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PubMed1987

Differential expression in murine somatic and germinal tissues of transcripts homologous to an abundant embryonal-carcinoma-cell mRNA.

EC1, a clone from a cDNA library of embryonal-carcinoma (EC) cells, was used to study the expression of the gene(s) homologous to this sequence in murine germ cells and somatic tissues. Northern-blot analysis was used to determine the size and relative abundance of ECl transcripts in EC cells, ovary, testis, liver, spleen, brain, thymus, and bone marrow. The stage-specific expression of EC1-related transcripts in meiotic cells was examined in enriched populations of testicular cells (meiotic prophase spermatocytes, early and late spermatids, and residual bodies). All tissues and cells examined contained mRNAs of approximately 1.3 and approximately 3.6 kb which were homologous to EC1. In RNA from liver and thymus, a faint, lower-molecular-weight transcript of 0.75 kb was seen. A unique pattern of expression was observed in male germ cells. In cells that had completed meiosis, two additional transcripts of 1.0 and 4.4 kb were visualized, as well as high levels of expression of the 1.3- and 3.6-kb species. Only the 1.3- and 3.6-kb transcripts were detected in ovarian RNAs. The high level and stage specificity of expression of EC1-related mRNAs in germ cells suggest that this gene may be important in meiosis or in germ-cell maturation.

Animals↗
PubMed1998

New splicing mutations in the human factor XIIIA gene, each producing multiple mutant transcripts of varying abundance.

Coagulation factor XIII, a transglutaminase which stabilises blood clots by covalently cross-linking fibrin, is essential for normal haemostasis. FXIII deficiency results in a life-long bleeding disorder with added complications in wound healing and tissue repair. Sequence changes in the human FXIIIA gene, largely missense mutations, are primarily responsible for inherited FXIII deficiency. We have carried out molecular analysis of the FXIIIA gene in two unrelated FXIII deficient individuals and identified three splice site mutations; a g-->a at the exon 6 acceptor splice site, a g-->a at the exon 7 donor splice site and a coding sequence T-->G at the exon 8 donor splice site. We have also examined the FXIIIA mRNA in these patients and find that each mutation gives rise to multiple transcripts which vary in their relative abundance. The precise molecular mechanisms which result in these variant transcripts, and their relative abundance in our FXIII deficient patients, are discussed.

Adolescent↗
PubMed2001

Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome.

Long terminal repeat (LTR) retrotransposons have been shown to make up much of the maize genome. Although these elements are known to be prevalent in plant genomes of a middle-to-large size, little information is available on the relative proportions composed by specific families of elements in a single genome. We sequenced a library of randomly sheared genomic DNA from maize to characterize this genome. BLAST analysis of these sequences demonstrated that the maize genome is composed of diverse sequences that represent numerous families of retrotransposons. The largest families contain the previously described elements Huck, Ji, and Opie. Approximately 5% of the sequences are predicted to encode proteins. The genomic abundance of 16 families of elements was estimated by hybridization to an array of 10,752 maize bacterial artificial chromosome (BAC) clones. Comparisons of the number of elements present on individual BACs indicated that retrotransposons are in general randomly distributed across the maize genome. A second library was constructed that was selected to contain sequences hypomethylated in the maize genome. Sequence analysis of this library indicated that retroelements abundant in the genome are poorly represented in hypomethylated regions. Fifty-six retroelement sequences corresponding to the integrase and reverse transcriptase domains were isolated from approximately 407,000 maize expressed sequence tags (ESTs). Phylogenetic analysis of these and the genomic retroelement sequences indicated that elements most abundant in the genome are less abundant at the transcript level than are more rare retrotransposons. Additional phylogenies also demonstrated that rice and maize retrotransposon families are frequently more closely related to each other than to families within the same species. An analysis of the GC content of the maize genomic library and that of maize ESTs did not support recently published data that the gene space in maize is found within a narrow GC range, but does indicate that genic sequences have a higher GC content than intergenic sequences (52% vs. 47% GC).

Chromosomes, Artificial, Bacterial↗
PubMed1994

Temporal patterns of human immunodeficiency virus type 1 transcripts in human fetal astrocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of the developing central nervous system results in a dementing process in children, termed HIV-1-associated encephalopathy. Infection of astroglial elements of the pediatric nervous system has been demonstrated and suggests that direct infection of some astrocytes may contribute to the neurologic deficit. In this model, HIV-1 establishes a persistent state of infection in astrocytes, which can be reactivated by the cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta). To better understand the natural history of viral persistence in astroglial cells, we characterized infection at the transcriptional level. The most abundant viral transcript during the establishment of persistence was the subgenomic multiply spliced 2-kb message, similar to mononuclear cell models of HIV-1 latency. Following reactivation with TNF-alpha or IL-1 beta the multiply spliced 2-kb message remained the most abundant viral transcript, in contrast to infected mononuclear cells in which reactivation leads to the reemergence of the 9- and 4-kb transcripts. Further characterization of the persistent 2-kb transcript by PCR amplification of in vitro-synthesized viral cDNA showed that, in the absence of cytokine stimulation, the most abundant multiply spliced transcripts were the Nef- and Rev-specific messages. However, following cytokine stimulation, double- and triple-spliced Tat-, Rev-, and Nef-specific messages could be identified. Immunohistochemical staining demonstrated that, during viral persistence, astrocytes expressed Nef protein but few or no viral structural proteins. These results demonstrate that viral persistence in astrocytes at the transcriptional level is fundamentally different from that seen in mononuclear cells and could account for the virtual absence of astroglial expression of viral structural antigens in vivo.

Astrocytes↗
PubMed1989

Increase in abundance of a transcript hybridizing to elongation factor I alpha during cellular senescence and quiescence.

We have isolated a senescence-specific clone (pSEN) from a cDNA library constructed from late passage WI-38 human diploid fibroblast that accounts for approximately 1% of the recombinants. Nucleotide sequence analysis of the partial cDNA clone has led to the identification of pSEN as elongation factor I alpha. Northern analysis of poly(A)+ RNA from various intermediate population doubling levels shows that a 2.2 kb transcript hybridizes to pSEN but is expressed prior to PDL-40 at very low levels. This transcript begins to accumulate at PDL-40 and is induced approximately 50-fold just prior to senescence. Furthermore, this transcript was shown to be specific to Go of the cell cycle whereas a second, lower molecular weight transcript (1.6 kb) was observed during S phase (Giordano and Foster, unpublished data). The 2.2 kb transcript is also detected in neonatal foreskin cells but very little increase in abundance is observed between early and late passage cells. Sucrose gradient fractionation of RNA from late passage WI-38 cells suggests that the lower molecular weight transcript is associated with the polysome fraction while the 2.2 kb transcript sediments with the nonpolysomal fraction. Thus, the possibility exists that the 1.6 kb transcript is derived from the 2.2 kb transcript.

Aging↗
PubMed2005

SuperSAGE combined with PCR walking allows global gene expression profiling of banana (Musa acuminata), a non-model organism.

Super-serial analysis of gene expression (SuperSAGE) was used to characterize, for the first time, the global gene expression pattern in banana (Musa acuminata). A total of 10,196 tags were generated from leaf tissue, representing 5,292 expressed genes. Forty-nine tags of the top 100 most abundantly expressed transcripts were annotated by homology to cDNA or EST sequences. Typically for leaf tissue, analysis of the transcript profiles showed that the majority of the abundant transcripts are involved in energy production, mainly photosynthesis. However, the most abundant tag was derived from a type 3 metallothionein transcript, which accounted for nearly 3% of total transcripts analysed. Furthermore, the 26-bp long SuperSAGE tags were applied in 3'-rapid amplification of cDNA ends (3'RACE) for the identification of unknown tags. In combination with thermal asymmetric interlaced PCR (TAIL-PCR), this allowed the recovery of a full gene sequence of a novel NADPH:protochlorophyllide oxidoreductase, the key enzyme in chlorophyll biosynthesis. SuperSAGE in conjunction with 3'RACE and TAIL-PCR will be a powerful tool for transcriptomics of non-model, but otherwise important organisms.

Base Sequence↗
PubMed1997

MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin.

MeCP2 is an abundant mammalian protein that binds to methylated CpG. We have found that native and recombinant MeCP2 repress transcription in vitro from methylated promoters but do not repress nonmethylated promoters. Repression is nonlinearly dependent on the local density of methylation, becoming significant at the density found in bulk vertebrate genomic DNA. Transient transfection using fusions with the GAL4 DNA binding domain identified a region of MeCP2 that is capable of long-range repression in vivo. Moreover, MeCP2 is able to displace histone H1 from preassembled chromatin that contains methyl-CpG. These properties, together with the abundance of MeCP2 and the high frequency of its 2 bp binding site, suggest a role as a global transcriptional repressor in vertebrate genomes.

Animals↗
PubMed2002

Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo.

Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.

Animals↗
PubMed1995

Human papillomavirus type 11 transcripts are present at low abundance in latently infected respiratory tissues.

Respiratory tract tissues containing latent human papillomavirus (HPV) 11 were analyzed by reverse transcription-polymerase chain reaction for the presence of viral-specific RNA from the early region of the genome and compared to a similar analysis of laryngeal papillomas. Latently infected tissue contained low-abundance transcripts that could code for E1 and E2 proteins, but lacked evidence of spliced transcripts for the E6 and E7 proteins. Both latently infected tissue and papilloma tissue contained low-abundance antisense transcripts. Cultured cells infected with HPV 11 virions or transfected with HPV DNA, and cells derived from latently infected tissue, expressed transcripts similar to those seen in papillomas, but at a lower abundance. We postulate that latency is determined by the absence of or limiting levels of critical viral proteins.

Cells, Cultured↗
PubMed1993

Sequence analysis of monkey acrosin-trypsin inhibitor transcripts and their abundant expression in the epididymis.

Seven independent cross-hybridizing clones have been isolated from a Macaca fascicularis epididymal cDNA library and their inserts shown to correspond to a secreted inhibitor of the sperm serine proteinase, acrosin. Hybridizing transcripts of approx. 0.6 kb have been shown by Northern blot analysis to be considerably more abundant in the epididymis than the testis. This finding is in surprising contrast to a previous report that human acrosin-trypsin inhibitor mRNA predominates in the testis.

Amino Acid Sequence↗
PubMed1988

An enhancer element in the short unique region of human cytomegalovirus regulates the production of a group of abundant immediate early transcripts.

A 275-bp sequence in the short unique region of human cytomegalovirus (HCMV) strain AD169 contains a series of five imperfect 18-bp repeats with homology to the SV40 and HCMV major immediate early enhancers. Plasmids containing these putative HCMV enhancer sequences linked to the human beta-globin gene were transiently transfected into HeLa cells, and the ability of the HCMV sequences to activate beta-globin transcription was assayed. The repeat-containing region stimulated transcription in a position- and orientation-independent manner, to approximately the same degree as that of the SV40 enhancer. A promoter located immediately 3' to the enhancer in the HCMV genome is active only in the presence of the enhancer sequences. Transcription from this HCMV promoter was analysed by a combination of S1 nuclease protection mapping and Northern blotting. At immediate early times postinfection, at least four extremely abundant differentially spliced mRNAs were detected; these RNAs constitute a previously unknown class of immediate early transcript.

Base Sequence↗
PubMed1992

cAMP post-transcriptionally diminishes the abundance of adrenodoxin reductase mRNA.

Adrenodoxin reductase (AR; ferridoxin: NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein that mediates electron transport from NADPH to all known mitochondrial forms of cytochrome P450. AR mRNA was found in all human adult and fetal tissues examined; however, it was vastly more abundant in tissues that synthesize steroid hormones. The ratio of the 18- form of mRNA lacking 18 alternately spliced bases to the 18+ form was approximately 100:1 and remained constant irrespective of the tissue or hormonal manipulation, indicating that the alternate splicing is a passive nonregulated event. AR protein was unchanged by forskolin treatment of human JEG-3 cytotrophoblast cells for 24 h, but the mRNA diminished. Phorbol 12-myristate 13-acetate and cycloheximide had no effect, even though these agents had the expected effects on P450scc and adrenodoxin mRNAs. cAMP decreased the abundance of AR mRNA expressed from both transfected plasmids and the endogenous gene, indicating the effect was post-transcriptional. AR gene transcription in JEG-3 cells and promoter-chloramphenicol acetyltransferase constructs transfected into JEG-3 cells were unresponsive to forskolin. Powerful basal transcription elements were identified between -46 and -214 bases from the principal transcriptional initiation site, a region containing six elements closely resembling the binding site for transcription factor SP1.

Cells, Cultured↗
PubMed2001

Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems.

The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.

Analysis of Variance↗
PubMed2005

SSH adequacy to preimplantation mammalian development: scarce specific transcripts cloning despite irregular normalisation.

BACKGROUND: SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. RESULTS: Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs) tester-specific transcripts. CONCLUSION: The close agreement between our hybridization and sequencing results shows that the addition and follow-up of exogenous reporter transcripts provides an easy and reliable means to check SSH performance. Despite some cases of irregular normalisation and subtraction failure, we have shown that SSH repeatedly enriches the libraries in very rare, tester-specific transcripts, and can thus be considered as a powerful tool to investigate situations where small amounts of biological material are available, such as during early mammalian development.

Animals↗
PubMed1998

Expression of the human papillomavirus type 11 E5A protein from the E1E4,E5 transcript.

The abundant human papillomavirus type 11 (HPV 11) E1E4,E5 transcript potentially encodes the E1E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3' end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1E4,E5a construct that lacked the 5' noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1wedgeE4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1E4,E5 transcript is a functional message that can support both E1E4 and E5a expression in eukaryotic cells.

Animals↗
PubMed2006

LucTrap vectors are tools to generate luciferase fusions for the quantification of transcript and protein abundance in vivo.

Proper plant growth and development strongly rely on the plant's ability to respond dynamically to signals and cues from the intra- and extracellular environment. Whereas many of these responses require specific changes at the level of gene expression, in recent years it has become increasingly clear that many plant responses are at least in part also controlled at the level of protein turnover. It is a challenge for signal transduction research to understand how distinct incoming signals are integrated to generate specific changes at the transcript or protein level. The activity of luciferase (LUC) reporters can be detected in nondestructive qualitative and quantitative assays in vivo. Therefore, LUC reporters are particularly well suited for the detection of changes at the transcript and protein level. To the best of our knowledge, the number of plant transformation vectors for LUC fusions is very limited. In this article, we describe the LucTrap plant transformation vectors that allow generation of targeted and random transcriptional and translational fusions with the modified firefly LUC reporter LUC+. We demonstrate that LucTrap-based fusions can be used to monitor rapid changes in gene expression and protein abundance in vivo.

Amino Acid Sequence↗
PubMed1998

Gibberellin dose-response regulation of GA4 gene transcript levels in Arabidopsis.

The gibberellins (GAs) are a complex family of diterpenoid compounds, some of which are potent endogenous regulators of plant growth. As part of a feedback control of endogenous GA levels, active GAs negatively regulate the abundance of mRNA transcripts encoding GA biosynthesis enzymes. For example, Arabidopsis GA4 gene transcripts encode GA 3beta-hydroxylase, an enzyme that catalyzes the conversion of inactive to active GAs. Here we show that active GAs regulate GA4 transcript abundance in a dose-dependent manner, and that down-regulation of GA4 transcript abundance is effected by GA4 (the product of 3beta-hydroxylation) but not by its immediate precursor GA9 (the substrate). Comparison of several different GA structures showed that GAs active in promoting hypocotyl elongation were also active in regulating GA4 transcript abundance, suggesting that similar GA:receptor and subsequent signal transduction processes control these two responses. It is interesting that these activities were not restricted to 3beta-hydroxylated GAs, being also exhibited by structures that were not 3beta-hydroxylated but that had another electronegative group at C-3. We also show that GA-mediated control of GA4 transcript abundance is disrupted in the GA-response mutants gai and spy-5. These observations define a sensitive homeostatic mechanism whereby plants may regulate their endogenous GA levels.

Arabidopsis↗