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PubMedDate not supplied

Early and transient osteopetrosis in microphthalmic MIB-rats.

Microphtalmic blanc mutation (mib/mib) displays a very mild form of osteopetrosis in rats. The autosomal recessive mib mutation shows pleiotropic expressions in homozygotes. Microphtalmia, absence of eye and skin pigmentation, retardation in the tooth eruption were observed in the mutants. Most bone abnormalities occurred in newborns. An increased radiological opacity of long bones, persistence of primitive bone in medullary cavities, reduced number of poorly differentiated osteoclasts in mandibulae, reduced number of mononuclear peritoneal cells as well as reduced number of mononuclear osteoclast precursors in peritoneal cell population were found. In 3 weeks old and in adult mutants, both bone structure and the number of mandible osteoclasts appear normal, but the number of blood monocytes, peritoneal cells and mononuclear osteoclast precursors in peritoneal cell population remain significantly lower than in the healthy littermates. These observations indicate that the early failure of osteoclast differentiation and maturation is transient in the mib/mib form of osteopetrosis.

Abnormalities, Multiple↗
PubMed1993

Hemophilia B Leyden: substitution of thymine for guanine at position -21 results in a disruption of a hepatocyte nuclear factor 4 binding site in the factor IX promoter.

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by an altered developmental expression of blood coagulation factor IX. This form of hemophilia B has been found to be associated with a variety of single point mutations in the factor IX promoter region. We now describe a novel point mutation, T-->G at position -21, in two related patients with the hemophilia B Leyden phenotype. This mutation lies within the factor IX promoter region (-40 to -9) that contains overlapping binding sites for hepatocyte nuclear factor 4 (HNF-4) and androgen receptor. Transient transfection assays in HepG2 cells show that the -21 mutation causes a significant reduction in factor IX promoter activity. Gel mobility shift assays and transient cotransfection experiments revealed that the HNF-4-binding site but not the androgen-responsive element is disrupted by the -21 mutation. A comparison of the -21 mutation with the previously described -20 T-->A mutation (associated with the hemophilia B Leyden phenotype) and -26 G-->C mutation (associated with severe hemophilia B throughout life) was made. It shows that the -21 mutation reduced HNF-4 binding and transactivation to a similar level as the -20 mutation, whereas the -26 mutation completely abolished HNF-4 binding and transactivation. Mobility shift experiments indicate that there was no significant difference in binding affinity of recombinant androgen receptor protein for oligonucleotides containing wild-type and -21 or -20 mutated DNA. The binding affinity for the oligonucleotide containing the -26 mutation was twofold lower. The results indicate that the disruption of the HNF-4-binding site by the -21 T-->G mutation is the cause of the bleeding disorder in these two patients. This study adds further support for the notion that the recovery from hemophilia at puberty may not only be related to an intact androgen-responsive element but also to the degree of disruption of the HNF-4-binding site.

Base Sequence↗
PubMed1995

Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene.

The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo.

Animals↗
PubMed1998

Nuclear corepressors enhance the dominant negative activity of mutant receptors that cause resistance to thyroid hormone.

The syndrome of resistance to thyroid hormone (RTH) is caused by multiple distinct mutations in the ligand-binding domain of the thyroid hormone receptor-beta (TRbeta). Although the mutant receptors are transcriptionally inactive, they inhibit normal receptor function in a dominant negative manner to cause hormone resistance. Recently, a group of transcriptional cofactors, referred to as corepressors (CoRs), was shown to induce ligand-independent silencing of genes that contain positive T3 response elements. CoRs also play a role in the ligand-independent basal activation of genes that are negatively regulated in response to T3. We hypothesized that CoR might play a role in the dominant negative inhibition by TRbeta mutants that cause RTH. In gel mobility shift assays, RTH mutants retained interactions with CoRs even in the presence of T3, whereas the ligand dissociated CoR from wild-type TRbeta. Using Gal4-TR chimeric receptors and a VP16-CoR fusion protein in an interaction assay, a strong positive correlation was found between mutant receptor interactions with CoR and transcriptional silencing activity. A mutation (P214R) that impairs CoR interactions with TR was introduced into the RTH mutants to assess the role of CoR in dominant negative activity. In transient transfection assays, introduction of the P214R CoR mutation decreased RTH mutant silencing of positively regulated genes and basal activation of negatively regulated genes. The dominant negative activity of several different RTH mutants, studied by cotransfection with wild-type receptor, was greatly diminished by the CoR mutation, and this effect was seen with both positively and negatively regulated genes. These results suggest that CoR interactions play a critical role in the dominant negative effect of RTH mutants and support the idea that these proteins are involved in the regulation of genes that are positively as well as negatively regulated by T3.

Amino Acid Sequence↗
PubMed1998

On the origin of multiple mutations in human cancers.

Tumor progression is a multi-step process, proceeding by multiple alterations from a normal cell to a localized tumor, and finally to one that acquires the ability to invade and metastasize. Tumors are characterized by many mutations in the form of base substitutions, deletions, chromosomal translocations, and gene amplifications, and these mutations are found to accumulate as tumors progress. In contrast, spontaneous mutations are very rare events. Considering the high fidelity of DNA replication in normal cells, it seems improbable that spontaneous mutations could be the source of the large numbers of genetic alterations that are observable in cancer cells. The question of how multiple mutations accumulate in tumor cells is one of considerable interest, since understanding the source of these mutations may facilitate the detection of tumors and may provide new approaches to cancer prevention. We have proposed that the multiple mutations detectable in cancer cells result from a mutator phenotype, in which loss of a genome stability function occurs early during tumor development and predisposes the tumor cell to the accumulation of further mutations. We will first consider the evidence that cancer cells manifest a mutator phenotype, and subsequently discuss the possibility that a mutator phenotype can be selected and can be transient as tumors progress.

Clone Cells↗
PubMed2003

Clinical and molecular characterization of 6 patients affected by severe deficiency of coagulation factor V: Broadening of the mutational spectrum of factor V gene and in vitro analysis of the newly identified missense mutations.

Severe factor V (FV) deficiency is a rare bleeding disorder, whose genetic bases have been characterized only in a limited number of cases. We investigated 6 unrelated patients with extremely reduced plasma FV levels, associated with a bleeding tendency ranging from moderately severe to severe. Clinical manifestations were substantially concordant with the previously established spectrum of hemorrhagic symptoms of the disease. Molecular analysis of FV gene identified 9 different mutations, 7 hitherto unknown, and 2 previously reported (Arg712ter and Tyr1702Cys). Four of 6 analyzed patients were compound heterozygotes, indicating the high allelic heterogeneity of this disease. Among novel mutations, 5 led to premature termination codons, because of nonsense (Arg1002ter, Arg1606ter, and Trp1854ter), or frameshift mutations (5127-5128insA and 6122-6123insAACAG). The remaining 2 were missense mutations (Cys472Gly and Val1813Met), located in FV A2 and A3 domains. Their effect on FV expression was studied by transient transfection experiments, demonstrating that the presence of each mutation impaired FV secretion. These data increase the number of severe FV deficiency-causing mutations by about 50%. The high number of "private" mutations identified in FV-deficient families indicates that full mutational screening of FV gene is still required for molecular diagnosis.

Adolescent↗
PubMed1991

Fixation of mutations at the VP1 gene of foot-and-mouth disease virus. Can quasispecies define a transient molecular clock?

The number of nucleotide (nt) substitutions found in the VP1 gene (encoding viral capsid protein) between any two of 16 closely related isolates of foot-and-mouth disease virus (FMDV) has been quantified as a function of the time interval between isolations [Villaverde et al., J. Mol. Biol. 204 (1988) 771-776]. One of them (isolate C-S12) includes some replacements found in isolates that preceded it and other replacements found in later isolates. The study has revealed alternating periods of rapid evolution and of relative genetic stability of VP1. During a defined period of acute disease, the rate of fixation of replacements at the VP1 coding segment was 6 x 10(-3) substitutions per nt per year. Only small differences in the rate of evolution were observed between subsegments within the VP1 gene. The observation of a relatively constant rate of evolution during a disease episode was unexpected. We propose that such constancy may be a consequence of random sampling of mutants from the FMDV quasispecies, followed by their amplification in susceptible hosts (to generate a new quasispecies). Successive sampling and amplification events may result in a steady accumulation of mutations.

Amino Acid Sequence↗
PubMed1997

Analysis of a temperature-sensitive vaccinia virus mutant in the viral mRNA capping enzyme isolated by clustered charge-to-alanine mutagenesis and transient dominant selection.

We have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994) Proc. Natl. Acad. Sci. USA 91, 4554-4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus capping enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis. In vitro assays of various capping enzyme activities revealed that in permeabilized virions, enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the capping enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis.

Alanine↗
PubMed2000

The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli population.

Connexin 26 (GJB2) mutations lead to hearing loss in a significant proportion of all populations studied so far, despite the fact that at least 50 other genes are also associated with hearing loss. The entire coding region of connexin 26 was sequenced in 75 hearing impaired children and adults in Israel in order to determine the percentage of hearing loss attributed to connexin 26 and the types of mutations in this population. Age of onset in the screened population was both prelingual and postlingual, with hearing loss ranging from moderate to profound. Almost 39% of all persons tested harbored GJB2 mutations, the majority of which were 35delG and 167delT mutations. A novel mutation, involving both a deletion and insertion, 51del12insA, was identified in a family originating from Uzbekistan. Several parameters were examined to establish whether genotype-phenotype correlations exist, including age of onset, severity of hearing loss and audiological characteristics, including pure-tone audiometry, tympanometry, auditory brainstem response (ABR), and transient evoked otoacoustic emissions (TEOAE). All GJB2 mutations were associated with prelingual hearing loss, though severity ranged from moderate to profound, with variability even among hearing impaired siblings. We have not found a significant difference in hearing levels between individuals with 35delG and 167delT mutations. Our results suggest that, in Israel, clinicians should first screen for the common 167delT and 35delG mutations by simple and inexpensive restriction enzyme analysis, although if these are not found, sequencing should be done to rule out additional mutations due to the ethnic diversity in this region.

Adult↗
PubMed2001

Nitric oxide synthase (NOS2) mutation in Dahl/Rapp rats decreases enzyme stability.

The pathogenesis of salt-sensitive hypertension remains poorly defined, but a role for nitric oxide (NO) has been suggested. The Dahl/Rapp salt-sensitive rat possesses a defect in NO synthesis that is overcome by supplementation with L-arginine, which increases NO and cGMP production and prevents salt-sensitive hypertension. An S714P mutation of inducible NO synthase (NOS2) was subsequently identified. The current report examined the functional significance of an S714P mutation in NOS2. COS-7 cells were transiently transfected with cDNA of wild-type NOS2 and S714P and S714A mutants of NOS2, and enzyme function was determined. Whereas steady-state mRNA levels did not differ, immunoblot analysis demonstrated decreased levels of NOS2 protein. Metabolic labeling experiments confirmed a reduced half-life of the S714P mutation. Nitrite production, which was dependent on the concentration of L-arginine in the medium, was diminished in cells transfected with the S714P mutant, compared with the wild type and the S714A mutant. These data provide a biochemical explanation of the physiological abnormalities of NOS2 in the Dahl/Rapp salt-sensitive rat and suggest that a posttranslational mechanism involving the proteasome may be responsible for the diminished NO production observed in response to increased dietary salt intake in these animals.

Amino Acid Substitution↗
PubMed1997

Region-specific meiotic recombination in Schizosaccharomyces pombe: the rec11 gene.

Mutations in the rec11 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies by as much as a factor of 300 on chromosome III but less than a factor of 4 in the intervals tested on chromosomes I and II. To gain insight into the function of this region- (or chromosome-) specific activator of recombination, we have cloned and sequenced the rec11 gene. Meiotic crosses with rec11 disruption mutations placed the rec11 gene 6 cM from ade6 on chromosome III. Transcripts of rec11 accumulated transiently at 2-3 h after induction of melosis in a pat1-114 (Ts) mutant. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of these transcripts revealed eight introns. The spliced RNA is predicted to encode a polypeptide of 923 amino acids with only very limited homology to reported proteins. The transient accumulation of rec11 transcripts and the phenotype of rec11 mutations suggest that the novel rec11 gene product acts early in meiosis to activate recombination preferentially on chromosome III.

Base Sequence↗
PubMed1991

Analysis of the herpes simplex virus type 1 OriS sequence: mapping of functional domains.

The herpes simplex virus type 1 (HSV-1) OriS region resides within a 90-bp sequence that contains two binding sites for the origin-binding protein (OBP), designated sites I and II. A third presumptive OBP-binding site (III) within OriS has strong sequence similarity to sites I and II, but no sequence-specific OBP binding has yet been demonstrated at this site. We have generated mutations in sites I, II, and III and determined their replication efficiencies in a transient in vivo assay in the presence of a helper virus. Mutations in any one of the sites reduced DNA replication significantly. To study the role of OriS sequence elements in site I and the presumptive site III in DNA replication, we have also generated a series of mutations that span from site I across the presumptive binding site III. These mutants were tested for their ability to replicate and for the ability to bind OBP by using gel shift analyses. The results indicate that mutations across site I drastically reduce DNA replication. Triple-base-pair substitution mutations that fall within the crucial OBP-binding domain, 5'-YGYTCGCACT-3' (where Y represents C or T), show a reduced level of OBP binding and DNA replication. Substitution mutations in site I that are outside this crucial binding sequence show a more detrimental effect on DNA replication than on OBP binding. This suggests that these sequences are required for initiation of DNA replication but are not critical for OBP binding. Mutations across the presumptive OBP-binding site III also resulted in a loss in efficiency of DNA replication. These mutations influenced OBP binding to OriS in gel shift assays, even though the mutated sequences are not contained within known OBP-binding sites. Replacement of the wild-type site III with a perfect OBP-binding site I results in a drastic reduction of DNA replication. Thus, our DNA replication assays and in vitro DNA-binding studies suggest that the binding of the origin sequence by OBP is not the only determining factor for initiation of DNA replication in vivo.

Animals↗
PubMed1998

Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line.

Previous reports have demonstrated that the U1 cell line, a model for postintegration latency, is defective at the level of Tat function and can be rescued by exogenously provided Tat protein. Sequence analysis of tat cDNAs from the U1 cell line identified two distinct forms of Tat, in agreement with the fact that this cell line contains two integrated human immunodeficiency (HIV) proviruses. One Tat cDNA lacked an ATG initiation codon, while the other contained an H-to-L mutation at amino acid 13 (H13-->L). Both tat cDNAs were defective in terms of transcriptional activation of long terminal repeat-luciferase reporter gene in transient-transfection experiments. Introduction of the H13-->L mutation in a wild-type tat background caused a severe reduction in transcriptional activation. Introduction of the same mutation in an infectious HIV molecular clone caused a severely defective phenotype which could be rescued when the HIV proviral DNA was transfected in a Jurkat cell line stably expressing the Tat protein (Jurkat-Tat) or in Jurkat cells treated with tumor necrosis factor alpha. Infectious virus stocks generated in Jurkat-Tat cells were used to infect Jurkat cells and exhibited severely impaired growth which could also be rescued by infecting Jurkat-Tat cells. These observations define tat mutations as a mechanism for HIV postintegration latency.

Amino Acid Sequence↗
PubMed1999

Enzymatic characterization of four new mutations in the glucose-6 phosphatase (G6PC) gene which cause glycogen storage disease type 1a.

Glycogen storage disease type 1a (GSD1a) is caused by mutations in the gene of glucose-6 phosphatase (G6PC), encoding the last enzyme of gluconeogenesis and glycogenolysis. To study the effect of mutations previously identified, but not yet enzymatically characterized, in French GSD1a patients, we used an in vitro expression system of the human glucose-6 phosphatase (hGlc6Pase) cDNA. Wild type hGlc6Pase expressed in COS-7 cells exhibited kinetic features comparable to microsomal Glc6Pase from normal human liver and kidney. Four new mutations inducing aminoacid changes in the coding sequence, e.g. W77R, A124T, G184E and L211P, were inserted into the Glc6Pase cDNA by site-directed mutagenesis, and studied after transient expression in COS-7 cells. All four mutations totally abolished Glc6Pase activity.

Amino Acid Sequence↗
PubMed1997

Characterization of the interaction between the baculovirus replication factors LEF-1 and LEF-2.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.

Animals↗
PubMed1992

Mutations of the human thyrotropin-beta subunit glycosylation site reduce thyrotropin synthesis independent of changes in glycosylation status.

In recent studies, site-directed mutagenesis has been used to alter the tripeptide glycosylation recognition sequences of glycoprotein hormone subunits, thereby affecting their structure and function. However, it is not known whether these effects result from changes in glycosylation status, amino acid sequence, or both. We therefore studied the synthesis of wild-type and mutant recombinant human thyrotropins produced by transient transfection of a human cell line. Mutating the TSH-beta subunit glycosylation recognition sequence, Asn-Thr-Thr (codons 23-25), to either Gln-Thr-Thr or Asn-Thr-Tyr abolished subunit glycosylation, as demonstrated by the inability to incorporate 3H-carbohydrates. However, a third mutation (Asn-Thr-Ser) contained an intact glycosylation recognition sequence site, and was shown to retain glycosylation. The mutations that abolished TSH-beta subunit glycosylation resulted in greater than 90% decreases in TSH synthesis. However, the glycosylation recognition sequence mutant that retained beta subunit glycosylation exhibited a 70% decrease in TSH production. These decreases were not attributable to the intracellular accumulation of TSH or its free beta subunit. We also engineered two TSH-beta subunit mutations that did not alter the glycosylation recognition sequence. A glycine to arginine mutation adjacent to the glycosylation recognition sequence, in a region thought to be critical for heterodimer formation, abolished TSH production. In contrast, shortening the TSH-beta subunit carboxyterminus by six amino acids increased TSH synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2004

Readthrough of dystrophin stop codon mutations induced by aminoglycosides.

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchenne's and Becker's muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (< 2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.

Aminoglycosides↗