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B Huang

Publications and source records attributed to B Huang.

At least 199 records · Page 11Linked to original sources
PubMed1988

Beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii.

Two beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii have been isolated on the basis of altered sensitivity to the growth-inhibitory effect of colchicine. The two mutations of colR4 and colR15 have been found to be tightly linked, mapping to a previously unmarked site in linkage group XII. The drug-resistance phenotypes of both mutations segregated in genetic crosses with the presence of distinct, acidic variant beta-tubulin isoforms found assembled into the microtubules of the flagella. Analysis of the in vitro translation products of total poly(A)+ RNA from the mutants provided evidence that the variant proteins are altered primary beta-tubulin gene products. Compared to wild type, strains carrying the mutations expressed an increased resistance to the inhibitory effects of colchicine in clonal growth, flagellar assembly, and germination of meiotic products, suggesting that the beta-tubulin altered in the mutants participates in multiple microtubule functions.

Chlamydomonas↗
PubMed1988

Purification and characterization of a basal body-associated Ca2+-binding protein.

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein: homology in its protein sequence with calmodulin and the yeast CDC31 gene product.

An extended synthetic oligonucleotide (59-mer) was used to isolate a Chlamydomonas cDNA containing the entire coding region for a novel basal body-associated 20-kD calcium-binding protein (CaBP). DNA and RNA blot analysis indicate that the 20-kD CaBP is encoded by a single copy gene from which is derived an approximately 1.1-kb-long transcript. The deduced amino acid sequence for the protein shows a linear relatedness with calmodulin from Chlamydomonas and other organisms (45-48% identity). The primary protein sequence of the 20-kD CaBP and its predicted secondary structure suggests that the protein is likely to contain four homologous calcium-binding domains that conform to the helix-loop-helix (or EF hand) structure found in calmodulin and related calcium-modulated proteins. The major difference between the protein and calmodulin is an amino-terminal domain of 21 amino acids present on the 20-kD CaBP. In addition to its relatedness to calmodulin, the Chlamydomonas 20-kD CaBP shows a strong sequence identity (50%) with the yeast Saccharomyces cerevisiae CDC31 gene product required for spindle pole body duplication. The association of these sequence-related calcium-binding proteins to microtubule-organizing centers of divergent structure suggests a potential conserved function for the proteins.

Amino Acid Sequence↗
PubMed1987

Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells.

Cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, produce phytochelatins (poly[gamma-glutamylcysteinyl]glycines) when exposed to cadmium. The synthesis of these peptides is accompanied by a decline in cellular levels of glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, inhibits the sustained production of phytochelatins. However, phytochelatin synthesis can occur in the presence of buthionine sulfoximine provided that sufficient glutathione is available. These results indicate that glutathione is a substrate for phytochelatin synthesis. The protein synthesis inhibitor cycloheximide does not affect the initial production of phytochelatin.

Journal Article↗
PubMed1985

Combinational diversity within variable regions bearing the predominant anti-p-azophenylarsonate idiotype of strain A mice.

The humoral immune response in strain A mice to protein conjugates of p-azophenylarsonate (Ars) is characterized by the presence of a major cross-reactive idiotype denoted as IdCR. Previous molecular analyses of monoclonal IdCR+ Ars-binding antibodies isolated from multiply immunized animals have indicated that these antibody variable (V) regions may be the expressed product of a single combination of VH, D, JH, V kappa, and J kappa gene segments. The basis of this apparent domination of the Ars response by V regions encoded by this single combination of gene segments is unclear, but is discussed in this report. Our structural analyses on five monoclonal IdCR+ antibodies that are unable to bind Ars show that in contrast to those of Ars-binding IdCR+ antibodies, these (Ars-nonbinding) IdCR+ V regions are encoded by multiple combinations of VH, D, JH, V kappa, and J kappa gene segments, but with the commonality that they all utilize a single VH gene segment (VHIdCR). We provide examples in which the VHIdCR gene segment is expressed with three different V kappa gene segments and with each of the four JH gene segments to produce serologically detectable IdCR+ Ars-nonbinding antibodies. It would thus appear that the previous failure to detect alternative IdCR+ V segment combinations was due to a sampling procedure requiring that the IdCR+ antibody bind Ars, and not the result of restricted assembly or expression of the VHIdCR gene segment with a particular combination of D, JH, V kappa, and J kappa gene segments. This bias in protocol, however, cannot completely account for the homogeneity in previously studied IdCR+ Ars-binding antibodies, because we were able to isolate, from primary immune responses, IdCR+ antibodies that do bind Ars but that utilize alternative V segment combinations. This finding suggests that combinations of V gene segments encoding IdCR+ antibodies are more numerous in primary as opposed to secondary immune responses, and raises the question of why a single combination of VH, D, JH, V kappa, and J kappa gene segments dominates the secondary strain A immune response to Ars.

Amino Acid Sequence↗
PubMed1984

Surgical management for recurrent pharyngoesophageal (Zenker's) diverticulum.

Recurrent pharyngoesophageal diverticulum is uncommon after a one-stage diverticulectomy. Among 888 operations at the Mayo Clinic, recurrence has been noted after only 32 (3.6%). The present report outlines our management during a 19-year period of 44 patients operated on previously, including management of various late complications seen in 13 patients after diverticulopexy and diverticulectomy performed at other institutions. The study then focuses on the results in 31 patients in whom reoperation for symptomatic recurrent diverticulum was performed at the Mayo Clinic during the same period. Although reoperation posed a technical challenge, there was only 1 operative death (3.2%). Surviving patients experienced highly satisfactory late results, with only two recurrences. Early surgical morbidity was considerably higher than for primary operations, but this did not interfere with most patients having excellent to good late results.

Adult↗
PubMed1984

Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex. Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes. This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule. The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pf16 axonemes. No structural gene product has been identified for the PF6 locus. The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments. Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule.

Chlamydomonas↗
PubMed1984

Mutant strains of Chlamydomonas reinhardtii that move backwards only.

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak-like projections found within the B-tubules of outer doublets 5 and 6.

Cell Movement↗
PubMed1982

Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for Flagellar function.

Reversion analysis of flagellar-motility mutants of Chlamydomonas reinhardtii yields an unusual class of intergenic suppressor mutations that restore flagellar activity to paralyzed radial-spoke or central-pair mutants without altering the structural or molecular defects associated with the original mutations. Four suppressors representing independent genetic loci were studied in detail. Two of the mutations, suppf1 and suppf2, restore flagellar motility to either radial-spoke or central-pair mutants of different genes. The mutants suppf3 and suppf 4 suppress flagellar paralysis associated only with mutants defective for the radial spokes. Analyses of the axonemal polypeptides of suppf1, suppf3 and suppf4 mutants indicate that the mutations restore flagellar activity to paralyzed radial-spoke or central-pair mutants by altering other components of the flagellar axoneme. suppf1 shows an altered electrophoretic migration for a 325,000 molecular weight polypeptide known to be a subunit of an outer-arm dynein. suppf3 and suppf4 are missing different axonemal polypeptides with molecular weights of 60,000 (in the case of suppf3), and 40,000 and 29,000 (in the case of suppf4). Genetic evidence has been obtained indicating that the polypeptides affected in suppf3 and suppf4 are components of a newly identified functional and/or structural compartment of the flagellar axoneme. The suppressor mutations described here reveal the operation of a control mechanism that inhibits the operations of flagellar movements in the presence of radial-spoke or central-pair defects. Suppressor mutations release the inhibition. The molecular defects of suppf1, suppf3 and suppf4 provide evidence that the inhibitory mechanism can be interrupted at two different levels of axonemal function.

Chlamydomonas↗
PubMed1982

Uniflagellar mutants of Chlamydomonas: evidence for the role of basal bodies in transmission of positional information.

A series of uniflagellar mutants isolated following mutagenesis of Chlamydomonas reinhardtii (strain 137c) with ICR-191 show a remarkable positional phenotype. The flagellum that fails to develop is cis to the eyespot in more than 95% of the cells examined. Both the positional and the uniflagellar phenotypes are transmitted stably through mitotic and meiotic divisions, and in backcrosses the meiotic segregation is two mutant to two wild-type progeny. Four of the mutants, uni1, uni2, uni3 and uni4, have been studied extensively. They appear to be alleles of a single gene locus or to be closely linked (less than or equal to 0.06 map units). The characteristic expression of the uniflagellar defect in cells under different growth conditions or in stable diploids indicates that the mutations alter the rate of development of the flagellum in the cis-eyespot flagellum. Electron microscopic studies suggest that the developmental defect resides in the basal body. Extensive recombination analysis to 33 nuclear markers representing the 16 linkage groups failed to establish linkage. The uni mutants, however, showed linkage to four unmapped mutant loci. Mutations for each of these loci also affect flagellar assembly.

Cell Division↗
PubMed1982

Analysis of the movement of Chlamydomonas flagella:" the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella.

The mutation uni-1 gives rise to uniflagellate Chlamydomonas cells which rotate around a fixed point in the microscope field, so that the flagellar bending pattern can be photographed easily. This has allowed us to make a detailed analysis of the wild-type flagellar bending pattern and the bending patterns of flagella on several mutant strains. Cells containing uni-1, and recombinants of uni-1 with the suppressor mutations, suppf-1 and suppf-3, show the typical asymmetric bending pattern associated with forward swimming in Chlamydomonas, although suppf-1 flagella have about one-half the normal beta frequency, apparently as the result of defective function of the outer dynein arms. The pf-17 mutation has been shown to produce nonmotile flagella in which radial spoke heads and five characteristic axonemal polypeptides are missing. Recombinants containing pf-17 and either suppf-2 or suppf-3 have motile flagella, but still lack radial-spoke heads and the associated polypeptides. The flagellar bending pattern of these recombinants lacking radial-spoke heads is a nearly symmetric, large amplitude pattern which is quite unlike the wild-type pattern. However, the presence of an intact radial-spoke system is not required to convert active sliding into bending and is not required for bend initiation and bend propagation, since all of these processes are active in suppfpf-17 recombinants. The function of the radial-spoke system appears to be to convert the symmetric bending pattern displayed by these recombinants into the asymmetric bending pattern required for efficient swimming, by inhibiting the development of reverse bends during the recovery phase of the bending cycle.

Chlamydomonas↗
PubMed1981

Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components.

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S-containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 170 axonemal components. By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8-7.1. Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads. The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild-type axonemes. Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question. Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1. No Ca++- or Mg++-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads. In vivo pulse 32P incorporation experiments provide evidence that greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.

Adenosine Triphosphatases↗
PubMed1981

Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function.

In addition to the previously studied pf-14 and pf-1 loci in Chlamydomonas reinhardtii, mutations for another five genes (pf-17, pf-24, pf-25, pf-26, and pf-27) have been identified and characterized as specifically affecting the assembly and function of the flagellar radial spokes. Mutants for each of the newly identified loci show selective alterations for one or more of the 17 polypeptides in the molecular weight range of 20,000-130,000 which form the radial spoke structure. In specific instances the molecular defect has been correlated with altered radial spoke morphology. Biochemical analysis of in vivo complementation in mutant X wild-type dikaryons has provided indirect evidence that mutations for four of the five new loci (pf-17, pf-24, pf-25, and pf-26) reside in structural genes for spoke components. In the case of pf-24, the identity of the mutant gene product was supported by analysis of induced intragenic revertants. In contrast to the other radial spoke mutants thus far investigated, evidence suggests that the gene product in pf-27 is extrinsic to the radial spokes and is required for the specific in vivo phosphorylation of spoke polypeptides.

Chlamydomonas↗
PubMed1981

Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants.

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.

Chlamydomonas↗