Of Messenger RNA Research Articles

Transsynaptic expression of a presynaptic glutamate receptor during hippocampal long-term potentiation.

Repetitive activation of excitatory synapses in the hippocampus produces a persistent enhancement of synaptic efficiency known as long-term potentiation (LTP). In anesthetized and in freely moving rats, the induction of LTP in the perforant path led to a transient increase in the amount of messenger RNA (mRNA) coding for a presynaptic glutamate receptor (GR33) in dentate granule cells. The amount of GR33 mRNA was increased for at least 5 hours after the induction of LTP but was indistinguishable from control values 1 day after induction. The N-methyl-D-aspartate receptor antagonist 2-aminophosphonovalerate prevented the induction of both LTP and the increase in GR33 mRNA. The amount of GR33 protein was increased in the mossy fiber terminal zone of dentate granule cells 5 hours after the induction of LTP. These results suggest that the induction of LTP in synapses at one stage in a neural network may lead to modification in synaptic function at the next stage in the network.

Regional codon randomization: defining a TATA-binding protein surface required for RNA polymerase III transcription.

The TATA-binding protein (TBP) is required for transcription by all three nuclear RNA polymerases. TBP was subjected to regional codon randomization, a codon-based mutagenesis method that generates complex yet compact protein libraries. Analysis of 186 temperature-sensitive TBP mutants yielded 65 specifically defective in transcription by RNA polymerase III (Pol III). These mutants map to a limited TBP surface that may interact with Tds4, a component of the Pol III transcription factor TFIIIB. Strains that contain the Pol III-defective derivatives have increased amounts of messenger RNA, which suggests that competition among TBP-interacting factors for limiting quantities of TBP determines the ratio of Pol II and Pol III transcription in vivo.

Molecular Abnormalities of a Phosphoglycerate Kinase Variant Generated by Spontaneous Mutation

A new case of X chromosome-linked phosphoglycerate kinase (PGK) abnormality is described. The male proband was mentally retarded, had behavior disorders, and displayed episodes of hemolytic anemia. The enzyme activity of red blood cells from the patient was about 10% of normal, and that of the cultured fibroblasts was about 50% of normal cells. The variant PGK was characterized by a lower affinity for the substrates, reduced thermostability, and increased anodal electrophoretic mobility. The pH activity profile of the variant enzyme was different from that of normal. The amount of messenger RNA (mRNA) in the variant fibroblasts was comparable to that of normal fibroblasts. The mRNA coding for PGK was subjected to coupled reverse transcription followed by amplification by the polymerase chain reaction. Nucleotide sequence of the variant cDNA showed a point mutation, T/A----C/G transition, in exon 9 of the variant gene. No other mutation was found in all coding regions of the variant. The mutation should cause Cys----Arg substitution at the 315th position from the NH2-terminal Ser of PGK, and it created an additional Ava II (or isoschimatic) cleavage site in the variant gene. Because the variant gene was not detected in the proband's mother and siblings, it must have been generated by spontaneous mutation during oogenesis.

Molecular abnormalities of a phosphoglycerate kinase variant generated by spontaneous mutation

A new case of X chromosome-linked phosphoglycerate kinase (PGK) abnormality is described. The male proband was mentally retarded, had behavior disorders, and displayed episodes of hemolytic anemia. The enzyme activity of red blood cells from the patient was about 10% of normal, and that of the cultured fibroblasts was about 50% of normal cells. The variant PGK was characterized by a lower affinity for the substrates, reduced thermostability, and increased anodal electrophoretic mobility. The pH activity profile of the variant enzyme was different from that of normal. The amount of messenger RNA (mRNA) in the variant fibroblasts was comparable to that of normal fibroblasts. The mRNA coding for PGK was subjected to coupled reverse transcription followed by amplification by the polymerase chain reaction. Nucleotide sequence of the variant cDNA showed a point mutation, T/A----C/G transition, in exon 9 of the variant gene. No other mutation was found in all coding regions of the variant. The mutation should cause Cys----Arg substitution at the 315th position from the NH2-terminal Ser of PGK, and it created an additional Ava II (or isoschimatic) cleavage site in the variant gene. Because the variant gene was not detected in the proband's mother and siblings, it must have been generated by spontaneous mutation during oogenesis.

Induction of tyrosine hydroxylase in the rat substantia nigra by local injection of forskolin

Forskolin (FSK) was locally injected into the substantia nigra (SN) of anesthetised rats. The day after injection (24 and 36 hr), tyrosine hydroxylase (TH) activity increased locally in this structure but remained unmodified in the ipsilateral caudate nucleus (CN). The amount of messenger RNA for TH (TH-mRNA) was also increased in the SN 24 hr after the injection. However, TH protein content was modified neither locally in the SN nor in the ipsilateral CN. In addition, the decrease of the ratio between dopamine and its first metabolite in the CN and the SN suggested a decreased activity of the dopaminergic nigral cells. The absence of increase of the protein synthesis in spite of the fact that TH-gene transcription was initiated could be the consequence of the inhibition of dopaminergic cells by the drug. These results confirm that, in vivo, TH induction is cAMP-dependent and demonstrate that the TH-gene activity is not strictly coupled to the activity of dopaminergic cells in the SN.

Requirement of ets -2 Expression for Xenopus Oocyte Maturation

A molecular clone of the Xenopus laevis ets-2 gene was isolated from an oocyte complementary DNA library. The amount of messenger RNA (mRNA) in each oocyte or embryo was almost constant during oogenesis and was maintained until the blastula stage of embryonic development, indicating that the observed 3.2-kilobase transcript is a maternal message. The only normal adult tissue in which ets-2 mRNA was detected was the ovary. Injection of antisense oligonucleotides homologous to the ets-2 sequence into oocytes led to degradation of the mRNA and blocked hormone-induced germinal vesicle breakdown. The ets-2 product is thus required for the meiotic maturation of Xenopus oocytes.

Transformation-Associated Alterations in Interactions Between Pre-B Cells and Fibronectin

Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2-10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2-10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co-cultured with M2-10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

Transformation-associated alterations in interactions between pre-B cells and fibronectin

Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

Quantitative analysis of myelin protein gene expression during development in the rat sciatic nerve

We determined the temporal profile of expression of the genes encoding the P 0 glycoprotein, the myelin-associated glycoprotein (MAG), the myelin basic protein (MBP), the proteolipid protein (PLP), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in the sciatic nerve of rats. The level of expression of the MAG gene occurred maximally in animals 13 days of age, approximately one week earlier than the peak expression of the MBP and P 0 genes. The genes encoding PLP and CNP were not expressed developmentally in a manner that correlated with the myelination of the sciatic nerve. Furthermore, using RNA synthesized in vitro, specific for each of the myelin protein genes, we have determined the absolute amounts of messenger RNA for the various myelin proteins in total RNA from sciatic nerves. P 0 and MBP RNA were present at very high levels, whereas the amount of MAG, PLP and CNP RNA were much less.

Limbic Seizures Increase Neuronal Production of Messenger RNA for Nerve Growth Factor

Nerve growth factor (NGF) produced by telencephalic neurons provides critical trophic support for cholinergic neurons of the basal forebrain. In situ hybridization and nuclease protection analyses demonstrate that limbic seizures dramatically increase the amount of messenger RNA for NGF in the neurons of the hippocampal dentate gyrus within 1 hour of seizure onset and in broadly distributed neocortical and olfactory forebrain neurons some hours later. The increased messenger RNA species is indistinguishable from messenger RNA for transcript B of the beta subunit of NGF from mouse submandibular gland. Thus, the expression of a known growth factor is affected by unusual physiological activity, suggesting one route through which trophic interactions between neurons in adult brain can be modified.

Rapid expression of protooncogenes c-fos and c-myc in B-chronic lymphocytic leukemia cells during differentiation induced by phorbol ester and calcium ionophore

The peripheral blood mononuclear cells from patients with B-chronic lymphocytic leukemia (B-CLL) were incubated for 0.5 h to 72 h in the presence of the phorbol ester TPA, the calcium ionophore A23187, or a combination of these reagents. Using Northern blot analysis, total cellular RNA was prepared from cells harvested at different time points and hybridized with DNA clones specific for the protooncogenes c-fos and c-myc. While untreated control cells lacked detectable amounts of messenger RNA (mRNA), increase in the level of c-fos mRNA was noted as early as 0.5 h after exposure to the inducers. Peaks of c-fos and c-myc transcript accumulation were seen at 1 h and 4 h after induction, respectively. The most effective inducer was double stimulation with TPA plus A23187. The kinetics of c-fos and c-myc mRNA accumulation in B- CLL appear to be similar to those reported for normal lymphocytes that have been either activated by physiologic external stimuli or by direct activators of protein kinase C and calcium flux (such as TPA and A23187). No direct link between oncogene expression and proliferation or differentiation parameters could be established. These results document that expression of c-fos and c-myc genes, which are among the earliest events following stimulation of the protein kinase signal transduction pathway, can be successfully induced in B-CLL cells. The data provide further evidence for the hypothesis that signal transmission downstream of protein kinase C is intact in B-CLL.

Rapid Expression of Protooncogenes c-fos and c-myc in B-Chronic Lymphocytic Leukemia Cells During Differentiation Induced by Phorbol Ester and Calcium Ionophore

The peripheral blood mononuclear cells from patients with B-chronic lymphocytic leukemia (B-CLL) were incubated for 0.5 h to 72 h in the presence of the phorbol ester TPA, the calcium ionophore A23187, or a combination of these reagents. Using Northern blot analysis, total cellular RNA was prepared from cells harvested at different time points and hybridized with DNA clones specific for the protooncogenes c-fos and c-myc. While untreated control cells lacked detectable amounts of messenger RNA (mRNA), increase in the level of c-fos mRNA was noted as early as 0.5 h after exposure to the inducers. Peaks of c-fos and c-myc transcript accumulation were seen at 1 h and 4 h after induction, respectively. The most effective inducer was double stimulation with TPA plus A23187. The kinetics of c-fos and c-myc mRNA accumulation in B-CLL appear to be similar to those reported for normal lymphocytes that have been either activated by physiologic external stimuli or by direct activators of protein kinase C and calcium flux (such as TPA and A23187). No direct link between oncogene expression and proliferation or differentiation parameters could be established. These results document that expression of c-fos and c-myc genes, which are among the earliest events following stimulation of the protein kinase signal transduction pathway, can be successfully induced in B-CLL cells. The data provide further evidence for the hypothesis that signal transmission downstream of protein kinase C is intact in B-CLL.

Circulating human blood platelets retain appreciable amounts of poly (A) + RNA

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A) + RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A) + RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A) + RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A) + RNA and that this RNA can be harvested by the described approach. The poly (A) + RNA provides templates for the synthesis of cDNA's that code for platelet proteins.

Growth factor expression in normal, benign, and malignant breast tissue

Several oncogenes seem to encode certain growth factors that may play a part in regulating cell growth in tumours. To assess whether such factors are synthesised endogenously by tumour cells the amounts of messenger RNA for several growth factors known to be synthesised by cancer cells of the breast in vitro were examined in biopsy specimens from 52 malignant and 15 non-malignant tumours of the breast and four samples of normal breast. Transforming growth factor beta messenger RNA was significantly more abundant in breast cancers (32 of 42 (76%) having appreciable amounts) than non-malignant breast tissue (five of 13 (38%) having similar amounts). Transcripts for both transforming growth factor alpha and its receptor, epidermal growth factor receptor, were found more commonly in carcinomas that were negative for oestrogen receptor (64% and 87%, respectively) than in those that were positive (27% and 30%, respectively). Insulin-like growth factor II messenger RNA was present in all 15 samples of non-malignant tissue but was found (in considerably lower amounts) in only 11 of 21 (52%) carcinomas. Epidermal growth factor receptor was also found in all non-malignant breast tissues, compared with 19 of 45 (42%) carcinomas. Platelet derived growth factor A and B chain transcripts coexisted in all normal and benign tissue and most carcinomas. This differing pattern of expression growth factors in tissue from malignant tumours compared with benign tumours and normal breast tissue suggests that some growth factors, particularly transforming growth factors alpha and beta, may have an important role in controlling growth of human breast cancers, particularly those that are hormone independent.

Measurement of the amount of mRNA for gonadotropins during an estradiol-induced preovulatory-like surge of LH and FSH in ovariectomized ewes

The amount of messenger RNA (mRNA) for luteinizing hormone β-subunit (LHβ), follicle-stimulating hormone β-subunit (FSHβ) and α-subunit was measured during estradiol-17β (E) positive feedback in ovariectomized (OVX) ewes. During the anestrous season, OVX ewes were given an i.m. injection of E (25 μg: n=5) or oil (control; n=4) and hourly blood samples were collected for 16 hr. After blood collection, ewes were killed and anterior pituitary glands were removed for analysis of hormone and mRNA content. Preovulatory-like increases in serum concentrations of LH and FSH were measured in E-treated OVX ewes. In two E-treated OVX ewes the serum concentrations of LH and FSH were still increasing, whereas in the remaining three E-treated OVX ewes, serum concentrations of LH were on the decreasing portion of the E-induced preovulatory-like surge. Pituitary content of LH was lower (P<.10) in E-treated OVX ewes when serum concentrations of LH were decreasing than that measured in control ewes or E-treated OVX ewes in which serum concentrations were still increasing. Pituitary content of FSH and prolactin were similar (P>.05) among all groups. The amount of mRNA for LHβ-subunit was similar (P>.05) in ewes in which serum concentrations of LH were increasing and in control ewes, but was lower (P<.05) in ewes with decreasing levels of LH. The amount of mRNA for FSHβ-subunit was lower (P<.05) in all E-treated OVX ewes (independent of whether serum concentrations of FSH were increasing or decreasing) than that measured in control ewes. There was no difference (P>.05) in the amount of mRNA for α-subunit among any groups. Thus, amounts of mRNA for the β-subunits of gonadotropins are reduced, while amounts of mRNA for α-subunit are unchanged during estradiol positive feedback in OVX ewes.

Photocontrol of Sorghum Leaf Phosphoenolpyruvate Carboxylase

The mechanism underlying the light effect on phosphoenolpyruvate carboxylase (PEPC) from the C(4) plant sorghum (Sorghum vulgare Pers., var Tamaran) leaves was investigated. Following exposure to light a new isozyme of PEPC, specific for the green leaf and responsible for primary CO(2) fixation in photosynthesis, was established. Northern blot experiments revealed the presence of PEPC mRNA showing a molecular weight of 3.4 kilobases. During the greening process, concomitant to enzyme activity, PEPC protein and PEPC messenger RNA amounts increased considerably. This photoresponse was shown to be under phytochrome control.

Necrobiosis Lipoidica: Ultrastructural and Biochemical Demonstration of a Collagen Defect

Ten patients with necrobiosis lipoidica lesions were studied. Five patients had diabetes mellitus. The age of the patients varied from 15 to 73 years and the duration of the skin lesions was from 2 to 20 years. Histologically, the lesions were characterized by degeneration of collagen and elastin. In some lesions elastin fibers could be seen in areas devoid of normal-looking collagen. Electron microscopy revealed loss of cross-striation of collagen fibrils and a marked variation in the diameter of individual collagen fibrils. The concentration of collagen, measured by assay of hydroxy-proline, a collagen-specific amino acid, was markedly decreased in the lesional skin, but the ratio of type I/III collagen was unchanged in the affected skin. Fibroblasts established from affected skin synthesized less collagen than cells derived from healthy-looking skin. The decreased collagen synthesis was due to a decreased amount of messenger RNA for type I procollagen, measured by hybridization with a specific human cDNA clone. The production of collagenase by these fibroblasts was not increased. Our results thus indicate that in necrobiosis lipoidica lesions, collagen fibrils are defective and the amount of collagen is reduced, probably due to decreased synthesis of collagen by affected fibroblasts.

Regulation of ferritin synthesis in malignant and non-malignant lymphoid cells

The different amounts of H-rich and L-rich isoferritins found in malignant and non malignant lymphoid cells are accompanied by proportional variations in the relative quantity of messenger RNAs for the H and L subunits of ferritin. The correlation between levels of messenger RNAs and proteins suggests that the amount of messenger RNA plays an important role in ferritin biosynthesis in these cells. The enhanced expression of ferritin messenger RNAs in some neoplastic cells is not caused by gross alterations in the structure of ferritin genes.

Molecular cloning and biosynthetic regulation of cry1 gene of Saccharomyces cerevisiae.

The cryptopleurine resistance gene, cry1, of Saccharomyces cerevisiae has been molecularly cloned using genetic complementation of cryptopleurine sensitivity by the cryptopleurine resistance gene contained in a clone library prepared from DNA of a cryptopleurine resistant strain. Analysis of RNA transcripts indicated that the cry1 gene is the template for a transcript of approximately 900 bases and that the primary transcript contains an intron of approximately 300 bases. In vitro hybrid selection translation experiments indicated that this transcript encodes a protein of molecular weight 17 kilodaltons which on two-dimensional SDS polyacrylamide gels exactly coincides with ribosomal protein rp59. Further analysis showed that when the gene was present on a plasmid of about five copies per cell the amount of messenger RNA was elevated approximately five-fold compared to a cell that had only a single chromosomal copy. The rate of synthesis of ribosomal protein rp59 was not detectably elevated. These data suggest that the cry1 gene is regulated, at least in part, post-transcriptionally.

29] Biosynthesis of acetylcholine receptor in Vitro

Torpedo acetylcholine receptor is accessible to analysis by cell-free protein synthesis, like other membrane proteins studied previously. The factors deserving particular attention in this system are the small amounts of messenger RNA contained in electroplax tissue, the inherent complexity of an oligomeric membrane protein (necessitating the use of subunit-specific antibodies), the anomalous electrophoretic behavior of the primary translation products (attributable in part to the high--SH content of sAChRs), and the multiple glycosylated forms that appear upon processing by microsomal membranes. Although not all these complexities can be satisfactorily explained, they have been empirically characterized and can be well controlled if the appropriate procedures are followed.