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.     

A simple method for detecting single base substitutions and its application to HLA-DPB1 typing.

We have developed a simple and reliable method, PCR-PHFA (polymerase chain reaction dependent preferential homoduplex formation assay), for detection of single base substitutions within PCR amplicons. This technique is based upon strand competition during hybridization between a double labeled amplicon, prepared from biotin and DNP labeled primers, and an unlabeled amplicon. Under the precisely controlled temperature gradient, the preferential formation of a homoduplex over a heteroduplex occurs. After annealing, the identical sequence of the double labeled and unlabeled amplicon resulted in a low population of regenerated double labeled dsDNA due to strand exchange between them. Even when the two differed by only a single base substitution, double labeled molecule was regenerated efficiently because of preferential homoduplex formation. The regenerated double labeled molecule was captured onto a streptavidin coated microtiter plate and quantified enzymatically with a chromogenic substrate. The technique has been successfully applied in HLA-DPB1 typing. Furthermore, we detected a mutated gene even in the presence of a large excess of the corresponding normal gene.     

Isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the C3H and RIII mouse strains.

Radioactive 60-70S RNA from the mouse mammary tumor virus (MMTV) produced by the C3H mouse mammary tumor cell line (Mm5mt) hybridized to a greater extent, and at a lower Cot1/2 value, to the DNA of C3H mammary tumor cells than to the DNA of C3H liver cells. The 125I-labeled MMTV (C3H) 60-40S RNA was annealed to a vast excess of DNA from C3H livers, and single-stranded RNA was eluted from hydroxylapatite and recovered. This "recycled RNA" did not hybridize to the DNA of the apparently normal organs tested from normal or from mammary tumor-bearing C3H mice, but hybridized extensively to both the DNA from the C3H mammary tumor cell line and the DNA from spontaneous C3H mammary tumors. This hybridization could be competed out by the addition of unlabeled MMTV 60-70S RNA but was unaffected by the addition of unlabeled 60-70S RNA of C3H type C virus. Similar experiments were conducted with the RIII mouse strain. We therefore report on the isolation of the sequences of the RNA genomes of the MMTVs from C3H and RIII mice that are transmitted by some mechanism other than via the germ line. These studies further define the differences, via molecular hybridization, between the MMTV-S and the MMTV-L in both C3H and RIII mice.     

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.     

Method for determination of nucleotide sequence homology between viral genomes by DNA reassociation kinetics.

A model and appropriate equations were derived for the quantitative estimation of nucleotide sequence homology between two partially related viral genomes by measurement of the initial rate of reassociation of one labeled DNA in the presence of a second unlabeled DNA. The validity and usefulness of this procedure were demonstrated by the analysis of the reassociation kinetics of labeled adenovirus 7 DNA in the presence of unlabeled adenovirus 2 DNA. Based on DNA reassociation, the extent of homology between adenovirus 2 and 7 genomes was found to be 10 to 12%. The duplex formed between adenovirus 2 and 7 DNA had the appropriate thermal stability for a well-matched DNA-DNA hybrid.     

Deoxyribonucleic acid homology in bacterial taxonomy: effect of incubation temperature on reaction specificity

Parameters affecting deoxyribonucleic acid duplex (DNA-DNA) formation on membrane filters were evaluated. The reference strains used were Cytophaga succinicans strain 8, which has a guanine plus cytosine (GC) content of 38%, and Myxococcus xanthus strain FB, which has a GC content of 70%. Both organisms are gliding bacteria classified among the myxobacteria. Among the parameters evaluated, the incubation temperature used during duplex formation was found to be the most important in terms of the physical nature of the reaction product. When an incubation temperature 25 C below the melting point (T(m)) of the native DNA was used, homologous duplexes exhibited a thermal stability similar to that of native DNA. At 35 C below the T(m), a considerable proportion of the duplexes were of much lower stability; at 40 C below the T(m), most of the duplexes were of much lower stability. Similar duplexes of low stability were also formed between DNA molecules from morphologically and nutritionally diverse organisms, provided the GC percentages of the DNA preparations were similar. Competition between unlabeled and labeled DNA fragments for binding sites on immobilized DNA was also greatly influenced by the incubation temperature. Heterologous DNA-DNA complexes exhibited thermal stabilities which correlated with measurements of DNA homology in experiments involving competition. In addition, the difference in thermal stabilities of heterologous and homologous DNA complexes (DeltaT'(m)) may provide a measure of divergence in nucleotide sequences.     

Sequence Homology between Chloroplast DNAs from Several Higher Plants

An estimate has been made of the amount of sequence homology present in the chloroplast DNA (ctDNA) of several higher plants by the technique of DNA-DNA hybridization. Approximately 85% of tomato, 60% of spinach, 45% of kale, and 15% of barley ctDNA sequences were found to hybridize with tobacco ctDNA under conditions in which maximum hybridization in homologous reactions reached 85%. All heteroduplexes contained significant amounts of sequence mismatch as indicated by a 3 to 9 C decrease in melting temperature as compared to homoduplex.     

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.     

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.     

Mutation detection by denaturing DNA chromatography using fluorescently labeled polymerase chain reaction products.

A specialized form of ion-pair reversed-phase high-performance liquid chromatography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modulated heteroduplex analysis (TMHA) by chromatographic separation of partially denatured DNA heteroduplexes from homoduplexes. Here, we demonstrate that fluorescent labeling is compatible with mutation analysis by this form of DNA chromatography and offers advantages over the use of unlabeled DNA fragments. Uniform labeling of wild-type and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels increase retention times but do not influence resolution of heteroduplexes from homoduplexes. They increase sensitivity and decrease the amount of DNA required for analysis; e.g., in the case presented here, one allele can be detected in the presence of a 500-fold excess of another allele. Furthermore, allele-specific wild-type probes, fluorescently labeled on one strand only, make it possible to selectively monitor specific homoduplexes and wild-type/mutant heteroduplexes. This, in combination with an internal homoduplex standard, greatly reduces the complexity of fluorescence chromatograms compared with chromatograms recorded in the UV. These simplified chromatograms, in which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detection and identification of mutant alleles.     

Specific interaction between mouse liver non-histone chromosomal proteins and mouse DNA demonstrated by a sequential DNA-protein binding procedure.

The binding of mouse liver chromosomal proteins to DNA has been investigated using the nitrocellulose filter binding technique. Careful purification of the DNA involving nuclease S1 digestion and prefiltration through a nitrocellulose filter is used to reduce background binding in the absence of protein to less than 1%. Procedures involving direct binding of protein to labeled DNA, competition of binding of labeled DNA by unlabeled DNA, and dissociation of DNA . protein complexes with time do not indicate significant preference for binding to mouse DNA relative to Escherichia coli DNA. This specificity is demonstrated much more clearly by a novel type of procedure, which we call a sequential binding procedure. In this procedure non-specific binding proteins are sequestered by incubation with an excess of unlabeled E. coli DNA prior to addition of labeled DNA. Under these conditions, labeled mouse DNA is bound to filters to a 3- to 4-fold greater extent than labeled E. coli DNA.     

Sequence homology between mitochondrial DNAs of different eukaryotes.

The sequence divergence of mitochondrial DNAs (mtDNA) from rat, mouse, guinea pig, monkey, and chicken has been examined by DNA-DNA hybridization. mtDNAs, isolated as closed circular molecules by propidium iodide-CsCl centrifugation, were labeled in vitro by use of Escherichia coli DNA polymerase I, and renatured (Tm-35 degrees) in the presence of a 2500-fold excess of heterologous mtDNA. Single-stranded and duples DNA were separated by hydroxylapatite chromatography. The thermal stability of heteroduplexes was compared to the homoduplex by thermal elution chromatography on hydroxylapatite columns. Heteroduplex fromation between the tritiated myDNAs and a 2500-fole excess of rar mtDNA were 70, 59, 37, and 22%, respectively, for mouse, guinea pig, monkey, and chicken. Similar results were obrained in reciprocal hybridizations where one of the other mtDNAs was present in excess. Considerable mismatching of sequences in all the heterohybrids was indicated by a 18-24 degrees depression in the te50 of the heteroduplexes compared with the homoduplex. There was no apparent change in heteroduplex formation when the concentration ratio of driving DNA in excess to [3H]mtDNA was varied between 1250 and 7500. Furthermore, a second renaturation with excess driving DNA after completion of the first reaction resulted in no detectable augmenting of heteroduplex formation. Similar sequences appear to be conserved preferentially in different organisms, since the presence of two of fouf different heterologous mtDNAs in excess resulted in only moderate and nonadditive increases in heteroduplex formation. Evolutionary divergence of mtDNA sequences appears to have occurred at rates similar to that for unique sequences nuclear DNA.     

Comparison of different cationic probes for electron microscopic visualization of bacterial polyanionic capsular material

We have isolated from the ovine rumen eight bacterial strains belonging to the speciesButyrivibrio fibrisolvens. DNA hybridization studies showed that the eight strains could be divided into four homology groups, of which none was closely related to the type strain ATCC 19171. Measurement of cross-hybridization between selected pairs of bacterial strains showed that DNA types which produced low, but significant, cross-hybridization on dot-blots were able to form heteroduplexes with between 8.4% and 32.9% of the efficiency of homoduplex formation. Thermal denaturation of the same heteroduplexes resulted in Tm values 6.4–7.5°C lower than those of the homologous duplexes formed under the same conditions. In some cases, hybridization between strains was below the level of reliable measurement. Similar experiments with ten recently isolated strains ofBacteroides ruminicola sub-sp.brevis revealed a similar degree of genetic divergence between isolates.     

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.     

Deoxyribonucleic acid hybridization betweenSpiroplasma citri and the corn stunt spiroplasma

The DNA base composition of the R8-A2 strain ofSpiroplasma citri and the I-747 and E strains of corn stunt spiroplasma was determined, by using the thermal denaturation temperature (T _m ), to be 26.8, 26.3, and 26.0 mol% (G+C), respectively. By the simple hybridization method, a measurement of the relative binding of homologous or heterologous labeled DNA to unlabeled, DNA-immobilized, nitrocellulose membrane, a homology of 56–60% was demonstrated betweenS. citri and two strains of corn stunt spiroplasma. A relatively higher DNA homology (66–71% between these two organisms was obtained in teh competition experiment, a measurement of the relative ability of homologous or heterologous competitor DNA to block the formation of homologous DNA-DNA duplex.     

Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells

Cytotoxic T lymphocytes (CTL) generated in C57BL/6 (H-2b) mice in response to infection with the serologically distinct herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were cross-reactive against target cells infected with either serotype. However, HSV-2-infected cells were shown to be much less susceptible to CTL-mediated lysis, and analysis through the use of HSV-1 X HSV-2 intertypic recombinants mapped the reduced susceptibility to a region contained within 0.82 to 1.00 map units of the HSV-2 genome. The study reported here was undertaken to determine the possible reasons for the reduced susceptibility of HSV-2-infected cells to lysis by CTL. Competition for the specific lysis of labeled HSV-1-infected cells by either HSV-1- or HSV-2-infected, unlabeled inhibitor cells and frequency analysis of the CTL precursor able to recognize HSV-1- and HSV-2-infected cells suggested that the reduced susceptibility of HSV-2-infected cells to lysis could be explained, at least in part, by reduced levels of target cell recognition. A determination of the surface expression of the critical elements involved in target cell recognition by CTL following infection with HSV-1 or HSV-2 revealed that all the major HSV-specific glycoprotein species were expressed. Infection with both HSV-1 and HSV-2 caused a reduction in the expression of the class I H-2 antigens. However, this reduction was much greater following infection with HSV-2. This suggested that one important factor contributing to reduced lysis of HSV-2-infected cells may be the altered or reduced expression of the class I H-2 self-antigens.     

单纯疱疹病毒2型特异性DNA片段的克隆和鉴定

用核酸限制性内切酶BamHI对单纯疱疹病毒2型(HSV—2)的DNA进行酶解,回收位于基因组中的反向重复序列区的Bam HIG片段,然后将其克隆在载体质粒PUC 8的Bam HI切点上,进一步用核酸限制性内切酶Eco RI和KPNI对这一重组质粒联合酶解,移去EcoRI—KPNI小片段,经末端修饰后,将其连接得到新的重组质粒pRC102,它含有一小段HSV—2的DNA序列。以此质粒为探针,分别与HSV—1、HSV—2及细胞DNA进行斑点杂交;与HSV—1和HSV—2酶解后的DNA片段进行Southern转印系交。两组实验结果显示,pRC102质粒DNA只与HSV—2 DNA特异性杂交,其HSV—2的型特异性良好。     

Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

Reticuloendotheliosis virus 60S RNA labeled with (125)I, or reticuloendotheliosis virus complementary DNA labeled with (3)H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and (125)I-labeled viral RNAs and by determination of melting temperatures. In contrast, (125)I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck.