ATG Codon Research Articles

Dysfunctional Clusterin-Positive Hematopoietic Stem Cells Expand with Aging

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes dysregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old), and aged (20-month-old) mice to gain insight into the dynamics of HSC aging. scRNA-seq data revealed that transcriptomic profiles of HSCs change with aging, although middle-aged and aged HSCs showed very similar profiles to each other. scRNA-seq data divided young and aged HSCs into 2 and 3 clusters, respectively. While both young and aged HSCs had clusters with myeloid or lymphoid gene signature, the cluster with myeloid signature dominated in aged HSCs, in which aged gene signature (Nupr1, Mt1, Mt2, Selp) was highly enriched. Clusterin (Clu) was also significantly up-regulated in this cluster. Clu encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis (Itakura et al., JCB 2020). We hypothesized that Clu would be useful as a marker of aged HSCs, and conducted further analysis using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory elements of the Clu gene. Bone marrow (BM) analysis of Clu-reporter mice revealed that Clu expression is confined to a part of HSCs in BM and significantly up-regulated within the HSC compartment with aging (10 and 70 % of HSCs are Clu-positive in young and middle-aged mice, respectively). In competitive repopulation assays, Clu-positive HSCs showed severely impaired repopulation capacity and preferentially differentiated into myeloid cells while Clu-negative HSCs largely retained repopulation capacity and showed balanced differentiation. We analyzed the transcriptomic profile of Clu-positive HSCs with aging. RNA-sequence analysis revealed that young and middle-aged Clu-positive HSCs were significantly enriched for aged HSC gene signature and myeloid-biased HSC signature compared to Clu-negative HSCs. We evaluated the transcriptional regulation mechanism of Clu by Assay for Transposase-Accessible Chromatin sequence (ATAC-seq) analysis. Chromatin accessibility at the Clu promoter was already open in young HSCs, and largely enhanced in Clu-positive HSCs but not in Clu-negative HSCs among middle-aged HSCs. The open chromatin region at the Clu promoter had binding motifs of AP-1 and STAT family transcription factors, suggesting that external signals or stresses activate Clu expression. These results suggest that Clu-positive cells represent myeloid-biased HSCs which expand with aging and that Clu serves as a faithful marker to trace alterations in HSC heterogeneity with aging.

The whole mitochondrial genome signature of Teressa goat, an indigenous goat germplasm of Andaman and Nicobar Islands, India

The domestic goat is a livestock species of global importance due to its ability to adapt to different agro-climatic conditions. The present study deals with the mitogenome signature of Teressa goat, a registered goat germplasm of Andaman and Nicobar Islands. Teressa goat mitogenome was 16,640 bp in length with 13 protein coding genes (PCGs), 22 tRNAs, 2 rRNAs and a non-coding region. Among the protein coding genes, only ND6 was present on light strand (L), other genes were encoded by heavy strand (H). All the PCGs had either ATG or ATA start codon whereas incomplete stop codons were detected in ND1, ND2, COX3, ND3 and ND4. Haplogroup assignment indicated that Teressa goat falls under Haplogroup A, most prevalent goat haplogroup. Mitogenome based phylogenetic analysis revealed that Teressa goat shared close genetic affinity with Xiangdong black goat, Longdong black goat, and Jintang black goat of China and Jamnapari goat of Bangladesh. The results of the study will be helpful to understand the origin and migration route of the goat breed and for sketching conservation plan of the indigenous breed.

ND2 Gene Sequencing of Sub fertile Patients Recovered from COVID-19 in Association with Toxoplasmosis

A total of (90) blood samples were collected from male patients infected with Toxoplasmosis who recovered from COVID- 19 and attended Kamal Alsamiraai Hospital from 15 January to 15 September 2021. We measured anti-Toxoplasma antibodies (IgG and IgM) detected by ELISA, whereas Anti-COVID-19 antibodies (IgG and IgM) were estimated using Elisa and Afilias. The semen characteristics were also studied among fertile, healthy individuals (control group) and sub-fertile patients. Results showed that the mean sperm count was high among the control group (40.5±1.3x 106/ml) compared with that of the sub-fertile patients (10.3±1.75 and 8.8±1.9 x 106/ml for oligozoospermia, and oligoasthenozoospermia respectively), and it was the highest (44.7±1.4 x 106/ml) among asthenozoospermia patients. Compared to the control group, there were highly significant differences between anti-Toxoplasma IgG antibodies and anti-COVID-19 IgG antibodies (P<0.001). The mean level of Toxoplasma IgM was (11.74±8.90) and for control was (0.05±0.10), while the mean level of COVID-19 IgM was (1.91±1.06) and for control was (0.04±0.03) in sub-fertile patients. The mutation occurred in IL-IB gene A to G transgene at site 4514 of the IL-IB gene (sample code, 6383) and in the case of an invalid sample code, 2409 and 5097. In the alanine codon, the GCA codon has mutated into GCG. Also, G to A transgene occurred at site 4514 of the IL-IB gene. (sample code, 6750) In the case of an invalid sample code, it happened in 010081 and 009593. In the alanine codon, the ATG codon has mutated into ATA. Keywords: ND2 Gene, sequence, Sub-fertile patients, COVID-19, Toxoplasmosis

Complete mitochondrial genomes of two catfishes (Siluriformes, Bagridae) and their phylogenetic implications.

The mitochondrial genome (mitogenome) has been widely used as a molecular marker to investigate phylogenetic analysis and evolutionary history in fish. However, the study of mitogenomes is still scarce in the family Bagridae. In this study, the mitogenomes of Tachysurusbrachyrhabdion and T.gracilis were sequenced, annotated, and analyzed. The mitogenomes were found to be 16,532 bp and 16,533 bp, respectively, and each contained 37 typical mitochondrial genes, which are 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and a control region. All PCGs begin with the codon ATG, except for the cytochrome c oxidase subunit 1 (COI) gene, while seven PCGs end with an incomplete termination codon. All tRNA genes can fold into their typical cloverleaf secondary structures, except for tRNASer(AGY), which lacks the dihydrouracil arm. The Ka/Ks ratios for all PCGs are far lower than one. Phylogenetic analyses based on Bayesian inference (BI) and maximum likelihood (ML) showed that the two clades in Bagridae excluded Ritarita. The monophyly of Tachysurus supports previous research and the traditional classification that Leiocassis, Pseudobagrus, Pelteobagrus, and Tachysurus belong to one genus (Tachysurus). These findings provide a phylogenetic basis for future phylogenetic and taxonomic studies of Bagridae.

Algorithm Model Determination of DNA Primer Design For The Success PCR Process

Deoxyribonucleic Acid Primer Design is an important factor in the success of the Polymerase Chain Reaction process because it will determine the value of Guanine and Cytosine content and Melting temperature for the genome amplification process in the adequacy of research data. To get a good primer design, a simulation process is needed to get the desired results, namely Guanine and Cytosine content between 50%-60%, Melting temperature 50o -65oC, a minimum length of 70 base pair sequences and the temperature difference between the forward primer and reverse primer less than 5oC. Usually researchers use the Primer3plus or NCBI Primer-BLAST software to get the Deoxyribonucleic Acid Primer Design results. Because there are limitations in the use of the software, the researchers, researchers want to use software that are suitable for current and future research needs. For this reason, a new algorithm model is needed to make software to support the needs of researchers. The method used in the algorithm model is to use Start Codon ATG and Stop Codon TAA, TAG and TGA with sequence lengths of 18, 21 and 24 base pairs. The raw data used is the Deoxyribonucleic Acid of the Typhonium Flagelliforme plant which has potential anti-cancer compounds. The new algorithm model resulted in 22 (twenty two) Deoxyribonucleic Acid Primer Design options, all of which met the data constraints and research requirements.

Comparative mitogenomics of the genus Motacilla (Aves, Passeriformes) and its phylogenetic implications.

The genus Motacilla belongs to Motacillidae (Passeriformes), where mitochondrial features are poorly understood and phylogeny is controversial. Whole mitochondrial genome (mitogenome) data and large taxon sampling are considered to be ideal strategies to obtain this information. We generated four complete mitogenomes of M.flava, M.cinerea, M.alba and Dendronanthusindicus, and made comparative analyses of Motacilla species combined with mitogenome data from GenBank, and then reconstructed phylogenetic trees based on 37 mitochondrial genes. The mitogenomes of four mitogenome sequences exhibited the same gene order observed in most Passeriformes species. Comparative analyses were performed among all six sampled Motacilla mitogenomes. The complete mitogenomes showed A-skew and C-skew. Most protein-coding genes (PCGs) start with an ATG codon and terminate with a TAA codon. The secondary structures of RNAs were similar among Motacilla and Dendronanthus. All tRNAs except for trnS(agy) could be folded into classic clover-leaf structures. Three domains and several conserved boxes were detected. Phylogenetic analysis of 90 mitogenomes of Passeriformes using maximum likelihood (ML) and Bayesian inference (BI) revealed that Motacilla was a monophyletic group. Among Motacilla species, M.flava and M.tschutschensis showed closer relationships, and M.cinerea was located in a basal position within Motacilla. These data provide important information for better understanding the mitogenomic characteristics and phylogeny of Motacilla.

Characterization of the mitochondrial GlutamyltRNAGln amidotransferase (GatCAB) as a new model for mitochondrial translation disorders.

Mitochondrial OXPHOS diseases are provoked by dysfunction of the OXPHOS system, showing heterogeneity from a genetic, biochemical and clinical perspective. This complexity is partly due to the involvement of proteins encoded in two genomes located in different cellular compartments, nuclear and mitochondrial DNA (mtDNA). mtDNA encodes part of the mtRNAs translation machinery, including 22 tRNAs. All these are charged by their corresponding aminoacyl-tRNA synthetases (ARS2), except mt-Gln-tRNAGln whose synthesis is carried out through an indirect pathway. The mt-tRNAGln is charged with glutamic acid (Glu) by a non-discriminating mitochondrial glutamyl-tRNA synthetase (EARS2), then the Glu is converted to Gln by a Glutamyl-tRNAGln amidotransferase, using free glutamine as an amide donor, yielding Gln- tRNAGln. This mitochondrial amidotransferase activity lies in the GatCAB complex, which is formed by three subunits: GatA (QSRL1), GatB (GATB), and GatC (GATC). Patients with mutations in these genes presented with severe cardiomyopathy and lactic acidosis, which underscores the importance of the GatCAB complex as an essential component in the translational machinery of mitochondrial protein synthesis. We have generated Knockout (KO) lines for all subunits using the genomic editing system CRISPR/Cas9 in HEK293T cells. We measured the levels of the GatCAB subunits and mitochondrial proteins by western blot. We studied the oxygen consumption levels using a Clark electrode and detected the GatCAB complex by Blue Native-western blotting. In all cases, KO cells show reduced levels of mitochondrial proteins, decreased oxygen consumption and an absence of the GatCAB complex compared to wildtype cells. In the QRSL1 and GATC KO cells the levels of all subunits diminish, but in GATB KO cells the levels of subunits A and C remain stable. Possibly, QRSL1 and GATC could form a stable but nonfunctional dimer, to which GATB would join. GATB has two potential ATG codons at the start of its sequence separated by 12 nucleotides. From over 90 clones analyzed, we only obtained one KO, by generating an upstream ORF, and two cell lines with very low expression of GATB. The latter present a duplicated region at the beginning of GATB which changes the reading frame from the first ATG; suggesting the downstream ATG as an alternative translation start codon for those ribosome small subunits that do not assemble on the first one, yielding a GATB protein missing 4 amino acids. Characterization of the molecular mechanisms that lead to the synthesis of mt-Gln-tRNAGln and its role in the physiology of the cell could allow us to comprehend the pathological manifestation of the defects in these genes and propose possible therapeutic avenues.

Machine learning predicts translation initiation sites in neurologic diseases with nucleotide repeat expansions.

A number of neurologic diseases associated with expanded nucleotide repeats, including an inherited form of amyotrophic lateral sclerosis, have an unconventional form of translation called repeat-associated non-AUG (RAN) translation. It has been speculated that the repeat regions in the RNA fold into secondary structures in a length-dependent manner, promoting RAN translation. Repeat protein products are translated, accumulate, and may contribute to disease pathogenesis. Nucleotides that flank the repeat region, especially ones closest to the initiation site, are believed to enhance translation initiation. A machine learning model has been published to help identify ATG and near-cognate translation initiation sites; however, this model has diminished predictive power due to its extensive feature selection and limited training data. Here, we overcome this limitation and increase prediction accuracy by the following: a) capture the effect of nucleotides most critical for translation initiation via feature reduction, b) implement an alternative machine learning algorithm better suited for limited data, c) build comprehensive and balanced training data (via sampling without replacement) that includes previously unavailable sequences, and d) split ATG and near-cognate translation initiation codon data to train two separate models. We also design a supplementary scoring system to provide an additional prognostic assessment of model predictions. The resultant models have high performance, with ~85-88% accuracy, exceeding that of the previously published model by >18%. The models presented here are used to identify translation initiation sites in genes associated with a number of neurologic repeat expansion disorders. The results confirm a number of sites of translation initiation upstream of the expanded repeats that have been found experimentally, and predict sites that are not yet established.

Complete mitochondrial genome of Stigmaeopsis miscanthi (Acari: Tetranychidae)

Complete mitochondrial genome of Stigmaeopsis miscanthi was first reported for the genus Stigmaeopsis in Tetranychidae. The circular mitochondrial genome of S. miscanthi is 15,171 bp in length, with 80.4% AT content, involving 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA, and main one of non-coding regions. The length of 16SrRNA and 12SrRNA gene is 973 bp and 636 bp, respectively. Seven PCGs started with ATT codon, and other PCGs started with ATG and ATA codons. Five PCGs stopped with TAA codon, and the other eight PCGs ended with incomplete stop codons (T). Phylogenetic analysis indicated that S. miscanthi branched with the genus Panonychus.

Fragment screening and structural analyses highlight the ATP-assisted ligand binding for inhibitor discovery against type 1 methionyl-tRNA synthetase.

Methionyl-tRNA synthetase (MetRS) charges tRNAMet with l-methionine (L-Met) to decode the ATG codon for protein translation, making it indispensable for all cellular lives. Many gram-positive bacteria use a type 1 MetRS (MetRS1), which is considered a promising antimicrobial drug target due to its low sequence identity with human cytosolic MetRS (HcMetRS, which belongs to MetRS2). Here, we report crystal structures of a representative MetRS1 from Staphylococcus aureus (SaMetRS) in its apo and substrate-binding forms. The connecting peptide (CP) domain of SaMetRS differs from HcMetRS in structural organization and dynamic movement. We screened 1049 chemical fragments against SaMetRS preincubated with or without substrate ATP, and ten hits were identified. Four cocrystal structures revealed that the fragments bound to either the L-Met binding site or an auxiliary pocket near the tRNA CCA end binding site of SaMetRS. Interestingly, fragment binding was enhanced by ATP in most cases, suggesting a potential ATP-assisted ligand binding mechanism in MetRS1. Moreover, co-binding with ATP was also observed in our cocrystal structure of SaMetRS with a class of newly reported inhibitors that simultaneously occupied the auxiliary pocket, tRNA site and L-Met site. Our findings will inspire the development of new MetRS1 inhibitors for fighting microbial infections.

Mitochondrial genome structure, phylogenetic analyses and substitution rate estimation of the Oedogoniales

The order Oedogoniales comprises three genera, Oedogonium, Oedocladium and Bulbochaete, which were classified based on traditional morphological criteria, and includes more than 600 described species. This group is economically important in astaxanthin production and the energy sector. However, only one mitochondrial genome (mitogenome) has been reported so far. This study determined the mitochondrial genomes of seven Oedogoniales species, including six Oedogonium species and Oedocladium prescottii. Comparative analyses between the newly determined mitogenomes and the previously reported Bulbochaete rectangularis var. hiloensis mitogenome showed that all eight mitogenomes comprised 12 protein-coding genes (PCGs) and two rRNAs; however, the mitogenomes differed in their genome sizes, GC content, tRNAs, non-coding regions and introns. Synteny analysis of the eight mitogenomes revealed a high degree of syntenic conservation in general, with some rearrangements and inversions. The average nucleotide identity (ANI) analysis of the eight mitogenomes indicated Oedogonium dentireticulatum showed high similarity with Oedogonium sp3 (ANI of 96.32%). Most of the PCGs of the eight mitogenomes presented the conventional start codon ATG and stop codon TAR (TAA/TAG/TGA), and the synonymous codon preferences were conserved. Phylogenetic results indicated that Oedogonium was polyphyletic, and species of Oedocladium clustered with Oedogonium, while the position of B. rectangularis var. hiloensis was uncertain for the incongruent phylogenetic results. Statistical analyses of substitution rates demonstrated no significant differences among the three genera, and the dN/dS ratios based on branch model showed that cob, cox1 and nad4 of Oedocladium prescottii and B. rectangularis var. hiloensis were putative fast-evolving genes. These findings suggested that the traditional taxonomy of Oedogoniales did not define natural groups, and that species of Oedocladium and Bulbochaete may have undergone rapid evolution.

The Emergence of a New Isoform of POU2F1 in Primates through the Use of Egoistic Mobile Genetic Elements

The emergence of new genes and functions is of paramount importance in the emergence of new animal species. For example, the insertion of the mobile element Tigger 2 into the sequence of the functional gene POU2F1 in primates led to the formation of a new chimeric primate-specific isoform POU2F1Z, the translation of which is activated under cellular stress. Its mRNA was found in all species of monkeys, starting with macaques. Analysis of the fragments of the Tigger2 copy corresponding to the human exon Z showed that the splicing sites of exon Z are homologous in humans and in most monkeys, with the exception of lemurs and galagos. The stop codon introduced into the mRNA by the Tigger2 sequence is present in all primates, starting with macaques. The internal ATG codon is also present in all primates, with the exception of lemurs and galagos. In the course of evolution, other MGEs, mainly of the SINE type, were inserted into the Tigger2 copy. In the course of evolution, both the location and the number of mobile SINE elements within the POU2F1 gene changed. Starting with macaques, the pattern of the arrangement of SINE elements within the Tigger2 copy in the studied region of the POU2F1 gene was fixed and then remained unchanged in other primates and humans, which may indicate its functional significance.

The mitochondrial genome of Tremoctopus violaceus (Octopoda, Tremoctopodidae) and its phylogenetic consideration

The complete mitochondrial genome of Tremoctopus violaceus was sequenced to analyze its organization and phylogenetic status within the order Octopoda. The mitochondrial genome of T. violaceus had a structure and organization similar to that of other Octopoda. The content of the nucleotides A, C, G, and T was 31.68 %, 7.71 %, 20.02 %, and 40.58 %, respectively. All protein-coding genes (PCG) began with the ATG codon, excluding ND4 and ATP6, which began with ATC and ATT, respectively, and terminated with TAG, TAA, TA, or T. Codons for isoleucine were the most used codons, whereas those for arginine were used the least. Two extra tRNAs, trnN and trnL, were found in the control region. These tRNAs have a D-armless structure. The control region had excess A + T content (83.16 %) and a stem-loop structure with two elements, which is reported for the first time in Octopoda by our study. Bayesian inference using 13 PCG revealed that Octopus and Octopodidae were polyphyletic, and that Tremoctopodidae diverged relatively earlier within Octopoda. The mitochondrial genome of T. violaceus and its characteristics may help to understand the evolutionary history of Octopoda and establish a marine biodiversity conservation strategy.

Complete sequence and characterization of the Mobula tarapacana (Sicklefin Devilray) mitochondrial genome and its phylogenetic implications

Mitochondrial genome sequences provide excellent insight into the study of molecular phylogeny and evolution. Mobula tarapacana (Sicklefin devil ray) has an olive-green to brown colored dorsal surface and darkly colored ventral markings. Due to their grey ventral stripes, fisherman and divers sometimes mistake these species for manta rays. To avoid the misidentification during bycatch and to increase the scientific knowledge of this resource as per the SDG Target 14.A, the complete mitogenome sequence of Sicklefin devil ray was sequenced, annotated, and submitted to the GenBank (Accession: MH669414.1). The mitogenome of M. tarapacana is a closed circular dsDNA of 5, 686 bp in size, which transcribes into 13 protein-coding genes (PCGs), 22 tRNAs, and 2 rRNAs. The percentage of protein-coding genes in the genome was 60.30%. Each protein-coding gene is initiated with an ATG codon excluding COX1 and ATP8 that begin with GTG and CCT respectively. Leu (L) and ser (S) were the most common abundant amino acids in the PCGs. The phylogenetic relationship and evolutionary distances were constructed by maximum likelihood approach using MEGA X software. The reported mitogenome was closely related to Mobula kuhlii (MKU MG10, KM364987) and Mobula eregoodootenkee (NC 025954).

5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.

ABSTRACTHepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5′ P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation.IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.

Manipulation of viral protein production using the PCNA of halovirus фCh1 via alternative start codon usage

• Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. • Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing majorly in the presence of an alternate GTG start codon in the фCh1 variant 5’ of the ATG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. • Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behavior, viral protein levels, and virus titer. • Deletion of фCh1-ORF59, encoding PCNA ΦCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. • Complementation and overexpression with WT, and mutant variants of ORF59revealed that the 5’ GTG to ATG sequence enables фCh1 to control its equilibrium with the host. The different variants have a broad range of variable effects on each of the phenotypes observed. • Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of φCh1 modulated by the 5’ GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the behavior of the virus фCh1. The 5’ region upstream of the primary ATG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship. Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing in the presence of an alternate GUG start codon 5’ of the AUG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behaviour, viral protein levels, and virus titer. Deletion of фCh1 ORF59, encoding PCNA ФCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. Complementation and overexpression with WT and mutant variants of ORF59 revealed that modification of the GTG start codon to ATG changes the life cycle in terms of production of progeny virus particles. The different variants have a broad range of variable effects on each of the phenotypes observed. Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of фCh1 modulated by the 5’GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the life cycle of the virus фCh1. The 5’ region upstream of the primary AUG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship.

EmbB Gene association with Ethambutol drug Resistance in Mycobacterium Tuberculosis Patients

Background: Tuberculosis (TB) is fatal and life threatening infectious disease. The transmission rate of tuberculosis is very high. Various drugs are used as treatment for TB. Recently it has been observed that one of the most important factor for fast TB spread is development of anti-TB drug resistant mycobacterium tuberculosis (MTB). Various combination of drugs like isoniazid (INH), rifampicin (RIF), Streptomycin(SM), pyrazinamide (PZA) or ethambutol (EMB) are in global use for TB treatment. Improper usage of these drugs makes the person prone to develop anti-TB drug resistant tuberculosis. Aim: To evaluate association of embB gene with ethambutol resistance in Mycobacterium Tuberculosis. Methods: 104 Specimens of sputum from suspected tuberculosis patients were processed for inoculation in Lowenstein J Medium after it has been decontaminated properly. Kit method by using QIAamp DNA Mini kit was utilized for extraction of DNA. Then region from base 6953 to 10249 of embB gene was amplified through PCR and then followed by sequencing with the aid of softwares blast2seq and ClustalW2. Three primer sets were utilized to amplify embB gene. Ethambutol (EMB) Resistant MTB specimens were processed to study mutation in embB gene. Results: Out of the total 104 sputum specimens, 14 samples were found to have ethambutol resistance. These 14 samples were then processed for mutational analysis. DNA sequence analysis of these 14 samples confirmed embB gene mutation in 10 samples. Mutational analysis revealed that 08 samples showed mutation at codon 306 and two samples showed mutation at 319 codon. The reported mutation Methionine →Isoleucine was seen in 07 samples with ATG codon replaced by ATA codon at codon position 306. One sample showed mutation as Methionine →Isoleucine with ATG codon replaced by ATC codon at codon position 306. Two samples showed mutation as Tyrosine →Serine with TAT codon replaced by TCT at 319 codon position in embB gene. Conclusion: This study concludes that mutation of certain genes particularly point mutation of embB gene at codon 306 and 319 is associated with drug resistance of ethambutol in ethambutol resistant mycobacterium tuberculosis patients. Keywords: Ethambutol, embB gene, Mycobacterium tuberculosis.

Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12

In the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifEQ100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.

Unraveling Heterogeneity of Aged Hematopoietic Stem Cells By Single-Cell RNA Sequence Analysis

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes deregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old) and aged (20-month-old) mice to gain insight into the dynamics of HSC aging.scRNA-seq analysis revealed three major clusters (A, B, C) and three minor clusters (D, E, F) in young HSCs. Of interest, aged HSCs also showed similar cluster formation. One of the minor clusters was characterized by gene signature associated with inflammatory response and significantly increased with age, while the remaining two minor clusters, which showed cell cycle gene signature, did not change in proportion. One of the major clusters, Cluster C, which showed the strongest expression of HSC-specific gene sets compared with other clusters, moderately increased with age.Our scRNA-seq analysis confirmed upregulation of age-related genes previously reported, such as Selp, Mt1, and Vwf, in a considerable portion of aged HSCs. von Willebrand factor (Vwf) encodes a blood glycoprotein produced by megakaryocytes and endothelial cells. Several groups have reported that Vwf-expressing HSCs show myeloid/platelet-biased differentiation (Joana C et al., Nature 2013; Sandra P et al., Developmental Cell 2018). Importantly, our scRNA-seq data identified that Clusterin (Clu) is rarely expressed in young HSCs and is dramatically upregulated in aged HSCs. Clu expression was predominantly increased in one of the major clusters, Cluster C. Clusterin encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis. We hypothesized that Clu would be useful as a marker of a unique subpopulation of aged HSCs, and thus conducted further analysis by using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory sequences of the BAC gene.Clu/GFP was preferentially expressed in HSCs and at lower frequencies in MPP1 than HSCs in Lineage -Sca-1 +c-Kit + (LSK) cell fraction. Clu-positive HSCs expressed high levels of CD150 and were detected in 10% and 60% of HSCs in 10-week-old young mice and 8-month-old middle-aged mice, respectively, indicating that Clu-positive HSCs increase with aging. We next assessed the function of Clu-positive HSCs in young mice. Clu-positive young HSCs established significantly lower chimerism than Clu-negative young HSCs and preferentially differentiated into myeloid cells in competitive transplantation assays. RNA-seq analysis of Clu-positive and Clu-negative HSCs from young mice confirmed that Clu-positive HSCs show the gene signature of myeloid-biased HSCs. Characterization of Clu-positive and negative subpopulations in aged HSCs is currently underway. These results suggest that Clu-positive HSCs represent myeloid-biased HSCs which expand with aging, thus Clu expression serves as a novel marker to monitor the alterations in HSC heterogeneity with aging. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

Molecular cloning and characterization of two distinct caffeoyl CoA O-methyltransferases (CCoAOMTs) from the liverwort Marchantia paleacea

Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.