Showing papers on "Mutant published in 1994"
TL;DR: It is reaffirm that p53 function is not required for normal mouse development and conclude that p 53 status can strongly influence tumor latency and tissue distribution.
2,034 citations
TL;DR: The developmental programs of lymphoid and myeloid lineages require a common genetic function likely acting at the level of a multipotential progenitor, and mice carrying a mutation in the PU.1 locus were generated by gene targeting.
Abstract: The transcription factor PU.1 is a hematopoietic-specific member of the ets family. Mice carrying a mutation in the PU.1 locus were generated by gene targeting. Homozygous mutant embryos died at a late gestational stage. Mutant embryos produced normal numbers of megakaryocytes and erythroid progenitors, but some showed an impairment of erythroblast maturation. An invariant consequence of the mutation was a multilineage defect in the generation of progenitors for B and T lymphocytes, monocytes, and granulocytes. Thus, the developmental programs of lymphoid and myeloid lineages require a common genetic function likely acting at the level of a multipotential progenitor.
1,546 citations
TL;DR: This work has shown that with the use of the bacteriophage-derived, site-specific recombinase Cre in a transgenic approach, the same mutation can be selectively introduced into a particular cellular compartment-in this case, T cells.
Abstract: Deletion of the promoter and the first exon of the DNA polymerase beta gene (pol beta) in the mouse germ line results in a lethal phenotype. With the use of the bacteriophage-derived, site-specific recombinase Cre in a transgenic approach, the same mutation can be selectively introduced into a particular cellular compartment-in this case, T cells. The impact of the mutation on those cells can then be analyzed because the mutant animals are viable.
1,542 citations
TL;DR: A role for PR genes in preventing the proximal spread of pathogens in addition to their suggested role in SAR is suggested after the insensitivity of npr1 to SA, INA, and avirulent pathogens in SAR induction indicates that these inducers share a common signal transduction pathway.
Abstract: Systemic acquired resistance (SAR) is a general defense response in plants that is characterized by the expression of pathogenesis-related (PR) genes. SAR can be induced after a hypersensitive response to an avirulent pathogen or by treatment with either salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA). To dissect the signal transduction pathway of SAR, we isolated an Arabidopsis mutant that lacks the expression of an SA-, INA-, and pathogen-responsive chimeric reporter gene composed of the 5[prime] untranslated region of an Arabidopsis PR gene, [beta]-1,3-glucanase (BGL2), and the coding region of [beta]-glucuronidase (GUS). This mutant, npr1 (nonexpresser of PR genes), carries a single recessive mutation that abolishes the SAR-responsive expression of other PR genes as well. While SA-, INA-, or avirulent pathogen-induced SAR protects wild-type plants from Pseudomonas syringae infection, the mutant cannot be protected by pretreatment with these inducers. The insensitivity of npr1 to SA, INA, and avirulent pathogens in SAR induction indicates that these inducers share a common signal transduction pathway. Moreover, in npr1, the localized expression of PR genes induced by a virulent Pseudomonas pathogen is disrupted, and the lesion formed is less confined. These results suggest a role for PR genes in preventing the proximal spread of pathogens in addition to their suggested role in SAR.
1,421 citations
TL;DR: The results suggest that ced-9 and bcl-2 are homologs and that the molecular mechanism of programmed cell death has been conserved from nematodes to mammals.
1,210 citations
TL;DR: A gene, reaper (rpr), that appears to play a central control function for the initiation of programmed cell death (apoptosis) in Drosophila was identified and suggests that the basic cell death program is intact although it was not activated in mutant embryos.
Abstract: A gene, reaper (rpr), that appears to play a central control function for the initiation of programmed cell death (apoptosis) in Drosophila was identified. Virtually all programmed cell death that normally occurs during Drosophila embryogenesis was blocked in embryos homozygous for a small deletion that includes the reaper gene. Mutant embryos contained many extra cells and failed to hatch, but many other aspects of development appeared quite normal. Deletions that include reaper also protected embryos from apoptosis caused by x-irradiation and developmental defects. However, high doses of x-rays induced some apoptosis in mutant embryos, and the resulting corpses were phagocytosed by macrophages. These data suggest that the basic cell death program is intact although it was not activated in mutant embryos. The DNA encompassed by the deletion was cloned and the reaper gene was identified on the basis of the ability of cloned DNA to restore apoptosis to cell death defective embryos in germ line transformation experiments. The reaper gene appears to encode a small peptide that shows no homology to known proteins, and reaper messenger RNA is expressed in cells destined to undergo apoptosis.
1,059 citations
TL;DR: Results suggest that a tumor-specific alteration of the EGFR plays a significant role in tumor progression perhaps by influencing interactions of tumor cells with their microenvironment in ways not easily assayed in vitro.
Abstract: The development and neoplastic progression of human astrocytic tumors appears to result through an accumulation of genetic alterations occurring in a relatively defined order. One such alteration is amplification of the epidermal growth factor receptor (EGFR) gene. This episomal amplification occurs in 40-50% of glioblastomas, which also normally express endogenous receptors. Moreover, a significant fraction of amplified genes are rearranged to specifically eliminate a DNA fragment containing exons 2-7 of the gene, resulting in an in-frame deletion of 801 bp of the coding sequence of the extracellular domain. Here we used retroviral transfer of such a mutant receptor (de 2-7 EGFR) into glioblastoma cells expressing normal endogenous receptors to test whether the mutant receptor was able to augment their growth and malignancy. Western blotting analysis showed that these cells expressed endogenous EGFR of 170 kDa as well as the exogenous de 2-7 EGFR of 140-155 kDa. Although holo-EGFRs were phosphorylated on tyrosine residues only after exposure of the cells to ligand, de 2-7 EGFRs were constitutively phosphorylated. In tissue culture neither addition of EGF nor expression of the mutant EGFR affected the rate of cell growth. However, when cells expressing mutant EGFR were implanted into nude mice subcutaneously or intracerebrally, tumorigenic capacity was greatly enhanced. These results suggest that a tumor-specific alteration of the EGFR plays a significant role in tumor progression perhaps by influencing interactions of tumor cells with their microenvironment in ways not easily assayed in vitro.
942 citations
TL;DR: The determination of a large number of three-dimensional structures of Glycosidases, both free and in complex with ligands, has provided valuable new insights into glycosidase catalysis, especially when coupled with results from studies of specifically labelled glyCosidases and kinetic analyses of point mutants.
807 citations
TL;DR: A crucial role for E2A products as central regulators in early B cell differentiation is suggested, since no immunoglobulin DJ rearrangements can be detected in homozygous mutant mice.
801 citations
TL;DR: Observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin.
Abstract: The eukaryotic fatty acid desaturases are iron-containing enzymes that catalyze the NAD-(P)H- and O2-dependent introduction of double bonds into methylene-interrupted fatty acyl chains. Examination of deduced amino acid sequences for the membrane desaturases from mammals, fungi, insects, higher plants, and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 or 4)H,HX(2 or 3)HH, and HX(2 or 3)HH. This motif is also present in the bacterial membrane enzymes alkane hydroxylase (omega-hydroxylase) and xylene monooxygenase. Hydropathy analyses indicate that these enzymes contain up to three long hydrophobic domains which would be long enough to span the membrane bilayer twice. The conserved His-containing regions have a consistent positioning with respect to these potential membrane spanning domains. Taken together, these observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin. In order to examine the functional role of these conserved His residues, we have made use of the ability of the rat delta 9 desaturase gene to complement a yeast strain deficient in the delta 9 desaturase gene function (ole1). By site-directed mutagenesis, eight conserved His residues in the rat delta 9 desaturase were individually converted to Ala. Each His-->Ala mutation failed to complement the yeast ole1 mutant. In contrast, mutation of three nonconserved flanking His residues or a partially conserved Arg residue within the conserved motif to Ala allowed for complementation of the ole1 phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)
782 citations
TL;DR: Multiple eye abnormalities are described which are found in various RAR double mutant fetuses and are similar to those previously seen in VAD fetuses, and further abnormalities not previously reported in Vad fetuses are found.
Abstract: Numerous congenital malformations have been observed in fetuses of vitamin A-deficient (VAD) dams [Wilson, J. G., Roth, C. B., Warkany, J., (1953), Am. J. Anat. 92, 189–217]. Previous studies of retinoic acid receptor (RAR) mutant mice have not revealed any of these malformations [Li, E., Sucov, H. M., Lee, K.-F., Evans, R. M., Jaenisch, R. (1993) Proc. Natl. Acad. Sci. USA 90, 1590–1594; Lohnes, D., Kastner, P., Dierich, A., Mark, M., LeMeur, M., Chambon, P. (1993) Cell 73, 643–658; Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M. P., Lemeur, M., Chambon, P. (1993) Proc. Natl. Acad. Sci. USA 90, 7225–7229; Mendelsohn, C., Mark, M., Dolle, P., Dierich, A., Gaub, M.P., Krust, A., Lampron, C., Chambon, P. (1994a) Dev. Biol. in press], suggesting either that there is a considerable functional redundancy among members of the RAR family during ontogenesis or that the RARs are not essential transducers of the retinoid signal in vivo. In order to discriminate between these possibilities, we have generated a series of RAR compound null mutants. These RAR double mutants invariably died either in utero or shortly after birth and presented a number of congenital abnormalities, which are reported in this and in the accompanying study. We describe here multiple eye abnormalities which are found in various RAR double mutant fetuses and are similar to those previously seen in VAD fetuses. Interestingly, we found further abnormalities not previously reported in VAD fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The ABI1 gene encodes a protein with high similarity to protein serine or threonine phosphatases of type 2C with the novel feature of a putative Ca2+ binding site that could mediate pleiotropic hormone responses.
Abstract: The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, the maintenance of seed dormancy, and the inhibition of plant growth. All three responses are affected in the ABA-insensitive mutant abi1 of Arabidopsis thaliana, suggesting that an early step in the signaling of ABA is controlled by the ABI1 locus. The ABI1 gene was cloned by chromosome walking, and a missense mutation was identified in the structural gene of the abi1 mutant. The ABI1 gene encodes a protein with high similarity to protein serine or threonine phosphatases of type 2C with the novel feature of a putative Ca2+ binding site. Thus, the control of the phosphorylation state of cell signaling components by the ABI1 product could mediate pleiotropic hormone responses.
TL;DR: Analysis of signalling mutant phenotypes indicates that there are at least two separate signal‐response pathways which converge to regulate expression of luminescence in V. harveyl.
Abstract: Summary
Density-dependent expression of luminescence in Vibrio harveyl is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably β-hydroxybutryl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyl.
TL;DR: Surprisingly, heterozygous embryos contain, on average, about half as many B cells as wild-type embryos, suggesting the existence of a counting mechanism that translates levels of E2A into numbers of B cells.
01 Jan 1994
TL;DR: Results demonstrate that the Notch1 gene plays a vital role during early postimplantation development in mice, and is not allelic to a mouse mutation described previously, Danforth's short tail (Sd).
Abstract: The Notch gene of Drosopbila encodes a large transmembrane protein involved in cell fate determination during embryonic and larval development. This gene is evolutionarily conserved, and Notch homologs have been cloned from several vertebrate species. To examine the in vivo role of the Notchl gene, a mouse homolog of Notch, a mutation was introduced by targeted disruption in embryonic stem cells, and these cells were used to generate mutant mice. Intercrosses of animals heterozygous for the Notchl mutation yielded no live-born homozygous mutant offspring. Homozygous mutant embryos died before 11.5 days of gestation. Morphological and histological analysis of the homozygous mutant embryos indicated that pattern formation through the first nine days of gestation appeared largely normal. However, histological analysis of mutant embryos subsequent to this stage revealed widespread cell death. Death of mutant embryos did not appear to be attributable to defects in placentation or vascularization. Examination of the RNA expression pattern of the Notch2 gene, another Notch gene family member, indicated that it partially overlapped the Notchl expression pattern. Genetic analysis of the Notchl mutation also demonstrated that it was not allelic to a mouse mutation described previously, Danforth's short tail (Sd). These results demonstrate that the Notchl gene plays a vital role during early postimplantation development in mice.
Abstract: The Notch gene of Drosophila encodes a large transmembrane protein involved in cell fate determination during embryonic and larval development. This gene is evolutionarily conserved, and Notch homologs have been cloned from several vertebrate species. To examine the in vivo role of the Notch1 gene, a mouse homolog of Notch, a mutation was introduced by targeted disruption in embryonic stem cells, and these cells were used to generate mutant mice. Intercrosses of animals heterozygous for the Notch1 mutation yielded no live-born homozygous mutant offspring. Homozygous mutant embryos died before 11.5 days of gestation. Morphological and histological analysis of the homozygous mutant embryos indicated that pattern formation through the first nine days of gestation appeared largely normal. However, histological analysis of mutant embryos subsequent to this stage revealed widespread cell death. Death of mutant embryos did not appear to be attributable to defects in placentation or vascularization. Examination of the RNA expression pattern of the Notch2 gene, another Notch gene family member, indicated that it partially overlapped the Notch1 expression pattern. Genetic analysis of the Notch1 mutation also demonstrated that it was not allelic to a mouse mutation described previously, Danforth's short tail (Sd). These results demonstrate that the Notch1 gene plays a vital role during early postimplantation development in mice.
TL;DR: It is suggested that Sp3 is an inhibitory member of the Sp family, and neither the glutamine‐rich domains A and B nor the D domain of Sp1 can be replaced by the homologous regions of Sp3.
Abstract: Sp1, Sp3 (SPR-2) and Sp4 (SPR-1) are human sequence-specific DNA binding proteins with very similar structural features In this report, we have analyzed Sp3 in direct comparison with Sp1 We have raised antibodies against both Sp1 and Sp3, and show that Sp3 protein, like Sp1, is expressed in various cell lines Co-transfection experiments in different mammalian cell lines reveal that in contrast to Sp1 and Sp4, Sp3 is not able to activate several Sp1 responsive promoters In addition, Sp3 also fails to activate reporter constructs in Drosophila SL2 cells lacking endogenous Sp factors Instead, we find that Sp3 represses Sp1-mediated activation in a linear dose-dependent manner A mutant of Sp3 lacking the DNA binding domain does not affect activation by Sp1, suggesting that the inhibition is most likely due to the competition with Sp1 for their common binding sites To determine if any structurally similar domain of Sp3 is able to replace partially homologous domains of Sp1, we have generated chimeric proteins and tested their activation characteristics in gene transfer experiments It appears that neither the glutamine-rich domains A and B nor the D domain of Sp1 can be replaced by the homologous regions of Sp3 Our results suggest that Sp3 is an inhibitory member of the Sp family
TL;DR: It is suggested that the catalytic activity of the Fet3 protein is required for cellular iron accumulation, similar to that of the blue multicopper oxidoreductases.
TL;DR: In this paper, the authors describe mutant A. thaliana plants that contain lesions in a single accelerated cell death (ACD) gene called ACD2 and that bypass the need for pathogen exposure to induce the hypersensitive response (HR) to pathogens.
TL;DR: It is reported here that this phenotype is caused by mutation of the zebrafish homologue of the T gene, and expression of mRNA in mutants, but not in wild types, is greatly reduced along the dorsal midline where the notochord normally forms.
Abstract: The mouse T (Brachyury) gene is required for normal mesoderm development and the extension of the body axis. Recently, two mutant alleles of a zebrafish gene, no tail (ntl), have been isolated (Halpern, M. E., Ho., R. K., Walker, C. and Kimmel, C. B. (1993) Cell 75, 99–111). ntl mutant embryos resemble mouse T/T mutant embryos in that they lack a differentiated notochord and the caudal region of their bodies. We report here that this phenotype is caused by mutation of the zebrafish homologue of the T gene. While ntl embryos express mutant mRNA, they show no nuclear protein product. Later, expression of mRNA in mutants, but not in wild types, is greatly reduced along the dorsal midline where the notochord normally forms. This suggests that the protein is required for maintaining transcription of its own gene.
TL;DR: These results ascribe an essential function for the RXR alpha gene in embryonic development and provide the first evidence of a requirement for RXR in one of its predicted hormone response pathways.
Abstract: We have established a targeted loss-of-function mutation in the RXR alpha gene in the mouse germ line that results in embryonic lethality between E13.5 and E16.5 when bred to homozygosity. The major defect responsible for lethality is hypoplastic development of the ventricular chambers of the heart, which is manifest as a grossly thinned ventricular wall with concurrent defects in ventricular septation. This phenotype is identical to a subset of the effects of embryonic vitamin A deficiency and, therefore, establishes RXR alpha as a genetic component of the vitamin A signaling pathway in cardiac morphogenesis. The cardiac outflow tracts and associated vessels, which are populated by derivatives of the neural crest and which are also sensitive to vitamin A deficiency, are normal in homozygous embryos, indicating the genetic independence of ventricular chamber development. Hepatic differentiation was dramatically but transiently retarded yet is histologically and morphologically normal. These results ascribe an essential function for the RXR alpha gene in embryonic development and provide the first evidence of a requirement for RXR in one of its predicted hormone response pathways.
TL;DR: In this paper, the authors reported the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5.
Abstract: Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.
TL;DR: For mutants such as G37R, either surprisingly modest losses in activity (involving only the mutant subunit) can yield motor neuron death, or alternatively, mutant SOD1 may acquire properties that injure motor neurons by one or more mechanisms unrelated to the metabolism of oxygen radicals.
Abstract: Familial amyotrophic lateral sclerosis (FALS) has been linked to mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1). Assay by transient expression in primate cells of six FALS mutant enzymes revealed a continuum of enzymatic activity bounded by the enzyme carrying the mutation Gly-85-->Arg, which was inactive, and mutant enzyme G37R carrying the Gly-37-->Arg change, which retained full specific activity but displayed a 2-fold reduction in polypeptide stability. The G37R mutant displayed similar properties in transformed lymphocytes from an individual heterozygous for the G37R and wild-type SOD1 genes; heterodimeric enzymes composed of mutant and wild-type subunits were detected, but there was no measurable diminution in the stability and activity of the wild-type subunits. Thus, for mutants such as G37R, either surprisingly modest losses in activity (involving only the mutant subunit) can yield motor neuron death, or alternatively, mutant SOD1 may acquire properties that injure motor neurons by one or more mechanisms unrelated to the metabolism of oxygen radicals.
TL;DR: The whn gene, designated whn, encodes a new member of the winged-helix domain family of transcription factors and is disrupted on mouse nu and rat rnuN alleles, the first member of this class of genes to be implicated in a specific developmental defect in vertebrates.
Abstract: Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 (refs 3, 4) within a region of < 1 megabase. Here we show that one of the genes from this critical region, designated whn, encodes a new member of the winged-helix domain family of transcription factors, and that it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates.
TL;DR: These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice.
Abstract: The neurofibromatosis (NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nf1 locus. Although heterozygous mutant mice, aged up to 10 months, have not exhibited any obvious abnormalities, homozygous mutant embryos die in utero. Embryonic death is likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice
TL;DR: It is proposed that mGluR1 is not "in line" in LTP production, but rather modulates the plasticity process, and hence affects context-specific associative learning.
TL;DR: This work has indicated that the ABI3 protein directly participates in the regulation of several developmental programs and that multiple regulatory pathways can lead to the simultaneous expression of distinct mRNA markers in seed.
Abstract: The accumulation kinetics of 18 mRNAs were characterized during Arabidopsis silique development. These marker mRNAs could be grouped in distinct classes according to their coordinate temporal expression in the wild type and provided a basis for further characterization of the corresponding regulatory pathways. The abscisic acid (ABA)-insensitive abi3-4 mutation modified the expression pattern of several but not all members of each of these wild-type temporal mRNA classes. This indicates that the ABI3 protein directly participates in the regulation of several developmental programs and that multiple regulatory pathways can lead to the simultaneous expression of distinct mRNA markers. The ABI3 gene is specifically expressed in seed, but ectopic expression of ABI3 conferred the ability to accumulate several seed-specific mRNA markers in response to ABA in transgenic plantlets. This suggested that expression of these marker mRNAs might be controlled by an ABI3-dependent and ABA-dependent pathway(s) in seed. However, characterization of the ABA-biosynthetic aba mutant revealed that the accumulation of these mRNAs is not correlated to the ABA content of seed. A possible means of regulating gene expression by developmental variations in ABA sensitivity is apparently not attributable to variations in ABI3 cellular abundance. The total content of ABI3 protein per seed markedly increased at certain developmental stages, but this augmentation appears to result primarily from the simultaneous multiplication of embryonic cells. Our current findings are discussed in relation to their general implications for the mechanisms controlling gene expression programs in seed.
TL;DR: Cp-iap and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA.
Abstract: Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA.
TL;DR: It is demonstrated that a missense mutation (Glu128Ala) in this gene causes familial hypocalcaemia in affected members of one family, and this extracellular domain mutation increases the receptor's activity at low Ca2+ concentrations, causing hypocalcemia in patients heterozygous for such a mutation.
Abstract: Defects in the human Ca(2+)-sensing receptor gene have recently been shown to cause familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. We now demonstrate that a missense mutation (Glu128Ala) in this gene causes familial hypocalcaemia in affected members of one family. Xenopus oocytes expressing the mutant receptor exhibit a larger increase in inositol 1,4,5-triphosphate in response to Ca2+ than oocytes expressing the wild-type receptor. We conclude that this extracellular domain mutation increases the receptor's activity at low Ca2+ concentrations, causing hypocalcaemia in patients heterozygous for such a mutation.
TL;DR: The formation ofClfA gene, when introduced into the chromosome of the mutant strains, fuily compiemented the ciumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen‐coated PMMA.
Abstract: Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fully complemented the clumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen-coated PMMA. In addition, the cloned clfA gene on a shuttle plasmid allowed the weakly clumping strain 8325-4 to form clumps with the same avidity as the wild-type strain Newman and also significantly enhanced the adherence of 8325-4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encodes a fibrinogen-binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the cell surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall-associated proteins. Significant homology was found between the ClfA protein and the fibronectin-binding proteins of S. aureus, particularly in the N- and C-termini.