scholarly journals Molecular cloning and sequence analysis of the Chlamydomonas gene coding for radial spoke protein 3: flagellar mutation pf-14 is an ochre allele.

1989 ◽  
Vol 109 (1) ◽  
pp. 235-245 ◽  
Author(s):  
B D Williams ◽  
M A Velleca ◽  
A M Curry ◽  
J L Rosenbaum
Keyword(s):  
Blot Analysis ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Mutant Protein ◽  
Coding Region ◽  
Expression Library ◽  
Translational Initiation ◽  
Radial Spoke ◽  
Cell Biol

Chlamydomonas reinhardtii flagellar motility mutant pf-14 fails to assemble radial spokes because of a deficiency for assembly-competent radial spoke protein 3 (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck. 1981. J. Cell Biol. 88:80-88). Here, we raise an antiserum to protein 3 and use it to isolate the corresponding structural gene from an expression library. Southern blot analysis indicates that the gene is single copy and has not undergone major rearrangement in mutant pf-14 cells. Northern blot analysis suggests that wild-type amounts of an apparently normal 2.3-kb transcript accumulate in mutant cells during flagellar regeneration. When this mutant RNA is hybrid selected and translated in vitro, however, it produces a slightly truncated polypeptide 3 with an altered charge. The mutant protein 3 fails to assemble into pf-14 flagella and is maintained within a cytoplasmic pool of unassembled radial spoke polypeptides, as indicated by immunoblot analysis of proteins from whole cells and isolated axonemes using antisera to several radial spoke polypeptides. Interestingly, amounts of the mutant protein are greatly diminished relative to other spoke components. Complete genomic and cDNA nucleotide sequences were determined, and the pf-14 mutation was identified. It is a C-to-T transition near the 5' end of the protein coding region, which changes codon 21 to the ochre termination signal UAA. The size and charge of the mutant protein, and its reduced levels in cells, suggest that it is produced by relatively inefficient translational initiation as codon 42. The unphosphorylated isoform of radial spoke protein 3 is identified, and the sequence similarities between intervening sequences of the radial spoke protein 3 gene and a conserved intervening sequence of the two Chlamydomonas beta-tubulin genes (Youngblom, J., J. A. Schloss, and C. D. Silflow. 1984. Mol. Cell. Biol. 4:2686-2696) are reported.

scholarly journals Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas.

1986 ◽  
Vol 103 (1) ◽  
pp. 1-11 ◽  
Author(s):  
B D Williams ◽  
D R Mitchell ◽  
J L Rosenbaum
Keyword(s):  
Heavy Chain ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
In Vitro Translation ◽  
Temperature Sensitive ◽  
Expression Library ◽  
Recombinant Phage ◽  
Accumulation Pattern ◽  
Radial Spoke

Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in lambda gt11. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26ts (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybrid-select mRNA from mutant pf-26ts cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26ts mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar alpha-tubulin mRNA.

scholarly journals Identification and functional characterization of a missense mutation in resistin in two patients with severe obesity and insulin resistance

2011 ◽  
Vol 164 (6) ◽  
pp. 927-936 ◽  
Author(s):  
Sigri Beckers ◽  
Fenna de Freitas ◽  
Doreen Zegers ◽  
An Verrijken ◽  
Armand V Peeters ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Functional Characterization ◽  
Mutant Protein ◽  
Morbidly Obese ◽  
Male Body ◽  
Coding Region ◽  
Obese Adults ◽  
Obese Mother

ObjectiveIn this study, we hypothesized that mutations in the resistin encoding gene, RETN, may cause a monogenic form of obesity.Design/methodsWe screened the coding region of RETN in 81 morbidly obese adults, 263 overweight and obese children/adolescents, and 116 healthy lean subjects. In vitro experiments include qPCR, ELISA, and western blot for WT and mutant resistin transfected into 3T3-L1 adipocytes.ResultsMutation analysis identified five sequence variants in our patient populations: 3′-UTR +87 G/A, 3′-UTR +100 A/G, T73T, IV3-61 C/A, and C78S. In our control population, we only found the 3′-UTR +87 G/A variant. We started functional experiments for the C78S mutation that was found in a 20-year-old obese male (body mass index (BMI)=39.7 kg/m2) and his obese mother (BMI=31.9 kg/m2). In vitro testing demonstrated that the mutation does not impair mRNA expression. We identified a 100-fold lower extracellular protein concentration for mutant resistin compared with WT levels using a resistin ELISA on cell culture medium (P=4.87×10−6). We also detected a decreased intracellular concentration for the mutant protein (tenfold lower relative levels, P=0.007). The plasma resistin levels of the proband and his mother, however, did not differ significantly from lean control individuals.ConclusionsIn conclusion, we identified the first missense mutation in resistin in a morbidly obese proband and his obese mother. Functional testing of the mutant protein suggests that the C78S mutant protein is degraded, possibly resulting in a decreased extracellular concentration, which may predispose to obesity.

Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

1990 ◽  
Vol 10 (2) ◽  
pp. 720-726
Author(s):  
K Mensa-Wilmot ◽  
D Hereld ◽  
P T Englund
Keyword(s):  
Phospholipase C ◽  
Blot Analysis ◽  
Developmental Regulation ◽  
Mrna Level ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.

scholarly journals Cloning and Characterization of an Ehrlichia canis Gene Encoding a Protein Localized to the Morula Membrane

2003 ◽  
Vol 71 (4) ◽  
pp. 2218-2225 ◽  
Author(s):  
Ching-Hao Teng ◽  
Raghavan U. M. Palaniappan ◽  
Yung-Fu Chang
Keyword(s):  
Recombinant Protein ◽  
Blot Analysis ◽  
Polyclonal Antibodies ◽  
Transmembrane Protein ◽  
Single Copy ◽  
Ehrlichia Canis ◽  
Chromosomal Dna ◽  
Expression Library ◽  
Gene Encoding ◽  
Host Cytoplasm

ABSTRACT A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening an Ehrlichia canis expression library with convalescent dog sera, which resulted in three positive clones. Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA. The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated the presence of a single copy of the mmpA gene in E. canis and Ehrlichia chaffeensis but not in the human granulocytic ehrlichiosis agent. The mmpA gene was amplified, cloned, and expressed as a fusion protein. Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits. Western blot analysis of E. canis and E. chaffeensis lysates with the anti-rMmpA serum resulted in the presence of an MmpA band only in E. canis, not in E. chaffeenesis. Sera from dogs which were either naturally or experimentally infected with E. canis recognized the recombinant protein. Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane of E. canis. Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.

scholarly journals A point mutation in the coding region of uroporphyrinogen decarboxylase associated with familial porphyria cutanea tarda

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 892-895
Author(s):  
JR Garey ◽  
JL Hansen ◽  
LM Harrison ◽  
JB Kennedy ◽  
JP Kushner
Keyword(s):  
Point Mutation ◽  
Half Life ◽  
Porphyria Cutanea Tarda ◽  
Amino Acid Position ◽  
Mutant Protein ◽  
Mrna Levels ◽  
Coding Region ◽  
Uroporphyrinogen Decarboxylase

Familial porphyria cutanea tarda (PCT) is inherited as an autosomal dominant trait caused by decreased activity of uroporphyrinogen decarboxylase (URO-D). In most families with PCT, URO-D mRNA levels are normal but both catalytic activity and immunologic reactivity of URO-D are half normal. We have cloned and sequenced 8 URO-D cDNA transcripts derived from a pedigree member with familial PCT. Three of the cDNAs had sequences encoding normal URO-D but five cDNA's contained a point mutation resulting in a gly----val substitution at amino acid position 281. An oligonucleotide probe complementary to the mutant sequence hybridized to DNA from affected individuals within the pedigree, but not to DNA from normal individuals. Measurements of pulse labeled URO-D in Epstein-Barr virus transformed lymphocytes indicated that the mutant protein has a half-life in vivo of less than four hours. In vitro measurements utilizing labeled URO-Ds generated in a reticulocyte lysate system revealed a 12-hour half-life for the mutant protein compared with a 102-hour half-life for normal URO-D. This is the first URO-D mutation to be characterized in a pedigree with familial PCT. This mutation was not detected in affected individuals from seven other PCT pedigrees, suggesting that PCT can result from different mutations.

Rainbow Trout (Salmo gairdneri) Growth Hormone: in vitro Translation of Pituitary RNA and Product Analysis

1986 ◽  
Vol 43 (7) ◽  
pp. 1327-1331 ◽  
Author(s):  
L. B. Agellon ◽  
T. T. Chen ◽  
R. J. Van Beneden ◽  
R. A. Sonstegard ◽  
G. F. Wagner ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Rainbow Trout ◽  
Blot Analysis ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Salmo Gairdneri ◽  
Western Immunoblot ◽  
Western Immunoblot Analysis ◽  
Rna Blot Analysis

We have used RNA blot analysis, in vitro translation, and protein immunoblot analysis to characterize mRNA in the pituitary glands of rapidly growing rainbow trout (Salmo gairdneri). Cell-free translation products of total RNA, analyzed by direct immunoprecipitation or western immunoblot analysis with an antiserum to chum salmon (Oncorphynchus keta) growth hormone (GH), indicate that rainbow trout pregrowth hormone (pre-GH) is a polypeptide of 25 000 Da. RNA blot analysis of total pituitary RNA to its cDNA, using total liver RNA as a competitor, revealed the presence of at least four size classes of pituitary mRNA sequences. One class, with a size smaller than 18S rRNA, is the predominant mRNA component. By in vitro translation and western immunoblot analysis, the pre-GH mRNA activity is associated with this class of mRNA sequences. These results suggest that rainbow trout pre-GH mRNA is in the same size range as that of mammalian GH mRNA.

scholarly journals A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo

2007 ◽  
Vol 88 (3) ◽  
pp. 895-902 ◽  
Author(s):  
G. Haqshenas ◽  
X. Dong ◽  
H. Netter ◽  
J. Torresi ◽  
E. J. Gowans
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hypervariable Region ◽  
Immunoblot Analysis ◽  
Post Inoculation ◽  
Sequencing Analysis ◽  
Coding Region ◽  
Hypervariable Region 1

Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.

scholarly journals Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein.

1986 ◽  
Vol 103 (3) ◽  
pp. 767-776 ◽  
Author(s):  
N M Kumar ◽  
N B Gilula
Keyword(s):  
Gap Junction ◽  
Rat Liver ◽  
Human Liver ◽  
Single Copy ◽  
In Vitro Translation ◽  
Coding Region ◽  
Human Cdna ◽  
Junction Protein ◽  
Gap Junction Protein

An extended synthetic oligonucleotide (58-mer) has been used to identify and characterize a human liver gap junction cDNA. The cDNA is 1,574 bases long and contains the entire coding region for a gap junction protein. In vitro translation of the RNA products of this cDNA is consistent with it coding for a 32,022-D protein. Southern blot analysis indicates that the gap junction gene is present as a single copy, and that it can be detected in a variety of organisms using the human liver cDNA as a probe. The human cDNA has been used to screen a rat liver cDNA library, and a rat liver junction cDNA clone has been isolated. The rat liver clone is 1,127 bases in length, and it has strong sequence homology to the human cDNA in the protein-coding region, but less extensive homology in the 3'-untranslated region.

scholarly journals Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

1990 ◽  
Vol 10 (2) ◽  
pp. 720-726 ◽  
Author(s):  
K Mensa-Wilmot ◽  
D Hereld ◽  
P T Englund
Keyword(s):  
Phospholipase C ◽  
Blot Analysis ◽  
Developmental Regulation ◽  
Mrna Level ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.

scholarly journals A point mutation in the coding region of uroporphyrinogen decarboxylase associated with familial porphyria cutanea tarda

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 892-895 ◽  
Author(s):  
JR Garey ◽  
JL Hansen ◽  
LM Harrison ◽  
JB Kennedy ◽  
JP Kushner
Keyword(s):  
Point Mutation ◽  
Half Life ◽  
Porphyria Cutanea Tarda ◽  
Amino Acid Position ◽  
Mutant Protein ◽  
Mrna Levels ◽  
Coding Region ◽  
Uroporphyrinogen Decarboxylase

Abstract Familial porphyria cutanea tarda (PCT) is inherited as an autosomal dominant trait caused by decreased activity of uroporphyrinogen decarboxylase (URO-D). In most families with PCT, URO-D mRNA levels are normal but both catalytic activity and immunologic reactivity of URO-D are half normal. We have cloned and sequenced 8 URO-D cDNA transcripts derived from a pedigree member with familial PCT. Three of the cDNAs had sequences encoding normal URO-D but five cDNA's contained a point mutation resulting in a gly----val substitution at amino acid position 281. An oligonucleotide probe complementary to the mutant sequence hybridized to DNA from affected individuals within the pedigree, but not to DNA from normal individuals. Measurements of pulse labeled URO-D in Epstein-Barr virus transformed lymphocytes indicated that the mutant protein has a half-life in vivo of less than four hours. In vitro measurements utilizing labeled URO-Ds generated in a reticulocyte lysate system revealed a 12-hour half-life for the mutant protein compared with a 102-hour half-life for normal URO-D. This is the first URO-D mutation to be characterized in a pedigree with familial PCT. This mutation was not detected in affected individuals from seven other PCT pedigrees, suggesting that PCT can result from different mutations.

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