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PubMed1997

Functional characterization of human nucleosome assembly protein-2 (NAP1L4) suggests a role as a histone chaperone.

Histones are thought to play a key role in regulating gene expression at the level of DNA packaging. Recent evidence suggests that transcriptional activation requires competition of transcription factors with histones for binding to regulatory regions and that there may be several mechanisms by which this is achieved. We have characterized a human nucleosome assembly protein, NAP-2, previously identified by positional cloning at 11p15.5, a region implicated in several disease processes including Wilms tumor (WT) etiology. The deduced amino acid sequence of NAP-2 indicates that it encodes a protein with a potential nuclear localization motif and two clusters of highly acidic residues. Functional analysis of recombinant NAP-2 protein purified from Escherichia coli demonstrates that this protein can interact with both core and linker histones. We demonstrate that recombinant NAP-2 can transfer histones onto naked DNA templates. Deletion mutagenesis of NAP-2 demonstrates that both NH3- and COOH-terminal domains are required for histone transfer activity. Subcellular localization studies of NAP-2 indicate that it can shuttle between the cytoplasm and the nucleus, suggesting a role as a histone chaperone. Given the potential role of the human NAP-2 gene (HGMW-approved symbol NAP1L4) in WT etiology, we have elucidated the exon/intron structure of this gene and have analyzed the mutational status of NAP-2 in sporadic WTs. Our results, coupled with tumor suppression assays in G401 WT cells, do not support a role for NAP-2 in the etiology of WT. A putative role for NAP-2 in regulating cellular differentiation is discussed.

Amino Acid Sequence↗
PubMed1993

Sequence analysis of the phage 21 genes for prohead assembly and head completion.

Phage 21 is a temperate lambdoid coliphage, and its head-encoding genes, as well as those of phage lambda, are descended from a common ancestral phage. The head protein-encoding genes of phage 21 have been sequenced, confirming earlier genetic studies indicating that the head-encoding genes of 21 and lambda are analogous in location, size, and function. The phage 21 head-encoding genes identified (and their lambda analogues) include: 3(W), 4(B), 5(C), 6(Nu3), shp (D), 7(E), and 8(FII), respectively. An open reading frame, orf1, is analogous in position and shares some sequence identity with FI, a phage lambda gene involved in DNA packaging. The phage 21 major head protein, gp7, is predicted to have strong sequence identity (65%) with the lambda major capsid protein, gpE, including amino acids known to be important for capsid form determination. The nested genes 5/6 of phage 21 and C/Nu3 of lambda differ by several rearrangements including deletions and a triplication. The possibility that lambda genes C/Nu3 evolved from ancestal nested genes containing a triplication is discussed.

Amino Acid Sequence↗
PubMed1998

The late-expressed region of the temperate coliphage 186 genome.

The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesize the complex viral particle and lyse the bacterial host. We report the completion of the DNA sequence of the late region and the assignment of 18 previously identified genes to open reading frames in the sequence. The 186 late region is similar to the late region of phage P2, sharing 26 genes of known function: the single gene for activation of late gene transcription, 6 genes for construction of DNA-containing heads, 16 for tail morphogenesis, and 3 for cell lysis. We identified two 186 late genes with unknown function; one is homologous to previously unrecognised genes in P2, HP1, and phiCTX, and the other may modulate DNA packaging. The 186 late region, like the rest of the genome, lacks the lysogenic conversion genes that are carried by P2, allowing the 186 late region to be transcribed from only three late promoters rather than four. The relative absence of lysogenic conversion genes in 186 suggests that the two phages have evolved to use the lytic and lysogenic reproductive modes to different extents.

Amino Acid Sequence↗
PubMed2003

Expression and purification of a putative H-NS nucleoid-associated protein from the phytopathogen Xylella fastidiosa.

The H-NS protein is one of the major constituents of the nucleoid structure that has been implicated in the DNA packaging and in the global regulation of gene expression. The study of this transcriptional regulator is an effort to fight Xylella fastidiosa, a citrus pathogen responsible for a range of economically important plant diseases, including the citrus variegated chlorosis (CVC). The putative H-NS ORF was cloned into a pET32-Xa/LIC vector in order to overexpress it coupled with fusion tags in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by immobilized metal affinity chromatography (Ni-NTA resin) and its identity verified by mass spectrometry (MALDI-TOF). Final purification was performed by cation-exchange chromatography (SP Sepharose Fast Flow) and the purified protein was found as a single band on SDS-PAGE. The folding and its DNA binding activity were verified by circular dichroism and fluorescence spectroscopy, respectively.

Amino Acid Sequence↗
PubMed2004

The genome of phiAsp2, an actinoplanes infecting phage.

The first genome of a virus infecting a representative of the eubacterial genus Actinoplanes is presented. Phage phiAsp2 has a circularly permutated chromosome that consists of 58,638 bp; its G/C-bias of 70.39% resembles the hosts G + C-content (71-73% within the genus). A total of 76 open reading frames (orfs) were identified, the majority of which (63) displaying equal transcriptional orientations. Functional gene clustering is obvious as orfs coding for head and tail proteins are located close to the center in the first half of the genome and putative DNA-modifying enzymes are encoded by centrally located genes; DNA repair and recombination functions are situated in the remaining part of the genome, adjacent to a small gene cluster, the predicted proteins of which are involved in DNA packaging. Close to the left terminus there are two small regions (approximately 4.5 kb each, separated by 2.8 kb) which are homologous to the recently sequenced mycobacteriophage rosebush, however, the unique overall structure of the phiAsp2-genome does not bear resemblance to any other known viral genome. The nucleotide sequence was deposited in GenBank with the accession no. AY576796.

Acarbose↗
PubMed1996

Engineering a mini-herpesvirus as a general strategy to transduce up to 180 kb of functional self-replicating human mini-chromosomes.

The engineering of therapeutic human artificial episomal chromosomes, HAECs, requires the development of strategies to deliver large functional self-replicating extrachromosomal DNA in target cells. Members of the herpesviral family are among the largest episomal double-stranded DNA viruses. As model systems of this family of endemic infectious agents, vectors derived from the human herpes 4 Epstein-Barr virus (EBV) were constructed which transferred up to 180 kb of DNA packaged as infectious virions. Such a transduction strategy was based on a non-oncogenic helper-dependent mini-EBV carrying minimal cis elements for latent replication and virus production. After exposure of human B lymphoma and lymphoblastoid cells to mini-EBVs transducing lacZ and human HPRT minigenes, stable cell transformants were selected which carried the delivered multimeric linear DNAs as circular episomes up to 160-180 kb in size. Following transduction of Lesch-Nyhan disease cells with a mini-EBV/HPRT, normal human HPRT function was restored in cells carrying large episomal HPRT minigenes. Direct visualization of the therapeutic mini-EBV by fluorescent in situ hybridization (FISH) on metaphase and interphase nuclei indicated that 99% (556/563) of the transduced mini-EBV DNA was episomal with an average copy number of one to two per nucleus. This system should allow the delivery of large genes in common diseases such as hemophilia A and codelivery of multiple genes in cells from polygenic diseases such as cancer. The extrachromosomal mini-EBV-based strategy offers an alternative to integrative or non-replicating gene therapy infectious vectors, which may be generally applicable to other herpesviruses characterized by different tropisms.

B-Lymphocytes↗
PubMed2005

Sequence analysis of the Lactobacillus plantarum bacteriophage PhiJL-1.

The complete genomic sequence of a Lactobacillus plantarum virulent phage PhiJL-1 was determined. The phage possesses a linear, double-stranded, DNA genome consisting of 36,677 bp with a G+C content of 39.36%. A total of 52 possible open reading frames (ORFs) were identified. According to N-terminal amino acid sequencing and bioinformatic analyses, proven or putative functions were assigned to 21 ORFs (41%), including 5 structural protein genes. The PhiJL-1 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication, DNA packaging, head and tail morphogenesis, and lysis. This type of modular genomic organization was similar to several other phages infecting lactic acid bacteria. The structural gene maps revealed that the order of the head and tail genes is highly conserved among the genomes of several Siphoviridae phages, allowing the assignment of probable functions to certain uncharacterized ORFs from phage PhiJL-1 and other Siphoviridae phages.

Amino Acid Sequence↗
PubMed2005

Analysis of the histone H3 gene family in Arabidopsis and identification of the male-gamete-specific variant AtMGH3.

Histones are major components of chromatin, the protein-DNA complex involved in DNA packaging and transcriptional regulation. Histone genes have been extensively investigated at the genome level in animal systems and have been classified as replication dependent, replication independent or tissue specific. However, no such study is available in a plant system. In this paper we report that there are 15 histone H3 genes in the Arabidopsis genome, including five H3.1 genes, three H3.3 genes and five H3.3-like genes. A gene structure analysis revealed that gene duplication causes redundancy of the histone H3 genes. The expression of one of the H3 genes, termed AtMGH3/At1g19890, is cell-specific, being restricted to the generative and sperm cells of Arabidopsis pollen as shown by in situ hybridisation and reporter gene analysis. Thus, we conclude that in Arabidopsis, AtMGH3 is a male-gamete-specific histone H3 gene. A T-DNA insertion line for AtMGH3 revealed decreased expression and ectopic RNA splicing. The T-DNA insertion lines for AtMGH3/At1g19890 and other H3 genes revealed a normal growth phenotype and reproductive fertility. These findings suggest that other H3 genes are likely to compensate for the T-DNA-insertion-induced loss of a single H3 gene because of the high redundancy of these genes in the Arabidopsis genome. These T-DNA mutant lines should be useful for accumulating different H3 gene mutations in a single plant and for studying replication-dependent and replication-independent H3 genes and the specific role of AtMGH3 in chromatin remodelling and transcriptional regulation during development of male gametes.

Amino Acid Sequence↗
PubMed1996

A protein linkage map of Escherichia coli bacteriophage T7.

Genome sequencing projects are predicting large numbers of novel proteins, whose interactions with other proteins must mediate the function of cellular processes. To analyse these networks, we used the yeast two-hybrid system on a genome-wide scale to identify 25 interactions among the proteins of Escherichia coli bacteriophage T7. Among these is a set of six interactions connecting proteins that function in DNA replication and DNA packaging. Remarkably, two genes, arranged such that one entirely overlaps the other and uses a different reading frame, encode interacting proteins. Several of the interactions reflect intramolecular associations of different domains of the same polypeptide, suggesting that the two-hybrid assay may be useful in the analysis of protein folding. This global approach to protein-protein interactions may be applicable to the analysis of more complex genomes whose sequences are becoming available.

Amino Acid Sequence↗
PubMed1998

Functional implications of the structure of the murine parvovirus, minute virus of mice.

BACKGROUND: Minute virus of mice (MVM) is a single-stranded (ss) DNA-containing, murine parvovirus with a capsid built up of 60 icosahedrally related polypeptide chains, each of which consists of the C-terminal region common to two structural proteins, VP1 and VP2. In infectious virions, most VP2 molecules are cleaved to VP3 by the removal of about 20 amino acids from the N terminus. Of the 587 amino acids in VP2, approximately half are identical to those in the analogous capsid protein of the antigenically distinct canine parvovirus (CPV), the crystal structure of which has previously been determined. The three-dimensional structure determination of MVMi (the immunosuppressive strain of MVM) was previously reported to 3.5 A resolution. RESULTS: We report here an analysis of the MVMi virus structure and provide insights into tissue tropism, antigenicity and DNA packaging. Amino acids determining MVM tissue tropism were found to cluster on, or near, the viral surface. A conserved, glycine-rich, N-terminal peptide was seen to run through a cylindrical channel along each fivefold axis and may have implications for antigenicity. Density within the virion was interpreted as 29 ssDNA nucleotides per icosahedral asymmetric unit, and accounts for over one-third of the viral genome. CONCLUSIONS: The presence of the glycine-rich sequence in the fivefold channels of MVMi provides a possible mechanism to explain how the unique N-terminal region of VP1 becomes externalized in infectious parvovirions. Residues that determine tropism may form an attachment recognition site for a secondary host-cell factor that modulates tissue specificity. The ordering of nucleotides in a similar region of the interior surface in the CPV and MVMi capsids suggests the existence of a genomic DNA-recognition site within the parvoviral capsid.

Amino Acid Sequence↗
PubMed1996

The complete DNA sequence and genomic organization of the avian adenovirus CELO.

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.

Adenoviruses, Human↗
PubMed2005

[Sequencing and analysis of Vibrio cholerae typing phage VP3 genome].

DNA sequence and the genome of phage VP3 (a typing phage of V. cholera) were analyzed. A random library of VP3 DNA was constructed by shot-gun library method. The VP3 genome sequence was assembled with contigs sequences, the gaps between different contigs were filled with sequencing data from primer walking. ORFs were predicted; Phylogeny of DNA polymerase sequences was analyzed to determine the class of VP3; The activity of putative promoter genes were analyzed using lacZ report system. VP3 genome is a 39504bp of circular double-stranded DNA. Twenty-seven out of forty-nine putative ORFs were annotated; twenty gene products were homologous with T7-like phages, including DNAP, DNA replicative protein, capsid, tail tubular, tail fiber protein, and DNA packaged protein. The activity of the putative promoter regions was confirmed through cloning those regions to LacZ-fuse plasmid pRS1274 and analysis of the expression of beta galactosidase. The complete genomic sequence of VP3 and phylogenetic tree analysis suggests VP3 is a member of T7 phage family.

Bacteriophage T7↗
PubMed2006

Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life.

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.

Amino Acid Sequence↗
PubMed1997

Subcellular compartmentalization of adeno-associated virus type 2 assembly.

Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformation-specific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.

Cell Compartmentation↗
PubMed1996

Isolation of Borrelia burgdorferi genes encoding homologues of DNA-binding protein HU and ribosomal protein S20.

Linear DNA with covalently closed ends is the predominant form of DNA in the spirochaete Borrelia burgdorferi. All bacteria examined to date have small DNA-binding proteins related to the Escherichia coli IHF and HU proteins that appear to play roles in DNA compaction and replication, but such proteins had not been isolated from bacteria with linear genomes. We found a single gene in B. burgdorferi (named hbb) whose product (named Hbb) complements the defects for gamma DNA packaging found in E. coli strains mutant in the genes for IHF and HU. The sequence of the predicted B. burgdorferi protein is similar to those of HU and IHF-like proteins in other bacteria. The gene appears to be in an operon with the order rpsT-hbb-orfH, where the rpsT gene is a homologue of the E. coli gene encoding ribosomal protein S20 and the orfH gene encodes a protein of unknown function. This operon is located upstream of the previously identified B. burgdorferi rho homologue.

Amino Acid Sequence↗
PubMed2006

Free and chromatin-associated mono-, di-, and trimethylation of histone H4-lysine 20 during development and cell cycle progression.

Methylation of specific amino acids in histone tails is responsible for packaging DNA into condensed, repressed chromatin, and into open chromatin that is accessible to the transcription machinery. Monomethylation and trimethylation of histone H4-lysine 20 (H4-K20) control the formation of repressed chromatin. Using antibodies that specifically recognize the three methyl marks of histone H4-K20, we characterized their regulation during the cell cycle and throughout development. We find free mono- and trimethylated histone H4-K20 in unfertilized Drosophila eggs and in S2 tissue culture cells. Soluble mono-. di-, and trimethylated H4-K20 are also found in HeLa cells. These soluble modified histones may represent a pool of free histones that can rapidly be incorporated into chromatin. The three methyl marks are each regulated differentially during development and their detection on western blots does not overlap with their detection on chromosomes. Monomethylated H4-K20 is detected on condensed chromosomes throughout development, while di- and trimethylated H4-K20 are detected on metaphase chromosomes at specific stages. Our results suggest that the detection of methylated H4-K20 on chromosomes may reveal chromatin packaging rather than the distribution of the methyl marks.

Animals↗
PubMed1996

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.

Animals↗
PubMed2003

H-NS in Gram-negative bacteria: a family of multifaceted proteins.

DNA-packaging and the control of gene expression constitute a major challenge for bacteria to survive and adapt to environmental changes. The use of multiple strategies to solve these problems could explain the presence of various nucleoid-associated proteins in bacteria. H-NS, one of these proteins, has been extensively studied in Escherichia coli, and a variety of phenotypes have been associated with a mutation in its structural gene. However, the role of H-NS in bacterial physiology and its mechanism of action are still a matter of debate. The expanding number of H-NS-related proteins identified in Gram-negative bacteria reveals interesting clues about their structure-function-evolution relationship.

Bacterial Proteins↗