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Being used in blot-hybridization and S1-analysis, a number of cDNA clones homologous to L1 made it possible to detect a long abundant transcript of 7 kb. It accumulates solely in polysomal non-polyadenylated RNA from rat brain cells.
A hybridization subtraction protocol that uses low ratios of RNA to cDNA has been developed to enrich for the cDNA of transcripts that are elevated in one cell population relative to another. This low-ratio hybridization subtraction protocol was found to yield substantial enrichment for the cDNA of low-abundance transcripts induced or increased only several fold. Conditions for the cloning of cDNA enriched by our hybridization subtraction and identification of clones coding for induced transcripts are presented. By screening the cDNA library with probes synthesized from the starting cDNA and cDNA enriched by low-ratio hybridization subtraction, clones coding for induced transcripts could be efficiently identified. The choice of reverse transcriptase used to synthesize the cDNA was found to be important for the enrichment of cDNA for longer length RNA. Low-ratio hybridization subtraction of cDNA synthesized with MMLV reverse transcriptase was effective for the enrichment of cDNA coding for RNA to at least 5 kb in length, while the AMV enzyme was effective only for the cDNA of shorter RNA (less than 1 kb). The characterization of several different low-ratio hybridization subtraction libraries is presented, and the advantages and disadvantages of various hybridization subtraction strategies are discussed.
The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box-containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs.
The Arabidopsis thaliana mutants de-etiolated3 (det3), pom-pom1 (pom1) and ectopic lignification1 (eli1) all deposit lignins in cells where these polymers would not normally be found. Comparison of these mutants provides an opportunity to determine if the shared mutant phenotype arose by perturbing a common regulatory mechanism in each of the mutants. The mutants were compared using a combination of genetics, histochemistry, chemical profiling, transcript profiling using both Northern blots and microarrays, and bioinformatics. The subset of cells that ectopically lignified was shared between all three mutants, but clear differences in cell wall chemistry were evident between the mutants. Northern blot analysis of lignin biosynthetic genes over diurnal and circadian cycles revealed that transcript abundance of several key genes was clearly altered in all three mutants. Microarray analysis suggests that changes in the expression of specific members of the R2R3-MYB and Dof transcription factor families may contribute to the ectopic lignification phenotypes. This comparative analysis provides a suite of hypotheses that can be tested to examine the control of lignin biosynthesis.
The newly identified sin gene affects late growth processes in Bacillus subtilis when it is overexpressed or inactivated in the chromosome. S1 nuclease mapping of the sin gene transcripts in vivo reveals the existence of three transcripts (RNAI, RNAII, and RNAIII). By correlating 5' ends of sin gene transcripts with DNA sequence, we have identified three different promoterlike sequences (P1, P2, and P3) for these transcripts. 3'-End mapping of these transcripts identified three prominent termination sites at the end of the sin gene. These termination sites are localized on two hairpin structures previously identified from the DNA sequence. The most abundant transcript, RNAIII, coded only for the sin gene, while the polycistronic transcripts RNAII and RNAI coded for the sin gene and ORF1 that precedes the sin gene. S1 mapping and translational lacZ fusion studies indicated that ORF1 and the sin gene are regulated differently. ORF1 expression is under developmental control, increasing at the end of vegetative growth, and requires functional spo0A and spo0H gene products. The sin gene is expressed at an almost constant and relatively low level throughout growth and remains largely unaffected by spo0A and spo0H mutations.
We examined the in vivo transcriptional and posttranscriptional regulation of various genes involved in hepatocyte growth and replication that exhibited changes in steady-state mRNA levels after 70% partial hepatectomy (PH). Of the 19 genes examined by nuclear run-on assay, 17 demonstrated no change in transcriptional activity through the first 96 h of regeneration. However, results from in vivo half-life determinations indicated that changes in mRNA stability played a critical role in regulating transcript levels during liver regeneration. For many of the genes, alterations in transcript abundance correlated with similar changes in mRNA half-lives. Inhibition of protein synthesis by cycloheximide was generally associated with increased levels of mRNA expression, but no detectable changes in transcriptional rates in both control and regenerating rat liver. Finally, genomic methylation status was investigated by Southern analysis for several genes that displayed changes in mRNA stability. Interestingly, increases in mRNA half-lives for the genes p53, c-myc, H-ras, and ornithine decarboxylase were associated with decreased genomic methylation. In conclusion, regulation of gene expression beyond the immediate early phase of te cell cycle during rat liver regeneration after PH occurs predominantly at the posttranscriptional level. mRNA stability appears to be a significant factor in this control, and may itself be modulated by the methylation status of the corresponding genomic DNA.
Cytokines and chemokines are likely to be involved in the pathogenesis of inflammatory diseases of the canine respiratory tract. The roles and relative amounts of these molecules have not yet been defined in the respiratory mucosa of normal dogs or dogs with naturally acquired respiratory inflammation. In the present study, real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays were employed to quantify messenger RNA (mRNA) encoding the chemokines monocyte chemotactic protein (MCP)-2, eotaxin-2 and eotaxin-3, and the cytokines interleukin-4 (IL-4), IL-5, IL-6, IL-10, IL-12p40, IL-18, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) in normal nasal, bronchial and pulmonary tissues from puppies (n = 4) and from adult dogs (n = 7). There was no significant difference in the expression of any transcript between puppies and adult dogs at any of the anatomical sites examined. The expression of mRNA encoding eotaxin-2 and eotaxin-3 increased significantly with progression from the nasal mucosa to pulmonary parenchyma but expression of MCP-2 mRNA did not show this trend. At all levels of the respiratory mucosa, the most abundant transcripts were those encoding IL-18 and TGF-beta. Transcripts encoding IL-6, IL-10, IL-12 and TNF-alpha were approximately ten-fold less abundant, and IL-4, IL-5 and IFN-gamma were the least abundant templates. There was significantly different amount of mRNA encoding IL-5, IL-18 and TNF-alpha between particular anatomical levels of the respiratory mucosa while the mRNA expression of the other cytokines was similar at all anatomical sites. The results of the present study will enable comparisons to be made with results obtained from similar samples obtained from dogs with nasal, bronchial or pulmonary diseases.
The expression of members of the heat shock protein 90 (hsp90) gene family during testicular and embryonic development was investigated. Two different hsp90 transcripts were detected in RNA from mouse testis, approximately 3.2 kb and 2.9 kb in size, and were shown to exhibit cellular and developmental stage specificity of expression. The larger, more abundant transcript was expressed at high levels in the germinal compartment of the testis, particularly in germ cells in meiotic prophase. The smaller hsp90 transcript was expressed predominantly in the somatic compartment of the testis. Expression of the two hsp90 transcripts was observed in testes of other species, suggesting an important role for hsp90 in mammalian testicular function. In addition, expression of both hsp90 transcripts was detected in the embryonic and extra-embryonic compartments of mid-gestation embryos.
We are developing molecular approaches to study the growth and development of woody plants. As part of our research efforts, we report the molecular cloning and characterization of PsRLK here, a cDNA from the conifer Scots pine (Pinus sylvestris L.) encoding a polypeptide similar to the receptor protein kinases described in angiosperms. A full-length clone was isolated from a cDNA library constructed with poly (A)+ enriched RNA prepared from germinating pine seeds. Characterization of the isolated sequence revealed that it contains multiple leucine-rich repeats in the N-terminal region and a characteristic Ser/Thr protein kinase domain in the C-terminal region. N- and C-terminal conserved domains are separated by a putative membrane spanning sequence. PsRLK protein is encoded by a single gene in the pine genome. A comparison of the pine sequence with the LRR-RLKs from Arabidopsis revealed that PsRLK is phylogenetically related to the LRR XI subfamily members. RT-PCR analyses of transcript abundance in pine tissues suggest that the gene expression pattern of PsRLK reflects the plant body formation programme, with increased levels during development of pine seedlings. The precise localization of PsRLK transcripts revealed that gene expression was restricted to specialized phloem cells suggesting a possible function of the putative receptor-like protein kinase in this particular vascular element.
Understanding mammalian spermatozoan development and the events surrounding fertilization has grown slowly, in part because of uncertainty about the number and identity of the cellular components involved. Determination of those transcripts expressed specifically by germ cells should provide an inclusive list of probable critical proteins. Here, total mouse testis transcript profiles were trimmed of transcripts found in cultures enriched in Sertoli or interstitial cells to yield a germ cell-enriched transcript profile. Monitoring of changes of this profile in the developing testis identified 1,652 genes whose transcript abundance increased markedly coincident with the onset of meiosis. Remarkably, 351 of these genes (approximately equal to 20%) appear to be expressed only in the male germline. Germ cell-specific transcripts are much less common earlier in testis development. Further analysis of the UniGene EST database coupled with quantitative PCR indicates that approximately 4% of the mouse genome is dedicated to expression in postmeiotic male germ cells. Most or many of the protein products of these transcripts are probably retained in mature spermatozoa. Targeted disruption of 19 of these genes has indicated that a majority have roles critical for normal fertility. Thus, we find an astonishing number of genes expressed specifically by male germ cells late in development. This extensive group provides a plethora of potential targets for germ cell-directed contraception and a staggering number of candidate proteins that could be critical for fertilization.
The human genome contains a family of endogenous retroviruses, HERV-K, with sequence homology to the B-type mouse mammary tumor virus (MMTV). We have detected HERV-K-LTR related cDNA clones by screening a human placenta cDNA library with a HERV-K LTR probe. Three of the isolated cDNA clones were characterized by nucleotide sequencing. The analyzed clones did not contain any retroviral sequences other than those related to HERV-K LTRs, but were found to be coexpressed with cellular sequences. Furthermore, transcripts containing HERV-K LTR sequences were demonstrated by Northern blotting and PCR in human leukemic and normal white blood cells, as well as in various tumor cell lines, indicating abundant transcription of solitary HERV-K LTRs in human tissues. In patients with lymphatic leukemias, a transcript of about 6 kb hybridizing with HERV-K LTR was detected that was not found in patients with myelogenous leukemias or in healthy persons.
The plant growth substance (-)-jasmonic acid methyl ester (methyl jasmonate, JaMe) affects plastid gene expression at the protein and mRNA levels when applied exogenously to detached leaf segments of Hordeum vulgare L. cv. Salome. Translation of the large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (LSU, rbcL gene products) and of the 65 and 68 kDa proteins of photosystem I (psaA and psaB gene products, respectively) ceased, whereas synthesis of the 32 kDa photosystem II protein (D1, psbA gene product) continued in JaMe-treated leaf tissues. These changes were not caused by corresponding alterations in transcript abundances. The loss of LSU protein synthesis, occurring within 24 h of JaMe treatment, correlated with a decline in the in vitro-translatable rbcL mRNA, but contrasted with an almost constant transcript level. The 5' ends of the rbcL transcripts shifted from '-59' in freshly harvested or water-treated leaves to '-94' in JaMe-treated leaf tissues. Transcripts ending at these positions presumably arise from alternative processing of the primary transcript ending at position '-316'. The '-94' transcript contains, within the 5' untranslated region, a 35-base motif with remarkable complementarity to the extreme 3' terminal part of the 16S rRNA, involved in intramolecular base pairing within the ribosome and can associate with 30S but not 70S complexes in organello, suggesting that intermolecular base pairing impairs translation initiation, probably by competing for ribosome binding at the Shine-Dalgarno sequence. In contrast, transcripts ending at '-59' lack the 5' terminal 'extra' sequence and are active in terms of translation initiation.
Dehydration is associated with altered vasopressin (VP) gene expression in the rat hypothalamus; an increase in both arginine-vasopressin mRNA abundance and size (due to 3' poly(A) tail extension) has been observed previously. We have now shown that the effects of dehydration are not restricted to the magnocellular AVP neuronal systems since VP mRNA in the suprachiasmatic nucleus (SCN) exhibits a progressive increase in poly(A) tail length during dehydration. No significant increase in VP mRNA abundance was found. Similar kinetics of poly(A) tract extension in the SCN and magnocellular supraoptic nucleus implies a common regulatory mechanism which appears to be distinct from osmotic upregulation of VP transcript abundance.
Escherichia coli was adapted to grow in medium containing substantially elevated concentrations of either Zn(II), Cd(II), Co(II) or Ni(II). Whole-genome transcriptional profiles were generated from adapted strains and analysed for significant alteration in transcript abundance with reference to a wild-type strain. Similar alterations in specific message levels were observed for strains adapted to the four metal ions. One unexpected trend was the increase in transcript level of genes involved in transposition of IS elements, particularly insA. Subsequent expression of insA-7 from a heterologous promoter in E. coli conferred tolerance to Zn(II).
Human mitochondrial 16 S rRNA 3'-end formation requires a tridecamer template sequence and a trans-acting protein of approximately 34 kDa. This protein binds tightly to its target sequence and further analysis of the protein-DNA complex revealed that the DNA is bent. Either T3, T7, Escherichia coli, or yeast mitochondrial RNA polymerase produced transcripts mapping at this termination site. With these heterologous RNA polymerase, RNA 3'-end formation was detected only in the transcription polarity opposite that of mitochondrial rRNA synthesis; the efficiency of termination in the homologous human RNA polymerase system is approximately 2-fold greater in this same opposite polarity. These results suggested the possible importance of biased bipolar transcription termination in vivo. For wild-type mtDNA, the apparent relative efficiency of termination in vivo reflected the values determined in vitro. Examination of a pathogenic human mtDNA mutation known to result in impaired termination in vitro showed no significant differences in relative transcript abundances in vivo, despite a loss of in vitro termination efficiency in both directions. Recently, six additional mitochondrial disease-associated point mutations have been reported that cluster at the human mitochondrial transcription termination site. None of these resulted in significantly impaired transcription termination in vitro.
Normal neonatal lung growth requires a substantial increase in microvascular endothelial cells. Oxygen injury to neonatal lung destroys endothelial cells and alters the normal process of alveolarization, including development of the microvasculature. The mechanisms that regulate lung alveolar capillary growth and development are not known. Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells that is often expressed by epithelial cells in close proximity to capillary beds. VEGF expression is induced by hypoxia and may be inhibited by hyperoxia. We examined the cell-specific expression of VEGF during normal postnatal lung development and the effects of hyperoxic lung injury on VEGF mRNA and protein in vivo. Normal newborn rabbits between 1 day and 5 wk of age had VEGF transcripts located mainly in alveolar epithelial cells, with little or no VEGF mRNA noted in smooth muscle or endothelial cells. A subpopulation of freshly isolated, normal type II cells, but not mesenchymal cells, expressed VEGF mRNA. Newborn rabbits exposed to 100% oxygen for 4 days had no change in VEGF mRNA abundance, transcript location, or immunostaining. Animals exposed to 100% oxygen for an average of 9 days had an 80% decrease in lung VEGF mRNA abundance, decreased alveolar epithelial cell VEGF expression, and decreased VEGF immunostaining. Recovery of VEGF expression to control levels occurred during a 5-day recovery period. We conclude that alveolar epithelial cells in postnatal lung express VEGF, suggesting epithelial regulation of alveolar capillary formation. Furthermore, hyperoxic injury decreases neonatal lung VEGF mRNA and protein, which may be a contributory mechanism of impaired postnatal microvascular development in oxygen injury.
PURPOSE: To document the expression of mRNA for transthyretin (TTR) and retinol binding protein (RBP) in native and cultured Rhesus monkey retinal pigmented epithelium (RPE); to compare mRNA transcripts for these two proteins expressed in RPE with those found in whole monkey liver and brain; to demonstrate the secretion of TTR by RPE during short-term maintenance in a protein-free, defined medium, as a manifestation of the differentiated state of these cells in vitro. METHODS: Total RNA was isolated from cultured RPE in first passage, after incubation for eight days in defined, protein-free medium. Conditioned medium was collected for western analysis at this time. Total RNA was also extracted from RPE/choroid freshly dissected from monkey eyes. Using cDNA probes for human TTR and RBP, northern analysis was performed on the total RNA from fresh and cultured RPE samples, together with poly(A+) mRNA purified from monkey liver and brain. RESULTS: Conditioned medium from RPE yielded TTR protein of the expected monomer subunit molecular size. The TTR secreted de novo from the cultured cells was detectable in the absence of biosynthetic labeling. With the exception of some extremely low abundance transcripts expressed in cultured RPE, all samples contained a single 900 bp transcript for TTR. Based on relative amounts of actual message, RPE ranks higher than liver in abundance of TTR mRNA. In contrast, both native monkey RPE and cultured RPE cells expressed comparatively low levels of mRNA for RBP. All samples displayed a single RBP mRNA transcript at 1100 bp. CONCLUSIONS: Our results indicate that TTR is a significant gene product of the RPE, and may be considered as a marker for a differentiated phenotype for these cells in culture. There is increased recognition of various forms of ocular pathology associated with mutations or other malfunctions involving TTR and RBP, warranting a greater understanding of mechanisms of transcriptional and translational control for these two proteins.
The problem is to discover which of the promoters of the insulin-like growth factor-II gene stimulate the transcription of mRNA which is translated into protein. Three alternative leader exons are attached to the coding sequences in RNA transcribed from this gene in other systems, and it is mainly the paternal allele which is expressed in mouse development. Transcripts bearing each of the three leader exons were found in the RNA from the chorio-allantoic placenta, visceral yolk sac, and embryo, starting at 9.5 days. A varying proportion of one abundant transcript was disengaged from the polysomes at different days of development. This transcript was prefixed by the longest of the three alternative untranslated 5' leader exons (exon 2), and it was consistently associated with polysomes in the choroid plexus and leptomeninges of the brain. Many exon 2 transcripts were abbreviated by endonucleolytic cleavage and lacked a poly(A) tail. In contrast, the transcripts with the shortest leader (exon 3) were mainly displayed on polysomes at all the stages of development which were examined. During mouse development, the production of IGF-II protein must be partly controlled by the mechanisms which regulate translation.