scholarly journals Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.

1989 ◽  
Vol 9 (3) ◽  
pp. 875-884 ◽  
Author(s):  
T S Hays ◽  
R Deuring ◽  
B Robertson ◽  
M Prout ◽  
M T Fuller
Keyword(s):  
Drosophila Melanogaster ◽  
Missense Mutation ◽  
Null Allele ◽  
Genetic Interaction ◽  
Structural Proteins ◽  
Interacting Proteins ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Gene Encoding ◽  
Alpha Tubulin

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.

Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster

1989 ◽  
Vol 9 (3) ◽  
pp. 875-884
Author(s):  
T S Hays ◽  
R Deuring ◽  
B Robertson ◽  
M Prout ◽  
M T Fuller
Keyword(s):  
Drosophila Melanogaster ◽  
Missense Mutation ◽  
Null Allele ◽  
Genetic Interaction ◽  
Structural Proteins ◽  
Interacting Proteins ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Gene Encoding ◽  
Alpha Tubulin

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.

scholarly journals Two types of genetic interaction implicate the whirligig gene of Drosophila melanogaster in microtubule organization in the flagellar axoneme.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 961-973 ◽  
Author(s):  
L L Green ◽  
N Wolf ◽  
K L McDonald ◽  
M T Fuller
Keyword(s):  
Drosophila Melanogaster ◽  
Genetic Interaction ◽  
Dominant Male ◽  
Loss Of Function ◽  
Male Sterile ◽  
Microtubule Organization ◽  
Beta Tubulin ◽  
Absolute Level ◽  
Alpha Tubulin ◽  
Flagellar Axoneme

Abstract The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an alpha-tubulin locus, alpha 1t, mutations in a beta-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known alpha- or beta-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrlnc4 and B2tn are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2tn, for a deficiency of alpha 1t, or for the haync2 allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrlnc4 or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules.

Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum

1989 ◽  
Vol 9 (5) ◽  
pp. 2042-2049
Author(s):  
G S Harris ◽  
E J Keath ◽  
J Medoff
Keyword(s):  
Phase Transitions ◽  
Histoplasma Capsulatum ◽  
Blot Hybridization ◽  
Dot Blot ◽  
Dimorphic Fungus ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Two Dimensional Gel Electrophoresis ◽  
Western Blots ◽  
Alpha Tubulin

Recent investigations have confirmed the presence of one alpha-tubulin gene (TUB1) and one beta-tubulin gene (TUB2) in the dimorphic fungus Histoplasma capsulatum. In the present study, Northern blot (RNA blot) analyses revealed multiple alpha-tubulin transcripts and a single beta-tubulin transcript in the yeast and mycelial phases of the high-virulence 217B strain and low-virulence Downs strain. S1 nuclease protection assays demonstrated one initiation start site and two major stop sites for the TUB1 transcripts, suggesting that variations in 3' processing generate the alpha-tubulin messages of 2.5 and 2.0 kilobases. Dot blot hybridization experiments indicated that tubulin gene expression is developmentally regulated during the dimorphic phase transitions. alpha- and beta-tubulin mRNAs increased six- to eightfold during the yeast-to-mycelium conversion and decreased two- to threefold during the reverse transition. These changes in tubulin mRNA content coincided with major morphological events associated with H. capsulatum development. Western blots (immunoblots) of H. capsulatum yeast-specific proteins resolved by two-dimensional gel electrophoresis demonstrated a single alpha- and a single beta-tubulin isoform. Multiple tubulin polypeptides expressed in mycelia are probably products of posttranslational modifications.

scholarly journals A codon change in beta-tubulin which drastically affects microtubule structure in Drosophila melanogaster fails to produce a significant phenotype in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (9) ◽  
pp. 4726-4731 ◽  
Author(s):  
V Praitis ◽  
W S Katz ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Eukaryotic Cells ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Cell Biol ◽  
Codon Change ◽  
Almost All ◽  
Beta Tubulin Gene ◽  
Microtubule Structure

The relative uniformity of microtubule ultrastructure in almost all eukaryotic cells is thought to be a consequence of the conserved elements of tubulin sequence. In support of this idea, a mutation in a beta-tubulin gene of Drosophila melanogaster, occurring at a highly conserved position, produces U-shaped microtubules, suggesting a defect in either nucleation or packing during assembly (M. T. Fuller, J. H. Caulton, J. A. Hutchens, T. C. Kaufman, and E. C. Raff, J. Cell Biol. 104:385-394, 1987, and J. E. Rudolph, M. Kimble, H. D. Hoyle, M. A. Subler, and E. C. Raff, Mol. Cell. Biol. 7:2231-2242, 1987). Surprisingly, we find that introducing the same mutation into the sole beta-tubulin gene of Saccharomyces cerevisiae has virtually no consequences for microtubule structure or function in that organism.

A codon change in beta-tubulin which drastically affects microtubule structure in Drosophila melanogaster fails to produce a significant phenotype in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (9) ◽  
pp. 4726-4731
Author(s):  
V Praitis ◽  
W S Katz ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Eukaryotic Cells ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Cell Biol ◽  
Codon Change ◽  
Almost All ◽  
Beta Tubulin Gene ◽  
Microtubule Structure

The relative uniformity of microtubule ultrastructure in almost all eukaryotic cells is thought to be a consequence of the conserved elements of tubulin sequence. In support of this idea, a mutation in a beta-tubulin gene of Drosophila melanogaster, occurring at a highly conserved position, produces U-shaped microtubules, suggesting a defect in either nucleation or packing during assembly (M. T. Fuller, J. H. Caulton, J. A. Hutchens, T. C. Kaufman, and E. C. Raff, J. Cell Biol. 104:385-394, 1987, and J. E. Rudolph, M. Kimble, H. D. Hoyle, M. A. Subler, and E. C. Raff, Mol. Cell. Biol. 7:2231-2242, 1987). Surprisingly, we find that introducing the same mutation into the sole beta-tubulin gene of Saccharomyces cerevisiae has virtually no consequences for microtubule structure or function in that organism.

Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation

1986 ◽  
Vol 6 (5) ◽  
pp. 1422-1429
Author(s):  
C Whitfield ◽  
I Abraham ◽  
D Ascherman ◽  
M M Gottesman
Keyword(s):  
Cho Cells ◽  
Single Gene ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Subunit ◽  
Steady State Levels

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

scholarly journals The beta 3-tubulin gene of Drosophila melanogaster is essential for viability and fertility.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 991-1005
Author(s):  
M Kimble ◽  
R W Dettman ◽  
E C Raff
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Minor Component ◽  
Cell Types ◽  
P Element ◽  
Chromosome 2 ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Cytoplasmic Microtubule ◽  
Tissue Organization

Abstract We have previously shown that the beta 3-tubulin gene of Drosophila melanogaster encodes a divergent isoform expressed in a complex developmental pattern. The beta 3 gene is transiently expressed in the embryo and again in the pupa at high levels in the developing musculature, and at lower levels in several different pupal tissues of ectodermal origin. Adult expression is confined to specific somatic cells in the gonads. In some of the cell types in which it is expressed, beta 3 is the sole or predominant beta-tubulin, while in others the beta 3 protein is a minor component of the beta-tubulin pool. The sites and timing of beta 3 expression demonstrated that beta 3-tubulin is utilized primarily in cytoplasmic microtubule arrays involved in changes in cell shape and tissue organization, and suggested to us that this isoform may be functionally specialized. To determine whether the expression of the beta 3 gene is essential for normal development, and to examine the specific functions of this divergent isoform, we have generated mutations within the gene. We determined that the small deficiency Df(2R)Px2, which deletes the 60C5,6-60D9,10 region of chromosome 2, removes all of the beta 3 coding sequences, and that the distal breakpoint of the deficiency is approximately 2 kb upstream from the start of transcription of the beta 3 gene. We have generated a total of 31 ethyl methanesulfonate- or diepoxybutane-induced recessive lethal or visible mutations which map within the deficiency. These mutations define 12 new lethal complementation groups, which together with two previously identified visible mutations, altogether identify 14 genes in this interval of the second chromosome. A lethal complementation group comprising mutations in the beta 3-tubulin gene (beta Tub60D) was identified by rescue of their lethality by a wild-type copy of the gene introduced into the genome via P element-mediated germ line transformation. Analysis of the homozygous and transheterozygous phenotypes of the five beta 3 mutations recovered (alleles designated B3t1-B3t5) demonstrates that beta 3-tubulin is essential for viability and fertility.

scholarly journals iRhom2 is required for the secretion of mouse TNFα

Blood ◽  
2012 ◽  
Vol 119 (24) ◽  
pp. 5769-5771 ◽  
Author(s):  
Owen M. Siggs ◽  
Nengming Xiao ◽  
Ying Wang ◽  
Hexin Shi ◽  
Wataru Tomisato ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Inflammatory Disease ◽  
Sodium Sulfate ◽  
Null Allele ◽  
Inflammatory Stimulus ◽  
Toll Like Receptors ◽  
Gene Encoding ◽  
Rhomboid Protease ◽  
Secretion Signal ◽  
Chemically Induced

Abstract TNFα is a powerful inflammatory stimulus, central both to the control of infection, and as an agent of inflammatory disease. The most potent inducers of TNFα secretion signal through the Toll-like receptors, and we describe here a chemically-induced mutation that impairs this response in macrophages. A missense mutation was revealed in the gene encoding the inactive rhomboid protease iRhom2, which was not complemented by a null allele of the same gene. Neither the missense nor the null allele affected TLR-induced secretion of IL-6. Moreover, unlike a mutation in TNFα, the iRhom2 missense mutation did not cause enhanced susceptibility to colitis induced by dextran sodium sulfate. These results establish a specific role for iRhom2 in the secretion of TNFα, and present a new target for the modulation of inflammation.

Transcriptional regulation of tubulin gene expression in differentiating trochoblasts during early development of Patella vulgata

Development ◽  
1994 ◽  
Vol 120 (10) ◽  
pp. 2835-2845
Author(s):  
W.G. Damen ◽  
L.A. van Grunsven ◽  
A.E. van Loon
Keyword(s):  
Gene Expression ◽  
Gene Product ◽  
Cell Lines ◽  
Upstream Region ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Cell Stage ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Tubulin Mrna

The expression of alpha- and beta-tubulin genes during the early development of the marine mollusk Patella vulgata has been investigated. From the 32-cell stage onwards, an enhanced expression of both alpha- and beta-tubulin mRNAs was detected in the primary trochoblasts. After one additional cleavage, these cells become cleavage-arrested and then form cilia. They are the first cells to differentiate during Patella development. Later, alpha- and beta-tubulin mRNA is also found in the accessory and secondary trochoblasts. Together these three cell-lines form the prototroch, the ciliated locomotory organ of the trochophore larva. The early and abundant expression of tubulin genes precede and accompany cilia formation in the trochoblasts and provides us with an excellent molecular differentiation marker for these cells. Apart from the trochoblasts, tubulin gene expression was also found in other cells at some stages. At the 88-cell stage, elevated tubulin mRNA levels were found around the large nucleus of the mesodermal stem cell 4d. In later stages, tubulin gene expression was detected in the cells that form the flagella of the apical tuft and in the refractive bodies. An alpha-tubulin gene was isolated and characterized. A lacZ fusion gene under control of the 5′ upstream region of this tubulin gene was microinjected into embryos at the two-cell stage. The reporter gene product was only detected in the three trochoblast cell-lines at the same time as tubulin genes were expressed in these cells. Reporter gene product was not detected in any other cells. Thus, this 5′ upstream region of this alpha-tubulin gene contains all the elements required for the correct spatiotemporal pattern of expression.

scholarly journals Interacting genes that affect microtubule function in Drosophila melanogaster: two classes of mutation revert the failure to complement between haync2 and mutations in tubulin genes.

Genetics ◽  
1990 ◽  
Vol 125 (1) ◽  
pp. 77-90
Author(s):  
C L Regan ◽  
M T Fuller
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Product ◽  
Genetic Interaction ◽  
Meiotic Spindle ◽  
Male Sterile ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Recessive Phenotype ◽  
Tubulin Mutations ◽  
Microtubule Function

Abstract The recessive male sterile mutation haync2 of Drosophila melanogaster fails to complement certain beta 2-tubulin and alpha-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by haync2, which may act as a structural poison. Based on this observation, we have isolated ten new mutations that revert the failure to complement between haync2 and B2tn. The revertants tested behaved as intragenic mutations of hay in recombination tests, and fell into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than haync2 in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the haync2 allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywirenc2 product to interact structurally with microtubules. Flies heterozygous for the original haync2 allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle.

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