Sequence arrangement in herpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease digests and evidence for inversions in redundant and unique sequences.

It has been proposed by Sheldrick and Berthelot (1974) that the terminal sequences of herpes simplex virus type 1 (HSV-1) DNA are repeated in an internal inverted form and that the inverted redundant sequences delimit and separate two unique sequences, S and L. In this study the sequence arrangement in HSV-1 DNA has been investigated with restriction endonuclease cleavage, end-labeling studies, and molecular hybridization experiments. The terminal fragments in digests with restriction endonucleases Hind III, Hpa-1, EcoRI and Bum were identified and shown to be consistent with the Sheldrick and Berthelot model. Inverted fragments which contain unique sequences as well as redundant sequences, and which the model predicts, were identified by DNA-DNA hybridization studies. Further cleavage of Bum fragments with Hpa-1 also revealed inversions of the terminal sequences that contained unique sequences. The results obtained showed that the unique sequences S and L are relatively inverted in different DNA molecules in the population, resulting in the presence of four related genomes with rearranged sequences in apparently equal amounts. The redundant sequences bounding S do not share complete sequence homology with those bounding L, but hybridization studies are presented which show that the terminal 0.3% of the genome is repeated in every redundant sequence.     

Organization of type C viral DNA sequences endogenous to baboons: analysis with cloned viral DNA.

Unintegrated linear and circular forms of baboon endogenous type C virus M7 DNA were prepared from M7-infected cells by chromatography on hydroxyapatite columns, and the circular DNAs were purified in cesium chloride-ethidium bromide equilibrium density gradients. The circular DNAs were linearized by digestion with EcoRI, which had a unique site on the viral DNA. The linearized DNA was then inserted into lambda gtWES. lambda B at the EcoRI site and cloned in an approved EK2 host. Molecularly cloned full-length M7 DNA was restricted with BamHI, and the resulting five subgenomic fragments were then subcloned individually in plasmid pBR322. The organization and sites of integration of the approximately 100 copies of M7 DNA sequences endogenous to baboons were investigated by digesting the DNA with restriction enzymes and identifying the virus-specific fragments by hybridization to labeled probes made by using the molecularly cloned full-length and subgenomic fragments of the viral DNA. We found that most of the endogenous sequences had sizes and organizations similar to those of the unintegrated viral DNA and therefore approximately similar to the RNA of the infectious virus. A few of the multiple sequences had deletions in the 3' end (envelope region), and some of the sequences either lacked or contained modified BamHI restriction sites on the 5' end of the viral DNA. The endogenous viral DNA sequences were nontandem, uninterrupted, and colinear with the DNA of the infectious virus, and they were integrated at different sites in the baboon DNA, like the M7 proviral DNA sequences acquired upon infection.     

The retrieval of ancient human DNA sequences.

DNA was extracted from approximately 600-year-old human remains found at an archaeological site in the southwestern United States, and mtDNA fragments were amplified by PCR. When these fragments were sequenced directly, multiple sequences seemed to be present. From three representative individuals, DNA fragments of different lengths were quantified and short overlapping amplification products cloned. When amplifications started from <40 molecules, clones contained several different sequences. In contrast, when they were initiated by a few thousand molecules, unambiguous and reproducible results were achieved. These results show that more experimental work than is often applied is necessary to ensure that DNA sequences amplified from ancient human remains are authentic. In particular, quantitation of the numbers of amplifiable molecules is a useful tool to determine the role of contaminating contemporary molecules and PCR errors in amplifications from ancient DNA.     

DNA nucleotide sequence heterogeneity between the Towne and AD169 strains of cytomegalovirus.

The results of reciprocal DNA-DNA reassociation kinetics indicated that although the DNAs of human cytomegalovirus (CMV) strains Towne and AD169 shared approximately 90% of their nucleotide sequences, about 10% heterogeneity did exist. The implication was that, with respect to one another, the DNAs of CMV Towne and CMV AD169 contained unique nucleotide sequences. To obtain more direct evidence, 32P-labeled DNA of one virus strain was reassociated in the presence of excess unlabeled DNA of the heterologous virus strain. Those 32P-labeled DNA sequences remaining single stranded were separated from double-stranded DNA on hydroxyapatite columns and incubated with Southern blots containing XbaI restriction enzyme fragments of the homologous virus DNA. This approach not only enriched for nucleotide sequences unique to each strain of virus, but also provided for the identification of the restriction enzyme fragments in which the unique sequences were contained. The CMV Towne unique sequences were found in XbaI fragments A, C, G, L, N, and Q of CMV Towne DNA. The CMV AD169 unique sequences were found in XbaI fragments A, C, G, and J of CMV AD169 DNA. The possible significance of these data with respect to variation among other CMV isolates is discussed.     

XbaI, PstI, and BglII restriction enzyme maps of the two orientations of the varicella-zoster virus genome.

Cleavage of varicella-zoster virus DNA with the restriction endonucleases PstI, XbaI, and BglII resulted in 18, 22, and 20 fragments, respectively. Based on the molecular weights and molarities of these fragments, a molecular weight of 84 x 10(6) could be calculated for the varicella-zoster virus genome. In both the XbaI and the BglII patterns, four 0.5 M fragments were identified. The arrangement of the fragments was determined by molecular hybridization techniques, and the terminal fragments were identified by lambda exonuclease digestion. The 0.5 M fragments, of which two were located at the same terminus of the genome, contained repeated sequences: one terminally and one inverted internally. These results were in agreement with the existence of two equimolar subpopulations of the varicella-zoster virus genome, differing in the relative orientation of a short region of unique sequences. This region was bounded by the repeated sequences. From the molecular weights of the submolar fragments, a maximal molecular weight of 5 x 10(6) for the repeated region and a minimal molecular weight of 3.5 x 10(6) for the short unique sequence could be calculated.     

cis Functions involved in replication and cleavage-encapsidation of pseudorabies virus.

Serial passage at high multiplicity of pseudorabies virus generates defective interfering particles (DIPs) whose genomes consist at least in part of reiterations of segments of DNA in which sequences originating from different regions of the genome have become covalently linked (F. J. Rixon and T. Ben-Porat, Virology 97:151-163). To determine whether some cis functions present in these reiterated DNA sequences may be responsible for the amplification of DIP DNA, BamHI restriction fragments of this DNA were cloned. These fragments were analyzed and tested for their ability to promote the amplification of covalently linked pBR325 DNA when cotransfected into cells with helper pseudorabies virus DNA. The cloned DIP BamHI DNA fragments consisted of various combinations of sequences originating from either one or both ends as well as sequences from the middle of the unique long (UL) segment of the genome. Only plasmids with inserts consisting of segments of defective DNA originating from the middle of the UL, as well as from both ends of the genome, were able to replicate and be encapsidated autonomously. This finding indicated that signals present at both ends of the genome may be necessary for efficient cleavage-encapsidation. To confirm this observation, we constructed plasmids in which DNA segments containing an origin of replication and sequences from either one or both ends of the virus genome were linked. These experiments showed that efficient cleavage-encapsidation requires the presence of sequences derived from both ends of the genome. Two origins of replication, one at the end of the UL segment and one in the middle of the UL segment, were also identified.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA III. Host Sequences

A preparation of serially passaged simian virus 40 (SV40) DNA, in which at least 66% of the molecules contain covalently linked cellular DNA sequences, was digested to completion with the Hemophilus influenzae restriction endonuclease. Polyacrylamide gel electrophoresis of the digest showed that the majority of the cleavage products migrated as nine classes of fragments, each class defined by a particular molecular weight. These classes of fragments differ in molecular weight from the fragments produced by the action of the same enzyme on plaque-purified virus DNA. Three classes of fragments were present in less than equimolar amounts relative to the original DNA. The remaining six classes of fragments each contain more than one fragment per original DNA molecule. DNA-DNA hybridization analysis (using the filter method) of the isolated cleavage products demonstrated the presence of highly reiterated cell DNA sequences in two of the nine classes of fragments. A third class of fragments hybridized with high efficiency only to serially passaged SV40 DNA; the level of hybridization to plaque-purified virus DNA was low and there was essentially no hybridization with cell DNA immobilized on filters. It is suggested that this class of fragments contains unique host sequences. It was estimated that at least 27% of the sequences in the substituted SV40 DNA molecules studied are host sequences. The majority of these are probably of the nonreiterated type.     

桥式PCR，一种简易连接DNA标签序列的方法

MAST方法采用人工文库的DNA标签序列鉴定mRNA的可接近位点。大量的标签序列通过扩增和克隆测序达到阐明mRNA结合位点图。设计了单一引物的PCR,其引物在标签序列两端结合搭桥,在扩增中DNA标签序列在搭桥引物的作用下进行连接,连接的标签序列再克隆和测序。十几条这样的连接产物包含了上千条标签序列。该PCR方法简单、高效以用于高通量的方式对标签序列测序。     

Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.     

Isolation and characterization of a 15-kilobase genomic sequence coding for part of the Pro alpha 2 chain of sheep type I collagen

DNA fragments, prepared by partial Eco RI digestion of fetal sheep liver genomic DNA, were used to prepare a "library" of amplified genomic sequences with the lambda vector Charon 4A. Several recombinant plaques were identified by their ability to hybridize to 32P-labeled cDNA prepared from fetal sheep tendon type I procollagen mRNA. Two of these recombinant DNA bacteriophages (SpC3 and SpC7) were identified as containing procollagen pro alpha 2 gene sequences by their ability to specifically anneal to procollagen pro alpha 2 mRNA. Restriction endonuclease and hybridization to a cloned pro alpha 2 cDNA demonstrated that approximately half (2.5 kilobases) of the pro alpha 2 mRNA sequence is distributed over 15 kilobases of genomic DNA. Restriction maps of SpC3 and SpC7 demonstrated that these two DNA fragments contain overlapping sequences of the pro alpha 2 gene. Electron microscopy and R-loop analysis of SpC3 revealed that at least 12 to 16 intervening sequences are distributed throughout the length of this gene fragment.     

Avian erythroblastosis virus produces two mRNA''s.

We analyzed the viral mRNA's present in fibroblast nonproducer clones transformed by avian erythroblastosis virus. Two size classes of mRNA (28 to 30S and 22 to 24S) were identified by solution hybridization with both complementary DNA strong stop and complementary DNA made against the unique sequences of avian erythroblastosis virus. Based upon the kinetics of hybridization with complementary DNA made against the unique sequences of avian erythroblastosis virus, we estimated that there were 400 to 500 copies of the 28 to 30S RNA per cell and 200 to 250 copies of the 22 to 24S RNA per cell. Both RNA species were packaged in the virion. In vitro translation of the 28 to 30S virion RNA yielded a 75,000-dalton protein which was the 75,000-dalton gag-related polyprotein found in avian erythroblastosis virus-transformed cells. In vitro translation of the 22 to 24S virion RNA yielded two proteins (46,000 and 48,000 daltons). This indicates that there may be two genes in avian erythroblastosis virus, one coding for the 75,000-dalton gag-related polyprotein and the second coding for the 46,000- or 48,000-dalton protein or both.     

Genetic instability and associated genome plasticity in Streptomyces ambofaciens: pulsed-field gel electrophoresis evidence for large DNA alterations in a limited genomic region.

Using pulsed-field gel electrophoresis (PFGE) analysis, the amplifiable units of DNA (AUD) loci AUD6 and AUD90 of Streptomyces ambofaciens DSM40697 could be mapped in the wild-type genome within two adjacent AseI restriction fragments estimated to be about 75 and 850 kb. In addition, the genetic instability and formation of very large deletions were strictly correlated. Their sizes were estimated to range from 250 to more than 2,000 kb. These deletions affected the DNA region overlapping both amplifiable loci. PFGE also allowed us to localize the amplified DNA sequences and to establish their structure: amplification takes place at the AUD locus as a tandem array of the wild-type AUD sequence.