Reversion analysis of dynein intermediate chain function

1993 ◽  
Vol 105 (4) ◽  
pp. 1069-1078 ◽  
Author(s):  
D.R. Mitchell ◽  
Y. Kang
Keyword(s):  
Sequence Analysis ◽  
Stop Codon ◽  
Function Analysis ◽  
Beat Frequency ◽  
Immunoblot Analysis ◽  
Wild Type ◽  
Direct Role ◽  
Reading Frame ◽  
Frame Shift ◽  
Intermediate Chain

The ODA6 locus of Chlamydomonas reinhardtii encodes a 70 kDa intermediate chain protein of the flagellar outer row dynein ATPase, and mutations at this locus prevent assembly of the entire outer row dynein arm complex. To initiate a structure-function analysis of the 70 kDa protein, we used transformation with chimeric mutant/wild-type genes to localize the defect in one assembly mutation, oda6-95. Sequence analysis revealed a frame-shift mutation in codon 53, which is followed by a stop codon after 13 amino acids in the new reading frame. By selecting intragenic pseudorevertants of this mutation we obtained 11 new oda6 alleles. Many of these pseudorevertants encode intermediate chain proteins that permit assembly of outer row arms but do not restore full wild-type motility. Revertant strains fall into two phenotypic classes, one with average beat frequencies of 54 Hz (similar to wild type) and one with average frequencies of 27 Hz (compared with 24 Hz for oda6-95) during normal forward swimming. Low beat frequency strains also display abnormalities during photophobic reversal (symmetric waveform). Amplification and sequence analysis of revertant alleles indicated that each reversion caused a second frame-shift, within a 115 nt interval, which restored the original reading frame, and that phenotypic severity was related to both direction (5′ or 3′) and distance between the original mutation and the reversion event. On the basis of immunoblot analysis of outer arm proteins, we conclude that revertant motility defects do not correlate with deficits in assembly of a specific dynein heavy chain or intermediate chain polypeptide, and electron microscopy confirms that revertants have normal outer arm structures. These results suggest that the 70 kDa intermediate chain plays a direct role in outer arm function distinct from its role in the assembly process.

A Frame Shift-Induced Stop Codon Causes Hemophilia a in Sheep.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3378-3378 ◽  
Author(s):  
Chad Sanada ◽  
Josh A Wood ◽  
Wansheng Liu ◽  
Jay Nelson Lozier ◽  
Graca Almeida-Porada ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Stop Codon ◽  
Coagulation Disorder ◽  
Large Animal ◽  
Wild Type ◽  
Coding Sequence ◽  
Frame Shift ◽  
A1 Domain ◽  
High Degree

Abstract Hemophilia A (HA), or Factor VIII (FVIII) deficiency, is the most common severe hereditary coagulation disorder, affecting 1 in 5000 males. We have successfully re-established and characterized a line of sheep with a bleeding disorder that closely mimics severe human HA. These animals have almost non-existent levels of FVIIIc and an extremely prolonged PTT, with normal levels of platelets, fibrinogne, FVII, FIX, and vWF. All animals that survived birth have exhibited prolonged tail and nail cuticle bleeding, multiple episodes of severe spontaneous bleeding including hemarthroses, muscle hematomas, and hematuria, all of which have responded to recombinant human FVIII. Thus far, low-titer inhibitors to human FVIII have been detected in 3 treated animals, further validating the clinical relevance of this model. Unfortunately, almost nothing is known about the sheep FVIII gene or about the nature of the mutation causing HA in these animals. We thus undertook to characterize the normal sheep FVIII mRNA and elucidate the nature of the hemophilia-inducing mutation. RT-PCR of mRNA from the spleen of normal sheep, followed by overlapping sequence analysis allowed us to walk along the mRNA and obtain the sequence of the complete coding sequence for factor VIII. The coding sequence for ovine FVIII is 6765 nucleotides, which is translated into a protein consisting of 2254 amino acids (a.a.). BLAST alignment of this sequence to that of human FVIII at the a.a. level revealed a high degree of identity in all regions except the B domain (which in humans is known to be dispensable for clotting activity). Specifically, the A1 domain (ovine a.a. 1–332) showed 81% identity, A2 (a.a. 333–709) 88%, A3 (a.a. 1596–1924) 87%, C1 (a.a. 1925–2074) 90%, and C2 (a.a. 2075–2254) 86%, while the B domain (a.a. 710–1595) exhibited only 47% identity. A comparison of these sequences taking into consideration conservative a.a. changes that maintain the chemical properties of sheep and human FVIII proteins indicated even higher levels of homology, with the A1 domain exhibiting 87% positivity, A2 94%, A3 93%, C1 94%, and C2 93%. Even with this approach, however, there was still very low conservation (59%) of the B domain between the two species. Analysis of mRNA isolated from the spleen of a deceased HA sheep revealed several conservative point mutations in the hemophiliac and identified 11bp in exon 14 that differed between the wild type and the hemophiliac. Importantly, this difference included a frame-shift that introduced a premature stop codon in exon 14, as is seen in some human patients with HA. Using a PCR-based RFLP analysis, we can unequivocally distinguish sheep that are wild-type, heterozygous, or homozygous for the HA mutation, and thus confirm the genotype of all 26 existing carriers and 8 hemophiliac sheep. This PCR-based assay will greatly facilitate studies using these sheep, since it is now possible to either screen embryos prior to implantation or screen fetuses in utero using a small volume of blood or amniotic fluid, as is done for prenatal diagnosis of human genetic diseases. These studies thus defined the molecular basis for the HA in these sheep that are a valuable model of human disease. Given the close physiologic similarity between sheep and humans, the high degree of identity in their FVIII protein, and the decades of experience using the sheep to study both normal physiology and a wide array of diseases, we hope that this large animal model will contribute to a better understanding of HA and the development of novel treatments such as stem cell transplantation and gene therapy-based approaches that can directly translate to human patients with hemophilia.

scholarly journals Three new dominant C1 suppressor alleles in Zea mays

1998 ◽  
Vol 71 (2) ◽  
pp. 127-132 ◽  
Author(s):  
TATJANA SINGER ◽  
ALFONS GIERL ◽  
PETER A. PETERSON
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
Stop Codon ◽  
Suppressive Effect ◽  
Wild Type ◽  
Activation Domain ◽  
Reading Frame ◽  
Transcriptional Activation Domain ◽  
Imprecise Excision ◽  
Carboxy Terminal

Three new dominant suppressor mutations of the C1 transcription regulator gene in maize – C1-IΔ1, C1-IΔ2 and C1-IΔ3 – are described that suppress anthocyanin colouration in kernels similar to the function of the C1-I standard inhibitor. The C1-IΔ mutations were induced by imprecise excision of an En/Spm transposon in the third exon of the C1 gene. These transposon footprints cause a frameshift in the C1 open reading frame that leads to truncated proteins due to an early stop codon 30 amino acids upstream of the wild-type C1 protein. Therefore, the C1-IΔ gene products lack the carboxy-terminal transcriptional activation domain of C1. The C1-I standard allele also lacks this domain and in addition differs in 17 amino acids from the wild-type C1 allele. The new C1-IΔ alleles provide evidence that deletion of the carboxy-terminal activation domain alone is sufficient to generate a dominant suppressive effect on the function of wild-type C1.

scholarly journals Role of the E1∧E4 Protein in the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 31

2005 ◽  
Vol 79 (11) ◽  
pp. 6732-6740 ◽  
Author(s):  
Regina Wilson ◽  
Frauke Fehrmann ◽  
Laimonis A. Laimins
Keyword(s):  
Life Cycle ◽  
Stop Codon ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Wild Type ◽  
Late Gene Expression ◽  
Semisolid Medium ◽  
Genome Amplification ◽  
Reading Frame

ABSTRACT The most highly expressed protein in the productive life cycle of human papillomaviruses (HPVs) is E1∧E4, but its function is not well understood. To investigate the role of E1∧E4, we undertook a genetic analysis in the context of the complete HPV type 31 (HPV31) genome. A mutant HPV31 genome (E4M9) was constructed that contained a stop codon in the E4 open reading frame at amino acid 9 and was silent in the overlapping E2 coding sequence. Wild-type and mutant genomes were transfected into normal human foreskin keratinocytes (HFKs) and selected for drug resistance, and pooled cultures were examined for effects of E1∧E4 on viral functions. Southern blot analyses of transfected HFKs demonstrated that cells carrying the E4M9 mutant genomes were maintained as episomes at copy numbers similar to those in keratinocytes transfected with wild-type HPV31. Both sets of cells grew at similar rates, exhibited comparable extensions of life spans, and had equivalent levels of early transcripts. Following suspension of the cells in a semisolid medium, differentiation-dependent genome amplification and late gene expression were significantly decreased in cells maintaining the E4M9 mutant genome compared to those with wild-type HPV31. One explanation for these effects could be a reduction in the number of cells harboring mutant genomes that enter S phase upon differentiation. An analysis of cells containing E4M9 mutant genomes in organotypic raft cultures indicated a reduction in bromodeoxyuridine incorporation in differentiated suprabasal cells compared to that seen in wild-type rafts. Our results indicate that the HPV31 E1∧E4 protein plays a significant role in promoting HPV genome amplification and S phase maintenance during differentiation.

scholarly journals Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.

1995 ◽  
Vol 128 (3) ◽  
pp. 383-392 ◽  
Author(s):  
B Drees ◽  
C Brown ◽  
B G Barrell ◽  
A Bretscher
Keyword(s):  
Sequence Analysis ◽  
Wild Type ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Over Expression ◽  
Sequence Identity ◽  
Bona Fide ◽  
Essential Function

Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.

scholarly journals Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains.

1991 ◽  
Vol 113 (2) ◽  
pp. 381-391 ◽  
Author(s):  
J E Collins ◽  
P K Legan ◽  
T P Kenny ◽  
J MacGarvie ◽  
J L Holton ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Adhesion Molecules ◽  
Sequence Data ◽  
Stop Codon ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Reading Frame ◽  
Calcium Dependent ◽  
Partial Clone

Desmosomal glycoproteins 2 and 3 (dg2 and 3) or desmocollins have been implicated in desmosome adhesion. We have obtained a 5.0-kb-long clone for dg3 from a bovine nasal epidermal lambda gt11 cDNA library. Sequence analysis of this clone reveals an open reading frame of 2,517 bases encoding a polypeptide of 839 amino acids. The sequence consists of a signal peptide of 28 amino acids, a precursor sequence of 104 amino acids, and a mature protein of 707 amino acids. The latter has the characteristics of a transmembrane glycoprotein with an extracellular domain of 550 amino acids and a cytoplasmic domain of 122 amino acids. The sequence of a partial clone from the same library shows that dg2 has an alternative COOH terminus that is extended by 54 amino acids. Genomic DNA sequence data show that this arises by splicing out of a 46-bp exon that encodes the COOH-terminal 11 amino acids of dg3 and contains an in-frame stop codon. The extracellular domain of dg3 shows 39.4% protein sequence identity with bovine N-cadherin and 28.4% identity with the other major desmosomal glycoprotein, dg1, or desmoglein. The cytoplasmic domain of dg3 and the partial cytoplasmic domain of dg2 show 23 and 24% identity with bovine N-cadherin, respectively. The results support our previous model for the transmembrane organization of dg2 and 3 (Parrish, E.P., J.E. Marston, D.L. Mattey, H.R. Measures, R. Venning, and D.R. Garrod. 1990. J. Cell Sci. 96:239-248; Holton, J.L., T.P. Kenny, P.K. Legan, J.E. Collins, J.N. Keen, R. Sharma, and D.R. Garrod. 1990. J. Cell Sci. 97:239-246). They suggest that these glycoproteins are specialized for calcium-dependent adhesion in their extracellular domains and, cytoplasmically, for the molecular interactions involved in desmosome plaque formation. Moreover this represents the first example of alternative splicing within the cadherin family of cell adhesion molecules.

scholarly journals Conundrum of the Lack of Defective RNAs (dRNAs) Associated with Tobamovirus Infections: dRNAs That Can Move Are Not Replicated by the Wild-Type Virus; dRNAs That Are Replicated by the Wild-Type Virus Do Not Move

2001 ◽  
Vol 75 (12) ◽  
pp. 5518-5525 ◽  
Author(s):  
Elisabeth Knapp ◽  
William O. Dawson ◽  
Dennis J. Lewandowski
Keyword(s):  
De Novo ◽  
Stop Codon ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
In Planta ◽  
Reading Frame ◽  
Defective Rnas ◽  
Two Factors ◽  
Associated Proteins

ABSTRACT Two classes of artificially constructed defective RNAs (dRNAs) ofTobacco mosaic virus (TMV) were examined in planta with helper viruses that expressed one (183 kDa) or both (126 and 183 kDa) of the replicase-associated proteins. The first class of artificially constructed dRNAs had the helicase and polymerase (POL) domains deleted; the second had an intact 126-kDa protein open reading frame (ORF). Despite extremely high levels of replication in protoplasts, the first class of dRNAs did not accumulate in plants. The dRNAs with an intact 126-kDa protein ORF were replicated at moderate levels in protoplasts and in planta when supported by a TMV mutant that expressed the 183-kDa protein but not the 126-kDa protein (183F). These dRNAs were not supported by helper viruses expressing both replicase-associated proteins. De novo dRNAs were generated in plants infected by 183F but not in plants infected with virus with the wild-type replicase. These novel dRNAs each contained a new stop codon near the location of the wild-type stop codon for the 126-kDa protein and had most of the POL domain deleted. The fact that only dRNAs that contained a complete 126-kDa protein ORF moved systemically suggests that expression of a functional 126-kDa protein or the presence of certain sequences and/or structures within this ORF is required for movement of dRNAs. At least two factors may contribute to the lack of naturally occurring dRNAs in association with wild-type TMV infections: an inability of TMV to support dRNAs that can move in plants and the inability of dRNAs that can be replicated by TMV to move in plants.

scholarly journals Bacteriophage P4 sut1: a mutation suppressing transcription termination

2007 ◽  
Vol 88 (3) ◽  
pp. 1041-1047
Author(s):  
Susanna Terzano ◽  
Ilaria Oliva ◽  
Francesca Forti ◽  
Claudia Sala ◽  
Francesca Magnoni ◽  
...  
Keyword(s):  
Stop Codon ◽  
Transcription Termination ◽  
Spontaneous Mutation ◽  
Wild Type ◽  
Stem Loop ◽  
Reading Frame ◽  
E Coli ◽  
Stem Loop Structure ◽  
Bacteriophage P4 ◽  
Sense Codon

In the Escherichia coli satellite phage P4, transcription starting from PLE is prevalently controlled via premature termination at several termination sites. We identified a spontaneous mutation, P4 sut1 (suppression of termination), in the natural stop codon of P4 orf151 that, by elongating translation, suppresses transcription termination at the downstream t151 site. Both the translational and the transcriptional profile of P4 sut1 differed from those of P4 wild-type. First of all, P4 sut1 did not express Orf151, but a higher molecular mass protein, compatible with the 303 codon open reading frame generated by the fusion of orf151, cnr and the intervening 138 nt. Moreover, after infection of E. coli, the mutant expressed a very low amount of the 1.3 and 1.7 kb transcripts originating at PLE and PLL promoters, respectively, and terminating at the intracistronic t151 site, whereas correspondingly higher amounts of the 4.1 and 4.5 kb RNAs arising from the same promoters and covering the entire operon were detected. Thus the sut1 mutation converts a natural stop codon into a sense codon, suppresses a natural intracistronic termination site and leads to overexpression of the downstream cnr and α genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t151 transcription termination site within the 7633–7361 region between orf151 and gene cnr. A potential stem–loop structure, resembling the structure of a Rho-independent termination site, was predicted by mfold sequence analysis at 7414–7385.

scholarly journals Spontaneous mutation at the mtr locus in neurospora: the molecular spectrum in wild-type and a mutator strain.

Genetics ◽  
1994 ◽  
Vol 138 (1) ◽  
pp. 61-74
Author(s):  
D Dillon ◽  
D Stadler
Keyword(s):  
Sequence Analysis ◽  
Spontaneous Mutation ◽  
Molecular Spectrum ◽  
Open Reading Frame ◽  
Wild Type ◽  
Spontaneous Mutation Rate ◽  
Reading Frame ◽  
Mutator Strain ◽  
Oak Ridge ◽  
Base Substitutions

Abstract Sequence analysis of 34 mtr mutations has yielded the first molecular spectrum of spontaneous mutants in Neurospora crassa. The great majority of the mutations are base substitutions (48%) or deletions (35%). In addition, sequence analysis of the entire mtr region, including the 1472-base pair open reading frame and 1205 base pairs of flanking DNA, was performed in both the Oak Ridge and Mauriceville strains of Neurospora, which are known to be divergent at the DNA level. Sixteen sequence differences between these two strains have been found in the mtr region, with 13 of these in DNA flanking the open reading frame. The differences consisted of base substitutions and small frameshifts at monotonic runs. This set of sequence differences has allowed a comparison of mutations in unselected DNA to those mutations that produce a phenotypic signal. We have isolated a mutator strain (mut-1) of Neurospora in which the spontaneous mutation rate at various loci is as much as 80-fold higher than in the non-mutator (wild type). Twenty-one mtr mutations in the mutator background have been sequenced and compared to the non-mutator spectrum, revealing a striking increase in -1 frameshift mutations. These frameshifts occur exclusively within or adjacent to monotonic runs and can be explained by small slippage events during DNA replication. This argues for a role of the mut-1 gene in this process.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

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