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PubMed1973

Ultrastructural characterization of genome of epizootic hemorrhagic disease virus.

Purified epizootic hemorrhagic disease (EHD) virus preparations were treated with urea and sodium perchlorate. The viral ribonucleic acid (RNA) released when spread on protein monolayers according to the Kleinschmidt technique was examined by rotary shadow-casting electron microscopy. The viral RNA released by urea treatment had filaments which frequently formed three to five loop-shaped figures of varied length. In 80% of the virus particles the lengths of the viral RNA released were 2.5 to 4.5 mum. The sodium perchlorate-released viral nucleic acid also appeared linear, and about 70% had lengths of 0.1 to 2.0 mum, the longest filament measuring 5.8 mum. Evidence was obtained that EHD virus contains double-stranded RNA as its genetic material and the molecular weight of the EHD viral RNA was calculated to be 12.2 x to 10(6) to 15.1 x 10(6) daltons.

Animals↗
PubMed2002

Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis.

The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to DL-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

Bacillus subtilis↗
PubMed1981

Avian reticuloendotheliosis virus: characterization of genome structure by heteroduplex mapping.

The genome structure of defective, oncogenic avian reticuloendotheliosis virus (REV) was studied by heteroduplex mapping between the full-length complementary DNA of the helper virus REV-T1 and the 30S REV RNA. The REV genome (5.5 kilobases) had a deletion of 3.69 kilobases in the gag-pol region, confirming the genetic defectiveness of REV. In addition, REV lacked the sequences corresponding to the env gene but contained, instead, a contiguous stretch (1.6 to 1.9 kilobases) of the specific sequences presumably related to viral oncogenicity. Unlike those of other avian acute leukemia viruses, the transformation-specific sequences of REV were not contiguous with the gag-pol deletion. Thus, REV has a genome structure similar to that of a defective mink cell focus-inducing virus or a defective murine sarcoma virus. An additional class of heteroduplex molecules containing the gag-pol deletion and two other smaller deletion loops was observed. These molecules probably represented recombinants between the oncogenic REV and its helper virus.

Base Sequence↗
PubMed1982

Characterization of genome structure of amphotropic and ecotropic wild mouse retroviruses.

We studied the RNA genomes of several wild mouse type C retroviruses by using RNase T1-oligonucleotide fingerprinting. The amphotropic and ecotropic viruses of field strain 1504 produced very similar oligonucleotide fingerprints, but each also had several unique oligonucleotides. All of these unique oligonucleotides were located in the env gene region and were probably responsible for the host range differences between these viruses, as well as the lymphomagenic and paralytogenic properties of the viruses. We obtained similar results with the amphotropic and ecotropic viruses of another field strain (4070), which was isolated from a mouse from a different trapping area. The amphotropic viruses of several field strains (strains 1504, 4070, and 1313) were more closely related than the ecotropic viruses of different strains (strains 1504, 4070, and 4996). These findings suggested that the genetic sequences of the amphotropic viruses are more conserved than those of ecotropic viruses isolated from the same wild mice.

Animals↗
PubMed1997

Characterization and genomic organization of Ty1-copia group retrotransposons in rye (Secale cereale).

The genomic organisation of the Ty1-copia retrotransposons in rye (Secale cereale) has been studied. We have used the polymerase chain reaction (PCR) to amplify sequences from a conserved domain of the reverse transcriptase gene of the Ty1-copia retrotransposons in this species. Sequence analysis of 26 of these PCR products shows them to be a highly heterogeneous population, a feature that is common in plants. Slot blot analysis shows that there are about 100,000 individual Ty1-copia retrotransposons in rye. In situ hybridization of a heterogeneous probe, representing the whole population of rye Ty1-copia retrotransposon sequences, to chromosome spreads of triticale (xTriticosecale), a rye-wheat hybrid, shows that these sequences are present throughout all the rye chromosomes but absent from the centromeric regions and, in particular, from the terminal heterochromatin. Southern analysis of oat, barley, wheat, and rye, using as a probe R9, one of the rye sequences that is closely similar to the BARE-1 element of barley, shows that close relatives of this retrotransposon subgroup are present in all these species in high copy number. Northern analysis on RNAs from seedlings shows that the BARE-1 subgroup is transcribed in all these cereal plants but in variable amounts: high in barley, moderate in wheat and rye, and extremely low in oat.

Amino Acid Sequence↗
PubMed2004

Structural characterization of genomes by large scale sequence-structure threading: application of reliability analysis in structural genomics.

BACKGROUND: We establish that the occurrence of protein folds among genomes can be accurately described with a Weibull function. Systems which exhibit Weibull character can be interpreted with reliability theory commonly used in engineering analysis. For instance, Weibull distributions are widely used in reliability, maintainability and safety work to model time-to-failure of mechanical devices, mechanisms, building constructions and equipment. RESULTS: We have found that the Weibull function describes protein fold distribution within and among genomes more accurately than conventional power functions which have been used in a number of structural genomic studies reported to date. It has also been found that the Weibull reliability parameter beta for protein fold distributions varies between genomes and may reflect differences in rates of gene duplication in evolutionary history of organisms. CONCLUSIONS: The results of this work demonstrate that reliability analysis can provide useful insights and testable predictions in the fields of comparative and structural genomics.

Computational Biology↗
PubMed2026

Genome-wide characterization of the sugar transporter protein family identifies candidate genes for bacterial wilt resistance breeding in tobacco.

Sugar transporter proteins (STPs) play pivotal roles in hexose allocation and plant stress responses. However, systematic characterization of the STP family in tobacco (Nicotiana tabacum) and its involvement in Ralstonia solanacearum resistance remains unclear. In this study, 37 NtSTP genes were identified and classified into six groups, with Group VI being the most conserved and Group V exhibiting dicot-specific expansion. Gene structure and conserved motif analyses revealed that most NtSTP members possess the typical MFS_STP domain, although variations in exon-intron organization and motif composition suggested functional divergence. Tandem duplication (TD) served as the primary driver of NtSTP family expansion, and Ka/Ks values of all paralogous pairs were less than 1, indicative of purifying selection. Promoter cis-element analysis revealed a complex regulatory network involving hormone signaling (ABA, JA, SA, GA, ET), stress responses, and light signaling. RT-qPCR expression profiling revealed that ten NtSTP genes (NtSTP1, 5, 7, 21, 22, 24, 26, 27, 28, and 29) exhibited significant transcriptional upregulation upon R. solanacearum infection. Specifically, NtSTP5, NtSTP7, NtSTP21, NtSTP22, NtSTP24, NtSTP26, and NtSTP27 peaked at 12 h post-inoculation (hpi), whereas NtSTP1, NtSTP28, and NtSTP29 reached their highest expression levels at 24 hpi. By contrast, NtSTP6, NtSTP13, and NtSTP30 displayed reduced expression upon R. solanacearum infection. These expression patterns indicate functional diversification within the NtSTP family and imply that these members may be transcriptionally modulated during plant responses to R. solanacearum. The present work provides preliminary and valuable candidate gene resources that may facilitate future disease resistance breeding programs in tobacco.

NtSTP gene family↗
PubMed2026

Genome-Wide Characterization of the ScLOR Gene Family in Wild Tomato Solanum lycopersicoides Reveals ScLOR16 as a Negative Regulator of Cold-and Drought-Stress Tolerance.

The LOR (LURP-one related) gene family encodes proteins containing conserved LOR domains; however, its functions in plant abiotic stress responses remain largely unexplored. In this study, we systematically identified and characterized the LOR gene family in the stress-tolerant wild tomato Solanum lycopersicoides using comprehensive bioinformatic analyses and conducted functional validation of the candidate gene ScLOR16. A total of 19 ScLOR members were identified and classified into eight phylogenetic subgroups. Numerous cis-acting elements associated with responses to abscisic acid (ABA), cold, and drought, including ABRE, LTR, and MBS, were detected in the promoter regions, suggesting that the ScLOR family may be broadly involved in ABA-mediated stress signaling pathways. RT-qPCR analysis revealed that ScLOR16 expression was significantly induced by both cold and drought treatments. Subcellular localization assays demonstrated that ScLOR16 is localized in both the nucleus and cytoplasm. Virus-induced gene silencing (VIGS) was subsequently employed to generate ScLOR16-silenced plants. Following 24 h of cold treatment at 4 °C and four days of drought stress, ScLOR16-silenced seedlings exhibited significantly less severe wilting symptoms than empty-vector controls. Physiological analyses showed that silenced plants exhibited enhanced superoxide dismutase (SOD) and peroxidase (POD) activities, increased proline accumulation, and decreased thiobarbituric acid-reactive substances (TBARS) content. Collectively, these results indicate that reduced ScLOR16 transcript levels are associated with enhanced cold and drought tolerance, accompanied by alterations in antioxidant defense and osmoprotection-related physiological markers. This study yields new insights into the evolution and stress-related functions of the ScLOR family.

LOR gene family↗
PubMed2026

Genome-Wide Characterization and Salt-Responsive Expression Divergence of Chromosome Group 2 and Group 6 TaBADH Genes in Wheat.

Betaine aldehyde dehydrogenase (BADH) catalyzes the final step in glycine betaine biosynthesis, but the evolutionary divergence and differential salt responsiveness of BADH homeologs in bread wheat remain unclear. We identified six TaBADH genes and analyzed their phylogenetic relationships, conserved motifs, gene structures, promoter cis-acting elements and synteny. RNA-seq and qRT-PCR were used to compare expression in salt-tolerant Jimai 60 and salt-sensitive Chinese Spring under 200 mM NaCl, and BADH activity, glycine betaine, H2O2 and malondialdehyde (MDA) were measured during treatment. The genes separated into chromosome group 2 and group 6 clades with distinct structural and transcriptional patterns. TaBADH-2B encoded a shorter protein and lacked several conserved motifs. Group 6 genes showed stronger salt-responsive expression in Jimai 60, with TaBADH-6D displaying the strongest and most sustained induction. Jimai 60 also showed higher BADH activity and glycine betaine accumulation and lower H2O2 and MDA contents at later time points. Expression of TaBADH-6D improved E. coli growth under 200 mM NaCl. These findings identify homeolog-specific divergence within the BADH wheat family and support TaBADH-6D as a candidate for plant-level functional validation.

TaBADH-6D↗
PubMed2000

Characterization of genomically amplified segments using PCR: optimizing relative-PCR for reliable and simple gene expression and gene copy analyses.

Gene amplification is one of the mechanisms for oncogene activation in solid tumors. The size of the amplified regions may vary considerably among individual tumors, and more than one gene may be affected within the same amplicon. The main objective in analyzing genomic amplifications has therefore been to map the shortest region involved and to identify genes with increased expression as a result of the increased gene copy number. To facilitate such an analysis, we have developed simple polymerase chain reaction (PCR) procedures using the internal standards beta-actin (ACTB) and L1Hs for gene expression and gene copy number analyses, respectively. We used cDNA derived from pancreatic carcinoma cell lines, and genomic DNA extracted from the same cell lines, as templates in the gene expression and in the gene copy number analyses, respectively. To determine the optimal number of PCR cycles, dilution series of the templates were made. Furthermore, competing primers were used to adjust for differences in target sequence levels. We show that by these simple means it is possible to determine optimal conditions for expression analyses. In addition, the procedure was adapted for the analysis of gene copy number changes at the genomic level using L1Hs as the internal standard. This PCR method makes it possible to produce detailed gene copy number profiles of amplified genomic regions.

DNA Primers↗
PubMed1990

The yeast genus Trichosporon spec. LS3;molecular characterization of genomic complexity.

The genomic structure of an industrial yeast strain Trichosporon spec. LS3 was compared with Trichosporon adeninovorans type strain CBS 8244. The cot values, the portion of single copy sequences (CBS 8244: 10.9 x 10(9) D, LS3: 10.6 x 10(9) D) as well as of repetitive sequences (CBS 8244: 6.0 x 10(9) D, LS3: 5.5 x 10(9) D) per haploid genome and genome complexity of these strains (CBS 2844: 16.9 x 10(9) D, LS3: 16.1 x 10(9) D) have been analysed. Both strains show a high genome complexity. The mitochondrial DNA content was measured and compared. No plasmidal DNA was identified for both strains. A survey of the data of genomic DNA made it possible to postulate for each of both strains a haploid set of chromosomes. The intraspecific reassoziation values of the nuclear DNA from the strain LS3 and CBS 8244 are interpreted to confirm classification of these strains based on physiological and genetical properties into one species Trichosporon adeninovorans.

Centrifugation, Density Gradient↗
PubMed1986

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes.

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome.

Animals↗
PubMed1985

Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component.

Complementary and genomic DNA clones corresponding to the human C-reactive protein (CRP) mRNA and structural gene have been analyzed and compared. Nucleotide sequencing of the coding regions of both cDNA and genomic DNA revealed an additional 19 amino acid peptide not described in the published CRP amino acid sequence. The CRP gene contains a single 278 base pair intron within the codon specifying the third residue of mature CRP. The intron contains a repetitive sequence (GT)15G(GT)3 which is similar to structures capable of adopting the Z-DNA form. A comparison of CRP coding and amino acid sequences with those of serum amyloid P component revealed striking overall homology which was not uniform: a region of limited conservation is bounded by two highly conserved regions.

Amino Acid Sequence↗
PubMed1984

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences.

An in vitro system which replicates plasmid DNA containing the replication origin of adenovirus DNA has been established. Replication of plasmid pLA1 DNA, which contains the left-hand terminus (0-9.4 map units) of adenovirus serotype 5 DNA but which lacks the 55,000-dalton terminal protein, is initiated by a protein-primed mechanism in a manner similar to that found with adenovirus DNA. Initiation of DNA replication using plasmid pLA1 as a template requires (i) that the cloned adenovirus sequence be present at the terminus of a linearized (form III) DNA molecule ( Tamanoi , F., and Stillman , B. W. (1982) Proc. Natl. Acad. Sci. U. S. A., 79, 2221-2225; van Bergen, B. G. M., van der Ley , P. A., van Driel , W., van Mansfield , A. D. M., and van der Vliet , P. A. (1983) Nucleic Acid Res. 11, 1975-1979), and (ii) the presence of the 80,000-dalton precursor to the 55,000-dalton terminal protein and the adenovirus coded DNA-dependent DNA polymerase. In the presence of the four deoxy-nucleoside triphosphates, the preterminal protein, the adenovirus coded DNA binding protein, and an extract prepared from uninfected HeLa nuclei, the adenovirus DNA polymerase can elongate the preterminal-protein dCMP initiation complex formed on pLA1 DNA to full length (6.6 kilobase) DNA molecules. These results suggest that the 55,000-dalton terminal protein covalently linked to the 5' termini of adenovirus DNA is not essential for the replication of this DNA.

Adenoviridae↗
PubMed1993

Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene.

Endothelial nitric oxide (NO) synthase is a unique NO synthase isoform that is expressed constitutively by vascular endothelium both in vivo and in vitro and is believed essential to local vascular homeostasis. This calcium/calmodulin-dependent isoform is distinct from neuronal NO synthase. Genomic clones encoding the human endothelial NO synthase were isolated and the structural organization of the gene was determined. The gene contains 26 exons spanning approximately 21 kilobases of genomic DNA, encodes a messenger RNA of 4052 nucleotides, and is present as a single copy in the haploid human genome. Characterization of 5'-flanking genomic regions indicates that the endothelial NO synthase promoter is "TATA-less" and exhibits proximal promoter elements consistent with a constitutively expressed gene that is found in endothelial cells, namely Sp1 and GATA motifs. The 5'-flanking region contains putative AP-1, AP-2, NF-1, heavy metal, acute-phase response shear stress, and sterol-regulatory cis-elements. The human endothelial NO synthase gene was assigned to the 7q35-->7q36 region of chromosome 7 by Southern blot hybridization of human-rodent somatic cell hybrid lines and fluorescence in situ hybridization, whereas human neuronal NO synthase localized to the 12q24.2 region of chromosome 12.

Amino Acid Oxidoreductases↗
PubMed1996

Characterization of genomic alterations associated with glioma progression by comparative genomic hybridization.

Genomic alterations associated with glioma progression were determined by comparative genomic hybridization (CGH) 30 tumors from 15 patients with primary gliomas of World Health Organization (WHO) grade II that on recurrence showed progression to malignant gliomas of WHO grades III or IV (five cases of astrocytoma grade II (A II) to grade III (AA III), five cases of A II to glioblastoma multiforme grade IV (GBM) and five cases of oligodendroglioma grade II (O II) to grade III (AO III)). All tumors were additionally screened for p53 mutations by single strand conformational polymorphism and heteroduplex analysis of exons 5-8, followed by direct sequencing. Mutations of p53 were found in the primary and recurrent tumors of all cases of A II progressing to GBM and three of five cases of A II recurring as AA III. Alterations identified by CGH in more than one primary A II included losses on Xp (3/10) and 5p (2/10), gains on 8q and 19p (2/10 each), and gain/amplification on 12p (2/10). Common progression associated changes found in AA III or GBM were losses on 4q, 9p, 10q, 11p, 13q (4/10 each) and gains on 1q, 6p, 20q (2/10 each). The most frequent amplification site was located on 12p13 (1/10 A II, 3/5 AA III, 1/5 GBM). Other amplified chromosomal regions were 13q32-q34 (1/10 AII, 2/5 GBM), 7q31-qter (1/5 AA III, 1/5 GBM), 12q22-qter and 18p (1/5 AA III). In contrast to the astrocytic gliomas, only one of five oligodendroglial cases showed a p53 mutation. Genetic abnormalities identified by CGH to occur more than once were restricted to four chromosomes (1, 4, 9 and 19). Our results provide a comprehensive overview of the genomic alterations associated with the progression of individual gliomas and substantiate the hypothesis that glioma progression is associated with a cumulative acquisition of multiple genetic changes.

Adult↗