scholarly journals Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster.

1993 ◽  
Vol 123 (6) ◽  
pp. 1797-1809 ◽  
Author(s):  
J K Lee ◽  
R S Coyne ◽  
R R Dubreuil ◽  
L S Goldstein ◽  
D Branton
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Cell Shape ◽  
Germ Line ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
P Element ◽  
Cell Contact ◽  
Base Pair Deletion ◽  
Alpha Spectrin

We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin-deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues.

scholarly journals A rare large duplication of MLH1 identified in Lynch syndrome

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Abhishek Kumar ◽  
Nagarajan Paramasivam ◽  
Obul Reddy Bandapalli ◽  
Matthias Schlesner ◽  
Tianhui Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lynch Syndrome ◽  
Splice Site ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Splice Donor Site ◽  
Laboratory Diagnostics ◽  
Mmr Genes ◽  
Base Pair Deletion ◽  
Splice Site Variant

Abstract Background The most frequently identified strong cancer predisposition mutations for colorectal cancer (CRC) are those in the mismatch repair (MMR) genes in Lynch syndrome. Laboratory diagnostics include testing tumors for immunohistochemical staining (IHC) of the Lynch syndrome-associated DNA MMR proteins and/or for microsatellite instability (MSI) followed by sequencing or other techniques, such as denaturing high performance liquid chromatography (DHPLC), to identify the mutation. Methods In an ongoing project focusing on finding Mendelian cancer syndromes we applied whole-exome/whole-genome sequencing (WES/WGS) to 19 CRC families. Results Three families were identified with a pathogenic/likely pathogenic germline variant in a MMR gene that had previously tested negative in DHPLC gene variant screening. All families had a history of CRC in several family members across multiple generations. Tumor analysis showed loss of the MMR protein IHC staining corresponding to the mutated genes, as well as MSI. In family A, a structural variant, a duplication of exons 4 to 13, was identified in MLH1. The duplication was predicted to lead to a frameshift at amino acid 520 and a premature stop codon at amino acid 539. In family B, a 1 base pair deletion was found in MLH1, resulting in a frameshift and a stop codon at amino acid 491. In family C, we identified a splice site variant in MSH2, which was predicted to lead loss of a splice donor site. Conclusions We identified altogether three pathogenic/likely pathogenic variants in the MMR genes in three of the 19 sequenced families. The MLH1 variants, a duplication of exons 4 to 13 and a frameshift variant, were novel, based on the InSiGHT and ClinVar databases; the MSH2 splice site variant was reported by a single submitter in ClinVar. As a variant class, duplications have rarely been reported in the MMR gene literature, particularly those covering several exons.

scholarly journals CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. 665-672 ◽  
Author(s):  
Samantha A M Young ◽  
Haruhiko Miyata ◽  
Yuhkoh Satouh ◽  
Masanaga Muto ◽  
Martin R Larsen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Male Fertility ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Cytoplasmic Tail ◽  
Binding Partner ◽  
C Terminus ◽  
Manipulation System

IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm–egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit. However, the cytoplasmic tail of IZUMO1 in many species is highly variable, ranging from 55 to one amino acid. Therefore, to understand the role of the cytoplasmic tail of IZUMO1 in mouse, we utilised the gene manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression level of IZUMO1, it is dispensable for fertilisation in the mouse.

scholarly journals Identification of the genomic mutation in Epha4rb-2J/rb-2J mice

Genome ◽  
2016 ◽  
Vol 59 (7) ◽  
pp. 439-448 ◽  
Author(s):  
Siti W. Mohd-Zin ◽  
Nor-Linda Abdullah ◽  
Aminah Abdullah ◽  
Nicholas D.E. Greene ◽  
Pike-See Cheah ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Stop Codon ◽  
Cell Signalling ◽  
Experimental Studies ◽  
Premature Stop Codon ◽  
Mouse Mutant ◽  
Functional Studies ◽  
Base Pair Deletion ◽  
Numerous Cell

The EphA4 receptor tyrosine kinase is involved in numerous cell-signalling activities during embryonic development. EphA4 has the ability to bind to both types of ephrin ligands, the ephrinAs and ephrinBs. The C57BL/6J-Epha4rb-2J/GrsrJ strain, denoted Epha4rb-2J/rb-2J, is a spontaneous mouse mutant that arose at The Jackson Laboratory. These mutants exhibited a synchronous hind limb locomotion defect or “hopping gait” phenotype, which is also characteristic of EphA4 null mice. Genetic complementation experiments suggested that Epha4rb-2J corresponds to an allele of EphA4, but details of the genomic defect in this mouse mutant are currently unavailable. We found a single base-pair deletion in exon 9 resulting in a frame shift mutation that subsequently resulted in a premature stop codon. Analysis of the predicted structure of the truncated protein suggests that both the kinase and sterile α motif (SAM) domains are absent. Definitive determination of genotype is needed for experimental studies of mice carrying the Epha4rb-2J allele, and we have also developed a method to ease detection of the mutation through RFLP. Eph-ephrin family members are reportedly expressed as numerous isoforms. Hence, delineation of the specific mutation in EphA4 in this strain is important for further functional studies, such as protein–protein interactions, immunostaining and gene compensatory studies, investigating the mechanism underlying the effects of altered function of Eph family of receptor tyrosine kinases on phenotype.

scholarly journals In VivoEffects of Leptin-Related Synthetic Peptides on Body Weight and Food Intake in Female ob/obMice: Localization of Leptin Activity to Domains Between Amino Acid Residues 106–140*

Endocrinology ◽  
1997 ◽  
Vol 138 (4) ◽  
pp. 1413-1418 ◽  
Author(s):  
Patricia Grasso ◽  
Matthew C. Leinung ◽  
Stacy P. Ingher ◽  
Daniel W. Lee
Keyword(s):  
Body Weight ◽  
Weight Gain ◽  
Amino Acid ◽  
Food Intake ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Residues ◽  
Inactive Form ◽  
In Vivo Effects

Abstract In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136–150), LEP-(146–160), or LEP-(156–167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106–120) or LEP-(126–140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116–130) were 3.4% lighter. Food intake by mice receiving LEP-(106–120), LEP-(116–130), or LEP-(126–140), but not by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106–140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505 ◽  
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Abstract Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3181-3189 ◽  
Author(s):  
Babette Gwynn ◽  
Jose A. Martina ◽  
Juan S. Bonifacino ◽  
Elena V. Sviderskaya ◽  
M. Lynn Lamoreux ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Trafficking ◽  
Protein Complexes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Identity ◽  
Organelle Biogenesis ◽  
Hermansky Pudlak Syndrome ◽  
Human Orthologue ◽  
Lysosome Related Organelles

Abstract Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

scholarly journals Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 988-992 ◽  
Author(s):  
Stephen T. Oh ◽  
Erin F. Simonds ◽  
Carol Jones ◽  
Matthew B. Hale ◽  
Yury Goltsev ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Stop Codon ◽  
Myeloproliferative Neoplasms ◽  
Premature Stop Codon ◽  
Adaptor Protein ◽  
Janus Kinase ◽  
Negative Regulator ◽  
Negative Feedback Regulation ◽  
Base Pair Deletion ◽  
Stat Signaling

Abstract Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

A new ferrochelatase mutation combined with low expression alleles in a Japanese patient with erythropoietic protoporphyria

2002 ◽  
Vol 102 (5) ◽  
pp. 501-506 ◽  
Author(s):  
Yumiko YASUI ◽  
Shikibu MURANAKA ◽  
Tsuyoshi TAHARA ◽  
Ryo SHIMIZU ◽  
Sonoko WATANABE ◽  
...  
Keyword(s):  
Base Pair ◽  
Japanese Patient ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Position ◽  
Molecular Defect ◽  
Wild Type ◽  
Erythropoietic Protoporphyria ◽  
Base Pair Deletion ◽  
Low Expression

We investigated the molecular defect of the ferrochelatase gene in a Japanese patient with erythropoietic protoporphyria (EPP), and identified a novel 16 base pair (574-589) deletion within exon 5. This deletion resulted in a frame-shift mutation and created a premature stop codon at amino acid position 198. The same molecular defect was also identified in his mother and a brother who had symptomatic EPP, but not in his father who was asymptomatic. The subjects with EPP were homozygous for the low expression haplotype, while his father was heterozygous for this haplotype. These results indicate that the combination of a 16 base pair deletion and low expression of the wild-type allelic variant is responsible for EPP in this pedigree.

Novel Identification of a Four-base-pair Deletion Mutation in PITX2 in a Rieger Syndrome Family

2003 ◽  
Vol 82 (12) ◽  
pp. 1008-1012 ◽  
Author(s):  
Y. Wang ◽  
H. Zhao ◽  
X. Zhang ◽  
H. Feng
Keyword(s):  
Base Pair ◽  
Stop Codon ◽  
Critical Role ◽  
Premature Stop Codon ◽  
Chinese Family ◽  
Loss Of Function ◽  
Rieger Syndrome ◽  
Mutational Screening ◽  
Base Pair Deletion ◽  
Pitx2 Gene

Rieger syndrome is one of the most serious causes of tooth agenesis. Mutations in the PITX2, FOXC1, and PAX6 genes have been associated with Rieger syndrome. We have studied a three-generation Chinese family affected with Rieger syndrome and showing prominent dental abnormalities. Mutational screening and sequence analysis of the PITX2 gene revealed a previously unidentified four-base-pair deletion of nucleotides 717-720 in exon 5 in all affected members. The mutation causes a frame shift after Thr44, the 7th amino acid of the homeo-domain, and introduces a premature stop codon in the gene sequence. This deletion is the first unquestionable loss-of-function mutation, deleting all the functionally important parts of the protein. Our novel discovery indicates that the oligodontia and other phenotypes of Rieger syndrome observed in this family are due to this PITX2 mutation, and these data further support the critical role of PIXT2 in tooth morphogenesis.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

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