Biomedical subjects

Christopher A Makaroff

Publications and source records attributed to Christopher A Makaroff.

13 recordsLinked to original sources

PubMed2006

The Arabidopsis SKP1 homolog ASK1 controls meiotic chromosome remodeling and release of chromatin from the nuclear membrane and nucleolus.

During early stages of meiotic prophase I the nucleus undergoes considerable reorganization, including the clustering of telomeres, the release of contacts between chromosomes and the nuclear membrane, the reorganization of the nucleolus, and chromatin remodeling. Using a light squashing technique for the analysis of meiotic chromosomes along with fluorescent in situ hybridization, transmission electron microscopy and immunolocalization studies with antibodies to modified histones, we demonstrate that ASK1 is essential for early nuclear reorganization events. A relatively large number of meiotic alterations have been identified in ask1-1 plants. We show that many of these defects, including alterations in homolog pairing, nucleolus migration and the missegregation of chromosomes, may arise from alterations in chromatin structure and the inability of chromosomes to resolve and release properly from the nuclear membrane and nucleolus during leptotene. These results raise the interesting possibility that ASK1 controls chromatin structure by targeting of either an early regulator of meiotic progression or possibly matrix attachment proteins for destruction.

Arabidopsis↗

PubMed2006

Structure of an ETHE1-like protein from Arabidopsis thaliana.

The protein product of gene At1g53580 from Arabidopsis thaliana possesses 54% sequence identity to a human enzyme that has been implicated in the rare disorder ethylmalonic encephalopathy. The structure of the At1g53580 protein has been solved to a nominal resolution of 1.48 Angstrom. This structure reveals tertiary structure differences between the ETHE1-like enzyme and glyoxalase II enzymes that are likely to account for differences in reaction chemistry and multimeric state between the two types of enzymes. In addition, the Arabidopsis ETHE1 protein is used as a model to explain the significance of several mutations in the human enzyme that have been observed in patients with ethylmalonic encephalopathy.

Amino Acid Sequence↗

PubMed2006

ASK1, a SKP1 homolog, is required for nuclear reorganization, presynaptic homolog juxtaposition and the proper distribution of cohesin during meiosis in Arabidopsis.

Nuclear reorganization and juxtaposition of homologous chromosomes at late leptotene/early zygotene are essential steps before chromosome synapsis at pachytene. We report the results of detailed studies, which demonstrate that nuclear reorganization and homolog juxtapositioning processes are defective in a null mutant, ask1-1. Our results from 4, 6-diamino-2-phenylindole (DAPI)-stained spreads showed that the "synizetic knot", which is typically found in wild type (WT) meiosis during late leptotene and zygotene, was missing in the ask1-1 mutant. Furthermore, ask1-1 meiocytes exhibited only limited homolog juxtaposition at centromere regions at early zygotene. Immunodetection of the cohesin protein SYN1 identified ask1 defects in cohesin distribution from zygotene to anaphase I. Analysis of meiotic chromosomes in ask1-1 and syn1 single mutants, as well as an ask1-1 syn1 double mutant indicate that ASK1 is required for normal SYN1 distribution during meiotic prophase I and suggest that ask1 associated defects may be primarily related to SYN1 mislocalization.

Arabidopsis↗

PubMed2006

Arabidopsis separase AESP is essential for embryo development and the release of cohesin during meiosis.

To investigate how and when sister chromatid cohesion is released from chromosomes in plants, we isolated the Arabidopsis thaliana homolog of separase (AESP) and investigated its role in somatic and meiotic cells. AESP is similar to separase proteins identified in other organisms but contains several additional structural motifs. The characterization of two Arabidopsis T-DNA insertion alleles for AESP demonstrated that it is an essential gene. Seeds homozygous for T-DNA insertions in AESP exhibited embryo arrest at the globular stage. The endosperm also exhibited a weak titan-like phenotype. Transgenic plants expressing AESP RNA interference (RNAi) from the meiosis-specific DMC1 promoter exhibited alterations in chromosome segregation during meiosis I and II that resulted in polyads containing from one to eight microspores. Consistent with its predicted role in the release of sister chromatid cohesion, immunolocalization studies showed that the removal of SYN1 from chromosome arms and the centromeres is inhibited in the RNAi mutants. However, the release of SYN1 during diplotene occurred normally, indicating that this process is independent of AESP. Therefore, our results demonstrate that AESP plays an essential role in embryo development and provide direct evidence that AESP is required for the removal of cohesin from meiotic chromosomes.

Amino Acid Motifs↗

PubMed2005

Structural studies on a mitochondrial glyoxalase II.

Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).

Amino Acid Sequence↗

PubMed2005

Characterization of Arabidopsis thaliana SMC1 and SMC3: evidence that AtSMC3 may function beyond chromosome cohesion.

Structural maintenance of chromosome (SMC) proteins are conserved in most prokaryotes and all eukaryotes examined. SMC proteins participate in many different aspects of chromosome folding and dynamics. They play essential roles in complexes that are responsible for sister chromatid cohesion, chromosome condensation and DNA repair. As part of studies to better understand SMC proteins and sister chromatid cohesion in plants we have characterized Arabidopsis SMC1 and SMC3. Although transcripts for AtSMC1 and AtSMC3 are present throughout the plant, transcript levels for the two genes vary between different tissues. Cell fractionation and immunolocalization results showed that AtSMC3 was present in the nucleus and cytoplasm. In the nucleus, it is primarily associated with the nuclear matrix during interphase and with chromatin from prophase through anaphase in both somatic and meiotic cells. During mitosis and meiosis the protein also co-localized with the spindle from metaphase to telophase. The distribution of AtSMC3 in syn1 mutant plants indicated that SYN1 is required for the proper binding of AtSMC3 to meiotic chromosomes, but not the spindle. Data presented here represent the first detailed cytological study of a plant SMC protein and suggest that SMC3 may have multiple functions in plants.

3' Untranslated Regions↗

PubMed2005

The AtRAD51C gene is required for normal meiotic chromosome synapsis and double-stranded break repair in Arabidopsis.

Meiotic prophase I is a complex process involving homologous chromosome (homolog) pairing, synapsis, and recombination. The budding yeast (Saccharomyces cerevisiae) RAD51 gene is known to be important for recombination and DNA repair in the mitotic cell cycle. In addition, RAD51 is required for meiosis and its Arabidopsis (Arabidopsis thaliana) ortholog is important for normal meiotic homolog pairing, synapsis, and repair of double-stranded breaks. In vertebrate cell cultures, the RAD51 paralog RAD51C is also important for mitotic homologous recombination and maintenance of genome integrity. However, the function of RAD51C in meiosis is not well understood. Here we describe the identification and analysis of a mutation in the Arabidopsis RAD51C ortholog, AtRAD51C. Although the atrad51c-1 mutant has normal vegetative and flower development and has no detectable abnormality in mitosis, it is completely male and female sterile. During early meiosis, homologous chromosomes in atrad51c-1 fail to undergo synapsis and become severely fragmented. In addition, analysis of the atrad51c-1 atspo11-1 double mutant showed that fragmentation was nearly completely suppressed by the atspo11-1 mutation, indicating that the fragmentation largely represents a defect in processing double-stranded breaks generated by AtSPO11-1. Fluorescence in situ hybridization experiments suggest that homolog juxtaposition might also be abnormal in atrad51c-1 meiocytes. These results demonstrate that AtRAD51C is essential for normal meiosis and is probably required for homologous synapsis.

Arabidopsis↗

PubMed2004

The binding of iron and zinc to glyoxalase II occurs exclusively as di-metal centers and is unique within the metallo-beta-lactamase family.

Cytosolic glyoxalase 2 (GLX2-2) from Arabidopsis thaliana is a metalloenzyme that has been shown to bind a mixture of Zn, Fe, or Mn when produced in cells grown in rich media. In an effort to prepare metal-enriched samples, GLX2-2 was over-expressed in minimal media containing either Zn, Fe, or Mn. The resulting enzymes bound an average of 1 equivalent of metal ion and were partially enriched with a specific metal ion. The enzymes produced in minimal media were active towards the substrate S-D-lactoylglutathione, yielding kcat/ Km values similar to those of rich media GLX2-2. EPR studies on minimal media GLX2-2 samples revealed spectra which were identical to those over-expressed in rich media that contained nearly 2 equivalents of metal. The EPR spectra showed the presence of antiferromagnetically and ferromagnetically coupled, dinuclear metal centers. EXAFS spectra on the minimal media GLX2-2 samples over-expressed in the presence of Fe or Zn were also very similar to those of the rich media GLX2-2 samples, indicating the presence of dinuclear metal centers. The EXAFS studies also demonstrate that Zn(II) and Fe (in the Fe-enriched sample) are distributed in the dinuclear site. These data indicate that the minimal media GLX2-2 samples are a mixture of fully loaded, dinuclear metal-containing enzyme and metal-free enzyme. This characteristic of A. thaliana GLX2-2 makes it unique among the other members of the metallo-beta-lactamase family in that it does not ever appear to exist as a mononuclear metal ion containing enzyme and that it exhibits positive cooperativity in metal binding.

Arabidopsis↗

PubMed2004

Characterization of an unusual Ds transposable element in Arabidopsis thaliana: insertion of an abortive circular transposition intermediate.

The maize Ac/Ds transposable elements, which belong to the hAT transposon superfamily, are widely used as insertional mutagens in numerous plant species. Molecular studies suggest that Ac/Ds elements transpose in a conservative non-replicative fashion; however the molecular mechanism of transposition remains unclear. We describe here the identification of an unusual Ds element, Ds-mmd1 , in a transgenic Arabidopsis line. Ds-mmd1 is rearranged relative to the original Ds element, such that the original 5' and 3' ends are internal and previously internal sequences are the new 5' and 3' termini of Ds-mmd1 . Short duplications of plant genomic DNA and Ds sequences are present at the Ds-mmd1 junctions, suggesting that a circular Ds molecule was part of the events that created the Ds-mmd1 element. In addition, a revertant analysis on mmd1 plants demonstrated that Ds-mmd1 can be eliminated from the genome in an Ac -dependent process.

Arabidopsis↗

PubMed2003

Explaining the inhibition of glyoxalase II by 9-fluorenylmethoxycarbonyl-protected glutathione derivatives.

In an effort to probe the inhibition of glyoxalase II (GLX2-2) from Arabidopsis thaliana, a series of N- and S-blocked glutathione compounds containing 9-fluorenylmethoxycarbonyl (FMOC) and Cbz protecting groups were synthesized and tested. The di-FMOC and di-Cbz compounds were the best inhibitors of GLX2-2 with K(i) values of 0.89+/-0.05 and 2.3+/-0.5 microM, respectively. The removal of protecting groups from either position resulted in comparable, diminished binding affinities. Analyses of site-directed mutants of GLX2-2 demonstrated that tight binding of these inhibitors is not due to interactions of the protecting groups with hydrophobic amino acids on the surface of the enzyme. Instead, MM2 calculations predict that the lowest energy structures of the unbound, doubly substituted inhibitors are similar to those of a bound inhibitor. These studies represent the first systematic attempt to understand the peculiar inhibition of GLX2 by N- and S-blocked glutathiones.

Arabidopsis↗

PubMed2003

The Arabidopsis SYN1 cohesin protein is required for sister chromatid arm cohesion and homologous chromosome pairing.

The faithful transmission of chromosomes during mitosis and meiosis requires the establishment and subsequent release of cohesion between replicated chromosomes. Sister chromatid cohesion is mediated, in large part, by the cohesin complex, which consists of four highly conserved proteins: SMC1, SMC3, SCC1/REC8 and SCC3. Mitotic cohesin complexes contain SSC1, whereas meiotic cohesin complexes contain the related REC8 protein. As part of studies to identify and characterize proteins required for meiosis in plants, we previously identified a putative Arabidopsis REC8 homolog, referred to as syn1. Preliminary cytological studies indicated that syn1 plants exhibit defects in meiotic chromosome cohesion and condensation that result in fragmentation of the chromosomes and the formation of polyads. In the experiments presented here we show that SYN1 encodes a protein that localizes to arms of meiotic chromosomes from approximately meiotic interphase to anaphase I. The protein is not detected at the centromeres or after metaphase I. Furthermore, fluorescence in situ hybridization experiments on microsporocytes from syn1 plants demonstrate that the mutation eliminates arm cohesion as early as interphase, whereas centromere cohesion is maintained until approximately anaphase I. These results indicate that although the main role of SYN1 is in chromosome arm cohesion, it is also important for maintaining cohesion at the centromeres during late stages of meiosis I.

Anaphase↗

PubMed2003

The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis.

We report the detailed characterization of SWITCH1 (SWI1) an Arabidopsis thaliana protein that has been linked with the establishment of sister chromatid cohesion during meiosis. Using a combination of cytological methods including immunolocalization of meiotic chromosome-associated proteins we show that SWI1 is required for formation of axial elements. Our studies reveal that the swi1-2 mutation prevents the formation of RAD51 foci during meiotic prophase and suppresses the chromosome fragmentation phenotype of the recombination-defective dif1-1 mutant. Together, these data suggest that SWI1 may be required for meiotic recombination initiation. Finally we raised an antibody against SWI1 and showed, by immunolocalization coupled with bromodeoxyuridine incorporation experiments, that SWI1 is expressed exclusively in meiotic G(1) and S phase. Thus, SWI1 appears to be required for early meiotic events that are at the crossroad of sister chromatid cohesion, recombination and axial element formation. The possible inter-relationship between these processes and the function of SWI1 are discussed.

Alleles↗

PubMed2003

The Arabidopsis MALE MEIOCYTE DEATH1 gene encodes a PHD-finger protein that is required for male meiosis.

In plants, reproductive development requires normal meiosis, which involved several highly coordinated events. Such meiotic events are regulated in a number of ways in yeast and animal systems, including transcriptional and checkpoint control mechanisms. Although a number of mutations that affect different aspects of meiosis have been characterized in plants, very little is known about the regulation of plant meiosis at the molecular level. In particular, no meiosis-specific transcriptional regulators have been identified in plants, and checkpoint control has not been observed during plant meiosis. We report here the isolation and characterization of a new Arabidopsis male-sterile mutant that exhibits meiotic defects. Meiocytes from mutant plants appeared normal up to diakinesis, when they exhibited signs of apoptosis, including defects in chromosome behavior, cytoplasmic shrinkage, and chromatin fragmentation, followed by cell death before cytokinesis. Therefore, the mutant was named male meiocyte death1 (mmd1). The MMD1 gene was cloned using Dissociation transposon tagging and encodes a plant homeo domain domain-containing protein. MMD1 is expressed preferentially during male meiosis. Our results suggest that MMD1 may be involved in the regulation of gene expression during meiosis and that the mmd1 mutation triggers cell death in male meiocytes.

Amino Acid Sequence↗