Relative RNA Levels Research Articles

Mitochondrial RNA abundance in differentiating human colonie epithelial tumor cells estimated through use of a mitochondrial genome map

We describe a procedure for quantification of mitochondrial (mt) RNA present in total RNA extracts of HT-29 human colonie adenocarcinoma cells grown under conditions for rapid growth (25 mM glucose) or differentiation (25 mM trehalose or 5 mM butyrate). Purified mt DNA was fragmented into specific coding regions using restriction endonuclease sites predicted from HeLa cell mt DNA sequence and probed with either 32P-labelled mt DNA or cDNA made from total RNA of HT-29 or HeLa cells. The amounts of probe that hybridized to various gene-encoded mt DNA fragments or RNA were quantified by laser densitometry. Use of 13 restriction endonucleases revealed that most if not all the mt DNA of HT-29 and K562 leukemic cells was comparable in size to that of HeLa cells. Relative levels of mt RNA from rapidly growing HT-29 and HeLa cells were lower than those measured for differentiated HT-29 cells induced by either trehalose or butyrate. In rapidly growing HT-29 cells and HeLa cells, the highest levels of specific mt RNAs were those encoded by mt DNA sequences immediately flanking the nested promoters and the heavy-strand replication origin ( O H). Expression patterns of specific mt RNAs from HT-29 cells treated with butyrate and with trehalose were similar, but not identical. In either case, the mt RNAs that increased the most were those coded by mt DNA sequences located downstream from the light-strand replication origin ( O L), suggesting a novel pattern of expression not seen before.

Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation.

cis-acting inhibitory region (IR) sequences were identified within the gag/pol gene of the human immunodeficiency virus type 1 (HIV-1) by using a novel feedback-stimulated, rev-independent tat reporter gene to screen HIV-1 sequences in transient expression assays. Two regions, a 1,295-nucleotide segment in the gag gene (IR-1) and a 1,932-nucleotide segment of the pol gene (IR-2), each inhibited reporter gene expression 10- to 20-fold. IR-1 and IR-2 both contained subsequences which inhibited reporter gene expression. Introduction of IR sequences into a heterologous reporter plasmid, pCMV-CAT, resulted in decreased chloramphenicol acetyltransferase expression, suggesting that the inhibitory effect was not restricted to a reporter gene under the control of the HIV-1 promoter. The presence of HIV IR sequences in cis did not alter relative levels of reporter gene RNA; however, fractionation studies revealed IR-containing RNA accumulated in the nucleus. These findings demonstrate that IR sequences within the gag/pol region affect gene expression by altering the cellular distribution of viral RNA.

A flow‐cytometric analysis of hybridoma growth and monoclonal antibody production

A flow cytometric kinetic study of hybridoma growth and monoclonal antibody production is presented, along with the influence of glutamine on intracellular responses such as (relative) cell size, and cell RNA and total protein content. Specific findings are: (1) RNA content remained constant throughout the growth phase, then fell drastically as the cells entered the stationary phase. Also, in stationary phase, RNA content of antibody-producing cells was higher than for those not secreting antibody. (2) The cell size was constant and maximal throughout exponential phase, and diminished monotonically during later stages. (3) Average protein and antibody cellular content declined dramatically upon glutamine exhaustion. Thus, relative RNA levels and cell size provided quantitative determinants of both cell growth state and antibody secretion conditions. These results encourage consideration of structured kinetic studies which recognize the quality of the biophase.

Transcriptional and post-transcriptional processes regulate expression of RNA encoding the small subunit of ribulose-1, 5-biphosphate carboxylase differently in petunia and in soybean

The effects of white light, far-red light and darkness on the in vitro transcription and RNA levels of the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) were investigated in petunia and in soybean. In petunia plants treated with 48 hours of darkness the in vitro transcription rate of two of the rbcS subfamilies of petunia, rbcS A and rbcS C, declined 32- and 8-fold respectively, whereas treatment of dark-adapted plants with light caused the in vitro transcription rate of these subfamilies to return to their light-grown levels. Relative RNA levels of rbcS A and rbcS C declined in parallel with in vitro transcription rate changes upon treatment of petunia plants with darkness. However, while relative RNA levels of rbcS C changed in parallel with in vitro transcription rate under all conditions of far-red light and white light tested, there were differences between the changes in rbcS A in vitro transcription rate and RNA levels which were consistent with post-transcriptional regulation of rbcS A RNA. In addition we observed that nuclei isolated from the leaves of plants which were exposed to darkness for periods of 72 hours or longer were transcriptionally inactive. Similar experiments on the in vitro transcription and relative levels of the rbcS RNA in soybean seedlings have lead to the hypothesis that rbcS RNA is less stable in light than in darkness. In contrast, small decreases in rbcS in vitro transcription rate in mature soybean plants treated with darkness were accompanied by large decreases in rbcS RNA, suggesting that rbcS RNA was degraded more rapidly in darkness than in light in these plants. We have shown that differences in the modulation of rbcS RNA levels by post-transcriptional mechanisms exist between plants which belong to different orders, and between different developmental states of the same plant species.

Temporal expression of genes encoding free radical–metabolizing enzymes is associated with higher mRNA levels during in utero development in mice

The interaction of reactive oxygen metabolites with DNA is well characterized and may result in mutagenesis, chromosome aberrations, and modulation of gene expression. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) catalyze enzymatic reactions to remove oxidant stresses, particularly O2- and H2O2. The role of these enzymes during in utero development of the embryo and the developmental pattern of expression of the embryonic genes encoding them is not known. We examined the in utero developmental expression and activity of the three free-radical-metabolizing enzymes in mice. We collected mouse fetuses at different stages of development and examined total RNA populations by Northern and slot blots using gene-specific cDNA probes. In addition to quantifying the probe-specific RNAs, activities of the three enzymes were also evaluated on the same tissue samples. The gene-specific RNAs and the associated enzyme activities are detectable with somite formation (day 8 postcoitus [p.c.]) in mice. The relative RNA values for each of the genes studied are higher in in utero stages as compared with the adult. The specific activities of these enzymes, on the other hand, follow a characteristic increase with development and growth. The relative RNA levels for each of the genes studied are higher during in utero growth and development than the relative enzyme activity values (between day 8 and day 18, third trimester) in the liver and carcass. This may suggest that the mRNA specific to these genes may accumulate in utero and are not translated immediately. Such accumulating transcripts are translated efficiently after birth, when these enzymes are particularly needed with the advent of aerobic respiration.

Post-transcriptional control of IL-1 gene expression in the acute monocytic leukemia line THP-1

The acute monocytic leukemia cell line THP-1 secretes predominantly IL-1 beta after treatment with bacterial lipopolysaccharide and tumour promoting phorbol ester (PMA). IL-1 alpha is also secreted, but represents less than 10% of the total IL-1 activity. This differential is reflected at the level of mRNA as IL-1 beta mRNA is more abundant than IL-1 alpha mRNA. Studies of transcription in isolated nuclei however indicate that each gene is transcribed at a similar rate, suggesting that post-transcriptional mechanisms regulate the relative abundance of IL-1 alpha and IL-1 beta mRNA. Measurement of RNA half life after addition of alpha-amanitin (an inhibitor of RNA polymerase II) indicate that IL-1 alpha mRNA is not as stable as IL-1 beta mRNA suggesting one mechanism for the different relative levels of RNA.

Differential expression of a gene for a methionine-rich storage protein in maize

A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A full-length cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.

Post-transcriptional regulation of the abundance of mRNAs encoding alpha-tubulin and a 94,000-dalton protein in teratocarcinoma-derived stem cells versus differentiated cells.

Changes in the expression of the genes encoding alpha-tubulin and a 94,000-dalton protein (p94) specified by a cDNA clone, p4-30, were examined in a differentiated teratocarcinoma-derived parietal endoderm cell line, PYS-2, and an undifferentiated teratocarcinoma stem cell line, F9. Relative to other proteins or mRNA species, the synthesis rate of the alpha-tubulins and of p94, as well as the levels of their corresponding cytoplasmic mRNAs, were lower in PYS-2 than in F9 cells. The decrease was greater for the relative abundance of cytoplasmic alpha-tubulin mRNA than for p94 mRNA. Similarly, induction of differentiation of F9 cells by simultaneous exposure to retinoic acid (RA) and dibutyryl cyclic AMP resulted in reduced relative levels of the cytoplasmic mRNAs for these proteins. The reduction in abundance of the two RNA species was not due to a decrease in growth rate since the differentiated cells, PYS-2, RA-treated F9, and RA plus dibutyryl cyclic AMP-treated F9 cells, grew at a rate similar to that of undifferentiated F9 cells. However, induction of differentiation of F9 cells by treatment with RA alone did not cause down-regulation of the two RNA species. The relative levels of total cellular RNA encoding alpha-tubulin and p94 in PYS-2 cells were also lower than those in F9 cells to an extent comparable to the decrease in the cytoplasmic RNAs. Since the apparent relative rates of RNA transcription were similar in both cell types, we conclude that the reduction in relative levels of the alpha-tubulin and p94 RNAs in the cell depends largely on the relative stability of the two RNAs and not on the relative rates of transcription. The faster disappearance of the two RNA species relative to other cellular RNAs from actinomycin D-treated PYS-2 compared with F9 cells is consistent with this interpretation.

A tissue-specific transcription enhancer element in the human immunoglobulin λ light chain locus

An 8.0-kb EcoRI fragment of the human immunoglobulin (Ig) λ light chain locus carrying the Ke −Oz − and Ke −Oz + constant region genes and flanking sequences was studied for the presence of a transcriptional enhancer. Two types of assays were used. In the first, we measured the ability of recombinant plasmids carrying the 8.0-kb Ig fragment covalently linked to the aminoglycoside phosphotransferase ( aph) gene to transform mouse myeloma or mouse fibroblast cells to geneticin resistance. In the second, we used RNA spot and Northern blot hybridization analyses to determine the relative levels of aph specific RNA transiently expressed after DNA transfection. In fibroblast cells, the transformation frequencies were independent of the presence of the Ig fragment in the vector and there was no difference in the level of transient expression of the aph gene. In myeloma cells, the Ig fragment enhanced at least 10-fold both the number of transformants and the level of aph gene expression over that obtained with vector alone. These results indicate the presence of a tissue-specific transcriptional enhancer in the 8.0-kb EcoRI fragment of the human Ig Cλ locus.

Verticillium wilt of chrysanthemum: relative water content and protein, RNA, and chlorophyll levels in leaves in relation to visible wilt symptoms

Shifts in host metabolism during early stages of foliar infection and symptom development are examined. Verticillium dahliae Kleb. invaded and colonized chrysanthemum leaf tissue before symptom expression. Similar numbers of fungal propagules were recovered from non-symptomatic and symptomatic parts of a leaf lobe. No changes in relative water content, tissue permeability, RNA, and protein levels and only a slight decrease in chlorophyll level were detected within infected leaf tissue before the appearance of visible wilt. These data are discussed in relation to the possible role of senescence in the Verticillium wilt syndrome in chrysanthemum.