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PubMed1994

Nucleotide sequence analysis of a 38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding human cytomegalovirus immediate-early gene homologs and transactivating functions.

Human herpesvirus 6 (HHV-6) is prevalent in the human population, with primary infection occurring early in life. Its predominant CD4+ T-lymphocyte tropism, its ability to activate human immunodeficiency virus type 1 (HIV-1) gene expression in vitro, and its upregulation of CD4 expression has led to speculation that HHV-6 may act as a positive cofactor in the progression of HIV infection to AIDS in individuals infected with both viruses. Previous sequencing studies of restricted regions of the 161.5-kbp genome of HHV-6 have demonstrated unequivocally that it is a member of the betaherpesvirus subgroup and have indicated that the HHV-6 genome is generally collinear with the unique long (UL) component of human cytomegalovirus (HCMV). In the work described in this report we have extended these sequencing studies by determining the primary structure of 38.5-kbp of the HHV-6 genome (genomic position 21.0 to 59.5 kbp). Within the sequenced region lie 31 open reading frames, 20 of which are homologous to positional counterparts in HCMV. Of particular significance is the identification of homologs of the HCMV UL36-38 and US22-type genes, which have been shown to encode transactivating proteins. We show that DNA sequences encoding these HHV-6 homologs were able to transactivate HIV-1 long terminal repeat-directed chloramphenicol acetyltransferase expression in cotransfection assays, thus demonstrating functional as well as structural conservation of these betaherpesvirus-specific gene products. Our data therefore confirm the close relationship between HHV-6 and HCMV and identify putative immediate-early regulatory genes of HHV-6 likely to play key roles in lytic replication and possibly also in the interactions between HHV-6 and HIV in dually infected cells.

Animals↗
PubMed2003

The diversity and evolution of the T4-type bacteriophages.

Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population. In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them. For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type. It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range. The genomes of all these phages have homology to the T4 genes that determine virion morphology. Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle. This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed. Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution. The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes. These non-essential genes are probably important for the adaptation of these phages to their particular life-style. Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family. Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses.

Bacteriophage T4↗
PubMed2025

ChromaFactor: Deconvolution of single-molecule chromatin organization with non-negative matrix factorization.

The investigation of chromatin organization in single cells holds great promise for identifying causal relationships between genome structure and function. However, analysis of single-molecule data is hampered by extreme yet inherent heterogeneity, making it challenging to determine the contributions of individual chromatin fibers to bulk trends. To address this challenge, we propose ChromaFactor, a novel computational approach based on non-negative matrix factorization that deconvolves single-molecule chromatin organization datasets into their most salient primary components. ChromaFactor provides the ability to identify trends accounting for the maximum variance in the dataset while simultaneously describing the contribution of individual molecules to each component. Applying our approach to two single-molecule imaging datasets across different genomic scales, we find that these primary components demonstrate significant correlation with key functional phenotypes, including active transcription, enhancer-promoter distance, and genomic compartment. Also, we find that some bulk trends exist at the single-cell level, but only in a small fraction of cells, suggesting that critical changes in genome organization may be driven by specific rare subpopulations rather than occurring uniformly across all cells. ChromaFactor offers a robust tool for understanding the complex interplay between chromatin structure and function on individual DNA molecules, pinpointing which subpopulations drive functional changes and fostering new insights into cellular heterogeneity and its implications for bulk genomic phenomena.

Animals↗
PubMed2001

Functions of RecQ family helicases: possible involvement of Bloom's and Werner's syndrome gene products in guarding genome integrity during DNA replication.

Escherichia coli RecQ helicase is a component of the RecF pathway of recombination whose components are required to reassemble a replisome complex at the site of the replication fork after the removal of a lesion. There are at least five RecQ homologues in human cells, including BLM and WRN. The genes encoding BLM and WRN are mutated in the cancer-prone disorder Bloom's syndrome (BS) and the plogeroid disorder Werner's syndrome (WS), respectively. These syndromes are characterized by a high degree of genomic instability, including chromosomal breaks, multiple large deletions, and translocations, and cells derived from BS and WS patients show defects in DNA replication. Recently, it has become clear that a Holliday junction-like structure is formed at stalled replication forks to result in the formation of double-stranded breaks, and recombination plays an important role in the repair of stalled or broken replication forks, leading to the reinitiation of replication. Defects in the processing of stalled replication forks could lead to aberrant recombination events resulting in genetic instability. Recent studies on BLM, WRN, and the RecQ homologue of Saccharomyces cerevisiae, Sgs1, indicate that these RecQ homologues interact with proteins involved in DNA replication, and function in a pathway from the DNA replication check point to homologous recombination.

Adenosine Triphosphatases↗
PubMed2006

Human genomic deletions mediated by recombination between Alu elements.

Recombination between Alu elements results in genomic deletions associated with many human genetic disorders. Here, we compare the reference human and chimpanzee genomes to determine the magnitude of this recombination process in the human lineage since the human-chimpanzee divergence approximately 6 million years ago. Combining computational data mining and wet-bench experimental verification, we identified 492 human-specific deletions (for a total of approximately 400 kb) attributable to this process, a significant component of the insertion/deletion spectrum of the human genome. The majority of the deletions (295 of 492) coincide with known or predicted genes (including 3 that deleted functional exons, as compared with orthologous chimpanzee genes), which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees. Overall, we found that Alu recombination-mediated genomic deletion has had a much higher impact than was inferred from previously identified isolated events and that it continues to contribute to the dynamic nature of the human genome.

Animals↗
PubMed1981

Membranes of EBV-carrying virus nonproducer cells inhibit leukocyte migration of EBV-seropositive but not seronegative donors.

We have previously shown that the leukocytes of healthy EBV-seropositive (but not seronegative) donors respond with migration inhibition (LMI) when confronted with extracts of EBV-carrying (but not EBV negative) cells. In the present study, we have examined whether this EBV-specific LMI response is capable of detecting a membrane antigen on the surface of EBV-carrying virus nonproducer cells. Crude membranes from EBV-genome carrying and EBV-negative cell lines were used as antigen. Contamination with the EBV-determined nuclear antigen (EBNA) was ruled out. Membranes from EBV-genome carrying nonproducer cells inhibited the migration of leukocytes from healthy seropositive donors, whereas membranes from EBV-negative lines had no such effect. Seronegative donors did not show any LMI. The clear difference between the EBV-negative Ramos line and its EBV-converted sublines was particularly conclusive in showing that the membrane component is determined or induced by the viral genome.

Antigens↗
PubMed2006

An amino acid "transmembrane tendency" scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: relationship to biological hydrophobicity.

Hydrophobicity analyses applied to databases of soluble and transmembrane (TM) proteins of known structure were used to resolve total genomic hydrophobicity profiles into (helical) TM sequences and mainly "subhydrophobic" soluble components. This information was used to define a refined "hydrophobicity"-type TM sequence prediction scale that should approach the theoretical limit of accuracy. The refinement procedure involved adjusting scale values to eliminate differences between the average amino acid composition of populations TM and soluble sequences of equal hydrophobicity, a required property of a scale having maximum accuracy. Application of this procedure to different hydrophobicity scales caused them to collapse to essentially a single TM tendency scale. As expected, when different scales were compared, the TM tendency scale was the most accurate at predicting TM sequences. It was especially highly correlated (r = 0.95) to the biological hydrophobicity scale, derived experimentally from the percent TM conformation formed by artificial sequences passing though the translocon. It was also found that resolution of total genomic sequence data into TM and soluble components could be used to define the percent probability that a sequence with a specific hydrophobicity value forms a TM segment. Application of the TM tendency scale to whole genomic data revealed an overlap of TM and soluble sequences in the "semihydrophobic" range. This raises the possibility that a significant number of proteins have sequences that can switch between TM and non-TM states. Such proteins may exist in moonlighting forms having properties very different from those of the predominant conformation.

Algorithms↗
PubMed1999

Gene therapy in the central nervous system: the use of recombinant retroviruses.

Only a handful of the hundreds of known vertebrate retroviruses have been deliberately subverted for use as carriers of recombinant genetic material. Retroviruses receive their name from the fact that their genome undergoes conversion from RNA to DNA following infection of a host cell. Also characteristic of retroviruses and uncommon for most other types of viruses is that the genome of the retrovirus integrates itself permanently into the DNA of the host cell. Once integrated into the host genome, the inserted provirus acts as a factory for producing more retroviral RNA genomes and expressing retroviral packaging proteins. Both components combine to form viral particles that bud from the surface of the infected cells.

Brain Neoplasms↗
PubMed2003

Role of elastin polymorphisms in panic disorder.

Panic disorder (PD) is a complex neuropsychiatric disorder that has been associated with an increased frequency of mitral valve prolapse. Elastin is a prominent component of mitral valves and, in a genome screen of 23 pedigrees with PD, we found evidence of linkage to the region of chromosome 7 that contains the elastin gene (ELN). Therefore, we examined the minimal essential promoter and coding regions of ELN in 23 independent probands from the families in our linkage studies using single strand conformational polymorphism analysis. We found three polymorphisms including one that coded for a non-conservative amino acid change. However, none of these polymorphisms were associated with panic disorder in a case-control analysis or linked to it in multiplex pedigrees. In our pedigrees, exonic polymorphisms in ELN do not play a major role in the genetic vulnerability to PD.

Base Sequence↗
PubMed2006

High-throughput localization of organelle proteins by mass spectrometry: a quantum leap for cell biology.

Cells are the fundamental building blocks of organisms and their organization holds the key to our understanding of the processes that control Development and Physiology as well as the mechanisms that underlie disease. Traditional methods of analysis of subcellular structure have relied on the purification of organelles and the painstaking biochemical description of their components. The arrival of high-throughput genomic and, more significantly, proteomic technologies has opened hereto unforeseen possibilities for this task. Recently two reports((1,2)) show how much can be gleaned from the combination of analytical centrifugation, mass spectrometry and advanced statistical techniques focused on a high-throughput analysis of the content and organization of plant and animal cells. The results reveal intriguing possibilities for the future and the possibility of mapping much of the known proteome onto our current map of the cell.

Mass Spectrometry↗
PubMed2005

Dissection of heterogeneous phenotypes for quantitative trait mapping.

We discuss analyses of Genetic Analysis Workshop 14 data from the Collaborative Study on the Genetics of Alcoholism (COGA) as well as from a simulated complex disease, Kofendrerd personality disorder (KPD), with both genetic and phenotypic heterogeneity. Both data sets included numerous related phenotypes in addition to disease definitions. All analyses either chose from the given selection of phenotypes or defined new ones, including traits that may not have been related to alcoholism or KPD. Some contributors evaluated the genetic components of the trait. Many investigated genome-wide linkage and/or association, using microsatellites and/or single-nucleotide polymorphism (SNP) chip data. Here we will focus on methodological issues that the investigators faced. Their results depended on phenotype selection, whether continuous or discrete, the covariates included, and ethnicity of the study population. For SNP chip data, members of our group detected no difference in results for Affymetrix or Illumina chips, although higher marker density for association studies appeared to be advantageous. Overall, there were some observations that different chromosomal segments, i.e., physical locations on the p-arm, q-arm, or middle segment, might lead to possible differences in type I error rates. This finding and others highlight the importance of empirical determination of P-values to determine significance.

Alcoholism↗
PubMed2001

Cloning of a fatty acid synthase component FAS1 gene from Saccharomyces kluyveri and its functional complementation of S. cerevisiae fas1 mutant.

A gene encoding a fatty acid synthase component, FAS1, has been cloned from a genomic library of the polyunsaturated fatty acid (PUFA)-producing yeast Saccharomyces kluyveri. This gene (named Sk-FAS1) was found to contain an open reading frame of 6150 bp, coding for 2049 amino acids. The deduced Sk-FAS1 protein showed significant (75-59%) homology with FAS proteins from the other yeasts, including S. cerevisiae, Candida albicans and Yarrowia lipolytica. The substrate-binding sites of the acetyl transferase and malonyl/palmitoyl transferase domains, and the FMN- and NADPH-binding sites of the enoyl reductase domain, were all highly conserved. Expression of the Sk-FAS1 gene in S. cerevisiae complemented genetic disruption of the S. cerevisiae FAS1 gene (Sc-FAS1), suggesting the formation of a heterogeneous complex of Sk-FAS1 (beta) and Sc-FAS2 (alpha), which is able to function to synthesize fatty acids. Compared with the isogenic wild-type of S. cerevisiae, as well as S. kluyveri, the S. cerevisiae fas1 mutant carrying the Sk-FAS1 gene showed an increase in the relative amount of 16-carbon fatty acids and a decrease in 18-carbon fatty acids.

Amino Acid Sequence↗
PubMed1986

Organization and chromosomal distribution of a novel repetitive DNA component from Muntiacus muntjak vaginalis with a repeat length of more than 40 kb.

The organization and chromosomal distribution of the repetitive DNA component IB from Muntiacus muntjak vaginalis (MMV) was investigated. DNA fragments of component IB were cloned in cosmids and their structure analysed using restriction nucleases and blot-hybridization experiments. Two cosmids were found to be practically identical by restriction enzyme mapping. The repeat unit of component IB DNA is more than 40 kb and contains the 11 and 18 kb Bam HI fragments, which have previously been shown to cross-hybridize with MMV satellite IA. In addition, the repeat unit contains long stretches of DNA sequences which are unique to component IB. In situ hybridization experiments showed that component IB has the properties characteristic of long interspersed repetitive DNA rather than tandemly repeated satellite DNA. Consistent with this conclusion, only a minor fraction of component IB is located on the X chromosome as demonstrated by the analysis of somatic cell hybrids. This is in marked contrast to satellite IA that is specific for the X chromosome. These results have interesting implications for the evolution of the component I DNA family of the MMV genome.

Animals↗
PubMed1992

Protective role of aqueous turmeric extract against mutagenicity of direct-acting carcinogens as well as benzo [alpha] pyrene-induced genotoxicity and carcinogenicity.

Turmeric (Curcuma longa Linn.) has been shown to inhibit chemical carcinogenesis. In this study, we compared the chemopreventive efficacy of an aqueous turmeric extract (AqTE) and its constituents, curcumin-free aqueous turmeric extract (CFAqTE) and curcumin, using the Salmonella typhimurium mutagenicity assay and the bone marrow micronucleus test in female Swiss mice. AqTE exhibited antimutagenic activity against direct-acting mutagens, 4-nitro-O-phenylenediamine and 1-methyl-3-nitro-1-nitrosoguanidine, in strains TA 98 and TA 100 respectively. Both AqTE and CFAqTE inhibited the mutagenicity of benzo [alpha]pyrene in the two strains in the presence of Aroclor-1254-induced rat liver homogenate. The inhibition in both studies was dose-dependent. Administration of AqTE, CFAqTE and curcumin at a dose of 3 mg/animal 18 h prior to i.p. benzo [alpha]pyrene injection (250 mg/kg) significantly inhibited bone marrow micronuclei formation in female Swiss mice by 43%, 76%, and 65% respectively. Furthermore, the incidence and multiplicity of forestomach tumours induced by benzo [alpha]pyrene (1 mg/animal, twice weekly, p.o. for 4 weeks) in female Swiss mice were significantly inhibited by AqTE, CFAqTE and curcumin given 2 weeks before, during and after the carcinogen treatment. These data indicate that the protection against genomic damage by turmeric extract and its components tested could be necessary for some aspects of its cancer chemoprevention.

Animals↗
PubMed1990

Rapid evolution in a fraction of the Drosophila nuclear genome.

Previous observations have indicated that Drosophila DNA contains a component that evolves so rapidly that it fails to hybridize between the DNAs of sibling species. To establish the reality of this component and study its properties, the fraction (about 20%) of Drosophila simulans (Dsim) DNA that fails to hybridize to Drosophila melanogaster (Dmel) DNA has been isolated. The majority of the hybridizable part of this isolated fraction (based on control tests on Dsim DNA) fails to hybridize with Dmel DNA under the conditions used for the initial fractionation. Clones of this fraction do hybridize with Dmel DNA at open criterion producing duplexes with greatly reduced thermal stability, indicating that the underlying process is rapid sequence divergence rather than loss of the homologous sequences by relatively large deletions. Cloned fragments from the nonhybridizing fraction from Dsim are more than 15% divergent from the Dmel homologues, whereas the fraction that does hybridize is only 3-5% divergent. In comparison, synonymous substitutions in the coding regions of five genes show a 9% average divergence between Dsim and Dmel. They appear to be intermediate in their degree of divergence between the hybridizing and nonhybridizing components.

Animals↗
PubMed2000

Bacillus sp. WW3-SN6, a novel facultatively alkaliphilic bacterium isolated from the washwaters of edible olives.

A novel Gram-positive facultatively alkaliphilic, sporulating, rod-shaped bacterium, designated as WW3-SN6, has been isolated from the alkaline washwaters derived from the preparation of edible olives. The bacterium is nonmotile, and flagella are not observed. It is oxidase positive and catalase negative. The facultative alkaliphile grows from pH 7.0 to 10.5, with a broad optimum from pH 8.0 to 9.0. It could grow in up to 15% (w/v) NaCl, and over the temperature range from 4 degrees to 37 degrees C, with an optimum between 27 degrees and 32 degrees C: therefore, it is both halotolerant and psychrotolerant. The bacterium is sensitive to a range of beta-lactam, sulfonamide, and aminoglycoside antibiotics, but resistant to trimethoprim. The range of amino acids, sugars, and polyols utilized as growth substrates indicates that this alkaliphile is a heterotrophic bacterium. D(+)-glucose, D(+)-glucose-6-phosphate, D(+)-cellobiose, starch, or sucrose are the substrates best utilized. The major membrane lipids are phosphatidylglycerol and diphosphatidylglycerol, with smaller amounts of phosphatidylethanolamine and an unknown phospholipid. During growth at high pH, the proportion of phosphatidylglycerol is increased relative to phosphatidylethanolamine. The fatty acyl components in the membrane phospholipids are mainly branched chain, with 13-methyl tetradecanoic and 12-methyl tetradecanoic acids as the predominant components. The G + C content of the genomic DNA is 41.1 +/- 1.0 mol%. The results of 16S ribosomal RNA sequence analysis place this alkaliphilic bacterium in a cluster, together with an unnamed alkaliphilic Bacillus species (98.2% similarity).

Alkalies↗
PubMed2005

Insect chitin synthases: a review.

Chitin is the most widespread amino polysaccharide in nature. The annual global amount of chitin is believed to be only one order of magnitude less than that of cellulose. It is a linear polymer composed of N-acetylglucosamines that are joined in a reaction catalyzed by the membrane-integral enzyme chitin synthase, a member of the family 2 of glycosyltransferases. The polymerization requires UDP-N-acetylglucosamines as a substrate and divalent cations as co-factors. Chitin formation can be divided into three distinct steps. In the first step, the enzymes' catalytic domain facing the cytoplasmic site forms the polymer. The second step involves the translocation of the nascent polymer across the membrane and its release into the extracellular space. The third step completes the process as single polymers spontaneously assemble to form crystalline microfibrils. In subsequent reactions the microfibrils combine with other sugars, proteins, glycoproteins and proteoglycans to form fungal septa and cell walls as well as arthropod cuticles and peritrophic matrices, notably in crustaceans and insects. In spite of the good effort by a hardy few, our present knowledge of the structure, topology and catalytic mechanism of chitin synthases is rather limited. Gaps remain in understanding chitin synthase biosynthesis, enzyme trafficking, regulation of enzyme activity, translocation of chitin chains across cell membranes, fibrillogenesis and the interaction of microfibrils with other components of the extracellular matrix. However, cumulating genomic data on chitin synthase genes and new experimental approaches allow increasingly clearer views of chitin synthase function and its regulation, and consequently chitin biosynthesis. In the present review, I will summarize recent advances in elucidating the structure, regulation and function of insect chitin synthases as they relate to what is known about fungal chitin synthases and other glycosyltransferases.

Amino Acid Sequence↗
PubMed2006

Ordered assembly of mitochondria during rice germination begins with pro-mitochondrial structures rich in components of the protein import apparatus.

Mitochondrial maturation during imbibition of rice embryos follows the transition of unstructured double membrane bound pro-mitochondria to the typical cristae-rich mitochondrial structures observed in mature plant cells. During the first 48 h following imbibition, an ordered increase in the abundance of transcripts encoding mitochondrial proteins was observed. Co-incident with these changes in transcript levels was dynamic and rapid changes in mitochondrial protein content and mitochondrial function. Proteins representing components of the mitochondrial protein import apparatus are strikingly abundant in dry seeds, and a functional import apparatus was shown to operate 2 h after imbibition. Interestingly, this import process was best driven by the oxidation of NADH from outside the mitochondrial inner membrane. In later developmental stages the capacity for matrix organic acid metabolism was evident, accompanied by the appearance of proteins for TCA cycle components, and coordination of electron transport chain assembly through components encoded in both mitochondrial and nuclear genomes. Together these events provide new insights into the understanding of mitochondrial maturation and the nature of pro-mitochondrial structures in plant cells.

Blotting, Western↗