Regulation of glycoprotein D synthesis: does alpha 4, the major regulatory protein of herpes simplex virus 1, regulate late genes both positively and negatively?

Earlier studies have described the alpha 4/c113 baby hamster kidney cell line which constitutively expresses the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1). Introduction of the HSV-1 glycoprotein B (gB) gene, regulated as a gamma 1 gene, into these cells yielded a cell line which constitutively expressed both the alpha 4 and gamma 1 gB genes. The expression of the gB gene was dependent on the presence of functional alpha 4 protein. In this article we report that we introduced into the alpha 4/c113 and into the parental BHK cells, the HSV-1 BamHI J fragment, which encodes the domains of four genes, including those of glycoproteins D, G, and I (gD, gG, and gI), and most of the coding sequences of the glycoprotein E (gE) gene. In contrast to the earlier studies, we obtained significant constitutive expression of gD (also a gamma 1 gene) in a cell line (BJ) derived from parental BHK cells, but not in a cell line (alpha 4/BJ) which expresses functional alpha 4 protein. RNA homologous to the gD gene was present in significant amounts in the BJ cell line; smaller amounts of this RNA were detected in the alpha 4/BJ cell line. RNA homologous to gE, presumed to be polyadenylated from signals in the vector sequences, was present in the BJ cells but not in the alpha 4/BJ cells. The expression of the HSV-1 gD and gE genes was readily induced in the alpha 4/BJ cells by superinfection with HSV-2. The BJ cell line was, in contrast, resistant to expression of HSV-1 and HSV-2 genes. The BamHI J DNA fragment copy number was approximately 1 per BJ cell genome equivalent and 30 to 50 per alpha 4/BJ cell genome equivalent. We conclude that (i) the genes specifying gD and gB belong to different viral regulatory gene subsets, (ii) the gD gene is subject to both positive and negative regulation, (iii) both gD and gE mRNAs are subject to translational controls although they may be different, and (iv) the absence of expression of gD in the alpha 4/BJ cells reflects the expression of the alpha 4 protein in these cells.     

Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes

To determine which viral molecule(s) is recognized by herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL), target cells were constructed which express individual HSV glycoproteins. A mouse L cell line, Z4/6, which constitutively expressed high levels of HSV type 2 (HSV-2) gD (gD-2) was isolated and characterized previously (D. C. Johnson and J. R. Smiley, J. Virol. 54:682-689, 1985). Despite the expression of gD on the surface of Z4/6 cells, these cells were not killed by anti-HSV-2 CTL generated following intravaginal infection of syngeneic mice. In contrast, parental Z4 or Z4/6 cells infected with HSV-2 were lysed. Furthermore, unlabeled Z4/6 cells were unable to block the lysis of HSV-2-infected labeled target cells. Cells which express HSV-1 gB (gB-1) were isolated by transfecting L cells with the recombinant plasmid pSV2gBneo, which contains the HSV-1 gB structural sequences and the neomycin resistance gene coupled to the simian virus 40 early promoter and selecting G418-resistant cell lines. One such cell line, Lta/gB15, expressed gB which was detected by immunoprecipitation and at the cell surface by immunofluorescence. Additionally, cells expressing HSV-1 gC (gC-1) or gE (gE-1) were isolated by transfecting Z4 cells, which are L cells expressing ICP4 and ICP47, with either the recombinant plasmid pGE15neo, which contains the gE structural sequences and the neomycin resistance gene, or pDC17, which contains the gC structural gene coupled to the gD-1 promoter. A number of G418-resistant cell lines were isolated which expressed gC-1 or gE-1 at the cell surface. Anti-HSV-1 CTL generated following footpad infection of syngeneic mice were unable to lyse target cells expressing gB-1 or gE-1. In contrast, target cells expressing very low levels of gC-1 were killed as well as HSV-1-infected target cells. Furthermore, infection of gC-1-transformed target cells with wild-type HSV-1 or a strain of HSV-1 that does not express gC did not result in a marked increase in susceptibility to lysis. These results suggest that murine class I major histocompatibility complex-restricted anti-HSV CTL recognize gC-1 but do not recognize gB, gD, or gE as these molecules are expressed in transfected syngeneic target cells. The results are discussed in terms of recent evidence concerning the specificity of antiviral CTL.     

Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line.

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody.     

Intracellular transport of herpes simplex virus gD occurs more rapidly in uninfected cells than in infected cells.

A mouse L cell line which expresses the herpex simplex virus type 1 immediate-early polypeptides ICP4 and ICP47 was cotransfected with a cloned copy of the BglII L fragment of herpes simplex virus type 2, which includes the gene for gD, and the plasmid pSV2neo, which contains the aminoglycosyl 3'-phosphotransferase (agpt) gene conferring resistance to the antibiotic G418. A G418-resistant transformed cell line was isolated which expressed herpes simplex virus type 2 gD at higher levels than were found in infected cells. The intracellular transport and processing of gD was compared in transformed and infected cells. In the transformed Z4/6 cells gD was rapidly processed and transported to the cell surface; in contrast, the processing and cell surface appearance of gD in infected parental Z4 cells occurred at a much slower rate, and gD accumulated in nuclear membrane to a greater extent. Thus, the movement of HSV-2 gD to the cell surface in infected cells is retarded as viral glycoproteins accumulate in the nuclear envelope, probably because they interact with other viral structural components.     

Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells.

The gene for glycoprotein gB1 of herpes simplex virus type 1 strain Patton was expressed in stable Chinese hamster ovary cell lines. Expression vectors containing the dihydrofolate reductase (dhfr) cDNA plus the complete gB1 gene or a truncated gene lacking the 194 carboxyl-terminal amino acids of gB1 were transfected into CHO DHFR-deficient cells. Radioimmunoprecipitation demonstrated that the complete gB1 protein expressed in CHO cell lines was cell associated, whereas the truncated protein was secreted from the cells due to deletion of the transmembrane and C-terminal domains of gB1. Cells expressing the truncated gB1 protein were subjected to stepwise methotrexate selection, and a cell line was isolated in which the gB1 gene copy number had been amplified 10-fold and the level of expression of gB1 had increased over 60-fold. The truncated gB1 protein was purified from medium conditioned by the amplified cell line. N-terminal amino acid sequence analysis of this purified protein identified the signal peptide cleavage site and predicted the cleavage of a 30-amino-acid signal sequence from the primary protein. The immunogenicity of the truncated gB1 protein was also tested in mice, and high levels of antibody and protection from virus challenge were observed.     

Expression of Myelin Protein Genes and Other Myelin Components in an Oligodendrocytic Cell Line Conditionally Immortalized with a Temperature-Sensitive Retrovirus

Abstract: We have conditionally immortalized oligodendrocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (G418) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2′,3′-cyclic nucleotide 3′-phosphodiesterase mRNA. These cell lines grew continuously at the permissive temperature (34°C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39°C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin-specific protein mRNA levels were observed in N20.1 cells grown at 39°C for >9 days compared with cells maintained at 34°C. Immunocytochemical staining revealed N20.1 cells to be positive for the oligodendrocyte surface markers—galactocerebroside, A007, and A2B5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell-free protein synthesis experiments indicated that the MBP mRNAs isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 18.5-, and 21.5-kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a “mature” oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of “maturation’. This cell line has now been passaged >40 times with fidelity of phenotype and genotype.     

Detection of antibodies to herpes simplex virus type 2 with a mammalian cell line expressing glycoprotein gG-2

The gene (US4) coding for herpes simplex virus type 2 (HSV-2) glycoprotein G (gG-2) was cloned and constitutively expressed in Chinese hamster ovary (CHO) cells. The expression vector containing the dihydrofolate reductase (dhfr) gene, and the HSV-2 US4 gene under the control of the Simian virus 40 early promoter (SV40 EP), was transfected into dhfr-deficient CHO cells. The transfected cells were selected and amplified using methotrexate (MTX). To demonstrate that the gG-2 produced in these transformed cells had antigenic determinants in common with the native glycoprotein, CHO cells expressing gG-2 were used in an immunofluorescent assay (IFA) for the detection of HSV-2 type-specific antibodies in human serum samples. Seven of eight serum samples from adults with prior episodes of culture proven HSV-2 infections were found to be positive by the IFA method whereas none of seven serum samples from young children with culture documented HSV-1 infections were positive by IFA. Thus the recombinant CHO : gG-2 cells have diagnostic utility in an HSV-2 specific serologic assay.     

Glycoprotein D of herpes simplex virus encodes a domain which precludes penetration of cells expressing the glycoprotein by superinfecting herpes simplex virus.

Earlier studies have shown that herpes simplex viruses adsorb to but do not penetrate permissive baby hamster kidney clonal cell lines designated the BJ series and constitutively expressing the herpes simplex virus 1 (HSV-1) glycoprotein D (gD). To investigate the mechanism of the restriction, the following steps were done. First, wild-type HSV-1 strain F [HSV-1(F)] virus was passaged blindly serially on clonal line BJ-1 and mutant viruses [HSV-1(F)U] capable of penetration were selected. The DNA fragment capable of transferring the capacity to infect BJ cells by marker transfer contains the gD gene. The mutant gD, designated gDU, differed from wild-type gD only in the substitution of Leu-25 by proline. gDU reacted with monoclonal antibodies which neutralize virus and whose epitopes encompass known functional domains involved in virus entry into cells. It did not react with the monoclonal antibody AP7 previously shown to react with an epitope which includes Leu-25. Second, cell lines expressing gDU constitutively were constructed and cloned. Unlike the clonal cell lines constitutively expressing gD (e.g., the BJ cell line), those expressing gDU were infectable by both HSV-1(F) and HSV-1(F)U. Lastly, exposure of BJ cells to monoclonal antibody AP7 rendered the cells capable of being infected with HSV-1(F). The results indicate that (i) gD expresses a specific function, determined by sequences at or around Leu-25, which blocks entry of virus into cells synthesizing gD, (ii) the gD which blocks penetration by superinfecting virus is located in the plasma membrane, (iii) the target of the restriction to penetration is the identical domain of the gD molecule contained in the envelope of the superinfecting virus, and (iv) the molecular basis of the restriction does not involve competition for a host protein involved in entry, as was previously thought.     

Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.     

The early expression of glycoprotein B from herpes simplex virus can be detected by antigen-specific CD8+ T cells

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of na?ve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.     

Defining the herpes simplex virus-specific CD8+ T cell repertoire in C57BL/6 mice

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (β), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ～84 HSV-1 proteins are recognized by CD8(+) T cells, and most (～80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 β and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.     

G418抗性HEK293细胞的培育

目的　培育具有G418抗性的HEK2 93细胞 ,用于建立猪内源性反转录病毒感染人HEK2 93细胞的模型。方法　通过脂质体转染的方法 ,将含有neo基因的质粒pIRESneo导入HEK2 93细胞中 ,利用G418的选择特性 ,对转染细胞进行压力筛选 ,并对其进行了PCR鉴定。结果　经 6 0 0 μg ml的G418压力筛选后 ,获得了抗性细胞克隆。抗性细胞的形态和生长速度与筛选前细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了G418抗性HEK2 93细胞 ,为建立猪内源性反转录病毒感染人HEK2 93细胞的模型奠定了基础。     

Genetically tagged putative differentiation intermediates derived from undifferentiated HT29 human colon carcinoma cells.

The HT29 colonic carcinoma cell line has proven to be a very practical tool for modelling aspects of colonic cell differentiation and toxification by chemotherapeutic agents. As an approach to subclone and clarify molecular events involved in sublineage maturation, non-differentiated HT29 cells were electroporated with a dominant marker gene (NeoR) to convey aminoglycoside resistance (G418R). Transfectants surviving passage in glucose-G418 medium were >200 times the abundance of transient G418R cells of controls. Genomic analysis showed that each clonal type was unique in NeoR integration pattern while mitochondrial DNA copy was relatively unchanged. All of the randomly generated NeoR clones resembled the parental phenotype, but some over-produced the mucin, secretory cell type or the cell death phenotype after culturing in 2 mM sodium butyrate medium. Re-exposure to glucose medium restored the parental-like phenotype.     

The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.     

Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus

To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque assays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides.     

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.