scholarly journals ISOLATION AND GENETIC ANALYSIS OF MUTANT STRAINS OF CHLAMYDOMONAS REINHARDI DEFECTIVE IN GAMETIC DIFFERENTIATION

Genetics ◽  
1976 ◽  
Vol 82 (2) ◽  
pp. 169-186
Author(s):  
Ursula W Goodenough ◽  
Carol Hwang ◽  
Howard Martin
Keyword(s):  
Genetic Analysis ◽  
Cell Fusion ◽  
Mating Type ◽  
Normal Growth ◽  
The Other ◽  
Genetic Dissection ◽  
Structural Studies ◽  
Tetrad Analysis ◽  
Mating Structure ◽  
Mutant Strains

ABSTRACT Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-6, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt  - or mt  + gametes; these strains have been induced to form rare zygotes with mt  - gametes and the mutations are shown to be unlinked to the mt locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-1 strain agglutinate well with mt  - gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt  + locus, and fine-structural studies reveal that imp-1gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.

scholarly journals The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

1997 ◽  
Vol 137 (7) ◽  
pp. 1537-1553 ◽  
Author(s):  
Nedra F. Wilson ◽  
Mary J. Foglesong ◽  
William J. Snell
Keyword(s):  
Cell Fusion ◽  
Mating Type ◽  
Plasma Membranes ◽  
Surface Proteins ◽  
Filamentous Actin ◽  
Mating Structure ◽  
Cell Protrusion ◽  
Isolation And Characterization ◽  
In Vitro Adhesion

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ∼3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 106 fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes.

scholarly journals SEX-LIMITED EXPRESSION OF GENE LOCI CONTROLLING FLAGELLAR MEMBRANE AGGLUTINATION IN THE CHLAMYDOMONAS MATING REACTION

Genetics ◽  
1978 ◽  
Vol 89 (2) ◽  
pp. 235-243
Author(s):  
Ursula W Goodenough ◽  
Carol Hwang ◽  
A Jane Warren
Keyword(s):  
Genetic Analysis ◽  
Cell Fusion ◽  
Mating Reaction ◽  
Sexual Agglutination ◽  
Flagellar Membrane ◽  
Mutant Strains

ABSTRACT Mutant strains of Chlamydomonas reinhardi that have lost their ability to undergo sexual agglutination via their flagellar tips have been induced to undergo zygotic cell fusion and meiosis, using a flagellar-directed antiserum. Genetic analysis of antiserum-mediated crosses involving five nonagglutinating mt  + mutant strains reveals the following: (1) None of the mutations is linked to the mt locus. (2) All of the mutations are "sex-limited," meaning that they can be carried and transmitted by, but not expressed in, mt  - cells. (3) Four of the mutations (imp-2, imp-5, imp-6, imp-7) are either allelic or closely linked to one another, with imp-8 defining a second locus.

scholarly journals MUTATIONS AFFECTING SEXUAL CONJUGATION AND RELATED PROCESSES IN SACCHAROMYCES CEREVISIAE. II. GENETIC ANALYSIS OF NONMATING MUTANTS

Genetics ◽  
1974 ◽  
Vol 76 (2) ◽  
pp. 273-288
Author(s):  
Vivian Mackay ◽  
Thomas R Manney
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Mating Type ◽  
Tetrad Analysis ◽  
Genetic Loci ◽  
Type Locus ◽  
Mating Type Locus

ABSTRACT Rare diploids formed by sterile mutants have been studied by tetrad analysis. Sixteen classes of mutants representing at least five distinct genetic loci have been defined. One group of mutations, isolated only in α, maps at the mating-type locus, while none of the others shows any linkage to mating type. Some of the mutations are nonspecific for mating type, while others act only on a or α. In addition, mutations were found that prevent sporulation when heterozygous in diploids. These appear to be mutations of the mating-type alleles.

scholarly journals LINKAGE OF MUTATIONS AFFECTING MINUS FLAGELLAR MEMBRANE AGGLUTINABILITY TO THE mt  - MATING-TYPE LOCUS OF CHLAMYDOMONAS

Genetics ◽  
1981 ◽  
Vol 99 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Carol J Hwang ◽  
Brian C Monk ◽  
Ursula W Goodenough
Keyword(s):  
Uv Irradiation ◽  
Mating Type ◽  
Zygote Formation ◽  
Wild Type ◽  
Tetrad Analysis ◽  
Type Locus ◽  
Mating Type Locus ◽  
Flagellar Membrane ◽  
Mutant Strains

ABSTRACT Two independently isolated mutant strains, imp-10 and imp-12, were obtained by UV irradiation of wild-type mating-type minus (wt-). Each fails to agglutinate sexually with gametes of either mating type, but mating and zygote formation can be elicited by agglutinating either strain to wt+ gametes by means of anti-flagellar antiserum. Tetrad analysis of the resultant zygotes shows that both imp-10 and imp-12 are very closely linked to mt  -, with no recombinants observed. Diploid strains constructed between imp-10 or imp-12 and wt+ gametes are wt-, that is, they agglutinate and fuse like normal minus cells. Tetrad analysis of triploids from imp-10 diploid x wt+ haploid crosses shows that only imp-10 and wt+ products are recovered. A model is proposed to account for these results.

scholarly journals ISOGENIC STRAINS OF PHYCOMYCES BLAKESLEEANUS SUITABLE FOR GENETIC ANALYSIS

Genetics ◽  
1983 ◽  
Vol 105 (4) ◽  
pp. 873-879
Author(s):  
M I Alvarez ◽  
A P Eslava
Keyword(s):  
Genetic Analysis ◽  
Mating Type ◽  
Type Strain ◽  
Wild Type ◽  
Tetrad Analysis ◽  
Phycomyces Blakesleeanus ◽  
Isogenic Strains ◽  
Type Strains

ABSTRACT The progeny of crosses between wild-type strains of Phycomyces usually do not exhibit all of the expected genotypes from meiosis. By backcrossing, we have isolated a new (+) mating-type strain, A56, which is nearly isogenic with the (-) wild-type NRRL1555 commonly used in Phycomyces research. Tetrad analysis of the backcrosses shows that meiosis becomes more regular as the parental (+) and (-) strains become more isogenic. In our two-factor crosses with unlinked markers, the regularity of meiosis is measured as the percent of reciprocal ditypes plus tetratypes in the progeny. We have shown that this percentage increases from about 15% for crosses between nonisogenic parents to 90% in the eighth backcross. The results indicate that routine, reliable recombination analyses are possible in P. blakesleeanus.

scholarly journals Phenotypic conversion of mating type specificity induced by transplantation of macronucleoplasm in Paramecium caudatum

1994 ◽  
Vol 63 (2) ◽  
pp. 101-107 ◽  
Author(s):  
Manabu Hori ◽  
Mihoko Takahashi
Keyword(s):  
Genetic Analysis ◽  
Mating Type ◽  
Null Allele ◽  
Gene Dosage ◽  
The Other ◽  
Paramecium Caudatum ◽  
Specific Expression ◽  
Classical Genetic ◽  
Transplantation Technique

SummaryAccording to the classical genetic analysis in Paramecium caudatum by Tsukii & Hiwatashi (1983), the E mating type of each syngen is expressed when the cell bears alleles specific for syngen at the Mt locus. The O mating type is expressed when cells are homozygous for the null allele, mt, at the Mt locus. In such mt/mt cells the O syngen specificity is determined by alleles at two other loci called MA and MB. Inthe study reported here, macronucleoplasmic transplantation technique was used to test the above hypothesis. When macronucleoplasm of type E3 (mating type E of syngen 3) was injected into a macronucleus of type O12 (mating type O of syngen 12), some recipients changed to type E of the donor syngen but some others changed to type E of the recipient syngen. Thus, syngen specificity of donor macronucleoplasm controlling type E was converted into that of the recipients, even though the latter has no gene that controls type E. When this transformant expressing type E of the recipiexnt syngen was re-injected back into E of the other syngen, the expression of the converted mating type in some way continued in the recipient. This suggests that syngen specificity of gene Mt of the donor was changed to that of the recipients by intersyngenic transplantation of macronucleoplasm. We also obtained results suggesting that the gene dosage ratio of Mt to mt or Mt to MA and MB may be important for syngen specific expression of type E.

scholarly journals Transient increase in calcium efflux accompanies fertilization in Chlamydomonas.

1983 ◽  
Vol 97 (2) ◽  
pp. 397-404 ◽  
Author(s):  
R A Bloodgood ◽  
E N Levin
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Mating Type ◽  
Transient Increase ◽  
Calcium Efflux ◽  
Efflux Rate ◽  
Mating Structure ◽  
Flagellar Membrane ◽  
Intracellular Storage ◽  
Cell Cell

Mating in Chlamydomonas is a complex process initiated by contact of gametic flagellar surfaces, resulting in transmission of a signal from the flagella to the cell bodies. This signal triggers later events of cell wall loss, mating structure activation, and cell-cell fusion. Little is known about the nature of the signal or the role of Ca in these events. It was found that extracellular Ca is not necessary for successful mating in Chlamydomonas. However, cells will take up Ca from the medium in a linear manner for many hours and will accumulate micromolar concentrations, presumably by sequestering Ca within intracellular storage sites. If gametic cells of one mating type (preloaded with 45Ca) are mated with gametes of the opposite mating type (preloaded with unlabeled calcium), there is a rapid, transient increase in calcium efflux rate (20 times that of the control) that lasts approximately 6 min. This effect is not associated with cell-cell fusion, since the same observation is made if (+) gametes preloaded with 45-Ca are agglutinated by isolated flagella from (-) gametes preloaded with unlabeled Ca. Other experiments have shown that the increased efflux rate is not a simple consequence of cell wall release. Ca efflux in unmated gametes is greatly reduced in deflagellated cells, suggesting that much of the Ca movement is associated with the flagellar membrane. Although signaling itself may involve Ca fluxes across the flagellar membrane, it is also possible that a consequence of signaling is release of Ca from intracellular storage sites (perhaps functional equivalents of the sarcoplasmic reticulum). The observed transient increase in Ca efflux rate may reflect a transient increase in the cytoplasmic free-Ca concentration. This increase in cytoplasmic Ca may regulate the later events in mating (such as cell wall release and mating structure activation).

scholarly journals GENETIC ANALYSIS OF MATING LOCUS LINKED MUTATIONS IN CHLAMYDOMONAS REINHARDII

Genetics ◽  
1985 ◽  
Vol 111 (3) ◽  
pp. 447-461
Author(s):  
R E Galloway ◽  
U W Goodenough
Keyword(s):  
Genetic Analysis ◽  
Mating Type ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Chloroplast Gene ◽  
Chlamydomonas Reinhardii ◽  
Mutant Strains ◽  
Sexual Fusion

ABSTRACT The mating-type (mt) locus of Chlamydomonas reinhardii has been analyzed using four mutant strains (imp-1, imp-10, imp-11 and imp-12). All have been shown, or are shown here, to carry mutations linked to either the plus (mt  +) or the minus (mt  -) locus, and their behavior in complementation tests has allowed us to define several distinct functions for each locus. Specifically, we propose that the mt  + locus contains the following genes or regulatory elements: a locus designated sfu, which is necessary for sexual fusion between gametes; a locus designated upp (uniparental plus), which controls aspects of chloroplast gene inheritance and perhaps also zygote maturation; and a locus designated sad, which functions in sexual adhesion. The mt  - locus also contains a sad locus as well as a gene or regulatory element designated mid, which is necessary for the minus dominance in mt  +/mt  - diploids.

scholarly journals Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.

1982 ◽  
Vol 92 (2) ◽  
pp. 378-386 ◽  
Author(s):  
U W Goodenough ◽  
P A Detmers ◽  
C Hwang
Keyword(s):  
Cell Fusion ◽  
Mating Type ◽  
Central Zone ◽  
Uranyl Acetate ◽  
Surface Coat ◽  
Wild Type ◽  
En Bloc ◽  
Mating Structure ◽  
Fusion Images ◽  
Coat Material

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.

scholarly journals Gametic differentiation in Chlamydomonas reinhardtii. III. Cell wall lysis and microfilament-associated mating structure activation in wild-type and mutant strains.

1975 ◽  
Vol 67 (3) ◽  
pp. 623-637 ◽  
Author(s):  
U W Goodenough ◽  
R L Weiss
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Mating Type ◽  
Experimental Manipulation ◽  
Wild Type ◽  
Opposite Mating Type ◽  
Mating Structure ◽  
Cell Wall Lysis ◽  
Gamete Formation ◽  
Mutant Strains

Cell fusion between mating type plus (mt+) and minus (mt-) gametes of Chlamydomonas reinhardtii is analyzed structurally and subjected to experimental manipulation. Cell wall lysis, a necessary prelude to fusion, is shown to require flagellar agglutination between competent gametes; glutaraldehyde-fixed gametes ("corpses") of one mating type will elicit both agglutination and cell wall lysis in the opposite mating type, whereas nonagglutinating impotent (imp) mutant strains are without effect. The fusion process is mediated by a narrow fertilization tubule which extends from the mt+ gamete and establishes contact with the mt- gamete. Formation of the tubule requires the "activation" of a specialized mating structure associated with the ml+ cell membrane; activation causes microfilaments to polymerize from the mating structure into the growing fertilization tubule. Mating structure activation is shown to depend on gametic flagellar agglutination; isoagglutination mediated by the lectin concanavalin A has no effect. Gametes carrying the imp-l mt+ mutation are able to agglutinate but not fuse with mt- cells; the imp-l gametes are shown to have structurally defective mating structures that do not generate microfilaments in response to gametic agglutination.

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