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PubMed1983

Topology of zigzag chromatin.

An enormous length of DNA is packaged in the nuclei of eukaryotic cells. This is achieved through several intermediate levels of compaction, ranging from the double helix to the chromosome. The nucleosome is now firmly established as the first level of chromatin structure. Next it appears that the nucleosomes are themselves stacked in a two-track array, with a dinucleosome repeat. Several winding patterns of DNA are compatible with such a structure. It is shown here that, compared to other feasible DNA paths, the observed winding pattern has remarkable topological properties. The possible biological significance of this peculiarity is discussed.

Chromatin↗
PubMed1999

Packaging cell lines for simian foamy virus type 1 vectors.

Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.

Animals↗
PubMed1986

The production of generalized transducing phage by bacteriophage lambda.

Generalized transduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage lambda to assess some of the factors that affect that process. The results of those experiments indicate: the production of generalized transducing particles by bacteriophage lambda is inhibited by the phage lambda exonuclease (Exo). Also inhibited by lambda Exo is the production of lambda docR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of lambda docL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing particles are not detected in induced lysis-defective (S-) lambda lysogens until about 60-90 min after prophage induction. Since wild-type lambda would normally lyse cells by 60 min, the production of lambda-generalized transducing particles depends on the phage being lysis-defective; if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10-20-fold range. In contrast, if transducing lysates are prepared by the induction of a lambda lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers; if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA. These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by lambda, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather recognize some feature of the DNA tht is sensitive to lambda exonuclease, such as a nick or a double-stranded cut.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage lambda↗
PubMed1993

Mutagenicity of methyl methanesulfonate (MMS) in vivo at the Dlb-1 native locus and a lacI transgene.

Methyl methanesulfonate (MMS) is an extraordinarily poor mutagen compared to ethylnitrosourea (ENU) or even X-rays. In lung fibroblasts in vivo, MMS has been shown to induce many micronuclei but few, if any, mutations at the hpt locus. We wondered if the lack of mutations might be due to the lack of division and DNA synthesis in fibroblasts in vivo, which would permit substantial time for differential repair of DNA lesions. This idea was tested in the small intestine, a tissue in which the cells are actively dividing. Two loci were examined: a native locus (Dlb-1) which determines the presence or absence of a lectin binding site on the surface of the epithelial cells, and a lacl transgene which controls beta-galactosidase synthesis. Locl mutations were detected after in vitro packaging of DNA isolated from the intestinal epithelium into lambda phage and expression in suitable bacteria. Although the epithelial cells are proliferating, acute treatments produced no significant increase in mutations at either locus. Subacute treatments produced low but significant increases in mutation frequency at both loci. The results confirm that MMS is a far more potent clastogen than it is a mutagen and should be regarded as a super-clastogen in the same manner as ENU is a super-mutagen. The carcinogenicity of MMS is probably the result of its potent clastogenicity rather than its weak activity as a point mutagen.

Animals↗
PubMed2003

Crystal structure of the SF3 helicase from adeno-associated virus type 2.

We report here the crystal structure of an SF3 DNA helicase, Rep40, from adeno-associated virus 2 (AAV2). We show that AAV2 Rep40 is structurally more similar to the AAA(+) class of cellular proteins than to DNA helicases from other superfamilies. The structure delineates the expected Walker A and B motifs, but also reveals an unexpected "arginine finger" that directly implies the requirement of Rep40 oligomerization for ATP hydrolysis and helicase activity. Further, the Rep40 AAA(+) domain is novel in that it is unimodular as opposed to bimodular. Altogether, the structural connection to AAA(+) proteins defines the general architecture of SF3 DNA helicases, a family that includes simian virus 40 (SV40) T antigen, as well as provides a conceptual framework for understanding the role of Rep proteins during AAV DNA replication, packaging, and site-specific integration.

Amino Acid Motifs↗
PubMed1992

Molecular cloning of the cDNA encoding a 42 kDa antigenic polypeptide of Anisakis simplex larvae.

The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from A. simplex larvae was ligated into phage vector lambda gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing cDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.

Amino Acid Sequence↗
PubMed2001

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer.

Two major pathways of recombination-dependent DNA replication, "join-copy" and "join-cut-copy," can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.

Amino Acid Sequence↗
PubMed1999

DNA-interacting proteins in the spermiogenesis of the mollusc Murex brandaris.

Sperm chromatin of Murex brandaris (a neogastropod mollusc) undergoes a series of structural transitions during spermiogenesis. The DNA-interacting proteins responsible for these changes as well as the mature protamines present in the ripe sperm nucleus have been characterized. The results reveal that spermiogenic nuclear proteins are protamine precursors that are subjected to a substantial number of small N-terminal deletions that gradually modify their overall charge. The composition of mature protamines is remarkably simple in turn, promoting an efficient and extremely tight packaging of DNA. The pattern of spermiogenic chromatin condensation in M. brandaris clearly departs from that corresponding to vertebrate chromatin.

Amino Acid Sequence↗
PubMed1966

Ultrastructure and cytochemistry of metabolic DNA in Tipula.

A DNA body is present in the females of the fly Tipula oleracea and is formed in contact with the sex chromosomes in the oogonial interphases. At each oogonial mitosis, the DNA body follows the chromosomes to one anaphase group and is included in one of the telophase nuclei. The body increases appreciably in size during the interphase of meiosis. All oocytes have the body, but only a few nurse cells possess it. The DNA body synthesizes its DNA at a different time than the chromosomes, as is shown by incorporation of tritiated thymidine, and contains 59% of the DNA of the nucleus, as is disclosed by spectrophotometric measurements. At late diplotene the DNA body disintegrates, releasing its DNA into either the nucleus or the cytoplasm. When studied in the electron microscope, the DNA body appears composed of a tight mass of intertwined fibrils. Demonstration that the main mass of the body is composed of DNA is obtained from cytochemical tests which reveal that the DNA body is Feulgen positive, stains green with azure B, incorporates H(3)-thymidine, and after digestion with DNase is Feulgen negative. The DNA of the body is complexed with histone, like the DNA of the chromosomes, as is revealed by an intense alkaline fast green staining. Electron microscope examination of oocytes reveals that one side of the DNA body is in close contact with the nuclear envelope and that the other side possesses an outer shell composed mainly of particles 150 to 250 A in diameter. Between the outer shell and the chromosomes there is a band of low electron opacity, 4000 to 7000 A thick. In the light microscope, this light band together with the outer shell is Feulgen negative and stains violet with azure B; this is confirmation of the presence of RNA. In the oocytes the nucleoli are found inside the DNA body. These nucleoli have a nucleolonema composed mainly of particles 150 to 250 A. The nucleoli are Feulgen negative, alkaline fast green negative, stain violet with azure B, and do not stain with azure B after RNase digestion, thus confirming their RNA content. The presence of the nucleoli inside the DNA body and of a band of RNA between the body and the chromosomes is indicative of a high RNA synthetic activity. Since the DNA of the body is complexed with histone, as in the chromosomes, and the nucleoli are located inside the body, the simplest interpretation of the DNA body is that it represents hundreds of copies of the operons of the nucleolar organizing region or neighboring regions. The situation found in Tipula has several basic features in common with the polytene chromosomes of other Diptera and with the hundreds of nucleoli present in Triturus oocytes. In all three cases, genes seem to be copied hundreds of times but are kept in different types of packages. A DNA body like the one in Tipula oleracea is found in other species of Diptera and in the Coleoptera. There is no indication, from the present investigation, that the DNA body is in any way associated with a virus.

Cell Division↗
PubMed1997

[Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121].

A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017.

Cloning, Molecular↗
PubMed1985

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light.

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct. On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v. mutagenesis.

Bacteriophage lambda↗
PubMed1986

DNA-supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin.

Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces 'packaging' of the DNA at higher concentrations. This was concluded from studies using the sensitive 4,5',8-trimethylpsoralen photobinding technique [Sinden, R. R., Carlson, J. O. & Pettijohn, D.-E. (1980) Cell 21, 773-783]. Relaxed DNA is not affected by tyrocidine whereas linearized molecules become packaged. The linear gramicidin synthesized by the same strain reverses the tyrocidine-induced relaxation as well as the packaging, an observation which might be of biological relevance.

DNA, Superhelical↗
PubMed2002

cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site.

BACKGROUND: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin. Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function. The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins. This centromeric heterochromatin is required for cohesion between sister centromeres. Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation. The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established. RESULTS: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast. Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus. This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site. In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9. CONCLUSIONS: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.

Binding Sites↗
PubMed1996

Species specificity for transduction of cultured cells by a recombinant LuIII rodent parvovirus genome encapsidated by canine parvovirus or feline panleukopenia virus.

We previously reported that a recombinant genome derived from the autonomous rodent parvovirus LuIII could be pseudotyped with capsids of the closely related viruses, H1 and minute virus of mice. To determine whether this was also possible with less related viruses, LuIII recombinant genomes containing a luciferase reporter were cotransfected into permissive cells together with plasmids expressing the capsid proteins of either feline panleukopenia virus (FPV) or its host range variant, canine parvovirus (CPV). We observed efficient packaging of the recombinant DNA into transducing virions that displayed the cell tropism of the virus that supplied the capsid. Thus, the FPV- and CPV-pseudotyped virions were able to transduce a feline cell line but they showed no transducing activity for the human NB324K line, which is permissive for LuIII. The transducing activity of the pseudotyped viruses was not inhibited by neuraminidase treatment of the permissive recipient cells, in contrast to that of virions packaged using LuIII capsid proteins. Furthermore, canine A72 cells (permissive for CPV but not FPV) were efficiently transduced by CPV-packaged but not by FPV-packaged LuIII recombinant genomes. Pseudotyped recombinants will be useful for elucidating parvovirus host range determinants since they enable the packaged DNA and each of the capsid proteins to be supplied independently. They should also facilitate control over the targeting of parvovirus vectors for gene transfer.

Animals↗
PubMed1994

[Construction of human glioma cDNA library].

Total RNA from a human glioma was extracted by acid guanidinium-thiocyanate-chloroform one step method, and poly(A+)RNA was separated by affinity chromatography on oligo (dT) cellulose. The cDNA was synthesized by using poly (A+) RNA as template, the length of cDNA being 0.2-5 kb. The glioma cDNA inserts could be cloned into lambda gt11 vector, the resulting recombinant DNA was packaged in vitro. The cDNA library of 1.12 x 10(5) pfu/ml was obtained, and the cloning efficiency was 4.8 x 10(3)/ng cDNA. This library might contribute an important basis to study of the structure and function of human glioma genes.

Brain Neoplasms↗
PubMed1991

Inhibition of 5S RNA transcription in vitro by nucleosome cores with low or high levels of histone acetylation.

Nucleosomes exert strong inhibitory effects on gene transcription in vitro and in vivo. Since most DNA is packaged in nucleosomes, there must exist mechanisms to alleviate this inhibition during gene activation. Nucleosomes could be destabilized by histone acetylation which is strongly correlated with gene expression. We have compared the effects of nucleosomes cores with low or high levels of histone acetylation on 5S RNA transcription with Xenopus nuclear extracts in vitro. Little or no difference was observed over a range of 1 to 15 nucleosome cores per plasmid template. This result suggests that nucleosomal DNA is not more accessible to transcription factors and to the transcription machinery in acetylated nucleosomes.

Acetylation↗
PubMed1995

Expression of an avian protamine in transgenic mice disrupts chromatin structure in spermatozoa.

The objective of this study was to determine the consequences of disrupting spermatozoal chromatin condensation on spermatozoal development and function. The avian protamine, galline, was targeted to spermatids of transgenic mice using the mouse protamine 1 gene promoter. Three transgenic mouse lines were established that expressed galline mRNA at 65%, 120%, and 185% of the level found in rooster testis. Galline mRNA accumulated in round spermatids to levels similar to that of mouse protamine and, as with the mammalian counterpart, translation was delayed until the elongating spermatid stage. Protein gels revealed that galline accumulated in mature spermatozoa whereas mouse protamines were reduced, suggesting that galline competes with protamines for binding to spermatozoal DNA. Acridine orange binding analysis indicated that DNA of the transgenic spermatozoa was not as tightly packed as that of controls. This was corroborated by electron microscopy, which revealed disruption of the normal dense chromatin structure of spermatozoal heads. Despite these perturbations of chromatin condensation, the transgenic spermatozoa were functionally normal, as the majority of transgenic mice had normal fertility. However, in mice that expressed excessive galline, there was a gradual destruction of seminiferous tubules leading to infertility. Our findings suggest that very precise packaging of DNA in germ cells may not be essential for subsequent unpackaging in the pronucleus of fertilized eggs and for subsequent normal development of the embryo.

Animals↗