End Of 18S Research Articles

Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5\u2032-phosphate-dependent exonuclease in Candida albicans and other yeasts

BackgroundMessenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5´-phosphate-dependent, 5´→3´ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5´-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell.ResultsWe have found that in the pathogenic yeast Candida albicans, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5′-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior.ConclusionsDigestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry.

Review of haploporid (Trematoda) genera with ornate muscularisation in the region of the oral sucker, including four new species and a new genus

Species of the Haploporidae Nicoll, 1914 with elaborate muscularisation of the oral sucker belong in three trematode genera, including three new species and a new genus from the intestine of fishes in Australian waters. Spiritestis Nagaty, 1948 is resurrected and S. herveyensis n. sp. is described from the mullet Moolgarda seheli (Forsskål) collected in Hervey Bay, Queensland, Australia; the latter differs from S. arabii Nagaty, 1948 in that the position of the genital pore is pharyngeal rather than post-pharyngeal and the geographical range is off Australia rather than the Red Sea. A new genus is proposed for two new species, with a uniquely ornamented oral sucker, which infect Australian scatophagids. Members of Capitimitta n. g. are distinguished from Waretrema Srivastava, 1937, species of which have a simple oral sucker with six radially arranged anterior muscular lobes, in that their oral sucker is V-shaped with six embedded muscular finger-like structures in the anteroventral portion. The relatively small C. darwinensis n. sp., collected from Selenotoca multifasciata (Richardson) at Darwin, Northern Territory, Australia, is distinguished from C. costata n. sp., collected from Scatophagus argus (Linnaeus) in the same locality and S. multifasciata off Brisbane, Australia, and by having smaller eggs, a vitellarium commencing at a level close to the ventral sucker rather than at greater than one ovarian length posterior to the ventral sucker, and shorter tegumental body spines. Sequence data of a c.2,500bp region of the 3' end of 18S, the entire ITS region and the 5' end of the 28S revealed that Spiritestis and Capitimitta are not as closely related as some morphological features would suggest and are probably not the closest relative of each other. What has been reported as Waretrema piscicolum Srivastava, 1937 probably consists of several species, some in different genera, and one, based on material collected by Dr Masaaki Machida, is proposed as Spiritestis machidai n. sp. from Crenimugil crenilabis (Forsskål) off Japan. Phylogenetic hypotheses, based on analysis of an alignment of partial 28S sequences with other haploporids, provide a framework for the evaluation of interrelationships within the Haploporidae. These analyses show that: (1) Spiritestis and Capitimitta are supported within the Haploporidae; (2) branches to Forticulcita Overstreet, 1982, Saccocoelioides Szidat, 1954, Spiritestis and Capitimitta create a clade that is sister to haploporines from the Mediterranean Sea; (3) the branch to Saccocoelioides, Spiritestis and Capitimitta create a polytomy; and (4) the two new species of Capitimitta, plus an immature specimen of an unnamed species, form a monophyletic clade.

Multiple Proteasome-Interacting Proteins Assist the Assembly of the Yeast 19S Regulatory Particle

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. The 26S proteasome consists of the proteolytic core particle (CP) and one or two 19S regulatory particles (RPs). Although the mechanisms of CP assembly are well described, the mechanism of RP assembly is largely unknown. Here, we show that four proteasome-interacting proteins (PIPs), Nas2/p27, Nas6/gankyrin, Rpn14/PAAF1, and Hsm3/S5b, bind specific Rpt subunits of the RP and interact each other genetically. Lack of these PIPs resulted in defective assembly of the 26S proteasome at an early stage, suggesting that these proteins are bona fide RP chaperones. Each of the RP chaperones formed distinct specific subassemblies of the base components and escorted them to mature RPs. Our results indicate that the RP assembly is a highly organized and elaborate process orchestrated by multiple proteasome-dedicated chaperones.

Characterization of Dermanyssus gallinae (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions

In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3'end of 18S and at the 5'start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.

Molecular differentiation of dermatophyte fungi.

Our previous studies of the ribosomal DNA variation in dermatophytes have shown that these fungi are monophyletic in origin. However, this approach did not allow us to differentiate all the species defined by classical means. Therefore, we studied the internal transcribed spacer 1 (ITS 1) region of 17 species of the fungal order Onygenales, comprising the pathogenic keratinophilic fungi. Interspecific nucleotide composition and sequence length variation of the ITS 1 region was high, mean identities were as low as 40% and sequence lengths varied from 169 to 293 basepairs. Each established dermatophyte species could be identified. In contrast, the flanking sequences at the 3' end of 18S and the 5.8S rDNA were conserved. Although the value of the ITS 1 region as a phylogenetic tool may be limited because of its high variability, it provides the information necessary to design species-specific probes, or polymerase chain reaction restriction fragment polymorphism systems useful for taxonomic or rapid diagnostic tests.

Intergenic spacers of rRNA genes in three species of theCynareae (Asteraceae)

Intergenic spacers of the rRNA genes of three species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Onopordum acanthium, andO. illyricum were cloned in the plasmid pGEM-7zf(+). Detailed restriction mapping and partial sequencing of the IGSs were carried out. The structural analysis showed a clear diversity betweenCynara andOnopordum, while a high degree of homology was found between the twoOnopordum spp. In all three species a fragment of about 450 bp from the 5′ end of 18S to the Acc I site with a high sequence homology was present. Nucleotide sequences upstream from the above mentioned Acc I site show a gradual decrease of homology betweenCynara andOnopordum.

PCR cloning and nucleotide sequence determination of the 18S rRNA genes and internal transcribed spacer 1 of the protozoan parasites Cryptosporidium parvum and Cryptosporidium muris

The genes encoding 18S rRNA and internal transcribed spacer 1 (ITS1) of Cryptosporidium parvum and Cryptosporidium muris were amplified from oocysts by PCR utilizing primers complementary to conserved regions of the 5′ end of 18S and 5.8S rRNA. PCR products were cloned and the complete nucleotide sequences of two clones of each Cryptosporidium species were determined. The 18S rRNA genes of C. parvum and C. muris showed more than 99% sequence identity.

An in vitro interaction between the human U3 snRNP and 28S rRNA sequences near the alpha-sarcin site.

Model transcripts containing mammalian pre-rRNA sequences were incubated with a HeLa cell extract, digested with T1 RNase, and immunoprecipitated with anti-(U3)RNP or control antibodies. Two overlapping fragments derived from the 3' domain of human 28S rRNA were specifically immunoprecipitated although transcripts which spanned the transcription initiation site, the ETS processing site, the 5' end of 18S, and both termini of 5.8S yielded no protected fragments. The sequence of these fragments was determined using a novel technique in which the [32P]-labeled fragment was co-finger-printed with [3H]-labeled total transcript serving as an internal marker. The fragments immunoprecipitated derive from nucleotides 4570-4590 and 4575-4590 of human 28S and are adjacent to the alpha-sarcin site. Protection most likely involves the U3 RNA since it is sensitive to pretreatment of the extract with micrococcal nuclease. Complementarity between U3 and this rRNA region is phylogenetically conserved in species ranging from human to S. cerevisiae. The possible significance of this finding is discussed.

Nucleotide sequence of the transcription initiation region for rRNA in the sea urchin Lytechinus variegatus

The 1191-bp sequence which includes the 5' end of 18S · rDNA and its adjacent spacer have been determined for a cloned fragment of sea urchin rDNA. The 5' end of 33S precursor rRNA (pre-rRNA) has been located by S1 nuclease mapping and primer extension. Pre-rRNA appears to be initiated at an A, 658 bp upstream from the 5' terminus of 18S rRNA. The first nucleotide of 18S rRNA was also analyzed by S1 nuclease mapping and found to correspond to T in the nontranscribed DNA strand. Comparisons of the transcription initiation region in rDNA and the 5' end of 18S · rDNA with the corresponding regions in other eukaryotes reveal no significant nucleotide sequence homology in the precursor portion while the 5' end of 18S · rDNA is highly conserved.