New Class Of Mutations Research Articles

FRAXA and FRAXE: new tools for the diagnosis of mental retardation.

In the era of prevention and early diagnosis, mental retardation (MR) represents one of the most important challenges to modern medicine. Much needs to be done to restrict the number of different forms of this vast category of chronic handicaps for which accurate diagnoses are not yet available. The goal is to reduce the social burden and provide better care for patients and families.The identification and characterisation of the molecular mechanisms which silence the FMR1 gene and which are responsible, in the majority of cases, for the fragile X syndrome (FRAXA) [1-4], the leading known cause of inherited mental retardation, led to the discovery of an extremely important new class of mutations: “dynamic mutations”. These are highly unstable interspersed repeats, located close to or within genes, which show a strong tendency to expand. This discovery has raised the possibility for direct molecular diagnosis of FRAXA and several other diseases based on the same molecular mechanism, including a different form of MR associated with a fragile site in Xq28, named FRAXE [5].With these tools, we have started to study the structural characteristics and pattern of transmission of these mutations in a population of mentally retarded individuals mainly coming from north-eastern Italy. The aims of our study were (a) to establish the true incidence of FRAXA and FRAXE full mutations as a cause of mental retardation in our population, and (b) to re-evaluate families in which at least one individual had a cytogenetic fra(X) diagnosis, in order to identify mosaicisms and premutations that could not be identified cytogenetically, and to establish the carrier status of relatives of affected individuals.

Complex Behavior of Simple Repeats: The Fragile X Syndrome

The fragile X syndrome of mental retardation is one of the most common genetic diseases. The mutation causing this disease was the first of a new class of mutations involving repeat sequences disturbing gene function. Fragile X mutations consist of an expansion of a CGG trinucleotide repeat in the FMR1 gene, which is inactivated as a result of this expansion. The lack of FMR1 protein is believed to be responsible for the mental retardation. The mechanism and the timing of the repeat amplification are still not known. Characterization of the repeat has clarified the genetics of fragile X syndrome, and has given tools to establish the diagnosis and to determine carrier status.

Mechanisms of DNA expansion.

Unstable transmission of repeating segments in genes is now recognized as a new class of mutations causing human disease. Genetic instability observed in disease is termed an "expansion mutation" when the mutation is an increase in the copy number of a repeated unit, commonly a di- or trinucleotide. While the expansion mutation is well characterized in disease, the mechanism by which expansion occurs is not clear. This article focuses on physical properties of expansion at repeating nucleotides that may provide clues to the mechanism. Both biochemical and genetic data indicate that DNA structure is part of the mechanism and the underlying cause for expansion.

A New Class of Histone H2A Mutations in Saccharomyces cerevisiae Causes Specific Transcriptional Defects In Vivo

Nucleosomes have been shown to repress transcription both in vitro and in vivo. However, the mechanisms by which this repression is overcome are only beginning to be understood. Recent evidence suggests that in the yeast Saccharomyces cerevisiae, many transcriptional activators require the SNF/SWI complex to overcome chromatin-mediated repression. We have identified a new class of mutations in the histone H2A-encoding gene HTA1 that causes transcriptional defects at the SNF/SWI-dependent gene SUC2. Some of the mutations are semidominant, and most of the predicted amino acid changes are in or near the N- and C-terminal regions of histone H2A. A deletion that removes the N-terminal tail of histone H2A also caused a decrease in SUC2 transcription. Strains carrying these histone mutations also exhibited defects in activation by LexA-GAL4, a SNF/SWI-dependent activator. However, these H2A mutants are phenotypically distinct from snf/swi mutants. First, not all SNF/SWI-dependent genes showed transcriptional defects in these histone mutants. Second, a suppressor of snf/swi mutations, spt6, did not suppress these histone mutations. Finally, unlike in snf/swi mutants, chromatin structure at the SUC2 promoter in these H2A mutants was in an active conformation. Thus, these H2A mutations seem to interfere with a transcription activation function downstream or independent of the SNF/SWI activity. Therefore, they may identify an additional step that is required to overcome repression by chromatin.

Developmental Analysis of the smellblind Mutants: Evidence for the Role of Sodium Channels in Drosophila Development

We have recently found evidence that two smellblind mutants, originally identified as olfactory mutants, define a new class of mutation in the para (paralytic) sodium channel gene (Lilly et al., in press). These two mutants have previously been shown to be heat-sensitive developmental lethals: animals raised at elevated temperatures die as embryos or as young larvae (Lilly and Carlson, 1990, Genetics 124, 293-302). This article shows, surprisingly, that both mutants are not only heat-sensitive lethals, but also cold-sensitive lethals: when raised at reduced temperatures, the mutants develop to the adult stage, but then die prematurely. Moreover, this effect is sexually dimorphic, in that lethality is substantially greater for females than males. The association of cold-sensitivity with mutations of a sodium channel gene has recently been found in the case of a cold-sensitive human muscle disease, paramyotonia congenita, in which sodium channel function is defective (Ptacek et al., 1992, Neuron 8, 891-897). The heat-sensitive and cold-sensitive periods for the two smellblind mutants are determined through a series of reciprocal temperature shift experiments and are found to be different. Most, if not all, of the contribution to the heat-sensitive lethality occurs before metamorphosis, whereas the cold-sensitivity is particularly pronounced after the onset of metamorphosis. These results are discussed in terms of the developmental role of the para sodium channel and the role of electrical activity in Drosophila development.

Genes with triplet repeats: a new class of mutations causing neurological diseases

Microsatellites, simple tamdem repeats of 2 to 4 nucleotide sequences, are widely distributed throughout the genome. Trinucleotide repeats are found every 300 to 500 kb. Recently, a new type of mutation was described involving a specific expansion of triplets within or in close proximity to a gene. Expanded triplets have been found in the genes causing six different neurological disorders: fragile X syndrome (FRAXA), spinal and bulbar muscular atrophy (SBMA), myotonic dystrophy (DM), Huntington's disease (HD), spinocerebellar ataxia type I (SCAT), and dentato-rubra-pallidoluysian atrophy (DRPLA). These neurological disorders have in common a variable age of onset and clinical severity, as well as a decrease in the age of onset over generations, known as anticipation. These unusual characteristics are related to the observation that expanded repeats are unstable both in meioses and mitoses. A younger age of onset and an increase in severity correlate with a higher number of repeats. Interestingly, particular haplotypes are in disequilibrium with the mutation for FRAXA, DM and HD, suggesting instability for selected chromosomes. How expanded triplets affect the expression and the function of genes is still unknown. Since neurodegenerative disorders are often variable in age of onset and clinical severity, the list of expanding triplet mutations should increase in the very near future.

A new class of mutations in Arabidopsis thaliana, acaulis1, affecting the development of both inflorescences and leaves

ABSTRACT We isolated and analyzed mutants of Arabidopsis thaliana, acaulis, with flower stalks that are almost absent or are much reduced in length. The mutations are divided between two loci, acaulisl (acll) and acaulis2 (acl2). The acll-1 mutation has been assigned to linkage group 4 in the vicinity of locus ap2. The acl1-1 mutant showed premature arrest of the inflorescence meristem after the onset of reproductive development, followed by consequent reduction in the number of flower-bearing phytomers and therefore flowers. The apical meristem of the inflorescences was morphologically normal but its radius was about half that of the wild type. The acl1 mutants are also defective in the development of foliage leaves. Both defects could be rescued by growth at a specific temperature (28°C). The length of the cells in acl1-3 mutant was less than that in the wild type but the numbers of cells in leaves and internodes of acl1 mutants were calculated to be the same as those of the wild type. Thus, the defects in inflorescences and leaves were attributed to defects in the process of elongation (maturation) of these cells. Temperature-shift experiments showed that the Acl1+ product was necessary at all developmental stages. A critical stage was shown to exist for recovery from the cessation of development of inflorescence meristems that was caused by the acl1-1 mutation. Grafting experiments showed that the acl1-1 mutation does not affect diffusible substances. An analysis of double mutants carrying both acl1-1 and one of developmental mutations, ap1, clv1, Ify, or tfl1, showed that ACL1 is a new class of gene.

Dynamic mutations: A new class of mutations causing human disease

Dynamic mutations: A new class of mutations causing human disease

A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation.

The cob operon of Salmonella typhimurium encodes enzymes required for synthesis of adenosyl-cobalamin (vitamin B12). The pdu operon encodes enzymes needed for use of propanediol as a carbon source, including an adenosyl-cobalamin-dependent enzyme, propanediol dehydratase. These two operons both map near min 41 of the S. typhimurium linkage map and are transcribed divergently. Here we report that the cob and pdu operons form a single regulon. Transcription of this regulon is induced by either glycerol or propanediol. The metabolism of these compounds is not required for induction. Propanediol induces the regulon either aerobically or anaerobically during growth on poor carbon sources. Aerobically glycerol induces only if its metabolism is prevented by a mutational block such as a glpK mutation. Under anaerobic conditions, glycerol induces in both glpK+ and glpK mutant strains during growth on poor carbon sources. A new class of mutations, pocR, prevents induction of the cob/pdu regulon by either propanediol or glycerol and causes a Cob- Pdu- phenotype. The pocR gene is located between the cob and pdu operons and appears to encode a trans-acting protein that acts as a positive regulator of both operons. Transcription of the pocR regulatory gene is induced, even without the PocR protein, during aerobic growth on poor carbon sources and during anaerobic respiration. With the functional PocR protein, transcription of the pocR gene is autoinduced by propanediol but not by glycerol. The growth conditions that increase pocR gene expression correlate with growth conditions that allow high induction of the cob/pdu regulon. A model for control of this regulon suggests that the PocR protein is a transcriptional activator of both the cob and pdu operons and that both glycerol and propanediol can individually serve as effectors of the PocR protein. We suggest that global control mechanisms cause variation in the level of the PocR protein; an increased level of the PocR protein permits higher induction by propanediol or glycerol.

Aphidicolin resistance in Herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site

We describe the mapping and sequencing of mutations within the DNA polymerase gene of herpes simplex virus type 1 which confer resistance to aphidicolin, a DNA polymerase inhibitor. The mutations occur near two regions which are highly conserved among DNA polymerases related to the herpes simplex enzyme. They also occur near other herpes simplex mutations which affect the interactions between the polymerase and deoxyribonucleoside triphosphate substrates. Consequently, we argue in favor of the idea that the aphidicolin binding site overlaps the substrate binding site and that the near-by conserved regions are functionally required for substrate binding. Our mutants also exhibit abnormal sensitivity to another DNA polymerase inhibitor, phosphonoacetic acid. This drug is thought to bind as an analogue of pyrophosphate. A second-site mutation which suppresses the hypersensitivity of one mutant to phosphonoacetic acid (but not its aphidicolin resistance) is described. This second mutation may represent a new class of mutations, which specifically affects pyrophosphate, but not substrate, binding.

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta.

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.

Mutations in rpoD, the gene encoding the σ70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP

We have isolated a new class of mutations in rpoD, the gene encoding the σ 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and β-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the σ 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of σ 70.

Mutations in rpoD that increase expression of genes in the mal regulon of Escherichia coli K-12

The σ subunits of eubacterial RNA polymerases determine the site selectivity of initiation of transcription at promoters. Mutations in rpoD, the gene that encodes σ 70, the major σ factor in Escherichia coli, should be useful in determining the molecular details of the process of transcription initiation. However, such mutations are likely to be deleterious or lethal, since σ 70 is an essential gene product. We designed a system for the rapid isolation and fine structure mapping of mutations in rpoD, which allows selection of mutations that would otherwise be deleterious to the cell. We used this system to isolate a new class of mutations in rpoD, mutations that relieve the requirement for CAP-cAMP for initiation at promoters in the mal regulon. These mutations, which we designate rpoD(Mal) mutations, occur in two clusters in the rpoD gene within regions previously suggested by amino acid sequence comparisons to be important for σ structure or function. We cannot distinguish whether the rpoD(Mal) mutations affect mal expression by altering interaction between RNA polymerase and mal promoters or between RNA polymerase and the accessory transcription factor MalT. However, the effects of the mutations on activator-independent transcription from the lac promoter (4 rpoD(Mal) mutations decrease CAP-independent expression of the lac promoter in vivo) suggest that the regions of σ identified by our mutations may be directly involved in promoter recognition.

Isolation and characterization of a pleiotropic glucose repression resistant mutant of Saccharomyces cerevisiae

A new mutation has been described which confers resistance to catabolite repression in Saccharomyces cerevisiae. The mutant allele, termed grr-1 for glucose repression-resistant, is characterized by insensitivity to glucose repression for the cytoplasmic enzymes invertase, maltase, and galactokinase, as well as the mitochondrial enzyme cytochrome c oxidase. Hexokinase levels in grr-1 mutants are approximately 3-fold higher than the corresponding activity of the parental strain. Although the grr-1 allele is expressed phenotypically similarly to the hex-1 (hxk-2) and hex-2 mutations described by Entian et al. (1977) and Zimmermann and Scheel (1977) respectively, we have shown genetically and physiologically that grr-1 represents a new class of mutation.

New class of mutations in Escherichia coli (uup) that affect precise excision of insertion elements and bacteriophage Mu growth.

We have used a papillation screening technique to isolate mutations that increase the precise excision of insertion elements. The three mutations isolated stimulated precise excision of Tn5, Tn10, and the IS elements. They had a large, 20- to 600-fold, effect on excision of Tn5 at various chromosomal sites. The varied stimulation for different Tn5 insertions showed that the mutations altered the relationship between a precise excision activity and the chromosomal sequence flanking an inserted Tn5. A much smaller stimulation was observed for insertions on the plasmid F'128. The stimulation was recA independent. The mutations also reduced the rate of production of bacteriophage Mu progeny. The mutations were mapped by two- and three-factor crosses with closely linked Tn10 insertions. They defined the uup locus, located at 21.3 min on the Escherichia coli map, next to pyrD.

A new class of mutations altering the response of the ribosome to streptomycin.

Mutants were isolated from high-level streptomycin dependent strains of Escherichia coli B, which do not spontaneously revert to antibiotic independence. In these mutants the requirement for streptomycin was much reduced, but not abolished. The relieving of the antibiotic dependence was caused by qui (for quasi-independent) mutations. These were analogous to the ramA (rpsD) mutations which relieve the streptomycin requirement of other classes of streptomycin dependent mutants, but strains harboring qui mutations exhibited novel streptomycin phenotypes in conjunction with all rpsL (strA) alleles. RamA mutations increase ribosomal misreading; qui mutations either did not significantly alter misreading, or else reduced it.

Temperature-sensitive prune (pn) mutations of Drosophila melanogaster

Temperature-sensitivemutations, at the prune locus ( pn ts) of Drosophila melanogaster, were induced with EMS. The majority of the new class of mutations, designated pn ts-e, do not interact with the mutation Killer of prune (Kpn) at all of the experimental temperatures, i.e., pn ts-e; Kpn flies do not die. One mutation, designated pn ts-ek, is temperature-sensitive with respect to both the eye coloration and the interaction with Kpn. pn tr (temperature-non-sensitive), pn ts-e, and pn ts-ek alleles were found to differ with respect to both quantity of drosopterines and the color of the eyes. The temperature-sensitive period (TSP) for pn eye color occurs at a late pupal stage. The TSP for the pn—Kpn interaction lasts from the beginning of the first laraval instar to eclosion [22]. It is concluded that all pn mutations known to date occupy the same functional unit. The various pn mutations affect the same metabolic step to various degrees: the eye color phenotype results from a reduction in the end product, while the interaction with Kpn depends on the amount of the accumulated precursor. No recombination between pn alleles was found among more than 10 6 zygotes that were scored. The reasons for this failure are disscussed.

Specific suppression of mutations in genes 46 and 47 by das, a new class of mutations in bacteriophage T4D.

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.

Temperature-sensitive mutation in Paramecium aurelia: I. Induction and inheritance

A new class of mutation was induced by X-radiation in Paramecium aurelia. These mutant segregants were called temperature-sensitive mutants ( ts mutants) since they can grow normally at 27° but are inviable at 36°. The maximum yield of ts mutant segregants after 2 kR of X-radiation was about 14% of the segregants viable at 27°. Crosses between persistent ts mutant segregants and wild-type stocks showed that the ts character is inheritable and recessive. Crosses between different ts mutant stocks revealed that these ts loci are widely spread over the genome of Paramecium. Certain subsidiary problems arising in the course of the work and the genetic applications of the ts mutations to genetic analysis of paramecia were discussed.

Mutagenesis of super-suppressors in yeast.

A new class of mutations, referred to as “nonsense” mutations, has been described in phages and bacteria in recent years (Benzer and Champe, 1962; Garen and Siddiqi, 1962). It is characterized by two major properties: a drastic effect on protein synthesis (A cistron of the rII region in phage T4, Benzer and Champe, 1962; alkaline phosphatase in E. coli, Garen and Siddiqi, 1962) produced by a polypeptide chain termination at the site of the mutational lesion (Sarabhai et al., 1964), and by responsiveness to external suppressors which can give rise to a wild or pseudo-wild phenotype (ambivalent rII mutants, Benzer and Champe, 1961; host-dependent defective mutations in phage λ, Campbell, 1961; alkaline phosphatase in E. coli, Garen and Siddiqi, 1962; amber mutations in phage T4, Epstein et al., 1963).