The role of spliced leader RNA (SL RNA) in trans-splicing in Caenorhabditis elegans has been studied through a combination of in vitro mutagenesis and in vivo complementation of rrs-1 mutant nematodes, which lack endogenous SL1 RNA. Three classes of mutant SL1 RNAs have been found-those that rescue the lethal phenotype at low concentration of transforming DNA, those that rescue at high but not low concentration, and those that do not rescue at all. These studies showed that some mutations in the otherwise highly conserved 22-nt spliced leader are tolerated for splicing and post-splicing events. A longer spliced leader also can be tolerated but only when present in high copy number. Changes in the first 16 nucleotides result in the appearance of no SL RNA, consistent with the in vitro studies by others showing that the SL1 RNA promoter partly resides within the spliced leader sequence.

The genome of the nematode Caenorhabditis elegans possesses an orthologous sequence to the Drosophila muscleblind (mbl) and mammalian muscleblind-like genes (MBNLs). This ortholog, K02H8.1, which has a high degree of homology (about 50%) to human MBNLs, encodes two zinc finger domains, as does the sequence of the Drosophila mbl gene. This distinguishes it from human MBNLs, which encode four zinc finger domains. In this study, we cloned six major isoforms of K02H8.1 using cDNA generated from C. elegans total RNA. All six of the cloned isoforms had an SL1 leader sequence at the 5'-position. Interestingly, one of the isoforms lacked a zinc finger domain-encoding sequence. To understand better the function of K02H8.1, we performed yeast three-hybrid experiments to characterize the binding of K02H8.1 to bait RNAs. K02H8.1 exhibited strong binding affinity for CUG and CCUG repeats, and the binding affinity was very similar to that of MBNLs. In addition, promoter analysis was performed using promoter-green fluorescent protein (GFP) fusion constructs. The expression of GFP driven by the K02H8.1 promoter was absent in muscle; however, significant GFP expression was detected in the neurons around the pharynx.

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the beta-tubulin amino acid sequences of other species. To complete the coding sequence, the 3' end was amplified with the 3' RACE, and for amplification of the 5' end the SL1-primer was used. The cDNA of the beta-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the beta-tubulin gene. The complete sequence of the genomic DNA of the beta-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 beta-tubulin gene of Haemonchus contortus range between 79% and 97%.

The novel levansucrase produced from Bacillus subtilis NRC1aza yielded two types of levan L1 and L2 with different molecular weights 85.23 kDa and 31.95 kDa, respectively. The levan identification was detected by paper chromatography and high-pressure liquid chromatography. The antioxidant activity of levan and their derivatives (SL1 and SL2) exhibited a strong free radical scavenging activity with DPPH. The antitumor activity of SL1 was tested against different human cancer cell lines. The cell death was explored mechanistically through evaluation of Apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase activity, mitochondrial transmembrane potential (Δψm), cytochrome C release, total caspases, caspase-3, and caspase-9, and cell cycle. SL1 showed high selective cytotoxicity against HepG2 cells. SL1 led to DNA damaging and fragmentation that was associated with induced apoptosis via mitochondrial pathway, which initiated by the impairment of Δψm and then increased mitochondria, released cytochrome c, that in turn activated caspase-9 and caspase-3 and induced apoptosis.

BACKGROUND

The Knops blood group system consists of antigens encoded by exon 29 of complement receptor 1 (CR1) gene. To better elucidate the complexity of Knops group system, the frequency of six single-nucleotide polymorphisms (SNPs) in three Brazilian populations is determined.

METHODS

A total of 118 individuals descendant from Europe, Asia, and Africa were studied. The genomic fragment of CR1 was amplified by polymerase chain reaction, and the SNPs and haplotypes were determined after DNA sequence analysis.

RESULTS

Among the six polymorphisms characterized, one of them was described for the first time. The analysis of allele frequency showed that these six SNPs did not differ between the European and Asian groups. The African group presented a higher frequency of alleles McC(b), Sl2, and KAM+. The six polymorphisms gave origin to 12 haplotypes that were defined for the first time. The haplotypes 1 (4646A, Kn(a), McC(a), Sl1, Sl4, KAM+), 2 (4646A, Kn(a), McC(a), Sl1, KAM-), and 3 (4646A, Kn(a), McC(a), Sl2, Sl4, KAM-) are the most frequent in all populations. The H2 presents similar frequency in all populations; however, whereas the H1 presented a higher prevalence in the European and Asian groups, in the African group H3 was present in a higher prevalence.

CONCLUSIONS

In this study, a new SNP substituting serine for asparagine at amino acid 1540 was identified. Moreover 12 haplotypes were identified. The differences in haplotype frequencies strongly suggest that the H1 and H2 might be the ancestral one while the H3 may have originated in Africa and may have fixed there by positive selection.

Specific packaging of human immunodeficiency virus type 1 (HIV-1) RNA is attributable to the high affinity of nucleocapsid (NC) sequence of Gag for the cis-acting RNA packaging signals located within the 5' un-translated region (5' UTR). Interestingly, we have previously reported that the T12I mutation (named MP2) within SP1 of Gag prevented incorporation of spliced viral RNA into mutated viruses that lacked the stem-loop 1 (SL1) RNA element (also named dimerization initiation site, DIS), suggesting a role for the SP1 sequence in viral RNA packaging. In this study, we have further tested this activity of MP2 in the context of a variety of mutations that affect viral RNA incorporation. The results showed that MP2 was able to effectively restrict packaging of spliced viral RNA into viruses containing either NC mutations R10A and K11A or mutated 5' UTR sequence, such as DeltaGU3 that lacked the 112-GUCUGUUGUGUG-123 sequence of U5, D1 that was deleted of a 27 nt fragment immediately downstream of the primer binding site (PBS), Delta(306-325) that had the SL3 RNA element removed and MD2 that was missing the 328-GGAG-331 sequence. As a result, MP2 contributed increased infectivity to the related viruses. Therefore, the MP2 mutation demonstrates a distinct role in HIV-1 RNA packaging that is neither pertained to the specific viral RNA packaging signal nor to the NC sequence.

The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity from Acanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460, 000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 +/- 3 pM. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promoter-containing DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex.

As high-throughput cDNA sequencing (RNA-Seq) is increasingly applied to hypothesis-driven biological studies, the prediction of protein coding genes based on these data are usurping strictly in silico approaches. Compared with computationally derived gene predictions, structural annotation is more accurate when based on biological evidence, particularly RNA-Seq data. Here, we refine the current genome annotation for the Meloidogyne hapla genome utilizing RNA-Seq data. Published structural annotation defines 14 420 protein-coding genes in the M. hapla genome. Of these, 25% (3751) were found to exhibit some incongruence with RNA-Seq data. Manual annotation enabled these discrepancies to be resolved. Our analysis revealed 544 new gene models that were missing from the prior annotation. Additionally, 1457 transcribed regions were newly identified on the ends of as-yet-unjoined contigs. We also searched for trans-spliced leaders, and based on RNA-Seq data, identified genes that appear to be trans-spliced. Four 22-bp trans-spliced leaders were identified using our pipeline, including the known trans-spliced leader, which is the M. hapla ortholog of SL1. In silico predictions of trans-splicing were validated by comparison with earlier results derived from an independent cDNA library constructed to capture trans-spliced transcripts. The new annotation, which we term HapPep5, is publically available at www.hapla.org.

BACKGROUND

The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1) sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants.

RESULTS

HIV-1 lacking SL1 (NLΔSL1) did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K) on day 18 postinfection and C1907T in the SP1 domain (P10L) on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1.

CONCLUSIONS

HIV-1 evolved to overcome a deletion in SL1 and restored infectivity by acquiring compensatory mutations in the N-terminal matrix or SP1 domain of Gag. These data shed light on the functions of the N-terminal matrix and SP1 domains and suggest that both regions may have a role in Gag interactions with spliced viral RNA.

Potato virus X (PVX) contains five viral proteins as well as cis-acting elements like stem-loop 1 (SL1) RNAs at the 5' region. SL1 RNAs are involved in PVX RNA replication, encapsidation, translation, and cell-to-cell movement. In this study, we performed two-dimensional electrophoresis Northwestern blot analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry and identified 24 tobacco proteins that interact with SL1 RNAs. Interestingly, one-third of the identified host proteins have been shown to interact with many plant viral proteins. In addition, we demonstrated that PVX capsid protein can bind to both SL1(+/-) RNAs. We further selected three Nicotiana benthamiana proteins including NbMPB2Cb, NbMBF1, and NbCPIP2a, to confirm results of Northwestern blot analysis. Electrophoretic mobility shift assay showed that NbMPB2Cb and NbMBF1 bind to both SL1(+/-) RNAs in vitro. In contrast, NbCPIP2a interacts only SL1(+) RNA. Taken together, we provide a list of host proteins interacting with PVX SL1 RNAs, which would be good candidates for the study of viral RNA-host protein interaction.

The 5'-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.

DNA-dependent protein kinase represses RNA polymerase I (Pol I) transcription in vitro. To investigate the mechanism underlying transcriptional repression, we compared Pol I transcription in extracts from cells that either contain or lack the catalytic subunit of DNA-PK (DNA-PKcs). ATP-dependent repression of Pol I transcription was observed in extracts from DNA-PKcs-containing but not -deficient cells, required templates with free DNA ends, and was overcome by exogenous SL1, the factor that nucleates initiation complex formation. Order-of-addition experiments demonstrate that DNA-PKcs does not inactivate component(s) of the Poll transcription machinery. Instead, phosphorylated Ku protein competes with SL1 for binding to the rDNA promoter and, as a consequence, prevents initiation complex formation. The results reveal a novel mechanism of transcriptional regulation by DNA-PK. Once targeted to DNA, autophosphorylated Ku may displace positive- or negative-acting factors from their target sites, thereby repressing or activating transcription in a gene-specific manner.

Alpha tubulin isotypes are encoded by at least four genes designated alpha-1 to alpha-4 in the nematode Caenorhabditis elegans. We describe here, molecular cloning of the alpha-2 tubulin gene, located on chromosome I, that encodes a protein of 449 amino acids that has high homology to human, mouse and Drosophila alpha tubulins, but relatively lower homology to the yeast alpha tubulins. The alpha-2 tubulin gene is trans-spliced to the SL1 leader sequence. Northern analysis shows that the gene is increasingly transcribed during the early (L1-L3) larval stages but has a lower level of transcription in L4 L4 larvae, adults, and embryos. Using an alpha-2-lacZ fusion gene expression in transgenic animals, we show that the gene is expressed in a tissue-specific manner in the intestine, pharyngeal muscle cells, and a subset of neurons which include a class of DB and VB motor neurons in the ventral nerve cord, posterior touch receptor neurons, PLML, PLMR, in the lumbar ganglia; PVT in the pre-anal ganglion, and ALA in the dorsal ganglion in the head. Our results support the notion that tubulin structure may contribute to the functional specialization of microtubules.

Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries.

An obligate anaerobic, mesophilic, motile and endospore-forming bacterium, designated 1D(T), was isolated from a subsurface soil sample. The young culture of strain 1D(T) was Gram-positive and formed oval spores that were central in position. Based on the biochemical, chemotaxonomic and physiological data, strain 1D(T) appears to be a member of the genus Clostridium. Strain 1D(T) was found to be capable of degrading p-nitrophenol (pNP) at a concentration of 0.5 mM under anaerobic conditions as revealed by HPLC analysis. The major fatty acids were C(16 : 0 )(28.02 %), iso-C(17 : 1) I/anteiso B (23.05 %) and C(14 : 0) (10.02 %). The major polar lipid content was diphosphatidylglycerol. Strain 1D(T) showed highest 16S rRNA gene sequence similarity to Clostridium aciditolerans JW/YJL-B3(T) (98.2 %) and similarity was less for Clostridium scatologenes ATCC 25775(T) (95.1 %), Clostridium drakei SL1(T) (95.0 %) and Clostridium carboxidivorans P7(T) (95.0 %). Phylogenetic analysis showed that it formed a coherent cluster with the species belonging to cluster I of the genus Clostridium. The DNA G+C content was 35.5 mol%. DNA-DNA hybridization analysis indicated a mean value of 36.4 % between strain 1D(T) and its closest relative C. aciditolerans. Several phenotypic differences from the closely related species were also revealed. On the basis of the polyphasic characteristics, strain 1D(T) represents a novel species of the genus Clostridium, for which the name Clostridium nitrophenolicum sp. nov. is proposed. The type strain is 1D(T) (=MTCC 7832(T)=JCM 14030(T)).

A Caenorhabditis elegans (Ce) homologue to the eukaryotic tcp-1 gene (encoding t-complex polypeptide-1) has been mapped, isolated and sequenced. Ce tcp-1 is a single-copy gene located on chromosome II. Nucleotide sequence analysis of the gene reveals the presence of four introns in the coding region and repetitive elements upstream from the start codon. The predicted Ce TCP-1 protein displays more than 60% amino-acid sequence identity to other eukaryotic TCP-1, suggesting a common origin and function for these proteins. The primary tcp-1 transcript undergoes trans-splicing to the spliced leader SL1 RNA, in addition to cis-splicing, to yield a single mRNA species of 1.9 kb. Northern blot analysis shows that unlike the evolutionarily related Hsp60 chaperonin genes, tcp-1 is not upregulated at elevated temperatures, but instead appears to be down-regulated. Additionally, the overall level of the tcp-1 transcript is approximately constant throughout the development of the nematode. The Ce chaperonin-containing TCP-1 (CCT) was identified. A protein extract made from Ce embryos was subjected to sucrose gradient fractionation and ATP-agarose chromatography. Western blot analysis of the purified protein fractions, using anti-mouse TCP-1 monoclonal antibody and antibodies raised against Ce TCP-1, reveals that Ce TCP-1 is a 57-kDa protein subunit of a high-molecular-mass complex capable of binding ATP.

rRNA transcription is a fundamental requirement for all cellular growth processes and is activated by the phosphorylation of the upstream binding factor (UBF) in response to growth stimulation. Even though it is well known that phosphorylation of UBF is required for its activation and is a key step in activation of rRNA transcription, as yet, there has been no direct mapping of the UBF phosphorylation sites. The results of the present studies employed sophisticated nano-flow HPLC-microelectrospray-ionization tandem mass spectrometry (nHPLC-muESI-MS/MS) coupled with immobilized metal affinity chromatography (IMAC) and computer database searching algorithms to identify 10 phosphorylation sites on UBF at serines 273, 336, 364, 389, 412, 433, 484, 546, 584, and 638. We then carried out functional analysis of two of these sites, serines 389 and 584. Serine-alanine substitution mutations of 389 (S389A) abrogated rRNA transcription in vitro and in vivo, whereas mutation of serine 584 (S584A) reduced transcription in vivo but not in vitro. In contrast, serine-glutamate mutation of 389 (S389E) restored transcriptional activity. Moreover, S389A abolished UBF-SL1 interaction in vitro, while S389E partially restored UBF-SL1 interaction. Taken together, the results of these studies suggest that growth factor stimulation induces an increase in rRNA transcriptional activity via phosphorylation of UBF at serine 389 in part by facilitating a rate-limiting step in the recruitment of RNA polymerase I: i.e., recruitment of SL1. Moreover, studies provide critical new data regarding multiple additional UBF phosphorylation sites that will require further characterization by the field.

NO-donating ability of nitrosyl [Fe-S] complexes, namely, mononuclear dinitrosyl complexes of anionic type [Fe(S2O3)2(NO)2]-(I) and neutral [Fe2(SL1)2(NO)2] with L1=1H-1,2,4-triazole-3-yl (II); tetranitrosyl binuclear neutral complexes [Fe2(SL2)2(NO)4] with L2=5-amino-1,2,4-triazole-3-yl (III); 1-methyl-1H-tetrazole-5-yl (IV); imidazole-2-yl (V) and 1-methyl-imidazole-2-yl (VI) has been studied. In addition, Roussin's "red salt" Na2[Fe2S2(NO)4] x 8H2O (VII) and Na2[Fe(CN)5NO] x H2O (VIII) have been investigated. The method for research has been based on the formation of Hb-NO adduct upon the interaction of hemoglobin with NO generated by complexes I-VIII in aqueous solutions. Kinetics of NO formation was studied by registration of absorption spectra of the reaction systems containing Hb and the complex under study. For determination of HbNO concentration, the experimental absorption spectra were processed during the reaction using standard program MATHCAD to determine the contribution of individual Hb and HbNO spectra in each spectrum. The reaction rate constants were obtained by analyzing kinetic dependence of Hb interaction with NO donors under study. All kinetic dependences for complexes I-VI were shown to be described well in the frame of formalism of pseudo first-order reactions. The effective first-order rate constants for the studied reactions have been determined. As follows from the values of rate constants, the rate of interaction of sulfur-nitrosyl iron complexes (I-VI) with Hb is limited by the stage of NO release in the solution.

Circumnutations of hypocotyls of sunflower (Helianthus annuus L. cv. Californicus) were studied under 1 g and 3 g conditions. Root mean square values of the hypocotyl deviation from the plumbline and period of the movements were determined from calculations of the autocorrelation functions of the movements. The amplitude and the period of the circumnutations increased under 3 g as compared to 1 g. A transition from 3 to 1 g or vice versa also caused changes in period and amplitude of the movements. The results are interpreted as a support for the idea that gravity influences the circumnutation parameters in this sunflower variety. A comparison is made with published results on the dwarf sunflower cv. Teddy Bear where the force influence is very small or negligible. Simulations of a model for circumnutations show movements which are in qualitative agreement with the experimental results, provided adaptation to g-levels is included in the model. Finally, the results are discussed with the recent Spacelab-experiment (SL1) as a background.

Halorhodospira halophila is among the most halophilic organisms known. It is an obligately photosynthetic and anaerobic purple sulfur bacterium that exhibits autotrophic growth up to saturated NaCl concentrations. The type strain H. halophila SL1 was isolated from a hypersaline lake in Oregon. Here we report the determination of its entire genome in a single contig. This is the first genome of a phototrophic extreme halophile. The genome consists of 2,678,452 bp, encoding 2,493 predicted genes as determined by automated genome annotation. Of the 2,407 predicted proteins, 1,905 were assigned to a putative function. Future detailed analysis of this genome promises to yield insights into the halophilic adaptations of this organism, its ability for photoautotrophic growth under extreme conditions, and its characteristic sulfur metabolism.

The gene tpa-1 on chromosome IV of the nematode Caenorhabditis elegans plays a major and definitive role in the adversary action of tumour-promoting phorbol esters, which induce growth arrest and locomotory distress in the animal. The gene was deduced to code for a protein kinase C (PKC) homologue by molecular cloning. We have now sequenced the complete genomic and complementary DNAs for tpa-1 and have analysed their structural features in detail: (1) tpa-1 spans over 20 kb consisting of eleven exons and ten introns; (2) two different-sized mRNAs are generated from the tpa-1 locus; (3) both mRNAs are trans-spliced to the trans-spliced leader SL1; (4) both mRNAs encode PKC isoforms, which are most similar to Ca(2+)-independent novel PKC0; (5) the two PKC isoforms differ from each other in that the smaller lacks the amino-terminal region of the larger corresponding to the first four exons and a portion of the fifth exon; and (6) three introns are located at; identical positions in the polypeptide sequences aligned between the C. elegans tpa-1 product and a PKC of the fruit fly Drosophila melanogaster.

Translational efficiency is subject to extensive regulation. However, the factors influencing such regulation are poorly understood. In Caenorhabditis elegans, 62% of genes are trans-spliced to a specific spliced leader (SL1), which replaces part of the native 5' untranslated region (5' UTR). Given the pivotal role the 5' UTR plays in the regulation of translational efficiency, we hypothesized that SL1 trans-splicing functions to regulate translational efficiency. With genome-wide analysis on Ribo-seq data, polysome profiling experiments, and CRISPR-Cas9-based genetic manipulation of trans-splicing sites, we found four lines of evidence in support of this hypothesis. First, SL1 trans-spliced genes have higher translational efficiencies than non-trans-spliced genes. Second, SL1 trans-spliced genes have higher translational efficiencies than non-trans-spliced orthologous genes in other nematode species. Third, an SL1 trans-spliced isoform has higher translational efficiency than the non-trans-spliced isoform of the same gene. Fourth, deletion of trans-splicing sites of endogenous genes leads to reduced translational efficiency. Importantly, we demonstrated that SL1 trans-splicing plays a key role in enhancing translational efficiencies of essential genes. We further discovered that SL1 trans-splicing likely enhances translational efficiency by shortening the native 5' UTRs, hence reducing the presence of upstream start codons (uAUG) and weakening mRNA secondary structures. Taken together, our study elucidates the global function of trans-splicing in enhancing translational efficiency in nematodes, paving the way for further understanding the genomic mechanisms of translational regulation.

BACKGROUND

This study describes phenotypic, genotypic and antibiotic susceptibility patterns of the strains isolated from the 2012 Sierra Leone cholera outbreak. Rectal swabs were collected from patients and cultured for Vibrio cholerae O1.

METHODS

The isolates were subjected to multiplex PCR, mismatch amplification mutation assay (MAMA) PCR, pulsed field gel electrophoresis (PFGE), and antibiotic sensitivity tests using disk diffusion and minimum inhibitory concentration (MIC) E-test following standard procedures.

RESULTS

Out of 17 rectal swabs tested, 15 yielded V. cholerae O1 biotype El Tor, serotype Ogawa. All the strains belonged to 'altered' variants as MAMA PCR result showed the presence of classical cholera toxin B. PFGE result revealed four pulse types. Using antibiotic disk diffusion, all the isolates were resistant to erythromycin, chloramphenicol, furazolidone, and trimethoprim/sulfamethoxazole (SXT) except SL1 which was sensitive to chloramphenicol and SXT. All the isolates were sensitive to nalidixic acid, tetracycline, doxycycline, azithromycin, and ciprofloxacin except SL2 which was resistant to nalidixic acid. However, variable sensitivity patterns were observed for kanamycin. The ranges of MIC were 0.125-0.50 mg/l, 0.003-0.023 mg/l and 0.38-0.75 mg/l for azithromycin, ciprofloxacin and tetracycline, respectively.

CONCLUSIONS

This study demonstrates that altered variants of V. cholerae O1 of four clonal types were responsible for the 2012 outbreak of cholera in Sierra Leone.