Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1.

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.     

Genetic relatedness of equine herpesvirus types 1 and 3.

The genetic relatedness of two types of equine herpesviruses (EHVs), 1 (EHV-1) and 3 (EHV-3), was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA probe was allowed to reassociate in the presence or absence of the second unlabeled viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating only 2 to 5% homology between the two EHV genomes. Moreover, labeled RNA extracted from EHV-3-infected cells hybridized to filter-immobilized EHV-1 DNA only 2 to 3 percent as efficiently as to the homologous EHV-3 DNA. These results demonstrate that the genital (EHV-3) and nongenital (EHV-1) types of EHVs exhibit very little genetic homology.     

Amount of viral DNA in the genome of cells transformed by adenovirus type 2

The number of copies of viral DNA in DNA of cells transformed by adenovirus type 2 has been determined by following the kinetics of reassociation of ³²P-labeled viral DNA in the presence of unlabeled DNA extracted from transformed and control cells. There is close to one copy of adenovirus 2 DNA for every diploid quantity of cell DNA.     

Genetic relatedness of the genomes of equine herpesvirus types 1, 2, and 3.

Genomic DNAs of equine herpesvirus type 1 (EHV-1), EHV-2 (equine cytomegalovirus), and EHV-3 were examined by reassociation kinetic and thermal denaturation analyses to determine the extent and degree of homology among the three viral DNAs. Results of reassociation analyses indicated a limited homology among the three EHV genomes. Homologous DNA sequences equivalent to 1.8 to 3.7 megadaltons between EHV-1 and equine cytomegalovirus, 7.6 to 8.2 megadaltons between EHV-1 and EHV-3, and 1.3 to 1.9 megadaltons between equine cytomegalovirus and EHV-3 were detected. Examination by thermal denaturation of the DNA homoduplexes and heteroduplexes formed during reassociation revealed a high degree of base pairing within the duplexes, suggesting that closely related sequences may be conserved among the genomes of EHV.     

Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

The genomes of phage I3 and its host Mycobacterium smegmatis have been compared. From thermal melting studies the GC contents of DNA from mycobacteriophage I3 and its host M. smegmatis were found to be 66%. A new method, based only on the initial rates of reassociation, has been developed for calculating the DNA homology. Analysis of DNA reassociation kinetics suggested the presence of one equivalent of the phage I3 genome within the M. smegmatis genome. Southern analysis revealed the presence of almost all of the phage I3 specific sequences within the host genome.     

Thermal Stability of Escherichia coli–Salmonella typhimurium Deoxyribonucleic Acid Duplexes

Single-stranded, labeled deoxyribonucleic acid (DNA) fragments from Escherichia coli were incubated at 60 and 66 C with a large excess of single-stranded, unlabeled DNA fragments from E. coli and Salmonella typhimurium. The resulting reassociated DNA was adsorbed to hydroxylapatite and eluted in a series of washes at increasing temperatures. The thermal stability of the reassociated DNA was determined by means of this procedure. Neither the extent of reassociation nor stability of the reassociated E. coli DNA was affected by increasing the incubation temperature from 60 to 66 C. The double-stranded molecules resulting from the reassociation of E. coli DNA with S. typhimurium DNA had a markedly lower thermal stability than reassociated E. coli DNA. More reassociation occurred between E. coli and S. typhimurium at 60 C than at 66 C. In addition, the product of interspecies reassociation occurring at 66 C had a higher thermal stability than that occurring at 60 C. Preliminary results indicate that the decreased thermal stability of the interspecies duplex is in part the result of unpaired bases.     

Isolation and partial characterization of single-stranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA.

Single-stranded fragments of adenovirus type 2 DNA were isolated from infected KB cells under conditions which retarded reassociation of complementary sequences but did not denature native viral DNA. Of the total intracellular, virus-specific DNA labeled during a 1-h pulse with tritiated thymidine begining 15 h after infection, about 20% was single stranded when fractionated on hydroxylapatite. This DNA shifted predominantly to the double-stranded fraction on hydroxylapatite during an extended chase incubation, suggesting that it may represent single-stranded DNA in replicating intermediates. Furthermore, the single-stranded DNA annealed nearly equally to both strands of the adenovirus genome. These findings indicate that at least portions of both complementary strands of adenovirus type 2 DNA are exposed as single strands during the period of viral DNA synthesis.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.     

Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis

Evidence is presented that crown gall tumors are caused by the incorporation of part of a virulence plasmid carried by the inciting bacterium, Agrobacterium tumefaciens. The rate of reassociation of labeled plasmid DNA was slightly accelerated in the presence of tobacco crown gall tumor DNA, but not normal tobacco DNA. Treatment of tumor DNA with DNAase abolished the acceleration. To determine whether all plasmid sequences are represented in tumor DNA, the labeled plasmid DNA was separated into specific fragments after digestion with restriction endonuclease Sma I. Renaturation rates for DNA from bands 1, 2, 7, 8, 9, 12 and 14 were not affected by tumor DNA. DNA from band 3 showed a slight rate increase in the presence of tumor DNA, indicating 21–27 copies of 14–18% of the DNA sequences in this (doublet) band. The band 3 doublet was separated by electrophoresis into bands 3a and 3b. Tumor DNA had little effect on the rate of reassociation of labeled band 3a DNA. Band 3b DNA renatured rapidly in the presence of tumor DNA, and its rate increase indicated that approximately 18 copies of 40% of band 3b DNA sequences are present per diploid tumor cell. This amounts to 3.7 × 10⁶ daltons of foreign genetic information and represents a contribution of 0.0011% to the DNA content of the tumor cell. The relationship between this plant tumor and virally induced animal tumor systems is discussed.     

Size and complexity of the nuclear genome of Colletotrichum graminicola.

DNA reassociation was used to estimate GC content, size, and complexity of the nuclear genomes of Colletotrichum from maize and sorghum. Melting-temperature analysis indicated that the GC content of the maize pathotype DNA was 51% and that the GC content of the sorghum pathotype was 52%. DNA reassociation kinetics employing S1 nuclease digestion and an appropriately modified second-order equation indicated that the genome sizes of the maize and sorghum pathotypes were 4.8 x 10(7) bp, and 5.0 x 10(7) bp, respectively. Genomic reconstruction experiments based on Southern blot hybridization between a cloned single-copy gene, PYR1 (orotate phosphoribosyl transferase), and maize-pathotype DNA confirmed the size of the nuclear genome. The single-copy component of the genomes of both pathotypes was estimated at about 90%. For both pathotypes, ca. 7% of the genome represented repetitive DNA, and 2 to 3% was foldback DNA.     

Detection of Viral DNA Sequences in Adenovirus-Transformed Cells by In Situ Hybridization

Cytological preparations of cells transformed by members of three groups of human adenoviruses, adenovirus 12, 7, and 2, were annealed with radioactive complementary RNA (cRNA) (4 x 10(7) to 4.5 x 10(7) dpm/mug) prepared by copying viral DNA with the Escherichia coli DNA-directed RNA polymerase. These in situ hybridizations detected adenovirus-specific DNA sequences in interphase nuclei when transformed cells were annealed with homologous viral cRNA, but not with heterologous viral cRNA. The highest autoradiographic grain counts were found over adenovirus 7-transformed cell nuclei, next over adenovirus 12-, and the lowest over adenovirus 2-transformed cell nuclei. This is the same order as found by reassociation kinetic measurements (K. Fujinaga and M. Green, unpublished data).     

Genetic Relatedness of Type 1 and Type 2 Herpes Simplex Viruses

The extent of homology between herpes simplex virus(1) and(2) (HSV-1 and HSV-2) deoxyribonucleic acid (DNA) was measured in two ways: (i) by determination of the relative rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA immobilized on filters and (ii) by determination of the rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA in solution. Approximately 40% of HSV-1 and HSV-2 DNA is homologous at hybridization temperatures 25 C below the melting temperature (T(m)) of HSV DNA (liquid-filter annealing). Lowering the temperature to 34 C below the T(m) increased the extent of homology to 46% (liquid annealing). The extent of base-pairing in HSV-1-HSV-2 heteroduplex DNA was determined by thermal chromatography on hydroxyapatite. Heteroduplexes of HSV-1 and HSV-2 DNA eluted in a single peak whose midpoint (Te(50)) was 10 C below that of the homoduplex. Conspicuously absent were heteroduplexes that eluted at more than 15 C below the Te(50) of the homoduplex. The data indicate the existence of a variable region of DNA (54%) with very little, if any, homology and an invariable region (46%) with relatively good (85%) matching of base pairs.     

Conserved polynucleotide sequences among the genomes of papillomaviruses.

The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.     

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.     

Conservation of transfer ribonucleic acid and 5S ribonucleic acid cistrons in Enterobacteriaceae.

The genes for tranfer ribonucleic acid (tDNA) and 5S ribonucleic acid (5SDNA) were isolated from the total deoxyribonucleic acid (DNA) of Escherichia coli. The relatedness of tDNA and 5S from E. coli and other species of Enterobacteriaceae was determined by reassociation of the isolated genes labeled with 32PO4 to unlabeled, unfractionated DNA. Double-stranded DNA was separated from unreacted DNA by hydroxyapatite chromatography. Thermal elution profiles were done to determine the amount of unpaired bases present in related DNA sequences. Relative to total DNA, both 5S DNA and tDNA were highly conserved throughout the Enterobacteriaceae, including the genera Yersinia and Proteus.     

Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

Quantitation of bovine papilloma viral DNA in viral-induced tumors.

Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching.     

Cytoplasmic RNA from normal and malignant human cells shows homology to the DNAs of Epstein-Barr virus and human adenoviruses.

Cytoplasmic RNA prepared from several human cell lines and tissues was hybridised to DNA from Epstein-Barr virus, human adenovirus types 2, 3 and 12 and human papovaviruses BK and JC. RNA from all the cells, regardless of whether or not they were virally infected, hybridised to specific regions of the Epstein-Barr virus or adenovirus genomes but not to papovavirus DNA. The cellular cross-hybridising species appear to be repetitive sequences which are conserved in higher eukaryotes. Mismatch estimations indicate a high degree of homology between the viral and host sequences. Detailed analysis of selected regions of viral DNA failed to reveal any primary-structural peculiarities.