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 Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

1993 ◽  
Vol 178 (1) ◽  
pp. 49-62 ◽  
Author(s):  
M J Fritzler ◽  
J C Hamel ◽  
R L Ochs ◽  
E K Chan
Keyword(s):  
Recombinant Proteins ◽  
Heavy Chain ◽  
Golgi Complex ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Cytoplasmic Dynein ◽  
In Vitro Translation ◽  
Rabbit Serum ◽  
Cell Extracts

Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.

scholarly journals Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation

2000 ◽  
Vol 348 (1) ◽  
pp. 189-199 ◽  
Author(s):  
Janice M. LAPLANTE ◽  
Flavia O'ROURKE ◽  
Xinghua LU ◽  
Alan FEIN ◽  
Anne OLSEN ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Types ◽  
In Vitro Translation ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Regulated Expression ◽  
Endoplasmic Reticulum Protein ◽  
Hel Cells ◽  
Expressed Sequence

A monoclonal antibody which blocks InsP3-induced Ca2+ release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank® of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca2+ homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of Mr 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP3 receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca2+ mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca2+ homoeostasis, growth and proliferation.

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster

1984 ◽  
Vol 4 (12) ◽  
pp. 2643-2652
Author(s):  
D K Burns ◽  
B C Stark ◽  
M D Macklin ◽  
W Y Chooi
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Genomic Library ◽  
Polytene Chromosomes ◽  
In Vitro Translation ◽  
Ribosomal Protein Genes ◽  
Recombinant Phage ◽  
Recombinant Clone ◽  
Isolation And Characterization

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.

scholarly journals Phage-Displayed T-Cell Epitope Grafted into Immunoglobulin Heavy-Chain Complementarity-Determining Regions: an Effective Vaccine Design Tested in Murine Cysticercosis

1999 ◽  
Vol 67 (9) ◽  
pp. 4764-4770 ◽  
Author(s):  
Karen Manoutcharian ◽  
Luis Ignacio Terrazas ◽  
Goar Gevorkian ◽  
Gonzalo Acero ◽  
Pavel Petrossian ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Cell Epitope ◽  
Effective Vaccine ◽  
Recombinant Phage ◽  
Free Peptide ◽  
New Type ◽  
Complementarity Determining Regions ◽  
Antigen Presenting

ABSTRACT A new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (VH) domain with the Taenia crassiceps epitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4+ and CD8+ T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig VH domains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction.

scholarly journals UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

2004 ◽  
Vol 167 (2) ◽  
pp. 315-325 ◽  
Author(s):  
Matthew Lord ◽  
Thomas D. Pollard
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Gliding Motility ◽  
Light Chains ◽  
Temperature Sensitive ◽  
Contractile Ring ◽  
In Vitro Motility

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

scholarly journals Eukaryotic Translation Initiation Factors 4G and 4A from Saccharomyces cerevisiae Interact Physically and Functionally

1999 ◽  
Vol 19 (8) ◽  
pp. 5557-5564 ◽  
Author(s):  
Carrie L. Neff ◽  
Alan B. Sachs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Eukaryotic Translation ◽  
Initiation Of Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT The initiation of translation in eukaryotes requires several multisubunit complexes, including eukaryotic translation initiation factor 4F (eIF4F). In higher eukaryotes eIF4F is composed of the cap binding protein eIF4E, the adapter protein eIF4G, and the RNA-stimulated ATPase eIF4A. The association of eIF4A withSaccharomyces cerevisiae eIF4F has not yet been demonstrated, and therefore the degree to which eIF4A’s conserved function relies upon this association has remained unclear. Here we report an interaction between yeast eIF4G and eIF4A. Specifically, we found that the growth arrest phenotype associated with three temperature-sensitive alleles of yeast eIF4G2 was suppressed by excess eIF4A and that this suppression was allele specific. In addition, in vitro translation extracts derived from an eIF4G2 mutant strain could be heat inactivated, and this inactivation could be reversed upon the addition of recombinant eIF4A. Finally, in vitro binding between yeast eIF4G and eIF4A was demonstrated, as was diminished binding between mutant eIF4G2 proteins and eIF4A. In total, these data indicate that yeast eIF4G and eIF4A physically associate and that this association performs an essential function.

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 Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

1984 ◽  
Vol 4 (12) ◽  
pp. 2643-2652 ◽  
Author(s):  
D K Burns ◽  
B C Stark ◽  
M D Macklin ◽  
W Y Chooi
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Genomic Library ◽  
Polytene Chromosomes ◽  
In Vitro Translation ◽  
Ribosomal Protein Genes ◽  
Recombinant Phage ◽  
Recombinant Clone ◽  
Isolation And Characterization

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.

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