bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi
Authors: Luporini Pierangelo , Miceli Cristina , Ortenzi Claudio , Vallesi Adriana
Published: May 2005

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat‐2, which controls the pheromone Er‐2, Er‐2 mRNA synthesis and mature Er‐2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5‐ to 10‐fold lower than at maturity. In addition, experiments of 125 I‐Er‐2 binding and crosslinking provided evidence that autocrine pheromone‐binding sites, showing values of the dissociation constant of the order of 10−9 M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 106 per cell of 5–7 fissions of age, to about 16 × 106 at maturity. These results were taken to suggest that a pheromone‐receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone‐receptor density high enough to transform the cell from “immature” to “adult,” that is competent to respond as well to pheromones of conspecific, genetically different cells. © 1992 Wiley‐Liss, Inc.
Suicide is not the inevitable outcome of “perpetual” selfing in tetrahymenines collected from natural habitats
Authors: Simon Ellen M. , Meyer E. Barbara
Published: May 2005

A significant fraction of the Tetrahymena clones isolated from natural habitats self (mating occurs within a clone). Early attempts to study such clones failed because stable subclones were rarely, if ever, observed, and isolated pairs all died. Isozyme analysis revealed that these wild selfers were a diverse group; some were very similar to T. australis, a species with synclonal mating type determination and to T. elliotti, shown recently to have a karyonidal mating type system. One originally stable clone of T. australis included some selfing clones after a few years in our laboratory. Other clones manifested unique zymograms.

Subclones isolated from 18 selfer strains were heterogeneous. All subclones of several selfers mated massively at each transfer through 100 fissions. Selfing among subclones of other selfers was highly variable or not observed. Although 77% of the pairs isolated died, and 9% of the pair cultures selfed, 15 selfers yielded some viable nonselfing “immature” progeny. Additional immature progeny were obtained by isolating pairs from macronuclear retention synclones. Although some “immature” progeny eventually selfed, most remained stable. Giemsa staining revealed macronuclear anlagen in nearly all mating pairs and some anomalies. Crosses among the F1 progeny clones of the T. elliotti selfers yield viability data comparable to those from crosses among normal strains. Perhaps perpetual selfing is a mechanism of getting rid of deleterious combinations of genes and uncovering better combinations in homozygous state by playing genetic roulette. © 1992 Wiley‐Liss, Inc.
Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: Early expression of the zygotic genotype
Authors: Kaczanowski Andrzej
Published: May 2005

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild‐type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1–2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them.

After addition of 10mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs.

During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macro‐nuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. © 1992 Wiley‐Liss, Inc.
A micronucleus‐limited sequence family in Tetrahymena thermophila: Organization and sequence conservation
Authors: Tsao Nora N‐G , Tsao Smiley G‐S , Pearlman Ronald E.
Published: May 2005

During mocronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus‐limited sequence families. We have previously identified a moderately repetitive micronu‐cleus‐limited sequence family called X‐H (family members hybridize to an approximately 450 bp Xbal‐HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel‐1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel‐1 terminal sequences. We have nucleotide sequence data for three cloned members of the X‐H family. This analysis has demonstrated that the longest cloned members of the X‐H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus‐limited. © 1992 Wiley‐Liss, Inc.
Tetrahymena Telomerase RNA levels increase during macronuclear development
Authors: Avilion Ariel A. , Harrington Lea A. , Greider Carol W.
Published: May 2005

Telomeres, the G‐rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady‐state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2‐to 5‐fold at 9–15 h, and 1.5‐ to 3.5‐fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley‐Liss, Inc.
Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated, heterozygous macronuclei of Tetrahymena thermophila
Authors: Orias Eduardo , Bradshaw Amy D.
Published: May 2005

Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4–8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification. We discuss the relevance of this stochastic developmental variability to classical genetic observations of Nanney and their collaborators on other T. thermophila loci. © 1992 Wiley‐Liss, Inc.
Bacteriophage λ DNA fragments replicate in the Paramecium macronucleus: Absence of active copy number control
Authors: Sookkim Chung , Preer John R. , Polisky Brry
Published: May 2005

We show that bacteriophage λ DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit‐length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. © 1992 Wiley‐Liss, Inc.
DNA elimination and its relation to quantities in the macronucleus of Tetrahymena
Authors: Bodenbender J. , Prohaska A. , Jauker F. , Hipke H. , Cleffmann G.
Published: May 2005

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level.

We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminate. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA.

We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.

The result indicates that in a given cell line at least one of the DNA molecules becoms extinct after 60 generations which we expect would cause the cell's extinction and restrict a clone's life to 60 generations. As this does not happen in nature, there must be some control of the copy numbers preventing their extinction during vegetative multiplication. Whether elimination increases or decreases the imbalance of genes remains to be investigated. © 1992 Wiley‐Liss, Inc.
Variable copy number of macronuclear DNA molecules in Tetrahymena
Authors: Brunk Clifford F. , Navas Patrick A.
Published: May 2005

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000‐kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley‐Liss, Inc.
Rate of phenotypic assortment in Tetrahymena thermophila
Authors: Doerder F. P. , Deak J. C. , Lief J. H.
Published: May 2005

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf/ the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with ∼45 assorting units, as has been asserted. © 1992 Wiley‐Liss, Inc.
Genetic characterization of the secretory mutant MS‐1 of Tetrahymena thermophila: Vacuolarization and block in secretion of lysosomal hydrolases are caused by a single gene mutation
Authors: Hünseler Peter , Tiedtke Arno
Published: May 2005

The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS‐1, of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitu‐tively in secretion, MS‐1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F‐2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS‐1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS‐1 with nullisomic strains, all secreting lysosomal hydrolases at wild‐type rates, was performed. The sec phenotype was expressed in monosomic‐4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila. © 1992 Wiley‐Liss, Inc.
Ultrastructure and cell size‐dependent regulation of the adoral zone of membranelles in the heterotrich ciliate Stentor coeruleus
Authors: Jacobson Patrice M. , Lynn Denis H.
Published: May 2005

The number and length of oral membranelles were determined for both large and small Stentor from well‐fed, growing cultures and nutrient‐deprived cultures, respectively. Small cells possess both significantly fewer and shorter membranelles than do large cells. For both large and small cells, each membranelle is composed of three rows of basal bodies. The membranelles closest to the gullet have a third row that is only slightly shorter than the other two. The third row becomes rapidly shorter as membranelles become increasingly distant from the gullet. A short distance from the gullet, and for the remainder of the band, the third row is composed ofonly one to four basal bodies. The first two rows consist of approximately 35 basal bodies each in large cells and approximately 26 basal bodies each in small cells. This indicates that Stentor regulates the number of basal bodies per row, but not the number of rows, in response to changes in cell size. © 1992 Wiley‐Liss, Inc.
Timing of formation of Tetrahymena contractile ring microfilaments investigated by inhibition with skeletal muscle actin
Authors: Ohba Hiroyoshi , Hirono Masafumi , Edamatsu Masaki , Watanabe Yoshio
Published: May 2005

Understanding the mechanism that determines the cell division plane is one of the most important problems in the fields of cell and developmental biology. Studying the timing and site of formation of contractile ring (CR) micro‐filaments provides key information for solving the problem. We tried to create a nonfunctional CR in Tetrahymena by microinjecting rabbit skeletal muscle actin, which can copolymerize with Tetrahymena actin but has properties different from those of Tetrahymena actin. When skeletal muscle actin was injected in a predivision stage, before the onset of furrow constriction, long‐term arrest of cell division was observed. Muscle actin did not cause any delay in cell division when the actin was injected at any stage other than the predivision stage. In all cases, muscle actin had little affect on other actin‐related functions. Injected skeletal muscle actin polymerized near the equatorial division plane in cases of cell division arrest; it polymerized at other nonspecific locations when cell division was observed. Arrest occurred when the microinjection took place in the 17‐min period just before the start of furrowing. This period coincides with the occurrence of equatorial deposits of p85, which is also suggested to be required for the determination of the division plane. The present experimental results are consistent with the idea that p85 is a crucial factor for determining the cell division plane and also functions as a polymerization nucleus for CR microfilaments. © 1992 Wiley‐Liss, Inc.
The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila
Authors: Kaczanowska J. , Buzanska L. , Frontczak M.
Published: May 2005

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature‐sensitive cell‐division‐arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35°C), some of the chains were arrested prior to the start of fission line formation (D‐shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B‐shaped chains). In their second cell cycle following the temperature shift, the D‐shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B‐shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre‐existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity. © 1992 Wiley‐Liss, Inc.
Nuclear functions in postconjugational development of Paramecium caudatum: Ability to form food vacuoles analyzed by nuclear elimination and implantation
Authors: Mikami Kazuyuki
Published: May 2005

Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley‐Liss, Inc.
Timing of micronuclear mitosis and its relation to commitment to division inParamecium tetraurelia
Authors: Adl Sina M. , Berger James D.
Published: May 2005

This study examines the timing of micronuclear mitosis during the vegetative cell cycle and shows that mitosis begins early in the division process and coincides approximately with the earliest stages of oral morphogenesis (about 0.6 in the cell cycle in synchronous cell samples). The cc1 mutation blocks cell cycle progression prior to the point of commitment to division. Although the cc1 mutation blocks macronuclear DNA synthesis under restrictive conditions, it does not block micronuclear DNA synthesis. However, absence of functional cc1 gene product leads to blockage of micronuclear mitosis prior to completion of anaphase. This point coincides with commitment to division and is also the point at which oral morphogenesis is blocked in cc1 cells. The tim‐ings of the transition points for micronuclear mitosis and oral morphogenesis in cc1 cells are closely associated in both synchronous cell samples and in asynchronous cultures. © 1992 Wiley‐Liss, Inc.
Molecular phylogeny of ciliates: What does it tell us about the evolution of the cytoskeleton and of developmental strategies?
Authors: Fleury Anne , Delgado Pilar , Iftode Francine , Adoutte André
Published: May 2005

An rRNA phylogeny of 22 species of ciliates belonging to seven of Small and Lynn's eight classes has been obtained by distance and parsimony methods. It displays good congruence with classical systematics at low taxonomic levels and several major surprises at higher levels: (1) The species analyzed group into five major branches, four of which emerge almost simultaneously: hypotrichs, oligohymenophorans, lito‐stomes, and nassophoreans corresponding to four of Small and Lynn's classes. The simultaneous emergence of these groups contradicts the long accepted view that litostomes (a group with “simple”, symmetrical, apical oral apparatus) are “primitive,” while hypotrichs are “highly evolved.” (2) Heterotrichs group with a karyorelictid, together forming the first emerging branch. While this supports the view that karyorelictids may be early‐emerging ciliates, it completely explodes the traditional “spirotrichs” taxon, which united heterotrichs and hypotrichs. Instead, this reinforces the concept of Postciliodesmatophora and suggests that asymmetric oral apparatuses (i.e., with distinct paroral and adoral ciliatures) may be primitive in ciliates. The global topology of the tree therefore does not fit with the classical views of ciliate evolution, from “simple” oral apparatus and stomatogenesis to “complex” ones. Instead, a rather striking agreement with the strategy adopted to construct the cortical framework was disclosed. We noted that the cytoskeletal elements used to strengthen the cell surface could be subdivided into four main types: epiplasm, filaments, continuous microtu‐bules, or basal body derived fibers. These four types fitted quite well with the major evolutionary lines disclosed by the molecular phylogeny. We therefore discuss unorthodox hypotheses assuming an early explosive radiation of ciliates into a small number of major lineages differing essentially in the solution adopted to subtend the cell surface and anchor the infraciliature. © 1992 Wiley‐Liss, Inc.
Early gene interaction during prepupal expression of Drosophila arginine kinase
Authors: James Judith M. , Collier Glen E.
Published: May 2005

Arginine kinase displays a distinctive rise and fall in specific activity and specific protein levels during the prepupal stage of Drosophila development with maximal activity occurring at morphological stage P3. This developmentally regulated peak is under the influence of ecdysone. Altered doses of the major ecdysone‐inducible “early” genes at cytological regions 75B and 2B5 alter this pattern of expression while altered doses of another major “early” gene at 74EF have no effect. We hypothesize that a product of the 2B5 locus and a product of the 75B locus interact to effect this developmental pattern of expression of Drosophila arginine kinase. © 1992 Wiley‐Liss, Inc.
The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement
Authors: Keller Anne‐Marie , Mouël Anne Le , Caron François , Katinka MichaëL , Meyer Eric
Published: May 2005

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed. © 1992 Wiley‐Liss, Inc.
Localization in situ of polyhomeotic transcripts in Drosophila embryos reveals spatially restricted expression beginning at the blastoderm stage
Authors: Deatrick Janet
Published: May 2005

polyhomeotic is a member of a group of genes, the Pc‐group responsible for the maintenance of gene expression during development. In particular, the Pc‐group of genes is involved in the correct expression of homeotic genes of the bithorax and Antennapedia complexes.

Molecular analysis reveals that the Pc‐group genes function relatively late in development, once homeotic gene expression has been correctly initiated. This initiation of homeotic gene expression depends on interaction between genes in the segmentation gene hierarchy, the gap and pair‐rule genes.

The in situ analysis presented here indicates that polyhomeotic transcripts are expressed in a spatially restricted fashion early in development. This blastoderm expression is under the control of genes in the segmentation hierarchy. Given these results, and the role of polyhomeotic in the correct maintenance of homeotic gene expression, I propose that polyhome otic expression may relay the initiation of homeotic gene expression with other mechanisms involved in the maintenance of this expression, involving the other Pc‐group genes. © 1992 Wiley‐Liss, Inc.
The rad3‐1 mutant is defective in axial core assembly and homologous chromosome pairing during meiosis in the basidiomycete Coprinus cinereus
Authors: Pukkila Patricia J. , Skrzynia Cécile , Lu Benjamin C.
Published: May 2005

We have utilized spreading methods as well as serial sectioning three‐dimensional reconstruction to examine meiotic chromosome behavior in cells homozygous for the rad3‐1 mutation in Coprinus cinereus. Comparison of 42 wild‐type nuclei that had been spread, stained with silver, and viewed by electron microscopy with 30 mutant nuclei treated in the same manner revealed several defects in the mutant. Axial core formation was defective in the mutant, although limited side‐by‐side association of axial cores was observed. To detect any differences in three‐dimensional architecture between the wild‐type and mutant nuclei, we reconstructed three of the former and six of the latter after serial sectioning. It was not possible to trace the expected number of axial cores from section to section in the mutant, although some tripartite synaptonemal complex was observed. Many axial core ends failed to terminate in the nuclear envelope in the mutant. This spectrum of defects (incomplete axial core assembly with some tripartite synaptonemal complex formation) had not been observed previously in either C. cinereus or other systems. We conclude that this combination of spreading and sectioning methods is very useful for analysis of meiotic mutants. © 1993 Wiley‐Liss, Inc.
Effects of several meiotic mutations on female meiosis in maize
Authors: Golubovskaya Inna , Avalkina Nadezhda A. , Sheridan William F.
Published: May 2005

A modified enzyme digestion technique of ovary isolation followed by staining and squash preparation has allowed us to observe female meiosis in normal maize meiotically dividing megaspore mother cells (MMCs). The first meiotic division in megasporogenesis of maize is not distinguishable from that in mi‐crosporogenesis. The second female meiotic division is characterized as follows: (1) the two products of the first meiotic division do not simultaneously enter into the second meiotic division; as a rule, the chalazal‐most cell enters division earlier than the micropylar one, (2) often the second of the two products does not proceed with meiosis, but degenerates, and (3) only a single haploid meiotic product of the tetrad remains alive, and this cell proceeds with three rounds of mitoses without any intervening cell wall formation to produce the eight‐nucleate embryo sac. This technique has allowed us to study the effects of five meiotic mutations (aml, aml‐pral, afdl, dsy *‐9101, and dvl) on female meiosis in maize. The effects of the two alleles of the aml gene (aml and aml‐pral) and of the afdl and dsy *‐9101mutations are the same in both male and female meiosis. The aml allele prevents the entrance of MMCs into meiosis and meiosis is replaced by mitosis; the aml‐pral permits MMCs to enter into meiosis, but their progress is stopped at early prophase I stages. The afdl gene is responsible for substitution of the first meiotic (reductional) division by an equational division including the segregation of sister chromatid centromeres at anaphase I. The dsy * ‐9101 gene exhibits abnormal chromosome pairing; paired homologous chromosomes are visible at pachytene, but only univalents are observed at diakinesis and metaphase I stages. These mutation specific patterns of abnormal meiosis are responsible for the bisexual sterility of these meiotic mutants.

The abnormal divergent shape of the spindle apparatus and the resulting abnormal segregation of homologous chromosomes observed in micro‐sporogenesis in plants homozygous for the dv1 mutation have not been found in meiosis of megasporogenesis. Only male sterility is induced by the dv1 gene in the homozygous condition. © 1993 Wiley‐Liss, Inc.
Temporal and spatial distribution of meiotin‐1 in anthers of Lilium longiflorum
Authors: Hasenkampf Clare , Qureshi Misbah , Horsch Andrea , Riggs C. Daniel
Published: May 2005

Meiotin‐1 is a chromatin associated, conserved protein found in meiocytes immediately preceding and during meiosis and is thought to have a role in determining the higher order structure of meiotic chromosomes [Riggs and Hasenkampf: Chromosoma 101:92–98, 1991]. In the studies reported here we utilized immunoblotting and immunocytochemical techniques to examine the temporal and spatial distribution of meiotin‐1 in the anthers of Lilium longiflorum. The results with the anti‐meiotin‐1 immune serum were compared with those obtained using an anti‐his‐tone Hl immune serum. The anti‐histone Hl immune serum gave constant immunostaining in all cell types of the anther at all of the stages tested. In contrast, the anti‐meiotin‐1 immune serum only gave immunostaining with the microsporocytes and to a lesser extent with the nutritive layer, the tapetum. It did not react with the cells of the anther wall. Meiotin‐1 immunostaining was first present in significant quantities in the microsporocytes as they accumulated in the G1 phase before the onset of premeiotic S phase and reached peak levels in the time interval between leptotene and pachytene—the same interval when chromosome synapsis occurs and when reciprocal genetic exchange is thought to occur. Immunostaining for both meiotin‐1 and histone H1 uniformly decorates the longitudinal axes of the chromosomes. Our data are consistent with the idea that the role of meiotin‐1 may be to tag certain sequences or to limit the degree of chromosome condensation that occurs during meiotic prophase. © 1993 Wiley‐Liss, Inc.
Promoter‐containing ribosomal DNA fragments function as X‐Y meiotic pairing sites in D. melanogaster males
Authors: Merrill Cynthia J. , Chakravarti Dhrubajyoti , Habera Ledare , Das Suchita , Eisenhour Lynn , McKee Bruce D.
Published: May 2005

The Drosophila melanogaster ribosomal DNA (rDNA) functions as an X‐Y meiotic pairing site. Deletions encompassing the X chromosomal rDNA block (located in the heterochromatin) disrupt X‐Y pairing and disjunction. Insertions of single, complete rRNA genes at ectopic locations on the heterochromatically deficient X partially restore X‐Y pairing capacity. This study was undertaken to test fragments of an rDNA repeat for the ability to stimulate X‐Y pairing and disjunction and to test for relationships between pairing capacity and two other phenotypes associated with rDNA insertions: transcription and the ability to organize a nucleolus. Insertions of three different fragments, all of which retained the rDNA promoter and upstream spacer sequences and which differed among each other in the length of downstream sequences, were obtained by P‐element mediated transformation. One of the fragments is truncated only 140bp downstream from the promoter. Insertions of all three fragments proved capable of stimulating X‐Y disjunction. Double insertions were substantially more effective than single insertions. RNA/PCR analysis was used to show that transcripts initiated at the inserted rDNA promoters are present in testis RNA from all insertions. Treatment with an antinucleolar antibody revealed that none of the insertions was associated with a mininucleolus. Thus promoter‐containing rDNA fragments are autonomously capable of being transcribed and of functioning as X‐Y pairing sites, but not of forming a mini‐nucleolus. © 1993 Wiley‐Liss, Inc.
Pulsed‐field gel analysis of the pattern of DNA double‐strand breaks in the Saccharomyces genome during meiosis
Authors: Game John C.
Published: May 2005

Pulsed‐field gel electrophoresis (PFGE) has been used to study the timing, frequency, and distribution of double‐strand breaks (DSBs) in chromosomal‐sized DNA during meiosis in yeast. It has previously been shown that DSBs are associated with some genetic hotspots during recombination, and it is important to know whether meiotic recombination events routinely initiate via DSBs. Two strains have been studied here—a highsporulating homothallic wild type and a congenic mutant strain carrying a rad50S mutation. This mutant has previously been reported to accumulate broken molecules in meiosis to much higher frequencies than wild type and to abolish the characteristic wild‐type processing of DNA that has been observed at the break sites. When whole chromosomes are resolved by PFGE, both strains show some broken molecules starting at the time that cells commit to genetic recombination. Breakage has been assessed primarily on Chromosome III and Chr. XV, using Southern hybridization to identify these chromosomes and their fragments. At any one time, break frequency in wild type is much lower than the cumulative frequency of recombination events that occur during meiosis. However, there is suggestive evidence that each break is short‐lived, and it is therefore difficult to estimate the total number of breaks that may occur. In rad50S, chromosome breaks accumulate to much higher levels, which are probably broadly consistent with the estimated number of recombination events in wild type. However, since rad50S is substantially defective in completing recombination, it is not known for certain if it initiates events at wild‐type frequencies.

A surprising feature of the data is that a strong banding pattern is observed in the fragment distribution from broken chromosomes in both strains, implying that at least much of the breakage occurs at specific sites or within short regions. However, with the exception of the rDNA region on Chr. XII, assessment of the genetic map indicates that recombination can occur almost anywhere in the genome, although some regions are much hotter than others. Possible reasons for this apparent paradox are discussed. It may in part result from breakage levels too low for adequate detection in cold regions but may also imply that recombination events are localized more than previously realized. Alternatively, there may be a more indirect relationship between break sites and the associated recombination events. © 1993 Wiley‐Liss, Inc.
Minimal extent of homology required for completion of meiotic recombination in Saccharomyces cerevisiae
Authors: Hayden Martha S. , Byers Breck
Published: May 2005

The minimal length of contiguous homology required for successful completion of meiotic recombination was investigated by using heterologous insertions to delimit homologous segments of chromosome III in the yeast Saccharomyces cerevisiae. Constructs created in vitro by insertion of selectable markers into the LEU2 locus were transplaced into haploid strains, which were then mated to create diploids containing pairs of insertion heterologies at various distances. Analysis of the meiotic products from these diploids revealed a gradient in the frequency of both reciprocal and nonreciprocal recombination declining monotonically from the 5′ end of LEU2. Both types of event were found to be restricted by the presence of the insertion heterologies. The spo 13 single division meiosis was exploited to develop a plating assay in which LEU2 diploid spores containing reciprocally recombinant strands derived from events occurring completely within the interval flanked by the insertion heterologies were selected by random spore methods. Reciprocal recombination frequencies measured with this assay decreased linearly with extent, extrapolating to a minimal homology requirement of 150–250 nucleotides. When homology was most severely restricted, unexpected flanking marker configurations among reciprocal recombinants within LEU2 demonstrated the occurrence of complex recombination events. In addition to detecting reciprocal recombinants, the system is capable of measuring the probability that a non‐reciprocal recombination event will have one endpoint between the heterologous inserts and the other lying outside the interval. The minimal length of homology required for this aspect of recombination was found to be 25–60 nucleotides. © 1993 Wiley‐Liss, Inc.
Regulation of two different hsp70 transcript populations in steroid hormone‐induced fungal development
Authors: Silver Julie C. , Brunt Shelley A. , Kyriakopoulou Garyfallia , Borkar Monica , Nazarian‐Armavil Vigen
Published: May 2005

In the filamentous oomycete fungus Achlya, the differentiation of gamete bearing structures on vegetative hyphae of the male mating type, is induced by the Achlya steroid hormone, antheridiol. Among the several metabolically labeled intracellular proteins whose synthesis or accumulation is altered by hormone treatment are steroid‐induced 85‐kDa and 68‐ to 78‐kDa proteins. The 85‐kDa protein was previously shown to be the Achlya heat shock protein hsp85 [Brunt et al., 1990; Brunt and Silver, 1991], a component of the putative Achlya steroid hormone receptor. It was of interest to determine if the antheridiol‐induced “70‐kDa” proteins were hsp70‐family heat shock proteins and if hormone treatment‐induced changes in the level of hsp70 transcripts. Two different Achlya hsp70 genomic sequences were cloned and used to investigate these questions. The two hsp70 sequences recognized two different mycelial transcript populations, one of which was regulated also by decreased glucose. Of note, both of the two hsp70 transcript populations were found to be regulated by antheridiol. The hormone‐induced chcnges in hsp70 transcript levels were temporally correlated with the onset of massive lateral hyphal branching and alterations in the pattern of secreted N‐linked glycoproteins which occur in hormone‐treated mycelia. To our kncwledge, this represents one of the first reports on changes in hsp70 proteins and transcripts during fungal differentiation. Our results may have implications for the role of heat shock proteins in hyphal branching and secretion in filamentous fungi and perhaps other cell types. © 1993Wiley‐Liss, Inc. Inc.
Characterization of two Maize HSP90 heat shock protein genes: Expression during heat shock, embryogenesis, and pollen development
Authors: Marrs Kathleen A. , Casey Elena Silva , Capitant Sherry A. , Bouchard Robert A. , Dietrich Paul S. , Mettler Irvin J. , Sinibaldi Ralph M.
Published: May 2005

We have isolated two genes from Zea mays encoding proteins of 82 and 81 kD that are highly homologous to the Drosophila 83‐kD heat shock protein gene and have analyzed the structure and pattern of expression of these two genes during heat shock and development. Southern blot analysis and hybrid select translations indicate that the highly homologous hsp82 and hsp81 genes are members of a small multigene family composed of at least two and perhaps three or more gene family members. The deduced amino acid sequence of these proteins based on the nucleotide sequence of the coding regions shows 64‐88% amino acid homology to other hsp90 family genes from human, yeast, Drosophila, and Arabidopsis. The promoter regions of both the hsp82 and hsp81 genes contain several heat shock elements (HSEs), which are putative binding sites for heat shock transcription factor (HSF) commonly found in the promoters of other heat shock genes. Gene‐specific oligonucleotide probes were synthesized and used to examine the mRNA expression patterns of the hsp81 and hsp82 genes during heat shock, embryogenesis, and pollen development. The hsp81 gene is only mildly heat inducible in leaf tissue, but is strongly expressed in the absence of heat shock during the premeiotic and meiotic prophase stages of pollen development and in embryos, as well as in heat‐shocked embryos and tassels. The hsp82 gene shows strong heat inducibility at heat‐shock temperatures (37–42°C) and in heat shocked embryos and tassels but is only weakly expressed in the absence of heat shock. Promoter‐GUS reporter gene fusions made and analyzed by transient expression assays in Black Mexican Sweet (BMS) Maize protoplasts also indicate that the hsp82 and hsp81 are regulated differentially. The hsp82 promoter confers strong heat‐inducible expression of the GUS reporter gene in heat‐treated cells (60‐ to 80‐fold over control levels), whereas the hsp81 promoter is only weakly heat inducible (5‐ to 10‐fold over control levels). © 1993Wiley‐Liss, Inc.