scholarly journals An Overlapping Bacterial Artificial Chromosome System That Generates Vectorless Progeny for Channel Catfish Herpesvirus

2008 ◽  
Vol 82 (8) ◽  
pp. 3872-3881 ◽  
Author(s):  
Dusan Kunec ◽  
Larry A. Hanson ◽  
Sandra van Haren ◽  
I. F. Nieuwenhuizen ◽  
Shane C. Burgess
Keyword(s):  
Channel Catfish ◽  
Infectious Clone ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Targeted Mutation ◽  
Infectious Clones ◽  
Viral Progeny ◽  
Herpesvirus 1

ABSTRACT Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild-type (wt) virus are extremely valuable tools for elucidating the roles of specific genes in virus pathophysiology as well as for making vaccines. Ictalurid herpesvirus 1 (channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here, we describe the cloning of the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BACs). These clones allowed us to regenerate vectorless wt CCVs with a phenotype that is indistinguishable from that of the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full-length CCV BAC by replacing the CCV ORF5 with the BAC cassette and cotransfecting CCO cells. The viral progeny that we used to transform Escherichia coli and the resulting BAC had only one of the 18-kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of the generation of an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants.

scholarly journals Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8320-8329 ◽  
Author(s):  
Eva-Maria Borst ◽  
Gabriele Hahn ◽  
Ulrich H. Koszinowski ◽  
Martin Messerle
Keyword(s):  
Escherichia Coli ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Reading Frame ◽  
E Coli ◽  
Artificial Chromosomes ◽  
Internal Repeat ◽  
The Stability

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

scholarly journals Chimeras Link to Tandem Repeats and Transposable Elements in Tetraploid Hybrid Fish

2016 ◽  
Author(s):  
Lihai Ye ◽  
Xiaojun Tang ◽  
Yiyi Chen ◽  
Li Ren ◽  
Fangzhou Hu ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Tandem Repeats ◽  
Artificial Chromosome ◽  
Strand Break ◽  
Bacterial Artificial Chromosomes ◽  
Distant Hybridization ◽  
Artificial Chromosomes ◽  
Hybrid Fish ◽  
The Relationship ◽  
Allotetraploid Hybrid

AbstractThe formation of the allotetraploid hybrid lineage (4nAT) encompasses both distant hybridization and polyploidization processes. The allotetraploid offspring have two sets of sub-genomes inherited from both parental species and therefore it is important to explore its genetic structure. Herein, we construct a bacterial artificial chromosome library of allotetraploids, and then sequence and analyze the full-length sequences of 19 bacterial artificial chromosomes. Sixty-eight DNA chimeras are identified, which are divided into four models according to the distribution of the genomic DNA derived from the parents. Among the 68 genetic chimeras, 44 (64.71%) are linked to tandem repeats (TRs) and 23 (33.82%) are linked to transposable elements (TEs). The chimeras linked to TRs are related to slipped-strand mispairing and double-strand break repair while the chimeras linked to TEs are benefit from the intervention of recombinases. In addition, TRs and TEs are linked not only with the recombinations, but also with the insertions/deletions of DNA segments. We conclude that DNA chimeras accompanied by TRs and TEs coordinate a balance between the sub-genomes derived from the parents which reduces the genomic shock effects and favors the evolutionary and adaptive capacity of the allotetraploidization. It is the first report on the relationship between formation of the DNA chimeras and TRs and TEs in the polyploid animals.

FISH of a maize sh2-selected sorghum BAC to chromosomes of Sorghum bicolor

Genome ◽  
1997 ◽  
Vol 40 (4) ◽  
pp. 475-478 ◽  
Author(s):  
Martha I. Gómez ◽  
M. Nurul Islam-Faridi ◽  
Sung-Sick Woo ◽  
Don Czeschin Jr. ◽  
Michael S. Zwick ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Sorghum Bicolor ◽  
Fluorescence In Situ Hybridization ◽  
Artificial Chromosome ◽  
Single Copy ◽  
Bacterial Artificial Chromosomes ◽  
Extra Copy ◽  
Artificial Chromosomes ◽  
Sorghum Plant

Fluorescence in situ hybridization (FISH) of a 205 kb Sorghum bicolor bacterial artificial chromosome (BAC) containing a sequence complementary to maize sh2 cDNA produced a large pair of FISH signals at one end of a midsize metacentric chromosome of S. bicolor. Three pairs of signals were observed in metaphase spreads of chromosomes of a sorghum plant containing an extra copy of one arm of the sorghum chromosome arbitrarily designated with the letter D. Therefore, the sequence cloned in this BAC must reside in the arm of chromosome D represented by this monotelosome. This demonstrates a novel procedure for physically mapping cloned genes or other single-copy sequences by FISH, sh2 in this case, by using BACs containing their complementary sequences. The results reported herein suggest homology, at least in part, between one arm of chromosome D in sorghum and the long arm of chromosome 3 in maize.Key words: sorghum, maize, shrunken locus, physical mapping, fluorescence in situ hybridization, bacterial artificial chromosomes.

scholarly journals Oncogenicity of Virulent Marek's Disease Virus Cloned as Bacterial Artificial Chromosomes

2004 ◽  
Vol 78 (23) ◽  
pp. 13376-13380 ◽  
Author(s):  
Lawrence Petherbridge ◽  
Andrew C. Brown ◽  
Susan J. Baigent ◽  
Ken Howes ◽  
Melanie A. Sacco ◽  
...  
Keyword(s):  
Disease Virus ◽  
Artificial Chromosome ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Wild Type Virus ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Bac Clones

ABSTRACT Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the US2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

scholarly journals Using Bacterial Artificial Chromosomes in Leukemia Research: The Experience at the University Cytogenetics Laboratory in Brest, France

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Etienne De Braekeleer ◽  
Nathalie Douet-Guilbert ◽  
Audrey Basinko ◽  
Frédéric Morel ◽  
Marie-Josée Le Bris ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Artificial Chromosome ◽  
Genome Project ◽  
Comparative Genomic ◽  
Cancer Genes ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Dna Libraries ◽  
Conventional Cytogenetics

The development of the bacterial artificial chromosome (BAC) system was driven in part by the human genome project in order to construct genomic DNA libraries and physical maps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300 kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions.

scholarly journals Cloning of the genome of Alcelaphine herpesvirus 1 as an infectious and pathogenic bacterial artificial chromosome

2006 ◽  
Vol 87 (3) ◽  
pp. 509-517 ◽  
Author(s):  
B. Dewals ◽  
C. Boudry ◽  
L. Gillet ◽  
N. Markine-Goriaynoff ◽  
L. de Leval ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Parental Strain ◽  
Recombinant Virus ◽  
Artificial Chromosome ◽  
Important Advance ◽  
Bac Clone ◽  
Coding Regions ◽  
Viral Genes ◽  
Herpesvirus 1 ◽  
Low Passage

Alcelaphine herpesvirus 1 (AlHV-1), carried asymptomatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order Artiodactyla. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, mainly due to the fact that AlHV-1 becomes attenuated during passage in culture. In this study, these difficulties were overcome by cloning the entire AlHV-1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC). A modified loxP-flanked BAC cassette was inserted in one of the two large non-coding regions of the AlHV-1 genome. This insertion allowed the production of an AlHV-1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. The loxP-flanked BAC cassette was excised from the genome of reconstituted virions by growing them in permissive cells stably expressing Cre recombinase. Importantly, BAC-derived AlHV-1 virions replicated comparably to the virulent (low-passage) AlHV-1 parental strain and induced MCF in rabbits that was indistinguishable from that of the virulent parental strain. The availability of the AlHV-1 BAC is an important advance for the study of MCF that will allow the identification of viral genes involved in MCF pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

scholarly journals Replication-Competent Bacterial Artificial Chromosomes of Marek's Disease Virus: Novel Tools for Generation of Molecularly Defined Herpesvirus Vaccines

2003 ◽  
Vol 77 (16) ◽  
pp. 8712-8718 ◽  
Author(s):  
Lawrence Petherbridge ◽  
Ken Howes ◽  
Susan J. Baigent ◽  
Melanie A. Sacco ◽  
Simon Evans ◽  
...  
Keyword(s):  
Dna Vaccine ◽  
Marek's Disease ◽  
Published Data ◽  
Marek’S Disease ◽  
Bacterial Artificial Chromosomes ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Bac Clones

ABSTRACT Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.

scholarly journals Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during CultureIn Vitro

2016 ◽  
Vol 90 (8) ◽  
pp. 3929-3943 ◽  
Author(s):  
Isa Murrell ◽  
Gavin S. Wilkie ◽  
Andrew J. Davison ◽  
Evelina Statkute ◽  
Ceri A. Fielding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Clinical Strain ◽  
Minimal Risk ◽  
Bacterial Artificial Chromosomes ◽  
Wild Type ◽  
Artificial Chromosomes

ABSTRACTClinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb′ (UL/b′) region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L.IMPORTANCEResearchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagatedin vitrowith minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.

scholarly journals Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

2006 ◽  
Vol 80 (21) ◽  
pp. 10900-10906 ◽  
Author(s):  
Fernando Almazán ◽  
Marta L. DeDiego ◽  
Carmen Galán ◽  
David Escors ◽  
Enrique Álvarez ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Cdna Clone ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Genetic System ◽  
Infectious Cdna Clone ◽  
Reverse Genetic System ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Processing Enzymes

ABSTRACT The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2′-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

scholarly journals Construction and Manipulation of an Infectious Clone of the Bovine Herpesvirus 1 Genome Maintained as a Bacterial Artificial Chromosome

2002 ◽  
Vol 76 (13) ◽  
pp. 6660-6668 ◽  
Author(s):  
Timothy J. Mahony ◽  
Fiona M. McCarthy ◽  
Jennifer L. Gravel ◽  
Lani West ◽  
Peter L. Young
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Infectious Clone ◽  
Bovine Herpesvirus ◽  
Artificial Chromosome ◽  
Gene Encoding ◽  
Kinase Gene ◽  
Bovine Herpesvirus 1 ◽  
Glycoprotein E ◽  
Herpesvirus 1

ABSTRACT The complete genome of bovine herpesvirus 1 (BoHV-1) strain V155 has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli strain DH10B, the BoHV-1 BAC was stably propagated over multiple generations of its host. BAC DNA recovered from DH10B cells and transfected into bovine cells produced a cytopathic effect which was indistinguishable from that of the parent virus. Analysis of the replication kinetics of the viral progeny indicated that insertion of the BAC vector into the thymidine kinase gene did not affect viral replication. Specific manipulation of the BAC was demonstrated by deleting the gene encoding glycoprotein E by homologous recombination in DH10B cells facilitated by GET recombination. These studies illustrate that the propagation and manipulation of herpesviruses in bacterial systems will allow for rapid and accurate characterization of BoHV-1 genes. In turn, this will allow for the full utilization of BoHV-1 as a vaccine vector.

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