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Results for “Complete genome characterization”

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PubMed2026

Genomic characterization of the attenuated human cytomegalovirus strain TR-VAC developed for subviral particle vaccine production.

We report the complete genome sequence of the attenuated human cytomegalovirus strain TR-VAC, developed for subviral particle vaccine production. Oxford Nanopore duplex sequencing confirmed all engineered modifications, including UL130 repair, UL25 stop codons, ddFKBP insertion, GFP deletion, and retention of the bacterial artificial chromosome backbone, without large-scale structural rearrangements.

Human cytomegalovirus↗

PubMed2002

Complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri.

Bacteriophage SfV is a temperate serotype-converting phage of Shigella flexneri. SfV encodes the factors involved in type V O-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. We now report on the complete sequence of the SfV genome (37,074 bp). A total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. The general organization of the genes in the SfV genome is similar to that of bacteriophage lambda, and numerous features of the sequence are described. The superinfection immunity system of SfV includes a lambda-like repression system and a P4-like transcription termination mechanism. Sequence analysis also suggests that SfV encodes multiple DNA methylases, and experiments confirmed that orf-41 encodes a Dam methylase. Studies conducted to determine if the phage-encoded methylase confers host DNA methylation showed that the two S. flexneri strains analyzed encode their own Dam methylase. Restriction mapping and sequence analysis revealed that the phage genome has cos sites at the termini. The tail assembly and structural genes of SfV show homology to those of phage Mu and Mu-like prophages in the genome of Escherichia coli O157:H7 and Haemophilus influenzae. Significant homology (30% of the genome in total) between sections of the early, regulatory, and structural regions of the SfV genome and the e14 and KpLE1 prophages in the E. coli K-12 genome were noted, suggesting that these three phages have common evolutionary origins.

Amino Acid Sequence↗

PubMed2004

Engineering the chloroplast encoded proteins of chlamydomonas.

Over a decade ago (1988), John Boynton and colleagues successfully transformed the chloroplast genome of chlamydomonas for the first time by complementation of a chloroplast deletion mutant. Since the first demonstration of chloroplast transformation the function and structure of many chloroplast encoded subunits of the photosynthetic apparatus has been characterized by site-directed mutagenesis. With the completion of the sequencing of the Chlamydomonas chloroplast genome the genetic tools are now in hand to characterize structure-function relationships for each of the chloroplast-encoded proteins of the photosynthetic apparatus.

Journal Article↗

PubMed2004

Functional characterization in Caenorhabditis elegans of transmembrane worm-human orthologs.

BACKGROUND: The complete genome sequences for human and the nematode Caenorhabditis elegans offer an opportunity to learn more about human gene function through functional characterization of orthologs in the worm. Based on a previous genome-wide analysis of worm-human orthologous transmembrane proteins, we selected seventeen genes to explore experimentally in C. elegans. These genes were selected on the basis that they all have high confidence candidate human orthologs and that their function is unknown. We first analyzed their phylogeny, membrane topology and domain organization. Then gene functions were studied experimentally in the worm by using RNA interference and transcriptional gfp reporter gene fusions. RESULTS: The experiments gave functional insights for twelve of the genes studied. For example, C36B1.12, the worm ortholog of three presenilin-like genes, was almost exclusively expressed in head neurons, suggesting an ancient conserved role important to neuronal function. We propose a new transmembrane topology for the presenilin-like protein family. sft-4, the worm ortholog of surfeit locus gene Surf-4, proved to be an essential gene required for development during the larval stages of the worm. R155.1, whose human ortholog is entirely uncharacterized, was implicated in body size control and other developmental processes. CONCLUSIONS: By combining bioinformatics and C. elegans experiments on orthologs, we provide functional insights on twelve previously uncharacterized human genes.

Animals↗

PubMed2004

HIV type 1 circulating recombinant form CRF09_cpx from west Africa combines subtypes A, F, G, and may share ancestors with CRF02_AG and Z321.

Two HIV-1 intersubtype recombinant forms are circulating widely in populations and have become important strains in the pandemic: CRF01_AE in Southeast Asia and CRF02_AG in West and West Central Africa, respectively. Several other circulating recombinant forms (CRF) have also been identified, but with fewer numbers of infections and/or more limited geographic spread. Here we expand knowledge of HIV-1 CRF using clinical samples, principally from West Africa, that were difficult to classify by partial genome sequencing. DNA was extracted from primary patient peripheral blood mononuclear cells (PBMC). The virtually complete HIV-1 genome was amplified by polymerase chain reaction (PCR) and directly sequenced. Additional strains were characterized by partial envelope sequencing. Phylogenetic analysis was used to identify and map intersubtype recombination breakpoints. Four virtually complete genome sequences and two partial envelope sequences represent CRF09_cpx, a newly identified complex recombinant HIV-1 whose principal focus seems to be in West Africa. This recombinant includes segments of subtypes A, F, G, and unclassified genetic material. It shares unique unclassified regions with the early Zaire strain Z321. There are similarities in structure, but considerable genetic distances, between CRF09_cpx and CRF02_AG IbNG. In conclusion, it is possible that this CRF shared common ancestors with both Z321 and CRF02_AG in the course of the pandemic, perhaps arising by recombination between earlier forms of these strains. Although newly identified, at least one infection with CRF09_cpx has already occurred outside of Africa.

Adult↗

PubMed1983

Detection of HSV-1 genome in central nervous system of latently infected mice.

Herpes simplex virus type 1 (HSV-1) can establish latent infections in peripheral nerve ganglia and the central nervous system (CNS) of experimentally infected mice. Latent infections of peripheral nervous tissue are characterized by the ability to recover infectious virus from explant cultures of most of the latently infected ganglia. In contrast, infectious virus is infrequently recovered from the CNS of latently infected mice following explant culture, although viral DNA can be detected in CNS tissue. The presence of incomplete or defective viral genomes during the latent CNS infections has been proposed to explain this difference between the two latent infections. To characterize the completeness and the physical state of the latent viral genome, we used Southern blot hybridization to analyse the viral DNA from latently infected mouse ganglia and brains. Our results, reported here, indicate that most, if not all, of the HSV-1 genome is present in latently infected CNS tissue and that the latent viral genome exists in a form other than linear, unit length DNA.

Animals↗

PubMed2001

Genomic and functional characterization of polyunsaturated fatty acid biosynthesis in Caenorhabditis elegans.

The biosynthetic pathway for polyunsaturated fatty acids in the model animal Caenorhabditis elegans was examined in the context of the completed genome sequence. The genomic organization and location of seven desaturase genes and one elongase activity, all previously identified by functional characterization, were elucidated. A pathway for the biosynthesis of polyunsaturated fatty acids in C. elegans was proposed based on these genes. The role of gene duplication in enzyme evolution and proliferation is discussed.

Animals↗

PubMed1989

Characterization of the complete human elastin gene. Delineation of unusual features in the 5'-flanking region.

Genomic clones encompassing the entire human elastin gene, including 11 kilobases flanking the ATG translation initiation codon, have been obtained and characterized by restriction enzyme analysis and extensive DNA sequencing. These analyses demonstrated that functionally distinct hydrophobic and cross-linking domains of the protein are segregated into separate exons throughout the gene. All exons are multiples of three nucleotides, and exon:intron borders always split codons in the same way which permits cassette-like alternative splicing. The 5'-flanking region lacks a canonical TATA sequence, is G + C-rich, and contains several SP1 binding sites and an AP2 binding site. Primer extension and S1 nuclease protection experiments indicate that transcription is initiated at multiple sites in the gene.

Amino Acid Sequence↗

PubMed2005

Identification of a novel HIV-1 circulating ADG intersubtype recombinant form (CRF19_cpx) in Cuba.

Circulating recombinant forms (CRFs) represent a substantial proportion of HIV-1 isolates in the global pandemic. Characterization of HIV-1 genetic forms, including CRFs, may be relevant to studies on molecular epidemiology, recombination, superinfection, vaccine development, and antiretroviral therapy. This study analyzes near complete genomes of 4 epidemiologically unlinked viruses from Cuba, originally characterized as D/A intersubtype recombinants in pol and env segments. The genomes of 3 viruses exhibited virtually coincident mosaic structures, with multiple segments of subtypes A, D, and G and uniform phylogenetic clustering with each other along the genome. These results allow us to define a new CRF (CRF19_cpx). The 4th analyzed Cuban virus was recombinant between CRF19_cpx and CRF18_cpx (which also circulates in Cuba). CRF19_cpx exhibited homology to an AG intersubtype recombinant virus from Cameroon (CM53392) along approximately 5 kb and clustered with a subtype D virus from Gabon (G109) in gag. Four other viruses from central or west Africa were also phylogenetically related to CRF19_cpx in env fragments. These results allow us to define CRF19_cpx as a second novel CRF of African origin circulating in Cuba, to identify putative representative viruses of its parental strains, and to characterize a unique CRF18/CRF19 recombinant virus.

Adult↗

PubMed2025

The Complete Chloroplast Genome and the Phylogenetic Analysis of Panicum bisulcatum (Thumb.) (Poaceae).

The chloroplast (cp) genome of Panicum bisulcatum (Thumb.), a significant agricultural weed, was sequenced and characterized to elucidate its genomic architecture, evolutionary dynamics, and phylogenetic relationships. The complete cp genome was assembled as a circular DNA molecule of 138,489 bp, exhibiting a typical quadripartite structure comprising a large single-copy (LSC, 82,260 bp), a small single-copy (SSC, 12,569 bp), and a pair of inverted repeats (IR, 21,830 bp each) regions. It encodes 135 genes, including 89 protein-coding genes, 49 tRNAs, and 8 rRNAs. Functional annotation revealed that most genes are involved in photosynthesis and genetic system. A total of 51 simple sequence repeats (SSRs) and 62 long repeats (LRs) were identified, providing potential molecular markers. Comparative analysis of IR boundaries highlighted both conserved features and species-specific expansion/contraction events among Panicum species. Phylogenomic analysis robustly placed P. bisulcatum within the genus Panicum, showing a closest relationship with P. incomtum and confirming the monophyly of the genus. Furthermore, single nucleotide polymorphism (SNP) analysis with its closest relative, P. incomtum, revealed 4659 SNPs, with a dominance of synonymous substitutions, indicating the action of purifying selection. This study provides the first comprehensive cp genomic resource for P. bisulcatum, which will facilitate future studies in species identification, phylogenetic reconstruction, population genetics, and the development of sustainable management strategies for this weed.

Phylogeny↗

PubMed2004

The complete nucleotide sequence, genome organization, and specific detection of Beet mosaic virus.

Beet mosaic virus (BtMV) was identified almost five decades ago but has not been fully characterized at the molecular level. In this study, we have determined for the first time the complete nucleotide sequence of BtMV genomic RNA and have developed a specific molecular means for its diagnosis. The viral genome of BtMV comprises 9591 nucleotides, excluding the 3' terminal poly (A) sequence, and contains a single open reading frame (ORF) that begins at nt 166 and terminates at nt 9423, encoding a single polyprotein of 3086 amino acid residues. A 3' untranslated region of 168 nucleotides follows the ORF. The deduced genome organization is typical for a member of the family Potyviridae and includes 10 proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and coat protein (CP). Nine putative protease cleavage sites were predicted computationally and by analogy with genome arrangements of other potyviruses. Conserved sequence motifs of homologous proteins of other potyviruses were found in corresponding positions of BtMV. BtMV is a distinct species of the genus Potyvirus with the most closely related species being Peanut mottle virus ( approximately 55% amino acid identity). Based on the nucleotide sequence obtained, we have developed a virus-specific RT-PCR assay for accurate diagnosis and differentiation of BtMV.

Amino Acid Sequence↗

PubMed1984

Characterization of the DNA of the hamster papovavirus: I. Genom length and molecular cloning.

The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of the Syrian hamsters was measured by electron microscopy and cloned in Escherichia coli using the certified plasmid vector pBR322. The cloned viral DNA were characterized by digestion of the recombinant DNA with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNA and by electron microscopy to determine the genome size of cloned HaPV DNA. The restriction enzyme analysis of the cloned HaPV DNA showed the same cleavage pattern as the corresponding fragments from the uncloned DNA. No major insertions or deletions could be detected by heteroduplex analysis between cloned HaPV DNA and the starting material. The estimated genome size of 5.52 kb for HaPV DNA is approx. 300 bases larger than those determined for other known papovaviruses as SV40 or polyoma.

Animals↗

PubMedDate not supplied

Oligonucleotide fingerprinting with (CAC)5: nonradioactive in-gel hybridization and isolation of individual hypervariable loci.

The first topic to be treated in this paper is the nonradioactive DNA fingerprinting by means of in-gel hybridization with digoxigenated (CAC)5. Besides the fact that time-consuming Southern blotting can be avoided, the dried agarose is an excellent matrix to produce background-free nonradioactive DNA fingerprints. There is no tendency of either the oligonucleotide probe or the antibody towards unspecific binding to the dried agarose. Prehybridization and blocking steps are therefore superfluous. Furthermore, we will discuss what effect the degree of crosslinking of the antibody-enzyme conjugates has. The second topic concerns the isolation and characterization of locus-specific probes from a human (CAC)5 fingerprint. The isolation and characterization of one variable probe, by screening complete genomic libraries, is described and discussed. This probe is compared to a hypervariable single-copy probe, isolated from a size-enriched genomic library. The sequence of the repeat flanking locus-specific probe is presented and a semi-specific, adaptor-mediated polymerase chain reaction was designed to amplify (CAC)n/(GTG)n flanking sequences.

Base Sequence↗

PubMed1993

Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole in Toxoplasma gondii.

The monoclonal antibody (mAb) TG17.179 recognizes an excreted-secreted antigen (ESA) of 28.5 kDa named Gra 2, which is stored in the dense granules of Toxoplasma cells and secreted into the parasitophorous vacuole after host cell invasion. Screening of an expression cDNA library with TG17.179 led to the isolation of several clones, the longest one (clone L) being of 1030 bp. Clone L cDNA was found to be homologous to a previously described composite cDNA encoding a P28 protein of Toxoplasma gondii. Characterization of one genomic clone indicates that the complete GRA 2 gene is about 1.3 kb in length, including an intron of 241 bp. Northern blot and primer extension analyses confirmed the size of the mature messenger (1.1 kb). Amino acid partial sequencing of the native antigen purified by HPLC and metabolic radiolabelings of ESAs perfectly matched the primary amino acid structure deduced from the clone L cDNA. This primary translation product consists of an 185 amino acid polypeptide (19.8 kDa) including a 23 amino acid signal sequence. The presence of many serine and threonine residues may indicate an O-glycosylation. The predicted mature polypeptide shows an internal helical domain with 2 amphipathic alpha-helices. These might be involved in the association of Gra 2 with the membranous network within the parasitophorous vacuole.

Amino Acid Sequence↗

PubMedDate not supplied

Identification of non-autonomous non-LTR retrotransposons in the genome of Trypanosoma cruzi.

As observed for most eukaryotic cells, trypanosomatids contains non-LTR retrotransposons randomly inserted in the nuclear genome. Autonomous retroelements which, code for their own transposition, have been characterized in Trypanosoma brucei (ingi) and Trypanosoma cruzi (L1Tc), whereas non-autonomous retroelements have only been characterized in T. brucei (RIME). Here, we have characterized in the genome of Trypanosoma cruzi four complete copies of a non-autonomous non-LTR retrotransposon, called NARTc. This 0.26 kb NARTc element has the characteristics of non-LTR retrotransposons: the presence a poly(dA) tail and of a short flanking duplicated motif. Analysis of the Genome Survey Sequence databases indicated that the Trypanosoma cruzi haploid genome contains about 140 NARTc copies and about twice as many L1Tc copies. Interestingly, the NARTc and L1Tc retroelements share, with the Trypanosoma brucei ingi and RIME retrotransposons, a common sequence (the first 45 bp with 91% identity), whereas the remaining sequences are very divergent. This suggests that these four trypanosome non-LTR retrotransposons were derived from the same common ancester and the sequence of their 5'-extremity may have a functional role. In addition, the genome of Leishmania major contains the same conserved motif present in the trypanosome retroelements, whicle no transposable elements have been detected so far in Leishmania sp.

Animals↗

PubMed2001

Informatics and multiplexing of intact protein identification in bacteria and the archaea.

Although direct fragmentation of protein ions in a mass spectrometer is far more efficient than exhaustive mapping of 1-3 kDa peptides for complete characterization of primary structures predicted from sequenced genomes, the development of this approach is still in its infancy. Here we describe a statistical model (good to within approximately 5%) that shows that the database search specificity of this method requires only three of four fragment ions to match (at +/-0.1 Da) for a 99.8% probability of being correct in a database of 5,000 protein forms. Software developed for automated processing of protein ion fragmentation data and for probability-based retrieval of whole proteins is illustrated by identification of 18 archaeal and bacterial proteins with simultaneous mass-spectrometric (MS) mapping of their entire primary structures. Dissociation of two or three proteins at once for such identifications in parallel is also demonstrated, along with retention and exact localization of a phosphorylated serine residue through the fragmentation process. These conceptual and technical advances should assist future processing of whole proteins in a higher throughput format for more robust detection of co- and post-translational modifications.

Algorithms↗

PubMed1989

A single DNA response element can confer inducibility by both alpha- and gamma-interferons.

Genomic and cDNA clones corresponding to 9-27, a member of the human 1-8 gene family highly inducible by alpha- and gamma-interferons (IFNs), have been isolated and characterized. A 1.7-kilobase genomic clone contains a complete functional gene with two exons, encoding a 125-amino acid polypeptide of unknown function. The 5' flanking region of the gene contains a 13-base-pair IFN-stimulable response element (ISRE), homologous to the ISREs of the 6-16, ISG 15, and ISG 54 genes, which are predominantly inducible by IFN-alpha, beta. Analysis of constructs containing native and mutated ISREs suggests that this motif is essential for the response of 9-27 to IFN-gamma as well as IFN-alpha. Furthermore, the 9-27 (GGAAATAGAAACT) and 6-16 (GGGAAAATGAAACT) ISREs can each confer a response to both types of IFN when placed on the 5' side of a marker gene. Since the 6-16 gene does not normally respond to IFN-gamma, the context of the ISRE must determine the specificity of the response.

Amino Acid Sequence↗

PubMed2002

Systematic G-protein-coupled receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles.

Leucokinins are insect neuropeptides that stimulate hindgut motility and renal fluid secretion. Drosophila has a single leucokinin gene, pp, encoding the longest known leucokinin, Drosokinin. To identify its receptor, a genome-wide scan for G-protein-coupled receptors was performed in silico and candidate receptors identified by similarity to known tachykinin receptors. The deduced peptides were expressed, with a transgene for the calcium reporter aequorin, in S2 cells and only one gene (CG10626) encoded a protein that responded to Drosokinin. The properties of the heterologously expressed receptor (action through intracellular calcium with an EC(50) of 4 x 10(-11) m and a t(1/2) <1 s) match closely those reported for the action of Drosokinin on Malpighian (renal) tubules. Antibodies raised against the receptor identified known sites of leucokinin action: stellate cells of the Malpighian tubule, two triplets of cells in the pars intercerebralis of the adult central nervous system, and additional cells in larval central nervous system. Western blots and reverse transcription-PCR confirmed these locations, but also identified expression in male and female gonads. These tissues also displayed elevated calcium in response to Drosokinin, demonstrating novel roles for leucokinin. A functional genomic approach has thus yielded the first complete characterization of a leucokinin receptor in an insect.

Amino Acid Sequence↗