Discovery of a 382-nt deletion during the early evolution of SARS-CoV-2

To date, the SARS-CoV-2 genome has been considered genetically more stable than SARS-CoV or MERS-CoV. Here we report a 382-nt deletion covering almost the entire open reading frame 8 (ORF8) of SARS-CoV-2 obtained from eight hospitalized patients in Singapore. The deletion also removes the ORF8 transcription-regulatory sequence (TRS), which in turn enhances the downstream transcription of the N gene. We also found that viruses with the deletion have been circulating for at least four weeks. During the SARS-CoV outbreak in 2003, a number of genetic variants were observed in the human population [1], and similar variation has since been observed across SARS-related CoVs in humans and bats. Overwhelmingly these viruses had mutations or deletions in ORF8, that have been associated with reduced replicative fitness of the virus [2]. This is also consistent with the observation that towards the end of the outbreak sequences obtained from human SARS cases possessed an ORF8 deletion that may be associated with host adaptation [1]. We therefore hypothesise that the major deletion revealed in this study may lead to an attenuated phenotype of SARS-CoV-2.

Exogenous RNAi mechanisms contribute to transcriptome adaptation by phased siRNA clusters in Paramecium

Extensive research has characterized distinct exogenous RNAi pathways interfering in gene expression during vegetative growth of the unicellular model ciliate Paramecium. However, role of RNAi in endogenous transcriptome regulation, and environmental adaptation is unknown. Here, we describe the first genome-wide profiling of endogenous sRNAs in context of different transcriptomic states (serotypes). We developed a pipeline to identify, and characterize 2602 siRNA producing clusters (SRCs). Our data show no evidence that SRCs produce miRNAs, and in contrast to other species, no preference for strand specificity of siRNAs. Interestingly, most SRCs overlap coding genes and a separate group show siRNA phasing along the entire open reading frame, suggesting that the mRNA transcript serves as a source for siRNAs. Integrative analysis of siRNA abundance and gene expression levels revealed surprisingly that mRNA and siRNA show negative as well as positive associations. Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Importantly, most SRCs depend on both RDRs, reminiscent to primary siRNAs in the RNAi against exogenous RNA, indicating mechanistic overlaps between exogenous and endogenous RNAi contributing to flexible transcriptome adaptation.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective: The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites. Design and setting: The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI. Method: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences. Results: The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them. Conclusion: Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Porcine phosphotyrosine interaction domain containing 1 modulates 3T3-L1 preadipocyte proliferation and differentiation

The phosphotyrosine interaction domain containing 1 (PID1) gene was firstly isolated from obese subjects and involved in obesity-associated insulin resistance. In the present study, Duroc×Landrace×Yorkshire (DLY) pig PID1 cDNA was cloned. The entire open reading frame of the cloned porcine PID1 is 654 bp. The predicted protein is composed of 217 amino acids residues with a molecular mass of 24,774 Da. Over-expression of porcine PID1 significantly accelerated the proliferation of 3T3-L1 preadipocyte, but inhibited preadipocyte differentiation by decreasing the numerous fat droplets appeared and down-regulating the mRNA expression levels of peroxisome proliferators-activated receptor-γ, CCAAT/enhancer binding protein α, fat acid synthase and lipoprotein lipase. Together, these results suggest that porcine PID1 plays a role in regulating adipose development.

MLL-SEPTIN gene fusions in hematological malignancies

The mixed lineage leukemia (MLL) locus is involved in more than 60 different rearrangements with a remarkably diverse group of fusion partners in approximately 10% of human leukemias.MLLrearrangements include chromosomal translocations, gene internal duplications, chromosome 11q deletions or inversions andMLLgene insertions into other chromosomes, or vice versa. MLL fusion partners can be classified into four distinct categories: nuclear proteins, cytoplasmatic proteins, histone acetyltransferases and septins. Five different septin genes (SEPT2,SEPT5,SEPT6,SEPT9, andSEPT11) have been identified asMLLfusion partners, giving rise to chimeric fusion proteins in which the N terminus of MLL is fused, in frame, to almost the entire open reading frame of the septin partner gene. The rearranged alleles result from heterogeneous breaks in distinct introns of bothMLLand its septin fusion partner, originating distinct gene fusion variants.MLL-SEPTIN rearrangements have been repeatedly identified inde novoand therapy related myeloid neoplasia in both children and adults, and some clinicopathogenetic associations are being uncovered. The fundamental roles of septins in cytokinesis, membrane remodeling and compartmentalization can provide some clues on how abnormalities in the septin cytoskeleton and MLL deregulation could be involved in the pathogenesis of hematological malignancies.

Recombinant GRA4 or ROP2 Protein Combined with Alum or the gra4 Gene Provides Partial Protection in Chronic Murine Models of Toxoplasmosis

ABSTRACT The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant.

Molecular and Genetic Characterization of a Taf1p Domain Essential for Yeast TFIID Assembly

ABSTRACT Yeast Taf1p is an integral component of the multiprotein transcription factor TFIID. By using coimmunoprecipitation assays, coupled with a comprehensive set of deletion mutants encompassing the entire open reading frame of TAF1, we have discovered an essential role of a small portion of yeast Taf1p. This domain of Taf1p, termed region 4, consisting of amino acids 200 to 303, contributes critically to the assembly and stability of the 15-subunit TFIID holocomplex. Region 4 of Taf1p is mutationally sensitive, can assemble several Tafps into a partial TFIID complex, and interacts directly with Taf4p and Taf6p. Mutations in Taf1p-region 4 induce temperature-conditional growth of yeast cells. At the nonpermissive temperature these mutations have drastic effects on both TFIID integrity and mRNA synthesis. These data are consistent with the hypothesis that Taf1p subserves a critical scaffold function within the TFIID complex. The significance of these data with regard to TFIID structure and function is discussed.

Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase

Biosynthesis of (6R)-5,6,7,8-tetrahydro-l-biopterin (H4-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H4-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H4-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H4-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.

Molecular Cloning, Sequencing, Expression, and Characterization of an Immunogenic 43-Kilodalton Lipoprotein of Bartonella bacilliformis That Has Homology to NlpD/LppB

ABSTRACT A recombinant clone expressing an immunoreactive antigen ofBartonella bacilliformis was isolated by screening a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis. The clone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein. Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clones that contain the entire open reading frame. The open reading frame (ORF-401) is capable of encoding a protein of 401 amino acids with a predicted molecular mass of 43 kDa. The deduced amino acid sequence of the encoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD) which has been associated with virulence. Evidence has been provided to show that the 43-kDa antigen of B. bacilliformis is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacterial lipoproteins. This is the first report to date that characterizes a lipoprotein of B. bacilliformis. The immunogenicity of the B. bacilliformis LppB homologue was demonstrated by Western blot analysis using sera from patients with clinical bartonellosis. Sera from patients who had a high titer forBartonella henselae, the causative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDa antigen, suggesting that a homologue of this antigen is present in B. henselae. Using a cocktail of synthetic peptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homologue of B. bacilliformis was generated. This antiserum did not recognize the NlpD homologue of Escherichia coli or the 43-kDa antigen ofB. henselae.

Probable Identification of a Membrane-Associated Repressor of Bacillus subtilis DNA Replication as the E2 Subunit of the Pyruvate Dehydrogenase Complex

ABSTRACT Two Bacillus subtilis lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of B. subtilis DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452–7456, 1988). Three clones that reacted strongly with the antibody contained an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound. This sequence was almost identical to one detected in Bacillus thuringiensis that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901–7906, 1999). However, the exact sequence was not as important in B. subtilis as the AT-rich core region. Binding would occur as long as most of the AT character of the core remained. Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered. The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay.