Random DNA Fragments Research Articles

Late-Onset Chronic Inflammatory Encephalopathy in Immune-Competent and Severe Combined Immune-Deficient (SCID) Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.

DNA organization and length polymorphism at the 2L telomeric region of Anopheles gambiae.

The molecular structure of the telomeric region at the left arm of the second chromosome of the mosquito Anopheles gambiae has been determined in the transformed strain G418 that contains a pUChsneo transgene attached at the 2L chromosome end, and in the Pink eye laboratory strain (PE). Both strains contain the same complex satellite positioned distal to a unique region. FIGE mapping of the telomeric region of the PE strain revealed distinct DNA fragment lengths that segregated with individual chromosomes. Genomic DNA fragments were cloned from the 2L telomeric region, which accounted for about half of 2L chromosomes in the PE population. In all three cases studied, long fragments of different middle repetitive sequences were found attached to the distal ends of the 2L satellite. We propose that random fragments of DNA may be occasionally added during recombination between complex satellite repeats at the chromosome ends.

Rapid cloning of mouse DNA as yeast artificial chromosomes by transformation-associated recombination (TAR).

Rapid cloning of mouse DNA as yeast artificial chromosomes by transformation-associated recombination (TAR).

Uptake and processing of DNA by Acinetobacter calcoaceticus--a review.

In natural transformation, DNA in the form of macromolecular fragments can be translocated across the cell envelope of prokaryotic microorganisms. During the past two decades, several, largely mutually contradictory, hypotheses have been forwarded to explain the molecular mechanism and bioenergetics of this translocation process. Other biomacromolecules are translocated across the bacterial cell envelope as well, such as polysaccharides and proteins, the latter for instance in the process of the assembly of type-IV pili. This brings up the question whether or not common components are involved. Here, we review analyses of DNA translocation in Acinetobacter calcoaceticus, a Gram-negative eubacterium that is able to migrate through twitching motility, and also shows a high frequency of natural transformation. DNA uptake in this organism is an energy-dependent process. Upon entry into the cells, the DNA fragments are integrated into the resident chromosome when a sufficiently large region of mutual homology is available (200 to 400 bp). However, this process is rather inefficient, and on the average 500 bp of each incoming fragment is degraded through exonuclease activity. Upon covalent attachment of a bulky protein molecule to the transforming DNA, the DNA-translocation machinery becomes blocked in further translocation activity. Since A. calcoaceticus is not well suited for transposon mutagenesis, a random mutagenesis procedure has been developed, based on the ligation of an antibiotic-resistance marker to random fragments of chromosomal DNA. This method was used to generate several mutants impaired in the natural transformation process. Three of these have been characterized in detail. No components, common to the translocation of macromolecules through the cell envelope of Acinetobacter, have been detected in this screen.

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes.

Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/micrograms of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional beta-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.

Determinants of chromatin disruption and transcriptional regulation instigated by the thyroid hormone receptor: hormone-regulated chromatin disruption is not sufficient for transcriptional activation.

Chromatin disruption and transcriptional activation are both thyroid hormone-dependent processes regulated by the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). In the absence of hormone, TR-RXR binds to nucleosomal DNA, locally disrupts histone-DNA contacts and generates a DNase I-hypersensitive site. Chromatin-bound unliganded TR-RXR silences transcription of the Xenopus TRbetaA gene within a canonical nucleosomal array. On addition of hormone, the receptor directs the extensive further disruption of chromatin structure over several hundred base pairs of DNA and activates transcription. We define a domain of the TR protein necessary for directing this extensive hormone-dependent chromatin disruption. Particular TR-RXR heterodimers containing mutations in this domain are able to bind both hormone and their thyroid hormone receptor recognition element (TRE) within chromatin, yet are unable to direct the extensive hormone-dependent disruption of chromatin or to activate transcription. We distinguish the hormone-dependent disruption of chromatin and transcriptional activation as independently regulated events through the mutagenesis of basal promoter elements and by altering the position and number of TREs within the TRbetaA promoter. Chromatin disruption alone on a minichromosome is shown to be insufficient for transcriptional activation of the TRbetaA gene.

Extracellular DNA from Serpulina hyodysenteriae consists of 6.5 kbp random fragments of chromosomal DNA

Preparations of chromosomal DNA from a number of Serpulina hyodysenteriae strains have shown, using agarose gel electrophoresis, the presence of an additional band with a mobility similar to that of a 6.5 kbp linear DNA fragment. Analysis showed that this is not a plasmid but rather a form of extracellular DNA like that observed for Gram-negative bacteria. However, unlike the extracellular DNA from Gram-negative bacteria, which showed a similar band profile to that of the DNA from whole cells, that from S. hyodysenteriae consisted primarily of fragments of a fixed 6.5 kbp.

Complete DNA sequence of canine adenovirus type 1.

The complete DNA sequence of a field strain of canine adenovirus type 1 was determined by sequencing random fragments of viral DNA cloned into pBluescript. The virus has a genome of 30536 bp flanked by two identical 161 bp inverted terminal repeats. Thirty ORFs have been identified, based on genomic location or sequence identity with published adenoviruses. These are arranged into similar discrete regions found in the human adenoviruses. ORFs in the late region show greatest identity with published human adenovirus sequences, whereas the E3 and E4 ORFs show little or none.

A survey of the Trypanosoma brucei rhodesiense genome using shotgun sequencing

A comparison of the efficiency of sequencing random genomic DNA fragments versus random cDNAs for the discovery of new genes in African trypanosomes was undertaken. Trypanosome DNA was sheared to a 1.5–2.5 kb size distribution, cloned into a plasmid and the sequences at both ends of 183 cloned fragments determined. Sequences of both kinetoplast and nuclear DNA were identified. New coding regions were discovered for a variety of proteins, including cell division proteins, an RNA-binding protein and a homologue of the Leishmania surface protease GP63. In some cases, each end of a fragment was found to contain a different gene, demonstrating the proximity of those genes and suggesting that the density of genes in the African trypanosome genome is quite high. Repetitive sequence elements found included telomeric hexamer repeats, 76 bp repeats associated with VSG gene expression sites, 177 bp satellite repeats in minichromosomes and the Ingi transposon-like elements. In contrast to cDNA sequencing, no ribosomal protein genes were detected. For the sake of comparison, the sequences of 190 expressed sequence tags (ESTs) were also determined, and a similar number of new trypanosomal homologues were found including homologues of another putative surface protein and a human leucine-rich repeat-containing protein. We conclude from this analysis and our previous work that sequencing random DNA fragments in African trypanosomes is as efficient for gene discovery as is sequencing random cDNA clones.

Preferential condensation of SAR-DNA by histone H1 and its SPKK containing octapeptide repeat motif

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA·dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI- SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3–4-fold higher than that of the random DNA fragment. A 16-mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C-terminus of histone H1d, which has recently been shown to possess DNA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1-SAR DNA interaction through SPKK containing peptide motifs and its competition by AT-hook peptides present in the nonhistone chromosomal proteins like HMG-I and HMG-Y.

A whole genome shotgun gene fusion method for isolation of translation initiation sites in Escherichia coli: identification of Haemophilus influenzae translation initiation sites in E. coli.

We have developed a new method for isolating translation initiation sites based on the expression of Haemophilus influenzae Rd gene fusions with the Escherichia coli galactokinase (galK) gene. We cloned random DNA fragments of H. influenzae Rd DNA into a plasmid vector containing the galK coding sequence from which the translation initiation site (the ribosome binding site and translation initiation codon) had been removed. A subset of the cloned DNA fragments contained translation initiation sites that, when fused to the galK gene, produced active galactokinase and complemented the host galK mutation. Molecules expressing galactokinase activity were isolated and characterized by DNA sequence analysis, and the sequences were aligned with the recently completed whole genomic sequence of H. influenzae Rd. Translation initiation sites for known, hypothetical, and new genes were identified. Translation initiation sites internal to the coding sequences of a number of genes were identified, suggesting that internal translation initiation sites are common, especially in large genes. This shotgun method provides functional information on translation initiation sites and helps to define gene coding sequences.

A novel SR-related protein specifically interacts with the carboxy-terminal domain (CTD) of RNA polymerase II through a conserved interaction domain.

The largest subunit of the RNA polymerase II (pol II) contains at the carboxy-terminus a peculiar repetitive sequence that consists of 52 tandem repeats of the consensus motif Tyr-Ser-Pro-Thr-Ser-Pro-Ser, referred to as the C-terminal domain (CTD). Upon transcriptional initiation/promoter clearance, the CTD becomes extensively phosphorylated and apparently remains so during elongation. While the underphosphorylated CTD plays a role in transcriptional initiation, recent evidence couples the highly phosphorylated CTD to RNA processing, namely polyadenylation and splicing. Using a yeast two-hybrid screen, we have selected for human proteins that interact with the CTD of RNA polymerase II. The CTD-GAL fusion protein used as a bait is highly phosphorylated in yeast and, accordingly, we did not isolate proteins implicated in transcriptional regulation but rather proteins with possible roles in RNA splicing. One major cDNA clone isolated this way encodes SRrp129/CASP11, a protein that contains a conserved CTD-interaction domain at the C-terminus and an internal serine-arginine rich domain (SR domain). Proteins of the SR family have been implicated in RNA splicing, notably in the regulation of alternative splicing. Thus we consider it likely that SRrp129 is an auxiliary splice factor. We also improved our method to quickly map domains involved in protein-protein interaction (Stagljar et al., 1996, BioTechniques 21, 430-432). Instead of using sonication for the production of a random DNA fragment library, we took advantage of the fact that DNAse I in the presence of manganese (II) produces double strand rather than single strand DNA breaks. The DNA fragment library of the SRrp129 clone was then used in the yeast two-hybrid system to identify the 100-amino acid domain that interacts with the CTD of RNA polymerase II.

Improved "activator trap" method for the isolation of transcriptional activation domains from random DNA fragments.

We have previously developed an "activator trap" method for selective isolation of activation domains from viral and cellular transcription factors. In this method, random sonicated DNA fragments were ligated next to the DNA binding domain (DBD) of the GAL4 factor in a plasmid that also contained a simian virus 40 (SV40) replication origin. A library of such random insert plasmids was transfected into a monkey cell line (CV-1-5GT), which had been stably transformed with a GAL4-inducible SV40 T-antigen gene. Chimeric GAL factors with a heterologous activation domain were harvested after selective replication in these CV-1-5GT cells. Here we report a simplification and generalization of the "activator trap" method. First, the time-consuming library construction step can be omitted by direct transfection of the sonicated DNA fragments and the linearized recipient plasmid vector into CV-1-5GT cells to obtain chimeric GAL-activation factors by in vivo ligations. Second, the dependence on CV-1-5GT cells can be bypassed by direct co-transfection of all components, including a plasmid carrying the T-antigen gene into cells other than CV-1-5GT. This latter step allows the application of the method to cultured human cells, as demonstrated with the human B-cell line BJA-B.

Conversion of bacterial gene products to secretion-competent fusion proteins.

We describe an efficient and easy procedure that allows the generation, detection and secretion of foreign proteins by the secretion apparatus of E. coli hemolysin. The gene (or gene fragment) encoding the foreign protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyAs). Generally, the expressed fusion is efficiently secreted into the culture supernatant of the producing strain. The new approach allows the direct generation of fusion proteins from genomic DNA fragments. The successful use of this method is demonstrated by cloning of random chromosomal DNA fragments from Salmonella typhimurium.

The cyanobacterial toxin microcystin-LR induced DNA damage in mouse liver in vivo

Microcystin-LR (MCLR) is a potent cyclic heptapeptidic hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. Hepatotoxic and other toxic manifestations of MCLR are well documented. However, information on genotoxicity of MCLR is limited. The present investigation addresses the DNA damage induced by MCLR in mouse liver in vivo. The DNA strand breaks were measured by the fluorimetric analysis of DNA unwinding (FADU). MCLR at 0.5, 1.0 and 2.0 LD 50 doses exhibited a dose and time-dependent DNA damage accompanied by similar effects on various enzymes of hepatic origin, e.g. lactate dehydrogenase, alkaline phosphatase and gamma glutamyl transferase. MCLR-induced genomic DNA fragmentation was also assessed qualitatively by agarose gel electrophoresis. MCLR induced random DNA fragmentation and DNA degradation. Glutathione (GSH) pretreatment significantly extended the survival time of animals exposed to 1.0 LD 50 MCLR but offered only partial protection with regard to DNA damage. The DNA damage observed in the present study can be ascribed to activation of endonucleases.

Characterization of four dispersed repetitive DNA sequences fromZea maysand their use in constructing contiguous DNA fragments using YAC clones

We have isolated four repetitive DNA fragments from maize DNA. Only one of these sequences showed homology to sequences within the EMBL database, despite each having an estimated copy number of between 3 x 104 and 5 x 104 per haploid genome. Hybridization of the four repeats to maize mitotic chromosomes showed that the sequences are evenly dispersed throughout most, but not all, of the maize genome, whereas hybridization to yeast colonies containing random maize DNA fragments inserted into yeast artificial chromosomes (YACs) indicated that there was considerable clustering of the repeats at a local level. We have exploited the distribution of the repeats to produce repetitive sequence fingerprints of individual YAC clones. These fingerprints not only provide information about the occurrence and organization of the repetitive sequences within the maize genome, but they can also be used to determine the organization of overlapping maize YAC clones within a contiguous fragment (contigs). Key words : maize, repetitive DNA, YACs.

DNA polymorphism among Fusarium oxysporum f.sp. elaeidis populations from oil palm, using a repeated and dispersed sequence ``Palm''

A worldwide collection, of 76 F. oxysporum f.sp. elaeidis isolates (Foe), and of 21 F. oxysporum isolates from the soil of several palm grove was analysed by RFLP. As a probe, we used a random DNA fragment (probe 46) from a genomic library of a Foe isolate. This probe contains two different types of sequence, one being repeated and dispersed in the genome "Palm", the other being a single-copy sequence. All F. oxysporum isolates from the palm-grove soils were non-pathogenic to oil palm. They all had a simple restriction pattern with one band homologous to the single-copy sequence of probe 46. All Foe isolates were pathogenic to oil palm and they all had complex patterns due to hybridization with "Palm". This repetitive sequence reveals that Foe isolates are distinct from the other F. oxysporum palm-grove soils isolates. The sequence can reliably discriminate pathogenic from non-pathogenic oil palm isolates. Based on DNA fingerprint similarities, Foe populations were divided into ten groups consisting of isolates with the same geographic origin. Isolates from Brazil and Ecuador were an exception to that rule as they had the same restriction pattern as a few isolates from the Ivory Coast, suggesting they may originated from Africa.

Application of linker-ligation-PCR for construction of phage display epitope libraries

An efficient method for construction of random epitope libraries using filamentous phage is described. Random DNA fragments generated by DNase I digestion were blunt ended by T4 DNA polymerase and ligated with a 12-mer linker, followed by PCR amplification using the same oligonucleotide linker as primers. The results showed that only the ligated product containing linker sequences on both ends was specifically amplified. When the linker ligated-PCR (LL-PCR) product was used for the construction of phage display epitope libraries, the total number of independent clones in the libraries was increased by 100 to 1000 fold in comparison to that obtained for libraries constructed using other methods. In addition, the LL-PCR strategy also increased the probability of isolating recombined DNA fragments which are derived by random in-frame ligation of two or more discontinuous peptide-coding sequences before being inserted into the display vector. Such randomly recombined DNA fragments might be useful in defining conformational epitopes.

Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis.

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alternations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385-388). We now show that a significant number of striatal and cortical neurons, exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

Target gene identification: target specific transcriptional activation by three murine homeodomain/VP16 hybrid proteins in Saccharomyces cerevisiae.

The mammalian homeodomain proteins encoded by Hox genes play an important role in embryonic development by providing positional queues which define developmental identities along the anteroposterior axis of developing organisms. These proteins bind DNA specifically through their homeodomain to sequences containing ATTA cores, and thereby are thought to exert their effect regulating downstream genes. Little is known about the specificity of binding of homeodomain proteins to their sequences and the identity of their target genes. We have developed a transcriptional activation assay in yeast which employs a homeobox/VP16 fusion gene as a transcriptional activator and a target construct in which test fragments of DNA are inserted upstream to a reporter gene. Using this assay, we compared transcriptional activation by three chimeric proteins containing the homeodomains of the mouse homeobox genes, Hoxa-5, Hoxb-6, and Hoxc-8. When tested on previously defined target sequences, strong differential specificities of activation were observed. In an effort to identify enhancers that normally respond to homeodomain transcriptional activators, random fragments of mouse genomic DNA were cloned upstream of the reporter gene. Genomic DNA fragments with distinct activation profiles were obtained and were found to share matches beyond the ATTA core with previously described enhancers. These results demonstrate that the transcriptional activation system in yeast can be used as a convenient system to detect DNA motifs which bind homeodomain proteins, and subsequently, to identify authentic target genes responsive to Hox gene proteins.