Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32EcoRI CIV DNA fragments), was used for transfection ofChoristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring theEcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535–0.582 m.u.), M (7.25 kbp, 0.310–0.345 m.u.), O (6.5 kbp, 0.196–0.228 m.u.), Q (5.9 kbp, 0.603–0.631 m.u.), and Y (2.0 kbp, 0.381–0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of theEcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within theEcoRI DNA fragments C and H at the genome coordinates 0.924–0.930 and 0.535–0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of theEcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661–675 and 677–691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of theEcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535–0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within theEcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661–691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within theEcoRI CIV DNA fragment Y.     

Molecular cloning and physical and translational mapping of the frog virus 3 genome

A library of cloned fragments representing nearly the entire frog virus 3 (FV 3) genome (99.65%) has been constituted. Individual plasmid recombinants, labeled by nick-translation, were hybridized to Southern blots of genomic FV 3 DNA fragments obtained with XbaI, HindIII, SmaI, and SalI. From these results physical maps were generated and the distribution of restriction sites in the genome was established by double digestion of the fragments. A preliminary translational map was likewise developed. The viral messages were selected by hybridization to the recombinant DNAs immobilized on nitrocellulose filters and were translated in the reticulocyte cell-free system. About 30 polypeptides were detected among the translation products of RNA synthesized in the presence of cycloheximide. It appears that these genes are not clustered but in several cases more than one polypeptide is encoded by a given fragment. The 15 new polypeptide obtained by translation of late mRNAs derive from genes located on one-half of the genome.     

Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus

A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.     

Physical mapping of the DNA genome of insect iridescent virus type 9 from Wiseana spp. larvae

A physical map for the DNA genome of insect iridescent virus type 9 isolated from Wiseana spp. larvae [Lepidoptera: Hepialidae] was constructed using the restriction enzymes BamHl, EcoRl, and Pstl. Viral DNA was cloned into the plasmids pBR328 and pUC8 using Escherichia coli strains HB101 and JM83, respectively. The physical map for BamHl, EcoRl, and Pstl was constructed by multiple enzyme analysis and Southern hybridization of cloned viral DNA. Statistical analysis of restriction data by computer-aided linear modeling supported the physical map produced and indicated a total genome size of 192.5 kb. Due to the cyclic permutation of iridescent virus genomes the map is presented in a circular form.     

Identification and characterization of the repetitive DNA element in the genome of insect iridescent virus type 6

The genome of the Chilo iridescent virus (CIV) was analyzed for existence of repetitive DNA sequences by DNA-DNA hybridization using a defined and complete gene library of the viral genome (209 kbp) and by heteroduplex mapping. These experiments revealed the presence of repetitive DNA elements in the CIV genome, which are located in the EcoRI fragment H and in the EcoRI DNA fragment C at the coordinates 0.535 to 0.548 (EcoRI/Pstl DNA fragment, 2.7 kbp) and 0.920 to 0.944 (PvuII CIV DNA fragment L, 5.1 kbp), respectively. The DNA nucleotide sequence (2708 bp) of the EcoRI/Pstl subfragment was determined. The comparative analysis of the DNA sequences of this particular region of the viral genome with the DNA sequences of the PvuII DNA fragment L (5064 bp) revealed the presence of several DNA sequences within the EcoRI/Pstl subfragment of the EcoRI CIV DNA fragment H which show homology to DNA sequences of the PvuII DNA fragment L. For example, a DNA element (box A, 91 bp) is located at nucleotide positions 1981 to 2072 of the EcoRI CIV DNA fragment H which are complementary (greater than 90%) to the nine regions of the PvuII DNA fragment L (L-boxes 1 to 9). Furthermore heteroduplex mapping revealed the existence of a stem-loop structure (stem, 65 +/- 10 bp and loop, 652 +/- 80 bp) at the genome coordinates 0.571 to 0.582 (2.5 kbp, HindIII/EcoRI subfragment of the EcoRI CIV DNA fragment H). This indicates that an inverted repeat sequence is located at this region of the viral genome. The DNA nucleotide sequence of this subfragment was determined (2555 bp) which confirmed the data obtained from electron microscopy. An inverted repeat DNA sequence located at nucleotide positions 304 and 1011 is able to form this type of stem-loop structure.     

Characterization of the third origin of DNA replication of the genome of insect iridescent virus type 6

The structure of the third origin of DNA replication (CIV-ori-M) of the genome (209 kbp) of Chilo iridescent virus (CIV) was determined by DNA nucleotide sequence analysis. The CIV-ori-M is located within the DNA sequences of theEcoRI CIV DNA fragment M (7 kbp; 0.310–0.345 viral map units) between the genome coordinates 0.310 (EcoRI site) and 0.317 (NcoI site). The DNA nucleotide sequence of theEcoRI/NcoI CIV DNA fragment (1601 bp) was determined for identifying the DNA sequence of the corresponding origin of DNA replication. The analysis of the DNA sequences of this region revealed the presence of a 12-mer inverted repeat at nucleotide positions 485–496 and 503–513 (485-AGATATTTGACT-496-TATGT-503-AGTCAAATATCT-513) that are able to form a hairpin-loop structure. A double-stranded DNA fragment was synthesized that corresponds to the nucleotide positions 485–513 that were cloned into the phages M13mp18 and M13mp19, and were screened for their ability to be amplified in CF-124 cell cultures infected with CIV. The successful amplification of the DNA sequence of the CIV-ori-M is strong evidence that this particular region of the CIV genome indeed serves as the origin of DNA replication.The analysis of the DNA sequence of CIV-ori-M in comparison to the DNA sequence of the two other characterized origins of DNA replication (CIV-ori-H and Y) of the CIV genome (9) was carried out, and the results are shown in Table 2. According to these data the DNA sequence homology between the DNA sequences of the CIV-ori-M and CIV-ori-Y, and between the CIV-ori-M or CIV-ori-H, was found to be 58% and 55%, respectively.     

Characterization, molecular cloning, and physical mapping of the Shope fibroma virus genome

Five strains of Shope fibroma virus (SFV), a strain of rabbit myxoma virus, and a strain of vaccinia virus were compared by restriction endonuclease digestion of their viral DNAs. Restriction digest patterns revealed that SFV and rabbit myxoma, both members of the Leporipoxvirus genus, were distinct from vaccinia, an Orthopoxvirus. All strains of SFV examined had a high degree of nucleotide sequence homology as shown by conservation of restriction sites within their genomes. However, restriction patterns of SFV and myxoma were quite different from one another suggesting that the genomes from these two viruses of the Leporipoxvirus genus do not share a large, highly conserved region of homology as do the viruses belonging to the Orthopoxvirus genus. Restriction mapping identified inverted terminal repeats of approximately 12 kb in length. Restriction fragments representing all but 400 bp of the termini were cloned in plasmid vectors.     

Infectivity and pathogenesis of iridescent virus type 22 in various insect hosts

Summary This paper reports the results of a series of laboratory experiments to determine the infectivity and pathogenesis of iridescent virus type 22 (IV 22) for six species of mosquitoes, phlebotomine sand flies and triatomid bugs. Following inoculation, IV 22 replicated in all of the species tested, without producing noticeable mortality within a 14 day observation period. Examination of the infected insects by immunofluorescence demonstrated large amounts of viral antigen in many different organs. Electron microscopy done on infected mosquitoes (Aedes aegypti) showed large numbers of virus particles within cells of the fat body, muscle tracheal and midgut epithelium. Virus replication in the mosquitoes was confined to host cell cytoplasm and was similar to that described in the natural blackfly (Simulium) host. Transovarial transmission of IV 22 could not be demonstraed inA. aegypti, and only a small percentage of mosquito larvae could be infected orally. Results of these experiments are compatible with observations of other iridescent viruses; IV 22 is highly infectious for a wide range of insects when introduced into their hemolymph, but it is not very infectious per os. These characteristics would appear to limit its value as a potential biocontrol agent for Diptera.     

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome

Summary To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.     

Molecular cloning of the PRCII sarcoma viral genome and the chicken proto-oncogene c-fps

The class II avian sarcoma viruses comprise PRCII, PRCIIp, PRCIV, URI, 16L, and Fujinami. The members of this class are all replication-defective viruses containing various amounts of a transforming sequence called v-fps. PRCII contains the smallest amount of fps-specific sequences, transforms fibroblasts in tissue culture, but is only weakly tumorigenic. As a first step in understanding variations in pathogenicity among the class II avian sarcoma viruses and the mechanism by which the oncogene of these viruses was transduced from a single cellular locus, we have molecularly cloned the viral genome of PRCII, its related helper PRCII-AV, and the chicken proto-oncogene (c-fps) from which v-fps derived. The fps-specific region within the cloned PRCII genome was shown to be 0.8-1.0 kb smaller than that of the Fujinami fps-specific region, in agreement with previous studies. Transfection of the cloned DNAs into primary chicken cells demonstrated that both clones (PRCII and PRCII-AV) are biologically active. The cloned PRCII genome is helper dependent and produces a gag-fusion phosphoprotein (P105) which is phosphorylated on a tyrosine residue. The cloned PRCII-AV genome produces infectious virus and can function as a helper for the cloned PRCII genome in transfection assays. Three overlapping recombinant lambda clones homologous to v-fps from a chicken genomic library have been isolated. One of these, lambda-c-fps(2), contains all of the cellular sequences homologous to v-fps. In the aggregate, the three molecular clones may represent the entirety of c-fps.     

Molecular cloning and nucleotide sequence of the genome of hog cholera virus

A cDNA clone derived from genomic RNA of hog cholera virus (HCV) was identified using an oligonucleotide complementary to the RNA encoding a hexapeptide from the putative RNA-dependent RNA polymerase of the closely related bovine viral diarrhea virus (BVDV). This clone served as a probe for screening different size-selected cDNA libraries. After molecular cloning and nucleotide sequencing the HCV genome was shown to consist of 12,284 nucleotides containing one long open reading frame. Sequence comparison revealed a high degree of homology between HCV and BVDV genomic RNAs. With respect to HCV the genome of BVDV contains an insertion coding for 90 amino acids.     

Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus

The RNA genome of the cytopathic NADL isolate of bovine viral diarrhea virus (BVDV) has been molecularly cloned and the nucleotide sequence determined. The cloned sequence was 12,573 nucleotides in length, corresponding to a molecular weight of 4.3 X 10(6), having a base composition of 32.2% A, 25.7% G, 22.1% U, and 20.0% C. However, the sequences at the 5' and 3' termini of the RNA have not been unequivocally established. A single major open reading frame extending the length of the molecule was found in the viral-sense (positive polarity) sequence. This open reading frame was capable of encoding 3988 amino acids, representing 449 kDa of protein.     

Molecular cloning and physical mapping of porcine adenovirus types 1 and 2

Summary The 25R strain of porcine adenovirus type 1 (PAV-1) and the A47 strain of PAV-2 were propagated in ST cells, and DNA was extracted from the infected cells by a modified Hirt method. The DNA of each virus was digested by each of nine restriction endonucleases, and restriction enzyme fragments representing the entire genome were cloned. The genomic size of each virus was approximately 33 kb. Physical maps for the nine restriction endonucleases were constructed from the results of double digestion and Southern blot hybridization experiments, and oriented with respect to the PAV-3 genome. PAV-1 and PAV-2 were found to be related genetically to PAV-3, and there was a closer relationship between PAV-1 and PAV-3 than between PAV-1 and PAV-2 or between PAV-2 and PAV-3.     

The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning,physical mapping and gene location

Summary The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80 500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.     

Structural and thermodynamic investigation of the Chilo iridescent virus (Iridovirus type 6)

Structural features and thermodynamic parameters of the complete Chilo iridescent virus (Iridovirus type 6) and its constituents, isolated from the larvae of Galleria mellonella, were evaluated by means of UV spectroscopy and microcalorimetry. It can be demonstrated that the viral DNA is attached to the coat protein in a chromatin-like fashion, which is preserved after disruption of the virus by low temperature or partial digestion of the coat protein with proteinase K. At elevated temperature however the viral DNA is denaturated irreversibly. The coat protein appears to remain in its native state during the DNA transition and its own thermal denaturation profile shows its independence from the DNA denaturation.     

Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1.

Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.     

Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith strain)

We have cloned EcoRI and HindIII fragments of the Smith strain of murine cytomegalovirus (MCMV) in the plasmid vector pACYC184. These cloned fragments were used to establish a restriction endonuclease map of the genome with respect to the EcoRI and HindIII sites. The map was constructed on the basis of data derived from cross-hybridizations of EcoRI and HindIII cloned fragments, double-digestions of the cloned fragments with EcoRI and HindIII, and hybridization of cloned HindIII fragments to Southern blots of MCMV DNA cleaved with EcoRI. From our mapping data, we have determined that the length of the MCMV genome is approximately 240 kbp. The genome does not appear to undergo inversions and lacks detectable repeated sequences. One HindIII cloned fragment was obtained which contained both HindIII termini. The existence of this fragment may be related to the mode of replication of the MCMV genome.