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PubMed2001

Mutations in the carboxyl-terminal domain of phospholipase C-beta 1 delineate the dimer interface and a potential Galphaq interaction site.

The carboxyl-terminal domain of phospholipase C-beta is required for its stimulation by Galpha(q) and for its Galpha(q)-specific GTPase-activating protein (GAP) activity. We subjected this domain to a combination of deletion and alanine/glycine scanning mutagenesis to detect mutations that would inhibit either responsiveness to G(q) or G(q) GAP activity. Most mutations that altered either response or GAP activity diminished both in parallel. Many of these mutations map at the interface at which the carboxyl-terminal domain was recently shown to form a dimer (Singer, A. U., et al. (2001) Nat. Struct. Biol., 9, 32-36). Most others clustered in an area that is a plausible Galpha(q) binding site. In addition, one mutation that differentially inhibited GAP activity relative to responsiveness to Galpha(q) mapped in this region at a location modeled to be in close contact with the switch II region of Galpha(q). This is the site at which RGS proteins are thought to exert their GAP activity. Last, a deletion mutation differentially inhibited the response of phospholipase C-beta1 to Galpha(q) without blocking GAP activity. Its location in the molecule suggests that moving the attachment point of the catalytic domain can disrupt its ability to be activated by Galpha(q).

Animals↗
PubMed1993

Calorimetric studies of the N-terminal half-molecule of transferrin and mutant forms modified near the Fe(3+)-binding site.

The effects of single amino acid substitution on the thermal stability of the N-terminal half-molecule of human transferrin and its iron-binding affinity have been studied by high-sensitivity scanning calorimetry. All site-directed mutations are located on the surface of the binding cleft, and they are D63-->S, D63-->C, G65-->R, H207-->E and K206-->Q. Differential scanning calorimetry results show that the mutations do not significantly alter the conformational stability of the apo-forms of the proteins. The changes in free energy of unfolding relative to the wild-type protein range from 0.83 to -2.4 kJ/mol. The D63-->S, G54-->R and H207-->E mutations slightly destabilize the apo-protein, while the D63-->C and K206-->Q mutations increase its stability by a small amount. However, there are large compensating enthalpy-entropy changes caused by all mutations. All mutants bind ferric ion, but with different affinities. Replacement of Asp-63 by either Ser or Cys decreases the apparent binding constant by 5-6 orders of magnitude. The G65-->R mutation also decreases the apparent binding constant by 5 orders of magnitude. The K206-->Q mutation increases the apparent binding constant by 20-fold, while the H207-->E mutation does not significantly change the apparent iron-binding affinity of the half-molecule.

Binding Sites↗
PubMed2003

Detection of a novel ryanodine receptor subtype 1 mutation (R328W) in a malignant hyperthermia family by sequencing of a leukocyte transcript.

BACKGROUND: To determine whether malignant hyperthermia (MH) susceptibility in a Canadian pedigree is associated with a mutation in the ryanodine receptor subtype 1 (RYR1) gene, the complete RYR1 transcript obtained from the leukocytes of one MH-susceptible family member was sequenced, using a newly developed protocol. METHODS: RNA was extracted from leukocytes and converted into complementary DNA. Overlapping fragments of RYR1 complementary DNA were amplified by the polymerase chain reaction and used for double-strand sequencing to find a single mutation likely to be causal of MH susceptibility. Inheritance of the mutation in the family was studied by restriction endonuclease analysis and/or sequencing of genomic DNA and compared to available caffeine halothane contracture test data. The mutation was introduced into rabbit RYR1 complementary DNA, the complementary DNA was expressed in human embryonic kidney line 293 cells, and Ca2+ release by the mutant Ca2+ release channel was measured following the addition of caffeine and halothane. RESULTS: A novel arginine 328 to tryptophan mutation in RYR1 was detected by direct sequencing of the RYR1 transcript from leukocytes of one MH-susceptible individual. A causal role for this mutation in MH is indicated by cosegregation of the mutation with the MH-susceptible phenotype within the family and by the demonstration that the mutant channel has increased sensitivity to both caffeine and halothane. CONCLUSIONS: The feasibility of using complete RYR1 transcripts from leukocytes for sequence analysis offers an efficient and noninvasive method for scanning RYR1 for novel mutations.

Amino Acid Substitution↗
PubMed2005

Hush puppy: a new mouse mutant with pinna, ossicle, and inner ear defects.

OBJECTIVES/HYPOTHESIS: Deafness can be associated with abnormalities of the pinna, ossicles, and cochlea. The authors studied a newly generated mouse mutant with pinna defects and asked whether these defects are associated with peripheral auditory or facial skeletal abnormalities, or both. Furthermore, the authors investigated where the mutation responsible for these defects was located in the mouse genome. METHODS: The hearing of hush puppy mutants was assessed by Preyer reflex and electrophysiological measurement. The morphological features of their middle and inner ears were investigated by microdissection, paint-filling of the labyrinth, and scanning electron microscopy. Skeletal staining of skulls was performed to assess the craniofacial dimensions. Genome scanning was performed using microsatellite markers to localize the mutation to a chromosomal region. RESULTS: Some hush puppy mutants showed early onset of hearing impairment. They had small, bat-like pinnae and normal malleus but abnormal incus and stapes. Some mutants had asymmetrical defects and showed reduced penetrance of the ear abnormalities. Paint-filling of newborns' inner ears revealed no morphological abnormality, although half of the mice studied were expected to carry the mutation. Reduced numbers of outer hair cells were demonstrated in mutants' cochlea on scanning electron microscopy. Skeletal staining showed that the mutants have significantly shorter snouts and mandibles. Genome scan revealed that the mutation lies on chromosome 8 between markers D8Mit58 and D8Mit289. CONCLUSION: The study results indicate developmental problems of the first and second branchial arches and otocyst as a result of a single gene mutation. Similar defects are found in humans, and hush puppy provides a mouse model for investigation of such defects.

Animals↗
PubMed2000

Genetic and neuroradiological heterogeneity of double cortex syndrome.

Mutations in the X-linked doublecortin gene appear in many sporadic cases of double cortex (DC; also known as subcortical band heterotopia), a neuronal migration disorder causing epilepsy and mental retardation. The purpose of this study was to examine why a significant percentage of sporadic DC patients had been found not to harbor doublecortin mutations and to determine whether clinical features or magnetic resonance imaging scan appearance could distinguish between patients with and without doublecortin mutations. Magnetic resonance imaging scan analysis differentiated patients into the following four groups: anterior biased/global DC with doublecortin mutation (16 of 30; 53%), anterior biased/global DC without mutation (8 of 30; 27%), posterior biased DC without mutation (3 of 30; 10%), and limited/unilateral DC without mutation (3 of 30; 10%). The presence of these atypical phenotypes suggests that other genetic loci or mosaicism at the doublecortin locus may be responsible for this diversity of DC cases.

Adolescent↗
PubMed1995

Mapping of an NH2-terminal ligand binding site of the insulin receptor by alanine scanning mutagenesis.

Affinity labeling studies and mutational analyses have implicated the involvement of a predicted domain of the insulin receptor (L1, amino acids 1-119) in ligand binding. In order to obtain a higher resolution localization of this ligand binding site, we have performed alanine scanning mutagenesis of this domain. Alanine mutant cDNAs encoding a secreted recombinant insulin receptor extracellular domain were expressed transiently in adenovirus transformed human embryonic kidney cells and the affinity of the expressed receptor for insulin was determined. Mutation of 14 amino acids located in four discontinuous peptide segments to alanine was disruptive of insulin binding: Segment 1, amino acids 12-15; Segment 2, amino acids 34-44; Segment 3, amino acids 64-67; and Segment 4, amino acids 89-91. The quantitative contribution of the four segments to the free energy of insulin binding was 1 > 3 > 2 > 4. Of the 14 amino acids whose mutation compromised insulin binding, 3 are charged, 3 hydrophobic, 5 aromatic, and 3 are amides.

Adenoviridae↗
PubMed2005

Characterization of a new allele of Ames waltzer generated by ENU mutagenesis.

Mutation in the protocadherin 15 (Pcdh15) gene causes hair cell dysfunction and is associated with abnormal stereocilia development. We have characterized the first allele (Pcdh15(av-nmf19)) of Ames waltzer (av) obtained by N-ethyl-N-nitrosourea (ENU) mutagenesis. Pcdh15(av-nmf19) was generated in the Neuroscience Mutagenesis Facility (NMF) at The Jackson Lab (Bar Habor, USA). Pcdh15(av-nmf19) mutants display circling and abnormal swimming behavior along with lack of auditory-evoked brainstem response at the highest intensities tested. Mutation analysis shows base substitution (A--> G) in the consensus splice donor sequence linked to exon 14 resulting in the skipping of exon 14 and the splicing of exon 13-15. This results in the introduction of a stop codon in the coding sequence of exon 15 due to shift in the reading frame. The effect of nmf19 mutation is expected to be severe since the expressed Pcdh15 protein is predicted to truncate in the 5th cadherin domain. Abnormalities of cochlear hair cell stereocilia are apparent in Pcdh15(av-nmf19) mutants near the time of birth and by about P15 (15 days after birth) there is evidence of sensory cell degeneration. Disorganization of outer hair cell stereocilia is observed as early as P2. Inner hair cell stereocilia are also affected, but less severely than those of the outer hair cells. These results are consistent with characteristics of the mutation in the Pcdh15(av-nmf19) allele and they support our previous finding that Protocadherin 15 plays an important role in hair-bundle morphogenesis.

Acoustic Stimulation↗
PubMed1998

Improved envelope function selected by long-term cultivation of a translation-impaired HIV-1 mutant.

The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple regulatory elements that fold into stable hairpin structures. Because extensive secondary structure can block the scanning of ribosomes, an alternative mechanism for HIV translation seems feasible. To study the mechanism of HIV-1 mRNA translation, a start codon was introduced in the leader region that will usurp scanning ribosomes. This upstream AUG mutation (uAUG) inhibited HIV gene expression, indicating that HIV-1 mRNA translation occurs via the regular scanning mechanism. Revertant viruses with increased replication capacity were obtained upon prolonged culturing of the mutant virus. To our surprise, the introduced start codon had not been inactivated in these phenotypic revertants. Instead, these revertants contain additional mutations in the envelope (Env) protein that stimulated HIV-1 replication. These second-site Env mutations did not specifically overcome the gene expression defect of the uAUG mutant, as the replication capacity of other HIV-1 mutants with an unrelated defect could also be improved. The uAUG construct appears to be a unique tool in forced HIV-1 adaptation studies because the deleterious uAUG mutation is stably maintained in the progeny, yielding phenotypic revertants with second-site mutations elsewhere in the viral genome.

Base Sequence↗
PubMed1994

sin 1, a mutation affecting female fertility in Arabidopsis, interacts with mod 1, its recessive modifier.

In Arabidopsis thaliana, a mutation in the SIN 1 gene causes aberrant ovule development and female-specific sterility. The effect of the sin 1 mutation is polymorphic and pleiotropic in different genetic backgrounds. The polymorphism concerns morphology of the mutant ovules. The pleiotropism involves internodal distance and inflorescence initiation time. The particular ovule phenotype and the length of internodes are dependent on an interaction of sin 1 with a second recessive gene, which we term mod 1. The recessive mod 1 allele in a homozygous sin 1 mutant plant reduces internode length and ovule integument size. The mutation sin 1, but not mod 1, has a demonstrable effect on ovule morphology when acting independently. In our crosses mod 1 was inseparably linked to the well known mutation erecta that is known to cause a reduction in internode and pedicle lengths.

Arabidopsis↗
PubMed2006

Autosomal recessive cerebellar ataxias.

Autosomal recessive cerebellar ataxias (ARCA) are a heterogeneous group of rare neurological disorders involving both central and peripheral nervous system, and in some case other systems and organs, and characterized by degeneration or abnormal development of cerebellum and spinal cord, autosomal recessive inheritance and, in most cases, early onset occurring before the age of 20 years. This group encompasses a large number of rare diseases, the most frequent in Caucasian population being Friedreich ataxia (estimated prevalence 2-4/100,000), ataxia-telangiectasia (1-2.5/100,000) and early onset cerebellar ataxia with retained tendon reflexes (1/100,000). Other forms ARCA are much less common. Based on clinicogenetic criteria, five main types ARCA can be distinguished: congenital ataxias (developmental disorder), ataxias associated with metabolic disorders, ataxias with a DNA repair defect, degenerative ataxias, and ataxia associated with other features. These diseases are due to mutations in specific genes, some of which have been identified, such as frataxin in Friedreich ataxia, alpha-tocopherol transfer protein in ataxia with vitamin E deficiency (AVED), aprataxin in ataxia with oculomotor apraxia (AOA1), and senataxin in ataxia with oculomotor apraxia (AOA2). Clinical diagnosis is confirmed by ancillary tests such as neuroimaging (magnetic resonance imaging, scanning), electrophysiological examination, and mutation analysis when the causative gene is identified. Correct clinical and genetic diagnosis is important for appropriate genetic counseling and prognosis and, in some instances, pharmacological treatment. Due to autosomal recessive inheritance, previous familial history of affected individuals is unlikely. For most ARCA there is no specific drug treatment except for coenzyme Q10 deficiency and abetalipoproteinemia.

Abetalipoproteinemia↗
PubMed1993

Pleiotropic and differential phenotypic expression of two sn (snowflake) mutant alleles of Neurospora crassa: analysis in homokaryotic and heterokaryotic cells.

Mutations sn (snowflake) JL301 and C136, in the centromere region of linkage group I in Neurospora crassa, are at 0.6-3.0 map units to the left of the os-4 locus. Strains carrying snJL301 produce very short aerial hyphae and only arthroconidia, and do not grow in high salt media. snC136 strains produce aerial hyphae, with abnormally large and rounded blastoconidia, at the top of the agar slant cultures, and revert to wild-type growth in high salt media. Studies with forced primary heterokaryons indicate that snJL301 is recessive while snC136 is a semi-dominant and gene-dose dependent allele, with respect to the wild-type. Taken together the results show that: (1) the sn mutations are allelic with a differential pleiotropic phenotype, and (2) snC136 may code for a partially functioning gene product while snJL301 appears to be a null allele.

Alleles↗
PubMed2002

Mutation profiling of mismatch repair-deficient colorectal cncers using an in silico genome scan to identify coding microsatellites.

Human colorectal, endometrial, and gastric cancers with defective DNA mismatch repair (MMR) have microsatellite instability, a unique molecular alteration characterized by widespread frameshift mutations of repetitive DNA sequences. We developed "Kangaroo," a bioinformatics program for searches in nucleotide and protein sequence databases, and performed an in silico genome scan for DNA coding microsatellites that may have novel mutations in MMR-deficient cancers. Examination of 29 previously untested coding polyadenines revealed widespread mutations in MMR-deficient colorectal cancers, with the highest frequencies in ERCC5, CASP8AP2, p72, RAD50, CDC25, RECQL1, CBF2, RACK7, GRK4, and DNAPK (range, 10-33%). This algorithm allows comprehensive mutation profiling of MMR-deficient cancers, an important step in understanding the pathogenesis of these neoplasms.

Algorithms↗
PubMed2005

Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects.

As part of a large-scale noninvasive fetal ultrasound screen to recover ethylnitrosourea (ENU)-induced mutations causing congenital heart defects in mice, we established a high-throughput ultrasound scanning strategy for interrogating fetal mice in utero utilizing three orthogonal imaging planes defined by the fetus' vertebral column and body axes, structures readily seen by ultrasound. This contrasts with the difficulty of acquiring clinical ultrasound imaging planes which are defined by the fetal heart. By use of the three orthogonal imaging planes for two-dimensional (2D) imaging together with color flow, spectral Doppler, and M-mode imaging, all of the major elements of the heart can be evaluated. In this manner, 10,091 ENU-mutagenized mouse fetuses were ultrasound scanned between embryonic days 12.5 and 19.5, with 324 fetuses found to die prenatally and 425 exhibiting cardiovascular defects. Further analysis by necropsy and histology showed heart defects that included conotruncal anomalies, obstructive lesions, and shunt lesions as well as other complex heart diseases. Ultrasound imaging also identified craniofacial/head defects and body wall closure defects, which necropsy revealed as encephalocele, holoprosencephaly, omphalocele, or gastroschisis. Genome scanning mapped one ENU-induced mutation associated with persistence truncus arteriosus and holoprosencephaly to mouse chromosome 2, while another mutation associated with cardiac defects and omphalocele was mapped to mouse chromosome 17. These studies show the efficacy of this novel ultrasound scanning strategy for noninvasive ultrasound phenotyping to facilitate the recovery of ENU-induced mutations causing congenital heart defects and other extracardiac anomalies.

Abnormalities, Multiple↗
PubMed2006

Phenotype and enamel ultrastructure characteristics in patients with ENAM gene mutations g.13185-13186insAG and 8344delG.

OBJECTIVE: The main clinical manifestations of amelogenesis imperfecta (AI) include alteration in the quality and quantity of enamel. AI is associated with different mutations in four genes: enamelin (ENAM), amelogenin (AMGX), kallikrein (KLK4) and enamelysin (MMP-20). Seven different mutations have been identified in the enamelin gene (ENAM). DESIGN: In this paper, we describe the phenotype and ultrastructure of enamel observed using scanning electron microscopy (SEM) in patients with two autosomal dominant (AD) mutations in the ENAM gene: g.13185-13186insAG and g.8344delG, each in one of two unrelated families. Mutations were confirmed by sequence analysis of PCR amplified products of all 10 exons and exon/intron boundaries of the ENAM gene. RESULTS: Phenotypic diversity was observed in patients with ENAM gene mutations g.13185-13186insAG with consecutive protein alteration designated as p.P422fsX488 within family 1. In the proband, the enamel of his entire dentition was chalky white with only mild local hypoplastic alteration, while the phenotypic appearance of his father's dentition was that of local hypoplastic AI. In patients with the ENAM gene mutation g.8344delG from family 2 with consecutive protein alteration designated as p.N197fsX277, generalised hypoplastic AI was observed. CONCLUSIONS: Ultrastructural enamel changes in the patient with the autosomal dominant ENAM g.13185-13186insAG mutation, described for the first time in this study, were less pronounced compared to ultrastructural changes in patients with the autosomal dominant ENAM mutation 8344delG. Ultrastructural characteristics of the g.13185-13186insAG mutation revealed deformed prisms, an oval shape on the cross-section and wider interprism spaces, while enamel with the ENAM mutation 8344delG was laminated, but prismless.

Amelogenesis Imperfecta↗
PubMed1991

Developmental analysis of the facets, a group of intronic mutations at the Notch locus of Drosophila melanogaster that affect postembryonic development.

The activity of the Notch locus of Drosophila melanogaster during embryogenesis is necessary for the correct segregation of neural from epidermal lineages. The action of Notch is not confined to embryogenesis but is also essential for normal development during the postembryonic stages. Its action is pleiotropic, as revealed by the existence of several classes of mutations which affect various imaginal structures. Here, we examine a group of six recessive mutations, the facets (fa, fa3, fag, fag-2, fafx and fasw), which affect eye and optic lobe morphology and have been previously shown to be associated with the insertion of transposable elements into an intronic region of Notch. Using both somatic recombination and gynandromorph analysis, we find that their behavior in a mosaic analysis is not identical. While in the majority of alleles abnormal Notch function in the retina is sufficient to induce optic lobe abnormalities, in the case of fag-2, a considerable number of individuals having mosaic retinas exhibit normal optic lobe structure. All the facet alleles appear to behave in a cell-autonomous manner. A developmental analysis of the eye and optic lobe defects associated with the facet mutations support the contention that Notch may be involved not only in the formation of certain structures but also in their maintenance.

Alleles↗
PubMed1993

Mutations of the fizzy locus cause metaphase arrest in Drosophila melanogaster embryos.

We describe the effects of mutations in the fizzy gene of Drosophila melanogaster and show that fizzy mutations cause cells in mitosis to arrest at metaphase. We show that maternally supplied fizzy activity is required for normal nuclear division in the preblastoderm embryo and, during later embryogenesis, that zygotic fizzy activity is required for the development of the ventrally derived epidermis and the central and peripheral nervous systems. In fizzy embryos, dividing cells in these tissues arrest at metaphase, fail to differentiate and ultimately die. In the ventral epidermis, if cells are prevented from entering mitosis by using a string mutation, cell death is prevented and the ability to differentiate ventral epidermis is restored in fizzy; string double mutant embryos. These results demonstrate that fizzy is a cell cycle mutation and that the normal function of the fizzy gene is required for dividing cells to exit metaphase and complete mitosis.

Animals↗
PubMed2005

Evidence-based algorithm for the evaluation of a child with bilateral sensorineural hearing loss.

OBJECTIVE: To develop an evidence-based algorithm for determining the etiology of bilateral sensorineural hearing loss (SNHL) in a child. METHODS: The frequency of different etiologies was previously determined. A systematic review of the literature for articles published between 1940 and January 2003 was performed for studies providing information on the diagnosis of each etiology relevant to their clinical presentation. RESULTS: Connexin mutation testing is highly sensitive and specific. CT scanning of the temporal bones is frequently valuable in detecting inner ear malformations. Routine laboratory studies are rarely helpful. ECG is particularly valuable when a history of syncope or arrhythmias or a family history of sudden death in a young child is elicited. There is no literature to support routine urinalysis for the diagnosis of Alport syndrome and thyroid studies lack specificity in the absence of physical findings (goiter). CONCLUSIONS: An evidence-based algorithm was developed that included: history, physical and audiological evaluation, and ophthalmological evaluation. Further directed investigations may include genetic testing for the Cx26 mutation, CT scan of the temporal bones, ECG and urinalysis.

Algorithms↗
PubMed1997

Identification of the functional regions required for hepatitis D virus replication and transcription by linker-scanning mutagenesis of viral genome.

To define the important cis-elements in hepatitis delta virus (HDV) RNA, the viral genome was mutated by a linker-scanning mutagenesis strategy that maintained the native rod-like structure of HDV RNA. Mutant HDV cDNAs or their corresponding RNA transcripts were transfected into a Huh-7-derived cell line which continuously expressed small hepatitis delta antigen to study the viral replication and transcription. Here we report the following findings. (i) Although most of the mutant RNAs could self-process to generate the 1.7-kb genomic RNA and all their stabilities were similar, positions which surround the genomic ribozyme domain were found to be important for the self-processing of the dimeric RNA. (ii) The replication of viral RNA was greatly diminished in many mutants, suggesting that multiple regions in HDV RNA were required for replication. (iii) In certain mutants, replication of the HDV antigenomic RNA was selectively abolished but that of the genomic RNA was not. Therefore, this was the first report to show that the cis-elements needed for the replication of genomic or antigenomic HDV RNA could be different. (iv) A continuous region (nt 1625 to nt 431), spanning the HDAg mRNA initiation site and containing the in vitro identified RNA promoter, was found to be important for mRNA production in vivo. (v) The HDV RNA replication and transcription was previously proposed to be governed by a single "double-acting promoter." However, two mutants which were deficient in mRNA synthesis still retained active viral RNA replication. It suggested that the HDV replication could initiate from sites other than this single promoter. This study therefore provided an insight into the cis-elements required for HDV RNA replication and transcription and further contributed to our understanding of HDV life cycle.

Genome, Viral↗