A Dictyostelium mutant with severe defects in alpha-actinin: its characterization using cDNA probes and monoclonal antibodies

1988 ◽  
Vol 90 (1) ◽  
pp. 59-71
Author(s):  
M. Schleicher ◽  
A. Noegel ◽  
T. Schwarz ◽  
E. Wallraff ◽  
M. Brink ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polypeptide Chain ◽  
Mutant Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Extract ◽  
Binding Activity ◽  
Partial Purification ◽  
Wild Type ◽  
Coding Region ◽  
In The Wild

Cells of a Dictyostelium discoideum mutant deficient in binding a monoclonal antibody to alpha-actinin have previously been shown to grow and develop similarly to the wild type and to exert unimpaired chemotaxis as well as patching and capping of membrane proteins. Here we show that the normal 3.0 kb message for alpha-actinin is replaced in the mutant by two RNA species of approximately 3.1 and 2.8 kb. The 3.1 kb RNA was recognized by DNA fragments from all parts of the coding region, while the 2.8 kb RNA hybridized to all but a 3′-terminal fragment. Proteins synthesized in the mutant were analysed using four monoclonal antibodies that in the wild type specifically recognize the 95 × 10(3) Mr polypeptide of alpha-actinin. Cleavage mapping indicated that the binding sites of these antibodies are distributed over a region comprising more than half of the alpha-actinin polypeptide chain. In the mutant, three of the antibodies faintly labelled two polypeptides of 95 × 10(3) Mr and 88 × 10(3) Mr; the fourth antibody, which binds closest to one end of the polypeptide chain, faintly labelled the 95 × 10(3) Mr polypeptide only. The 88 × 10(3) Mr polypeptide most probably lacks the C-terminal portion of alpha-actinin. The binding of an antibody that recognized both polypeptides was quantified by a radio-immuno competition assay using wild-type alpha-actinin as a reference. In a mutant cell extract containing total soluble proteins the antibody binding activity was decreased to 1.1% when compared with wild-type extract. After their partial purification and SDS-polyacrylamide gel electrophoresis the mutant 95 × 10(3) Mr and 88 × 10(3) Mr polypeptides were barely detectable as Coomassie Blue-stained bands, indicating that in the mutant not only certain epitopes of alpha-actinin were altered but the entire molecule is almost completely lacking. When the fitness of mutant cells relative to wild type was determined during growth in nutrient medium, a slight disadvantage for the mutant was indicated, by finding selection coefficients between 0.03 and 0.05.

scholarly journals Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

1991 ◽  
Vol 11 (2) ◽  
pp. 1133-1137 ◽  
Author(s):  
Y You ◽  
K Aufderheide ◽  
J Morand ◽  
K Rodkey ◽  
J Forney
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Mutant Cell ◽  
Restriction Pattern ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Coding Region ◽  
Cytoplasmic Factor ◽  
In The Wild ◽  
Dna Restriction

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

scholarly journals Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia

1991 ◽  
Vol 11 (2) ◽  
pp. 1133-1137
Author(s):  
Y You ◽  
K Aufderheide ◽  
J Morand ◽  
K Rodkey ◽  
J Forney
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Mutant Cell ◽  
Restriction Pattern ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Coding Region ◽  
Cytoplasmic Factor ◽  
In The Wild ◽  
Dna Restriction

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

scholarly journals Replication Properties of Clade A/C Chimeric Feline Immunodeficiency Viruses and Evaluation of Infection Kinetics in the Domestic Cat

2008 ◽  
Vol 82 (16) ◽  
pp. 7953-7963 ◽  
Author(s):  
Sohela de Rozìeres ◽  
Jesse Thompson ◽  
Magnus Sundstrom ◽  
Julia Gruber ◽  
Debora S. Stump ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Domestic Cat ◽  
Fine Tuning ◽  
Surface Glycoprotein ◽  
Wild Type ◽  
Coding Region ◽  
Mild Disease ◽  
Clade C ◽  
In The Wild

ABSTRACT Feline immunodeficiency virus (FIV) causes progressive immunodeficiency in domestic cats, with clinical course dependent on virus strain. For example, clade A FIV-PPR is predominantly neurotropic and causes a mild disease in the periphery, whereas clade C FIV-C36 causes fulminant disease with CD4+ T-cell depletion and neutropenia but no significant pathology in the central nervous system. In order to map pathogenic determinants, chimeric viruses were prepared between FIV-C36 and FIV-PPR, with reciprocal exchanges involving (i) the 3′ halves of the viruses, including the Vif, OrfA, and Env genes; (ii) the 5′ end extending from the 5′ long terminal repeat (LTR) to the beginning of the capsid (CA)-coding region; and (iii) the 3′ LTR and Rev2-coding regions. Ex vivo replication rates and in vivo replication and pathologies were then assessed and compared to those of the parental viruses. The results show that FIV-C36 replicates ex vivo and in vivo to levels approximately 20-fold greater than those of FIV-PPR. None of the chimeric FIVs recapitulated the replication rate of FIV-C36, although most replicated to levels similar to those of FIV-PPR. The rates of chloramphenicol acetyltransferase gene transcription driven by the FIV-C36 and FIV-PPR LTRs were identical. Furthermore, the ratios of surface glycoprotein (SU) to capsid protein (CA) in the released particles were essentially the same in the wild-type and chimeric FIVs. Tests were performed in vivo on the wild-type FIVs and chimeras carrying the 3′ half of FIV-C36 or the 3′ LTR and Rev2 regions of FIV-C36 on the PPR background. Both chimeras were infectious in vivo, although replication levels were lower than for the parental viruses. The chimera carrying the 3′ half of FIV-C36 demonstrated an intermediate disease course with a delayed peak viral load but ultimately resulted in significant reductions in neutrophil and CD4+ T cells, suggesting potential adaptation in vivo. Taken together, the findings suggest that the rapid-growth phenotype and pathogenicity of FIV-C36 are the result of evolutionary fine tuning throughout the viral genome, rather than being properties of any one constituent.

MicroRNA signature to predict sensitivity to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 3521-3521
Author(s):  
Federico Cappuzzo ◽  
Andrea Sacconi ◽  
Lorenza Landi ◽  
Francesca Biagioni ◽  
Vienna Ludovini ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Messenger Rna ◽  
Validation Cohort ◽  
Progression Free Survival ◽  
Wild Type ◽  
Braf Mutations ◽  
Small Noncoding Rnas ◽  
Mirna Array ◽  
High Signature ◽  
In The Wild

3521 Background: MicroRNAs (MiRNAs) are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts, usually resulting in gene silencing. Microarray technology is a powerful high-throughput tool capable of monitoring the expression of thousands of small noncoding RNAs at once. In the present study we aimed to investigate whether MiRNA signature was predictive for sensitivity to anti-EGFR monoclonal antibodies in mCRC. Methods: A total of 183 mCRC from two independent cohorts (cohort 1: 74 cases; validation cohort: 109 cases) treated with cetuximab/panitumumab either alone (n=19) or in combination with chemotherapy (n=164) were included onto the study. MiRNA arrays were analysed using Agilent’s miRNA platform. Results: MiRNA array analyses identified the cluster miR-99a/Let7c/miR-125b located on 21p11.1 as associated with different outcome to anti-EGFR therapies. In the first cohort, individuals with high signature (n=25, 33.8%) had a significantly longer progression free survival (PFS, 6.1 versus 2.3 months, p=0.01, HR=0.42) and overall survival (OS, 29.8 versus 7.0 months, p=0.04, HR=0.39) than patients with low signature (n=25, 33.8%). Similar results were observed in the validation cohort. Patients with high signature (n=60, 32.8%) had a significantly longer PFS and longer OS than individuals with low signature (PFS 8.2 versus 4.2 months, p=0.04, HR=0.52; OS 12.8 versus 6.8 months, p=0.09, HR=0.65). To further assess the potential confounding effect of KRAS and BRAF mutations, we analyzed the outcome of patients with high and low signature in the 75 cases with KRAS or BRAF mutation and in 90 cases KRAS and BRAF wild-type. In the wild-type population, high signature patients (n=29, 32.2%) had a significantly longer PFS (8.8 versus 4 months, p=0.002, HR:0.46) and longer OS (17.7 versus 10 months, p=0.015, HR=0.62) than low signature individuals, with no difference in the KRAS or BRAF mutated patients. Conclusions: MiR-99a/Let7c/miR-125b signature is useful for improving selection of KRAS/BRAF wild-type mCRC patients candidate for anti-EGFR strategies.

scholarly journals The msh2 Gene ofSchizosaccharomyces pombe Is Involved in Mismatch Repair, Mating-Type Switching, and Meiotic Chromosome Organization

1999 ◽  
Vol 19 (1) ◽  
pp. 241-250 ◽  
Author(s):  
Claudia Rudolph ◽  
Christophe Kunz ◽  
Sandro Parisi ◽  
Elisabeth Lehmann ◽  
Edgar Hartsuiker ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mating Type ◽  
Meiotic Chromosome ◽  
Binding Activity ◽  
Mutation Rates ◽  
Wild Type ◽  
Cell Extracts ◽  
In The Wild ◽  
Mating Type Switching ◽  
Postmeiotic Segregation

ABSTRACT We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup. msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers. In bandshift assays performed with msh2Δ cell extracts, a general mismatch-binding activity is absent. By complementation assays, we showed that S. pombe msh2 is allelic with the previously identified swi8 andmut3 genes, which are involved in mating-type switching. The swi8-137 mutant has a mutation in the msh2gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain. Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type. Our data show that besides having a function in mismatch repair,S. pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.

scholarly journals A Mutation in the Flanking 5′-TA-3′ Dinucleotide Prevents Excision of an Internal Eliminated Sequence From the Paramecium tetraurelia Genome

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 597-604 ◽  
Author(s):  
Kimberly M Mayer ◽  
James D Forney
Keyword(s):  
Inverted Repeat ◽  
Mutant Cell ◽  
Consensus Sequence ◽  
Terminal Inverted Repeat ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Coding Region ◽  
Discrete Elements ◽  
Additional Information ◽  
Gene Excision

Abstract The germline chromosomes in Paramecium and other ciliated protozoa contain regions of DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5′-TA-3′ dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5′-TA-3′ and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) defective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5′-TA-3′ dinucleotide. Failure to excise IES6649 also revealed a 29-bp IES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal IES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal IES is extremely rare or nonexistent. These results indicate that IESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an IES with its excision partner.

scholarly journals The major intracellular alkaline deoxyribonuclease activities expressed in wild-type and Rec-like mutants of Neurospora crassa

1979 ◽  
Vol 57 (6) ◽  
pp. 889-901 ◽  
Author(s):  
T. Y.-K. Chow ◽  
M. J. Fraser
Keyword(s):  
Molecular Weight ◽  
Neurospora Crassa ◽  
Apparent Molecular Weight ◽  
Partial Purification ◽  
Mitochondrial Enzyme ◽  
Wild Type ◽  
Dna And Rna ◽  
Dependent Endonuclease ◽  
In The Wild

Over 95% of the deoxyribonuclease (DNase) activity of log-phase mycelia of Neurospora crassa is expressed as single-strand (ss) specific endonucleolytic activity. This activity is associated with three nucleases (D1, D2, and D3) which, after partial purification from extracts, express activity with double-strand (ds) DNA as well. All three enzymes also degrade RNA at approximately the same rates that they degrade ss-DNA. D3 has been identified as endo–exonuclease, an enzyme previously shown to have endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA, both of which are inhibited by ATP. D3 is inhibited by ATP, is relatively resistant to p-hydroxymercuribenzoate (PHMB), and sediments with an apparent molecular weight of 75 000. D2 has the properties of the previously described mitochondrial nuclease. It is a relatively unstable Mg2+-dependent endonuclease with no appreciable strand specificity for DNA. In addition, it is not inhibited by ATP and is strongly inhibited by PHMB and by ethylenediamine tetraacetic acid (EDTA). It also sediments with an apparent molecular weight of 75 000. The properties of D1 are quite variable from one preparation to another. Freshly isolated D1 sediments with an apparent molecular weight of 180 000. It often shows some inhibition by ATP, but is relatively resistant to both PHMB and EDTA. However, on 'ageing,' the properties of D1 gradually convert to those of D2 with concomitant decrease in molecular weight, loss of inhibition by ATP, and increase in sensitivities to PHMB and EDTA. The results indicate that D1 is very likely a second form of the mitochondrial enzyme. Evidence was obtained for the presence of protein inhibitor(s) in crude extracts which may account for the masking of the ds-DNase activities of these enzymes in extracts.Two Rec-like mutants of Neurospora (uvs-3, and nuh-4) are deficient mainly in expressed levels of D3, the endo–exonuclease. However, the levels of inactive endo–exonuclease precursor in these two mutants are higher than in the wild type. There may, therefore, be some defect in the conversion of precursor to active enzyme in these two mutants. Another mutant, which is not sensitive to mutagens relative to the wild (nuh-3), has depressed levels of both endo–exonuclease and the mitochondrial enzyme. Nuh-3 has some defect in the conversion of D1 to D2. Proteinases probably play some role in vivo in these enzyme conversions.

scholarly journals Sensitivity of SARS-CoV-2 Variants to Neutralization by Convalescent Sera and a VH3-30 Monoclonal Antibody

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuai Yue ◽  
Zhirong Li ◽  
Yao Lin ◽  
Yang Yang ◽  
Mengqi Yuan ◽  
...  
Keyword(s):  
South Africa ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Binding Activity ◽  
Wild Type ◽  
The Past ◽  
Global Pandemic ◽  
Neutralizing Capacity ◽  
Novel Coronavirus ◽  
Medical Countermeasures

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel coronavirus disease (COVID-19). Though vaccines and neutralizing monoclonal antibodies (mAbs) have been developed to fight COVID-19 in the past year, one major concern is the emergence of SARS-CoV-2 variants of concern (VOCs). Indeed, SARS-CoV-2 VOCs such as B.1.1.7 (UK), B.1.351 (South Africa), P.1 (Brazil), and B.1.617.1 (India) now dominate the pandemic. Herein, we found that binding activity and neutralizing capacity of sera collected from convalescent patients in early 2020 for SARS-CoV-2 VOCs, but not non-VOC variants, were severely blunted. Furthermore, we observed evasion of SARS-CoV-2 VOCs from a VH3-30 mAb 32D4, which was proved to exhibit highly potential neutralization against wild-type (WT) SARS-CoV-2. Thus, these results indicated that SARS-CoV-2 VOCs might be able to spread in convalescent patients and even harbor resistance to medical countermeasures. New interventions against these SARS-CoV-2 VOCs are urgently needed.

scholarly journals The occurrence of long ribosomal transcripts homologous to type I insertions in bobbed mutants of Drosophila melanogaster

1989 ◽  
Vol 54 (2) ◽  
pp. 127-135 ◽  
Author(s):  
Mohamed Makni ◽  
Mohamed Marrakchi ◽  
Nicole Prud'Homme
Keyword(s):  
Drosophila Melanogaster ◽  
Ribosomal Genes ◽  
Type I ◽  
Wild Type ◽  
Coding Region ◽  
Rdna Genes ◽  
Sandwich Hybridization ◽  
Number Of Genes ◽  
In The Wild ◽  
Two Temperatures

SummaryIn Drosophila melanogaster up to two thirds of the rDNA genes contain insertion sequences of two types in the 28S coding region. Comparison of the ribosomal insertion transcripts in the wild type and in two bobbed mutants reared at two temperatures showed that the level of type I transcripts is dependent on both the number of genes with type I insertions in the bobbed loci and the intensity of bobbed phenotype. Importantly, a long transcript of 8·7 kb hybridized to the ribosomal probe, the INS I probe and also to the restriction fragment of the rDNA downstream of the point of insertion was found in one bobbed mutant. This result and also those from sandwich hybridization indicate that some interrupted ribosomal genes are functional.

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