Small genomes: sequencing, functional characterization and comparative genomics. Hilton Head, South Carolina, USA. January 25-28, 1997. Abstracts.
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Animals↗
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Results for “Genomic characterization”
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Expression, characterization, and genomic structure of carp JAK1 kinase gene.
A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3, and human TYK2 is 57%, 35.5%, 31.3%, and 42.4%, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JH1) domains (approximately 70% identity). Therefore, carp JAK1 is a homolog of mammalian JAK1. To investigate the possible function of JH2 domain, full-length, and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JH1 and c-JH2 associate with each other and c-JH2 can be tyrosine-phosphorylated by c-JAK1 and by c-JH(1 + 2). The JAK1 gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. The 2.5-kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, AP1, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT-Basic. Deletion analysis defined a positive regulatory region between -1,023 and -528. A smaller region (-181 to +59) without any typical TATA-box sequences, G + C-rich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK1 gene.
Amino Acid Sequence↗
[Characterization of genome of A/Guangzhou/333/99(H9N2) virus].
BACKGROUND: To understand the characterization of genome of a strain of avian influenza A H9N2 virus repeatedly isolated from a child with influenza illness. Thereafter to reveal the origin of this H9N2 virus. METHODS: Viruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares. RESULTS: Genome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment derived from human influenza A virus. However, the genes encoding the HA,NA,NP and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA) proteins were derived from G1 lineage strain. CONCLUSIONS: A/Guangzhou/333/99 virus was a reassortant derived from reassortment betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most possibility is that it is derived from avian influenza A virus directly. The results do not only demonstrate that avian influenza A (H9N2) virus could infect men, but also firstly prove that the genetic reassortment could be occurred between different genetic lineages of avian influenza A (H9N2) viruses in the nature.
Animals↗
Isolation and characterization of bacteriophages from clinical enterohemorrhagic Escherichia coli strains.
Temperate bacteriophages play a pivotal role in the biology of their bacterial host. Of particular interest are bacteriophages infecting enterohemorrhagic E. coli (EHEC) due to their significant contribution to the pathogenicity of its host, most notably by encoding the key virulence factor of this pathogen, the Shiga toxin. To better understand the role of EHEC phages on the functionality of its host, we isolated eight temperate phages from clinical EHEC isolates and characterized their genomic composition, morphology, and receptor targeting. Morphological analysis identified one long-tailed siphophage, targeting the OmpC receptor for host recognition, whereas the other seven phages are short-tailed podophages and target the essential BamA protein. Genomic characterization revealed significant variations between the long- and short-tailed phages. Five of the eight isolated phages encode the potent Shiga toxin. Comparative analysis displays the typical lambdoid mosaicism, indicative of horizontal gene transfer driving evolution. These findings provide insights into the genetic and morphologic diversity and receptor specificity of EHEC phages, highlighting their role in the evolution and pathogenicity of clinical EHEC strains.IMPORTANCECharacterizing bacteriophages from clinical EHEC isolates is crucial in understanding the mechanisms underlying bacterial evolution and virulence. Despite the clinical relevance of EHEC bacteriophages, they remain underexplored, and particularly phage receptors are often not characterized. Studying temperate EHEC phages is essential in the development of strategies to address the global burden of these foodborne infections. Notably, identifying the phage receptors is critical in unraveling the specific interaction between phage and host. Knowledge of the phage receptors can provide insights into the mechanisms of phage infection, host range, and bacterial resistance and is fundamental in the design of targeted therapies like new antimicrobials, phage therapy, or prevention of those infections.
Humans↗
Clusters of proteins in archaeal and bacterial proteomes using compositional analysis.
In silico proteomics complements computational genomics in characterizing genome evolution. Here we examine cluster patterns in archaeal and bacterial proteomes using compositional properties of protein sequences in contrast to the traditionally used sequence alignment procedures. Application of standard Principal Component Analysis to the multi-dimensional data identified cluster patterns. Two types of cluster patterns exist in bacterial proteomes. Proteomes of type I have one major cluster with few isolated points in space revealing an underlying largely homogeneous compositional structure. In type II proteomes two clusters of protein distribution were discernible. The two clusters differ in size and were separated from each other although the boundary was somewhat fuzzy. Proteins falling in the major cluster were labeled as 'typical' and proteins of the minor cluster were called 'atypical'. The atypical proteins were mapped to Cluster of Orthologous Groups. Species distribution in COGs maps with respect to atypical proteins illuminated the biological relationships of extreme diversity among the archaeal members and of diversity among bacteria in relation to their niche. Amino acids that were over-represented in the atypical proteins had higher biosynthetic cost compared to 'typical' ribosomal proteins. However, archaea and bacteria economize by preferring the less costly amino acid to others closely related in chemical structure. Further, over-representation of serine in atypical proteins of archaeal members suggests re-examining these proteomes for the presence of Serine/Threonine phosphatases and kinases in Archaea. Our computational procedure can serve as a useful addition to the existing tools for carrying out in silico proteomics.
Archaeal Proteins↗
Characterization of genomic poly(dT-dG).poly(dC-dA) sequences: structure, organization, and conformation.
Hybridization studies suggest the abundant presence of poly(dT-dG).poly(dC-dA) (TG-element), a potential Z-DNA sequence, in eucaryotic genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TG-elements: the elements consist of 20 to 60 base pairs of (dT-dG)n.(dC-dA)n, the sequences characterized in our study were not flanked by direct or inverted repeats, the sequences are interspersed rather than in satellite blocks, the elements are not usually associated with other repeated elements, and some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: it is in the Z-form only at low ionic strength, S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.
Actins↗
Microbial genomes III: Sequencing, functional characterization, and comparative genomics.
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Genetics, Microbial↗
Microbial Genomes III: Sequencing, Functional Characterization and Comparative Genomics. Chantilly, Virginia, USA. January 29-February 1, 1999. Abstracts.
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Genetics, Microbial↗
Genome-wide characterization of the tomato PERK gene family and its expression profiling under abiotic stresses.
UNLABELLED: This study presents the first systematic genome-wide characterization of the proline-rich extensin-like receptor kinases (PERK) gene family in tomato (Solanum lycopersicum) and their transcriptional responses under abiotic stresses. Using the latest SL4.0/ITAG4.0 genome assembly, we identified six SlPERK genes, all harboring the conserved Ser/Thr protein kinase domain. Evolutionary and structural analyses revealed strong purifying selection (Ka/Ks < 1), distinct exon-intron organizations, and the presence of stress- and hormone-responsive cis-regulatory elements in their promoters. Furthermore, post-transcriptional regulation by 57 miRNAs and complex protein-protein interaction networks were predicted. To validate their stress-responsive roles, two tomato cultivars (GMOTL-1 and Roma) were subjected to cold, heat, and salinity treatments. Quantitative RT-PCR analysis revealed cultivar-specific expression dynamics: SlPERK4 exhibited strong transient induction under cold and heat stress, while SlPERK6 was highly responsive to salinity. Notably, the GMOTL-1 cultivar displayed significantly higher and broader stress-responsive expression profiles compared to Roma, indicating a potential role of these SlPERK genes in cultivar-specific stress tolerance. These findings provide a comprehensive genomic resource and establish a critical foundation for the functional validation and molecular breeding for stress-resilience tomato cultivars. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05044-y.
Abiotic stress↗
Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene.
Thymic stromal derived lymphopoietin receptor (TSLPR) is a novel receptor subunit that is related in sequence to the interleukin (IL)-2 receptor common gamma chain. TSLPR forms a heterodimeric complex with the IL-7 receptor alpha chain to form the receptor for thymic stromal derived lymphopoietin, a cytokine involved in B- and T-cell function. We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human. As in the mouse, TSLP receptor is widely expressed in rats suggesting that TSLPR may have roles in signaling outside the hematopoietic system. A zooblot analysis revealed that TSLPR is expressed in all vertebrate species examined. The absence of TSLPR in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans genomes is similar to the expression of several other cytokine receptors that have been characterized thus far. We have also characterized the genomic structure of the murine Tslpr gene which shows that in addition to primary sequence homology, it shares a common genomic organization of coding exons with the murine IL-2 receptor common gamma chain (Il2rg). Use of an alternative splice acceptor site leads to two alternatively spliced transcript variants of murine TSLPR, both of which are functional receptors. Finally, using linkage analysis, we mapped the murine Tslpr gene to mouse chromosome 5 between the Ecm2 and Pxn genes.
Alternative Splicing↗
Evidence for substantial fine-scale variation in recombination rates across the human genome.
Characterizing fine-scale variation in human recombination rates is important, both to deepen understanding of the recombination process and to aid the design of disease association studies. Current genetic maps show that rates vary on a megabase scale, but studying finer-scale variation using pedigrees is difficult. Sperm-typing experiments have characterized regions where crossovers cluster into 1-2-kb hot spots, but technical difficulties limit the number of studies. An alternative is to use population variation to infer fine-scale characteristics of the recombination process. Several surveys reported 'block-like' patterns of diversity, which may reflect fine-scale recombination rate variation, but limitations of available methods made this impossible to assess. Here, we applied a new statistical method, which overcomes these limitations, to infer patterns of fine-scale recombination rate variation in 74 genes. We found extensive rate variation both within and among genes. In particular, recombination hot spots are a common feature of the human genome: 47% (35 of 74) of genes showed substantive evidence for a hot spot, and many more showed evidence for some rate variation. No primary sequence characteristics are consistently associated with precise hot-spot location, although G+C content and nucleotide diversity are correlated with local recombination rate.
Genome, Human↗
Characterization of genomic clones specifying rabbit alpha- and beta-ventricular myosin heavy chains.
We have isolated gene sequences coding for the alpha- and beta-myosin heavy chains (HC) of rabbit ventricular muscle. A rabbit genomic library was screened with previously characterized cDNA clones specifying part of the light meromyosin and the entire subfragment 2 portion of alpha- and beta-myosin HCs, as well as with a clone containing the 3' nontranslated sequences of the alpha-myosin HC mRNA. Seven strongly hybridizing clones were analyzed in detail. One genomic clone encoded all of the 3' nontranslated sequences of an alpha-cDNA clone and, therefore, contained the 3' end of the alpha-myosin HC gene. Electron microscopic heteroduplex analysis and DNA sequence analysis showed that this clone overlapped a second genomic clone providing more than 25 kilobase pairs of the alpha-myosin HC gene. The exons within this region corresponded to approximately equal to 85% of the mRNA and were separated by at least 28 introns. A clone for the beta-myosin HC gene was also identified by Southern blot hybridization, by heteroduplex mapping, and by comparing the DNA sequence of a subfragment 2 exon to sequences of the alpha- and beta-cDNA clones. The introns of the alpha- and beta-myosin HC genes were in the same position but showed marked variation in length. These results conclusively showed that the alpha- and beta-myosin HCs are products of separate genes.
Animals↗
5' Long serial analysis of gene expression (LongSAGE) and 3' LongSAGE for transcriptome characterization and genome annotation.
Complete genome annotation relies on precise identification of transcription units bounded by a transcription initiation site (TIS) and a polyadenylation site (PAS). To facilitate this process, we developed a set of two complementary methods, 5' Long serial analysis of gene expression (LS) and 3'LS. These analyses are based on the original SAGE and LS methods coupled with full-length cDNA cloning, and enable the high-throughput extraction of the first and the last 20 bp of each transcript. We demonstrate that the mapping of 5'LS and 3'LS tags to the genome allows the localization of TIS and PAS. By using 537 tag pairs mapping to the region of known genes, we confirmed that >90% of the tag pairs appropriately assigned to the first and last exons. Moreover, by using tag sequences as primers for RT-PCRs, we were able to recover putative full-length transcripts in 81% of the attempts. This large-scale generation of transcript terminal tags is at least 20-40 times more efficient than full-length cDNA cloning and sequencing in the identification of complete transcription units. The apparent precision and deep coverage makes 5'LS and 3'LS an advanced approach for genome annotation through whole-transcriptome characterization.
Animals↗
Cloning of the RhoB gene from the mouse genome and characterization of its promoter region.
Rho proteins have been implicated in a variety of cytoskeletal functions, but it is unclear how Rho proteins regulate these cellular functions and how Rho proteins are regulated. In this study, we cloned the rhoB gene from the mouse genome and characterized its promoter region. The predicted amino acid sequence was identical to that encoded by the human rhoB gene. A site for initiation of transcription was found at position -376 relative to the site for initiation of translation. Deletion analysis of the 5'-flanking region of the rhoB gene revealed that the minimum region of the promoter was located between positions -507 and -376. Northern blotting analysis showed that the expression of the mouse rhoB gene was induced by serum, suggesting that expression of the rhoB gene might be controlled by some signal-responsive element(s).
3T3 Cells↗
Genome-wide characterization of tetrahymena thermophila chromosome breakage sites. I. Cloning and identification of functional sites.
The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.
Animals↗
Antigenic and genomic relation between human influenza viruses that circulated in Argentina in the period 1995-1999 and the corresponding vaccine components.
BACKGROUND: The analysis of epidemic influenza virus has been focused on antigenic and genomic characterization of the hemagglutinin (HA) glycoprotein in order to detect new variants for the recommendation of the vaccine strains in each season. Since October 1998, WHO organized a second meeting to evaluate the vaccine formula for the southern hemisphere. OBJECTIVES: (a) Present the antigenic and genomic characterization of influenza strains obtained from the Argentina surveillance network, (b) compare between strains collected in Argentina and other countries with the vaccine formula strains used in each season. STUDY DESIGN: Influenza strains were collected during a 5-year period (1995-1999). Initially, laboratory diagnosis was done by immunofluorescence (IF) assay on clinical samples, followed by viral isolation in Madin-Darby canine kidney (MDCK) cells. The isolates were characterized antigenically by hemagglutination-inhibition (HI) assay with post-infection ferret antisera. The genomic characterization consisted on RT-PCR followed by sequencing of the HA1 portion of the HA gene. The comparison between reference and circulating strains was analyzed by the construction of phylogenetic trees. RESULTS: The H3N2 circulating strains matched the corresponding vaccine component only in 1999, the first year when a vaccine recommended specifically for the southern hemisphere was used. Besides, H1N1 circulating strains matched the corresponding vaccine component only in 1998. Regarding to influenza B, only in 1995, the circulating strains showed no match to the B vaccine component. CONCLUSIONS: The results showed the usefulness of an intensified influenza laboratory surveillance to access the correct vaccine and the importance of having the necessary tools to characterize the circulating strains.
Animals↗
[Characterization of genomic structure and mutation analysis of SMARCA1 gene in a Smith-Fineman-Myers syndrome family].
The study is to determine the genomic structure and the role of SMARCA1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member1, SMARCA1) in the etiology of Smith-Fineman-Myers syndrome (SFMS). By comparing the cDNA sequence of SMARCA1 with the genomic sequences, genomic structure of SMARCA1 was determined, and conformed by amplifying and sequencing the sequences of exons and splicing junction. The results show that the genomic sequence of SMARCA1 gene exceeds 71.7 kb in length, and contains 24 exons and 23 introns. All the exon/intron boundaries follow the GT-AG rule and are in good agreement with the exon/intron consensus sequence. The characterization of genomic structure of SMARCA1 gene allows us to detect disease-causing mutation within the gene and further study its biological function. The open reading frame of SMARCA1 was detected for mutation by PCR amplification and direct sequencing in affected males from SFMS family in Shandong China. The disease in SFMS family from Shandong is not caused by the mutation within open reading frame of SMARCA1 gene.
Abnormalities, Multiple↗
Characterization of genomic organization of the adenosine A2A receptor gene by molecular and bioinformatics analyses.
The adenosine A(2A) receptor (A(2A)R) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A(2A)R gene expression, we have characterized the genomic organization of the mouse and human A(2A)R genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5' untranslated regions (5'-UTRs) of mouse A(2A)R mRNA were identified by rapid amplification of 5' cDNA end (5' RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding "exon 1" (h1A, h1B, h1C, h1D, h1E and h1F) in the human A(2A)R gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5' oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A(2A)R transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A(2A)R gene by 5' oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A(2A)R gene expression. Finally, our analysis indicates that A(2A)R mRNA and a novel transcript partially overlapping with the 3' exon h3, but in opposite orientation to the A(2A)R gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A(2A)R genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5'-UTR. This raises the possibility of generating multiple tissue-specific A(2A)R mRNA species by alternative promoters with varying regulatory susceptibility.
Animals↗