Translocation of repetitive RNA sequences with the germ plasm in Xenopus oocytes.

Xlsirts are a family of interspersed repeat RNAs from Xenopus laevis that contain from 3 to 13 repeat units (each 79 to 81 nucleotides long) flanked by unique sequences. They are homologous to the mammalian Xist gene that is involved in X chromosome inactivation. Xlsirt RNA appears first in the mitochondrial cloud (Balbiani body) in stage 2 oocytes and is then translocated as island-like structures to the vegetal cortex at early stage 3 coincident with the localization of the germ plasm. Exogenous Xlsirt RNA injected into oocytes translocates to the location of the endogenous RNA at that particular stage. The Xlsirt RNA repeat sequences are required for translocation and can cause the translocation of heterologous unique RNAs to the vegetal cortex.

Regulation of Xenopus c-myc promoter activity in oocytes and embryos.

We have studied the regulation of transcription of the Xenopus c-myc I gene in oocytes and embryos. Various 5' and internal deletions of a 1310-bp-long c-myc I promoter fragment have been ligated upstream of the chloramphenicol acetyl transferase (CAT) reporter gene and microinjected into oocytes and fertilized eggs. Activity was determined by CAT assay and primer extension. The c-myc promoter drives transcription very efficiently, and a truncated promoter -158/+46 essentially retains full activity. This region contains an overlapping E2F/SP1 site and two tandem Sp1 sites homologous to those found in the c-myc gene of mouse. Internal deletions show that both elements are equally active in oocytes in driving the expression of CAT. A germinal vesicle extract contains a DNA-binding activity specific for an Sp1 consensus sequence but not the E2F site. The data suggest that the high transcription level of the endogenous c-myc gene in Xenopus oocytes is mediated by Sp1 or a related transcription factor. In embryos a different mechanism emerges and the functional role of the Sp1 binding sites appears to be less important.

Messenger RNA synthesis in synchronized Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G1, S or G2 phase by incubation for 90 min with [3H]- OR [14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Poly-adenylated [3H]uridine-labelled mRNA from the G1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [14C] uridine-labelled polyadenylated nRNA from the S or G2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7-28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.

Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos.

A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.

Id gene activity during Xenopus embryogenesis.

The activity of bHLH transcription factors that are involved in cell determination and differentiation is inhibited by Ids, HLH proteins lacking the basic amino acid sequence element. In order to determine the role of Id during development, we have isolated and characterized the Id genes expressed in Xenopus embryos. Three cDNAs were characterized: XIdIa and XIdIb, which are transcribed from one gene but differentially spliced in the 3' untranslated part, and XIdII which is transcribed from a second copy of the gene. One of the two forms of the differentially spliced mRNAs exhibits, 30 nucleotides upstream from the AATAAA site, a sequence box homologous to the cytoplasmic polyadenylation element (CPE) which is present also in Id2 and Id3 mRNAs from higher vertebrates. This raises the question of whether this CPE-like element may link Id mRNA polyadenylation and translation to the cell cycle metabolism. The Xenopus Id gene is transcribed at low level in oocytes and at high level in embryos, after midblastula transition, in a large number of tissues, including the notochord, neural tube, eye, ear, neural crest cells, presomitic mesoderm, myotomes, tailbud and dorsal fin. In myotomes, expression is high in the areas of proliferating myoblasts and decreases as terminal differentiation proceeds, consistent with a function in cell determination and differentiation and possibly also in cell cycle regulation.

Localized XId3 mRNA activation in Xenopus embryos by cytoplasmic polyadenylation.

In Xenopus development, during meiosis and cleavage, the extent of polyadenylation plays a central role in regulating the expression of transcripts and this is mediated by cis regulatory cytoplasmic polyadenylation elements (CPE) in the 3'-UTRs. We have identified a palindromic CPE in the mRNA of Xenopus Id3 which is conserved in the Id genes from other vertebrates. It promotes cytoplasmic polyadenylation and is negatively regulated by sequences further upstream in the 3'-UTR. This palindromic CPE promotes polyadenylation in both the epithelial and sensorial layers of the dorsal ectoderm in early embryos, but association with the upstream negative element blocks this effect in the epithelial layer. The asymmetric polyadenylation may be important for establishing a prepattern of transcriptional regulators.

17-Iodine-123 iodoheptadecanoic acid for metabolic liver studies in humans.

(17-123I)-Iodoheptadecanoic acid ([123I]HA) was used for dynamic planar scintigraphy of the liver in normal individuals (control I), in patients without liver disease but with elevated serum cholesterol and/or triglycerides (control II), and in patient groups with alcohol-induced fatty liver (PG I), fatty liver not due to alcohol (PG II), alcohol-induced liver cirrhosis (PG III), or liver cirrhosis of the posthepatitic type (PG IV). Tracer uptake and elimination time were assayed in different liver regions; mean elimination time was expressed for total liver. In control I, tracer uptake was homogeneous, and mean elimination time was 20.7 +/- 5.3 min without significant local variations. In control II, tracer uptake was reduced but homogeneous and mean elimination time was 59.4 +/- 35.8 min with some local variations. In PG I, uptake was reduced and inhomogeneous and elimination time was the same as in control I, irrespective of cholesterol and triglyceride values. In PG II, uptake was the same as in PG I but mean elimination time was 48 +/- 8.1 min with some local variations. In PG III, uptake was extremely reduced and spotty and elimination time correlated with the severity of disease from 19 to 881 min in different liver regions.

Absolute quantification of pharmacokinetic distribution of RES colloids in individuals with normal liver function.

Estimates of the radiation dose resulting from liver-spleen scintigraphy 99Tcm-labelled colloids are based on pharmacokinetic data mainly determined in animals. The aim of this study was to check the pharmacokinetic data by direct, absolute in vivo quantification in man. For this purpose appropriate methods of measurement were developed, or procedures taken over from literature were modified. Liver and spleen activities were directly measured using a double-energy window technique. Activities in other organs were quantified by conjugate whole-body scans. All measurement procedures were checked using the whole-body Alderson phantom. Pharmacokinetic data for sulphur colloid, tin colloid, human serum albumin (HSA) millimicrospheres, and phytate were obtained in 13 to 20 normal subjects for each type of colloid. Depending on the colloid type liver uptake was between 54 and 75% of the total administered dose (TAD) and spleen uptake was 3.5 to 21% TAD. Activity measured in blood, urine, lung and thyroid proved to be far from negligible. The results of this work suggest a correction of the animal-based data of colloid distribution and radiation dose on the basis of the direct measurement of absolute uptake in man.

15-(ortho-123I-phenyl)-pentadecanoic acid, a new myocardial imaging agent for clinical use.

The result of previous experiments in rodents indicated different kinetics for the para- and ortho-isomers of 15-(iodophenyl)-pentadecanoic acid (p-IPPA, o-IPPA), with o-IPPA showing an enhanced rate of washout. To test the relevance of this phenomenon for clinical diagnosis, 15 fasting male patients with confirmed coronary heart disease (1-VD/7, 2-VD/4, 3-VD/4) were investigated under exercise. Serial images were recorded at a rate of 3 frames min-1 for 70 to 90 min, corrected for tracer in blood and compared with thallium-201 images obtained from these patients within less than 2 weeks. Time-activity curves were also taken from the peripheral blood. Ortho-IPPA was well taken up by healthy myocardium and, contrary to rodents, retained with elimination half times longer than 200 min. A decreased myocardial uptake was seen which was very similar to the pattern obtained with thallium. Ortho-IPPA was eliminated from the blood to less than 10% at 4 min. Almost all radioactivity was in the organic phase (greater than 95% at 5 min) and chromatography showed only one major peak (o-IPPA) indicative of minimal organic catabolism.

Myocardial fatty acid metabolism after acute ethanol consumption.

This study was undertaken to assess the effect of ethanol ingestion on myocardial fatty acid metabolism in man. Nine individuals with informed consent and with a habitual ethanol consumption of approximately 40 g per day, but without any clinical signs of heart and metabolic disease, were examined after i.v. injection of omega-123I-heptadecanoic acid (IHA). Eight days later, these individuals were similarly examined after 2 h of continuous ingestion of a body weight dependent amount of ethanol, which was calculated to produce a blood level of 100 mg per 100 ml (1%). Then the subjects had been asked to reduce their ethanol consumption rigorously for 15 months. Subsequently after 2 weeks of abstinence a follow-up investigation without ethanol loading was carried out. The investigations were performed with an Anger scintillation camera in LAO-45 degrees projection. The measurement period was 40 min. Tracer accumulation and regional elimination half-times of IHA were analysed. In all patients, acute ethanol loading produced significant changes in pattern of accumulation and/or regional elimination half-times. Ethanol-induced alterations in segmental accumulation did not appear to be predictably correlated with changes in segmental elimination half-times. After rigorous reduction of ethanol consumption followed by 2 weeks of abstinence a normalization of the tracer uptake was observed; the distribution pattern was almost homogeneous. Also the regional elimination half-times became normal. The data demonstrate the significant effects of both chronic ethanol consumption and particularly acute ethanol loading on myocardial fatty acid metabolism and the reversibility of the effects.

Xenopus laevis c-myc I and II genes: molecular structure and developmental expression.

The structure of the two Xenopus laevis c-myc I and c-myc II genes has been investigated by isolating and sequencing genomic and cDNAs clones. In oocytes, c-myc I mRNAs represent 80-90% of the overall amount of c-myc transcripts. The c-myc I expression is controlled primarily by two differentially regulated tandem promoters P1 and P2 which are separated by 50 bases. During oogenesis, maternal c-myc I mRNAs, are transcribed from both promoters whereas zygotic transcripts seem to initiate only from the P2 promoter. Sequence comparison between the promoter regions of c-myc I and II genes reveals the insertion in the c-myc I promoter region, between positions -831 and -389 relative to the P1 start site of a repetitive element. Comparison of X.laevis and mammalian c-myc promoter sequences reveals furthermore the conservation of cis-regulatory elements, including a motif known to be a negative regulator of the human c-myc transcription, a purine rich region, a binding site for the E2-F transcription factor and three SP1 binding sites. Finally, we report characterization of a new c-myc I mRNA which differ at the 5' end. Transcripts are possibly initiated at a putative alternative promoter located further upstream in the genome, and undergoes alternative splicing.

A processed gene coding for a sarcomeric actin in Xenopus laevis and Xenopus tropicalis.

A processed gene potentially coding for a sarcomeric actin has been identified in Xenopus laevis and in the more primitive species X. tropicalis. The peptides encoded in these two species differ by two out of 377 amino acid residues. On the basis of the amino acid substitutions, the encoded peptide was identified as an alpha-skeletal actin in X. laevis and as an alpha-cardiac actin in X. tropicalis. Northern blot analysis and S1 mapping experiments suggest that in X. tropicalis the gene is expressed in embryos and adult heart. In X. laevis, transcripts homologous to this gene were found in embryos and in adult tissues, but predominantly in skeletal muscle rather than in heart.

Frequency distribution of mRNA and pre-mRNA in growing and differentiated Friend cells.

The frequency distribution of poly(A)+-mRNA in growing and in differentiated Friend cells has been measured by mRNA-cDNA hybridization and their differences established by heterologous hybridization of mRNA of one type and cDNA of the other. It was shown that induction of Friend cells involves an increase in abundance of a small number of mRNAs, while no specific pattern of messenger disappearance could be detected. The frequency distribution of pre-mRNA was determined by hybridizing nuclear RNA with the cDNA probes complementary to mRNA. In uninduced Friend cells, it was shown that most precursor messenger sequences are present at a single frequency of about 3 molecules per nucleus, independently of their final frequency in polysomal mRNA. In induced Friend cells, the frequency distribution of pre-mRNA is more heterogeneous and correlated to some extent with the corresponding mRNA frequency distribution.

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells.

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.

Identification of Xenopus laevis mRNAs with homology to repetitive sequences.

Hybrid selection translation experiments have been carried out with genomic and cDNA relatives of two repetitive sequence families. On the basis of the in vitro translation products detected, it was found that transcripts complementary to these repeats are linked to several different mature mRNAs in stage 40 embryos of Xenopus laevis. One repeat hybridizes to mRNAs that direct the synthesis of 17 proteins. The second is present on mRNAs coding for 3 proteins. By estimating the abundance of these proteins among the translation products of total embryonic mRNA, it is inferred that all of the repeat bearing mRNAs are rare, less than one in 20,000 mRNA molecules.

Electron microscope study of duck globin mRNA precursor crosslinked in situ.

Double-stranded RNA segments present in duck globin pre-mRNA were crosslinked in situ with aminomethyltrioxalen and UV light. The secondary structure of the crosslinked pre-mRNA was then studied by electron-microscopic analysis of pre-mRNA . cDNA hybrids. The data suggest that duck globin pre-mRNAs contain intervening sequences that are excised stepwise. Excision and subsequent ligation appears to occur on precursor molecules that are stabilized by base-paired regions.

Synthesis and processing of nuclear precursor-messenger RNA in avian erythroblasts and HeLa cells.

The kinetics of synthesis and turnover of animal cell nuclear precursor-mRNA fractions all of which, in the case of avian erythroblast RNA, are shown by specific complementary DNA hybridization to contain globin mRNA sequences, were analyzed by exponential polyacrylamide gel electrophoresis. Three metabolically distinct size-fractions were characterized: (1) nascent precursor-mRNA (apparent molecular weight 5 to 20 x 10(6), approximate half-life 30 min), (2) intermediate-size precursor-mRNA (molecular weight 1 to 5 x 10(6), approximate half-life 3 hr), (3) small precursor-mRNA (molecular weight 0.5 to 1.5 x 10(6), half-life more than 15 hr). Nascent precursor-mRNA behaves kinetically as a precursor to the smaller precursor-mRNAs that accumulate in the nucleus, as well as to cytoplasmic mRNA; however, no stringent proof can be given that the two smaller nuclear precursor-mRNA fractions are direct physical precursors of functional mRNA. In terms of total mass, more precursor-mRNA accumulates in the nucleus than there is translated mRNA in the cytoplasm. Globin mRNA of final size (9 S) does not accumulate in the nuclei of avian erythroblasts.

Globin mRNA sequences in polyadenylated and nonpolyadenylated nuclear precursor-messenger RNA from avian erythroblasts.

Nuclear RNA from immature duck erythrocytes was fractionated into polyadenylated and nonpolyadenylated fractions, and globin mRNA sequences were determined by hybridization to DNA complementary to globin mRNA. 80--90% of labeled nuclear RNA is found to be nonpolyadenylated, and 70--80% of the globin mRNA sequences present in the nucleus are found in nonpolyadenylated molecules. These data suggest that polyadenylation does not specifically select for globin mRNA sequences. The nonpolyadenylated globin mRNA sequences present in the nucleus are found mostly in molecules of small size, close to the size of polyribosomal globin mRNA, suggesting that polyadenylation is a later event in globin mRNA formation.

Haemodynamic effects of ketamine and thiopentone during anaesthetic induction for caesarean section.

Ketamine (1 mg . kg-1) or thiopentone (4 mg . kg-1) was used to induce anaesthesia for Caesarean section in 62 normotensive patients. During induction of anaesthesia and before laryngoscopy, blood pressure did not change in either group (preinduction systolic blood pressure, 131 mmHg, and diastolic blood pressure, 75 mmHg). When laryngoscopy and intubation were performed, mean blood pressures of both patient groups increased 20-30 per cent. With ketamine (n = 30) heart rate was unchanged from the preinduction rate of 85 beats/min before laryngoscopy and increased significantly by 15 per cent during laryngoscopy and intubation. With thiopentone (n = 32), heart rate increased significantly to 20 per cent above the preinduction rate of 87 beats/min during induction and increased further (to 35 per cent above the preinduction rate) during laryngoscopy and intubation. The average maximal rate-pressure product calculated for the thiopentone group was over 18,000, which was significantly higher than the 15,000 calculated for the ketamine group. Neonatal outcome as assessed by Apgar score and umbilical blood gas analysis was good and did not differ significantly between groups.