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PubMed2004

Structural and functional analysis of ataxin-2 and ataxin-3.

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are autosomal-dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding regions of the genes encoding ataxin-2 and ataxin-3, respectively. To provide a rationale for further functional experiments, we explored the protein architectures of ataxin-2 and ataxin-3. Using structure-based multiple sequence alignments of homologous proteins, we investigated domains, sequence motifs, and interaction partners. Our analyses focused on presumably functional amino acids and the construction of tertiary structure models of the RNA-binding Lsm domain of ataxin-2 and the deubiquitinating Josephin domain of ataxin-3. We also speculate about distant evolutionary relationships of ubiquitin-binding UIM, GAT, UBA and CUE domains and helical ANTH and UBX domain extensions.

Amino Acid Sequence↗
PubMed2006

Functional analysis of glucan binding protein B from Streptococcus mutans.

Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these, Streptococcus mutans glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from S. agalactiae and S. pneumoniae, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true gbpB mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable gbpB null mutants supports the notion that gbpB is an essential gene. Northern blot and real-time PCR analyses suggested that induction of gbpB expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division.

Bacterial Proteins↗
PubMed1990

Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis.

The B subunit of Shiga toxin and the Shiga-like toxins (SLTs) mediates receptor binding, cytotoxic specificity, and extracellular localization of the holotoxin. While the functional receptor for Shiga toxin, SLT type I (SLT-I), and SLT-II is the glycolipid designated Gb3, SLT-II variant (SLT-IIv) may use a different glycolipid receptor. To identify the domains responsible for receptor binding, localization in Escherichia coli, and recognition by neutralizing monoclonal antibodies, oligonucleotide-directed site-specific mutagenesis was used to alter amino acid residues in the B subunits of Shiga toxin and SLT-IIv. Mutagenesis of a well-conserved hydrophilic region near the amino terminus of the Shiga toxin B subunit rendered the molecule nontoxic but did not affect immunoreactivity or holotoxin assembly. In addition, elimination of one cysteine residue, as well as truncation of the B polypeptide by 5 amino acids, caused a total loss of activity. Changing a glutamate to a glutamine at the carboxyl terminus of the Shiga toxin B subunit resulted in the loss of receptor binding and immunoreactivity. However, the corresponding mutation in the SLT-IIv B subunit (glutamine to glutamate) did not reduce the levels of cytotoxicity but did affect extracellular localization of the holotoxin in E. coli.

Amino Acid Sequence↗
PubMed1995

Functional analysis of sequences adjacent to dapE of Corynebacterium glutamicum reveals the presence of aroP, which encodes the aromatic amino acid transporter.

An initially nonclonable DNA locus close to a gene of L-lysine biosynthesis in Corynebacterium glutamicum was analyzed in detail. Its stepwise cloning and its functional identification by monitoring the amino acid uptakes of defined mutants, together with mechanistic studies, identified the corresponding structure as aroP, the general aromatic amino acid uptake system.

Amino Acid Sequence↗
PubMed2002

Functional analysis of the signal recognition particle in Escherichia coli by characterization of a temperature-sensitive ffh mutant.

The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42 degrees C as well as show reduced growth at the permissive temperature of 30 degrees C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42 degrees C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.

Alleles↗
PubMed2002

Functional analysis of 44 mutant androgen receptors from human prostate cancer.

Mutations of the androgen receptor gene are believed to contribute to the androgen-independent growth of prostate cancer cells. To date, 56 missense mutations of the androgen receptor have been identified in human prostate cancer. The functional status of most of these mutants has not yet been investigated. To address their functional properties, we generated 44 androgen receptor mutants that have been identified in human prostate cancer and used a colorimetric yeast reporter assay to analyze their transactivational activities in response to seven different ligands. We found that these mutant androgen receptors exhibited diverse transactivational activity: seven (16%) showed loss of function, three (7%) had wild-type function, 14 (32%) had partial function, and 20 (45%) had gains of function. Five of 20 gain-of-function mutants had promiscuous activity, being transactivated by non-androgens. We also found that the combination of estradiol and progesterone at physiological concentrations weakly or moderately activated an additional seven mutant androgen receptors. Our findings provide essential information for understanding the role of mutant androgen receptors in prostate cancer.

Estradiol↗
PubMed2004

[Expression establishment and functional analysis of breast cancer resistance protein with doxycycline induced tet regulating system in mouse fibroblast cell line PA317].

BACKGROUND & OBJECTIVE: Breast cancer resistance protein (BCRP), discovered in 1998, is a novel member of ATP-binding cassette (ABC) membrane transporters superfamily. This study was to establish the functional expression of BCRP with doxycycline (Dox) induced Tet regulating system in mouse fibroblast cell line PA317, provide an ideal experimental platform for understanding the mechanism of BCRP-mediated drug resistance, and develop effective methods to reverse the drug-resistance. METHODS: Tet-on regulating plasmid was transferred into PA317 cells under the Dox induced Tet-on regulating system, and stable expression of Tet-on was established in PA317 cells through G418 selection. The response plasmid of recombinant pTRE-BCRP was transferred into positive PA317/Tet-on cells, and stable expression of BCRP was established through hygromycin selection. Six single cellular clones were taken and cultivated in amplification. Positive PA317/Tet-on/TRE-BCRP cells, which showed well dose-response in expression of BCRP with different concentration of Dox induction, were selected by both reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The inhibitory effects of mitoxantrone on positive PA317/Tet-on/TRE-BCRP cells with various concentrations of Dox induction were detected by MTT method. Ko143, a specific inhibitor of BCRP, was used to detect the drug sensitivity of positive PA317/Tet-on/TRE-BCRP cells to mitoxantrone. Fluorescence intensity of remaining intracellular mitoxantrone in positive PA317/Tet-on/TRE-BCRP cells with different expression of BCRP was analyzed by flow cytometry (FCM). RESULTS: The positive No.5 PA317/Tet-on /TRE-BCRP cell clone showed well dose-response in expression of BCRP with different concentration of Dox induction; its drug-resistance against mitoxantrone has positive correlation with BCRP expression (r=0.995, P=0.002); its drug sensitivity to mitoxantrone was significantly enhanced by Ko143 (P< 0.05). Furthermore, different expression of BCRP induced by various concentrations of Dox caused different intracellular mitoxantrone retention in this cell clone. CONCLUSION: Functional expression of BCRP under Dox induced Tet regulating system was successfully established in PA317 cells, which provided an ideal experimental platform for further study of BCRP.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1980

Functional analysis of human T cell subsets defined by monoclonal antibodies. I. Collaborative T-T interactions in the immunoregulation of B cell differentiation.

T-B and T-T interactions involved in the regulation of PWM-triggered human B cell differentiation were studied in vitro. Functionally distinct human T cell subsets were isolated by C-mediated lysis by using the monoclonal antibodies OKT4 and OKT8. Graded numbers of either untreated or irradiated T cell subsets were added to autologous B cells, and total antibody synthesis was measured after 5 to 6 days of culture by using a highly sensitive reverse hemolytic plaque assay. The data indicate that a) the helper activity that is exclusively contained within the OKT4+ population is radiosensitive. Only at high T/B ratios can this radiosensitivity be overcome; b) the OKT8+ population contains radiosensitive cells important in suppressing B cell differentiation, and c) the suppression induced with OKT8+ cells requires the presence of radiosensitive OKT4+ cells. Thus, OKT8+ cells added to cultures containing B cells and irradiated OKT4+ cells do not suppress the PFC response. Addition of unirradiated OKT4+ cells to these cultures permits reexpression of suppression by OKT8+ cells. It is concluded that two radiosensitive cells, one within the OKT4+ population and the other within the OKT8+ population, collaborate to induce suppression. Possible mechanisms for this suppressive interaction including induction of suppressor precursor cells within the OKT4+ population or inhibition of OKT4+ helper cells by OKT8+ cells are discussed.

Antibodies↗
PubMed1999

The ferric uptake regulator (Fur) homologue of Helicobacter pylori: functional analysis of the coding gene and controlled production of the recombinant protein in Escherichia coli.

A homologue of the ferric uptake regulator protein Fur has recently been identified within the Helicobacter pylori genome. The promoterless gene on a plasmid did partially complement a fur-negative mutant of Escherichia coli, and was strongly positive in the Fur titration assay (FURTA). The genetic and functional characterization of the complete fur homologue performed in this study revealed that the gene is conserved among H. pylori strains ( > 95% identity), and does not carry nucleotide transitions in iron-resistant mutants of H. pylori. The fur homologue on a plasmid mediated full iron-dependent ferric uptake regulator activity in the fur-deficient mutant strains H1681 and H1780 of E. coli. Immunoblot analysis revealed that Fur from H. pylori cross-reacts with antibodies raised against Fur from E. coli. The fact that inactivation of the fur gene abolished the FURTA-positive phenotype in the E. coli indicator strain H1717, indicated that this phenotype is rather caused by the encoded protein than by real Fur titration. Subcloning of the fur gene into an expression vector allowed controlled production in E. coli, and purification of a recombinant version of the H. pylori Fur protein. In summary, the results confirm the function of the H. pylori Fur homologue as iron-dependent transcriptional repressor by its ability to interact with the Fur-regulated promoters of the genes fiu and fhuF in E. coli.

Amino Acid Sequence↗
PubMed1997

Functional analysis of mouse keratin 8 in polyoma middle T-induced mammary gland tumours.

Keratin 8 and 18 are commonly used as tumorigenic markers for various types of carcinomas. They are known to be involved in cell migration, cell invasiveness, plasminogen activity and drug and radiation resistance. To ascertain a potential function for simple epithelium keratins in mammary adenocarcinoma in vivo, keratin-8-deficient mice (mK8) were mated with transgenic mice carrying the middle T oncogene driven by the MMTV promoter. The resulting mK8 knockout and control progeny carrying the middle T transgene developed mammary gland tumours with the same incidence. However, the onset of palpable mammary gland tumours occurred earlier in mK8 mutant than in control mice. This effect was prominent in males where the onset in control animals is delayed overall, because of the lower hormonal inducibility of the MMTV promoter. Metastatic foci were observed in the lungs of all females and of a few males, independently of the genotype. Histological analysis revealed no morphological differences of the tumorigenic cells in primary tumours nor in metastatic foci. As expected, keratin 8 was absent in the mK8 tumours. Keratin 7 (mK7), keratin 18 (mK18) and keratin 19 (mK19) protein were observed in both primary and metastatic foci. These results constitute the first in vivo analysis of the role of simple epithelium keratins in mammary carcinogenesis. It demonstrates that the latency, but not the incidence nor the morphological features, of PyV middle T-induced mammary gland tumours is affected by keratin 8 deficiency.

Age Factors↗
PubMed1997

Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: evidence for the role of Sp1 and not of c-Myb or PU.1 in myelomonocytic lineage-specific expression.

The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelomonocytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in bone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to -16) that did not include the cluster of c-Myb sites. A 4-bp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22) including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4-bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super-shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene.

Antigens, Differentiation, Myelomonocytic↗
PubMed2001

Structure and function analysis of the calcium-related gene spray in Neurospora crassa.

The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame plus seven introns. The spray mutant had a T --> G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca(2+) via calcineurin.

Amino Acid Sequence↗
PubMed2006

Functional analysis of Arabidopsis NCED6 and NCED9 genes indicates that ABA synthesized in the endosperm is involved in the induction of seed dormancy.

The cleavage of 9-cis-epoxycarotenoids to xanthoxin, catalyzed by 9-cis-epoxycarotenoid dioxygenases, is considered to be the key regulatory step of abscisic acid (ABA) biosynthesis. In Arabidopsis, genes for these enzymes form a multigene family with nine members, only five of which are thought to be involved in ABA production. In contrast to the prominent function of AtNCED3 in stress responses, the physiological and developmental role of the other 9-cis-epoxycarotenoid dioxygenases (NCEDs) remain unknown. Our functional and expression analyses have revealed that AtNCED6 and AtNCED9 are required for ABA biosynthesis during seed development. Reverse genetic analysis showed that ABA levels were reduced in Atnced6 and Atnced9 mutant seeds. In addition, transgenic plants overexpressing the AtNCED6 gene overproduced ABA. In accordance with mutant phenotypes, both AtNCED6 and AtNCED9 exhibited seed-specific expression. Detailed cytological studies were carried out, either by using transcriptional fusions of the promoter with GUS and GFP reporter genes, or by in situ hybridization. Expression of AtNCED6 was observed exclusively in the endosperm during seed development, that of AtNCED9 in both embryo and endosperm at mid-development. In addition to reduced ABA levels, Atnced6 and Atnced9 mutant seeds were also resistant to paclobutrazol, a gibberellin biosynthesis inhibitor. Although seeds of single mutants were still dormant, reduced dormancy was observed in the Atnced6 Atnced9 double-mutant seeds. These demonstrate that ABA synthesized in both the endosperm and the embryo participates in the hormonal balance that controls seed dormancy and germination.

Abscisic Acid↗
PubMed2002

Functional analysis of cone-rod homeobox (CRX) mutations associated with retinal dystrophy.

Mutations in the photoreceptor transcription factor cone-rod homeobox (CRX) have been identified in patients with several forms of retinal degenerative disease. To investigate the mechanisms by which these mutations cause photoreceptor degeneration, CRX constructs representing eleven known mutations, as well as a set of C-terminal deletions, were generated and tested for their ability to activate a rhodopsin-luciferase reporter in a transient cell transfection assay. To further define functional domains, several Gal4dbd-Crx fusions were similarly tested using a Gal4 response element containing heterologous promoter. This analysis demonstrated that the C-terminal region, between amino acids 200 and 284, is essential for CRX-mediated transcriptional activation. Consistent with this, four mutants carrying C-terminal truncations demonstrated significantly reduced transcriptional activation. Confirming the importance of the homeodomain (HD), four of the five mutants carrying HD missense mutations displayed altered transactivating activity, either decreased (three) or increased (one). In vitro protein-DNA binding assays (EMSAs) with CRX-HD peptides representing the three HD mutants with decreased transactivating activity, indicated that the alteration was due to reduced, but not abolished, DNA binding to CRX targets. Taken together, these results support the hypothesis that CRX mutations involved in human photoreceptor degeneration act by impairing CRX-mediated transcriptional regulation of the photoreceptor genes. However, a clear relationship between the magnitude of biochemical abnormality and degree of disease severity was not observed, suggesting that other genetic and environmental modifiers may also contribute to the disease phenotype.

Amino Acid Sequence↗
PubMed1993

Acute inflammatory response to sheep red blood cell challenge in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): phenotypic and functional analysis of peritoneal exudate cells.

TCDD is a widespread environmental contaminant of concern to human health because of its well-recognized immunotoxicity in laboratory animals. Suppression of the murine antibody response to xenogeneic erythrocytes has been shown to be one of the most sensitive assays for TCDD immunotoxicity. However, the cellular mechanisms underlying the suppressed immune function have not been fully elucidated. In the present studies, peritoneal macrophage recruitment, activation, and antigen-presenting function in response to sheep red blood cell (SRBC) injection were compared in C57Bl/6 mice treated with a single oral dose of 0 or 5 micrograms TCDD/kg. In vehicle-treated mice, SRBC injection induced a typical inflammatory response in the peritoneal cavity. Within 6 hr, the number of neutrophils increased and remained elevated until 40 hr. Macrophage numbers increased at 24 hr and remained elevated through 72 hr. In TCDD-treated mice, a hyperinflammatory response to SRBC was observed. The total number of peritoneal exudate cells was significantly greater at 16, 24, and 40 hr after SRBC challenge when compared to that of vehicle-treated mice. The increased number of peritoneal cells reflected significant increases in both neutrophils and macrophages. Mac-1+ peritoneal cells were examined by two-color flow cytometric analysis on Days 0-3 after SRBC injection for expression of the activation markers F4/80 and I-A. The intensity of F4/80 fluorescence significantly decreased 24-72 hr following SRBC challenge, while fluorescence associated with I-A significantly increased at 72 hr. These changes are consistent with macrophage activation. TCDD did not significantly alter F4/80 expression on Mac-1+ cells, whereas I-A expression was increased earlier on cells from TCDD-treated mice. However, TCDD treatment did not alter the antigen presentation function of peritoneal cells, assessed by their ability to induce the proliferation of SRBC-primed T cells in vitro. The antigen-presenting function of adherent spleen cells was also not altered by TCDD exposure. To test the hypothesis that an excess number of phagocytes in TCDD-treated mice were clearing the antigen more efficiently, leading to a smaller (e.g., suppressed) antibody response, we attempted to overcome TCDD suppression by increasing the amount of SRBC antigen used for challenge. However, the magnitude of the anti-SRBC response in TCDD-treated mice was not significantly altered by increasing the antigen challenge dose, suggesting that enhanced clearance of antigen by macrophage is not a mechanism for TCDD-induced suppression of the anti-SRBC response.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2001

Disabling erbB receptors with rationally designed exocyclic mimetics of antibodies: structure-function analysis.

Overexpression of the HER2 receptor is observed in about 30% of breast and ovarian cancers and is often associated with an unfavorable prognosis. We have recently designed an anti-HER2 peptide (AHNP) based on the structure of the CDR-H3 loop of the anti-HER2 rhumAb 4D5 and showed that this peptide can mimic some functions of rhumAb 4D5. The peptide disabled HER2 tyrosine kinases in vitro and in vivo similar to the monoclonal antibody (Park, B.-W. et al. Nat. Biotechnol. 2000, 18, 194--198). AHNP has been shown to selectively bind to the extracellular domain of the HER2 receptor with a submicromolar affinity in Biacore assays. In the present paper, we demonstrate that in addition to being a structural and functional mimic of rhumAb 4D5, AHNP can also effectively compete with the antibody for binding to the HER2 receptor indicating a similar binding site for the peptide and the parental antibody. To further develop AHNP as an antitumor agent useful for preclinical trials and as a radiopharmaceutical to be used for tumor imaging, a number of derivatives of AHNP have been designed. Structure--function relationships have been studied using surface plasmon resonance technology. Some of the AHNP analogues have improved binding properties, solubility, and cytotoxic activity relative to AHNP. Residues in the exocyclic region of AHNP appear to be essential for high-affinity binding. Kinetic and equilibrium analysis of peptide-receptor binding for various AHNP analogues revealed a strong correlation between peptide binding characteristics and their biological activity. For AHNP analogues, dissociation rate constants have been shown to be better indicators of peptide biological activity than receptor-binding affinities. This study demonstrates a possibility of mimicking the well-documented antibody effects and its applications in tumor therapy by much smaller antibody-based cyclic peptides with potentially significant therapeutic advantages. Strategies used to improve binding properties of rationally designed AHNP analogues are discussed.

Animals↗
PubMed1998

Structural and functional analysis of the homing endonuclease PI-sceI by limited proteolytic cleavage and molecular cloning of partial digestion products.

PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the catalytic activity, we performed limited proteolytic digestion experiments with various proteases. Two protease-resistant fragments spanning the N- and C-terminal halves of the nuclease were identified using different proteases which cleave the protein in the same region. Each fragment contains one of the two conserved LAGLIDADG motifs. The products of the limited proteolytic digests were shown to remain associated and to exhibit specific DNA binding but to be inactive in DNA cleavage. Different from what is observed with native PI-SceI, only one complex is formed as shown in an electrophoretic mobility shift assay. Expression clones for the N- and C-terminal protein fragments obtained by tryptic digestion were constructed, and the proteins PI-SceI-N and PI-SceI-C were purified. Only PI-SceI-N exhibits DNA binding activity. Bending experiments with PI-SceI-N, a mixture of PI-SceI-N and PI-SceI-C, as well as the products of the limited tryptic digest show that a DNA substrate with the full length recognition sequence is bent by 45;. This degree of bending is also observed with a DNA containing only the right side of the recognition sequence, corresponding to one of the DNA cleavage products of PI-SceI. Our results demonstrate that the N-terminal half of PI-SceI which lacks one of the two LAGLDADG motifs is able to bind to DNA specifically and to induce one of the distortions observed to occur in the process of DNA binding by PI-SceI. These results are discussed in light of the recently solved crystal structure of PI-SceI and used to refine a model for the mechanism of DNA binding and cleavage by PI-SceI.

Amino Acid Sequence↗
PubMed1998

Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.

Adaptor Proteins, Signal Transducing↗