Mutational properties of the primary aflatoxin B1-DNA adduct.

The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G --> T mutations of AFB1-N7-Gua, unlike those (if the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin. It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.

Mutation detection by highly sensitive methods indicates that p53 gene mutations in breast cancer can have important prognostic value.

Human cancer cells with a mutated p53 tumor-suppressor gene have a selective growth advantage and may exhibit resistance to ionizing radiation and certain chemotherapeutic agents. To examine the prognostic value of mutations in the p53 gene, a cohort of 90 Midwestern Caucasian breast cancer patients were analyzed with methodology that detects virtually 100% of all mutations. The presence of a p53 gene mutation was by far the single most predictive indicator for recurrence and death (relative risks of 4.7 and 23.2, respectively). Direct detection of p53 mutations had substantially greater prognostic value than immunohistochemical detection of p53 overexpression. Analysis of p53 gene mutations may permit identification of a subset of breast cancer patients who, despite lack of conventional indicators of poor prognosis, are at high risk of early recurrence and death.

Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/- ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.

Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis.

The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression.

Binding in the growth hormone receptor complex.

Binding reactions between human growth hormone (hGH) and its receptor provide a detailed account of how a polypeptide hormone activates its receptor and more generally how proteins interact. Through high-resolution structural and functional studies it is seen that hGH uses two different sites (site 1 and site 2) to bind two identical receptor molecules. This sequential dimerization reaction activates the receptor, presumably by bringing the intracellular domains into close proximity so they may activate cytosolic components. As a consequence of this mechanism it is possible to build antagonists to the receptor by introducing mutations in hGH that block binding at site 2 and to build even more potent antagonists by combining these with mutants that enhance binding at site 1. Alanine-scanning mutagenesis of all contact residues at the site 1 interface shows that only a small and complementary set of side chains clustered near the center of the interface affects binding. The most important contacts are hydrophobic, and these are surrounded by polar and charged interactions of lesser importance. Kinetic analysis shows for the most part that the important side chains function to maintain the complex, not to guide the hormone to the receptor. Hormone-induced homodimerization or heterodimerization reactions are turning out to be pervasive mechanisms for signal transduction. Moreover, the molecular recognition principles seen in the hGH-receptor complex are likely to generalize to other protein-protein complexes.

Differentiation of B cells in the nonlymphoid tissue of the synovial membrane of patients with rheumatoid arthritis.

In patients with rheumatoid arthritis the synovial membrane of the affected joint is infiltrated with lymphoid cells which may be arranged in structures resembling germinal centers. We have directly isolated such infiltrates to determine whether B-cell clones within them are selected and expanded in a process analogous to that which normally takes place in the germinal centers in secondary lymphoid organs. The data suggest that an antigen-driven process leads to the accumulation of B cells in the synovial membrane. The finding of identical sequences in consecutive sections suggests that under conditions of chronic stimulation, memory B cells may enter a stage of differentiation in which they proliferate without further accumulation of somatic mutations. Further we see intraclonal diversity which underlines the germinal center-like character of these infiltrates and demonstrates that a microenvironment is built up in this nonlymphoid tissue which supports antigen-dependent differentiation of B cells. This is the first demonstration, to our knowledge, of a germinal center-like reaction outside lymphoid tissue.

14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae.

Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas.

Malignant mesotheliomas (MMs) are aggressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been described in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofibromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neuroectodermal origin. Although MMs have a different histogenic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from 15 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2-encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene.

Electrophoretic variation in adenylate kinase of Neisseria meningitidis is due to inter- and intraspecies recombination.

In prokaryotic and eukaryotic organisms, the electrophoretic variation in housekeeping enzymes from natural populations is assumed to have arisen by the accumulation of stochastic predominantly neutral mutations. In the naturally transformable bacterium Neisseria meningitidis, we show that variation in the electrophoretic mobility of adenylate kinase is due to inter- and intraspecies recombination rather than mutation. The nucleotide sequences of the adenylate kinase gene (adk) from isolates that express the predominant slow electrophoretic variant were rather uniform, differing in sequence at an average of 1.1% of nucleotide sites. The adk sequences of rare isolates expressing the fast migrating variant were identical to each other but had a striking mosaic structure when compared to the adk genes from strains expressing the predominant variant. Thus the sequence from the fast variants was identical to those of typical slow variants in the first 158 bp of the gene but differed by 8.4% in the rest of the gene (nt 159-636). The fast electrophoretic variant appears to have arisen by the replacement of most of the meningococcal gene with the corresponding region from the adk gene of a closely related Neisseria species. The adk genes expressing the electrophoretic variant with intermediate mobility were perfect, or almost perfect, recombinants between the adk genes expressing the fast and slow variants. Recombination may, therefore, play a major role in the generation of electrophoretically detectable variation in housekeeping enzymes of some bacterial species.

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.

Mutations in the gene encoding the alpha subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa.

Mutations in the genes encoding two proteins of the retinal rod phototransduction cascade, opsin and the beta subunit of rod cGMP phosphodiesterase, cause retinitis pigmentosa (RP) in some families. Here we report defects in a third member of this biochemical pathway in still other patients with this disease. We screened 94 unrelated patients with autosomal dominant RP and 173 unrelated patients with autosomal recessive RP for mutations in the gene encoding the alpha subunit of the rod cGMP-gated cation channel. Five mutant sequences cosegregated with disease among four unrelated families with autosomal recessive RP. Two of these were nonsense mutations early in the reading frame (Glu76End and Lys139End) and one was a deletion encompassing most if not all of the transcriptional unit; these three alleles would not be expected to encode a functional channel. The remaining two mutations were a missense mutation (Ser316Phe) and a frameshift [Arg654(1-bp del)] mutation truncating the last 32 aa in the C terminus. The latter two mutations were expressed in vitro and found to encode proteins that were predominantly retained inside the cell instead of being targeted to the plasma membrane. We conclude that the absence or paucity of functional cGMP-gated cation channels in the plasma membrane is deleterious to rod photoreceptors and is an uncommon cause of RP.

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.

Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.