Brillouin-Raman microspectroscopy for the morpho-mechanical imaging of human lamellar bone.

Bone has a sophisticated architecture characterized by a hierarchical organization, starting at the sub-micrometre level. Thus, the analysis of the mechanical and structural properties of bone at this scale is essential to understand the relationship between its physiology, physical properties and chemical composition. Here, we unveil the potential of Brillouin-Raman microspectroscopy (BRaMS), an emerging correlative optical approach that can simultaneously assess bone mechanics and chemistry with micrometric resolution. Correlative hyperspectral imaging, performed on a human diaphyseal ring, reveals a complex microarchitecture that is reflected in extremely rich and informative spectra. An innovative method for mechanical properties analysis is proposed, mapping the intermixing of soft and hard tissue areas and revealing the coexistence of regions involved in remodelling processes, nutrient transportation and structural support. The mineralized regions appear elastically inhomogeneous, resembling the pattern of the osteons' lamellae, while Raman and energy-dispersive X-ray images through scanning electron microscopy show an overall uniform distribution of the mineral content, suggesting that other structural factors are responsible for lamellar micromechanical heterogeneity. These results, besides giving an important insight into cortical bone tissue properties, highlight the potential of BRaMS to access the origin of anisotropic mechanical properties, which are almost ubiquitous in other biological tissues.

Mechanical mapping with chemical specificity by confocal Brillouin and Raman microscopy.

We describe the first application of confocal Brillouin and Raman microscopy to ex vivo human epithelial tissue - Barrett's oesophagus. This non-invasive label-free approach provides high-resolution mechanical mapping with chemical specificity, opening the route to a new integrated method for multiple biomedical and bioengineering applications, and potentially in vivo real-time diagnostics.

Intratumor RNA interference of cell cycle genes slows down tumor progression.

Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge.

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.

Hydrogen bonding in liquid and supercritical 1-octanol and 2-octanol assessed by near and midinfrared spectroscopy.

The near and midinfrared spectra of 1-octanol (and 2-octanol) have been measured along the liquid-gas coexistence curve from room temperature up to the critical point and in the supercritical domain along the isotherm T=385 degrees C (and T=365 degrees C) above the critical point of both 1-octanol and 2-octanol for pressure ranging from 0.5 up to 15 MPa. The density values of SC 1- and 2-octanol have been estimated by analysing the near infrared (NIR) spectra in the 3nu(a)(CH) region. A quantitative analysis of the absorption band associated with the OH stretching vibration [nu(OH)] and its first and second overtones [2nu(OH) and 3nu(OH)] was carried out in order to estimate the percentage of "free" OH groups in both alcohols in the whole thermodynamic domain investigated here. Very consistent results have been obtained from the independent analysis of these three different absorption bands which gave us a good confidence in the degree of hydrogen bonding reported here for 1- and 2-octanol. Thus, the percentage of free OH groups which is around 5% in liquid 1-octanol under ambient conditions strongly increase up to 70%-80% at a temperature of about 340 degrees C. Then, in the supercritical domain, upon a decrease of the density from 0.4 to 0.1 g cm(-3), the fraction of free hydroxyl groups is nearly constant presenting a plateaulike regime around 80%. As the density decreases again, this plateau regime is followed by a further increase of X(nb) which reaches a value of 96% for the system in the gaseous phase (0.01 g cm(-3); P=0.45 MPa). Finally, it comes out from this study that the percentage of free OH groups is always greater in 2-octanol than in 1-octanol at the same density.

Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2(s)-M2 tetracycline transactivator and repressor system.

Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2(s)-M2) alone or in combination with Tet-repressor (tTS-Kid) were explored in the context of helper-dependent adenovirus vector. Various genetic elements were assembled in a series of vectors and the ability to control secreted alkaline phosphatase expression evaluated in vitro in HeLa cells and in vivo by intramuscular injection in both C57/B6 and Balb/C nude mice. The results allow us to draw some general conclusions about the combination of transcription regulators and their relative orientation to the transgene to achieve maximal induction, while minimizing leakiness of expression.

Tamarin alpha-interferon is active in mouse liver upon intramuscular gene delivery.

BACKGROUND: The hepatitis C virus (HCV) is responsible for a severe and widespread form of hepatitis for which a durable and effective therapy has not yet been established. The only approved therapy against hepatitis C, alpha-interferon protein intramuscular administration, presents numerous drawbacks that might be overcome by adopting a gene therapy approach. HCV exclusively infects humans and chimpanzees, hence an acceptable animal model for hepatitis C pharmacological studies is not available. Recently, tamarins infected by GB virus B (GBV-B) have been proposed as a surrogate animal model for HCV infection. The aim of the present study was the production of tamarin interferon (tIFN) through delivery of tIFN-coding DNA to evaluate the feasibility of a gene therapy approach based on IFN electro-gene transfer (EGT) in future studies with primates. METHODS: Production and biological activity of cloned tamarin interferon was monitored in cultured cells upon transfection and in mice upon muscle EGT of the corresponding plasmid DNA, respectively. RESULTS: A tamarin gene encoding a protein homologous to human interferon-alpha2 (hIFN-alpha2) has been cloned. The tamarin IFN-alpha (tIFN-alpha) protein shows antiviral activity in a cell-based assay. Upon EGT of the corresponding gene in mouse muscles, tIFN-alpha is detectable at high levels in serum for at least 4 months. Most important, activity of tIFN, measured as enhancement of mRNA levels of genes induced by type I IFNs, is also detectable in the liver of EGT-treated mice. CONCLUSION: The present study demonstrates that the delivery of tIFN-alpha DNA via intramuscular injection yields a functional protein able to produce biological effects inside a remote target organ, the liver. This finding, besides the specific purpose of the present study, is of general relevance with a view to establishing therapeutic protocols based on EGT.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

Regulated and prolonged expression of mIFN(alpha) in immunocompetent mice mediated by a helper-dependent adenovirus vector.

A major goal in gene therapy is to develop efficient gene transfer protocols that allow tissue-specific, long-term and tightly regulated expression of the desired transgene. This objective is becoming more attainable through the co-evolution of gene transfer vectors and regulation systems. The ideal vector should efficiently transduce non-dividing cells with minimal toxicity, thus endowing the system with persistent transgene expression. The helper-dependent adenovirus vectors meet these requirements, as demonstrated in various studies in the literature. The most promising regulation system is the tet-on system, which has low basal transcriptional activity and high inducibility. To explore the regulated transgene expression in the context of a helper-dependent vector, we constructed the HD-TET-IFN vector, containing the mIFN(alpha) gene under the control of the tetracycline inducible transactivator rtTA2(s)-S2. Mice injected with HD-TET-IFN showed high levels of serum mIFN(alpha) only upon transcriptional activation. The transgene expression was reinducible to the same high level up to 3 months p.i., and the amount of expressed cytokine could be regulated by dosing doxycycline. Transcriptional activation of mIFN(alpha) induced by doxycycline resulted in prolonged survival and reduced liver damage in HD-TET-IFN-injected mice challenged with a lethal dose of coronavirus. Activation of antiviral genes mediated by doxycycline-dependent mIFN(alpha) expression was also observed at low HD-TET-IFN doses. The possibility of controlling gene expression by the combination of HD vectors and the latest tet-on transactivator also holds promise for studying gene function in other animal models.

Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis.

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.

Effect of hMSH6 cDNA expression on the phenotype of mismatch repair-deficient colon cancer cell line HCT15.

Mismatch recognition in human cells is mediated primarily by a heterodimer of hMSH2 and hMSH6. Cells mutated in both alleles of the hMSH6 gene are deficient in the correction of base/base mispairs and insertion/deletion loops of one nucleotide and thus exhibit a strong mutator phenotype, evidenced by elevated mutation rates and microsatellite instability, as well as by tolerance to methylating agents. The decrease in replication fidelity associated with a loss of mismatch correction implies that with each division, these cells are likely to acquire new mutations throughout their genomes. Should such secondary mutations occur in genes linked to replication fidelity or involved in the maintenance of genomic stability, they might contribute to the observed mutator phenotype. The human colon tumour line HCT15 represents one such case. Although it carries inactivating mutations in both hMSH6 alleles, it has also been shown to contain a missense mutation in the coding sequence of the proofreading domain of the polymerase-delta gene. In an attempt to find out whether the phenotype of HCT15 cells was indeed brought about solely by the lack of hMSH6, we stably transfected them with a vector carrying the wild-type hMSH6 cDNA. Our results show that although the levels of transgenic hMSH6 were low, expression of the wild-type protein resulted in a substantial restoration of mismatch binding, mismatch repair capacity and the stability of mononucleotide repeats, as well as in the reduction of mutation rates. Although methylation tolerance of the hMSH6-expressing cells was not markedly affected, the G2 cell cycle checkpoint, absent in N-methyl-N'-nitro-N-nitrosoguanidine-treated control cells, was restored.

Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector.

Adenovirus (Ad) and adeno-associated virus (AAV) have attractive and complementary properties that can be exploited for gene transfer purposes. Ad vectors are probably the most efficient vehicles to deliver foreign genes both in vitro and in vivo. AAV exhibits the unique ability to establish latency by efficiently integrating at a specific locus of human chromosome 19 (AAVS1). Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs). In this study, we report the development of two helper-dependent adenoviral (HD) vectors, one carrying the Rep78 gene, the other an AAV-ITR-flanked transgene. Although Rep proteins have been demonstrated to interfere with Ad replication, HD Rep78 vector was successfully amplified on serial passages in 293CRE4 cells with a yield of 50-100 transducing units per cell. DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs. The high transduction efficiency, large cloning capacity, and high titer of the HD, combined with the site-specific integration machinery provided by AAV-derived components, make the Ad/AAV hybrid viruses a promising vehicle for gene therapy.

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.

hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutSalpha.

In extracts of human cells, base-base mismatches and small insertion/deletion loops are bound primarily by hMutSalpha, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160). Recombinant hMutSalpha bound a G/T mismatch-containing oligonucleotide with an apparent dissociation constant Kd = 2.6 nM, while its affinity for a homoduplex substrate was >20-fold lower. In the presence of ATP, hMutSalpha dissociated from mismatched oligonucleotide substrates, and this reaction was attenuated by mutating the conserved lysine in the ATP-binding domains of hMSH6, hMSH2 or both to arginine. Surprisingly, this reaction required only ATP binding, not hydrolysis. The ATPase activity of hMutSalpha variants carrying the Lys-->Arg mutation in hMSH2 or in hMSH6 was severely affected, but these mutants were still proficient in mismatch binding and were able to complement, albeit to different extents, mismatch repair-deficient cell extracts. The mismatch binding-proficient, ATPase-deficient double mutant was inactive in the complementation assay and its presence in repair-proficient extracts was inhibitory. We conclude that although the ATPase activity of hMutSalpha is dispensible for mismatch binding, it is required for mismatch correction.

Increased somatic recombination in methylation tolerant human cells with defective DNA mismatch repair.

We have studied whether spontaneous intrachromosomal recombination is altered in methylation tolerant human cells with a defect in mismatch repair. Somatic recombination was analysed in HeLaMR cells containing the vector pTPSN, which carries two copies of the gene for hygromycin resistance. The hygromycin genes are both inactivated by an inserted HindIII linker but hygromycin-resistant clones can arise by recombination. The spontaneous rate of recombination in a clone of HeLaMR cells containing a single integrated copy of pTPSN (HeLaG1) was 3.1x10(-6)/cell per generation. Two methylation tolerant variants from HeLaG1 cells (clone 12 and clone 15) were isolated by exposure to MNNG. Clone 12 cells exhibited a 16-fold increase in spontaneous mutation rate at the HPRT gene and extensive microsatellite instability at both mono- and dinucleotide repeats. Microsatellite instability limited to mononucleotide repeats was found in clone 15, whereas the mutation rate at HPRT was not significantly affected. A mismatch binding defect in extracts of clone 15 could be complemented by exogenous GTBP but not by purified hMSH2 protein. These data suggest that clone 15 is defective in GTBP. Extracts of clone 12 were unable to correct a single C:T mispair and complementation by extracts of human colorectal carcinoma cells with known deficiencies in mismatch repair indicated a defect in hMutLalpha. Western blotting with antibodies against different human mismatch repair proteins showed that clone 12 cells did not express hPMS2 protein, but expression of hMLH1, hMSH2 and GTBP appeared normal. The spontaneous recombination rate of clone 12 was 19-fold higher than the parental HeLaG1 cells, whereas no increase was observed in clone 15. Analysis of individual recombinants showed that hygromycin resistance arose exclusively by gene conversion. Our data indicate that mismatch correction regulates somatic recombination in human cells.

Site-specific integration in mammalian cells mediated by a new hybrid baculovirus-adeno-associated virus vector.

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the beta-galactosidase (beta-Gal) reporter gene and the hygromycin resistance (Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR-Hygr-beta-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.

Expression of long-patch and short-patch DNA mismatch repair proteins in the embryonic and adult mammalian brain.

Expression of the DNA mismatch repair (MMR) pathway was examined in the adult and developing rat brain. Rat homologues of human GTBP and MSH2, which are essential components of the post-replicative DNA MMR system, were identified in nuclear extracts from the adult and developing rat brain. Developmental studies showed that both GTBP and MSH2 levels were higher in nuclei isolated from the embryonic brain (day 16) than adult brain. However, this difference was not as dramatic as the difference in the number of proliferating cells. Levels of thymine DNA glycosylase (TDG), the enzyme which catalyzes the first step in short patch G:T mismatch repair, were also decreased in adult compared to embryonic brain. In the adult brain, MMR proteins were elevated in nuclear extracts enriched for neuronal nuclei. These results suggest that adult brain cells have the capacity to carry out DNA mismatch repair, in spite of a lack of ongoing DNA replication.

hMutSbeta, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA.

In human cells, mismatch recognition is mediated by a heterodimeric complex, hMutSalpha, comprised of two members of the MutS homolog (MSH) family of proteins, hMSH2 and GTBP [1,2]. Correspondingly, tumour-derived cell lines defective in hMSH2 and GTBP have a mutator phenotype [3,4], and extracts prepared from these cells lack mismatch-binding activity [1]. However, although hMSH2 mutant cell lines showed considerable microsatellite instability in tracts of mononucleotide and dinucleotide repeats [4,5], only mononucleotide repeats were somewhat unstable in GTBP mutants [4,6]. These findings, together with data showing that extracts of cells lacking GTBP are partially proficient in the repair of two-nucleotide loops [2], suggested that loop repair can be GTBP-independent. We show here that hMSH2 can also heterodimerize with a third human MSH family member, hMSH3, and that this complex, hMutSbeta, binds loops of one to four extrahelical bases. Our data further suggest that hMSH3 and GTBP are redundant in loop repair, and help explain why only mutations in hMSH2, and not in GTBP or hMSH3, segregate with hereditary non-polyposis colorectal cancer (HNPCC) [7].

Misincorporation rate and type on the leading and lagging strands of UV-damaged DNA.

We have examined the fidelity of replication of the leading and lagging strands of UV-irradiated DNA by using an EBV-derived shuttle vector system which contains as marker gene for mutation analysis the bacterial gpt gene in both orientations relative to the EBV oriP. Human cells stably transformed with this vector were UV irradiated and gpt mutation rate and type were analysed. An increased mutagenicity associated with UV irradiation was observed, but the average error frequency was unaffected by the direction of replication of the target gene. Some variability by position and sequence context of leading and lagging strand errors was detected, suggesting that the different architecture of the replication complex for the two strands might, to some extent, affect mutation spectra. The comparable fidelity of translesion replication on the leading and lagging strands is in agreement with the current model for eukaryotic replication that postulates the simultaneous synthesis of both strands by a DNA polymerase with a proof-reading exonuclease.

MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2.

The process of post-replicative DNA-mismatch repair seems to be highly evolutionarily conserved. In Escherichia coli, DNA mismatches are recognized by the MutS protein. Homologues of the E. coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals. Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) and human hMSH2 proteins have been shown to bind to mismatch-containing DNA in vitro. However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutS alpha isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP. It has been reported that S. cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2. We show here that, as in human cells, the S. cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP.