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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↗
PubMed2004

Patients homozygous for the T435N mutation of succinyl-CoA:3-ketoacid CoA Transferase (SCOT) do not show permanent ketosis.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; locus symbol OXCT; E.C. 2.8.3.5) is the main determinant of the ketolytic capacity of tissues. Hereditary SCOT deficiency causes episodic ketoacidosis. Permanent ketosis has been regarded as a pathognomonic feature of SCOT deficiency. There are three SCOT-deficient patients from a small region in Japan and they have not manifested permanent ketosis, even though their ketoacidotic crises were as severe as those of other SCOT-deficient patients. All three were homozygous for the T435N mutation. Transient expression analysis of wild-type and mutant cDNA showed that the T435N mutant retained significant residual SCOT activities (20% for that of the wild-type at 39.5 degrees C, 25% at 37 degrees C, and 50% at 30 degrees C). The difference of residual SCOT activities at these temperatures in expression analyses was due to differences in the level of the mutant protein. SCOT activity of the T435N protein was more vulnerable than the wild-type to heat treatment at 42 degrees C and 55 degrees C. These temperature-sensitive characteristics of the mutant protein may explain, in part, why the patients developed ketoacidotic crises during febrile illness. In SCOT-deficient patients retaining some residual activity, permanent ketosis may be absent.

Child↗
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↗
PubMed2005

Diversify or die: generation of diversity in response to stress.

When challenged with unfavorable conditions, microorganisms can develop a stress response that allows them to adapt to or survive in the new environment. A common feature of the numerous specific stress response pathways that have been described in a wide range of bacteria is that they are energy demanding and therefore often transient. In addition, stress responses may come too late or be insufficient to protect the cell or the population against very sudden or severe stresses. However, it seems that microorganisms can also enhance their chances of survival under stress by increasing the generation of diversity at the population level. This can be achieved either by creating genetic diversity by a variety of mechanisms involving for example constitutive or transient mutators and contingency loci, or by revealing phenotypic diversity that remained dormant due to a mechanism called genetic buffering. This review gives an overview of these emerging diversity-generating mechanisms, which seem to play an important role in the ability of microbial populations to overcome stress challenges.

Adaptation, Biological↗
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↗
PubMed2003

Congenital hypothyroidism clinical aspects and late consequences.

Congenital hypothyroidism (CH) is the commonest treatable cause of mental retardation. The prevelance is 1/3000 - 1/4000 live births worldwide. The importance of CH is that, the longer the diagnosis of CH is delayed, the higher the risk of mental retardation and neurologic sequale; such as poor motor coordination, ataxia, spastic diplegia, muscular hypotonia, strabismus, learning disability and diminished attention span. The most common cause of permenant CH is thyroid dysgenesis (85-90%) in which the transcription factors TTF1,TTF2 and PAX8 would appear to be obvious candidate genes in the aetiology. Especially cardiac defects and some other birth defects are described in patients with CH. Inborn errors of thyroid hormonogenesis are responsible for 10-15% of CH cases and usually have autosomal recessive inheritance, consistent with a single gene mutation. Transient CH is very common in prematures with an estimate of 10% of CH babies identified on newborn screening, or 1 in 40,000 neonates. CH neonates are usually symptom-free and the most encountered symptoms are prolonged jaundice, large fontanelles and umbilical hernia. In general, the extent of clinical findings depends on the cause, severity and duration of hypothyroidism. An elevated TSH>20 microm Iu/L and a decreased concentration of T4 confirms the diagnosis of CH. Infants with permanant abnormalities of thyroid function mostly have a serum TSH concentration > 50 microm Iu/L. Ultrasonography, thyroid scintigraphy, bone x ray of the knee and serum thyroglobulin concentration are the other essentials after diagnosis to clarify the status of the thyroid and the severity of hypothyroidism. The higher doses of 10- 15 microm g/kg/day and the commencement of treatment before 2 weeks gave rise to better long term outcome of CH patients. In the follow up of the patients noncompliance is the most important problem and serum freeT4 or T4 and TSH should be obtained at each visit to adjust the doses of L-thyroxine. Still a small number of patients with severe hypothyroidism in utero or reflected by clinical signs and symptoms extremely low T4 levels and delayed bone age may have intellectual deficits despite normal intelligence.

Congenital Hypothyroidism↗
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↗
PubMed2006

On the effect of transient expression of mutated eIF2alpha and eIF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock.

There are a growing number of reports on the beneficial effects of subphysiological temperature in vitro culturing (27-35 degrees C) of mammalian cells on recombinant protein yield. However, this effect is not conserved across cell lines and target products, and our understanding of the molecular mechanism(s) responsible for increased recombinant protein yield upon reduced temperature culturing of mammalian cells is poor. What is known is that mammalian cells respond to cold-shock by attenuating global cap-dependent translation. Here, we have investigated the hypothesis that the cap-dependent attenuation of mRNA translation upon cold-stress of in vitro-cultured mammalian cells can be prevented, or at least alleviated, by overexpressing mutant translation initiation factors in Chinese hamster ovary and HeLa cells. We have shown that the transient coexpression of either an eIF2alphaSer51 Ala51 mutant or an eIF4ESer209 Glu209 mutant with firefly luciferase affects luciferase expression levels in a cell line and temperature dependent manner. Further, regardless of the coexpression of initiation factors, transient reporter gene expression was enhanced at subphysiological temperatures (<37 degrees C), suggesting that reduced temperature cultivation can be used to improve the yield of recombinant protein during transient expression. The implications of these results upon cell engineering strategies involving manipulation of the translational apparatus for the enhancement of recombinant protein synthesis upon cold-shock are discussed.

Amino Acid Substitution↗
PubMed1998

Probing immunoglobulin gene hypermutation with microsatellites suggests a nonreplicative short patch DNA synthesis process.

As the rate of Ig gene hypermutation approximates the level of nucleotide discrimination of DNA polymerases (10(-3) to 10(-4)), a local inhibition of proofreading and mismatch repair during semiconservative replication could generate the mutations introduced by the process. To address this question, we have constructed transgenic mice that carry a hypermutation substrate containing a "polymerase slippage trap": an Ig gene with a mono or dinucleotide tract inserted in its V region. The low amount of slippage events as compared to the number of mutations, the absence of transient misalignment mutations at the border of the repeats, and the dissociation between the amount of frameshifts and mutations when the transgene is put on mismatch repair-deficient genetic backgrounds, suggest that Ig gene hypermutation occurs by an error-prone short patch DNA synthesis taking place outside global DNA replication.

Animals↗
PubMed1999

Dominant negative allele (N47D) in a compound heterozygote for a variant of 6-pyruvoyltetrahydropterin synthase deficiency causing transient hyperphenylalaninemia.

Mutations in the 6-pyruvoyltetrahydropterin synthase (PTPS) gene result in persistent hyperphenylalaninemia and severe catecholamine and serotonin deficiencies. We investigated at the DNA level a family with a PTPS-deficient child presenting with an unusual form of transient hyperphenylalaninemia. The patient exhibited compound heterozygosity for the PTPS-mutant alleles N47D and D116G. Transfection studies with single PTPS alleles in COS-1 cells showed that the N47D allele was inactive, while D116G had around 66% of the wild-type activity. Upon co-transfection of two PTPS alleles into COS-1 cells, the N47D allele had a dominant negative effect on both the wild-type PTPS and the D116G mutant with relative reduction to about 20% of control values. Whereas the mother and the father had reduced enzyme activity in red blood cells (34.7% and 51.7%, respectively) and skin fibroblasts (2.8% and 15.4%, respectively), the clinically normal patient had in these cells activities at the detection limits, although PTPS-cross-reactive material was present in the fibroblasts. The specifically low PTPS activity in the mother's cells corroborated the evidence of a dominant negative effect of the maternal N47D allele on wild-type PTPS.

Animals↗