Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag+--Cs2SO4 density gradient centrifugation.--According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2X10(4) copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).--Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides.--Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides.--The organization of mouse genome analyzed by Ag+--Cs2SO4 density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.     

Retrotransposon-like sequences integrated into the genome of pineapple, Ananas comosus

The efficacy of transcervical recanalization of obstructed postoperative Fallopian tubes was evaluated in 29 patients who were referred for recanalization. Nineteen had strictures at the site of Fallopian tube reconstruction, and five had strictures, three had fistulae, and two had fistulae and strictures at the site of reversal surgery. A 0.014-inch highly flexible guidewire was passed through the obstruction into the ampullary segment, followed by a 1. 1-2.2 Fr bougie catheter to dilate the stricture. After recanalization, the distal tube was studied by selective salpingography. The method was technically successful in 17 of 19 patients with underlying inflammatory disease and resultant postoperative strictures. The tubes remained patent in 12 patients for a period of 12-48 months; three patients conceived, all delivering healthy babies. Significant disease of the distal tubes was present in seven patients. The technique succeeded in three of five patients with postoperative strictures following reversal surgery. One patient subsequently conceived and delivered a healthy baby. The technique failed in all five patients with fistulae complicating reversal surgery. Transcervical recanalization is thus recommended in the management of patients with postoperative strictures with underlying inflammatory obstruction and strictures complicating reversal surgery.     

Integration of foreign DNA and its consequences in mammalian systems

The insertion of foreign DNA into the genomes of established cells and organisms and the consequences of this integration event are of significance for viral oncology, reverse genetics, transgenic organisms, human somatic gene therapy and evolution. This review summarizes recent experimental findings and focuses on the alteration of cellular DNA methylation at regions remote from the site of insertion. We also discuss experimental data demonstrating that foreign DNA ingested by mice is not completely degraded in their gastrointestinal tracts; fragments of this DNA have been found to be covalently linked to DNA with 70% homology to the mouse IgE receptor gene.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Cytosine methylation of repeated sequences in eukaryotes: the role of DNA pairing

In eukaryotic genomes, methylation of cytosine residues commonly occurs in repetitive sequences. This methylation correlates with reduced gene expression and suppression of recombination, and is thus thought to serve as a genome-defense mechanism that guards against the deleterious effects of multicopy transposable elements and aberrant gene duplications. Analysis of methylation in fungi and plants suggests that the ability of DNA repeats to pair with one another is a key to their selection for methylation. Recent data have outlined the substrate requirements for the establishment and maintenance of methylation in eukaryotic repeated sequences. Substrate-methylation patterns could help us to understand the way in which methyltransferase enzymes recognize their substrates.     

Cosmid library of the turkey herpesvirus genome constructed from nanogram quantities of viral DNA associated with an excess of cellular DNA

A protocol was designed for the rapid and efficient construction of cosmid libraries from cell-associated viral genomes available in very low quantities. Purification of viral DNA from cellular DNA was unnecessary. The vast excess of cellular DNA compensated for the limited amount of available viral DNA, enabling titration of the restriction endonuclease partial digest. A cosmid library of the turkey herpesvirus DNA genome was constructed from 1.5 micrograms of cellular DNA containing approximately 6 nanograms of viral DNA.     

p53 DNA binding can be modulated by factors that alter the conformational equilibrium

The p53 tumor suppressor protein is a dimer of dimers that binds its consensus DNA sequence (containing two half-sites) as a pair of clamps. We show here that after one wild-type dimer of a tetramer binds to a half-site on the DNA, the other (unbound) dimer can be in either the wild-type or the mutant conformation. An equilibrium state between these two conformations exists and can be modulated by two types of regulators. One type modifies p53 biochemically and determines the intrinsic balance of the equilibrium. The other type of regulator binds directly to one or both dimers in a p53 tetramer, trapping each dimer in one or the other conformation. In the wild-type conformation, the second dimer can bind to the second DNA half-site, resulting in drastically enhanced stability of the p53-DNA complex. Importantly, a genotypically mutant p53 can also be in equilibrium with the wild-type conformation, and when trapped in this conformation can bind DNA.     

Interspersion of different repeated sequences in the wheat genome revealed by interspecies DNA/DNA hybridisation

The repeated sequences in oats DNA have been used to study chromosomal repeated sequence organisation in wheat. Approximately 75% of the wheat genome consists of repeated sequences but only approximately 20% will form heteroduplexes with repeated sequences from oats DNA at 60 degrees C in 0.18 M Na+. The proportion of wheat DNA that forms heteroduplexes with oats DNA is shown to be independent of the wheat DNA fragment length. However, the proportion of wheat DNA that is retained with the heteroduplexes when fractionated on hydroxyapatite is very dependent upon the wheat fragment length up to 3500 nucleotides. This is because more non-renatured wheat DNA is attached to the heteroduplexes with longer fragments. The results indicate that the repeated sequences in the wheat genome homologous to repeated sequences in oats are not clustered in the chromosomes but distributed amongst other repeated and possible non-repeated sequences.     

Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA

Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35-280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci.     

E-cadherin expression can alter the specificity of gap junction formation

Specificity of gap junction formation produces communication compartments, groups of cells joined to each other by gap junctions (homologous communication) but more rarely to cells in adjacent compartments (heterologous communication). Specificity of junction formation can be studied in mixed cultures of different cell types. In these model systems, compartmentation is often associated with sorting out, a process that produces separate domains of the different cells. The borders of the physically distinct domains correlate with the functional boundaries of the communication compartments. Compartments have also been observed in vivo where they are believed to play a role in separating groups of cells following different differentiation pathways. Two classes of cell surface molecule, connexins and cell adhesion molecules, are candidates for a role in the control of specificity. A representative of each class appears to be necessary for gap junction formation and both are expressed in a tissue specific manner. We have shown that mixed cultures of rat epithelial (BRL) cells and rat (BICR) fibroblasts show specificity, form communication compartments and sort out. Both cell types express the same connexin (connexin 43) but different cell adhesion molecules (BRL, P-cadherin and 125-kDa N-cadherin; BICR, 140-kDa N-cadherin). Transfection of both cell types with E-cadherin results in a 10-fold increase in heterologous communication. These data suggest that E-cadherin plays a role in the control of specificity of gap junction formation.     

The CDNA and genomic DNA sequences of a mammalian neurotoxin from the scorpion Buthus martensii Karsch

The cDNA library of venomous glands of the scorpion Buthus martensii Karsch (BmK) was constructed. A cDNA encoding a mammalian neurotoxin corresponding to the known alpha-type toxin, BmK M1, was amplified by polymerase chain reaction (PCR) and cloned, and its full-length sequence was determined. The open reading frame encoded the precursor of BmK M1 with 84 amino acid residues, including a signal peptide of 19 residues, a mature toxin of 64 residues and an additional C-terminal residue Arg which might be cleaved off by proteinase postprocessing immediately after protein synthesis. Based on the determined cDNA sequence and using the total DNA of the scorpion as a template, the gene of BmK M1 was also amplified by PCR and sequenced. The genomic DNA sequence revealed an intron of 408 base pairs present within the signal peptide region. Both the intron and exon of BmK M1 share about 75% similarity with those of AaH I' another alpha-type mammalian neurotoxin in the scorpion Androctonus australis Hector.     

DNA methylation in mycobacteria: absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

The histopathology of low-grade primary gastric lymphoma re-capitulates the structure of Peyer's patches (mucosa-associated lymphoid tissue--MALT) rather than lymph nodes, characterised by an increased frequency of trisomy 3. Gastric B-cell lymphoma shows a favourable clinical behaviour, possibly as its growth appears to be influenced by a local antigen in the form of Helicobacter pylori. There is no lymphoid tissue in the normal stomach, but lymphoid tissue accumulates in gastric mucosa almost exclusively as a consequence of H. pylori infection, which has MALT characteristics, and H. pylori is found in over 90% of cases of gastric MALT lymphoma. Laboratory studies have shown that the growth of tumour cells from low-grade gastric lymphomas can be stimulated by H. pylori, and that the effect is strain-specific and mediated by contact-dependent help from H. pylori-specific T-cells. Cases of low-grade gastric lymphoma, when confined to the mucosa, may regress following eradication of H. pylori from the patient's stomach. It remains to be shown whether deeply penetrating or high-grade tumours will respond in the same way. Other outstanding questions relate to the optimum interval between eradication of H. pylori and final evaluation and expected duration of the response. Based on these laboratory and clinical findings it is possible to suggest a scheme for the pathogenesis of gastric MALT lymphoma.     

An in vivo assay for measuring the recombination potential between DNA sequences in mammalian cells

Mammalian intermolecular recombination vectors that place the recombination junction within the intron of a selectable marker gene are presented. Many of the previously reported recombination assays require that recombination occur homologously and that they occur within the coding region of the selectable marker. This vector system involves the use of a human thymidine kinase (tk) minigene and measures the recombination frequency between any chosen DNA sequences, in mammalian thymidine kinase negative cells. The tk minigene is divided into a 5' vector and a 3' vector. In the 5' vector, the DNA sequence of interest is inserted in the proximal portion of tk intron 2. In the 3' vector, the DNA sequence of interest is inserted in the intron sequence between the proteolipid protein exon 2 and tk exons 3-7. Recombination through the DNA sequences of interest, either homologous or illegitimate, will reconstruct a functional tk minigene. The recombination junction is spliced out of the transcribed mRNA and thymidine kinase positive cells can be selected in hypoxanthine-aminopterin-thymidine medium. We have tested these vectors to measure the recombination potential of two Alu repetitive sequences (BLUR 8 and BLUR 11) against a control DNA sequence. BLUR 8 and BLUR 11 do not seem to recombine at a significantly higher frequency over that of the control DNA sequence. These recombination vectors display similar sensitivity to previous recombination systems, but allow tremendous flexibility in the choice of potentially recombinogenic sequences.     

Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits

Molecular medicine began with Pauling's seminal work, which recognized sickle-cell anemia as a molecular disease, and with Ingram's demonstration of a specific chemical difference between the hemoglobins of normal and sickled human red blood cells. During the four decades that followed, investigations have focused on the gene--how mutations specifically alter DNA and how these changes affect the structure and expression of encoded proteins. Recently, however, the advances of the human genome project and the completion of total genome sequences for yeast and many bacterial species, have enabled investigators to view genetic information in the context of the entire genome. As a result, we recognize that the mechanisms for some genetic diseases are best understood at a genomic level. The evolution of the mammalian genome has resulted in the duplication of genes, gene segments and repeat gene clusters. This genome architecture provides substrates for homologous recombination between nonsyntenic regions of chromosomes. Such events can result in DNA rearrangements that cause disease.