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PubMed1990

Suppression of the malignant phenotype in somatic cell hybrids between Burkitt's lymphoma cells and Epstein-Barr virus-immortalized lymphoblastoid cells despite deregulated c-myc expression.

To approach the question whether the absence of specific cellular gene functions may be involved in Burkitt's lymphoma pathogenesis, somatic cell hybrids were established between a malignant Epstein-Barr virus (EBV) positive Burkitt's lymphoma cell line (BL 60) and a nonmalignant EBV-immortalized lymphoblastoid cell line (IARC 277) derived from the same individual. The hybrids revealed a near tetraploid karyotype including one copy of the 8q+ chromosome resulting from the Burkitt's lymphoma-specific translocation t(8;22) in addition to three apparently normal copies of chromosome 8. Although the hybrid cells exhibited the deregulated c-myc expression pattern of the parental Burkitt's lymphoma cell line with highly abundant transcripts originating from the 8q+ chromosome, their growth characteristics in tissue culture as well as in nude mice were identical to that of the parental nonmalignant lymphoblastoid cell line. These data indicate that, at least in the system described here, the malignant phenotype of Burkitt's lymphoma cells can be suppressed by introduction of an additional set of apparently normal chromosomes from the same individual and that EBV infection and c-myc deregulation may not be sufficient for maintenance of the malignant phenotype.

Animals↗
PubMed1989

Independent regulation of neuropeptide mRNA level and poly(A) tail length.

Osmotic stimulation increases arginine-vasopressin (AVP) and oxytocin (OT) mRNA levels and poly(A) tail size concurrently. We now show that these changes are independently regulated. Whereas depletion of serotonin with p-chlorophenylalanine (PCPA) blocked the increase in hypothalamic levels of AVP and OT mRNA following osmotic stimuli in rats (2% saline drinking or dehydration), the increase in mRNA size was similar in PCPA and control animals. PCPA alone did not modify mRNA size. In addition to demonstrating an apparent obligatory role for serotonin in osmotically induced AVP and OT mRNA accumulation, the results provide evidence for different signaling pathways mediating transcript abundance and poly(A) tail length. Direct investigation of the regulatory mechanisms should be amenable to study in this system.

Animals↗
PubMed1989

Examination of the effects of epidermal growth factor on the production of urokinase and the expression of the plasminogen activator receptor in a human colon cancer cell line.

The expression of the plasminogen activator, urokinase, and the display of its receptor in response to growth factors were examined in a serum-free adapted colon cancer cell line, CBSsf. Cells propagated in protein-free medium secreted 6.5 +/- 1.0 ng/ml of urokinase/10(6) cells in a 3-day period as determined by enzyme-linked immunosorbent assay. Inclusion of insulin or transferrin into the protein-free medium was without effect on this parameter. However, addition of epidermal growth factor (EGF) to the protein-free medium resulted in a 50% reduction in this parameter. This change was also reflected in the plasminogen-dependent solubilization of immobilized radioactive laminin. Plasminogen-supplemented conditioned medium derived from CBSsf cells grown in protein-free medium solubilized 135,000 +/- 25,000 dpm/10(6) cells of radioactive substrate. This value was decreased to 59,000 +/- 6,000 when conditioned medium was collected in the presence of EGF. Dose-response curves indicated that, while 0.5 ng/ml of EGF were suboptimal for the suppression of urokinase secretion, a concentration of 5.0 ng/ml had a maximum effect on this measurement. Northern hybridization studies indicated that the reduced plasminogen activator reflected, at least in part, translation of a less abundant transcript. Examination of the colon carcinoma cell line for altered urokinase receptor display revealed that EGF caused a dose-dependent increase in the amount of radioactive urokinase bound. This did not reflect reduced occupation of binding sites with endogenous ligand. Scatchard manipulation of the binding data indicated that the increased amount of radioactive plasminogen activator bound to cells cultured with EGF reflected an increase in receptor number from 7,500 to 13,000 sites/cell. Time course studies revealed that the decrease in urokinase secretion precedes changes in receptor display by 5 h. A 60% reduction in assayable urokinase was demonstrated in the conditioned medium from cells treated with the growth peptide for 10 h. However, a 24-h period was required to observe an increase (80%) in the amount of radioligand bound to EGF-treated cells. These data suggest EGF to be a regulator of both urokinase production and urokinase receptor display in a colon cancer cell line.

Colonic Neoplasms↗
PubMedDate not supplied

Activation of protein kinase C by serotonin: biochemical evidence that it participates in the mechanisms underlying facilitation in Aplysia.

1.) Application of serotonin to Aplysia sensory neurons can result in facilitated synaptic transmission, both short- and long-term. This facilitation is likely to be produced by a complex set of molecular mechanisms: serotonin activates adenylate cyclase, increasing cAMP and protein kinase (Cedar and Schwartz, 1972); serotonin also changes the subcellular distribution of the Ca2+/calmodulin-dependent protein kinase (Saitoh and Schwartz, 1983). Recently, phorbol esters also have been shown to produce facilitation. We have therefore investigated how protein kinase C (PKC) participates in serotonin-mediated synaptic facilitation. 2.) We found that the Aplysia genome encodes PKC, which is expressed in nervous tissue as at least two abundant transcripts (about 0.003% of the total message). Its inferred amino acid sequence is 85% homologous to that of enzymes from mammals and Drosophila, and over 95% homologous if compared to both. The specific activity of the Aplysia kinase is comparable to that found in rat brain, with similar reaction parameters and dependencies on phosphatidylserine (PS), Ca2+, diacylglycerol and phorbol esters. While PKC is found on neuronal membrane in the basal state, the PKC activators, Ca2+ and phorbol esters, further translocate the kinase to membrane in crude extracts of neuronal tissue. The amounts of membrane-bound PKC, as determined by 3H-phorbol-ester binding, are greatest in neuropil and nerve. 3.) Exposure of sensory neurons and their terminals in Aplysia pleural-pedal ganglia to facilitating doses of either phorbol ester or serotonin results in the translocation of PKC from cytosol to membrane, activating the enzyme. cAMP does not produce this translocation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Selective cloning of cDNA for secretory proteins of early embryos. Identification of a transiently expressed kunitz domain protein from preimplantation sheep trophoblast.

The preimplantation ovine conceptus transiently secretes several proteins, including a type I interferon (IFN-tau), that are likely involved in establishment of pregnancy. A method has been developed to identify proteins produced simultaneously with IFN-tau. An antiserum against total ovine conceptus secretory proteins, from which IFN-tau and proteins common to maternal uterine tract secretions had first been removed, was used to immunoscreen cDNA libraries created from mRNA of days 13 and 15 ovine conceptuses. This approach has allowed several unique cDNA to be identified, including one particularly abundant transcript for a novel member of the Kunitz family of serine protease inhibitors. This cDNA encodes a 265-amino acid protein with a 20-amino acid signal sequence. A 64-amino acid Kunitz domain occupies the carboxyl terminus. It is preceded by two similar repeats of 84 residues that bear no obvious similarity to any sequences present in the protein data banks. The protein present in conceptus secretions (M(r) of approximately 14,000) represents only the carboxyl terminus of the molecule. The mRNA for this putative proteinase inhibitor was confined to trophectoderm and was highly expressed for only a few days (approximately 13-18) of development. A similar transcript was detected during the days 17-21 period in cattle embryos. Despite their high expression, no proteinase-inhibitory activity can so far be ascribed to either the ovine or bovine proteins. The P1 residue, an asparagine, is not represented in any other known Kunitz inhibitors.

Amino Acid Sequence↗
PubMed1995

Expression of extracellular matrix genes in adult human dermal microvascular endothelial cells and their regulation by heparin and endothelial cell mitogens.

BACKGROUND: Microvascular alterations are prominent features of systemic sclerosis (SSc) and often precede the appearance of clinically detectable fibrosis. The mechanism leading to selective microvascular injury in SSc is not known; however, microvascular endothelial cell (EC) activation has been demonstrated in SSc skin and is considered to be an early event in the pathogenesis of SSc. EXPERIMENTAL DESIGN: The expression of genes encoding extracellular matrix (ECM) proteins was examined in adult human dermal microvascular EC (HDMVEC), human iliac vein EC (HIVEC), and human umbilical vein EC (HUVEC) using indirect immunofluorescence (IIF) and Northern hybridization analysis. The effects of heparin and the endothelial cell mitogens, endothelial cell growth factor (ECGF) supplement and acidic and basic fibroblast growth factors (aFGF and bFGF), on the expression of ECM genes by these cells were also studied. RESULTS: Abundant transcripts for collagen types I, IV, VI, and fibronectin (FN) and weak expression of the type III collagen gene were detected in HDMVEC cultures in the absence of ECGF and heparin. In contrast, in the presence of these factors, no mRNA for types I, III, and VI collagens and marked down-regulation (more than twofold) of mRNA levels for collagen type IV and FN were observed. These results were confirmed at the protein level by IIF staining. In contrast to HDMVEC, HIVEC and HUVEC did not show expression of genes encoding types I, III, and VI collagens under any culture conditions examined. Next we studied the separate effect of heparin and aFGF or bFGF on the expression of ECM genes in HDMVEC. In contrast to the maximal expression of types I and VI collagens and FN detected in the absence of growth factors, aFGF decreased mRNA levels by 43% for type I collagen, by 52% for type VI collagen, and by 47% for FN. The decreases in mRNA levels caused by bFGF were 37, 41, and 36%, respectively. Heparin alone decreased the mRNA levels for these genes by 60, 77, and 65%, respectively; however, FGF potentiated the negative effect of heparin on ECM gene expression. CONCLUSIONS: These results demonstrate that HDMVEC display a unique pattern of expression of ECM genes that is different from that displayed by EC from medium and large vessels. The data also demonstrate that heparin, ECGF supplement, aFGF, and bFGF regulate ECM gene expression in HDMVEC in vitro and suggest that these growth factors may modulate the expression of matrix genes in vivo. Altered expression of ECM genes by HDMVEC may play an important role in diseases affecting the microvasculature, such as SSc.

Adult↗
PubMed1994

Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily.

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.

3T3 Cells↗
PubMed1996

Invertebrate synapsins: a single gene codes for several isoforms in Drosophila.

Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed.

Amino Acid Sequence↗
PubMed1996

Differential regulation of cyclin B1 RNA and protein expression during hepatocyte growth in vivo.

Cyclin genes and their products are important regulatory participants in the eukaryotic cell cycle. It is well established that cyclin B1 protein forms a complex with cyclin-dependent kinase 1 (CDK1), which, when activated, initiates mitosis. We have previously established that cyclin B1 gene expression is posttranscriptionally regulated in regenerating rat liver after 70% partial hepatectomy (PH). We now report further characterization of cyclin B1 gene expression, as well as that of CDK1 and cdc25B, in this unique in vivo model of cell proliferation. Cyclin B1 transcripts were detected by RNase protection through 96 h of liver regeneration and exhibited dramatic changes in steady-state levels. Peak expressions occurred at 24-30 h, more significantly at 42-48, and at 72 h. By Northern blot analysis, single transcripts for CDK1 and cdc25B were detected, and the temporal expression of both transcripts during liver regeneration mirrored that of cyclin B1. By Western blot and immunohistochemical analyses, cyclin B1 protein levels did not change significantly in either nuclear or cytoplasmic fractions, whereas CDK1 protein levels paralleled their associated RNA expression. Cdc25B protein levels steadily decreased from 0 to 96 h after PH. In addition, cytoplasmic protein levels of cyclin B1 exhibited a constant distribution in subfractions of microsome- and polysome-associated and free proteins. Cyclin B1 RNA also localized to these three cytoplasmic subfractions. Finally, the apparent translational activity of cyclin B1 transcripts was very similar at both 24 and 48 h after PH, in contrast to their respective mRNA half-lives. In a peroxisome proliferation model of hepatocyte growth and apoptosis, cyclin B1 and CDK1 proteins were induced in the absence of transcript up-regulation. Our results demonstrate that cyclin B1 mRNA steady-state levels are regulated posttranscriptionally in regenerating rat liver. Furthermore, the pattern of cyclin B1 transcript expression is paralleled by that of the CDK1 gene, whereas their respective protein steady-state levels provide a striking contrast. Finally, cyclin B1 is differentially regulated by an uncoupling of transcript abundance and translational processing in two in vivo models of hepatocyte growth. The abundance of cyclin B1 protein in nonreplicating cells suggests that cyclin B1 may be available for other cellular pathways in the hepatocyte.

Animals↗
PubMedDate not supplied

Regulation of IGFBP-1 and -4 expression by triiodothyronine (T3) in cultured hepatocytes is cell- and gene-specific.

Evidence suggests that thyroid hormone plays a role in the regulation of hepatic IGF/IGFBP expression both in human and rats. In this study we compared the effect of T3 on IGFBP-1 and -4 expression in rat hepatocyte primary cultures and in the human hepatoma cell line HepG2. Northern blot analysis revealed that IGFBP-1 mRNA levels were not affected by T3 in cultured rat hepatocytes, whereas a net increase of IGFBP-1 transcript abundance was induced by the hormone in HepG2 cells. On the contrary, IGFBP-4 mRNA levels were increased in rat hepatocytes cultured in the presence of T3, but unaffected in T3-treated HepG2 cells. Therefore, thyroid hormone seems to regulate hepatic IGFBP expression in a direct and gene-specific way. Moreover, the effects of thyroid hormone depend strictly on the source of target hepatocyte.

Animals↗
PubMed1998

Expression cloning and characterization of NSIST, a novel sulfotransferase expressed by a subset of neurons and postsynaptic targets.

Synapses are distinguished by localized concentrations of specific proteins, many of which bear the marks of posttranslational processing such as glycosylation and sulfation. One strategy to elucidate this posttranslational tailoring is to identify the enzymes that create these modifications. Monoclonal antibody 3B3 recognizes a carbohydrate-containing epitope expressed on dystroglycan and other constituents of Torpedo electric organ synaptic membranes. We used mAb 3B3 in an immunofluorescence-based expression-cloning method and isolated a cDNA clone conferring mAb-3B3 immunoreactivity to transfected COS cells. The deduced polypeptide has a predicted molecular weight of 51 kDa, a type II transmembrane topology, and four potential N-linked glycosylation sites. The polypeptide, which we term NSIST (nervous system involved sulfotransferase), shows extensive, although not complete, homology to a chondroitin-6-sulfotransferase and limited homology to other sulfotransferases. In NSIST-transfected COS cells, 35SO4 incorporation and chondroitin-sulfate-like immunoreactivity are increased. In vivo, NSIST occurs as a single 2.4 kb transcript abundant in Torpedo electric organ, moderately expressed in spinal cord and electric lobe, and undetectable in non-neural tissues. Immunohistochemistry shows that NSIST is expressed in a punctate distribution in the innervated portion of electrocytes. In the CNS, NSIST-like immunoreactivity is localized within the somas of motor neurons and neurons of the electromotor nucleus, whereas mAb-3B3 immunostaining is associated with cell surfaces and neuropil. Neuronal NSIST is therefore likely to exert its effects extracellularly; although NSIST is synthesized by neurons, its product, the 3B3 epitope, is found outside neuronal cell bodies. Our evidence indicates that NSIST participates in nervous system specific posttranslational modifications, perhaps including those at synapses.

Animals↗
PubMed2001

The abundance of sterile transcripts in Giardia lamblia.

The protozoan parasite Giardia lamblia synthesizes a diverse and surprisingly abundant array of sterile transcripts unable to code for proteins. Random sampling of cDNAs from two evolutionarily divergent Giardia strains indicates that approximately 20% of cDNAs in the libraries represent polyadenylated sterile transcripts. RNase protection analysis and northern blot hybridization of three sterile transcript loci demonstrated that both the sterile transcript and a complementary mRNA were made in each case, further categorizing these sterile transcripts as antisense transcripts. Investigation of the genomic loci for these same three sterile antisense transcripts showed typical transcription units for the sense transcripts, but still failed to reveal a usable open reading frame for the sterile antisense transcripts. 5'-RACE mapped the transcription start site for one of the sterile antisense transcripts to an AT-rich region, as is typical for GIARDIA: It is unclear whether these sterile transcripts represent errors in transcription or whether they have regulatory functions within the cell, although preliminary investigations failed to reveal evidence for a role in developmental gene regulation. In either case, the presence of such a large pool of sterile antisense transcripts is dramatic evidence of the unusual molecular machinery of the early diverging protist G.lamblia.

Animals↗
PubMed2007

Development and pattern of mRNA relative abundance of bovine embryos cultured in the isolated mouse oviduct in organ culture.

The aim of this study was to examine the development of bovine zygotes in isolated mouse oviducts (IMO) and the quality of the blastocysts produced. In vitro produced bovine zygotes were transferred into the ampullae of the IMO and cultured in SOF or KSOM. Control embryos were cultured in droplets of the same media. Following 6 days of culture, blastocysts were processed for nuclei counts or mRNA abundance. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 17.7 +/- 3.2% vs. 18.8 +/- 2.7%; KSOM: 20.7 +/- 2.6% vs. 22.2 +/- 2.8%). Culture in the IMO in KSOM resulted in an increased number of inner cell mass (ICM) nuclei; however, total nuclei number or incidence of apoptosis was unaffected. Culture in the IMO in SOF resulted in an increase (P < 0.05) in abundance of transcripts in blastocysts for Oct-4 and SOX, and reduced abundance of Glut-1, Na/K, Cx43, and survivin compared to blastocysts derived from culture in SOF alone. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and reduced expression of Na/K and SOX compared to KSOM alone. Transcripts for G6PDH, IFN-tau, and E-Cad were unaffected. These data confirm that the IMO is capable of supporting development of bovine embryos. Depending on the basal medium used, the pattern of transcript abundance in embryos derived from the IMO is similar to that of in vivo derived embryos.

Animals↗
PubMed1985

Alterations in early adenovirus transcription and mRNA abundance induced by translational inhibitors.

At least six viral genes are activated early after adenovirus 2 infection of HeLa cells. In an effort to define more clearly the effects of protein synthesis inhibition on early viral gene expression, the rate of transcription and the cytoplasmic abundance of viral mRNA from each transcription unit were studied in infected cells treated with translational inhibitors added at different times during infection and at concentrations known to inhibit 95 to 99% of protein synthesis. Tritiated uridine-labeled nuclear and cytoplasmic viral RNA molecules synthesized in infected HeLa cells in both the presence and absence of inhibitors were analyzed and compared by hybridization using specific DNA probes. Translational inhibitors (25 micrograms/ml of cycloheximide or 10 microM anisomycin) added 30 min to 1 h after infection stimulated the over-accumulation of cytoplasmic viral mRNA from 3- to 20-fold, early regions E1A, E2, and E4 being affected most. This overproduction of viral mRNA was found to result from enhanced transcription of the respective early genes. Treatment of cells with 100 microM anisomycin 1 h prior to and during infection did not prevent the accumulation of early viral mRNA molecules; however, total cellular RNA synthesis was drastically inhibited in 100 microM anisomycin-treated cells. These results support the hypothesis that a cellular factor may function in the regulation of early viral gene expression at a transcriptional level.

Adenoviridae↗
PubMed1988

Phytochrome regulation of greening in barley : effects on mRNA abundance and on transcriptional activity of isolated nuclei.

Red light treatment of etiolated barley (Hordeum vulgare L.) seedlings causes an increase in the relative abundance of the mRNA for light-harvesting, chlorophyll a/b-binding polypeptides, and a decrease in the relative abundance of the mRNA for the NADPH:protochlorophyllide oxidoreductase. It also increases transcriptional activity of subsequently isolated nuclei for the mRNA for the chlorophyll-binding polypeptides and reduces it for the reductase. These results confirm those published previously. Fluence-response and kinetic studies support the hypothesis that the abundance of mRNA for the chlorophyll-binding polypeptides may be transcriptionally limited. They do not support the same hypothesis for the reductase. Red light treatments in the very low fluence range significantly decrease transcriptional activity in isolated nuclei for RNAs hybridizable by the reductase probe, but have little effect on mRNA abundance. By contrast, red light treatments in the low fluence range bring about a sharp decrease in reductase mRNA abundance with little further effect on transcription, suggesting light regulation at the level of mRNA stability rather than transcription. The fluence-response relationships for increase in abundance of mRNA for the chlorophyll-binding polypeptides is similar to that published elsewhere for elimination of the lag period of chlorophyll accumulation in barley. However, kinetic studies published elsewhere show that for elimination of the lag in chlorophyll accumulation, the dependence on the far red-absorbing form of phytochrome concentration is significantly different from the dependence of the transcriptional changes, suggesting that although transcription might be what limits mRNA abundance for the chlorophyll-binding proteins, the mRNA abundance can not be what limits chlorophyll accumulation per se.

Journal Article↗
PubMed2005

The maize root transcriptome by serial analysis of gene expression.

Serial Analysis of Gene Expression was used to define number and relative abundance of transcripts in the root tip of well-watered maize seedlings (Zea mays cv FR697). In total, 161,320 tags represented a minimum of 14,850 genes, based on at least two tags detected per transcript. The root transcriptome has been sampled to an estimated copy number of approximately five transcripts per cell. An extrapolation from the data and testing of single-tag identifiers by reverse transcription-PCR indicated that the maize root transcriptome should amount to at least 22,000 expressed genes. Frequency ranged from low copy number (2-5, 68.8%) to highly abundant transcripts (100-->1,200; 1%). Quantitative reverse transcription-PCR for selected transcripts indicated high correlation with tag frequency. Computational analysis compared this set with known maize transcripts and other root transcriptome models. Among the 14,850 tags, 7,010 (47%) were found for which no maize cDNA or gene model existed. Comparing the maize root transcriptome with that in other plants indicated that highly expressed transcripts differed substantially; less than 5% of the most abundant transcripts were shared between maize and Arabidopsis (Arabidopsis thaliana). Transcript categories highlight functions of the maize root tip. Significant variation in abundance characterizes transcripts derived from isoforms of individual enzymes in biochemical pathways.

Base Sequence↗
PubMed1998

Analysis of variation in relative mRNA abundance for specific gene transcripts in single bovine oocytes and early embryos.

Variation in the abundance of a specific gene transcript was assessed in single bovine oocytes and in vitro-derived blastocysts. Transcripts encoding the Na+,K(+)-ATPase alpha 1 subunit were detected by reverse-transcription polymerase chain reaction (RT-PCR) and quantified relative to an exogenously supplied rabbit alpha-globin mRNA using laser-induced fluorescence capillary electrophoresis (LIF-CE). The precision of this relative abundance (RA) calculation was predicted and shown to resolve 2-fold differences in transcript abundance between individual blastocysts and predicted in oocytes to resolve 3-fold differences. The RA of the alpha 1 subunit transcript differed by 2- to 3-fold among blastocysts, and 3- to 6-fold among oocytes. Comparison of a general population of oocytes with blastocysts revealed little overlap in RA values between the two groups, with a 8- to 14-fold increase in the mean RA for each group with development observed in two successive experiments (P < or = 0.05). In contrast, oocytes selected for their developmental competence on the basis of morphologic criteria exhibited only a 1.6- to 1.7-fold developmental increase when the assay was performed on cDNA generated from either embryo pools (n = 6 versus 6) or individuals (n = 7 versus 7), respectively. These results provide the first characterization of the degree of heterogeneity in the abundance of a specific mRNA transcript among individual mammalian oocytes and preimplantation embryos and demonstrate that transcript relative abundance can be correlated with bovine oocyte morphology.

Actins↗
PubMed1999

Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA.

In preimplantation bovine embryos, the relative abundance of various developmentally important gene transcripts was determined by a semi-quantitative RT-PCR assay to analyze the effects of two medium supplements, serum or polyvinyl alcohol (PVA). Development to morula, blastocyst, and hatched blastocyst stages was higher (P < or = 0.05) in medium supplemented with serum than in medium supplemented with PVA. Connexin43 mRNA expression virtually disappeared from the 8-16 cell stage onward, but reappeared in the hatched blastocyst in serum-supplemented medium, whereas it was detected in PVA-derived embryos throughout development. No differences were found for plakophilin mRNA between both culture groups. Desmocollin II mRNA showed a sharp increase at the blastocyst stage in both groups with a higher transcription level in PVA-generated embryos. A significant difference in desmocollin III transcripts was detectable at the 8-16-cell stage between serum- and PVA-derived embryos. Transcripts for desmoglein 1 and desmocollin I were not detected at any preimplantation stage, irrespective of medium supplementation. The relative abundance of glucose-transporter-1 mRNA was significantly increased at the 8-16-cell stage in embryos produced in medium supplemented with PVA, but not serum. Heat shock protein and poly(A)polymerase mRNA were continuously expressed during preimplantation development in both culture groups. Although poly(A)polymerase mRNA was significantly elevated in PVA- over serum-derived embryos, heat shock protein mRNA expression was significantly enhanced in serum-generated embryos over PVA-derived embryos. Interferon tau mRNA showed a significant increase at the hatched blastocyst stage only in PVA-supplemented medium. These data suggest that alterations in mRNA expression are associated with culture environment. Timing and magnitude of the alterations varied among the different transcripts and were significantly affected by the presence of exogenous protein in a stage-specific manner, predominantly at critical developmental time points.

Animals↗