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S A Gerbi

Publications and source records attributed to S A Gerbi.

At least 19 recordsLinked to original sources
PubMed2001

Origin recognition complex binding to a metazoan replication origin.

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.

Adenosine Triphosphatases↗
PubMed2001

Maintenance of the DNA puff expanded state is independent of active replication and transcription.

The maintenance of the expanded state of DNA puffs II/2B and II/9A in polytene chromosomes from stage 14 x 7 Sciara coprophila salivary glands was assayed after inhibition of RNA synthesis, DNA synthesis, or both processes together. Heat shock conditions were established in order to inhibit transcription. Polypeptides of Mr 72,000 and 36,000 were produced in Sciara after heat shock. The gene encoding the Mr 72,000 polypeptide, the homolog of Drosophila hsp70, was cloned. In situ hybridization detected Sciara hsp70 at bands 4A and 17C on chromosome IV. Sciara hsp70 encodes a 2.3 kb heat shock mRNA. DNA puffs (e.g., DNA puffs 2B and 9A on chromosome II) remained fully expanded even after inhibition of transcription by heat shock or actinomycin D, or after inhibition of DNA replication by aphidicolin, or inhibition of both RNA synthesis and DNA synthesis together by actinomycin D plus aphidicolin. Therefore, maintenance of the DNA puff expanded state in Sciara does not require ongoing transcription and/or replication. Mechanisms for initiation and for maintenance of puffs (open chromatin structure) are discussed.

Animals↗
PubMed2001

Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing.

Mutations in the 5' portion of Xenopus U3 snoRNA were tested for function in oocytes. The results revealed a new cleavage site (A0) in the 3' region of vertebrate external transcribed spacer sequences. In addition, U3 mutagenesis uncoupled cleavage at sites 1 and 2, flanking the 5' and 3' ends of 18S rRNA, and generated novel intermediates: 19S and 18.5S pre-rRNAs. Furthermore, specific nucleotides in Xenopus U3 snoRNA that are required for cleavages in pre-rRNA were identified: box A is essential for site A0 cleavage, the GAC-box A' region is necessary for site 1 cleavage, and the 3' end of box A' and flanking nucleotides are required for site 2 cleavage. Differences between metazoan and yeast U3 snoRNA-mediated rRNA processing are enumerated. The data support a model where metazoan U3 snoRNA acts as a bridge to draw together the 5' and 3' ends of the 18S rRNA coding region within pre-rRNA to coordinate their cleavage.

Animals↗
PubMed2001

Where it all starts: eukaryotic origins of DNA replication.

Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery. During the past decade much progress has been made in identifying replication origins in eukaryotic genomes. More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely. Beyond giving us the precise positions of start sites, the application of RIP mapping in yeast and human cells has revealed a single, defined start point at which replication initiates, a scenario very reminiscent of transcription initiation. More importantly, studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Therefore, in our pursuit of identifying ORC-binding sites in higher eukaryotes, RIP mapping may lead the way.

Animals↗
PubMed2000

The spacing between functional Cis-elements of U3 snoRNA is critical for rRNA processing.

The sequences and structural features of Xenopus laevis U3 small nucleolar RNA (snoRNA) necessary for pre-rRNA cleavage at sites 1 and 2 to form 18 S rRNA were assayed by depletion/rescue experiments in Xenopus oocytes. Mutagenesis results demonstrated that the putative stem of U3 domain I is unnecessary for 18 S rRNA processing. A model consistent with earlier experimental data is proposed for the structure of domain I when U3 is not yet bound to pre-rRNA. For its function in rRNA processing, a newly discovered element (5' hinge) was revealed to be important but not as critical as the 3' hinge region in Xenopus U3 snoRNA for 18 S rRNA formation. Base-pairing is proposed to occur between the U3 5' hinge and 3' hinge and complementary regions in the external transcribed spacer (ETS); these interactions are phylogenetically conserved, and are homologous to those previously described in yeast (5' hinge-ETS) and trypanosomes (3' hinge-ETS). A model is presented where the base-pairing of the 5' hinge and 3' hinge of U3 snoRNA with the ETS of pre-rRNA helps to correctly position U3 boxes A'+A for their function in rRNA processing. Like an earlier proposal for yeast, boxes A' and A of Xenopus may base-pair with 18 S sequences in pre-rRNA. We present the first direct experimental evidence in any system that box A' is essential for U3 snoRNA function in 18 S rRNA formation. The analysis of insertions and deletions indicated that the spacing between the U3 elements is important, suggesting that they base-pair with the ETS and 18 S regions of pre-rRNA at the same time.

Animals↗
PubMed2000

Transient nucleolar localization Of U6 small nuclear RNA in Xenopus Laevis oocytes.

Recent studies on the 2'-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

Animals↗
PubMed1999

U3 small nucleolar RNA is essential for cleavage at sites 1, 2 and 3 in pre-rRNA and determines which rRNA processing pathway is taken in Xenopus oocytes.

A molecular dissection of U3 small nucleolar RNA (snoRNA) was performed in vivo in Xenopus oocytes and the effects on rRNA processing were analyzed. Oocyte injection of antisense oligonucleotides against parts of U3 snoRNA resulted in specific fragmentation of U3 by endogenous RNase H. Fragmentation of U3 domain II correlated with a decrease in 20 S pre-rRNA and a concomitant increase in 36 S pre-rRNA, indicating reduced cleavage at site 3. Conversely, fragmentation of U3 domain I completely blocked 18 S rRNA formation, increased the 20 S rRNA precursor, and decreased 36 S pre-rRNA, indicating inhibition of cleavage at sites 1+2. rRNA processing defects at sites 1+2 or 3 after destruction of intact endogenous U3 snoRNA were rescued by injection of in vitro transcripts of U3 snoRNA or certain U3 fragments. Thus, cleavage at sites 1+2 and 3 is U3 snoRNA dependent. Moreover, U3 snoRNA has two functional modules: domain I for sites 1+2 cleavage and domain II for site 3 cleavage. The data suggest that whichever of these U3 domains acts first determines which rRNA processing pathway will be taken: cleavage first at site 3 of pre-rRNA leads to pathway A, whereas cleavage first at sites 1+2 leads to pathway B for rRNA processing. Predictions of this model were validated by rescue of site 3 cleavage by injection of just domain II after U3 depletion. Rescue of sites 1+2 cleavage required covalent continuity of domain I with the hinge region and non-covalent association with domain II. We could experimentally shift which rRNA processing pathway was taken by injecting fragments of U3 to compete with endogenous U3 snoRNA.

Animals↗
PubMed1999

Chromosomal ARS1 has a single leading strand start site.

Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.

Base Sequence↗
PubMed1999

Box H and box ACA are nucleolar localization elements of U17 small nucleolar RNA.

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevis oocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin-hinge-hairpin-tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.

Animals↗
PubMed1998

Conserved boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs.

Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein-labelled wild-type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates.

Animals↗
PubMed1998

Discrete start sites for DNA synthesis in the yeast ARS1 origin.

Sites of DNA synthesis initiation have been detected at the nucleotide level in a yeast origin of bidirectional replication with the use of replication initiation point mapping. The ARS1 origin of Saccharomyces cerevisiae showed a transition from discontinuous to continuous DNA synthesis in an 18-base pair region (nucleotides 828 to 845) from within element B1 toward B2, adjacent to the binding site for the origin recognition complex, the putative initiator protein.

Base Composition↗
PubMed1998

Nucleolar localization elements in U8 snoRNA differ from sequences required for rRNA processing.

U8 small nucleolar RNA (snoRNA) is essential for metazoan ribosomal RNA (rRNA) processing in nucleoli. The sequences and structural features in Xenopus U8 snoRNA that are required for its nucleolar localization were analyzed. Fluorescein-labeled U8 snoRNA was injected into Xenopus oocyte nuclei, and fluorescence microscopy of nucleolar preparations revealed that wild-type Xenopus U8 snoRNA localized to nucleoli, regardless of the presence or nature of the 5' cap on the injected U8 snoRNA. Nucleolar localization was observed when loops or stems in the 5' portion of U8 that are critical for U8 snoRNA function in rRNA processing were mutated. Therefore, sites of interaction in U8 snoRNA that potentially tether it to pre-rRNA are not essential for nucleolar localization of U8. Boxes C and D are known to be nucleolar localization elements (NoLEs) for U8 snoRNA and other snoRNAs of the Box C/D family. However, the spatial relationship of Box C to Box D was not crucial for U8 nucleolar localization, as demonstrated here by deletion of sequences in the two stems that separate them. These U8 mutants can localize to nucleoli and function in rRNA processing as well. The single-stranded Cup region in U8, adjacent to evolutionarily conserved Box C, functions as a NoLE in addition to Boxes C and D. Cup is unique to U8 snoRNA and may help bind putative protein(s) needed for nucleolar localization. Alternatively, Cup may help to retain U8 snoRNA within the nucleolus.

Animals↗
PubMed1998

Nucleolar localization elements of Xenopus laevis U3 small nucleolar RNA.

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected into Xenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5' region containing Boxes A and A', known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5' cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box C' led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing.

Animals↗
PubMed1998

Education and employment patterns of U.S. Ph.D.'s in the biomedical sciences.

During most of the 1970s and 1980s, the number of biomedical Ph.D.'s conferred in the United States was fairly constant. From 1987 to 1995, however, there was an increase of almost 50% in the number of biomedical Ph.D.'s awarded by U.S. institutions; nearly 70% of this increase can be accounted for by the increase in the number of noncitizens receiving a Ph.D. in the U.S. Although unemployment among U.S. citizens with biomedical Ph.D.'s is now extremely low--less than 2.0%--there have been some important changes in the job market for biomedical Ph.D.'s. The total number of biomedical scientists has grown, whereas the number of faculty positions has remained stable, causing a decline in faculty positions as a percentage of total employment for biomedical scientists. Jobs in industry have increased, and in the future might surpass academic jobs as the most prevalent form of employment for U.S. biomedical scientists.

Academies and Institutes↗
PubMed1997

Replication initiation point mapping.

Replication in eukaryotes is bidirectional and semi-discontinuous. This asymmetry provides the basis for mapping the origin of bidirectional replication (OBR), which is the transition point from discontinuous to continuous synthesis. The regions of each DNA strand complementary to the leading strand or lagging strand can be measured by the methods of imbalanced DNA synthesis or Okazaki fragment distribution, respectively. The resolution of both of these hybridization-based procedures is a few hundred base pairs. Nucleotide resolution was previously achieved for viral origins by mapping the initiation sites of Okazaki fragments on sequencing gels. To overcome the background caused by nicked DNA, all DNA ends were phosphorylated, RNA primers were removed from the Okazaki fragments by NaOH hydrolysis, and the hydroxyl ends thus created were phosphorylated with 32P. Unfortunately, this method was not sensitive enough to map eukaryotic cellular origins. A new method, replication initiation point (RIP) mapping, that is 1000-fold more sensitive and has been applied to yeast ARS1 where the OBR is mapped to and 18-bp region from within element B1 toward B2 is described here. RIP mapping utilizes Vent (exo-) polymerase to extend from a labeled primer to the DNA/RNA junctions of nascent strand template in an asynchronous population of replicating molecules. The DNA is digested with lambda-exonuclease prior to primer extension to remove nicked contaminating DNA.

Animals↗
PubMed1997

U3 snoRNA may recycle through different compartments of the nucleolus.

A model is proposed in which U3 small nucleolar RNA (snoRNA) is recruited from an inactive, stored form in the dense fibrillar component (DFC) of the nucleolus to an active form that is associated with the initial ribosomal RNA (rRNA) precursor. The initial steps of rRNA processing occur in the DFC, and then it is proposed that the U3 snoRNA moves with intermediates in rRNA processing from the DFC to the granular component (GC) of the nucleolus. The nucleolar protein fibrillarin is located primarily in the DFC, and it is suggested that the complex of fibrillarin and U3 snoRNA dissociates when U3 snoRNA transits to the GC. Finally, when U3 snoRNA is released from the processed rRNA, the tether holding the rRNA in the nucleolus is broken and rRNA can then be exported from the nucleolus to the cytoplasm. U3 snoRNA is hypothesized to recycle back from the GC to the DFC where it is stored until future association with another initial rRNA precursor. Data supporting this model are summarized. U3 snoRNA is also stored in the coiled body of interphase cells and in the nucleolar remnants and prenucleolar bodies of mitotic cells, and there may be some similarity in the binding sites for stored U3 snoRNA in the DFC and in these structures.

Animals↗