scholarly journals Mutual Correction of Faulty PCNA Subunits in Temperature-Sensitive Lethal mus209 Mutants of Drosophila melanogaster

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1721-1733
Author(s):  
Daryl S Henderson ◽  
Ulrich K Wiegand ◽  
David G Norman ◽  
David M Glover
Keyword(s):  
Germ Line ◽  
Position Effect ◽  
Three Dimensional ◽  
Nuclear Antigen ◽  
Dimensional Structure ◽  
Female Sterility ◽  
Temperature Sensitive ◽  
Position Effect Variegation ◽  
Dna Double Strand Breaks ◽  
Mutant Genotype

Abstract Proliferating cell nuclear antigen (PCNA) functions in DNA replication as a processivity factor for polymerases δ and ε, and in multiple DNA repair processes. We describe two temperature-sensitive lethal alleles (mus209B1 and mus2092735) of the Drosophila PCNA gene that, at temperatures permissive for growth, result in hypersensitivity to DNA-damaging agents, suppression of position-effect variegation, and female sterility in which ovaries are underdeveloped and do not produce eggs. We show by mosaic analysis that the sterility of mus209B1 is partly due to a failure of germ-line cells to proliferate. Strikingly, mus209B1 and mus2092735 interact to restore partial fertility to heteroallelic females, revealing additional roles for PCNA in ovarian development, meiotic recombination, and embryogenesis. We further show that, although mus209B1 and mus2092735 homozygotes are each defective in repair of transposase-induced DNA double-strand breaks in somatic cells, this defect is substantially reversed in the heteroallelic mutant genotype. These novel mutations map to adjacent sites on the three-dimensional structure of PCNA, which was unexpected in the context of this observed interallelic complementation. These mutations, as well as four others we describe, reveal new relationships between the structure and function of PCNA.

New Insights Into the Three-Dimensional Structure of the Nucleus and Chromosomes

1997 ◽  
Vol 3 (S2) ◽  
pp. 213-214
Author(s):  
J. Sedat ◽  
W. Marshall ◽  
A. Dernburg ◽  
J. Fung ◽  
D. Agard
Keyword(s):  
Chromosome Structure ◽  
Position Effect ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Position Effect Variegation ◽  
Genetic Loci ◽  
Interphase Nuclei ◽  
Intact Cells ◽  
3 Dimensional ◽  
Specific Localization

Recent methodology, to be described, now makes possible specific localization and analysis of genetic loci within 3-Dimensional interphase nuclei in intact cells and tissues with minimal perturbation of the chromosome structure (Dernburg and Sedat, 1997). These techniques define genetic loci that specifically interact with the nuclear envelope and interior structures; we are able to map all loci to highly localized 3-dimensional positions within Drosophila embryonic nuclei (Marshall et al, 1996). S-Dimensions-as-a-function-of-time (4-D) studies of live nuclei, from Yeast and Drosophila, allow dynamic chromosome interactions to be probed and quantitated. Our results suggest a very dynamic but highly determined and organized nucleus. Using these approaches, we can now study specific mechanisms leading to homologue chromosome pairing and position-effect variegation (Dernburg et al., 1996).

pitkinD, a Novel Gain-of-Function Enhancer of Position-Effect Variegation, Affects Chromatin Regulation During Oogenesis and Early Embryogenesis in Drosophila

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1227-1244 ◽  
Author(s):  
Steffi Kuhfittig ◽  
János Szabad ◽  
Gunnar Schotta ◽  
Jan Hoffmann ◽  
Endre Máthé ◽  
...  
Keyword(s):  
Germ Line ◽  
Position Effect ◽  
Early Embryogenesis ◽  
Position Effect Variegation ◽  
Chromatin Regulation ◽  
Gain Of Function ◽  
Position Effects ◽  
Dominant Female ◽  
Effect Variegation ◽  
Female Germ Line

Abstract The vast majority of the >100 modifier genes of position-effect variegation (PEV) in Drosophila have been identified genetically as haplo-insufficient loci. Here, we describe pitkinDominant (ptnD), a gain-of-function enhancer mutation of PEV. Its exceptionally strong enhancer effect is evident as elevated spreading of heterochromatin-induced gene silencing along euchromatic regions in variegating rearrangements. The ptnD mutation causes ectopic binding of the SU(VAR)3-9 heterochromatin protein at many euchromatic sites and, unlike other modifiers of PEV, it also affects stable position effects. Specifically, it induces silencing of white+ transgenes inserted at a wide variety of euchromatic sites. ptnD is associated with dominant female sterility. +/+ embryos produced by ptnD/+ females mated with wild-type males die at the end of embryogenesis, whereas the ptnD/+ sibling embryos arrest development at cleavage cycle 1-3, due to a combined effect of maternally provided mutant product and an early zygotic lethal effect of ptnD. This is the earliest zygotic effect of a mutation so far reported in Drosophila. Germ-line mosaics show that ptn+ function is required for normal development in the female germ line. These results, together with effects on PEV and white+ transgenes, are consistent with the hypothesis that the ptn gene plays an important role in chromatin regulation during development of the female germ line and in early embryogenesis.

The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation.

Genetics ◽  
1992 ◽  
Vol 131 (2) ◽  
pp. 345-352 ◽  
Author(s):  
J C Eissenberg ◽  
G D Morris ◽  
G Reuter ◽  
T Hartnett
Keyword(s):  
Germ Line ◽  
Position Effect ◽  
Inducible Promoter ◽  
Specific Protein ◽  
P Element ◽  
Position Effect Variegation ◽  
Chromosomal Protein ◽  
Gene Encoding ◽  
Position Effects ◽  
Effect Variegation

Abstract Chromosome rearrangements which place euchromatic genes adjacent to a heterochromatic breakpoint frequently result in gene repression (position-effect variegation). This repression is thought to reflect the spreading of a heterochromatic structure into neighboring euchromatin. Two allelic dominant suppressors of position-effect variegation were found to contain mutations within the gene encoding the heterochromatin-specific chromosomal protein HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for one of the mutant alleles (Su(var)2-504), a truncated HP-1 protein was detectable by Western blot analysis. An HP-1 minigene, consisting of HP-1 cDNA under the control of an Hsp70 heat-inducible promoter, was transduced into flies by P element-mediated germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression thus appear to depend upon the level of expression of a heterochromatin-specific protein. The implications of these observations for mechanism of heterochromatic position effects and heterochromatin function are discussed.

scholarly journals Identification of a positive regulator of the cell cycle ubiquitin-conjugating enzyme Cdc34 (Ubc3).

1996 ◽  
Vol 16 (2) ◽  
pp. 677-684 ◽  
Author(s):  
J A Prendergast ◽  
C Ptak ◽  
D Kornitzer ◽  
C N Steussy ◽  
R Hodgins ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Temperature Sensitive ◽  
Reading Frame ◽  
Positive Regulator ◽  
Morphological Defects ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.

scholarly journals Structure–Function Relationships in Yeast Tubulins

2000 ◽  
Vol 11 (5) ◽  
pp. 1887-1903 ◽  
Author(s):  
Kristy L. Richards ◽  
Kirk R. Anders ◽  
Eva Nogales ◽  
Katja Schwartz ◽  
Kenneth H. Downing ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Cold Sensitivity ◽  
Temperature Sensitive ◽  
Large Patch ◽  
Tubulin Dimer ◽  
The Core ◽  
Synthetically Lethal ◽  
Β Tubulin

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major α-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitivetub1 alleles were synthetically lethal in combination with tub3Δ, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of α-tubulin. The systematictub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast α–β-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of β-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of α-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the α–β interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201allele.

scholarly journals The effects of the pink-eyed unstable gene on the retinal pigment epithelium of the mouse

Development ◽  
1983 ◽  
Vol 78 (1) ◽  
pp. 291-298
Author(s):  
M. S. Deol ◽  
Gillian M. Truslove
Keyword(s):  
Retinal Pigment Epithelium ◽  
Germ Line ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Position Effect ◽  
Autosomal Gene ◽  
Stable Gene ◽  
Position Effect Variegation ◽  
Unstable Gene ◽  
Chromosomal Alteration

Pink-eyed unstable (pun), an autosomal gene in the mouse, causes variegation of the coat. In some melanocytes it functions as the normal allele p+, producing dark pigment, and in others as the mutant p, producing light pigment. As a study of another unstable gene at a different locus had shown that the instability was strongly influenced by the tissue environment, it seemed desirable to find out whether this also applied to pun. An examination of the retinal pigment epithelium, the only structure in mammals in which it is practicable to determine the position of individual melanocytes, showed that the distribution of dark and light cells in pun pun animals was not random. The dark cells increased in frequency with the distance from the optic nerve, suggesting that the tissue environment was a factor in the instability of the gene (i.e. in its rate of mutation), although the increase was less striking than in the other mutant. It is generally assumed that when pun behaves as p+ it is a case of reversion, and that reversion can also occur in germ cells, the revertant p+ allele subsequently behaving as a stable gene. It is here argued that it is unlikely to be a case of reversion, and that the evidence for the involvement of the germ line is inconclusive. Further, it is suggested that the phenotype of pun pun animals is probably an instance of Position Effect variegation, the instability resulting from some chromosomal alteration, which is too small to be cytologically detectable.

scholarly journals Super-Resolution Microscopy Reveals Structural Mechanisms Driving the Nanoarchitecture of a Viral Chromatin Tether

2017 ◽  
Author(s):  
Margaret J. Grant ◽  
Matthew S. Loftus ◽  
Aiola P. Stoja ◽  
Dean H. Kedes ◽  
Malcolm Mitchell Smith
Keyword(s):  
Genetic Material ◽  
Three Dimensional ◽  
Super Resolution ◽  
Nuclear Antigen ◽  
Dimensional Structure ◽  
Scaling Exponent ◽  
Three Dimensional Model ◽  
Latently Infected ◽  
Optical Reconstruction ◽  
Host Chromatin

By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the three-dimensional structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C-termini to the tandem array of episomal terminal repeats (TRs) and with their N-termini to host chromatin. Super-resolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power-law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a three-dimensional model of the episomal half of the tether that integrates and extends previously established findings from epi-fluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

scholarly journals Telomeric Position Effect Variegation in Saccharomyces cerevisiae by Caenorhabditis elegans Linker Histones Suggests a Mechanistic Connection between Germ Line and Telomeric Silencing

2003 ◽  
Vol 23 (10) ◽  
pp. 3681-3691 ◽  
Author(s):  
Monika A. Jedrusik ◽  
Ekkehard Schulze
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromatin Structure ◽  
Germ Line ◽  
Budding Yeast ◽  
Position Effect ◽  
Linker Histone ◽  
Position Effect Variegation ◽  
Linker Histones ◽  
C Elegans ◽  
Effect Variegation

ABSTRACT Linker histones are nonessential for the life of single-celled eukaryotes. Linker histones, however, can be important components of specific developmental programs in multicellular animals and plants. For Caenorhabditis elegans a single linker histone variant (H1.1) is essential in a chromatin silencing process which is crucial for the proliferation and differentiation of the hermaphrodite germ line. In this study we analyzed the whole linker histone complement of C. elegans by telomeric position effect variegation in budding yeast. In this assay an indicator gene (URA3) placed close to the repressive telomeric chromatin structure is subject to epigenetically inherited gene inactivation. Just one out of seven C. elegans linker histones (H1.1) was able to enhance the telomeric position effect in budding yeast. Since these results reflect the biological function of H1.1 in C. elegans, we suggest that chromatin silencing in C. elegans is governed by molecular mechanisms related to the telomere-dependent silencing in budding yeast. We confirmed this hypothesis by testing C. elegans homologs of three yeast genes which are established modifiers of the yeast telomeric chromatin structure (SIR2, SET1, and RAD17) for their influence on repeat-dependent transgene silencing for C. elegans.

Effect of 2-(dimethylamino) Ethyl Methacrylate on Nanostructure and Properties of pH and Temperature Sensitive Cellulose-Based Hydrogels

2020 ◽  
Vol 20 (3) ◽  
pp. 1799-1806
Author(s):  
Ping Jiang ◽  
Shaowei Chen ◽  
Linda Lv ◽  
Hongmin Ji ◽  
Gen Li ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Temperature Response ◽  
Light Transmittance ◽  
Dimensional Structure ◽  
Temperature Sensitive ◽  
The Novel ◽  
Ethyl Methacrylate ◽  
3D Network ◽  
Swelling Rate ◽  
Equilibrium Swelling Ratio

The novel double responsive cellulose/poly 2-(dimethylamino) ethyl methacrylate (PDMAEMA) hydrogel was synthesized via in situ free radical polymerization. The results from light transmittance measurements, scanning electron microscopy (SEM), mechanical property testing as well as swelling experiments demonstrated that DMAEMA played a significant impact on in hydrogel’s nanostructure.With increasing DMAEMA concentration, the transparency of hydrogels dropped and texture became softer. The incorporation of relatively hard cellulose and increase in crosslink density significantly enhanced 3D network structure of hydrogel. The tensile strength, compression and bending capabilities of the composite responsive hydrogels were fine with the DMAEMA in a certain range. When the DMAEMA concentration increased to 6 g/g(cellulose), the mechanical strength decreased noticeably. An increase in DMAEMA concentration resulted in a faster initial swelling rate and higher equilibrium swelling ratio. Further, three-dimensional structure and increase of DMAEMA concentration can also improve pH and temperature response sensitivity of hydrogels. Thus, the mechanical, swelling and responsive properties of hydrogel could be adjusted by the DMAEMA and polymerization modes.

Nuclear distribution of proliferating cell nuclear antigen (PCNA) in fertilized eggs of the starfish Asterina pectinifera

1994 ◽  
Vol 107 (12) ◽  
pp. 3291-3300 ◽  
Author(s):  
A. Nomura
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Three Dimensional ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Simultaneous Occurrence ◽  
Limited Region ◽  
Proliferating Cell

Previous studies (Nomura et al. (1991) Dev. Biol. 143, 289–296 (1993) Dev. Biol. 159, 288–297) determined the time of DNA replication period (S phase) in starfish eggs fertilized either during or after oocyte maturation. Here proliferating cell nuclear antigen (PCNA) localized within nuclei of starfish eggs was detected with an anti-PCNA human antiserum. Using a confocal laser scanning microscope, a three-dimensional structure of the PCNA region was analyzed. In eggs fertilized during maturation, PCNA started to localize within the nuclei at the same time as the initiation of the first S phase. During the S phase, the distribution of localized PCNA in a three-dimensional view coincided with the chromatin distribution. After the S phase, PCNA remained localized within the nuclei, but its distribution no longer coincided with the chromatin distribution. In eggs fertilized after maturation, however, PCNA started to localize within the female pronuclei about 10 minutes ahead of the first S phase. Localized PCNA occupied only a limited region of the nuclei without diffusing over the whole nuclear area. Chromatin distributed around the peripheral region of the nuclei mostly outside the PCNA region. When the first S phase was initiated, the chromatin distribution became coincident with the PCNA region. Later behavior of PCNA was the same as that of the eggs fertilized during maturation. The precocious localization of PCNA in those eggs fertilized after maturation simply demonstrates that the ‘postactivation process’ for preparing DNA replication is triggered by fertilization and PCNA localization and S phase are sequentially initiated with a time-lapse. On the other hand, the simultaneous occurrence of them seen in those eggs fertilized during maturation indicates that the postactivation process must be going on in parallel with the maturation process.

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