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F Amaldi

Publications and source records attributed to F Amaldi.

At least 55 records · Page 3Linked to original sources
PubMed1989

Expression of ribosomal protein genes and regulation of ribosome biosynthesis in Xenopus development.

Studies on ribosome biosynthesis in developing Xenopus oocytes and embryos, and after microinjection of cloned ribosomal-protein genes, have revealed that the synthesis of ribosomal proteins (r-proteins) is controlled by two types of regulation: (1) a post-transcriptional regulation, operated by feedback of the r-proteins themselves, controls processing and stability of r-protein transcripts and thus the amount of the corresponding mRNA present in the cell; and (2) a translational regulation controls the efficiency of utilization of r-protein mRNA (rp-mRNA) in response to the cellular needs for new ribosomes.

Animals↗
PubMed1989

Nucleolin from Xenopus laevis: cDNA cloning and expression during development.

Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis.

Amino Acid Sequence↗
PubMed1988

Isolation and nucleotide sequences of cDNAs for Xenopus laevis ribosomal protein S8: similarities in the 5' and 3' untranslated regions of mRNAs for various r-proteins.

Recombinant clones specific for ribosomal protein (r-protein) S8 have been isolated from a Xenopus laevis cDNA bank. Sequence analysis shows that they are of two types, derived from two different gene copies originating from gene duplication. The two cDNAs differ in several silent sites and code for the same S8 protein whose complete amino acid sequence has been derived. Sequence comparison of S8 mRNAs with those for other X. laevis r-proteins, has revealed interesting similarities in the 5' and 3' untranslated regions. These could be involved in r-protein synthesis regulation which we have previously shown to occur mainly at post-transcriptional and translational levels.

Amino Acid Sequence↗
PubMed1986

Sequences coding for the ribosomal protein L14 in Xenopus laevis and Xenopus tropicalis; homologies in the 5' untranslated region are shared with other r-protein mRNAs.

In the haploid genome of Xenopus laevis there are two genes coding for the r-protein L14. It is not known if they are located on the same chromosome. cDNA clones deriving from the transcripts of the two genes have been isolated from an oocyte messenger cDNA bank showing that they are both expressed. We have studied the structure of one of the L14 genes by Electron Microscopy, restriction mapping and sequencing. An allelic form of the L14 gene was also isolated. It contains a large deletion covering the 5' end region up to the middle of the third intron. The 5' end of the X. laevis L14 gene was compared to that of the corresponding gene in the closely related species X. tropicalis and found to be highly conserved. The L14 gene has multiple initiation sites, but the large majority of the transcripts start in the middle of a pyrimidine tract not preceded by a canonical TATA box as in other eukaryotic housekeeping genes. The X. laevis L1 and L14 genes have a common decanucleotide in the first exon in the same position with regard to the initiator ATG which just precedes the first intron. The decanucleotide shows homology with the X. laevis 18S rRNA.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of the L1 ribosomal protein gene of Xenopus laevis: remarkable sequence homology among introns.

Ribosomal protein L1 is encoded by two genes in Xenopus laevis. The comparison of two cDNA sequences shows that the two L1 gene copies (L1a and L1b) have diverged in many silent sites and very few substitution sites; moreover a small duplication occurred at the very end of the coding region of the L1b gene which thus codes for a product five amino acids longer than that coded by L1a. Quantitatively the divergence between the two L1 genes confirms that a whole genome duplication took place in Xenopus laevis approximately 30 million years ago. A genomic fragment containing one of the two L1 gene copies (L1a), with its nine introns and flanking regions, has been completely sequenced. The 5' end of this gene has been mapped within a 20-pyridimine stretch as already found for other vertebrate ribosomal protein genes. Four of the nine introns have a 60-nucleotide sequence with 80% homology; within this region some boxes, one of which is 16 nucleotides long, are 100% homologous among the four introns. This feature of L1a gene introns is interesting since we have previously shown that the activity of this gene is regulated at a post-transcriptional level and it involves the block of the normal splicing of some intron sequences.

Animals↗
PubMed1985

Ribosomal-protein synthesis is not autogenously regulated at the translational level in Xenopus laevis.

Whether ribosomal-protein synthesis in Xenopus laevis is autogenously controlled at the translational level as is known to occur in prokaryotes has been studied. For this purpose ribosomal (r) proteins were prepared from X. laevis ribosomal subunits and group fractionated by ion-exchange chromatography. They were then added to an in vitro translation system directed by an oocyte mRNA fraction which contains template activity for r proteins. The synthesized radioactive products were analyzed by 2D gel electrophoresis and compared with controls. Similarly in vivo experiments were performed by microinjection of the fractionated proteins into the cytoplasm of Xenopus oocytes followed by incubation with [35S]methionine for different times. In all the experiments no evident effect of r proteins on the translation of their own mRNA was observed.

Animals↗
PubMed1985

Ribosomal protein production in normal and anucleolate Xenopus embryos: regulation at the posttranscriptional and translational levels.

We have studied the regulation of ribosomal protein (r-protein) synthesis in Xenopus anucleolate mutants, which lack the genes for rRNA. The accumulation of mRNA for the two r-proteins analyzed parallels the controls up to stage 30. This mRNA is mobilized onto polysomes and is translated as in normal embryos, but r-proteins are unstable in the absence of rRNA to assemble with. A translational control of rp-mRNA distribution between polysomes and mRNPs is observed, but this is not due to an autogenous regulation by r-proteins. After stage 30 the amount of rp-mRNA declines specifically in the mutants because the transcripts are unstable. Considering the temporal correlation between this event and the onset of r-protein synthesis we suggest that an autogenous control operates at the level of transcript stability.

Animals↗
PubMed1984

Expression of two Xenopus laevis ribosomal protein genes in injected frog oocytes. A specific splicing block interferes with the L1 RNA maturation.

The expression of two Xenopus laevis ribosomal protein genes (L1 and L14) has been analysed by microinjection of the cloned genomic sequences into frog oocyte nuclei. While the injection of the L14 gene causes the accumulation of the corresponding protein in large excess with respect to that synthesized endogenously, the L1 gene does not. Analysis of the RNA shows that both genes are actively transcribed. The seven-intron-containing L14 transcript is completely processed to a mature form, while two out of nine intron sequences persist in the L1 transcript. This precursor RNA is confined to the nucleus; its accumulation is due to a specific block of splicing operating at the level of two defined introns and not to saturation of the processing apparatus of the oocyte. The different behaviour of the two genes may reflect different mechanisms of regulation which, in the case of the L1 gene, could operate at the level of splicing.

Animals↗
PubMed1984

Splicing of Xenopus laevis ribosomal protein RNAs is inhibited in vivo by antisera to ribonucleoproteins containing U1 small nuclear RNA.

The activity of antisera against ribonucleoproteins containing U1 small nuclear RNA (Sm and RNP) has been analysed on pol II transcripts in an in vivo system. Xenopus laevis ribosomal protein gene transcripts are accumulated in the form of precursor RNA when either of the two kinds of antisera are injected into the germinal vesicles of X. laevis oocytes before the injection of purified L1 and L14 ribosomal protein genes. No effect on the accumulation of mature histone mRNA is detected when X. laevis histone genes are injected together with the RNP antiserum. These results strongly suggest that U1-RNP complexes play an essential role in intron removal in vivo.

Animals↗
PubMed1982

Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.

Amino Acid Sequence↗
PubMed1982

Expression of ribosomal-protein genes in Xenopus laevis development.

Using probes to Xenopus laevis ribosomal-protein (r-protein) mRNAs, we have found that in the oocyte the accumulation of r-protein mRNAs proceeds to a maximum level, which is attained at the onset of vitellogenesis and remains stable thereafter. In the embryo, r-protein mRNA sequences are present at low levels in the cytoplasm during early cleavage (stages 2-5), become undetectable until gastrulation (stage 10) and accumulate progressively afterwards. Normalization of the amount of mRNA to cell number suggests an activation of r-protein genes around stage 10; however, a variation in mRNA turnover cannot be excluded. Newly synthesized ribosomal proteins cannot be found from early cleavage up to stage 26, with the exception of S3, L17 and L31, which are constantly made, and protein L5, which starts to be synthesized around stage 7. A complete set of ribosomal proteins is actively produced only in tailbud embryos (stages 28-32), several hours after the appearance of their mRNAs. Before stage 26 these mRNA sequences are found on subpolysomal fractions, whereas more than 50% of them are associated with polysomes at stage 31. Anucleolate mutants do not synthesize ribosomal proteins at the time when normal embryos do it very actively; nevertheless, they accumulate r-protein mRNAs.

Animals↗
PubMed1982

Nucleotide sequences of cloned cDNA fragments specific for six Xenopus laevis ribosomal proteins.

We have previously constructed and selected six recombinant plasmids containing cDNA sequences specific for different ribosomal proteins of Xenopus laevis (Bozzoni et al., 1981). DNA cloned in these plasmids have been isolated and sequenced. Amino acid sequences of the corresponding portions of the proteins have been derived from DNA sequences; they are arginine- and lysine-rich as expected for ribosomal proteins. One of the cDNA sequences has an open reading frame also on the strand complementary to the one coding for the ribosomal protein; this fragment has inverted repeats twenty nucleotides lone at the two ends. The codon usage for the six sequences appears to be non-random with some differences among the ribosomal proteins analysed.

Animals↗
PubMed1981

Replication of ribosomal DNA in Xenopus laevis.

The study of the localization of the replication origins of rDNA in Xenopus laevis has been approached by two different methods. 1. The DNA of X. laevis larvae was fractionated by CsCl gradient centrifugation in bulk and ribosomal DNA and examined in the electron microscope. In bulk DNA, clusters of microbubbles, which are related with the origins of replication, appear to be spaced along the DNA molecules at intervals comparable with the size of the 'average' replicon of X. laevis. In ribosomal DNA, the distance between adjacent clusters is much shorter and corresponds to the size of the rDNA repeating unit. When ribosomal DNA was submitted to digestion with restriction enzymes (Eco RI and HindIII) the microbubbles are observed in the non-transcribed spacer-containing fragment. 2. Cultured cells of X. laevis were synchronized by mitotic selection and incubated with 5-fluoro-2-deoxyuridine for a time longer than the G1 phase. This treatment synchronizes the replicons and allows them to start replicating very slowly. It was thus possible to obtain a preferential labelling of the regions containing the origins. The analysis by gel electrophoresis of the Eco Ri-digested rDNA showed that the radioactivity was preferentially incorporated in the fragments which contain the non-transcribed spacer. The results of these two approaches indicate that the rRNA gene cluster consists of multiple units of replication, possibly one per gene unit. Furthermore they show that the origins of replication are localized into the non-transcribed spacer.

Animals↗
PubMed1981

Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization.

Poly-A+ mRNA from Xenopus laevis oocytes, partially enriched for r-protein coding capacity has been used as starting material for preparing a cDNA bank in plasmid pBR322. The clones containing sequences specific for r-proteins have been selected by translation of the complementary mRNAs. Clones for six different r-proteins have been identified and utilized as probes for studying their genomic organization. Two gene copies per haploid genome were found for r-proteins L1, L14, S19, and four-five for protein S1, S8 and L32. Moreover a population polymorphism has been observed for the genomic regions containing sequences for r-protein S1, S8 and L14.

Animals↗
PubMed1978

Electron microscopic analysis of replicating DNA of sea urchin embryos.

DNA was extracted from Paracentrotus lividus embryos at the third S phase afer fertilization and analyzed with the electron microscope. The most relevant structures observed in this actively replicating DNA are clusters of short, closely spaced microbubbles (about 0.1 micron long on the average), partially or entirely single-stranded molecules and few linear forks. Unexpectedly, no long eye forms were observed. The analysis of DNA purified from gastrulae and from adult somatic tissues has revealed the same structures, although at a low frequency. A quantitative analysis has been carried out to determine the size distribution and spacing of microbubbles. A number of control experiments have been performed to characterize these structures better. Various possiblities are discussed to account for the presence of the observed forms and the absence of larger eyes.

Animals↗
PubMed1978

Differently sized rDNA repeating units of Xenopus laevis are arranged as internally homogeneous clusters along the nucleolar organizer.

The organization of differently sized genes coding for rRNA in Xenophus laevis has been investigated by partial EcoRI or HindIII digestion. The electrophoretic patterns obtained revealed that most adjacent repeating units are equally sized. This conclusion is in agreement with a previous suggestion that the nucleolar organizer is made up of internally homogeneous blocks of rRNA genes.

Animals↗
PubMed1977

Length and interspersion of repetitive and non repetitive DNA sequences in four amphibian species with different genome sizes.

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.

Animals↗