Larger mRNA Species Research Articles

Analysis of RNA species of various sizes from stationary-phase planktonic yeast cells of Candida albicans

We initiated a comparison of Candida albicans stationary-phase gene expression with other growth states. The widely used hot acid phenol method for RNA extraction did not extract rRNA from late stationary-phase cells. The RNA from growing yeast cells, hyphae and biofilm was biased towards small-sized RNA. The 2 : 1 ratio between the two large rRNA bands was rarely obtained. Real-time reverse transcriptase PCR was used to determine mRNA extraction by several methods for OXR1, IRA2, RAD50, PNC1 and CHS2, which have 300 bp-8 kb coding regions, and ACT1, EFB1 and TDH3, sometimes used as internal standards. Only smaller-sized cDNA species were amplified from some extracts. Crushing cells with glass beads in liquid nitrogen before RNA extraction by the hot phenol method (CGB) yielded an unbiased distribution for rRNA and mRNA as verified by real-time reverse transcriptase PCR. With the CGB method, the large mRNA species, RAD50, IRA2 and OXR1, were present throughout the stationary phase, whereas the CSH2 transcript increased. The ACT1, EFB1 and TDH3 transcripts decreased in the stationary phase, making them unsuitable for standardization. The CGB method yielded high-quality RNA with the various growth conditions and permitted the comparison of stationary-phase transcripts with those obtained under other conditions.

Mydj2 as a potent partner of hsc70 in mammalian cells.

Dj2 is a member of the DnaJ family of proteins, which regulate the chaperoning function of the hsp70s. We isolated a monkey cDNA dj2 clone corresponding to the large mRNA species encoded by the gene. This mRNA differs from the small mRNA produced by the same gene in that it contains a long 3' untranslated region. Both messages were found to be equally stable and to produce the same protein, which is susceptible to farnesylation. Studies in mouse tissues and various cell lines revealed that these messages and their products are differentially expressed. Surprisingly, we found that only the nonfarnesylated form of dj2 is capable of translocating to the cell nucleus, especially after heat shock. Finally, based on protein interaction studies, our results indicate that dj2 is a specific partner for hsc70 and not for hsp70.

Temporal patterns of feline immunodeficiency virus transcripts in peripheral blood cells during the latent stage of infection

We have investigated the in vivo state of feline immunodeficiency virus (FIV) transcription in peripheral blood mononuclear cells (PBMC) of chronically FIV-infected, asymptomatic cats. FIV was detected in a high percentage of PBMC but not in the plasma of these cats. By quantitative reverse transcription-PCR (RT-PCR) analysis. FIV transcriptional status in the PBMC was characterized by extremely low or undetectable levels of unspliced or singly spliced mRNAs and predominantly multiply spliced mRNAs. Upon stimulation in vitro, however, the larger mRNA species and infectious virus production were rapidly induced in the PBMC. Furthermore, we demonstrated that viral production was induced in association with differential increases in the levels of each multiply spliced mRNA coding for FIV regulatory proteins. From these results, we suggest that replication of FIV is blocked at an early stage of gene expression in vivo, as described in asymptomatic human immunodeficiency virus (HIV) -infected patients, and that FIV infection in cats may be a useful model for clinical latency of HIV infection in man. Moreover, we propose that the replication of FIV in vivo may be controlled by the differential expression of each multiply spliced mRNA.

The mRNA for alpha 1(XIX) collagen chain, a new member of FACITs, contains a long unusual 3' untranslated region and displays many unique splicing variants.

We have isolated cDNAs and completed for the first time the primary structure for a novel collagenous chain that was partially characterized earlier and named alpha 1(Y) chain [Yoshioka, H. et al. (1992) Genomics 13, 884-886]. The size of the coding region was unexpectedly small compared with the length of the mRNA (> 10 kb), owing to the presence of a long 3' untranslated region (> 5 kb). The predicted polypeptide contained 1,142 amino acid residues with a 23-residue signal peptide consisting of 5 collagenous domains of 70-224 residues in length, interspersed and flanked with 6 noncollagenous (NC) domains. The primary structure is distinct from those of the 32 known collagen alpha-chains of types I through XVIII. Therefore, we designate this newly discovered collagen chain the alpha 1 chain of type XIX collagen. Sequence analysis suggested that this chain belongs to the recently discovered group of collagens known as FACITs (fibril associated collagens with interrupted triple-helices). Northern blotting analysis demonstrated hybridization of the cDNA to a large mRNA species (> 10 kb) extracted from a rhabdomyosarcoma cell line (CCL 136). We also isolated numerous truncated cDNA clones of which the 3' parts were different from the "proto" type of the mRNA of > 10-kb size. Sequence comparison between cDNAs and corresponding genomic DNA fragments indicated that unusual splicing events occurred through insufficient recognition at acceptor sites. Expression of the gene was extremely infrequent in the rhabdomyosarcoma cell line; it could be restricted to certain animal tissues both temporally and spatially during early development.

Identification and characterization of a novel high-molecular-weight form of the integrin α3 subunit

The integrin α 3 β 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti- α 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected α 3 β 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa α 3 and 115-kDa β 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti- α 3 antibody—Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa α 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa α 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa α 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the α 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa α 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human α 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin α 3 subunit consisting of the α 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative “light” chain to the 25-kDa light chain of the α 3 subunit.

Growth hormone-releasing hormone-like messenger ribonucleic acid and immunoreactive peptide are present in human testis and placenta.

Although the sequence of human GH-releasing hormone (GHRH) has been determined, all of the information concerning gene expression has been based on pathological sources of ectopic GHRH, since the only established physiological source of GHRH in humans is the hypothalamus. We recently reported the presence of extrahypothalamic GHRH-like mRNA and immunoreactive material in rat testis and placenta. To determine if human testis and placenta also contain immunoreactive GHRH-like peptides, tissue extracts were analyzed using enzyme-linked immunosorbent assay methodology. Both tissues had detectable quantities of immunoreactive peptide recognized by a monoclonal antibody to synthetic human GHRH-(1-44) (testis, 2.4 ng/g tissue, 0.68 ng/mg protein; placenta, 2.6 ng/g tissue, 0.36 ng/mg protein). The origin of these peptides was confirmed by extracting total RNA from human testis and placenta, with analysis on Northern blots probed with riboprobes for rat hypothalamic GHRH cDNA and human pancreatic tumor GHRH cDNA. Human testis and placenta total RNA both contain an approximately 790-nucleotide RNA species similar in size to that reported in ectopic GHRH-producing human tumors. In addition, two larger hybridization signals were seen at 3000 and 4900 nucleotides. These data suggest that testis and placenta are extrahypothalamic sites of expression of the human GHRH gene. Normal expression of the GHRH gene in extrahypothalamic sites may include transcription of larger mRNA species than those observed in ectopic pathological sources of GHRH expression.

Fastidious human adenovirus type 40 can propagate efficiently and produce plaques on a human cell line, A549, derived from lung carcinoma

Human adenovirus type 40 (Ad40) cannot propagate in conventional established human cell lines such as KB or HeLa cells. However, it has been shown that Ad40 DNA replicates in KB18 cells which express Ad2 E1B genes, suggesting that Ad40 is defective in the E1B gene function in KB or HeLa cells. We show here that Ad40 can propagate and produce plaques on A549 cells which do not contain Ad E1B genes. Our experiments show that the levels of replication of Ad40 DNA and production of infectious Ad40 virus in A549 cells are the same as or higher than those in 293 or KB18 cells. Dot blot analysis shows that the levels of Ad40 E1A and E1B mRNAs expressed in A549 cells at early to intermediate times postinfection are at least 10-fold higher than those in KB or KB18 cells. Northern (RNA) blot analysis shows that large E1B mRNA species (approximately 24S to 26S) are synthesized prior to the onset of DNA replication in A549 cells. No E1B mRNA species are synthesized in KB or KB18 cells at early times postinfection, and no differences in the expression of E1B mRNAs are seen between KB and KB18 cells. The experiment suggests that A549 cells have a cellular factor(s) which activates Ad40 E1B mRNA synthesis and that the E1B mRNA synthesis helps Ad40 propagation. In contrast, Ad40 can propagate in KB18 cells by using Ad2 E1B gene products that are constitutively expressed in this cell line. Furthermore, this result shows that Ad40 cannot propagate in KB cells because of the failure in the expression of E1B genes at early times postinfection.

Tissue-Specific Transcription Initiation and Effects of Growth Hormone (GH) Deficiency on the Regulation of Mouse and Rat GHReleasing Hormone Gene in Hypothalamus and Placenta

Hypothalamic GRH gene expression has been shown to be negatively regulated by GH in both rat and mouse. The recent reports of different 5' untranslated sequences in mouse GRH cDNA from hypothalamus and placenta have raised the possibility of tissue-specific regulation of the GRH gene. To provide support for this possibility, we have studied rodent models with GH deficiency due to genetic defects in the pituitary. Complementary DNA probes for the hypothalamic and placental 5' regions were used to determine the tissue specificity of each mRNA. Although the hypothalamic form of GRH mRNA was detected in placenta, it constituted less than 0.7% of total placental GRH mRNA. A placental 5' probe (based on the previously reported sequence) hybridized only with a larger mRNA species and was not tissue specific, indicating that it was not related to GRH and was derived possibly from a cloning artifact. The correct 5' sequence of mouse placental GRH cDNA was determined and shown to be distinct from both that previously reported and the hypothalamic sequence. Although the placental form of GRH mRNA was detected in hypothalamus using the polymerase chain reaction, its levels were undetectable by Northern blotting. The 5' end of rat placental GRH cDNA was similarly sequenced and shown to exhibit no homology with the rat 5' hypothalamic sequence, but a high degree of homology with the corresponding mouse placental sequence. In GH-deficient dwarf (dw/dw) rats, hypothalamic GRH mRNA levels were significantly increased above control levels in both females and males, and pregnancy did not alter the levels in either (dw) or control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of omeprazole on gene expression in canine gastric parietal cells

Stimulation of gastric parietal cells by carbachol induces coordinate expression of the genes for two enzymes involved in the process of acid secretion, H(+)-K(+)-ATPase and carbonic anhydrase II (CA II). The basis of this coordinate expression was examined in experiments using parietal cells that had been pretreated with omeprazole. We observed a twofold increase in the steady-state mRNA levels of both H(+)-K(+)-ATPase and CA II after cells were treated with the inhibitor. The induction of CA II mRNA by carbachol followed the same kinetics in omeprazole-pretreated cells as in those that were not pretreated, suggesting that the induction of CA II gene expression by carbachol was not dependent on activation of the gastric H(+)-K(+)-ATPase. In addition, carbachol stimulation of omeprazole-pretreated cells resulted in an induction of one or more larger mRNA species that hybridized with the H(+)-K(+)-ATPase probe. The observation that carbachol-induced increases in steady-state levels of beta-actin mRNA in parietal cells could be inhibited by omeprazole pretreatment suggests a possible linkage between increased beta-actin gene expression and the process of acid secretion.

Expression and stability of insulin-like growth factor-I (IGF-I) mRNA splicing variants in the GH 3 rat pituitary cell line

We have employed Northern blot analyses and solution hybridization/RNase protection assays to evaluate the presence and stability of IGF-I mRNA splicing variants in the GH 3 rat pituitary cell line. All of the IGF-I mRNA size classes and IGF-I mRNAs with alternately-spliced 5′-untranslated regions and E-peptide coding regions seen in adult rat liver also were present in GH 3 cells, although the proportions of the 5′ splicing variants were significantly altered. In actinomycin D-treated cells, all IGF-I mRNA splicing variants were equally stable; thus, changes in the levels of some splicing variants were not due to differential mRNA stability. Additionally, all IGF-I mRNA size classes seen on Northern blots were equally stable; this data suggests that the large IGF-I mRNA species is not a precursor of the smaller species.

Nucleotide sequence of testis-derived c-abl cDNAs: implications for testis-specific transcription and abl oncogene activation.

The c-abl gene codes for a protein-tyrosine kinase and is expressed in most examined murine cell types as two distinct mRNA species of 5.5 kilobases (kb) and 6.5 kb. In mouse testis, an additional species of 4.0 kb is expressed in very high levels. To study the interrelationship between various c-abl transcripts and to compare their sequence with the v-abl transcript, we prepared c-abl-specific cDNA clones from mouse testis and determined the complete nucleotide sequence of the 4.0-kb cDNA that appears to be the reverse transcript of the testis-specific mRNA. In addition, we have determined the 3' sequence of an additional clone derived from the larger mRNA species that is expressed in somatic as well as germ-line cells. These cDNA sequences have been compared with the v-abl sequences to understand the mechanism of activation of this oncogene. The results demonstrate that (i) testis-specific c-abl mRNAs arise as a result of 3' truncation, and (ii) the v-abl gene has arisen from its cellular homologue as a result of an extensive deletional/mutational process.

Multiple mRNAs from rat kidney and brain encode a single Na+,K+-ATPase beta subunit protein.

To determine whether multiple forms of the Na+,K+-ATPase beta subunit exist in rat kidney and brain, we constructed cDNA libraries from both tissues and isolated clones carrying inserts coding for this subunit. Characterization of these cDNAs by restriction endonuclease mapping and DNA sequencing suggests that a single form of the beta subunit protein is present in these tissues. We did, however, find that the beta subunit was encoded by multiple mRNAs which can be grouped into classes based on the lengths of their 5' and 3' untranslated regions. Size variation at the 3' end is due to the use of at least two of the five potential polyadenylation sites, whereas variation at the 5' end appears to be due to the use of different initiation sites. Northern blot analysis demonstrates the presence of multiple mRNA species which correlate with the sizes observed for the different cDNA classes and also demonstrates that the larger mRNA species are present at different levels in kidney and brain. S1 and primer extension analyses suggest that the observed tissue differences in the expression of these mRNAs are due to transcription from multiple initiation sites, whose strength is tissue-dependent. In the 3' untranslated region, comparison of the nucleotide sequence of the rat beta subunit cDNAs to those from other species reveals regions of high sequence similarity.

Characterisation of rat 9-kDa cholecalcin (CaBP) messenger RNA using a complementary DNA. Absence of homology with 28-kDa cholecalcin mRNA.

The rat possesses two cholecalciferol-induced calcium-binding proteins, the cholecalcins (CaBP). The 9-kDa CaBP is mainly concentrated in the duodenum while 28-kDa CaBP is located in the kidney and cerebellum. The mRNA encoding 9-kDa CaBP has been characterised using the cloned cDNA, pC109, synthesised from rat duodenal 9-kDa CaBP mRNA [Desplan et al. (1983) J. Biol. Chem. 258, 13502-13505]. Nucleotide sequence analysis of this cDNA shows the presence of two stop codons, TGA and TAG, at positions 207 and 271, respectively, of the 3' untranslated region. The cDNA-hybridised mRNA, isolated from rat duodenum, directs the cell-free synthesis of two proteins precipitable by antisera to 9-kDa intestinal CaBP. A major protein comigrates with 9-kDa CaBP whereas a minor product corresponds to a protein which is larger by 2000 Da. The minor protein appears to result from read-through of the 'leaky' UGA stop signal. No protein band which was immunoprecipitable with 28-kDa CaBP antiserum was detected when cDNA-hybridized mRNA from rat kidney and cerebellum was translated in a cell-free system. Northern blots show that the cDNA pC109 sequence hybridizes to a homogeneous mRNA species 500-600 nucleotides long from rat duodenum. Larger mRNA species encoding 28-kDa CaBP are undetectable in rat kidney and cerebellum even under low stringency conditions. All these findings demonstrate that there is no cross-hybridisation between 9-kDa and 28-kDa CaBP mRNAs. Southern blot analysis of rat genomic DNA, that shows only one homologous 9-kDa gene, is consistent with these findings. Thus, all our data indicate that there are distinct genes coding for each rat cholecalcin.

Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene

The analysis of RNA isolated from maize leaves indicates that there are two mRNA transcripts which are homologous to the chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The 5' end of the smaller transcript, 1.62 Kb in length, begins at a position which is 60 nucleotides upstream from the coding sequence of the gene, corresponding to the position mapped by earlier workers. The larger transcript, 1.86 Kb in length, has not been previously described and originates from a site on the gene which is 302 nucleotides upstream from the coding sequence. The increased size of the largerbcL mRNA transcript from maize, as compared to the transcripts from spinach and tobacco, results from the presence of a 130 nucleotide insert between the two maizerbcL gene mRNA start sites. The DNA sequence adjacent to the start site for the large mRNA transcript is shown to have greater than 90% homology to the DNA sequences adjacent to the mRNA start sites for the spinach and tobaccorbcL gene. Discounting the presence of the insert in the maizerbcL gene, the nucleotides upstream from the coding sequence in the maize, spinach and tobaccorbcL genes, all share approximately the same amount of homology to each other. This homology, along with other evidence, suggests that the large mRNA species are the primary transcripts and that the smaller RNA species are the result of post-transcriptional processing. The finding that spinach also contains two different mRNA transcripts for therbcL gene is consistent with this model.

Polyribosomes from Pear Fruit

Detailed analyses of polysome profiles from lyophilized pulp tissues of pear fruits (Pyrus communis L. cv. Passe-Crassane) at different stages of ripening and senescence, and estimates of the amount of polysomal-associated mRNA, lead to the conclusions that during senescence (ripening), the ripening and the over-ripening processes can clearly be separated and respectively linked to the first and the second increase in the large mRNA species. Ethylene synthesis which occurs at the beginning of a normal ripening at 15 degrees C after a cold storage or an ethephon treatment is related to an increase in mRNA and ribosomal material found only in pulp tissues. Finally, we suggest that in the pear fruit, the sequence of events which occurs during senescence (ripening) is initiated by two systems which regulate ethylene biogenesis, and that the first system is efficient only at low temperatures (from 0 to 4 degrees C).

In vitro synthesis of messenger RNA by a defective interfering particle of vesicular stomatitis virus.

A defective interfering particle derived from the heat-resistant strain of vesicular stomatitis virus was analyzed for the presence of virion-associated RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) activtiy. The RNA synthesizing capacity of the defective particles in vitro was similar to that of the wild-type virus. Characterization of the RNA produced in vitro indicated that the defective particles were able to synthesize vesicular stomatitis virus leader RNA and four virus mRNA species that sediment at 12-18 S. These RNA products were identical to the mRNAs synthesized in vitro by the wild-type virus in regard to size, polyadenylation, capping, and methylation. In contrast to the wild-type virus, the purified defective particles did not synthesize the large mRNA species sedimenting at 31 S in vitro. Possible mechanisms of homotypic and heterotypic interferences shown by this defective particle are discussed.