Cloning and physical mapping of enteric adenoviruses (candidate types 40 and 41)

We have studied the DNAs of fastidious enteric adenoviruses recovered from the stools of infants with gastroenteritis. By endonuclease analysis, the strains examined represent candidate adenovirus types 40 and 41, which are thought to comprise new adenovirus subgroups F and G. Cloning of DNA from representative enteric adenovirus isolates, together with hybridization and subcleavage analysis, permitted the mapping of restriction enzyme cleavage sites. Although the restriction profiles are different for the two strains, they appear to have several cleavage sites in common. Cross hybridization studies show considerable homology between the subgroup F and G strains but much less homology to adenovirus 2. In addition, regions on both ends of enteric adenovirus genomes (map units, 2.9 to 11.3 and 75 to 100) possess little or no homology to adenovirus 2. Restriction enzyme digests reveal submolar fragments that map to the terminal regions of the genome. Electron micrographic studies of denatured and renatured DNA strands suggest that the submolar fragments may derive from cleavage of defective molecules. Inverted terminal repeat sequences were shown to comprise 0 to 3.2% of the length of complete (greater than or equal to 22 megadaltons) enteric adenovirus DNA molecules but 4 to 69% of incomplete-length (less than 22-megadalton) molecules.     

Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.     

The location of the genes coding for hexon and fiber proteins in adenovirus DNA.

A serological analysis has been made of the capsid antigens hexon and fiber from 17 Ad5-Ad2+ND1 recombinants that enables us to determine the phenotype of the recombinants. By correlation of this data with the genetic and physical maps of the adenovirus genome, obtained by recombination and restriction endonuclease analysis, the genes coding for the hexon and fiber have been assigned to specific locations on the adenovirus DNA.     

In silico analysis of the restriction fragments length distribution in the human genome.]

The Restriction On Computer (ROC) program (freely available at http://www.mcb.harvard.edu/gilbert/ROC) was developed and used to analyze the restriction fragment length distribution in the human genome. In contrast to other programs searching for restriction sites, ROC simultaneously analyzes several long nucleotide sequences, such as the entire genomes, and in essence simulates electrophoretic analysis of DNA restriction fragments. In addition, this program extracts and analyzes DNA repeats that account for peaks in the restriction fragment length distribution. The ROC analysis data are consistent with the experimental data obtained via in vitro restriction enzyme analysis (taxonomic printing). A difference between the in vitro and in silico results is explained by underrepresentation of tandem DNA repeats in genomic databases. The ROC analysis of individual genome fragments elucidated the nature of several DNA markers, which were earlier revealed by taxonomic printing, and showed that L1 and Alu repeats are nonrandomly distributed in various chromosomes. Another advantage is that the ROC procedure makes it possible to analyze the nonrandom character of a genomic distribution of short DNA sequences. The ROC analysis showed that a low poly(G) frequency is characteristic of the entire human genome, rather than of only coding sequences. The method was proposed for a more complex in silico analysis of the genome. For instance, it is possible to simulate DNA restriction together with blot hybridization and then to analyze the nature of markers revealed.     

In Silico Analysis of the Restriction Fragment Length Distribution in the Human Genome

The Restriction On Computer (ROC) program (freely available at http://www.mcb.harvard.edu/ gilbert/ROC) was developed and used to analyze the restriction fragment length distribution in the human genome. In contrast to other programs searching for restriction sites, ROC simultaneously analyzes several long nucleotide sequences, such as the entire genomes, and in essence simulates electrophoretic analysis of DNA restriction fragments. In addition, this program extracts and analyzes DNA repeats that account for peaks in the restriction fragment length distribution. The ROC analysis data are consistent with the experimental data obtained via in vitro restriction enzyme analysis (DNA taxonoprint). A difference between the in vitro and in silico results is explained by underrepresentation of tandem DNA repeats in genomic databases. The ROC analysis of individual genome fragments elucidated the nature of several DNA markers, which were earlier revealed by DNA taxonoprint, and showed that L1 and Alurepeats are nonrandomly distributed in various chromosomes. Another advantage is that the ROC procedure makes it possible to analyze the nonrandom character of a genomic distribution of short DNA sequences. The ROC analysis showed that a low poly(G) frequency is characteristic of the entire human genome, rather than of only coding sequences. The method was proposed for a more complex in silico analysis of the genome. For instance, it is possible to simulate DNA restriction together with blot hybridization and then to analyze the nature of markers revealed.     

Specific interaction of a protein(s) at or near the termini of adenovirus 2 DNA

When human adenovirus type 2 DNA was extracted from the virions, and the sedimentation profile of the DNA-protein complex in a linear sucrose gradient was analyzed by the nitrocellulose filter binding assay, it was found that 90–95% of adenovirus 2 DNA molecules was tightly associated with the protein(s). The analyses of the binding site of the protein(s) on the DNA by using the restriction endonucleases suggested that the protein(s) interact with the DNA at or near the termini of the viral genome. The stability of the linkage between the DNA and the protein(s) was tested in the presence of various reagents. From the available data, it is suggestive that the protein(s) is linked to the DNA covalently.     

Underrepresentation of short palindromes in Selenomonas ruminantium DNA: evidence for horizontal gene transfer of restriction and modification systems?

Molecular analysis of isolates of the rumen bacterium Selenomonas ruminantium revealed a high variety and frequency of site-specific (restriction) endonucleases. While all known S. ruminantium restriction and modification systems recognize hexanucleotide sequences only, consistently low counts of both 6-bp and 4-bp palindromes were found in DNA sequences of S. ruminantium. Statistical analysis indicated that there is some correlation between the degree of underrepresentation of tetranucleotide words and the number of known restriction endonucleases for a given sequence. Control analysis showed the same correlation in lambda DNA but not in human adenovirus DNA. Based on the data presented, it could be proposed that there is a much higher historical occurrence of restriction and modification systems in S. ruminantium and (or) frequent horizontal gene transfer of restriction and modification gene complexes.     

Multiple Vibrio vulnificus strains in oysters as demonstrated by clamped homogeneous electric field gel electrophoresis.

Clamped homogeneous electric field gel electrophoresis and a computer program for managing electrophoresis banding patterns (ELBAMAP) were used to analyze genomic DNA of 118 Vibrio vulnificus strains, isolated from three oysters by direct plating. Analysis with SfiI resulted in 60 restriction endonuclease digestion profiles (REDP), while analysis with SrfI produced 53 different REDP. Similarities between REDP ranged from 7 to 93%. Principal-component analysis showed that the strains were heterogeneous.     

Adenovirus homologous recombination does not require expression of the immediate-early E1a gene.

To investigate whether early genes other than those involved directly in DNA replication are required for efficient adenovirus recombination, pairs of viruses with deletions in E1a, E1b 496R, E1b 196R, or E4 and containing differing restriction site markers were used to infect both permissive and non- or semipermissive cells. Recombination was assayed among intracellular and extracellular genomes by restriction digestion and blot hybridization. Recombination was delayed in infections of nonpermissive cells with E1a- viruses until a time consistent with the late onset of DNA replication characteristic of the cell type. This shows that E1a expression is not absolutely required for adenovirus recombination. Similar tests with deletion mutations in E1b and E4 also show that these genes are not required for efficient recombination. Taken together with earlier results showing that recombination depends on DNA replication, it is likely that adenovirus recombination is a consequence of cellular repair functions acting on the substrates produced by replication.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

Restriction analysis and homology studies of the bovine adenovirus 7 genome

We located the cleavage sites for restriction endonucleases EcoRI, HindIII, and BamHI on the genome of bovine adenovirus 7. Cross-hybridization at reduced stringency revealed two regions of homology shared by the DNA of human adenovirus 2 and bovine adenoviruses 7 and 3. These regions correspond to the hexon and the IVa2 protein genes of the human adenovirus. Another region of homology shared only by the human adenovirus and bovine adenovirus 7 corresponded to the penton or the polypeptide IIIa genes. These results allowed us to align the restriction map of bovine adenovirus 7 with respect to the other adenoviruses.     

香菇栽培种线粒体DNA和核糖体DNA多态性研究初探

本文应用RFLP技术研究了10个香菇主栽品种的线粒体DNA（mtDNA）和核糖体DNA（rDNA）的部分小区段,利用PCR技术扩增了rDNA5．8＋ITS区段及mtDNA的小区段,分析这些片段的限制性酸切图谱,并进行菌株间的遗传相似系数的估算。结果显示：菌株间的rDNA在5．8＋ITS区段差异很小,表明同一种内菌株间的rDNA具有相对的遗传稳定性;不同菌株间未检出mtDNA的差异,表明菌株间在所研究的区段具有很高的遗传相似性。     

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.     

Restriction endonuclease mapping of adenovirus 35, a type isolated from immunocompromised hosts.

The DNA of adenovirus 35 (Ad35), a type recently associated with infections in immunocompromised hosts, was mapped by the use of BamHI, SmaI, PstI, EcoRI, and HpaI restriction endonucleases. In addition to standard mapping procedures, we used the in vitro adenovirus DNA replication system with origins at both physical ends of the linear molecule to determine the terminal fragments. Deletions of single restriction endonuclease sites in a group of closely related adenovirus isolates from patients with acquired immunodeficiency syndrome helped determine the location of some DNA fragments on the genome.     

Intracellular forms of adenovirus DNA. V. Viral DNA sequences in hamster cells abortively infected and transformed with human adenovirus type 12.

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.     

Silver staining of DNA restriction fragments for the ultrarapid identification of pseudorabies virus strains.

Restriction endonuclease analysis of pseudorabies virus DNA has been used to study various virus strains. To make use of a rapid technique for the identification of viral strains, studies have been undertaken to facilitate purification of the DNA from viral particles present in cytoplasmic fractions. The ultrasensitive photochemical silver staining of nucleic acid, described by Beidler et al. (Analytic Biochemistry 1982: 126) has been adapted and applied to the detection of pseudorabies virus restriction fragments. In a period of 5 h more than 1 microgram of DNA can be extracted from a 25 cm2 plastic flask containing infected cells and purified without ultra centrifugation. Low molecular weight DNA was separated from high molecular weight DNA by polyacrylamide gel electrophoresis. The restriction fragments were selectively visualized by silver staining which can detect 0.025 micrograms of total pseudorabies virus DNA. The electrophoretic DNA pattern of vaccine and wild strains has been studied using these techniques and the results agree with previously published data.     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

A size analysis of the adenovirus replicon

The linear double-stranded genome of adenovirus DNA replicates semiconservatively from two origins of replication at either of the two molecular ends. Using an in vitro replication system which is able to initiate de novo DNA synthesis we have mapped the origin of DNA replication within the terminal 19 bp of the viral genome. Our conclusions are based on the use of different natural DNA templates, i.e., adenovirus type 2 and mouse adenovirus Fl DNA. In addition, we have employed linearized plasmid DNA templates which contain cloned terminal restriction enzyme fragments as well as chemically synthesized adenovirus termini of different length.     

The photobinding of 5,7-dimethoxycoumarin to adenovirus type-2 DNA. A method for in vitro mutagenesis

The photobinding of 5,7-dimethoxycoumarin to isolated adenovirus-type 2 DNA has been investigated with respect to the influence of the ionic environment, and varying molar ratios of DNA_(p): 5,7-dimethoxycoumarin. In particular, the ultraviolet radiation-induced covalent addition of 5,7-dimethoxycoumarin to adenovirus DNA was increased by reducing the concentration of Na⁺. The maximum photobinding of 5,7-[³H]dimethoxycoumarin to adenovirus DNA under the given ionic condition was one 5,7-dimethoxycoumarin per 101 nucleotides. Moreover, restriction enzyme analysis of the 5,7-dimethoxycoumarin-DNA photoadduct versus unmodified viral DNA, suggested that the sequence d(A-T) is the preferential site for intercalation and subsequent photobinding of 5,7-dimethoxycoumarin. This susceptibility of d(A-T) sequences to 5,7-dimethoxycoumarin interaction has a corresponding influence on the survival of adenovirus because of the A-T-rich sequences that occur in some of the early gene regions of the adenovirus genome. Specifically, 5,7-dimethoxycoumarin per 800 nucleotides in adenovirus DNA reduced the surviving fraction of adenovirus to a value of 0.1 after DNA infectivity (transfection) into human 293 cells. Results suggest that 5,7-dimethoxycoumarin may be used for generating a limited ‘library’ of mutations in each of the five early gene regions of the adenovirus genome.