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Genome analysis of South American Elymus (Triticeae) and Leymus (Triticeae) species based on variation in repeated nucleotide sequences.

Variation in repeated nucleotide sequences (RNSs) at the level of entire families assayed by Southern blot hybridization is remarkably low within species and is a powerful tool for scrutinizing the origin of allopolyploid taxa. Thirty-one clones from RNSs isolated from different Triticeae genera were used to investigate the genome constitution of South American Elymus. One of these clones, pHch2, preferentially hybridized with the diploid H genome Hordeum species. Hybridization of this clone with a worldwide collection of Elymus species with known genome formulas showed that pHch2 clearly discriminates Elymus species with the H genome (StH, StHH, StStH, and StHY) from those with other genome combinations (StY, StStY, StPY, and StP). Hybridization with pHch2 indicates the presence of the H genome in all South American Elymus species except Elymus erianthus and Elymus mendocinus. Hybridization with additional clones that revealed differential restriction fragments (marker bands) for the H genome confirmed the absence of the H genome in these species. Differential restriction fragments for the Ns genome of Psathyrostachys were detected in E. erianthus and E. mendocinus and three species of Leymus. Based on genome constitution, morphology, and habitat, E. erianthus and E. mendocinus were transferred to the genus Leymus.

DNA, Plant↗

Full-length HIV type 1 genome analysis showing evidence for HIV type 1 transmission from a nonprogressor to two recipients who progressed to AIDS.

Epidemiologically-linked HIV-1 transmission cohorts serve as excellent models to study HIV disease progression. The actual relationship between viral variability and HIV disease outcome can be extrapolated only through such rare epidemiologically linked HIV-1-infected cohorts. We present here a cohort of three patients with the source termed donor A (a nonprogressor) and two recipients B and C. Both recipients acquired HIV through blood transfusion from donor A and have progressed to AIDS. By analyzing 15 near full-length HIV- 1 genomes (8.7 kb each genome) from longitudinally collected peripheral blood cell samples (four time points for patient A, four for patient B, and seven from patient C), we were able to demonstrate transmission of HIV from donor A and epidemiologic linkage among members A, B, and C after 10 years of HIV infection. These analyses are novel in demonstrating that HIV-1-infected nonprogressing individuals bear the potential to transmit HIV-1 variants and that HIV variants, which led to a benign disease in a nonprogressor donor, were able to cause disease in other individuals. Overall, these studies highlight the utility of full genome sequencing in establishing epidemiologic linkage in a chronically infected HIV cohort after 10 years of initial infection.

Amino Acid Sequence↗

Whole-genome analysis to identify type III-secreted effectors.

Many Gram-negative plant and animal pathogens share a common virulence strategy that relies on the specialized type III secretion system. This apparatus is used to secrete virulence factors, called effectors, into the extracellular host environment and directly into the cytoplasm of host cells. Effectors interfere with host signaling and host metabolism to create an optimal environment for pathogen replication. The identification of effectors in plant pathogens was limited for many years to those effectors that elicit strong plant defenses on some hosts. The members of this subset, called avirulence proteins, can be readily identified because they dominantly confer strong defense-inducing properties to a heterologous virulent strain. This chapter describes two methods to identify type III-secreted effectors in plant pathogens independently of their phenotype. The first method consists of an in vivo molecular genetic screen that uses the activity of an avirulence protein to identify effectors without avirulence activity. It should be possible to apply this method to most Gram-negative plant pathogens. The second method consists of a bioinformatic approach applicable to those pathogens for which at least a draft genome sequence is available.

Arabidopsis↗

Genomic analysis in lymphoid leukemias.

The introduction of microarray analysis represents a revolution in the scientific field, allowing to investigate thousand of genes in a single experiment. Important biological insights have been revealed by this technique in several tumors: in acute lymphoblastic leukemia (ALL), these studies have allowed to identify specific patterns associated with known molecular abnormalities, as well as with phenotypic characteristics and with different prognostic features. In chronic lymphocytic leukemia (CLL), this approach has helped to dissect that this disease is a single entity with distinct variants that are characterized by a diverse IgVH mutational status, that can be discriminated by a small set of genes, has allowed to define a similarity between this disease and memory B cells and has also led to hypothesize that CLL cells from IgVH unmutated patients may be continuously stimulated in vivo, thus showing a gene profile that is reminiscent of the B cell receptor. In multiple myeloma (MM), gene expression profiles has provided insights into the disease and has offered the opportunity of stratifying patients according to the degree of aggressiveness of the disease. Current efforts are directed to the identification of patterns that may distinguish patients with a different outcome, thus providing useful prognostic information and to design experiments that may allow the identification of new therapeutic targets.

Gene Expression Profiling↗

Long and short forms of the ovine prolactin receptor: cDNA cloning and genomic analysis reveal that the two forms arise by different alternative splicing mechanisms in ruminants and in rodents.

Prolactin is a pituitary hormone that binds to specific receptors in numerous tissues. Depending on the size of their cytoplasmic domain, long and short prolactin receptors (l-PR, s-PR) have been described. Up to now, s-PR were found in rodents only. We report here the cloning of full-length coding sequences for short and long ovine prolactin receptors (s-oPR, l-oPR). The only difference between s- and l-oPR coding sequences was, respectively, the presence or absence of a 39 base pair insert at the beginning of the cytoplasmic domain, with two contiguous inframe stop codons at its 3' end. Sequence comparison revealed that the alternative splicing producing s- and l-oPR was different from that of rodents, although the resulting proteins were very similar. PCR experiments on ovine genomic DNA showed that the 39 base pair insert was directly linked to the downstream exon, and separated from the upstream exon by an 800 base pair intron. Thus, the alternative splicing used a single intron with one 5' and two 3' sites. The same organization was found in bovine and caprine genomes, suggesting that this feature is general in ruminants and different from rodents, which use mutually exclusive exons to produce s-PR and l-PR.

Alternative Splicing↗

Genomic analysis of the H-2 complex region associated with mouse t haplotypes.

Naturally occurring t haplotypes suppress recombination over a region of mouse chromosome 17 that includes the H-2 complex. Each of these t haplotypes is associated with a specific set of H-2 alleles and can be placed into one of a limited number of complementation groups. Genetic studies have demonstrated the existence of a basic homology in genomic organization among all t haplotypes. We used an H-2 cDNA probe to investigate, at the molecular level, possible relationships among the H-2 regions of different t haplotypes. We identified a family of t haplotype-specific restriction fragments that carry DNA sequences homologous to the H-2-like genes. Surprisingly, the H-2-defined restriction patterns from all five complete t haplo-types analyzed are highly homologous, even though H-2 gene products expressed are antigenically distinct. These data lead to two major conclusions. First, all t haplotypes were derived from a small number of closely related ancestors. Second, the H-2 complex region associated with each primordial t chromosome has been maintained within at least the five present-day t haplotypes analyzed here. Hence the H-2 complex is an integral component of naturally occurring t haplotypes.

Animals↗

An outbreak of the polyomavirus infection in budgerigars and cockatiels in Slovakia, including a genome analysis of an avian polyomavirus isolate.

In winter 2003-04, large numbers of budgerigars (Mellopsitacus undulatus) and cockatiels (Nymphicus hollandicus) fell ill and died in a large parrot-breeding aviary in Slovakia. In budgerigars, the disease outbreak occurred at the age of 2-3 weeks; cockatiels died within their first 7 days of life. In budgerigars, symptoms of the disease included delayed growth, tremor, darkish discoloration of skin, quill bleeding, and feathering defects. cockatiels often died without any symptoms and with a full crop; feathering defects occurred sporadically. Electron microscopy with negative staining of aqueous lysates of the affected skin and of bleeding quills showed isolated or clustered polyomavirus particles 45-50 nm in size. Long filamentous forms of the virus were also found in virion clusters of skin lysates from the budgerigars. In ultrathin sections through the pathologically altered skin tissue of budgerigars, virus particles were present in both nuclei and cytoplasm of epidermal cells, often in crystalline form. In infected cells, enlarged nuclei showed an extensive chromatin margination. On the DNA level, presence of a polyomavirus infection was conclusively proved by the polymerase chain reaction using avian polyomavirus (APV)-specific primers. A sequence analysis of the gene encoding viral protein (VP)1 and of the combined region for VP2 and VP3 proteins revealed a previously undescribed synonymous mutation in this isolate. This report extended the knowledge of the area of APV occurrence and of the spectrum of hosts in the context of genomic and morphologic variability of APV isolates.

Animals↗

Human genome analysis system.

An automated DNA sequencing system, the collaborative effort of Japanese scientists and engineers, which has a potential output of up to 108,000 bases per day is described.

Base Sequence↗

Beta-sarcoglycan: genomic analysis and identification of a novel missense mutation in the LGMD2E Amish isolate.

The sarcoglycan complex is involved in the etiology of four autosomal recessive limb-girdle muscular dystrophies (LGMD2C-F). A missense mutation (T151R) in the beta-sarcoglycan gene on chromosome 4q12 has been shown to cause a mild form of LGMD2E in 11 families from a Southern Indiana Amish community sharing a common haplotype. We now report that two sibs from another Amish family with mild LGMD2E are compound heterozygotes for chromosome 4q12 markers. In order to characterize the genetic defect in this new family, we determined the genomic organization of the beta-sarcoglycan gene. A second missense mutation (R91C) has now been identified in this LGMD2E Amish family. This mutation is also present in the homozygous state in another family of probable Amish ancestry. Finally, analysis of all the components of the dystrophin-glycoprotein complex was performed for the first time on a biopsy from a patient homozygous for the beta-sarcoglycan mutation (T151R). Interestingly, in addition to the loss of the entire sarcoglycan complex, we detected a reduction of alpha-dystroglycan which suggests a role for the sarcoglycan complex in stabilizing alpha-dystroglycan at the sarcolemma.

Adolescent↗

Comparative genomic analysis of the sigB operon in Listeria monocytogenes and in other Gram-positive bacteria.

The stress-responsive, alternative sigma factor sigmaB has been described in members of three Gram-positive genera, Bacillus, Listeria, and Staphylococcus. In these bacteria, sigmaB appears to play an important role in facilitating rapid adaptation to and survival in stressful environments. sigmaB activity is regulated through a complex system of phosphatases and kinases encoded by rsb (regulator of sigma B) genes. We describe the sigB operon structure for the facultative intracellular pathogen Listeria monocytogenes and apply this sequence as well as other previously described sigB operon sequences to probe the evolution and functional conservation of the sigmaB stress response system among different Gram-positive bacteria. While sigmaB as well as two Rsbs (RsbS and RsbT) are highly conserved (73%, 84%, and 79% average amino acid [aa] identities, respectively), the predicted aa sequences of the other Rsb proteins showed less conservation (62-71% aa identities). Furthermore, the sigB operon structure varies among bacterial species. Bacterial species differ in the numbers and identities of rsb genes encoded in their genomes. We thus conclude that the sigmaB stress-response system as represented by the sigB operon has diverged in both its overall components as well as in the sequences of its individual proteins, even among closely related bacterial species. Differential evolution of this stress response system among various genera may represent a strategy that enables bacteria to adapt cellular response and survival systems to a variety of stress conditions.

Amino Acid Sequence↗

Genome analysis: Assigning protein coding regions to three-dimensional structures.

We describe the results of a procedure for maximizing the number of sequences that can be reliably linked to a protein of known three-dimensional structure. Unlike other methods, which try to increase sensitivity through the use of fold recognition software, we only use conventional sequence alignment tools, but apply them in a manner that significantly increases the number of relationships detected. We analyzed 11 genomes and found that, depending on the genome, between 23 and 32% of the ORFs had significant matches to proteins of known structure. In all cases, the aligned region consisted of either >100 residues or >50% of the smaller sequence. Slightly higher percentages could be attained if smaller motifs were also included. This is significantly higher than most previously reported methods, even those that have a fold-recognition component. We survey the biochemical and structural characteristics of the most frequently occurring proteins, and discuss the extent to which alignment methods can realistically assign function to gene products.

Algorithms↗

Avian polymavirus in wild birds: genome analysis of isolates from Falconiformes and Psittaciformes.

Avian polyomavirus (APV) infections have been reported to cause fatal disease in a wide range of psittacine species. Here we demonstrate APV infections in buzzards (Buteo buteo) and in a falcon (Falco tinnunculus) found dead in Germany, and in lovebirds (Agapornis pullaria) with fatal disease, wild-caught in Moçambique. APV infection in buzzards was determined by PCR amplification of parts of the viral genome followed by Southern blot hybridisation. The genomes of the isolates obtained from the falcon and one of the lovebirds proved to be very closely related to those of Budgerigar Fledgling Disease Virus (BFDV)-1, BFDV-2 and BFDV-3, isolated from budgerigar, chicken, and parakeet, respectively. A consensus sequence was delineated from the known nucleotide sequences of APV isolates. The significance of some nucleotide changes is discussed. Infectivity of all of these isolates was neutralized by antibodies directed against BFDV-1. Data presented in this investigation show that the polyomavirus isolates obtained from different avian species so far all belong to one genotype and one serotype within the proposed subgenus Avipolyomavirus of the family Papovaviridae. The designation Budgerigar Fledgling Disease Virus (BFDV) is, therefore, misleading as this virus type infects different species of birds. The name Avian Polymavirus and the abreviation APV should be adopted to all of the isolates investigated in detail at present. The possible role of birds of passage in the epidemiology in APV infections is discussed.

Animals↗

Genomic analysis of the 67-kDa laminin receptor in normal and pathological tissues: circumstantial evidence for retroposon features.

We have cloned two cDNAs for the human 67-kDa laminin receptor (LR). In the present report we show that these clones hybridize to many restriction fragments in Southern experiments in human. This particular pattern is accounted for by the presence of up to 16 and 21 copies of the laminin receptor gene per haploid genome in human and mouse, respectively. In contrast, a single gene copy is found in chicken. Chromosomal localization reveals four main loci: LAMRP1, laminin receptor pseudogene 1 (Chr 3); LAMRP2, laminin receptor pseudogene 2 (Chr 12); LAMRP3, laminin receptor pseudogene 3 (Chr 14); LAMRP4, laminin receptor pseudogene 4 (Chr X). Comparison of our experimental results to the known features of processed retropseudogenes enabled us to conclude that the LR gene belongs to a retroposon family in mammals.

Animals↗

Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion.

Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year. However, standard export technologies, based on the well-characterised general secretory (Sec) pathway, are frequently inapplicable for the production of proteins. The recently discovered twin-arginine translocation (Tat) pathway offers additional potential to transport proteins. Here we review the use of functional genomic and proteomic approaches to explore the Tat pathway of Bacillus subtilis. The properties of Tat pathway components and the twin-arginine signal peptides that direct proteins into this pathway are discussed. Where appropriate, a comparison is made with Tat systems from other organism, such as Escherichia coli. Recent findings with the latter organism in particular provide proof-of-principle that the Tat pathway can be exploited for the production of Sec-incompatible proteins.

Amino Acid Sequence↗

Ancient expansion of the ribonuclease A superfamily revealed by genomic analysis of placental and marsupial mammals.

Members of the ribonuclease (RNase) A superfamily participate in a diverse array of biological processes, including digestion, angiogenesis, innate immunity, and possibly male reproduction. The superfamily is vertebrate-specific, with 13-20 highly divergent members in primates and rodents, but only a few members in chicken and fish. This has led to the proposal that the superfamily started off from a progenitor with structural similarities to angiogenin and that the superfamily underwent a dramatic expansion during mammalian evolution. To date this evolutionary expansion and understand the functional diversification of the superfamily, we here determine its entire repertoire in the sequenced genomes of dog, cow, and opossum. We identified 7, 20, and 21 putatively functional RNase genes from these three species, respectively. Many of the identified genes are highly divergent from all previously known RNase genes, thus representing new lineages within the superfamily. Phylogenetic analysis indicates that the superfamily expansion predated the separation of placental and marsupial mammals and that differential gene loss and duplication occurred in different species, generating a great variation in gene number and content among extant mammals.

Amino Acid Sequence↗

Whole-genome analysis of Brevibacterium sanguinis AZMABM HM27: a bacterial isolate from the sea anemone Radianthus magnifica and exhibiting promising multi-therapeutic properties.

BACKGROUND: The marine anemone Radianthus magnifica harbors symbiotic microbes with promising biomedical potential, yet their diversity and therapeutic properties remain underexplored. This study aimed to characterize a symbiotic bacterium isolated from R. magnifica collected from Samalona Island, Indonesia, and to evaluate its multi-therapeutic potential. METHODS: Strain AZMABM HM27 was characterized using whole-genome sequencing, functional annotation, biosynthetic gene cluster prediction, molecular docking, and in vitro bioactivity assays. RESULTS: Phylogenetic and genome-based analyses confirmed AZMABM HM27 as Brevibacterium sanguinis, with an OrthoANI value of 97.37% and a dDDH value of 76.50% against the type strain. The genome comprises a 3,834,082 bp chromosome encoding 3,362 protein-coding genes, including 95 genes involved in secondary metabolite biosynthesis. Five biosynthetic gene clusters were predicted, including those associated with ectoine, terpene, and siderophore production. The crude extract demonstrated antioxidant activity (IC₅₀ = 0.87 mg/mL), anti-inflammatory activity (up to 60% inhibition), antidiabetic activity through α-glucosidase inhibition (up to 40% inhibition), and dose-dependent antiproliferative activity against MCF-7 breast cancer cells (74.10% viability at 1 mg/mL). Molecular docking identified a lead compound, 8,9,9,10,10,11-hexafluoro-4,4-dimethyl-3,5-dioxatetracyclo [5.4.1.0(2,6)0.0(8,11)] dodecane, with strong binding affinities to selected therapeutic targets. CONCLUSIONS: B. sanguinis AZMABM HM27 represents a marine symbiotic strain associated with R. magnifica and a promising source of bioactive compounds with antioxidant, anti-inflammatory, antidiabetic, and antiproliferative potential. Further purification, structural elucidation, and in vivo studies are warranted to validate its therapeutic potential.

Animals↗

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis.

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.

Antigens, Bacterial↗

I-CeuI recognition sites in the rrn operons of the Bacillus subtilis 168 chromosome: inherent landmarks for genome analysis.

The Bacillus subtilis 168 circular chromosome yielded ten fragments on I-CeuI endonuclease digestion. I-CeuI recognizes a 26 bp sequence that is located within the gene encoding the 23S subunit of the rRNA in Chlamydomonas eugametos, Escherichia coli and Salmonella typhimurium. The precise locations of the I-CeuI sites of the B. subtilis chromosome were determined on a NotI-SfiI physical map by (i) double digestion analyses with I-CeuI and SfiI, (ii) comparison of mutant strains lacking a specific rrn operon, (iii) using an I-CeuI linking clone and (iv) analysis of nucleotide sequence data of some rrn operons. In conclusion, all the I-CeuI sites were located within the B. subtilis rrn operons and the I-CeuI sites were conserved in all the B. subtilis 168 derivatives tested. Thus, variations in size of the I-CeuI fragments must be due to genome alterations. A B. subtilis 168 strain was investigated with I-CeuI. We demonstrated that the aberrant structure was the outcome of the inversion of an approximately 1700 kb DNA segment.

Bacillus subtilis↗