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PubMed1983

Absence in glucocorticoid-resistant mouse lymphoma P1798 of a glucocorticoid receptor domain responsible for biological effects.

Glucocorticoid-resistant (CR), in contrast to glucocorticoid-sensitive (CS), mouse lymphoma P1798 was shown to lack antiglucocorticoid receptor immunoactivity. Antibodies raised against the purified rat liver glucocorticoid receptor (GR) cross-reacted with the GR from CS, but not with the GR from CR, P1798 lymphoma. Using highly specific antisera against the GR in an indirect competitive enzyme-linked immunosorbent assay, it was demonstrated that alpha-chymotrypsin digestion of the GR from CS P1798 lymphoma caused a separation of a "resistant-like" nonimmunogenic steroid and DNA-binding domain (Stokes' radius, 3.3 nm) from an immunoactive domain (Stokes' radius, 2.6 nm). In contrast to CS P1798 lymphoma, neither before nor after alpha-chymotrypsin digestion, immunoactivity could be found in the cytosol from CR P1798 lymphoma. This was assayed after chromatography on DNA-cellulose or gel filtration on Agarose A (0.5 m). These results suggest that the domain of the CS GR containing the immunoactive determinant(s), normally removed by limited proteolysis by alpha-chymotrypsin, appears to be missing in CR P1798 lymphoma cytosol. It seems that this domain plays an important role in the mechanism of action of glucocorticoids. This might suggest that a mutation has occurred affecting the genome resulting in defective transcription of the receptor gene(s) in CR P1798 lymphoma.

Animals↗
PubMed1995

The genetics of cerebrovascular disease.

Genetics contribute to all aspects of cerebrovascular disease from the underlying pathology, through acute neuronal injury, to recovery and long-term prognosis. By understanding better the genetic contribution to the risk for cerebrovascular disease we can work toward the day when simple genetic tests can be used to assess an individual's risk for stroke (and cardiovascular disease) in the same way that we now use routine screening of blood pressure or cholesterol (Harrap, 1994b). Finally, it is worth noting that genetic effects do not work 'in vacuo'. They are subject to environmental influences which impact on the individual's phenotypic characteristics. Modification of these environmental factors may have a significant effect on the action of certain expressed genes and their putative mutations.

Cerebrovascular Disorders↗
PubMed1993

Dominant-negative mutations of platelet-derived growth factor (PDGF) receptors. Inhibition of receptor function by ligand-dependent formation of heterodimers between PDGF alpha- and beta-receptors.

We showed previously that a truncated form of the platelet-derived growth factor (PDGF) beta-receptor lacking its kinase region can form a nonfunctional heterodimer with the wild-type beta-receptor and thereby inhibit its signal transduction. In this paper we investigated whether the truncated form of either alpha- or beta-receptor could block the function of the other type of wild-type PDGF receptor. When the truncated alpha-receptor was expressed in Xenopus oocytes in excess over either the wild-type alpha- or beta-receptor, the Ca2+ mobilization signal elicited by either the wild-type alpha- or beta-receptor was completely blocked. The truncated beta-receptor abolished signaling by the wild-type alpha-receptor in response to PDGF-AB or -BB. However signal transduction by the alpha-receptor in response to PDGF-AA was not affected by the truncated beta-receptor. In the presence of PDGF-AB or -BB, both the wild-type and truncated beta-receptors formed a heterologous complex with the alpha-receptor in intact cells. A kinase-inactive beta-receptor (an ATP-binding site mutation) became cross-phosphorylated on tyrosine residues by the co-expressed wild-type alpha-receptor in response to PDGF-AB or -BB but not in response to PDGF-AA. These findings indicate that the alpha-and beta-receptors interact in response to PDGF-AB or -BB and are consistent with the hypothesis that the truncated alpha-receptor inhibits function of the wild-type beta-receptor through formation of a ligand-dependent nonfunctional alpha beta-heterodimer. A similar heterodimer can form between the truncated beta-receptor and the wild-type alpha-receptor. These observations provide useful information for future studies using dominant-negative mutations of PDGF receptors to selectively inhibit the actions of specific PDGFs in animals.

Animals↗
PubMed1995

Structural requirements for the efficient regulation of the Src protein tyrosine kinase by Csk.

Protein tyrosine kinases of the Src family are negatively regulated by phosphorylation in the C-terminal tail of the molecule. A different protein tyrosine kinase, Csk, is largely responsible for this regulation. The phosphorylated tail of c-Src engages with the SH2 domain in a conformation that is associated with low kinase activity and which involves stabilization by the SH3 domain. Inducible expression of c-Src in fission yeast is lethal unless Csk is coexpressed. Using this assay we present evidence that Src regulation by C-terminal phosphorylation does not require the myristylation signal or the unique domain at the N-terminus of the Src protein. Mutagenesis of the SH3 and SH2 domains of Csk show that neither are necessary in yeast or in vitro for efficient regulation of Src. Mutation of Tyr416 of Src, a site of autophosphorylation common to most protein tyrosine kinases, abolished the ability of Src to arrest growth of phosphorylate endogenous proteins. Tyr416 had the same effect on a shorter form of Src consisting of the kinase domain only, indicating that the mutation affects a property intrinsic to the catalytic domain. The residual activity of full-length Src mutated at Tyr416 is efficiently repressed by Csk action, suggesting that regulation by C-terminal phosphorylation does not act by preventing phosphorylation at Tyr416.

CSK Tyrosine-Protein Kinase↗
PubMed1996

Analysis of the negative transcriptional regulatory element in the angiotensin-converting enzyme gene.

We have characterized the sequence requirements and the protein binding properties of the previously identified transcriptional negative element present in the rabbit angiotensin-converting enzyme (ACE) gene. DNase footprinting experiments revealed that within the negative element (-715 to -610) several regions interact with proteins present in the nuclear extracts of ACE-expressing and -nonexpressing cell lines. Transfection analysis using the heterologous beta-actin promoter and mutated negative elements demonstrated that the SP1 site, the collagen-silencer-like sequence, and the inverted repeat elements are dispensable for their functioning. Deletion of the region between -692 to -668, however, completely eliminated the activity of the negative element, and mutation of the synapsin-silencer-like sequence present within this region vastly reduced it. This region (-692 to -668) by itself, when present in two copies, could effectively repress the activity of the beta-actin promoter. The same point mutations in the silencer element that destroyed its action on the beta-actin promoter greatly increased the transcriptional efficiency of the native ACE promoter. Electrophoretic mobility shift assay using the -692 to -668 ACE silencer sequence demonstrated the formation of a DNA/protein complex. UV cross-linking of the components of this complex revealed the presence of one prominent protein of approximately 21.5 kDa. This protein may be responsible for mediating the transcriptional-repressing activity of the ACE negative element. Homology between the ACE silencer and neuronal silencer consensus sequence, together with the promoter- and tissue-independent function of the the ACE silencer, suggests this element may bind a member of a large family of common negative regulatory transcription factors.

Animals↗
PubMedDate not supplied

[Chromosome aberrations, chromatin diminution and their significance in understanding the molecular genetic organization of eukaryotic chromosomes].

The paper analyzes the significance of new data obtained in the study of mechanism of formation of radiation-induced chromosome aberrations and in the investigation of chromatin diminution for understanding of the principles of the molecular genetical organization of eucaryotic chromosome. It is concluded that the structure of eucaryotic chromosome follows a certain plan. For its formation, a large number of various innovations were necessary, which could not been prepared simultaneously under the directed action of natural selection based upon the stochastic mutation process.

Animals↗
PubMed2003

[Inhibitory effect of p53 with deletion of c-terminal 356 - 393 amino acids on malignant phenotype of human lung cancer cell line].

OBJECTIVE: To study the effect of extraneous p53 gene with deletion of c-terminal 356 - 393 amino acids on inhibition of malignant phenotype of human lung cancer cell line. METHODS: Recombinant plasmid pEGFP-p53 (del) with codon deletion of c-terminal 37 amino acids from 393 to 356 region and pEGFP-p53 (wild type) were constructed. The human lung cancer cell line 801D served as a receipt cell had p53 deletion and mutation at 248 codon. 801D cells, having been transfected by pEGFP-p53 (wild type), pEGFP-p53 (del) or pEGFP, were selected by G418. Growing transfected cells were cloned respectively by method of dilution. Presence of extraneous gene was detected by PCR, their expression in cells was examined by fluorescence microscopy. Cloning efficiency was in vitro tested to examine the cellular proliferating ability. The xenograft in nude mice was performed and xenograft tumors were weighed one month later. Expression of GFP in tumor and transplanted cellular mass were detected by blot slices. RESULTS: pEGFP-p53 (del)-801D, pEGFP-p53-801D and pEGFP-801D were established. Extraneous p53 gene and expression of GFP were found in pEGFP-p53 (del)-801D and pEGFP-p53-801D. Inhibitory rate of colony was 99.6% for pEGFP-p53 (del)-801D and 81.0% for pEGFP-p53-801D. Inhibition of malignant proliferation of extraneous p53 (del) was higher than that of p53 (wild type) (P < 0.01). Even when inhibition of malignant proliferation extraneous pEGFP-p53 (del) was obvious, 0.2% colonies were formed, extraneous p53 and expression of GFP were observed. Animal test showed that tumor on the nude mice was positive (4/4, 4/4) in the control group (801D and pEGFP-801D), but negative (0/4, 0/4) in the experiment group [pEGFP-p53 (del) 801D and pEGFP-p53 (wild type) 801D]. Expression of GFP in the cells of cellular mass transplanted by pEGFP-p53 (del) 801D or pEGFP-p53 (wild type) 801D was observed. CONCLUSION: In vitro inhibitory effect of extraneous p53 gene with deletion of C-terminal 356 - 393 amino acids on malignant growth of lung cancer cell with p53 mutation or deletion at 248 codon is marked. Inhibitory action of p53 on malignant proliferation of cancer cells is heterogeneous.

Animals↗
PubMed1995

The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways.

The recessive mutations aux1 and axr1 of Arabidopsis confer resistance to the plant hormone auxin. The axr1 mutants display a variety of morphological defects. In contrast, the only morphological defect observed in aux1 mutants is a loss of root gravitropism. To learn more about the function of these genes in auxin response, the expression of the auxin-regulated gene SAUR-AC1 in mutant and wild-type plants has been examined. It has been found that axr1 plants display a pronounced deficiency in auxin-induced accumulation of SAUR-AC1 mRNA in seedlings as well as rosette leaves and mature roots. In contrast, the aux1 mutation has a modest effect on auxin induction of SAUR-AC1. To determine if the AUX1 and AXR1 genes interact to facilitate auxin response, plants which are homozygous for both aux1 and axr1 mutations have been constructed and characterized. The two mutations are additive in their effects on auxin response, suggesting that each mutation confers resistance by a different mechanism. However, the morphology of double mutant plants indicates that there is an inter-action between the AXR1 and AUX1 genes. In mature plants, the aux1-7 mutation acts to partially suppress the morphological defects conferred by the axr1-12 mutation. This suppression is not accompanied by an increase in auxin response, as measured by SAUR-AC1 expression, suggesting that the interaction between the AUX1 and AXR1 genes is indirect.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1984

Radiation-induced mutagenicity and lethality in ames tester strains of salmonella.

Mutation and killing induced by X radiation and 60CO gamma radiation were studied in six different histidine-requiring auxotrophs of Salmonella typhimurium. Strain TA100, which is sensitive to base-pair substitutions, and strains TA2637 and TA98, which are sensitive to frameshifts, carry the pKM101 plasmid and exhibit significantly higher radiation-induced mutations compared to their plasmidless parent strains TA1535, TA1537, and TA1538, respectively. Among the plasmid-containing strains, TA98 and TA2637 are much more sensitive to the mutagenic action of radiation than is TA100 based on a comparison with their respective spontaneous mutation rates; however, no uniformity was observed in the responses of the strains to the lethal action of ionizing radiation. The pKM101 plasmid provides partial protection against lethality in TA100 and TA2637, whereas the same plasmid enhances the lethal action of ionizing radiation in TA98. The following conclusions are consistent with these observations: (1) the standard Ames Salmonella assay correctly identifies ionizing radiation as a mutagenic agent; (2) frameshift-sensitive parent strains are more sensitive to the mutagenic effects of ionizing radiation than is the only strain studied that is sensitive to base-pair substitutions; and (3) enhancement of mutagenesis and survival is related to plasmid-mediated repair of DNA damage induced by ionizing radiation and does not involve damage induced by Cerenkov-generated uv radiation which is negligible for our irradiation conditions.

Cell Survival↗
PubMed1977

Apollo-Soyuz Test Project on biorhythm of zone-forming fungi: preliminary work.

The purpose of the experiment was to study general and local effects of space flight factors on the rhythm of cellular activity and on the morphological and genetic properties of biological objects. The Pushchino strain, Actinomyces levoris Kras 17-225A-IBFM, isolated at the Institute of Biological Physics, Moscow, was chosen as the main biological object. Under appropriate conditions it gives distinct and continuous rings of spore formation reflecting its intrinsic high degree of synchronism in changing its reproduction forms seen with the unaided eye as transparent rings (vegetative growth) alternate with convex white rings (spore-formation growth). As an additional test object, a film culture of bacteriophage T4Br+ developed at the institute was used. The strains were placed together in one bioblock together with plastic detectors for detecting nuclear particles. The film culture of bacteriophage enabled us to amplify the area of registration of local radiation effects by studying the genetic effects of these: frequency of mutations, induced radiation, their spectrum, subsequent revertability under the action of chemical mutagens with known mechanisms of action on DNA molecules.

Actinomyces↗
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

recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities.

Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination.

Amino Acid Sequence↗
PubMed1986

Neurogenetic analysis of Drosophila mutations affecting sodium channels: synergistic effects on viability and nerve conduction in double mutants involving tip-E.

In previous work it was shown that parats (paralyzed, temperature-sensitive, 1-53.9) and napts (no action potential, temperature-sensitive, 2-56.2), two temperature-sensitive paralytic mutations that block nerve conduction at restrictive temperatures, interact synergistically in double mutants causing unconditional lethality. This interaction is now shown to include tip-E (temperature-induced paralysis, 3-13.5), another temperature-sensitive paralytic mutation. There is an allele-dependent interaction between tip-E and various para alleles resulting in the unconditional lethality of the most extreme double mutant combinations. The pattern of this allele-dependency is strikingly different from that previously reported for napts and para in that the para alleles that interact strongest with napts interact weakest with tip-E and vice-versa. Double mutants of tip-E with napts also display greatly reduced viability. Surviving double mutants of tip-E with either parats1 or napts are weak and exhibit enhanced temperature sensitivity for both paralysis and nerve conduction failure. In addition, in a tip-E background, mutant para alleles enhance temperature-sensitive paralysis even when heterozygous with para+. The results of these studies suggest that tip-E shares related function with para and nap. It is proposed that tip-E, like para and nap exerts an effect at some level on the structure, function, or stability of sodium channels.

Alleles↗
PubMed1994

The mating of a fly.

Courtship in Drosophila is influenced by a wide variety of genes, in that many different kinds of pleiotropic mutations lead to defective courtship. This may seem to be a truism, but the broad temporal and spatial expression of most of the fly's "neuro genes" makes it difficult to exclude elements of such genes' actions as materially underlying reproductive behavior. "Courtship genes" that seem to play more particular roles were originally identified as sensory, learning, or rhythm mutations; their reproductive abnormalities have been especially informative for revealing components of male or female actions that might otherwise have gone unnoticed. Further behavioral mutations seemed originally to be courtship-specific, turned out not to have that property, and have led to a broadened perspective on the nature and action of Drosophila's sex-determination genes.

Animals↗
PubMed1999

The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating.

Hyperekplexia (startle disease) results from mutations in the glycine receptor chloride channel that disrupt inhibitory synaptic transmission. The Q266H missense mutation is the only hyperekplexia mutation located in the transmembrane domains of the receptor. Using recombinant expression and patch-clamping techniques, we have investigated the functional properties of this mutation. The ability of glycine and taurine to open the channel was reduced in the mutated channel, as shown by a 6-fold shift in the concentration-response curve for both agonists. This was not accompanied by similar changes in agonist displacement of strychnine binding, suggesting that the mutation affects functions subsequent to ligand binding. Taurine was also converted to a weak partial agonist and antagonized the actions of glycine, consistent with changes in its channel gating efficacy. Because the Q266H mutation is within the pore-forming second transmembrane domain, we tested for a direct interaction with permeating ions. No change in either the cation/anion selectivity ratio or in single channel conductance levels was observed. No differential effects of Zn++, pH, and diethylpyrocarbonate were observed, implying that the histidine side chain is not exposed to the channel lumen. Single-channel recordings revealed a significant reduction in open times in the mutant receptors, at both high and low agonist concentrations, consistent with the open state of the channel being less stable. This study demonstrates that residues within the second transmembrane domain of ligand-gated ion channel receptors, even those whose side chains do not directly interact with permeating ions, can affect the kinetics of channel gating.

Anions↗
PubMed2002

Clinical, genetic, and biophysical characterization of SCN5A mutations associated with atrioventricular conduction block.

BACKGROUND: Three distinct cardiac arrhythmia disorders, the long-QT syndrome, Brugada syndrome, and conduction system disease, have been associated with heterozygous mutations in the cardiac voltage-gated sodium channel alpha-subunit gene (SCN5A). We present clinical, genetic, and biophysical features of 2 new SCN5A mutations that result in atrioventricular (AV) conduction block. Methods and Results- SCN5A was used as a candidate gene in 2 children with AV block. Molecular genetic studies revealed G to A transition mutations that resulted in the substitution of serine for glycine (G298S) in the domain I S5-S6 loop and asparagine for aspartic acid (D1595N) within the S3 segment of domain IV. The functional consequences of G298S and D1595N were assessed by whole-cell patch clamp recording of recombinant mutant channels coexpressed with the beta1 subunit in a cultured cell line (tsA201). Both mutations impair fast inactivation but do not exhibit sustained non-inactivating currents. The mutations also reduce sodium current density and enhance slower inactivation components. Action potential simulations predict that this combination of biophysical abnormalities will significantly slow myocardial conduction velocity. CONCLUSIONS: A distinct pattern of biophysical abnormalities not previously observed for any other SCN5A mutant have been recognized in association with AV block. These data provide insight into the distinct clinical phenotypes resulting from mutation of a single ion channel.

Action Potentials↗
PubMed1995

The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes.

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.

3T3 Cells↗
PubMed1993

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Alleles↗