Comparison of the genomic short regions of a vaccine strain (SA-2) and a virulent strain (CSW-1) of infectious laryngotracheitis virus (Gallid herpesvirus 1)

Restriction enzyme linkage maps were produced for the genomic short region of the virulent infectious laryngotracheitis virus (CSW-1 strain). After comparison with the equivalent restriction enzyme linkage maps for the infectious laryngotracheitis virus SA-2 strain (a vaccine strain), it was determined that the maps for the short regions of the two strains were identical, apart from a single section in each of the inverted terminal repeats. Each inverted terminal repeat of the SA-2 strain was discovered to contain 467 base pairs more DNA than the CSW-1 strain's inverted terminal repeats. This extra DNA was more precisely mapped entirely within the EcoRI fragments D and d of SA-2, which were found to form part of the SmaI fragments U and P of SA-2 and Q and b of SA-2 and to contain one SmaI restriction enzyme site.     

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.     

Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida

A new insertion sequence (IS element), IS1411, was identified downstream of the phenol degradation genes pheBA that originated from plasmid DNA of Pseudomonas sp. strain EST1001. According to sequence analysis, IS1411 belongs to a new family of IS elements that has recently been named the ISL3 family (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725-774, 1998). IS1411 generates 8-bp duplication of the target DNA and carries 24-bp inverted repeats (IRs), highly homologous to the IRs of other IS elements belonging to this family. IS1411 was discovered as a result of insertional activation of promoterless pheBA genes in Pseudomonas putida due to the presence of outward-directed promoters at the left end of IS1411. Both promoters located on the IS element have sequences that are similar to the consensus sequence of Escherichia coli sigma70. IS1411 can produce IS circles, and the circle formation is enhanced when two copies of the element are present in the same plasmid.     

Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.     

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.     

IS481 and IS1002 of Bordetella pertussis create a 6-base-pair duplication upon insertion at a consensus target site

The insertion sequence IS481 and its isoform IS1002 have been observed to transpose into the bvgAS locus of Bordetella pertussis, for which the DNA sequence has previously been determined. Upon insertion of IS481 at three different sites and IS1002 at one site, a 6-bp sequence originally present was found at the junction of bvg and insertion sequence DNA. This indicates that, contrary to prior reports, IS481 and IS1002 do create a duplication upon insertion. In this light, examination of these and other examples of IS481 and IS1002 reported in the literature leads to the observation that the 6-bp recognition sequence usually fits the consensus NCTAGN. The near-palindromic nature of this sequence, when directly repeated at the ends of IS481 or IS1002, apparently led to the interpretation that 5 of these base pairs were part of the terminal inverted repeats flanking these elements.     

Circularization and cleavage of guinea pig cytomegalovirus genomes

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.     

Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT

The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.     

A deleted retroviral insertion at the ev21-K complex locus in Indonesian chickens

Very poor feather development has been observed in chickens of the Nunukan strain, originating from Indonesia. The wing of the newly hatched chick does not show any primary or covert feathers; this phenotype will be referred to as very-late feathering (VLF). As adults, chickens are feathered but tail feathers are short and fragile. An experimental population was set up at the National Institute of Agronomic Research (INRA), Jouy-en-Josas, from one Nunukan male and four Nunukan females. Preliminary observations did not support the hypothesis of a sex-linked dominant mode of inheritance for the VLF phenotype. A restriction fragment length polymorphism (RFLP) study using five restriction enzymes and two probes, RAV-2 and endogenous virus (ev) ev21-int specific for the endogenous viral locus ALVE21, showed the presence of the expected 3' junction fragments for the ev21 occupied site but failed to reveal the expected 5' junction fragments for ev21 in Nunukan chickens. The unoccupied site corresponded to the ev21 unoccupied repeat (UR) of type a (URa). A deletion in the 5' region of the provirus and of the insertion site was indicated by the RFLP analysis and confirmed by a PCR study. Primers were designed in order to amplify a 5' junction fragment specific to the modified ev21 found in the Nunukan chickens. The sequence of this amplified product showed that the deletion started 652 bp upstream of the insertion site of ev21 and ended within the pol gene of the viral genome. This deletion represents a new allele, OSD, at the ev21 insertion site (locus ALVE21), that appears insufficient to produce a complete virus. Current data do not show a clear causal relationship between OSD and the VLF phenotype. The presence of OSD may be required but is not in itself sufficient to obtain the VLF phenotype. The genetic relationships between OSD and the altered feathering phenotype of Nunukan chickens will be investigated further in families segregating for the VLF phenotype, using the locus-specific PCR test developed as part of this study.     

The genome of salmonid herpesvirus 1

Salmonid herpesvirus 1 (SalHV-1) is a pathogen of the rainbow trout (Oncorhynchus mykiss). Restriction endonuclease mapping, cosmid cloning, DNA hybridization, and targeted DNA sequencing experiments showed that the genome is 174.4 kbp in size, consisting of a long unique region (U(L); 133.4 kbp) linked to a short unique region (U(S); 25.6 kbp) which is flanked by an inverted repeat (R(S); 7.7 kbp). U(S) is present in virion DNA in either orientation, but U(L) is present in a single orientation. This structure is characteristic of the Varicellovirus genus of the subfamily Alphaherpesvirinae but has evidently evolved independently, since an analysis of randomly sampled DNA sequence data showed that SalHV-1 shares at least 18 genes with channel catfish virus (CCV), a fish herpesvirus whose complete sequence is known and which is unrelated to mammalian herpesviruses. The use of oligonucleotide probes demonstrated that in comparison with CCV, the conserved SalHV-1 genes are located in U(L) in at least five rearranged blocks. Large-scale gene rearrangements of this type are also characteristic of the three mammalian herpesvirus subfamilies. The junction between two SalHV-1 gene blocks was confirmed by sequencing a 4,245-bp region which contains the dUTPase gene, part of a putative spliced DNA polymerase gene, and one other complete gene. The implications of these findings in herpesvirus taxonomy are discussed.     

Differential effects of X-irradiation and cyclosporin-A administration on the thymus with respect to the generation of cyclosporin-A-induced autoimmunity

A new insertion sequence in Streptococcus pneumoniae was identified as a 1435-bp segment of the genome containing 24-bp terminal inverted repeats and flanked by 8-bp direct repeats. A copy of the element, named IS1167, adjacent to the comAB genes was sequenced; seven additional copies were found in the genome of strain CP1200 and relatives descended from strain R36A. Among 22 independent pneumococcal isolates, 11 contained copies of elements hybridizing to IS1167 in nine distinct restriction fragment patterns, with 3-12 copies each. The bulk of the element was occupied by two overlapping open reading frames, encoding basic proteins which together exhibited strong similarity to the full length of the putative transposase of the staphylococcal transposable element, IS1181, as well as significant similarity to those of seven additional known or putative insertion sequences related to the mycobacterial element, IS1096.     

Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria

A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.     

Protection of chickens with or without maternal antibodies against both Marek's and Newcastle diseases by one-time vaccination with recombinant vaccine of Marek's disease virus type 1

We constructed a stable recombinant Marek's disease virus type 1 (rMDV1) expressing the fusion protein (F) of Newcastle disease virus (NDV) by inserting the coding sequence within the US10 gene of MDV1 by homologous recombination and designated this as rMDV1-US10L(F). The NDV-F protein was significantly expressed under control of the SV40 late promoter in cultured cells infected with the rMDV1. To examine the protective efficacy of the rMDV1, specific pathogen-free (SPF) chickens were vaccinated with rMDV1 at one-day-old. Almost all birds (> 95%) were protected from NDV challenge via intramuscular, ocular, intranasal and intratrachial routes at 4 weeks after vaccination. The rMDV1 showed 100% protection against virulent MDV1 challenge in SPF chickens. Antibody responses against NDV-F and MDV1 antigens were observed at least up to 11 weeks after immunization. When the sera from chickens vaccinated with the rMDV1 were examined for the presence of anti-NDV-F antibody on the day of NDV challenge, the vaccinated bird group which did not survive from NDV challenge were found to show lower antibody titers than the surviving group. The rMDV1 also provided sufficient protection against NDV and MDV1 challenges in commercial chickens with maternal antibodies against NDV-F and MDV1 antigens.     

Molecular structure of a wheat chromosome end healed after gametocidal gene-induced breakage

In the progeny of the monosomic addition line of common wheat, Triticum aestivum, carrying the gametocidal chromosome of Aegilops cylindrica, deletion chromosomes carrying the break point within the nucleolar organizing region of chromosome 1B appeared. Attempts were made to amplify the break points by PCR using primers of telomere and rDNA (rRNA gene). In one deletion line, specific amplification of DNA fragments including the 18S rRNA gene, telomere repeats, and their junction occurred. At the junction of telomere and rRNA gene there was a 31-bp inverted duplication of the rRNA gene. Telomere sequences were initiated from the sequence TAG in the duplication. Between the duplications a small sequence was also inserted. This novel DNA sequence at the break point indicates that the breakage-fusion-bridge cycle(s) took place after the first chromatin breakage by the gametocidal gene.     

Non-random deletions in human foamy virus long terminal repeat during viral infection

We have characterized a new form of human foamy virus (HFV) non-random deleted long terminal repeat (LTR) sizing 1078-bp, deleted in its U3 region, sensitive to the viral transactivator and functional in an infectious proviral clone. Besides two known HFV LTRs of 1260-bp and 1123-bp, this LTR represents the smallest, designed S. Analysis of the LTR sequence shows the presence of short direct repeats surrounding the deletions, suggesting a mechanism generating deletion by misalignment of the growing strand during replication. Our data suggest that the deleted LTRs, preferentially associated with chronic viral infection, could be related with viral persistence.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Molecular cloning and expression of a novel human cDNA containing CAG repeats

A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.     

Targeted integration of adeno-associated virus-derived plasmids in transfected human cells

Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.