Clusters Of Open Reading Frames Research Articles

Hidden treasure in the microbiome

Bioinformatics We know that the human microbiome contains a wealth of largely unknown genetic diversity. Whole metagenome shotgun sequencing is needed to make the links between genes and phenotypes. Sberro et al. have computationally analyzed thousands of small protein-coding genes in the Human Microbiome Project data. More than 400,000 clusters of open reading frames (ORFs) of less than 150 nucleotides were constructed de novo. Within the approximately 4000 small ORFs likely to be protein-coding, the majority have no homology with known protein domains, and some are evolutionarily conserved, indicating that they have essential functions. Some peptides are transmembrane and secreted, which hints that they are used in signaling between bacteria and host. Others are implicated in bacterial immunity and horizontal gene transfer. Thus, this effort provides a rich resource for understanding the body's relationship with its microbiota. Cell 178 , 1245 (2019).

Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection.

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

Multiple products from microRNA transcripts

A single transcript sometimes codes for more than one product. In bacteria, and in a few exceptional animal lineages, many genes are organized into operons: clusters of open reading frames that are transcribed together in a single polycistronic transcript. However, polycistronic transcripts are rare in eukaryotes. One notable exception is that of miRNAs (microRNAs), small RNAs that regulate gene expression at the post-transcriptional level. The primary transcripts of miRNAs commonly produce more than one functional product, by at least three different mechanisms. miRNAs are often produced from polycistronic transcripts together with other miRNA precursors. Also, miRNAs frequently derive from protein-coding gene introns. Finally, each miRNA precursor can produce two mature miRNA products. We argue, in the present review, that miRNAs are frequently hosted in transcripts coding for multiple products because new miRNA precursor sequences that arise by chance in transcribed regions are more likely to become functional miRNAs during evolution.

Crucial Role for Insertion Sequence Elements in Lactobacillus helveticus Evolution as Revealed by Interstrain Genomic Comparison

Lactobacillus helveticus is a versatile dairy bacterium found to possess heterogeneous genotypes depending on the ecosystem from which it was isolated. The recently published genome sequence showed the remarkable flexibility of its structure, demonstrated by a substantial level of insertion sequence (IS) element expansion in association with massive gene decay. To assess this diversity and examine the level of genome plasticity within the L. helveticus species, an array-based comparative genome hybridization (aCGH) experiment was designed in which 10 strains were analyzed. The aCGH experiment revealed 16 clusters of open reading frames (ORFs) flanked by IS elements. Four of these ORFs are associated with restriction/modification which may have played a role in accelerated evolution of strains in a commercially intensive ecosystem undoubtedly challenged through successive phage attack. Furthermore, analysis of the IS-flanked clusters demonstrated that the most frequently encountered ISs were also those most abundant in the genome (IS1201, ISL2, ISLhe1, ISLhe2, ISLhe65, and ISLhe63). These findings contribute to the overall viewpoint of the versatile character of IS elements and the role they may play in bacterial genome plasticity.

Contribution of mobile genetic elements to Desulfovibrio vulgaris genome plasticity

The genome of Desulfovibrio vulgaris strain DePue, a sulfate-reducing Deltaproteobacterium isolated from heavy metal-impacted lake sediment, was completely sequenced and compared with the type strain D. vulgaris Hildenborough. The two genomes share a high degree of relatedness and synteny, but harbour distinct prophage and signatures of past phage encounters. In addition to a highly variable phage contribution, the genome of strain DePue contains a cluster of open-reading frames not found in strain Hildenborough coding for the production and export of a capsule exopolysaccharide, possibly of relevance to heavy metal resistance. Comparative whole-genome microarray analysis on four additional D. vulgaris strains established greater interstrain variation within regions associated with phage insertion and exopolysaccharide biosynthesis.

In Vivo Gene Expression Analysis Identifies Genes Required for Enhanced Colonization of the Mouse Urinary Tract by Uropathogenic Escherichia coli Strain CFT073 dsdA

Deletional inactivation of the gene encoding d-serine deaminase, dsdA, in uropathogenic Escherichia coli strain CFT073 results in a hypermotile strain with a hypercolonization phenotype in the bladder and kidneys of mice in a model of urinary tract infection (UTI). The in vivo gene expression profiles of CFT073 and CFT073 dsdA were compared by isolating RNA directly from the urine of mice challenged with each strain individually. Hybridization of cDNAs derived from these samples to CFT073-specific microarrays allowed identification of genes that were up- or down-regulated in the dsdA deletion strain during UTI. Up-regulated genes included the known d-serine-responsive gene dsdX, suggesting in vivo intracellular accumulation of d-serine by CFT073 dsdA. Genes encoding F1C fimbriae, both copies of P fimbriae, hemolysin, OmpF, a dipeptide transporter DppA, a heat shock chaperone IbpB, and clusters of open reading frames with unknown functions were also up-regulated. To determine the role of these genes as well as motility in the hypercolonization phenotype, mutants were constructed in the CFT073 dsdA background and tested in competition against the wild type in the murine model of UTI. Strains with deletions of one or both of the two P fimbrial operons, hlyA, fliC, ibpB, c0468, locus c3566 to c3568, or c2485 to c2490 colonized mouse bladders and kidneys at levels indistinguishable from wild type. CFT073 dsdA c2398 and CFT073 dsdA focA maintained a hypercolonization phenotype. A CFT073 dsdA dppA mutant was attenuated 10- to 50-fold in its colonization ability compared to CFT073. Our results support a role for d-serine catabolism and signaling in global virulence gene regulation of uropathogenic E. coli.

Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Stimulates the Transcriptional Activity of Cellular IRF-3 and IRF-7

Kaposi's sarcoma-associated herpesvirus has been linked to Kaposi's sarcoma, body cavity-based lymphoma, and Castleman's disease. The Kaposi's sarcoma-associated herpesvirus genome contains a cluster of open reading frames encoding proteins (vIRFs) with homology to the cellular transcription factors of the interferon regulatory factor family. vIRF-3, also called LANA2, is a latently expressed nuclear protein. Here we demonstrate that vIRF-3 directly interacts with cellular interferon regulatory factor (IRF) IRF-3, IRF-7, and the transcriptional co-activator CBP/p300. The mapping of the vIRF-3 binding domain revealed that vIRF-3 associates with both IRF-3 and IRF-7 through its C-terminal region. The p300 domain, which interacts with vIRF-3, is distinct from the previously identified IBiD domain, to which both vIRF-1 and IRF-3 bind. Thus, in contrast to vIRF-1, vIRF-3 neither blocks the interaction between IRF-3 and p300 nor inhibits the histone acetylation. Although vIRF-3 is not a DNA-binding protein, it is recruited to the IFNA promoters via its interaction with IRF-3 and IRF-7. The presence of vIRF-3 in the enhanceosome assembled on the IFNA promoters increases binding of IRF-3, IRF-7, and acetylated histone H3 to this promoter region. Consequently, vIRF-3 stimulates the IRF-3- and IRF-7-mediated activation of type I interferon (IFNA and IFNB) genes and the synthesis of biologically active type I interferons in infected B cells. These studies illustrate that vIRF-3 and vIRF-1 have clearly distinct functions. In addition to its co-repressor activity, vIRF-3 can also act as a transcriptional activator on genes controlled by cellular IRF-3 and IRF-7.

Base Frequencies at the Second Codon Position of Vibrio cholerae Genes Connect with Protein Function

In this paper, the base frequency at the second codon position of the 3839 open reading frames (ORFs) in the Vibrio cholerae genome is analyzed. It is shown that according to the base content at this codon site, the ORFs can be divided into two clusters, each containing 673 and 3166 ORFs, respectively. ORFs in the smaller cluster usually have significantly higher T frequency than that of A at the second codon position. For the two clusters of ORFs, there are significant differences in the frequencies for 18 of the 20 amino acids in the encoding proteins. The two clusters of ORFs are also significantly different in their functions. More than half of the known genes involved in transport and binding are included in the smaller cluster, while few genes involved in amino acid biosynthesis, protein synthesis, and so on are included in this cluster.

Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations.

The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

Characterization of a novel human herpesvirus 8-encoded protein, vIRF-3, that shows homology to viral and cellular interferon regulatory factors.

The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-3, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10. 6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-3-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-3 mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-3 ORF encodes a 73-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-3 functioned as a dominant-negative mutant of both IRF-3 and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-3 in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active interferons. These results suggest that by targeting IRF-3 and IRF-7, which play a critical role in the activation of alpha/beta interferon (IFN) genes, HHV-8 has evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity.

Expression of the Insect Parvovirus GmDNV in Vivo: The Structural and Nonstructural Proteins Are Encoded by Opposite DNA Strands

The nucleotide sequence of GmDNV, an insect parvovirus, reveals large open reading frames (ORFs) on both strands of the viral replicative form DNA. Previously, we identified two viral transcripts within the polyadenylated RNA fraction of infected host larvae (Gross et al., 1990, J. Invertebr. Pathol.56, 175–180). In this work we used hybridization of single-stranded, unidirectional probes to RNA blots to show that the two transcripts, synthesized in vivo in GmDNV-infected Galleria mellonella larvae, are of antiparallel orientation. To determine their coding specificities, polyadenylated RNAs were isolated from hybrids with DNA from the left and right halves of the viral genome and translated in a rabbit reticulocyte system. The “right,” 2.4-kb hybrid-selected RNA was shown to direct the synthesis of four polypeptides that comigrated with the four viral capsid proteins and were immunoprecipitated with anti-GmDNV serum. Translation of the “left,” 1.8-kb RNA yielded three polypeptides, none of which was detected among the viral capsid proteins. This type of expression strategy is unique among vertebrate and most invertebrate parvoviruses, which use only one DNA strand to encode all their proteins. On the other hand, the basic organization of parvoviruses, in which the regulatory and structural proteins are encoded, respectively, by two clusters of ORFs located at the left and right halves of the genome, is conserved.

The Genomic Sequence Analysis of the Left and Right Species-Specific Terminal Region of a Cowpox Virus Strain Reveals Unique Sequences and a Cluster of Intact ORFs for Immunomodulatory and Host Range Proteins

Sequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure–function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity to immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precisein vivorole(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied orthopoxviruses.

Bacteriophage B103: complete DNA sequence of its genome and relationship to other Bacillus phages

The genome of Bacillus subtilis bacteriophage B103 consists of double-stranded linear DNA 18 630 bp long. The DNA was sequenced, and the sequence was compared with DNA sequences of closely related phages, namely the members of the phage ϕ29 family. Among them, phage Nf was shown to be the most closely related to B103. Comparisons of several open reading frames (ORFs) among the familly members helped to identify genes 1 and 5. A cluster of ORFs between genes 16 and 17 contains two ORFs with partial homology with two ϕ29 ORFs located in the same region. There are three more ORFs in this region of B103 with good ribosome binding sites (RBS) and optimal codon usage that are not homologous to any of the ϕ29 ORFs. The function of these five ORFs remains unexplained. It was shown that major promoters characterized in ϕ29 are retained in B103. Where many substitutions occur in the vicinity of a promoter, at least the −10 and −35 boxes are conserved.

Structure and organization of the hisA gene of the thermophilic archaebacterium Methanococcus thermolithotrophicus.

A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli. Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M. thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M. voltae and M. vannielii, respectively. The ORF (ORF 206) immediately 5' to the hisA gene of M. thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.