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PubMed2000

Adaptive mutation: implications for evolution.

Adaptive mutation is defined as a process that, during nonlethal selections, produces mutations that relieve the selective pressure whether or not other, nonselected mutations are also produced. Examples of adaptive mutation or related phenomena have been reported in bacteria and yeast but not yet outside of microorganisms. A decade of research on adaptive mutation has revealed mechanisms that may increase mutation rates under adverse conditions. This article focuses on mechanisms that produce adaptive mutations in one strain of Escherichia coli, FC40. These mechanisms include recombination-induced DNA replication, the placement of genes on a conjugal plasmid, and a transient mutator state. The implications of these various phenomena for adaptive evolution in microorganisms are discussed.

Adaptation, Physiological↗
PubMed2001

Tau proteins with frontotemporal dementia-17 mutations have both altered expression levels and phosphorylation profiles in differentiated neuroblastoma cells.

The inherited form of frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) has been attributed to mutations in the tau gene. Pathologically, affected FTDP-17 brains share tau aggregates with other tauopathies, the most common being Alzheimer's disease. FTDP-17 mutations may therefore affect tau function leading to tau aggregation and cell loss. Interaction of tau with microtubules is thought to be regulated by phosphorylation. Investigating FTDP-17 mutations transiently expressed as enhanced green fluorescent protein (EGFP)-tagged proteins for the first time in differentiated neuronal cells, we found that two out of three missense mutations showed surprisingly decreased phosphorylation at the pathologically relevant S202/T205 site, mutant EGFP-tau being completely dephosphorylated in most cells. Moreover, phosphorylation at the S396/S404 site was moderately decreased for all mutant isoforms. Although microtubule integrity was not affected, with all mutants tested we demonstrated an increase in cellular tau protein level, some of which is microtubule-bound. Further enhancing this EGFP-tau accumulation by inhibition of tau degradation resulted in the previously less phosphorylated mutant EGFP-tau becoming highly phosphorylated. We conclude that the missense tau mutations primarily result in an excess of neuronal tau, which may interfere with important cellular functions such as axonal transport.

Alzheimer Disease↗
PubMed1997

Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling.

Members of the Smad family of intracellular signal transducers are essential for transforming growth factor-beta (TGF-beta) to exert its multifunctional effects. After activation of TGF-beta receptors, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. Thereafter, these activated Smad complexes translocate to the nucleus, where they may direct transcriptional responses. Here we report that TGF-beta mediates phosphorylation of Smad2 at two serine residues in the C terminus, i.e. Ser465 and Ser467, which are phosphorylated in an obligate order; phosphorylation of Ser465 requires that Ser467 be phosphorylated. Transfection of Smad2 with mutation of Ser465 and/or Ser467 to alanine residues into Mv1Lu cells resulted in dominant-negative inhibition of TGF-beta signaling. These Smad2 mutants were found to stably interact with an activated TGF-beta receptor complex, in contrast to wild-type Smad2, which interacts only transiently. Mutation of Ser465 and Ser467 in Smad2 abrogated complex formation of this mutant with Smad4 and blocked the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Moreover, peptides from the C terminus of Smad2 containing phosphorylated Ser465 and Ser467 were found to bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides were less effective. Thus, phosphorylated Ser465 and Ser467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation.

Amino Acid Sequence↗
PubMed2000

Characterization of beta-galactosidase mutations Asp332-->Asn and Arg148-->Ser, and a polymorphism, Ser532-->Gly, in a case of GM1 gangliosidosis.

We have identified and characterized three missense mutations in a patient with type 1 G(M1) gangliosidosis, namely a substitution of G for A at nucleotide position 1044 (G1044-->A; in exon 10) on one allele, which converts Asp(332) into asparagine, and both a mutation (C492-->A in exon 4, leading to the amino acid change of Arg(148)-->Ser) and a polymorphism (A1644-->G in exon 15, leading to a change of Ser(532)-->Gly) on the other allele. This patient had less than 1% residual beta-galactosidase activity and minimally detectable levels of immunoreactive beta-galactosidase protein in fibroblasts. To account for the above findings, a series of expression and immunolocalization studies were undertaken to assess the impact of each mutation. Transient overexpression in COS-1 cells of cDNAs encoding Asp(332)Asn, Arg(148)Ser and Ser(532)Gly mutant beta-galactosidases produced abundant amounts of precursor beta-galactosidase, with activities of 0, 84 and 81% compared with the cDNA clone for wild-type beta-galactosidase (GP8). Since the level of vector-driven expression is much less in Chinese hamster ovary (CHO) cells than in COS-1 cells, and we knew that exogenous beta-galactosidase undergoes lysosomal processing when expressed in these cells, transient expression studies were performed of Arg(148)Ser and Ser(532)Gly, which yielded active forms of the enzyme. In this case, the Arg(148)Ser and Ser(532)Gly products gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg(148)Ser mutation introduced a major conformational change into the protein, and we anticipated that it would be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp(332)Asn mutation on the catalytic activity, we isolated CHO clones permanently transfected with the Asp(332)Asn and Asp(332)Glu constructs, purified the enzymes by substrate-analogue-affinity chromatography, and determined their kinetic parameters. The V(max) values of both mutant recombinant enzymes were markedly reduced (less than 0.9% of the control), and the K(m) values were unchanged compared with the corresponding wild-type enzyme isolated at the same time. Both the Arg(148)Ser beta-galactosidase in CHO cells and Asp(332)Asn beta-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal beta-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg(148)Ser mutation has no effect on catalytic activity, whereas the Asp(332)Asn mutation seriously reduces catalytic activity, suggesting that Asp(332) might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg(148)Ser and Asp(332)Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in G(M1) gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most G(M1) gangliosidosis patient fibroblasts.

Amino Acid Substitution↗
PubMed2003

Molecular characterization of coagulation factor XII deficiency in a Japanese family.

We report the identification in a Japanese family of a novel homozygous W486C mutation in the protease domain of coagulation factor XII (FXII), which was associated with the reduction of plasma FXII activity and antigen level to less than 5% of normal. Sequences of each exon for FXII gene was analysed in family members by polymerase chain reaction (PCR) amplification followed by a direct sequencing method. Sequence analysis showed a homozygous substitution of G to C at nucleotide position 10587 (cDNA position 1458) in proband's FXII gene, resulting in a Trp to Cys substitution in the catalytic domain of FXII. PCR-fragment length polymorphism analysis of 55 healthy volunteers showed no such mutation. Transient expression of FXII in HK-293T cells and analysis of FXII antigen in culture media and cell lysates showed reduced secretion of mutant protein by more than 84% relative to that of wild type protein although the intracellular contents were similar. Our results suggest that the reduced secretion of FXII protein was due to incorrect folding caused by the introduction of Cys486. We designated this mutation as FXII Mie-1.

Amino Acid Substitution↗
PubMed1985

Molecular basis of an inherited retinal defect in Drosophila.

Fruitflies carrying the autosomal recessive mutation transient receptor potential (trp) are blind in bright light because the receptor potential of such a mutant decays almost completely during an intense stimulus. The trp gene has been localized and a set of partially overlapping genomic clones that include the trp gene has been isolated. The stretch of DNA represented by these genomic clones is found to contain genes that encode for four RNA species. Two of these RNA species are missing in the mutant. This observation is consistent with the notion that the mutation alters the DNA sequence in a region containing signals necessary for the expression of the gene. Accordingly, the molecular basis of the mutant phenotype may be due to the lack of a protein(s) that is/are important for normal visual transduction.

Animals↗
PubMed1998

Transient and heritable mutators in adaptive evolution in the lab and in nature.

Major advances in understanding the molecular mechanism of recombination-dependent stationary-phase mutation in Escherichia coli occurred this past year. These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient. We find that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously. We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab. We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.

Adaptation, Biological↗
PubMed1980

Membrane currents of pawn mutants of the pwA group in Paramecium tetraurelia.

Membrane currents were recorded from the wild type and two pawn mutants of the pwA complementation group in Paramecium tetraurelia under a voltage clamp. Most currents are not changed by the mutations. Transient inward currents of a leaky mutant, pwA132, upon step depolarizations are less than those in the wild type. The inward transient is completely lacking in a non-leaky mutant, pwA500. The time course of the residual inward currents in the leaky mutant is not significantly different from that of wild type. The voltage sensitivity of the Ca channels in the leaky mutant is also similar to that of wild type. The inward currents upon membrane hyperpolarizations in the mutants show normal characteristics in the presence or absence of external K+. With sufficiently large, prolonged depolarization, outward currents progressively develop in the wild type but decay in the mutants. The simplest conclusion we can draw is that the pwA mutations reduce the number of functional Ca channels but do not change the channel characteristics. From the conductance measurements, 45% of the Ca channels remain in the leaky mutant pwA132, and none remain in the non-leaky mutant pwA500. By subtracting the outward currents of pwA500 from the slow and prolonged outward currents of the wild type, we have tentatively separated a Ca-induced K+ current from the voltage-dependent K+ current. The time courses of these two currents differ by two orders of magnitude.

Animals↗
PubMed1998

Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the traffic ATPase family that includes multiple proteins characterized by (1) ATP binding, (2) conserved transmembrane (TM) motifs and nucleotide binding domains (NBDs), and (3) molecular transport of small molecules across the cell membrane. While CFTR NBD-1 mediates ATP binding and hydrolysis, the membrane topology and function of this domain in living eukaryotic cells remains uncertain. In these studies, we have expressed wild-type CFTR NBD-1 (amino acids 433-586) or NBD-1 containing the DeltaF508 mutation transiently in COS-7 cells and established that the domain is situated across the plasma membrane by four independent assays; namely, extracellular chymotrypsin digestion, surface protein biotinylation, confocal immunofluorescent microscopy, and functional measurements of cell membrane anion permeability. Functional studies indicate that basal halide permeability is enhanced above control conditions following wild-type or DeltaF508 NBD-1 expression in three different epithelial cell lines. Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are expressed, surface localization and enhanced halide permeability are again established. Together, these findings suggest that isolated CFTR NBD-1 (with or without the DeltaF508 mutation) is capable of targeting the epithelial cell membrane and enhancing cellular halide permeability. Furthermore, CFTR truncated at position 553 or 542 and possessing the majority of NBD-1 demonstrates surface localization and also confers increased halide permeability. These findings indicate that targeting to the plasma membrane and assumption of a transmembrane configuration are innate properties of the CFTR NBD-1. The results also support the notion that components of the halide-selective pore of CFTR reside within NBD-1.

Adenosine Triphosphate↗
PubMed1993

Molecular basis of aromatase deficiency in an adult female with sexual infantilism and polycystic ovaries.

We identified two mutations in the CYP19 gene responsible for aromatase deficiency in an 18-year-old 46,XX female with ambiguous external genitalia at birth, primary amenorrhea and sexual infantilism, and polycystic ovaries. The coding exons, namely exons II-X, of the CYP19 gene were amplified by PCR from genomic DNA and sequenced directly. Direct sequencing of the amplified DNA from the patient revealed two single-base changes, at bp 1303 (C-->T) and bp 1310 (G-->A) in exon X, which were newly found missense mutations and resulted in codon changes of R435C and C437Y, respectively. Subcloning followed by sequencing confirmed that the patient is a compound heterozygote. The results of restriction fragment length polymorphism analysis and direct sequencing of the amplified exon X DNA from the patient's mother indicate maternal inheritance of the R435C mutation. Transient expression experiments showed that the R435C mutant protein had approximately 1.1% of the activity of the wild type, whereas C437Y was totally inactive. Cysteine-437 is the conserved cysteine in the heme-binding region believed to serve as the fifth coordinating ligand of the heme iron. To our knowledge, this patient is the first adult to have described the cardinal features of a syndrome of aromatase deficiency. Recognition that such defects exist will lead to a better understanding of the role of this enzyme in human development and disease.

Adolescent↗
PubMed1990

A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene.

The chicken skeletal alpha-actin gene promoter region provides at least a 75-fold-greater transcriptional activity in muscle cells than in fibroblasts. The cis-acting sequences required for cell type-restricted expression within this 200-base-pair (bp) region were elucidated by chloramphenicol acetyltransferase assays of site-directed Bg/II linker-scanning mutations transiently transfected into primary cultures. Four positive cis-acting elements were identified and are required for efficient transcriptional activity in myogenic cells. These elements, conserved across vertebrate evolution, include the ATAAAA box (-24 bp), paired CCAAT-box-associated repeats (CBARs; at -83 bp and -127 bp), and the upstream T+A-rich regulatory sequence (at -176 bp). Basal transcriptional activity in fibroblasts was not as dependent on the upstream CBAR or regions of the upstream T+A-rich regulatory sequence. Transfection experiments provided evidence that positive regulatory factors required for alpha-actin expression in fibroblasts are limiting. In addition, negative cis-acting elements were detected and found closely associated with the G+C-rich sequences that surround the paired CBARs. Negative elements may have a role in restricting developmentally timed expression in myoblasts and appear to inhibit promoter activity in nonmyogenic cells. Cell type-specific expression of the skeletal alpha-actin gene promoter is regulated by combinatorial and possibly competitive interactions between multiple positive and negative cis-acting elements.

Actins↗
PubMed1997

Mobile elements inserted in the distant past have taken on important functions.

Current evidence on the long-term evolutionary effect of insertion of sequence elements is reviewed. There are three criteria for inclusion of an example: (i) the element was inserted far in the past and thus the event is not a transient mutation; (ii) the element is a member of a large group of similar sequences; (iii) the element now serves a useful function. There are 21 examples from Drosophila, sea urchin, human and mouse genomes that meet these criteria. Taken together, these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.

Animals↗
PubMed2002

A simple method for genome-wide screening for advantageous insertions of mobile DNAs in Escherichia coli.

Laboratory evolution in Escherichia coli has revealed that fitness typically increases in experimental populations. These changes are sometimes associated with changes in insertion sequence positions, some of which may themselves cause advantageous phenotypes. We have a novel and general method for identifying genes in Escherichia coli, whose knockout by mobile DNA insertions is beneficial in experimental evolution. Insertion sites in favored clones can be identified by reference to genomic information. We have implemented the method using modified Tn10 transposons bearing kanamycin and chloramphenicol resistance cassettes. Results are consistent across replicated experiments, demonstrating that the insertions are themselves creating selective advantages, rather than hitch-hiking with favorable base substitutions. The successful clones have subsequently been confirmed to have a fitness advantage relative to the progenitor strain. In experiments in shaking culture, we find that advantageous insertions usually fall in operons required in the pathways creating flagella. The method allows a rapid genome-wide screening for advantageous insertions in arbitrary environmental conditions. It allows investigation of the extent to which transient mutations generating environment-dependent selective advantages may help to explain the persistence of mobile DNAs in primarily clonal organisms, such as E. coli.

Chloramphenicol Resistance↗
PubMed1998

SOS and Mayday: multiple inducible mutagenic pathways in Escherichia coli.

Environmental and physiological stress conditions can transiently alter the fidelity of DNA replication. The DNA damage-mediated SOS response in Escherichia coli is the best-known example of such an 'inducible mutagenesis' or 'transient mutator' pathway. Emerging evidence suggests the existence of a number of other stress-inducible pathways that also affect the fidelity of replication. Among the more provocative recent findings are UVM, an SOS-independent damage-inducible mutagenic pathway, and a new recA-dependent but umuD/C-independent pathway that appears to be provoked by translational stress. These findings alter our view of inducible mutagenesis, and anticipate the existence of previously unrecognized links between protein synthesis and DNA replication.

Bacterial Proteins↗
PubMed2001

Hypermutation in bacteria and other cellular systems.

A temporary state of hypermutation can in principle arise through an increase in the rate of polymerase errors (which may or may not be triggered by template damage) and/or through abrogation of fidelity mechanisms such as proofreading and mismatch correction. In bacteria there are numerous examples of transient mutator states, often occurring as a consequence of stress. They may be targeted to certain regions of the DNA, for example by transcription or by recombination. The initial errors are made by various DNA polymerases which vary in their error-proneness: several are inducible and are under the control of the SOS system. There are several structurally related polymerases in mammals that have recently come to light and that have unusual properties, such as the ability to carry out 'accurate' translesion synthesis opposite sites of template damage or the possession of exceedingly high misincorporation rates. In bacteria the initial errors may be genuinely spontaneous polymerase errors or they may be triggered by damage to the template strand, for example as a result of attack by active oxidative species such as singlet oxygen. In mammalian cells, hypermutable states persisting for many generations have been shown to be induced by various agents, not all of them DNA damaging agents. A hypermutable state induced by ionizing radiation in male germ cells in the mouse results in a high rate of sequence errors in certain unstable minisatellite loci; the mechanism is unclear but believed to be associated with recombination events.

Animals↗
PubMed1997

Mutations resulting in transient and localized degeneration in the developing zebrafish brain.

In a large-scale mutagenesis screen in the zebrafish, Danio rerio, we have identified a heterogeneous group of 30 recessive, embryonic lethal mutations characterized by degeneration in the developing central nervous system that is either transient or initially localized to one area of the brain. Transient degeneration is defined as abnormal cell death occurring during a restricted period of development. Following degeneration, the affected structures do not appear to regenerate. In each case degeneration is identified after somitogenesis is complete and is not associated with visually identified patterning defects. These 30 mutations, forming 21 complementation groups, have been classified into four phenotypic groups: group 1, transient degeneration (13 mutations); group 2, spreading degeneration, early onset, in which degeneration is initially confined to the optic tectum but subsequently spreads to other areas of the central nervous system (7 mutations); group 3, late-onset degeneration, initially identified after 4 days (6 mutations); and group 4, degeneration with abnormal pigmentation (4 mutations). Although apoptotic cells are seen in the retina and tectum of all mutants, the distribution, temporal progression, and severity of degeneration vary between mutations. Several mutations also show pleiotropic effects, with degeneration involving extraneural structures including the pharyngeal arches and pectoral fins. We discuss some of the pathways important for cell survival in the nervous system and suggest that these mutations will provide entry points for identifying genes that affect the survival of restricted neural populations.

Animals↗
PubMed1992

Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding.

Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.

Animals↗
PubMed1984

Partial purification and characterization of the mRNA complementing a temperature-sensitive S-phase cell cycle mutation.

E36ts24 is a temperature-sensitive (ts) cell cycle mutant derived from the Chinese hamster lung cell line E36. At the restrictive temperature for growth (40.3 degrees C), the mutant cells are arrested at early S phase. We have microinjected poly(A)+ RNA isolated from the wild-type E36 cells into the cytoplasm of E36ts24 cells arrested at early S phase after 24 hr of incubation at 40.3 degrees C. The ts mutation was transiently complemented in a significant fraction of the microinjected cells as evidenced by the incorporation of [3H]thymidine assayed by autoradiography. Microinjection of mRNA fractionated by methylmercuric hydroxide/agarose gel showed that the mRNA capable of transiently complementing the mutation in E36ts24 contains about 940 nucleotides. Hence, it can code at most for a protein containing about 230 amino acids. We estimate that the partial purification by fractionation of the mRNA active in the transient complementation is on the order of 100- to 200-fold.

Animals↗