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Biomedical subjects

S A Gerbi

Publications and source records attributed to S A Gerbi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Delocalization of some small nucleolar RNPs after actinomycin D treatment to deplete early pre-rRNAs.

Retention of some components within the nucleolus correlates with the presence of rRNA precursors found early in the rRNA processing pathway. Specifically, after most 40S, 38S and 36S pre-rRNAs have been depleted by incubation of Xenopus kidney cells in 0.05 microg/ml actinomycin D for 4 h, only 69% U3 small nucleolar RNA (snoRNA), 68% U14 snoRNA and 72% fibrillarin are retained in the nucleolus as compared with control cells. These nucleolar components are important for processing steps in the pathway that gives rise to 18S rRNA. In contrast, U8 snoRNA, which is used for 5.8S and 28S rRNA production, is fully retained in the nucleolus after actinomycin D treatment. Therefore, U8 snoRNA is in a different category than U3 and U14 snoRNA and fibrillarin. It is proposed that U3 and U14 snoRNA and fibrillarin, but not U8 snoRNA, bind to the external transcribed spacer or internal transcribed spacer 1, and when these binding sites are lost after actinomycin D treatment some of these components cannot be retained in the nucleolus. Other binding sites may also exist, which would explain why only some and not all of these components are lost from the nucleolus.

Animals↗
PubMed1994

Analysis of an origin of DNA amplification in Sciara coprophila by a novel three-dimensional gel method.

The replication origin region for DNA amplification in Sciara coprophila DNA puff II/9A was analyzed with a novel three-dimensional (3D) gel method. Our 3D gel method involves running a neutral/neutral 2D gel and then cutting out vertical gel slices from the area containing replication intermediates, rotating these slices 90 degrees to form the third dimension, and running an alkaline gel for each of the slices. Therefore, replication intermediates are separated into forks and bubbles and then are resolved into parental and nascent strands. We used this technique to determine the size of forks and bubbles and to confirm the location of the major initiation region previously mapped by 2D gels to a 1-kb region. Furthermore, our 3D gel analyses suggest that only one initiation event in the origin region occurs on a single DNA molecule and that the fork arc in the composite fork-plus-bubble pattern in neutral/neutral 2D gels does not result from broken bubbles.

Animals↗
PubMed1993

Developmental progression of DNA puffs in Sciara coprophila: amplification and transcription.

DNA amplification for two major DNA puffs (II/2B and II/9A) of the fungus fly Sciara coprophila increases steeply from 17 to 19 days after hatching (18 degrees C), resulting in almost 20-fold more DNA at these loci in mature larval salivary glands than in adult tissues. At 19 days after hatching when gene amplification reaches a plateau, there is a burst in the amount of mRNA encoded by these two DNA puffs. Expansion of the two puffs coincides with the increase in transcription rather than reinitiation of DNA replication. In contrast, an RNA puff (III/9B) undergoes no DNA amplification, and the burst in RNA produced by this locus occurs slightly later, at 20 days after hatching. The progression of cytological puffing and associated molecular events of DNA amplification and transcription correlate with features of the external phenotype of the larvae, especially the number of pigment granules in the eyespots that are the anlage to the adult eyes. The sequential and coordinated regulation of DNA puff replication and transcription is discussed.

Animals↗
PubMed1993

Replication initiates at a confined region during DNA amplification in Sciara DNA puff II/9A.

Two independent two-dimensional (2D) gel methods were used to map an origin of replication that is developmentally regulated by the steroid hormone ecdysone, namely an origin for DNA puff amplification in the fungus fly Sciara coprophila. Initiation of replication was found to occur within a small region of no larger than 6 kb by use of the neutral/neutral 2D gel method. Neutral/alkaline 2D gel analyses support the results of the neutral/neutral 2D gels and further define within the origin region an approximately 1-kb area where the majority of replication initiates. This is the first example of an origin of replication in multicellular eukaryotes that has been mapped by 2D gels to such a small defined region. Moreover, replication can be seen by the neutral/alkaline 2D gel method to proceed bidirectionally outward from this replication origin region. These data are consistent with an onion-skin mechanism whereby multiple rounds of DNA replication initiate at a specific origin of replication for Sciara DNA puff amplification.

Animals↗
PubMed1992

Genes for Xenopus laevis U3 small nuclear RNA.

Genomic Southern blots showed there are only 14 to 20 copies of U3 snRNA genes per somatic genome in Xenopus laevis, unlike the highly repetitive, tandem arrangement of other snRNA genes in this organism. Sequencing of two U3 snRNA genes from lambda clones of a genomic library revealed striking similarity upstream, but much more divergence downstream. Consensus motifs common to other U snRNA genes were also found: a distal sequence element (DSE, octamer motif at -222 to -215), a proximal sequence element (PSE, at -62 to -52) and a 3' Box (15 or 16 bp downstream of the U3 genes). The DSE of mammals also has an inverted CCAAT motif specific for U3 snRNA genes, and we find this is conserved in the amphibian U3 snRNA genes. The Xenopus inverted CCAAT motif is exactly one helical turn further upstream of the octamer motif than its mammalian counterpart, suggesting interaction of putative transcription factors bound to these motifs. Mutation of the inverted CCAAT motif and part of an adjacent Sp1 site greatly depresses transcription of the mutant U3 snRNA gene in Xenopus oocytes, implying a role in transcriptional efficiency. Electrophoretic mobility shift assays implicate transcription factor binding to this region.

Animals↗
PubMed1992

Chorion gene cis-regulatory DNA restricts tissue specificity of reporter gene expression in transformed Drosophila.

P element mediated germ-line transformation was used to study the developmental specificity of Drosophila chorion gene regulatory sequences directing expression of the bacterial reporter genes for chloramphenicol acetyltransferase (CAT) and beta-galactosidase (lacZ). DNA fragments containing 5' flanking plus the entire 5' untranslated and the beginning of the coding region of either the s36 or the s15 chorion gene are able to confer on the reporter genes normal tissue as well as temporal specificity of expression, exclusively in the ovary of transformed female flies. However, if 5' untranslated and coding regions are omitted, normal ovarian expression is maintained but tissue specificity is relaxed: expression of the reporter gene is detected both in the ovary and in specific non-ovarian tissues of transformed females and males. The evidence suggests that the missing 5' untranslated and coding sequences may include negative elements that normally suppress expression in non-ovarian tissues, and that these putative elements are distinct from those that prevent premature expression in the ovarian follicles. The exact location of ectopic lacZ expression within the internal male genitalia depends on the constellation of 5' flanking chorion regulatory sequences included in the P element constructs. Ectopic expression of the CAT gene in the male genitalia under s15 promoter control can be abolished by mutating the hexamer TCACGT, a sequence previously shown to be essential for the normal expression of this chorion gene in the ovary.

Animals↗
PubMed1991

Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis.

Accessible single-strand bases in Xenopus laevis 28 S ribosomal RNA (rRNA) Domain V, the peptidyl transferase region, were determined by chemical modification with dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulfonate and kethoxal, followed by primer extension. The relative accessibilities of three rRNA substrates were compared: deproteinized 28 S rRNA under non-denaturing conditions (free 28 S rRNA), 60 S subunits and 80 S ribosomes. Overall, our experimental results support the theoretical secondary structure model of Domain V derived by comparative sequence analysis and compensatory base-pair changes, and support some theoretical tertiary interactions previously suggested by covariation. The 60 S subunits and 80 S ribosomes generally show increasing resistance to chemical modification. Bases which are sensitive in free 28 S rRNA but protected in 60 S subunits may be sites for ribosomal protein binding or induced structural rearrangements. Another class of nucleotides is distinguished by its sensitivity in 60 S subunits but protection in 80 S ribosomes; these nucleotides may be involved in subunit-subunit interactions or located at the interface of the ribosome. We found a third class of bases, which is protected in free 28 S rRNA but sensitive in 60 S subunits and/or 80 S ribosomes, suggesting that structural changes occur in Domain V as a result of subunit assembly and ribosome formation. One such region is uniquely hypersensitive in eukaryotic ribosomes but is absent in Escherichia coli ribosomes. Sites that we determined to be accessible on empty 80 S ribosomes could serve as recognition sites for translation components.

Aldehydes↗
PubMed1991

Changes in 7SL RNA conformation during the signal recognition particle cycle.

The structure of 7SL RNA has been probed by chemical modification followed by primer extension, using four substrates: (i) naked 7SL RNA; (ii) free signal recognition particle (SRP); (iii) polysome bound SRP; and (iv) membrane bound SRP. Decreasing sensitivity to chemical modification between these different substrates suggests regions on 7SL RNA that: bind proteins associated with SRP might interact with ribosomes; and are protected by binding to membranes. Other areas increase in chemical sensitivity, exemplified by a tertiary interaction present in naked 7SL RNA but not in free SRP. Such changes suggest that 7SL RNA changes its conformation during the SRP cycle. These conformational changes could be a necessary component to move through the SRP cycle from one stage to the next.

Animals↗
PubMed1991

Preribosomal RNA processing in Xenopus oocytes does not include cleavage within the external transcribed spacer as an early step.

Recently it has been reported that U3 snRNA is necessary for: (a) internal cleavage at +651/+657 within the external transcribed spacer (ETS) of mouse precursor ribosomal RNA (pre-rRNA); and (b) cleavage at the 5' end of 5.8S rRNA in Xenopus oocytes. To study if U3 snRNA plays a role at more than one processing site in the same system, we have investigated whether internal cleavage sites exist within the ETS of Xenopus oocyte pre-rRNA. The ETS of Xenopus pre-rRNA contains the consensus sequence for the mammalian early processing site (+651/+657 in mouse pre-rRNA), but freshly prepared RNA from Xenopus oocytes has no cuts in this region. The only putative cleavage sites we found in the ETS of Xenopus oocyte pre-rRNA are a cluster further downstream of the mouse early processing site consensus sequence. This cluster is not homologous to the mouse +651/+657 sites because unlike the latter it is (a) not abolished by disruption of U3 snRNA, (b) not cleaved during early steps of pre-rRNA processing, and (c) lacks sequence similarity to the +651/+657 consensus. Therefore, pre-rRNA of Xenopus oocytes does not cleave within the ETS as an early step in rRNA processing. We conclude that cleavage within the ETS is not an obligatory early step needed for the rest of rRNA maturation.

Animals↗
PubMed1991

The promoter of DNA puff gene II/9-1 of Sciara coprophila is inducible by ecdysone in late prepupal salivary glands of Drosophila melanogaster.

DNA puffs occur in Sciarid salivary gland chromosomes; they are sites of DNA amplification and intense transcription and they appear to encode secreted structural proteins needed for pupation. In this report we have used P-element transformation of Drosophila to study regulation of a Sciara DNA puff gene. We found that a 718-bp promoter fragment of DNA puff gene II/9-1 from Sciara coprophila directs expression of the bacterial reporter gene CAT in late prepupal salivary glands of transgenic Drosophila melanogaster. The identical tissue and analogous stage specificity indicate that some aspects of the ecdysone response are evolutionarily conserved between Drosophila and Sciara. When transgenic salivary glands are cultured in vitro, CAT activity is rapidly induced by ecdysone, suggesting direct control of gene expression by the ecdysone receptor. Putative stage-specific factors limit expression of the chimeric Sciara-CAT gene in transgenic Drosophila to late prepupae but not to third instar larvae when ecdysone titers are also high.

Age Factors↗
PubMed1990

In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing.

DNA oligonucleotide complementary to sequences in the 5' third of U3 snRNA were injected into Xenopus oocyte nuclei to disrupt endogenous U3 snRNA. The effect of this treatment on rRNA processing was examined. We found that some toads have a single rRNA processing pathway, whereas in other toads, two rRNA processing pathways can coexist in a single oocyte. U3 snRNA disruption in toads with the single rRNA processing pathway caused a reduction in 20S and '32S' pre-rRNA. In addition, in toads with two rRNA processing pathways, an increase in '36S' pre-rRNA of the second pathway is observed. This is the first in vivo demonstration that U3 snRNA plays a role in rRNA processing. Cleavage site #3 is at the boundary of ITS 1 and 5.8S and links all of the affected rRNA intermediates: 20S and '32S' are the products of site #3 cleavage in the first pathway and '36S' is the substrate for cleavage at site #3 in the second pathway. We postulate that U3 snRNP folds pre-rRNA into a conformation dictating correct cleavage at processing site #3.

Animals↗
PubMed1989

Molecular characterization of DNA puff II/9A genes in Sciara coprophila.

A cDNA clone, pSDII/9, that hybridizes in situ to ecdysone-regulated DNA puff II/9A in Sciara coprophila was used as a probe to isolate a Sciara genomic clone. lambda pSDII/9, which contains a 14.7 x 10(3) base-pair DNA insert. The full-length cDNA insert was sequenced and mapped to gene II/9-1 on the genomic clone. A second gene (II/9-2), transcribed in the same direction as II/9-1, was also mapped to lambda pSDII/9, and its nucleic acid sequence was found to be 85% similar to that of gene II/9-1. An RNase protection assay demonstrates that gene II/9-1 contains a single intron that also exists in gene II/9-2 according to sequencing analysis and primer extensions of RNA encoded by this gene. Computer analyses of the deduced amino acid sequences of genes II/9-1 and II/9-2 indicate that the two DNA puff-encoded proteins are mostly alpha-helical coiled-coils. The 5'-flanking sequences of both genes contain regions that are similar to other ecdysone-regulated genes from Drosophila melanogaster.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of ribosomal DNA variants from Sciara coprophila.

The ribosomal RNA multigene family in the fungus fly Sciara coprophila contains a total of only 65 to 70 repeat units. We explored the types and frequencies of variant repeats in this small multigene family by characterizing different cloned rDNA variants from Sciara. Although we did not observe any intergenic spacer length variants in Sciara, we found a variant due to the insertion of a putative mobile element (lambda Bc11), and variants containing ribosomal insertion elements. By DNA sequence analysis of rDNA/non-rDNA junctions, there are three distinct types of ribosomal insertion elements found in Sciara rDNA: two correspond to the R1 and R2 insertion elements found in other dipterans (clones lambda Bc5 and pBc1L1, respectively), and one is a novel class of ribosomal insertion elements (R3, exemplified by clone pBc6D6) which so far is unique to Sciara. Together, the several different rDNA variants make up from 12 to 20% of the rDNA in Sciara. These results are discussed in the context of evolution of the ribosomal RNA multigene family.

Animals↗
PubMed1988

Nucleotide sequence determination and secondary structure of Xenopus U3 snRNA.

Using a combination of RNA sequencing and construction of cDNA clones followed by DNA sequencing, we have determined the primary nucleotide sequence of U3 snRNA in Xenopus laevis and Xenopus borealis. This molecule has a length of 219 nucleotides. Alignment of the Xenopus sequences with U3 snRNA sequences from other organisms reveals three evolutionarily conserved blocks. We have probed the secondary structure of U3 snRNA in intact Xenopus laevis nuclei using single-strand specific chemical reagents; primer extension was used to map the positions of chemical modification. The three blocks of conserved sequences fall within single-stranded regions, and are therefore accessible for interaction with other molecules. Models of U3 snRNA function are discussed in light of these data.

Animals↗