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We describe gene targeting experiments involving a human cell line (RAN10) containing, in addition to its endogenous alleles, two ectopic alleles of the interferon-inducible gene 6-16. The frequency of gene targeting at one of the ectopic 6-16 alleles (H3.7) was 34-fold greater than the combined frequency of gene targeting involving endogenous 6-16 alleles in RAN10. Preference for H3.7 was maintained when the target loci in RAN10 were transcriptionally activated by interferon. Despite the 34-fold preference for H3.7, the absolute gene targeting efficiency in RAN10 was only 3-fold higher than in the parental HT1080 cell line. These data suggest that different alleles can compete with each other, and perhaps with non-homologous loci, in a step which is necessary, but not normally rate-limiting, for gene targeting. The efficiency of this step can therefore be more sensitive to chromosomal position effects than the rate-determining steps for gene targeting. The nature of the position effects involved remains unknown but does not correlate with transcription status, which in our system has a very modest influence on the frequency of gene targeting. In summary, our work unequivocally identifies a position effect on gene targeting in human cells.

Overexpression of the RAD52 epistasis group of gene products is a convenient way to investigate their in vivo roles in homologous recombination (HR) and DNA repair. Overexpression has the further attraction that any associated stimulation of HR may facilitate gene-targeting applications. Rad51p or Rad52p overexpression in mammalian cells have previously been shown to enhance some forms of HR and resistance to ionising radiation, but the effects of Rad52p overexpression on gene targeting have not been tested. Here we show that Rad52p overexpression inhibits gene targeting while stimulating extrachromosomal HR. We also find that Rad52p overexpression affects cell-cycle distribution, impairs cell survival and is lost during extensive passaging. Therefore, we suggest that excess Rad52p can inhibit the essential RAD51-dependent pathways of HR most likely to be responsible for gene targeting, while at the same time stimulating the RAD51-independent pathway thought to be responsible for extrachromosomal HR. The data also argue against Rad52p overexpression as a means of promoting gene targeting, and highlight the limitations of using a single HR assay to assess the overall status of HR.

Gene targeting allows the introduction of specific modifications into the eukaryotic genome by homologous recombination, but its efficiency is low in many mammalian systems. We are exploring different ways to increase the efficiency of gene targeting and we report here the effect of uracil incorporation in the targeting construct. Plasmids containing uracil substituting for a fraction of thymine residues are hyperrecombinogenic in some bacterial systems. To test whether a similar stimulation of recombination occurs in mammalian cells, we have prepared a uracil-rich HPRT targeting construct and quantified its homologous and nonhomologous recombination frequencies compared to the same plasmid lacking uracil. The uracil-rich plasmid led to reductions in both homologous and nonhomologous recombination in human cells.

The ideal therapy for single gene disorders would be repair of the mutated disease genes. Homologous recombination is one of several cellular mechanisms for the repair of DNA damage. Recombination between exogenous DNA and homologous chromosomal loci (gene targeting) can be used to repair an endogenous gene, but the low efficiency of this process is a serious barrier to its therapeutic potential. Recent progress in the isolation and characterisation of mammalian genes and proteins involved in DNA recombination has raised the possibility that the cellular biochemistry of recombination can be manipulated to improve the efficiency of gene targeting. As an initial test of this approach, we have overexpressed the gene encoding hRAD51, a protein with homologous DNA pairing and strand exchange activities, in human cells and measured its effect on gene targeting. We report a two- to three-fold increase in gene targeting, and enhanced resistance to ionising radiation in hRAD51-overexpressing cells with no obvious detrimental effects. These observations provide valuable genetic evidence for the involvement of hRAD51 in both gene targeting and DNA repair in human cells. Our data also establish overexpression of recombination genes as a viable approach to improving gene targeting efficiencies.

Gene targeting can be used for genetic studies of human cell lines and has significant potential for somatic cell gene therapy. These applications are however restricted by the low frequency of homologous recombination in higher eukaryotes compared to the relatively efficient nonhomologous integration of transfected DNA into the genome. As part of our attempts to overcome this problem, we compared two widely used transfection methods for their efficiency in gene targeting. To our surprise we found that, for conditions that render similar frequencies of nonhomologous integrants, lipofection is much less efficient than electroporation in generating targeted clones. This suggests that nonhomologous and homologous recombination have different requirements for DNA delivery in human cells.

African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3'-5'-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase beta (pol beta), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol beta-type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.

The present study describes the characterization of an African swine fever virus gene homologous to prenyltransferases. The gene, designated B318L, is located within the EcoRI B fragment in the central region of the virus genome, and encodes a polypeptide with a predicted molecular weight of 35,904. The protein is characterized by the presence of a putative hydrophobic transmembrane domain at the amino end. The gene is expressed at the late stage of virus infection, and transcription is initiated at positions -118, -119, -120, and -122 relative to the first nucleotide of the translation start codon. Protein B318L presents a colinear arrangement of the four highly conserved regions and the two aspartate-rich motifs characteristic of geranylgeranyl diphosphate synthases, farnesyl diphosphate synthases, and other prenyltransferases. Throughout these regions, the percentages of identity between protein B318L and various prenyltransferases range from 28.6 to 48.7%. The gene was cloned in vector pTrxFus without the amino-terminal hydrophobic region and expressed in Escherichia coli. The recombinant protein, purified essentially to homogeneity by affinity chromatography, catalyzes the sequential condensation of isopentenyl diphosphate with different allylic diphosphates, farnesyl diphosphate being the best allylic substrate of the reaction. All-trans-polyprenyl diphosphates containing 3-13 isoprene units are synthesized, which identifies the B318L protein as a trans-prenyltransferase.

We present an analysis of the complete genome of African swine fever virus (ASFV) strain BA71V, including 80 kbp of novel sequence and 90 kbp previously reported by several authors. The viral DNA is 170,101 nucleotides long and contains 151 open reading frames. Structural and/or functional information is available on 113 viral proteins. ASFV encodes five multigene families, putative membrane and secreted proteins, and enzymes involved in nucleotide and nucleic acid metabolism (including DNA repair) and protein modification. Database comparisons have provided clues about genes that may modulate the virus-host interaction, thus, possibly controlling ASFV virulence and persistence. The virus possesses genes similar to CD2, IkappaB, C-type lectins, MyD116/gadd34/gamma, 34.5, bcl-2/bax, iap, NifS, and ERV1, which may allow a viral regulation of cell adhesion, apoptosis, and redox metabolism, as well as of the host immune response against ASFV infection. The proteins encoded by different ASFV isolates are highly similar, the most variable ones being those belonging to multigene families, some membrane proteins, and those containing tandem repeats. DNA sequence data confirm the intermediate characteristics of ASFV between poxviruses and iridoviruses, supporting the notion that ASFV belongs to an independent virus family.

It has been reported that the propagation of African swine fever virus (ASFV) in cell culture generates viral subpopulations differing in protein p54 (C. Alcaraz, A. Brun, F. Ruiz-Gonzalvo, and J. M. Escribano, Virus Res. 23:173-182, 1992). A recombinant bacteriophage expressing a 328-bp fragment of the p54 gene was selected in a lambda phage expression library of ASFV genomic fragments by immunoscreening with antibodies against p54 protein. The sequence of this recombinant phage allowed the location of the p54 gene in the EcoRI E fragment of the ASFV genome. Nucleotide sequence obtained from this fragment revealed an open reading frame encoding a protein of 183 amino acids with a calculated molecular weight of 19,861. This protein contains a transmembrane domain and a Gly-Gly-X motif, a recognition sequence for protein processing of several ASFV structural proteins. In addition, two direct tandem repetitions were also found within this open reading frame. Further characterization of the transcription and gene product revealed that the p54 gene is translated from a late mRNA and the protein is incorporated to the external membrane of the virus particle. A comparison of the nucleotide sequence of the p54 gene carried by two virulent ASFV strains (E70 and E75) with that obtained from virus Ba71V showed 100% similarity. However, when p54 genes from viral clones generated by cell culture passage and coding for p54 proteins with different electrophoretic mobility were sequenced, they showed changes in the number of copies of a 12-nucleotide sequence repeat. These changes produce alterations in the number of copies of the amino acid sequence Pro-Ala-Ala-Ala present in p54, resulting in stepwise modifications in the molecular weight of the protein. These duplications and deletions of a tandem repeat sequence array within a protein coding region constitute a novel mechanism of genetic diversification in ASFV.

The putative DNA polymerase-encoding gene of African swine fever virus has been sequenced. The gene, designated G1207R, is located in the central region of the viral genome, and encodes a protein of 1207 amino acids (aa) with a predicted M(r) of 139,835. The gene is transcribed at both early and late stages of infection into a 4.1-kb RNA. Transcription is initiated at tsp, 8 nucleotides (nt) upstream from the start codon. Open reading frame (ORF) G1207R contains four direct repeats in tandem close to the 3'-end. Each repeat consists of 12 nt, coding for the reiterated sequence, K/NPAG. The deduced aa sequence of G1207R shows significant similarity with DNA polymerases from cellular and viral origin, belonging to the alpha-like family of DNA polymerases. In particular, the G1207R protein presents a colinear arrangement of all the 3'-->5' exonuclease and polymerization highly conserved aa regions characteristic of this group of DNA-dependent DNA polymerases.

Two open reading frames (ORFs) of African swine fever virus (ASFV) encoding putative helicases have been sequenced. The two genes, termed D1133L and B962L, are located in the central region of the viral genome, but are separated by about 40 kb of DNA. Both genes are expressed late during ASFV infection of Vero cells, after replication of viral DNA has begun. Contiguous to D1133L, three other ORFs (D129L, D79L and D339L), encoding putative proteins of unknown function, have been sequenced. Proteins D1133L and B962L contain the amino acid motifs that characterize helicases of superfamily II. D1133L is most similar to a group of putative helicases which includes two proteins of vaccinia virus (D11L and D6R) involved in transcription of the viral genome, their homologues in other poxviruses, the protein encoded by ORF 4 of the yeast plasmids, pGKL2 and pSKL, and the previously identified ASFV protein, Q706L. B962L resembles a group of RNA-helicase-like proteins which includes three proteins of Saccharomyces cerevisiae involved in pre-mRNA splicing (PRP2, PRP16 and PRP22), Drosophila melanogaster KURZ and MLE, and vaccinia virus 18R.

We have identified an open reading frame, EP402R, within the EcoRI E' fragment of the African swine fever virus genome that encodes a polypeptide of 402 amino acid residues homologous to the adhesion receptor of T cells, CD2. Transcription of EP402R takes place during the late phase of virus replication. The disruption of EP402R, achieved through the replacement of a 354-bp-long fragment from within EP402R by the marker gene lacZ, does not affect the virus growth rate in vitro but abrogates the ability of the virus to induce the adsorption of pig erythrocytes to the surface of infected cells. This result demonstrates that the protein encoded by EP402R is directly involved in the hemadsorption phenomenon induced by the infection of susceptible cells with African swine fever virus.

A putative thymidylate kinase gene of African swine fever virus has been identified at the left end of the SalI I' fragment of the virus genome. The gene, designated A240L, has the potential to encode a protein of 240 amino acids with an M(r) of 27,754 and is transcribed early after infection. Primer extension analysis indicates that transcription is initiated a short distance from the first ATG codon of open reading frame A240L. The deduced amino acid sequence of this open reading frame shows significant similarity with the human, yeast and vaccinia virus thymidylate kinases, the degree of identity being 23.7, 25 and 23.5%, respectively. The putative African swine fever virus thymidylate kinase sequence is essentially collinear with the other thymidylate kinase sequences, but contains a carboxy-terminal extension of 37 amino acids rich in glutamic and aspartic acids. The A240L protein conserves the ATP-binding and nucleotide/nucleoside-binding domains characteristic of thymidylate kinases.

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication.

Sequencing of the EcoRI N' fragment of African swine fever virus (ASFV) DNA revealed an open reading frame encoding a protein similar to ATP-dependent DNA ligases. When the gene encoding this protein was expressed in Escherichia coli, a protein of the expected molecular mass was labeled in bacterial extracts upon incubation with [alpha-32P]ATP. The recombinant protein comigrated in SDS-PAGE with the putative viral DNA ligase detected in extracts of infected cells. We demonstrate that the recombinant protein is a DNA ligase by dissociation of the protein-[32P]AMP adduct with pyrophosphate and nicked DNA. The putatively adenylylated lysine in ASFV is surrounded by two arginine residues, instead of by two hydrophobic amino acids as in the other ATP-dependent DNA ligases. This might explain the high concentration of pyrophosphate necessary to revert the DNA ligase--AMP adduct in ASFV, 10- to 100-fold higher than that required for other DNA ligases. A comparison of the amino acid sequences reported for ATP-dependent DNA ligases disclosed three new amino acid motifs around the adenylylation site of these enzymes. ASFV DNA ligase has little similarity to the other enzymes at the ends of the molecule, but conserves the amino acid motifs of the central region.

The gene coding for the guanylyltransferase of African swine fever virus has been identified and sequenced. The gene, designated NP868R, is located within fragments EcoRI N' and D of the virus genome (BA71V strain) and encodes a protein with a predicted molecular mass of 99.9 kDa that shares significant similarity with the large subunit of both vaccinia and Shope fibroma virus capping enzymes, with percentages of identity of 20.6 and 21.8%, respectively. A protein of 95 kDa was induced in Escherichia coli cells transformed with a recombinant plasmid carrying the NP868R gene. The E. coli expressed protein, as well as a protein of the same molecular weight present in African swine fever virus particles, form a covalent complex with GTP that can be reversed by pyrophosphate, two characteristic reactions of guanylyltransferases. An examination of the amino acid sequences of the African swine fever virus, poxvirus, and yeast guanylyltransferases has revealed a common motif around a lysine residue at the amino-terminal part of the proteins [Y(V, A)X2K(T, A)DG] which resembles the adenylylation site of DNA ligases (Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991). Proc. Natl. Acad. Sci. USA 88, 400-404). This lysine residue could be the guanylylation site in these enzymes.

Two African swine fever virus (ASFV) recombinant plasmids containing large inserts of DNA have been sequenced at random, and translations of the DNA sequence have been compared to libraries of vaccinia virus protein sequences. Among other genes identified by their extensive homology with vaccinia virus genes were the large and small subunits of ribonucleotide reductase. A 5.5-kb fragment from the Malawi (LIL20/1) strain of ASFV was identified as containing the genes for both these subunits. The fragment has been sequenced and the two genes have been found to be in a head-to-head orientation. The sequences are compared to other sequenced ribonucleotide reductase genes, and the evolutionary implications discussed.

Repetitive nucleotide sequencing of a dispensable genomic segment of a clonal population of African swine fever (ASF) virus has been carried out to estimate the mutant frequency to neutral alleles. Since no mutations have been detected in a total of 54026 nucleotides screened, the maximum mutant frequency is 5.5 x 10(-5) substitutions/nucleotide (95% confidence level). The result renders very unlikely the occurrence of hypermutational events during ASF virus DNA replication, at least within the selected DNA fragment.