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Integrated Optimization, Genomic Characterization, and Functional Evaluation of Biogenic Selenium Nanoparticles from Bacillus licheniformis BLN313: Antibacterial and Anticancer Potential.

Microbial synthesis of selenium nanoparticles (SeNPs) offers a sustainable alternative to chemical routes, but the genetic basis of selenium handling in Bacillus remains poorly defined, which limits rational strain selection. Here, SeNP production, physicochemical characterization, and closed-genome sequencing are combined for Bacillus licheniformis BLN313. Selenite reduction peaked at 500 µg/mL Na2SeO3 (88.8% conversion; 444 ± 27 µg/mL Se0); at higher concentrations, conversion efficiency and viability diverged, indicating that tolerance and reductive capacity are distinct traits. Purified SeNPs were spherical and partially crystalline trigonal Se0 (TEM 190 ± 52 nm; DLS 166 nm, PDI 0.03; zeta potential -20.8 mV), carrying a proteinaceous capping layer confirmed by XPS, EDS, and FTIR and shown by LC-MS to be enriched in cell wall-derived metabolites. The particles were bactericidal against Micrococcus luteus (MIC 62.5 µg/mL) and Klebsiella pneumoniae (MIC 250 µg/mL) and reduced MCF-7 viability (IC50 2.7 µg/mL) while sparing MCF-10A cells. The 4.11 Mb genome (46.3% GC; ANI 99.7%, dDDH 97.8%) encodes SulP and Pit transporters, multiple trxB copies, and sulfur-metabolism and oxidative-stress genes, defining a candidate gene set for selenium uptake, reduction and detoxification. BLN313 thus provides a genetically defined platform for SeNP production in biomedical and environmental applications.

Selenium↗

Genomic characterization of the lysophosphatidic acid receptor gene, lp(A2)/Edg4, and identification of a frameshift mutation in a previously characterized cDNA.

To understand the regulation, evolution, and genetics of lp(A2)/Edg4, a second lysophosphatidic acid receptor gene, we characterized its complete cDNA sequence, genomic structure, and chromosomal location. The full-length mouse transcript sequence was determined using rapid amplification of cDNA ends. Southern blot and restriction fragment length polymorphism segregation analyses revealed that the mouse gene was present as a single copy and located at the middle of Chromosome 8 near the mutations for myodystrophy (myd) and "kidney-anemia-testes" (kat). This region is syntenic with human chromosome 19p12, where the human genomic clone containing the lp(A2) gene (EDG4) was mapped. Sequence analysis of genomic clones demonstrated that both mouse and human transcripts were encoded by three exons, with an intron separating the coding region for transmembrane domain VI. Reverse transcriptase-PCR demonstrated that the three exons were spliced in all mouse tissues shown to express the transcript. Finally, in a comparison of all human lp(A2) sequences present in the database, we identified several sequence variants in multiple tumors. One such variant (a G deletion) in the initially characterized Edg4 cDNA clone (derived from an ovarian tumor) results in a frameshift mutation near the 3' end of the coding region. In addition to increasing our understanding of the mechanisms underlying lysophosphatidic acid signaling and lysophospholipid receptor gene evolution, these results have important implications regarding the genomic targeting and oncogenic potential of lp(A2).

Animals↗

Molecular cloning of the Mason-Pfizer monkey virus genome: characterization and cloning of subgenomic fragments.

The molecular characterization of the proviral DNA genome of Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, is described. An analysis of unintegrated viral DNAs present in acutely infected cells revealed open and closed circular molecules and linear species. The size of the M-PMV linear proviral DNA is determined to be 8.1 kbp in length. A preliminary screening of restriction enzymes indicated that many of those commonly used for cloning (EcoRI, SalI, ClaI, XhoI) did not cut the provirus. Digestion of a mixture of linear and circular forms of unintegrated DNA with HindIII produced a set of restriction fragments 2.3-3 kbp in length. These subgenomic fragments where cloned into the bacterial plasmid pAT153, and two classes of M-PMV subgenomic clones isolated. The first of these contained fragments that spanned the ends of the linear genome and presumably were derived from circular proviruses. Six of the seven clones in this class contained a single long terminal repeat (LTR), represented by pMP6, while the seventh, pMP9, contains two LTRs. Digestion of the latter clone with an enzyme that cleaves once within the LTR allowed the length of the M-PMV LTR to be determined as 350 bp. Both the LTR containing clones and the second class of subgenomic clones have been used in developing a detailed restriction map of the M-PMV proviral DNA and in orienting it with regard to transcription of viral RNA. Thus, pMP6/pMP9 contain sequences from the LTR-gag region of the genome and the second class of subclones (represented by pMP1) span the env-coding region. No clones containing the pol-coding region have been isolated. In order to determine the nature of M-PMV-related endogenous sequences in the chromosomal DNA of Old World primates, EcoRI-digested primate DNA was hybridized at low stringency to the subgenomic clones and then washed under conditions of low, moderate, and high stringencies. Multiple sequences closely related to the LTR-gag region of the M-PMV genome, were detected. Sequences more distantly related to the env region were also found in Old World monkeys. Ape and human DNAs were shown to contain sequences related to the LTR-gag region of the M-PMV genome, but were only weakly detectable at low stringency.

Animals↗

Genomic characterization of a novel HIV type 1 B/G intersubtype recombinant strain from an injecting drug user in Germany.

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study we characterize the full-length genome of a novel B/G intersubtype recombinant strain from a patient with a history of injection drug use. This is the first evidence of a B/G recombinant among injecting drug users in Germany. Moreover, this genetically divergent strain has important clinical implications since it was undetectable in the NucliSens HIV-1 QT assay and significantly underquantified by the COBAS MONITOR version 1.5 test relative to the LCx HIV RNA Quantitative assay. Phylogenetic analysis revealed that this recombinant virus is ancestrally related to CRF14_BG, but carries two additional subtype B-derived segments within vif and pol integrase.

Base Sequence↗

Nucleotide sequence and genome characterization of rice yellow mottle virus RNA.

The genome of rice yellow mottle virus (RYMV) is a single-stranded positive-sense RNA that is not polyadenylated, and has an M(r) of 1.4 x 10(6). We present here the 4550 nucleotide (nt) sequence of RYMV RNA, and its predicted genomic organization. The RYMV genomic RNA contains four open reading frames (ORFs). The first (nt 80 to 553) encodes a protein containing 157 amino acids with a predicted M(r) of 17.8K. No function has yet been attributed to this product. ORF2 (nt 608 to 3607) encodes a polyprotein of 999 amino acids, with a predicted M(r) of 110.7K. The first 134 amino acids of ORF2 are predicted to be the genome-linked protein, VPg, followed by the viral protease, the helicase and the RNA-dependent RNA polymerase. ORF3 is within the boundaries of ORF2 and is predicted to encode a polypeptide with 126 amino acids and an M(r) of 13.7K. No function has yet been attributed to this protein. ORF4 (nt 3447 to 4166), which overlaps the 3' terminus of ORF2, encodes a 26K protein. This polypeptide has been identified as the RYMV coat protein. The data presented here confirm that RYMV belongs to the sobemovirus group and thus is a member of the picorna-like family of plant viruses.

Amino Acid Sequence↗

Genomic characterization of Molluscum contagiosum virus type 1: identification of the repetitive DNA sequences in the viral genome.

The genomes (188 kbp) of the prototype Molluscum contagiosum virus type 1 (MCV-1) and a variant strain (MCV-1v) were characterized by construction of the physical maps of the viral DNA for the restriction enzymes BamHI, ClaI, EcoRI, and HindIII using a defined gene library harboring the DNA sequences of the MCV-1 genome and by DNA-DNA hybridizations. It was found that the genomes of both MCV strains are identical, with the exception of very few changes in the DNA fragmentation patterns of restriction endonuclease BamHI as a consequence of naturally occurring nucleotide exchanges in the genome of the variant strain. Detailed hybridization experiments revealed the existence of repetitive DNA sequences, which are located within the terminal regions of the viral genome at the map coordinates 0 to 0.027 and 0.973 to 1.

Cloning, Molecular↗

Strategies for proteomics with incompletely characterized genomes: the proteome of Bos taurus serum.

A reference map for Bos taurus serum was obtained using proteomic tools: 21 proteins, plus several serum albumin fragments, have been identified in 47 spots. One of the major acute-phase reactants, haptoglobin, was also detected in a pathological serum. A number of technical problems had to be solved. (i) Spot resolution in two-dimensional electrophoresis (2-DE) is not easily optimized, as several proteins have similar molecular mass; different polyacrylamide concentration gradients were used for the analysis of various size ranges. (ii) Identification of proteins through mass spectrometry (MS) procedures is also difficult as the genome of Bos taurus is incompletely characterized. The program FASTS proved particularly useful, since it allows simultaneous searching of several unordered sequence fragments, which may be individually too short to provide a statistically valid match using BLAST.

Amine Oxidase (Copper-Containing)↗

Karyotype and genome characterization in four cartilaginous fishes.

Different approaches can be used to elucidate the unsolved questions concerning taxonomic evolution in cartilaginous fish. The study of the karyological characteristics of these vertebrates by combining molecular and traditional techniques of chromosome preparation and banding has been demonstrated to be a very effective method. In this paper we studied the localization and the composition of the constitutive heterochromatin by using C- and restriction endonuclease-banding in four selachian species, belonging to two of the four superorders. We also characterized two different types of repetitive genomic sequences in these species: satellite DNA and (TTAGGG)(n) telomeric sequences. Finally, we analysed the nuclear ribosomal gene to determine the number of the nucleolar organizers and their position on chromosomes by using silver staining, chromomycin A(3), and FISH (fluorescent in situ hybridization). The results showed a prevailingly telomeric localization of constitutive heterochromatin in the Galeomorphii, the presence of additional nucleolar organizer sites in Raja asterias, an exclusively telomeric localization of the (TTAGGG)(n) sequences in Scyliorhinus stellaris and both telomeric and interstitial in Taeniura lymma. These data, together with those concerning the conservation of the satellite DNA, seem to support the hypothesis that Chondrichthyes have an evolutionary history leading them to the acquisition of large genomes rich in highly repeated sequences and subjected to some selective pressures favoring the conservation of this DNA fraction.

Animals↗

Genomic characterization of a slow/low maedi visna virus.

The complete genomic sequence of a sheep lentivirus isolate that presents a slow/low phenotype in vitro has been determined. The virus, designated P1OLV, was isolated from lung cells of a naturally infected sheep in Portugal. Three overlapping DNA fragments amplified by PCR, and encompassing the entire viral genome were cloned and sequenced. This isolate has an overall similarity of approximately 80% with the K1514 Maedi Visna virus (MVV) and approximately 70% with the caprine arthritis encephalitis virus (CAEV) Co strain. Phylogenetic analysis based on SU and RT nucleotide sequences grouped P1OLV with previously reported ovine MVV. To determine the virus replication rate, sheep choroid plexus (SCP) and lung cells, macrophages (MØ), and goat synovial membrane (GSM) cells were inoculated with either P1OLV or with the lytic North American strain WLC-1. Viral RNA in culture supernatants was measured by one-tube real time quantitative RT-PCR. Significant differences were observed between the replication rates of the two viruses, with WLC-1 growing rapidly and to high levels in all the cells tested, while P1OLV replicated more slowly and to lower levels inducing persistent infections in lung and SCP cells. The U3 region of the LTR of P1OLV lacks the sequence repeats that are present in the LTRs of WLC-1 and MVV prototype K1514 and that contain additional binding sites for the AML(vis) transcriptional factor. To evaluate the contribution of LTR in the virus replication rate in vitro, we measured the basal activity of the promoter from P1OLV and WLC-1 in a luciferase-driven gene expression assay and lower levels of expression were achieved for P1OLV. The genetic and biological properties of P1OLV will be useful for the study of virus transcriptional factors and genes that may be responsible for the slow/low phenotype.

Animals↗

Genomic characterization of Rim2/Hipa elements reveals a CACTA-like transposon superfamily with unique features in the rice genome.

The availability of huge amounts of rice genome sequence now permits large-scale analysis of the structure and molecular characteristics of the previously identified transposase-encoding Rim2 (also called Hipa) element, which is transcriptionally activated by infection with the fungal pathogen Magnaporthe grisea and by treatment with the corresponding fungal elicitor. Based on genomic cloning and data mining from 230 Mb of rice genome sequence, 347 Rim2 elements, with an average size of 5.8 kb, were identified. This indicates that an estimated total of 600-700 Rim2 elements are present in the whole genome. Rim2 insertions occur non-randomly on the chromosomes, as visualized by fluorescence in situ hybridization. The elements harbor 16-bp terminal inverted repeats with the core sequence CACTG, 16-bp sub-terminal repeats, internal variable regions, 3-bp target sequence duplications in the flanking regions, and genes coding for Rim2 proteins (the putative transposase) and hydroxyproline-rich glycoproteins. High levels of insertion into genic regions are observed for members of this family, and the transposition history of the family can be deduced from the high level of shared sequences and analysis of repeat target sites of the elements. Phylogenetic analysis indicates that the putative RIM2 proteins fall into a subgroup distinct from the TNP2-like subgroup of transposases. Southern hybridization with genomic DNA from monocotyledonous and dicotyledonous plants demonstrates that the RIM2-coding sequence is unique to the Oryza genome. Our results demonstrate that the Rim2 elements from rice belong to a distinct superfamily of CACTA-like elements with evolutionary diversity.

Amino Acid Sequence↗

Genomic characterization of an endemic Mycobacterium tuberculosis strain: evolutionary and epidemiologic implications.

In a study of 302 Mycobacterium tuberculosis clinical isolates from the low-incidence Canadian-born population of Quebec, we characterized a large endemic strain family by using genomic deletions. The DS6(Quebec) deleted region (11.4 kb) defined a strain family of 143 isolates encompassing two subgroups: one characterized by pyrazinamide (PZA) susceptibility and the other marked by a PZA-monoresistant phenotype. A second deletion (8 bp) in the pncA gene was shared by all 76 isolates with the PZA resistance phenotype, whereas a third DRv0961 deletion (970 bp) defined a further subset of 15 isolates. From their deletion profiles, we derived a most parsimonious evolutionary scenario and compared multiple standard genotyping modalities (using IS6110 restriction fragment length polymorphism [RFLP], spoligotyping, and mycobacterial interspersed repetitive units [MIRU]) across the deletion-based subgroups. The use of a single genotyping modality yielded an unexpectedly high proportion of clustered isolates for a high IS6110 copy strain (27% by IS6110 RFLP, 61% by MIRU, and 77% by spoligotyping). By combining all three modalities, only 14% were genotypically clustered overall, a result more congruent with the epidemiologic profile of reactivation tuberculosis, as suggested by the older age (mean age, 60 years), rural setting, and low proportion of epidemiologic links. These results provide insight into the evolution of genotypes in endemic strains and the potential for false clustering in molecular epidemiologic studies.

Biological Evolution↗

Genomic characterization of the mouse inhibitor of apoptosis protein 1 and 2 genes.

Genomic and cDNA clones encoding mouse inhibitor of apoptosis protein 1 and 2 (Miap1 and Miap2) were isolated and characterized. The genes encoding the 602-amino-acid MIAP1 protein and the 612-amino-acid MIAP2 protein are contained within a 57-kb locus in a tandem head-to-tail arrangement. The Miap1 gene consists of nine exons spanning 24 kb, and the Miap2 gene consists of seven exons spanning 21 kb. Fluorescence in situ hybridization analysis mapped the locus to chromosome 9A2, which is syntenic with portions of the human 11q22-q23 region containing the human homologues HIAP1 and HIAP2. Sequencing of the complete Miap1 and Miap2 cDNAs revealed an unusually long 5' untranslated region in the Miap2 transcript, which may indicate nonscanning ribosomal initiation of translation.

Amino Acid Sequence↗

Genomic characterization of two predominant genotypes of herpes simplex virus type 1.

Genomic profiles of 66 strains of herpes simplex virus type 1 (HSV-1) isolated in Japan were investigated with regard to restriction fragment length polymorphism (RFLP) and length variation of fragments containing reiterations. There were two predominant genotypes of F1 and F35, and the genomic characteristics of each were studied. The nucleotide change between F1 and F35 was estimated to be 1.5%. An RFLP marker (VR23) peculiar to genotype F35 was identified as the first case of genomic marker specific to a predominant genotype of HSV-1, and is the diagnostic marker of F35. The a sequences (repeating in an HSV-1 genome and containing reiterations) of F35 were cleaved by SacII on the DR4 (direct repeat 4) stretch, while a sequences of F1 had a rearranged DR4 and were resistant to SacII digestion. Thus, analyses of fragments containing reiterations, such as a sequences, can serve to classify HSV-1 strains as well as for purpose of differentiation. The proportion of strains derived from primary infection to those from recurrent infection was higher in strains of F35 than in those of F1, and this genotypic difference within HSV-1 may possibly influence clinical manifestations.

Animals↗

Genomic characterization of the Neurofibromatosis Type 1 gene of Fugu rubripes.

The genomic structure of the Neurofibromatosis Type1 (NF1) gene of Fugu rubripes was investigated by sequence analysis of two overlapping cosmids. The Fugu NF1 gene spans 27 kb and is 13 times smaller than the human counterpart owing primarily to reduced intron size. The predicted amino acid sequence is highly related to that of human neurofibromin, exhibiting an overall similarity of 91.5%. Nearly all exons described for the human NF1 gene could be identified, except exon 12b and the alternatively spliced exons 9br and 48a. With the exception of the splice acceptor site in front of exon 16, all splice sites are in identical positions to those found in the human gene. Intron 1, which is 100-140 kb long in humans, spans 2575 bp in the Fugu NF1 gene. Another large intron of the human NF1 gene, intron 27b (45-50 kb), is 3942 bp of size in Fugu. Sequences related to the OMgp gene (Oligodendrocyte-Myelin-glycoprotein) or the EVI2A gene (ecotropic viral integration site), which are inserted into human NF1 intron 27b, were not detected in the corresponding Fugu intron. However, a single exon gene with similarity to the human EVI2B gene has been found on the reverse strand of Fugu intron 27b. This suggests that the human EVI2B gene and the Fugu gene in intron 27b have a common ancestor. We found the expression of this inserted gene in liver and kidney, but not in brain tissue of Fugu rubripes.

Amino Acid Sequence↗

Near full-length genome characterization of an HIV type 1 CRF05_DF virus from Spain.

We report the near full-length sequence characterization of a HIV-1 DF intersubtype recombinant virus from Spain, X492, directly amplified from peripheral blood mononuclear cells' DNA. This isolate shares an identical mosaic structure and exhibits consistent phylogenetic clustering along the genome with VI961, a previously characterized DF recombinant virus. By contrast, VI1310, which may represent the same recombinant form as VI961 (CRF05_DF), is only partially homologous to VI961 and X492. Of three additional DF recombinant viruses previously characterized in gag-pol, only one, VI1267, clusters uniformly with VI961 and X492; the other two branch separately in a segment of pol. These results allow us to define an HIV-1 circulating recombinant form (CRF05_DF), characterized in near full-length genomes of two isolates (VI961 and X492) and in partial gag-pol sequences of a third virus (VI1267). Three other reported DF recombinant viruses, including the fully sequenced VI1310, exhibit incomplete homology to VI961 and X492.

Genome, Viral↗

Genomic characterization of a novel poxvirus contributing to the decline of the red squirrel (Sciurus vulgaris) in the UK.

The genome of a virulent squirrelpox virus (SQPV) isolate was characterized in order to determine its relationship with other poxviruses. Restriction enzyme analysis suggested a genome length of approximately 158 kb, whilst sequence analysis of the two ends of the genome indicated a G + C composition of approximately 66 %. Two contiguous stretches of 23 and 37 kb at the left-hand and right-hand ends of the genome, respectively, were sequenced allowing the identification of at least 59 genes contained therein. The partial sequence of a further 15 genes was determined by spot sequencing of restriction fragments located across the genome. Phylogenetic analysis of 15 genes conserved in all the recognized genera of the subfamily Chordopoxvirinae confirmed that the SQPV does not group within the family Parapoxvirinae, but instead partitions on its own in a separate clade of the poxviruses. Analysis of serum from British woodland rodents failed to find any evidence of SQPV infection in wood mice or bank voles, but for the first time serum samples from grey squirrels in the USA were found to contain antibody against SQPV.

Animals↗

Genome characterization and identification of viral-associated dsDNA component of banana bunchy top virus.

The single-stranded (ss) and double-stranded (ds) viral-associated DNAs (VADs) of banana bunchy top virus (BBTV) were characterized. The ssVADs were shown to be the viral genomic DNA and the dsVADs are the likely replication forms. Both dsVADs and ssVADs contain DNA species that cross-hybridize to each other. Under the electron microscope, both circular and linear DNAs were observed from these VAD species. Libraries were constructed from VADs to further characterize them. Two overlapping VAD clones were isolated and their combined sequence indicated that their corresponding VAD, named as BBTV component 2, is circular with a size of 1095 nt. The largest ORF spans from nucleotides 82 to 390, which is sandwiched by a putative TATA box, 54 nt upstream from the first ATG codon, and a AATAAA motif, 7 nt downstream from the UAA codon. Although the sequence of this VAD is different from the previously reported BBTV component 1 (Harding et al., J. Gen. Virol. 74, 323-328, 1993) and the genome of coconut foliar decay virus (Rohde et al., Virology 176, 648-651, 1990), stretches of highly homologous sequences were found between BBTV component 2 and the other two viral genomes by computer analysis. The sequence of BBTV component 1 is highly homologous to one of the clones, pBTs26, in the VAD library. Nevertheless, our libraries still contain many clones which hybridized with ss viral genomic DNAs but not with pBTs26 and BBTV component 2, indicating that the viral genome consists of more than two components.

Base Sequence↗

Genomic characterization of M and S RNA segments of hantaviruses isolated from bats.

Genomic analysis of three Hantaan-like virus isolates from bats was performed. Cleavage patterns of reverse transcription (RT)-polymerase chain reaction (PCR) products and nucleotide sequences of G2 region of M RNA segment and N protein region of S RNA segment of the isolates were compared to that of Hantaan 76-118 strain. Genomic characteristics of the bat isolates were identical to that of Hantaan virus.

Animals↗