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PubMed1998

Caught in the act: deducing meiotic function from protein immunolocalization.

Meiotic division comprises a complex series of events, many of which are unique in the life cycle of the organism. The process utilizes both proteins that participate in normal mitotic cell cycle progression and DNA damage repair and proteins expressed only during meiosis. Until recently, few meiotic protein participants had been identified and characterized, but several recent developments have changed this situation. Proteins can be selected for study based on their cDNA sequence and similarity to known proteins with "suspicious" repair/recombination or cell cycle activity and antibodies against these proteins applied to meiotic nuclei to test for activity. With the development of gene sequence data bases from many organisms, similarity to a known protein need not be based on the same or even a closely related species. Potential interactions between two or more proteins can be identified and involvement in a common process inferred based on antibody colocalization. The gene sequence can be disrupted and the effect on meiotic progression directly examined. Previously identified structures, the synaptonemal complex (SC) and both early and late recombination nodules (RNs), provide structural and temporal landmarks that assist in inferring meiotic activity of the protein being studied. Mammalian meiosis is especially attractive for these kinds of studies since spermatocyte and oocyte nuclei are large with distinct nuclear organelles and since meiosis is highly protracted, occurring over a period of several days. In this chapter, an approach to the study of mammalian meiosis based on use of specific antibodies is outlined and methods of coupling this approach to other techniques, such as targeted gene disruption or chromosome aberrations, are described. Some of the proteins already identified as participants in meiotic prophase are reviewed and their presumed functions discussed.

Animals↗
PubMed1997

Nucleoprotein-based nanoscale assembly.

A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-fluorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M-HhaI and M x MspI. When 5-fluorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M x HhaI or FCGG for M x MspI), we show that the first recognition sequence becomes an address for M x HhaI, while the second sequence becomes an address for M x MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M-HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.

Base Sequence↗
PubMed1997

Chromosome rearrangements in Neurospora and other filamentous fungi.

Knowledge of fungal chromosome rearrangements comes primarily from N. crassa, but important information has also been obtained from A. nidulans and S. macrospora. Rearrangements have been identified in other Sordaria species and in Cochliobolus, Coprinus, Magnaporthe, Podospora, and Ustilago. In Neurospora, heterozygosity for most chromosome rearrangements is signaled by the appearance of unpigmented deficiency ascospores, with frequencies and ascus types that are characteristic of the type of rearrangement. Summary information is provided on each of 355 rearrangements analyzed in N. crassa. These include 262 reciprocal translocations, 31 insertional translocations, 27 quasiterminal translocations, 6 pericentric inversions, 1 intrachromosomal transposition, and numerous complex or cryptic rearrangements. Breakpoints are distributed more or less randomly among the seven chromosomes. Sixty of the rearrangements have readily detected mutant phenotypes, of which half are allelic with known genes. Constitutive mutations at certain positively regulated loci involve rearrangements having one breakpoint in an upstream regulatory region. Of 11 rearrangements that have one breakpoint in or near the NOR, most appear genetically to be terminal but are in fact physically reciprocal. Partial diploid strains can be obtained as recombinant progeny from crosses heterozygous for insertional or quasiterminal rearrangements. Duplications produced in this way precisely define segments that cover more than two thirds of the genome. Duplication-producing rearrangements have many uses, including precise genetic mapping by duplication coverage and alignment of physical and genetic maps. Typically, fertility is greatly reduced in crosses parented by a duplication strain. The finding that genes within the duplicated segment have undergone RIP mutation in some of the surviving progeny suggests that RIP may be responsible for the infertility. Meiotically generated recessive-lethal segmental deficiencies can be rescued in heterokaryons. New rearrangements are found in 10% or more of strains in which transforming DNA has been stably integrated. Electrophoretic separation of rearranged chromosomal DNAs has found useful applications. Synaptic adjustment occurs in inversion heterozygotes, leading progressively to nonhomologous association of synaptonemal complex lateral elements, transforming loop pairing into linear pairing. Transvection has been demonstrated in Neurospora. Beginnings have been made in constructing effective balancers. Experience has increased our understanding of several phenomena that may complicate analysis. With some rearrangements, nondisjunction of centromeres from reciprocal translocation quadrivalents results in 3:1 segregation and produces asci with four deficiency ascospores that occupy diagnostic positions in linear asci. Three-to-one segregation is most frequent when breakpoints are near centromeres. With some rearrangements, inviable deficiency ascospores become pigmented. Diagnosis must then depend on ascospore viability. In crosses between highly inbred strains, analysis may be handicapped by random ascospore abortion. This is minimized by using noninbred strains as testers.

Chromosome Aberrations↗
PubMed1996

Distribution of gamma-tubulin differs in primary and secondary oocytes of Ephestia kuehniella (Pyralidae, Lepidoptera).

In a previous study, barrel-shaped spindles were found in metaphase I oocytes of Ephestia kuehniella (Pyralidae, Lepidoptera). Aster microtubules (MTs) were missing (Wolf, 1993: Cell Motil Cytoskeleton 24:200-204). This points to an acentriolar organization of the spindle apparatus. The present study was aimed at the question of whether gamma-tubulin, a newly detected member of the tubulin superfamily that has often been identified in microtubule-organizing centers, plays a role in the nucleation of MTs in meiotic spindles of the moth. To this end, the distribution of gamma-tubulin was examined in oocytes of E. kuehniella using an antibody against gamma-tubulin in combination with indirect immunofluorescence. The antibody evenly decorated spindle MTs in metaphase I oocytes of the moth. Enhanced staining of the spindle poles was not detectable. In subsequent stages of meiosis, gamma-tubulin was gradually lost from spindle MTs and was then found at the surface of the so-called elimination chromatin. Female meiosis in Lepidoptera is achiasmatic. The elimination chromatin, i.e., modified and persisting synaptonemal complexes, is believed to keep homologous chromosomes linked until the onset of anaphase I. In meiosis I of female Lepidoptera, the elimination chromatin persists at the spindle equator between the segregating chromatin masses. It is plausible to assume that gamma-tubulin is involved in spindle organization in the absence of canonical centrosomes. In MTs of metaphase II spindles of E. kuehniella, gamma-tubulin was no longer detectable with our immunological approach. This points to a far-reaching change in spindle organization during transition from meiosis I to meiosis II.

Animals↗
PubMed1997

Analysis of expression and function of topoisomerase I and II during meiosis in male mice.

Topoisomerases are nuclear enzymes that remove torsional stress in DNA. Their function is important for replication, transcription, chromosome condensation, and chromosome segregation during mitosis and meiosis. The goal of this work is to analyze both expression and function of topoisomerases during the meiotic stages of mammalian spermatogenesis. The patterns of expression of topoisomerase I and topoisomerase II alpha genes were followed on Northern blots of RNA from testes of mice of different ages and from specific germ cell populations. The transcript of the topoisomerase I gene was highest in somatic cells of the testis and in the mitotically proliferating spermatogonia and meiotic prophase spermatocytes, with the level of transcript decreasing dramatically in postmeiotic spermatids. In contrast, the levels of topoisomerase II alpha transcript were negligible in germ-cell free testes and highest in late meiotic prophase cells and round spermatids. Enzyme activity for both topoisomerase I and topoisomerase II was detected in both pachytene spermatocytes and in round spermatids; topoisomerase II exhibited a higher level of activity in meiotic spermatocytes than in round spermatids. In cultured cells, camptothecin, an inhibitor of topoisomerase I, caused some abnormalities of paired meiotic homologs, but did not inhibit the transition to metaphase. In contrast, teniposide and ICRF-193, inhibitors of topoisomerase II, dramatically inhibited the formation of metaphase chromosomes in cells induced to progress from prophase to metaphase. However, the disassembly of the synaptonemal complex was not inhibited, indicating that this process could be uncoupled from condensation of chromatin to form chromosomes. These studies constitute evidence for a functional requirement for topoisomerase II activity in the transition from meiotic prophase to meiotic metaphase I in mammalian spermatocytes.

Animals↗
PubMed1998

Cre expression in primary spermatocytes: a tool for genetic engineering of the germ line.

Transgenic mice were generated expressing a testicular Cre recombinase driven by promoter sequences derived from the gene encoding Synaptonemal Complex Protein 1 (Sycp1), expressed at an early stage of the male meiosis (leptotene to zygotene). Recombination at target LoxP sites was examined during germinal differentiation in mice harboring Sycp1-Cre and a second transgene where LoxP sites flank either the beta geo coding region, the Pgk1 promoter, or a tk-neo cassette inserted into the Rxr alpha locus. The LoxP-flanked transgenes were stably maintained in the somatic tissues of the double transgenic animals, as well as in the progeny of the females. Mice born after mating the double-transgenic males with normal females showed extensive deletions of the LoxP-flanked sequences. When the males were hemizygous for the Sycp1-Cre transgene, the deletions were observed even in the fraction of the offspring which had not inherited the Cre gene, thus demonstrating that expression occurred in the male parent during spermatogenesis. The high efficiency of excision at the LoxP sites makes the Sycp1-Cre transgenic males suitable for evaluating the role of defined gene functions in the germinal differentiation process.

Animals↗
PubMed1988

New mutation causing sterility in the mouse.

A new murine mutation, skeletal fusions with sterility, sks, has been identified. This mutation causes arrest during the pachytene stage of virtually all spermatogenic cells. Defects in chromosome pairing and appearance of the synaptonemal complex during meiosis in the male are apparent, but defective pairing is probably not the cause of sterility. Affected females are functionally infertile. Oocytes are capable of undergoing meiotic maturation in vitro but cannot be fertilized in vitro. Affected individuals of both sexes are characterized by fusions of vertebrae and of ribs. The sks gene has been mapped to Chromosome 4, 16.6 cM distal to the brown locus.

Animals↗
PubMed2004

Tesmin transcription is regulated differently during male and female meiosis.

Tesmin is a protein with homology to the metal-binding motif of the metallothionein protein family. Tesmin has been described as a testis-specific transcript, which starts to accumulate in 8-day-old mouse spermatocytes. Herein, a differential display comparing meiotic gene expression in embryonic ovaries and mature testes also revealed the presence of the Tesmin transcript in fetal ovaries as well as in fetal and adult heart. Time-course experiments showed that Tesmin was expressed in a characteristic development-related manner in fetal ovaries. Only a weak expression was observed at E12(1/2), the strongest signal was reached at E14(1/2), whereas the signal declined between E14(1/2) and E16(1/2). This transitional expression coincides with the early stages of the female meiotic prophase I. In the male, however, Tesmin was expressed in all stages of meiotic prophase I except preleptonema and leptonema. In situ hybridization further showed that the mRNA level increased during prophase I in the male, with the strongest expression seen at the transition from mid- to late pachytema (Stage VII-VIII). Furthermore, initiation of Tesmin transcription paralleled that of the synaptonemal complex protein 1 transcript (Scp1) in the fetal ovary and prepubertal testis. We, therefore, propose that Tesmin is likely to have a function in both the male and female meiotic prophase I. Moreover, the distinct difference in both the timing and the level of mRNA accumulation in the two gender's meiotic prophase I suggests that Tesmin transcription may be controlled by two different mechanisms during male and female meiosis. Mol. Reprod. Dev. 67: 116-126, 2004.

Amino Acid Sequence↗
PubMed2006

Capillary morphogenesis gene (CMG)-1 is among the genes differentially expressed in mouse male germ line stem cells and embryonic stem cells.

We recently established a technique to expand male germ line stem (GS) cells in long-term culture without losing their spermatogenic capacity. To gain insight into the genetic program of these cells, we compared the mRNA expression profile of GS cells with that of embryonic stem (ES) cells using DNA microarrays. We found 79 genes that were upregulated in GS cells compared to ES cells, including synaptonemal complex protein-1, deleted in azoospermia-like, ubiquitin-conjugating enzyme E2B, and ubiquitin carboxy-terminal hydrolase L1, all of which are functionally important for spermatogenesis. In addition, we identified a cDNA encoding the mouse ortholog of capillary morphogenesis gene (CMG)-1. CMG-1 transcripts were predominantly produced in spermatogonia and spermatocytes in mouse testis. When CMG-1 expression was attenuated in a mouse spermatocyte-derived cell line, GC-2spd(ts), by a target-specific short interfering RNA, the morphology of the cells was changed and the expression of cyclin D2 was abrogated. A reporter assay using a genomic region upstream of the mouse cyclin D2 gene revealed that this downmodulation occurs at the transcriptional level. We detected FLAG-tagged CMG-1 protein in the nuclei of transfected COS7 cells, suggesting that CMG-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene. The upregulated GS genes identified in this study will provide useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation.

Animals↗
PubMed2005

Proteomics profiling of nuclear proteins for kidney fibroblasts suggests hypoxia, meiosis, and cancer may meet in the nucleus.

Proteomics methods were used to characterize proteins that change their form or abundance in the nucleus of NRK49F rat kidney fibroblasts during prolonged hypoxia (1% O(2), 12 h). Of the 791 proteins that were monitored, about 20% showed detectable changes. The 51 most abundant proteins were identified by mass spectrometry. Changes in nuclear receptor transcription factors (THRalpha1, RORalpha4, HNF4alpha, NUR77), other transcription factors (GATA1, AP-2alpha, OCT1, ATF6alpha, ZFP161, ZNF354A, PDCD2), and transcription cofactors (PC4, PCAF, MTA1, TCEA1, JMY) are indicative of major, co-ordinated changes in transcription. Proteins involved in DNA repair/recombination, ribosomal RNA synthesis, RNA processing, nuclear transport, nuclear organization, protein translation, glycolysis, lipid metabolism, several protein kinases (PKCdelta, MAP3K4, GRK3), as well as proteins with no established functional role were also observed. The observed proteins suggest nuclear regulatory roles for proteins involved in cytosolic processes such as glycolysis and fatty acid metabolism, and roles in overall nuclear structure/organization for proteins previously associated with meiosis and/or spermatogenesis (synaptonemal complex proteins 1 and 2 (SYCP1, SYCP2), meiosis-specific nuclear structural protein 1 (MNS1), LMNC2, zinc finger protein 99 (ZFP99)). Proteins associated with cytoplasmic membrane functions (ACTN4, hyaluronan mediated motility receptor (RHAMM), VLDLR, GRK3) and/or endocytosis (DNM2) were also seen. For 30% of the identified proteins, new isoforms indicative of alternative transcription were detected (e.g., GATA1, ATF6alpha, MTA1, MLH1, MYO1C, UBF, SYCP2, EIF3S10, MAP3K4, ZFP99). Comparison with proteins involved in cell death, cancer, and testis/meiosis/spermatogenesis suggests commonalities, which may reflect fundamental mechanisms for down-regulation of cellular function.

Animals↗
PubMed1995

Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis.

Little is known about the control of events ending the lengthy prophase of meiosis I and leading to the G2/M-phase transition in mammalian spermatocytes, primarily because the relevant late pachytene, diplotene, and MI cells are present in low numbers in the testis and it is not possible to isolate them in significant numbers. We have utilized short-term cultures of pachytene spermatocytes from the mouse to study events of the G2/M cell-cycle transition induced by the protein phosphatase inhibitor okadaic acid (OA). Treatment of cultured pachytene spermatocytes with OA induced a rapid and premature onset of events leading to the M phase, visualized cytologically by nuclear envelope breakdown and chromosome condensation. After OA treatment, condensed chromosomes were seen as bivalents, not as univalents. Treatment with OA induced disassembly of synaptonemal complexes and resolution of crossovers as cytologically visible chiasmata. Chiasmata counts were similar in treated cells and control cells. Thus, surprisingly, even though the treated cells were in the pachytene substage of meiotic prophase, events of recombination were apparently completed to the point of chiasma formation in the majority of these cells. The sex chromosomes, forming the sex body of the pachytene spermatocyte, lagged behind the autosomal chromosomes in their condensation and progression toward the M phase. Treatment with OA induced an increase in histone H1 kinase activity, generally used as an indicator of metaphase-promoting factor (MPF) activity; furthermore, the OA-induced cell-cycle transition does not require new protein synthesis. These results suggest that OA treatment overrides a cell-cycle checkpoint control that normally keeps pachytene spermatocytes in a lengthy prophase and that this control may be exerted by regulation of protein phosphorylation status.

Animals↗
PubMed2002

Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: insights into regulation of spermatogenic progress.

The MLH1 protein is required for normal meiosis in mice and its absence leads to failure in maintenance of pairing between bivalent chromosomes, abnormal meiotic division, and ensuing sterility in both sexes. In this study, we investigated whether failure to develop foci of MLH1 protein on chromosomes in prophase would lead to elimination of prophase spermatocytes, and, if not, whether univalent chromosomes could align normally on the meiotic spindle and whether metaphase spermatocytes would be delayed and/or eliminated. In spite of the absence of MLH1 foci, no apoptosis of spermatocytes in prophase was detected. In fact, chromosomes of pachytene spermatocytes from Mlh1(-/-) mice were competent to condense metaphase chromosomes, both in vivo and in vitro. Most condensed chromosomes were univalents with spatially distinct FISH signals. Typical metaphase events, such as synaptonemal complex breakdown and the phosphorylation of Ser10 on histone H3, occurred in Mlh1(-/-) spermatocytes, suggesting that there is no inhibition of onset of meiotic metaphase in the face of massive chromosomal abnormalities. However, the condensed univalent chromosomes did not align correctly onto the spindle apparatus in the majority of Mlh1(-/-) spermatocytes. Most meiotic metaphase spermatocytes were characterized with bipolar spindles, but chromosomes radiated away from the microtubule-organizing centers in a prometaphase-like pattern rather than achieving a bipolar orientation. Apoptosis was not observed until after the onset of meiotic metaphase. Thus, spermatocytes are not eliminated in direct response to the initial meiotic defect, but are eliminated later. Taken together, these observations suggest that a spindle assembly checkpoint, rather than a recombination or chiasmata checkpoint, may be activated in response to meiotic errors, thereby ensuring elimination of chromosomally abnormal gamete precursors.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Change of karyoskeleton during mammalian spermatogenesis: expression pattern of nuclear lamin C2 and its regulation.

Nuclear lamins are a multigene family of major karyoskeletal proteins. The expression pattern of members of the lamin family has been shown to be developmentally regulated. Of particular interest have been the findings that mammalian spermatogenic cells express two small lamin isoforms (B3 and C2) which are shorter splicing variants of their somatic counterparts (the lamins B2 and C, respectively). Although lamins B3 and C2 appear to be specific for the germ line, the expression pattern and localization of these proteins is not known. In the present study, we present evidence that during rat spermatogenesis lamin C2 is selectively expressed in spermatocytes, i.e., the cells undergoing meiosis. As in the case of other members of the family, lamin C2 was detected at the nuclear periphery. Northern blotting and in situ hybridization indicate that meiotic expression of lamin C2 is regulated at the transcriptional level. This is in contrast to the situation during amphibian oogenesis, where lamin expression is largely posttrascriptionally regulated. Interestingly, the expression pattern of lamin C2 temporally coincides with that of structural protein components of another meiosis-specific karyoskeletal structure, the synaptonemal complex (SC). Taken together, we conclude that pairing and recombination of homologous chromosomes during meiotic prophase is accompanied by significant changes in the organization of the karyoskeleton which are accomplished by the expression of stage-specific proteins.

Amino Acid Sequence↗
PubMed1997

Mapping of eight testis-specific genes to mouse chromosomes.

We previously identified eight testis-specific genes using antibodies raised against testicular germ cells. They are expressed during spermatogenesis and are presumed to be involved in testicular germ cell differentiation and sperm formation. We have mapped the genomic loci for these testis-specific genes using restriction fragment length variants in interspecific backcross mice. The calmegin gene (Clgn) was mapped to Chr 8. The synaptonemal complex protein gene 1 (Sycp1) probe hybridized with two sequences on different chromosomes; Sycp1-rs2 was mapped to Chr 3, whereas Sycp1-rs3 was mapped to Chr 7. The relaxin-like factor gene (Rlnl) was mapped to Chr 8, and collapsin response mediator protein 1 (Crmp1) was mapped to Chr 5. Three novel genes encoding testis-specific proteins A2 (Tsga2), A8 (Tsga8), and A12 (Tsga12) were mapped to chromosomes 3, X, and 10, respectively.

Animals↗
PubMed2002

A new gene family (FAM9) of low-copy repeats in Xp22.3 expressed exclusively in testis: implications for recombinations in this region.

Illegitimate recombinations between low-copy repetitive elements (LCR) have been implicated in the pathogenesis of various chromosomal rearrangements. Two such duplicons have been reported previously on Xp22.3, the CRI-S232 elements, involved in the generation of deletions in the steroidsulfatase gene and five members of the G1.3 (DXF22S) repetitive sequence family. By molecular characterization of an Xp22/10q24 translocation, we identified one duplicon of the G1.3 family in the breakpoint region in Xp22.3. We show that G1.3 elements harbor at least three expressed genes, FAM9A, FAM9B, and FAM9C, and three putative pseudogenes, all mapped to Xp22.33-p22.31. The deduced amino acid sequence of the three novel proteins shows homology to SYCP3, a component of the synaptonemal complex located along the paired chromosomes during meiosis. FAM9A, FAM9B, and FAM9C are expressed exclusively in testis; their proteins are located in the nucleus, and FAM9A localizes to the nucleolus. The presence of genes within duplicons may represent putative recombination-promoting factors for actively transcribed genes in meiotic cells, with the resulting open chromatin structure facilitating unequal crossing-over events and chromosomal rearrangements.

Amino Acid Sequence↗
PubMed1998

Affinity chromatographic purification of antibodies to a biotinylated fusion protein expressed in Escherichia coli.

HOP1, a protein component of the synaptonemal complex in Saccharomyces cerevisiae which is believed to play an important role in meiotic synapsis, was expressed in Escherichia coli as a fusion protein incorporating a "tag" polypeptide which is biotinylated naturally in the bacteria. The HOP1 fusion protein was produced in an insoluble form within the bacteria; once solubilized using a denaturing agent, the protein was purified by avidin monomer affinity chromatography. The recombinant protein was used to immunize rabbits and produce polyclonal antibodies. Procedures for affinity purification of antibodies using the recombinant protein attached to the avidin column and a magnetic method for concentration of antibodies are described. Antibody elution conditions in these procedures do not affect the affinity of the column for the recombinant protein, which can be recovered afterward. Affinity-purified antibodies show high binding capacity to HOP1 recombinant protein in immunoblotting experiments, but reduced background compared with crude antiserum or purified IgG fraction. The affinity-purified antibodies recognize a major band around 70 kDa in Western blots of yeast protein extracts following meiotic induction.

Animals↗
PubMed1999

Heterologously expressed polypeptide from the yeast meiotic gene HOP1 binds preferentially to yeast DNA.

HOP1 protein, present in sporulating cells of Saccharomyces cerevisiae and believed to be a component of the synaptonemal complex, has been expressed in Escherichia coli fused to a biotinylated tag protein. Once solubilized from bacterial inclusion bodies, the HOP1 fusion protein was purified by using a combination of avidin-affinity chromatography and gel filtration FPLC and refolded. Sequence comparisons indicate that the HOP1 gene product contains a zinc finger motif, which may confer DNA binding properties, and the recombinant polypeptide was used to assess the putative DNA binding properties of the product of native HOP1 protein using a gel-shift assay. Protein and protein-DNA complexes were detected by exploiting the affinity of streptavidin-alkaline phosphatase for the biotinylated tag protein after Western blotting. The HOP1 fusion protein bound unambiguously to digested genomic yeast DNA. This binding possessed some degree of specificity, was maintained under a wide range of salt concentrations, and was unaffected by the presence of high concentrations of competitor DNA (synthetic poly[dI-dC].poly[dI-dC]). In contrast, no shift was detected when the fusion protein was incubated with digested genomic DNA from Arabidopsis, or with lambda/HindIII DNA. Incubation with digested genomic DNA from Lilium produced a small change in the mobility of the protein. The biotinylated tag protein failed to show any DNA binding activity. Scatchard analysis indicated an apparent yeast genomic DNA:HOP1 fusion protein dissociation constant of K(d) = 5 x 10(-7) M.

Amino Acid Sequence↗
PubMed1994

Transgenerational, ultrastructural analysis on the antioxidative effects of tocopherol on early gametogenesis in Caenorhabditis elegans grown in 100% oxygen.

The random, free-radical-mediated oxidations of biological molecules result in membrane degradation leading to cellular deterioration (B. Halliwell, Free Radical Res. Commun. 9, 1-32, 1990). External oxygen, prooxidants, and internally produced oxygen free radicals (oxyradicals), interact and alter the nature of biomembranes. Antioxidants, e.g., tocopherol (Vitamin E), inhibit such oxidative damage of free radicals. In the present study, the nematode Caenorhabditis elegans was grown under hyperoxia (100% oxygen) with or without the addition of Vitamin E to the growth media. The nematodes were viable under such conditions for at least eight generations, although fecundity gradually decreased through successive generations, presumably due to genetic load. Vitamin E was also shown to have a protective effect against paraquat, which is a strong, intracellular, oxidizing agent. Ultrastructural observations of early meiosis showed that the formation of synaptonemal complexes was compromised and that the telomeres failed to attach to the nuclear envelope. Those nematodes grown in 100% oxygen with 200 micrograms/ml Vitamin E had normal meiotic structures and normal fecundity. Thus, the presence of enhanced levels of intracellular Vitamin E resulted in protection against oxidative stress during gametogenesis.

Animals↗